Note: Descriptions are shown in the official language in which they were submitted.
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TRUNCATED PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE
FIELD OF THE INVENTION
The present invention relates generally to platelet-activating factor
acetylhydrolase and more specifically to novel purified and isolated
polynucleotides
encoding human plasma platelet-activating factor acetyihydrolase, to the
platelet-
activating factor acetylhydrolase products encoded by the polynucleoddes, to
materials
and methods for the recombinant production of platelet-activating factor
acetylhydrolase products and to antibody substances specif c for platelet-
activating
factor acetylhydrolase.
BACKGROUND
Platelet-activating factor (PAF) is a biologically active phospholipid
synthesized by various cell types. In vivo and at normal concentrations of 10-
10 to
10-9 M, PAF activates target cells such as platelets and neutrophils by
binding to
specific G protein-coupled cell surface receptors [Venable et al. , J. Lipid
Res. , 34:
691-701 (1993)]. PAF has the structure I-O-alkyl-2-acetyl-sn-glycero-3-
phosphocholine. For optimal biological activity, the sn-1 position of the PAF
glycerol backbone must be in an ether linkage with a fatty alcohol and the sn-
3
position must have a phosphocholine head group.
PAF functions in normal physiological processes (e.g., inflammation,
hemostasis and parturition) and is implicated in pathological inflammatory
responses
(e. g. , asthma, anaphylaxis, septic shock and arthritis) [Venable et al. ,
supra, and
Lindsberg et al. , Ann. lVeurol. , 30: I 17-129 ( 1991 )] . The likelihood of
PAF
involvement in pathological responses has prompted attempts to modulate the
activity
of PAF and the major focus of these attempts has been the development of
antagonists
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of PAF activity which interfere with binding of PAF to cell surface receptors.
See,
for example, Heuer et al., Clin. Exp. Allergy, 22: 980-983 (1992).
The synthesis and secretion of PAF as well as its degradation and
clearance appear to be tightly controlled. To the extent that pathological
inflammatory actions of PAF result from a failure of PAF regulatory mechanisms
giving rise to excessive production, inappropriate production or lack of
degradation,
an alternative means of modulating the activity of PAF would involve mimicing
or
augmenting the natural process by which resolution of inflammation occurs.
Macrophages [Stafforini et al. , J. Biol. Chem. , 265( 17): 9682-9687 (
1990)],
hepatocytes and the human hepatoma cell line HepG2 [Satoh et al., J. Clin.
Invest.,
87: 476-481 (1991) and Tarbet et al., J. Biol. Chem., 265(25): 16667-16673
(1991)]
have been reported to release an enzymatic activity, PAF acetylhydrolase (PAF-
AH),
that inactivates PAF. In addition to inactivating PAF, PAF-AH also inactivates
oxidatively fragmented phospholipids such as products of the arachidonic acid
cascade
that mediate inflammation. See, Stremler et al. , J. Biol. Chem. , 266(17):
11095-
11103 (1991). The inactivation of PAF by PAF-AH occurs primarily by hydrolysis
of the PAF sn-2 acetyl group and PAF-AH metabolizes oxidatively fragmented
phospholipids by removing sn-2 acyl groups. Two types of PAF-AH have been
identified: cytoplasmic forms found in a variety of cell types and tissues
such as
endothelial cells and erythrocytes, and an extracellular form found in plasma
and
serum. Plasma PAF-AH does not hydrolyze intact phospholipids except for PAF
and
this substrate specificity allows the enzyme to circulate in vivo in a fully
active state
without adverse effects. The plasma PAF-AH appears to account for all of the
PAF
degradation in human blood ex vivo [Stafforini et al., J. Biol. Chem., 262(9):
4223-
4230 (1987)].
While the cytoplasmic and plasma forms of PAF-AH appear to have
identical substrate specificity, plasma PAF-AH has biochemical characteristics
which
distinguish it from cytoplasmic PAF-AH and from other characterized lipases.
Specifically, plasma PAF-AH is associated with lipoprotein particles, is
inhibited by
diisopropyl fluorophosphate, is not affected by caicium ions, is relatively
insensitive
to proteolysis, and has an apparent molecular weight of 43,000 daltons. See,
Stafforini et al. (1987), supra. The same Stafforini et al. article describes
a
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procedure for partial purification of PAF-AH from human plasma and the amino
acid
composition of the plasma material obtained by use of the procedure.
Cytoplasmic
PAF-AH has been purified from erythrocytes as reported in Stafforini et al. ,
J. Biol.
Chem., 268(6): 3857-3865 (1993) and ten amino terminal residues of cytoplasmic
PAF-AH are also described in the article. Hattori et al. , J. Biol. Chem. ,
268(25):
18748-18753 {1993) describes the purification of cytoplasmic PAF-AH from
bovine
brain. Subsequent to filing of the parent application hereto the nucleotide
sequence
of bovine brain cytoplasmic PAF-AH was published in Hattori et al. , J. Biol.
Chem. ,
269(237): 23150-23155 (1994). On January 5, 1995, three months after the
filing
date of the parent application hereto, a nucleotide sequence for a lipoprotein
associated phospholipase A2 (Lp-PLA2) was published in Smithkline Beecham PLC
Patent Cooperation Treaty (PCT) International Publication No. WO 95/00649. The
nucleotide sequence of the Lp-PLA2 differs at one position when compared to
the
nucleotide sequence of the PAF-AH of the present invention. The nucleotide
difference {corresponding to position 1297 of SEQ ID NO: 7) results in an
amino acid
difference between the enzymes encoded by the polynucleotides. The amino acid
at
position 379 of SEQ ID NO: 8 is a valine while the amino acid at the
corresponding
position in Lp-PLA2 is an alanine. In addition, the nucleotide sequence of the
PAF-
AH of the present invention includes 124 bases at the 5' end and twenty bases
at the
3' end not present in the Lp-PLA2 sequence. Three months later, on April 10,
1995,
a Lp-PLA2 sequence was deposited in GenBank under Accession No. U24577 which
differs at eleven positions when compared to the nucleotide sequence of the
PAF-AH
- of the present invention. The nucleotide differences (corresponding to
position 79,
81, 84, 85, 86, 121, 122, 904, 905, 911, 983 and 1327 of SEQ ID NO: 7) results
in
four amino acid differences between the enzymes encoded by the
polynucleotides.
The amino acids at positions 249, 250, 274 and 389 of SEQ ID NO: 8 are lysine,
aspartic acid, phenylalanine and leucine, respectively, while the respective
amino acid
at the corresponding positions in the GenBank sequence are isoleucine,
arginine,
ieucine and serine.
The recombinant production of PAF-AH would make possible the use
of exogenous PAF-AH to mimic or augment normal processes of resolution of
inflammation in vivo. The administration of PAF-AH would provide a
physiological
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advantage over administration of PAF receptor antagonists
because PAF-AH is a product normally found in plasma.
Moreover, because PAF receptor antagonists which are
structurally related to PAF inhibit native PAF-AH activity,
the desirable metabolism of PAF and of oxidatively
fragmented phospholipids is thereby prevented. Thus, the
inhibition of PAF-AH activity by PAF receptor antagonists
counteracts the competitive blockade of the PAF receptor by
the antagonists. See, Stremler et al., supra. In addition,
in locations of acute inflammation, for example, the release
of oxidants results in inactivation of the native PAF-AH
enzyme in turn resulting in elevated local levels of PAF and
PAF-like compounds which would compete with any exogenously
administered PAF receptor antagonist for binding to the PAF
receptor. In contrast, treatment with recombinant PAF-AH
would augment endogenous PAF-AH activity and compensate for
any inactivated endogenous enzyme.
There thus exists a need in the art to identify
and isolate polynucleotide sequences encoding human plasma
PAF-AH, to develop materials and methods useful for the
recombinant production of PAF-AH and to generate reagents
for the detection of PAF-AH in plasma.
SUMMARY OF THE INVENTION
The present invention provides novel purified and
isolated polynucleotides (i.e., DNA and RNA both sense and
antisense strands) encoding human plasma PAF-AH or
enzymatically active fragments thereof.
The invention also provides a purified and
isolated human plasma platelet-activating factor
acetylhydrolase (PAF-AH) polypeptide product which is
lacking up to the first twelve N-terminal amino acids of the
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mature human PAF-AH amino acid sequence set out in
SEQ ID NO: 8.
Preferred DNA sequences of the invention include
genomic and cDNA sequences as well as wholly or partially
chemically synthesized DNA sequences. The DNA sequence
encoding PAF-AH that is set out in SEQ ID NO: 7 and DNA
sequences which hybridize to the noncoding strand thereof
under standard stringent conditions or which would hybridize
but for the redundancy of the genetic code, are contemplated
by the invention. Also contemplated by the invention are
biological replicas (i.e., copies of isolated DNA sequences
made in vivo or in vi tro) of DNA sequences of the invention.
Autonomously replicating recombinant constructions such as
plasmid and viral DNA vectors incorporating PAF-AH sequences
and especially vectors wherein DNA encoding PAF-AH is
operatively linked to an endogenous or exogenous expression
control DNA sequence and a transcription terminator are also
provided.
The invention also contemplates a human PAF-AH
polypeptide product which has an amino acid replacement in
the sequence of SEQ ID NO: 8 selected from the group
consisting of: (a) D 286 A; (b) D 286 N; and (c) D 304 A.
The invention also contemplates an isolated
polynucleotide encoding a human PAF-AH polypeptide product
having Met46 of SEQ ID NO: 8 as the N-terminal residue and
Ile4zs or Asn441 as the C-terminal residue .
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According to another aspect of the invention, procaryotic or eucaryotic
host cells are stably transformed with DNA sequences of the invention in a
manner
allowing the desired PAF-AH to be expressed therein. Host cells expressing PAF-
AH products can serve a variety of useful purposes. Such cells constitute a
valuable
S source of immunogen for the development of antibody substances specifically
immunoreactive with PAF-AH. Host cells of the invention are conspicuously
useful
in methods for the large scale production of PAF-AH wherein the cells are
grown in
a suitable culture medium and the desired polypeptide products are isolated
from the
cells or from the medium in which the cells are grown by, for example,
immunoaffinity purification.
A non-immunological method contemplated by the invention for
purifying PAF-AH from plasma includes the following steps: (a) isolating low
density lipoprotein particles; (b) solubilizing said low density lipoprotein
particles in
a buffer comprising IOmM CHAPS to generate a first PAF-AH errayme solution;
(c)
applying said first PAF-AH enzyme solution to a DEAF anion exchange column;
(d)
washing said DEAF anion exchange column using an approximately pH 7.5 buffer
comprising 1mM CHAPS; (e) eluting PAF-AH enzyme from said DEAF anion
exchange column in fractions using approximately pH 7.5 buffers comprising a
gradient of 0 to 0.5 M NaCI; (f) pooling fractions eluted from said DEAF anion
exchange column having PAF-AH enzymatic activity; (g) adjusting said pooled,
active
fractions from said DEAF anion exchange column to lOmM CHAPS to generate a
second PAF-AH enzyme solution; (h) applying said second PAF-AH enzyme solution
to a blue dye ligand affinity column; (i) eluting PAF-AH enzyme from said blue
dye
ligand affinity column using a buffer comprising lOmM CHAPS and a chaotropic
salt;
(j) applying the eluate from said blue dye ligand affinity column to a Cu
ligand
affinity column; (k) eluting PAF-AH enzyme from said Cu ligand aff nity column
using a buffer comprising IOmM CHAPS and imidazole; (1) subjecting the eluate
from said Cu figand affinity column to SDS-PAGE; and (m) isolating the
approximately 44 kDa PAF-AH enzyme from the SDS-polyacrylamide gel.
Preferably, the buffer of step (b) is 25 mM Tris-HCI, IOmM CHAPS, pH 7.5; the
buffer of step (d) is 25 mM Tris-HCI, 1mM CHAPS; the column of step (h) is a
Blue
Sepharose Fast Flow*column; the buffer of step (i) is 25mM Tris-HCI, IOmM
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CHAPS, 0.5M KSCN, pH 7.5; the column of step (j) is a Cu Chelating Sepharose
column; and the buffer of step (k) is 25 mM Tris-HCI, lOmM CHAPS; 0.5M NaCI,
50mM imidazole at a pH in a range of about pH 7.5-8Ø
A method contemplated by the invention for purifying enzymatically-
active PAF-AH from E. coli producing PAF-AH includes the steps of: (a)
preparing
a centrifugation supernatant from lysed E. coli producing PAF-AH enzyme; (b)
applying said centrifugation supernatant to a blue dye ligand affinity column;
(c)
eluting PAF-AH enzyme from said blue dye ligand affinity column using a buffer
comprising lOmM CHAPS and a chaotropic salt; (d) applying said eluate from
said
blue dye ligand affinity column to a Cu Iigand affinity column; and (e)
eluting PAF-
AH enzyme from said Cu )rgand affinity column using a buffer comprising lOmM
CHAPS and imidazole. Freferably, the column of step (b) is a Blue Sepharose
Fast
Flow ~ column; the buffer of step (c) is 25mM Tris-HCI, lOmM CHAPS, 0.5M
KSCN, pH 7.5; the column of step (d) is a Cu Chelating Sepharose column; and
the
buffer of step (e) is 25mM Tris-HCI, l OmM CHAPS, 0.5M NaCI, 100mM imidazole,
pH 7.5.
Another method contemplated by the invention for purifying
enzymatically-active PAF-AH from E. coli producing PAF-AH includes the steps
of:
(a) preparing a centrifugation supernatant from lysed E. coli producing PAF-AH
enzyme; (b) diluting said centrifugation supernatant in a low pH buffer
comprising
IOmM CHAPS; (c) applying said diluted centrifugation supernatant to a cation
exchange column equilibrated at about pH 7.5; (d) eluting PAF-AH enzyme from
said
cation exchange column using 1M salt; (e) raising the pH of said eluate from
said
canon exhange column and adjusting the salt concentration of said eluate to
about
0.5M salt; (f) applying said adjusted eluate from said cation exchange column
to a
blue dye ligand affinity column; (g) eluting PAF-AH enzyme from said blue dye
ligand affinity column using a buffer comprising about 2M to about 3M salt;
and (h)
dialyzing said eluate from said blue dye ligand affinity column using a buffer
comprising about 0.1 ~ Tween Preferably, the buffer of step (b) is 25mM MES,
l OmM CHAPS, 1 mM EDTA, pH 4.9; the column of step (c) is an S sepharose
column equilibrated in 25mM MES, lOmM CHAPS, 1mM EDTA, 50mM NaCI, pH
5.5; PAF-AH is eluted in step (d) using 1mM NaCI; the pH of the eluate in step
(e)
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is adjusted to pH ?.5 using 2M Tris base; the column in step (f) is a
sepharose
column; the buffer in step (g) is 25mM Tris, lOmM CHAPS, 3M NaCI, 1mM
EDTA, pH 7.5; and the buffer in step (h) is 25mM Tris, 0.5M NaCI, 0.1 % Tween
80, pH 7.5.
Still another method contemplated by the invention for purifying
enzymatically-active PAF-AH from E. coli includes the steps of: (a) preparing
an
E. coli extract which yields solubilized PAF-AH supernatant after lysis in a
buffer
containing CHAPS; (b) dilution of said supernatant and application to a anion
exchange column equilibrated at about pH 8.0; (c) eluting PAF-AH enzyme from
said
anion exchange column; (d) applying said adjusted eluate from said anion
exchange
column to a blue dye ligand affinity column; (e) eluting the said blue dye
ligand
affinity column using a buffer comprising 3.OM salt; (f) dilution of the blue
dye
eluate into a suitable buffer for performing hydroxylapatite chromatography;
(g)
performing hydroxylapatite chromatography where washing' and elution is
accomplished using buffers (with or without CHAPS); . (h) diluting said
hydroxylapatite eluate to an appropriate salt concentration for canon exchange
chromatography; (i) applying said diluted hydroxylapatite eluate to a ration
exchange
column at a pH ranging between approximately 6.0 to 7.0; (j) elution of PAF-AH
from said ration exchange column with a suitable formulation buffer; (k)
performing
ration exchange chromatography in the cold; and (1) formulation of PAF-AH in
liquid
or frozen form in the absence of CHAPS.
Preferably in step (a) above the lysis buffer is 25mM Tris, 100mM
NaCI, 1mM EDTA, 20mM CHAPS, pH 8.0; in step (b) the dilution of the
supernatant for anion exchange chromatography is 3-4 fold into 25mM Tris, 1mM
EDTA, lOmM CHAPS, pH 8.0 and the column is a Q-Sepharose column equilibrated
with 25mM Tris, 1mM EDTA, 50mM NaCI, IOmM CHAPS, pH 8.0; in step (c) the
anion exchange column is eluted using 25mM Tris, 1mM EDTA, 350mM NaCI,
IOmM CHAPS, pH 8.0; in step (d) the eluate from step (c) is applied directly
onto
a blue dye affinity column; in step (e) the column is eluted with 3M NaCI,
lOmM
CHAPS, 25mM Tris, pH 8.0 buffer; in step (f) dilution of the blue dye eluate
for
hydroxylapatite chromatography is accomplished by dilution into lOmM sodium
phosphate, 100mM NaCI, lOmM CHAPS, pH 6.2; in step (g) hydroxylapatite
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chromatography is accomplished using a hydroxylapatite column equilibrated
with
IOmM sodium phosphate, 100mM NaCI, IOmM CHAPS and elution is accomplished
using SOmM sodium phosphate, 100mM NaCI (with or without) lOmM CHAPS, pH
7.5; in step (h) ~ dilution of said hydroxylapatite eluate for ration exchange
chromatography is accomplished by dilution into a buffer ranging in pH from
approximately 6.0 to 7.0 comprising sodium phosphate (with or without CHAPS);
in
step (i) a S Sepharose column is equilibrated with SOmM sodium phosphate,
(with or
without) lOmM CHAPS, pH 6.8; in step (j) elution is accomplished with a
suitable
formulation buffer such as potassium phosphate SOmM, l2.SmM aspartic acid,
I25mM NaCI, pH 7.5 containing 0.01 ~ Tween-80; and in step {k) ration exchange
chromatrography is accomplished at 2-8' C. Examples of suitable formulation
buffers
for use in step (1) which stabilize PAF-AH include SOmM potassium phosphate,
l2.SmM Aspartic acid, 125mM NaCI pH 7.4 (approximately, with and without the
addition of Tween-80 and or Pluronic F68) or 25mM potassium phosphate buffer
containing (at least) 125mM NaCI, 25mM arginine and 0.01 ~ Tween-80 (with or
without Pluronic F6~ at approximately 0.1 and 0.5 ~o).
Yet another method contemplated by the invention for purifying
enzymatically active rPAF-AH products from E. roll includes the steps of: (a)
preparing an E. roll extract which yields solubilized rPAF-AH product
supernatant
after lysis in a buffer containing Triton X-100, (b) dilution of said
supernatant and
application to an immobilized metal affinity exchange column equilibrated at
about
pH 8.0; (c) eluting rPAF-AH product from said immobilized metal affinity
exchange
column with a buffer comprising imidazole; (d) adjusting the salt
concentration and
applying said eluate from said immobilized metal affinity column to an
hydrophobic
interaction column (HIC#1); (e) eluting said HIC#1 by reducing the salt
concentration
and/or increasing the detergent concentration; (f) titrating said HIC#1 eluate
to a pH
of about 6.4; (g) applying said adjusted HIC#1 eluate to a ration exchange
column
(CF.X#I) equilibrated at about pH 6.4; (h) eluting said.CEX#1 with
concentration?
sodium chloride; (i) adjusting said CEX#1 eluate with sodium chloride to a
concentration of about 2.OM; (j) applying said adjusted CEX#I eluate to a
hydrophobic interaction column (HIC#2) equilibrated at about pH 8.0 and about
2.OM
sodium chloride; (k) eluting said HIC#2 by reducing the salt concentration
andlor
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increasing the detergent concentration; (1) diluting said HIC/!2 eluate and
adjusting
to a pH of about 6.0; (m) applying said adjusted HIC//2 eluate to a ration
exchange
column (CEX#2) equilibrated at about pH 6.0; (n) eluting the rPAF-AH product
from
said CEX//2 with a suitable formulation buffer.
Preferably, in step (a) above the lysis buffer is .90mM TRIS, 0.125 %
Triton X-100, 0.6M NaCI, pH 8.0, and lysis is carried out in a high pressure
homogenizer; in step (b) the supernatant is diluted into equilibration buffer
(20mM
TRIS, O.SM NaCI, 0.196 Triton X-100, pH 8.0), a zinc chelate column (Chelating
Sepharose Fast Flow, Pharmacia, Uppsala, Sweden) is charged, equilibrated with
. equilibration buffer, loaded with the diluted supernatant, and washed with
20mM
TRIS, O.SM NaCI, 4M urea, 0.196 Triton X-100, pH 8.0, followed by washing with
20mM TRIS, O.SM NaCI, 0.02 % Triton X-100, pH 8.0; in step (c) elution is
accomplished with 20mM Tris, SOmM imidazole, 0.02 % Triton X-100, pH 8.0; in
step (d) the eluate is adjusted to 1 mM EDTA and 2M NaCI, a Phenyl Scphamse 6
Fast Flow (Pharmacia) is equilibrated with equilibration buffer (2.OM NaCI,
25mM
Tris, 0.02 % Triton X-100, pH 8.0), loaded with the adjusted eluate from step
(c) at
room temperature, washed with equilibration buffer, arid washed with 25mM
NaP04,
0.02 % Triton X-100, pH6.5 at a flow rate of 34cm/hr; in step (e) elution is
accomplished with 25mM NaP04, 3 96 Triton X-100, pH 6.5; in step (g) a Macro-
Prep High S Column (Bio-Rad Labs, Richmond, CA) is equilibrated with
equilibration buffer (20mM NaP04, 0.02 9b Triton X-100, pH 6.4), loaded with
the
adjusted eluate from step (f), washed with equilibration buffer, and washed
with
25mM Tris, 0.02 % Triton X-100, pH 8.0; in step (h) elution is accomplished
with
25mM Tris, 0.02 f6 Triton X-100, 1.3M NaCI, pH 8.0; in step (j) a Bakerbond
Wide
Pore Hi-Propyl C3 (Baker, Phillipsburg, Nn is equilibrated with equilibration
buffer
(2.OM NaCI, 25mM Tris, 0.02 % Triton X-100, pH 8.0), loaded with adjusted
eluate
from step (i) at room temperature, washed with equilibration buffer, and
washed with
25mM Tris, 0.02 % Triton X-100, pH 8.0 at 30 cm/hr; in step (k) elution is
accomplished with l OmM Tris, 3.0 % Triton X-100, pH 8.0; in step (1) dilution
is in:o
equilibration buffer (20mM succinate, 0.1 % PLURONIC F68, pH 6.0); in step (m)
a SP Sepharose Fast Flow (Pharmacia) column is equilibrated with the
equilibration
buffer of step (1), loaded with eluate from step (I), and washed with
equilibration
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buffer; and in step (n) elution is accomplished with SOmM NaP04, 0.7M NaCI,
0.1
PLURONIC F68, 0.02 % TWEEN 80, pH 7.5.
PAF-AH products may be obtained as isolates from natural cell sources
or may be chemically synthesized, but are preferably produced by recombinant
procedures involving procaryotic or eucaryotic host cells of the invention.
PAF-AH
products having part or all of the amino acid sequence set out in SEQ ID NO: 8
are
contemplated. Specifically contemplated are fragments lacking up to the first
twelve
N-terminal amino acids of the mature human PAF-AH amino acid sequence set out
in SEQ ID NO: 8, particularly those having Met46, A1a47 or A1a48 of SEQ ID NO:
8 as the initial N-terminal amino acid. Also contemplated are fragments
thereof
lacking up to thirty C-terminal amino acids of the amino acid sequence of SEQ
ID
NO: 8, particularly those having I1e429 and Leu431 as the C-terminal residue.
Further contemplated are variants of PAF-AH or PAF-AH or which have an amino
acid replacement in the sequence of SEQ ID NO: 8 selected from the group
consisting
of S 108 A, S 273 A, D 286 A, D 286 N, D 296 A, D 304 A, D 338 A, H 351 A,
H 395 A, H 399 A, C 67 S, C 229 S, C 291 S, C 334 S, C 407 S, D 286 A, D 286
N and D 304 A. As noted above, polynucleotides (including DNA) encoding such
fragments or variant fragments are provided by the invention, as well as
methods of
recombinantly producing such fragments or variants by growing host cells
comprising
such DNA. Presently preferred PAF-AH products include the prokaryotic
polypeptide expression products of DNA encoding amino acid residues Met46
through
Asn441 of SEQ ID NO: 8, designated rPH.2, and the prokaryotic polypeptide
expression products of DNA encoding amino acid residues Met46 through I1e429
of
SEQ ID NO: 8, designated rPH.9. Both the rPH.2 and rPH.9 products display less
amino-terminal heterogeneity than, for example, the corresponding prokaryotic
expression products of DNA encoding the full mature sequence of PAF-AH
preceded
by a translation initiation codon. Moreover, the rPH.9 product displays
greater
carboxy terminal homogeneity (consistency). The use of mammalian host cells is
expected to provide for such post-translational modifications (e.g.,
myristolation,
glycosylation, truncation, lipidation and tyrosine, serine or threonine
phosphorylation)
as may be needed to confer optimal biological activity on recombinant
expression
products of the invention. PAF-AH products of the invention may be full length
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polypeptides, fragments or variants. Variants may comprise PAF-AH analogs
wherein one or more of the specified (i. e. , naturally encoded) amino acids
is deleted
or replaced or wherein one or more nonspecified amino acids are added: (1)
without
loss of one or more of the enzymatic activities or immunological
characteristics
specific to PAF-AH; or (2) with specific disablement of a particular
biological activity
of PAF-AH. Proteins or other molecules that bind to PAF-Al;i may be used to
modulate its activity.
Also comprehended by the present invention are antibody substances
(e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric
antibodies, CDR-grafted antibodies and the like) and other binding proteins
specific
for PAF-AH. Specifically illustrating binding proteins of the invention are
the
monoclonal antibodies produced by hybridomas 90G 11 D and 90F2D which were
deposited with the American Type Culture Collection (ATCC), 12301 Parklawn
Drive, Rockville, MD 20852 on September 30, 1994 and were respectively
assigned
Accession Nos. HB 11724 and HB 11725. Also illustrating binding proteins of
the
invention is the monoclonal antibody produced by hybridoma I43A which was
deposited with the ATCC on June 1, 1995 and assigned Accession No. HB 11900.
Proteins or other molecules (e.g., lipids or small molecules) which
specifically bind
to PAF-AH can be identified using PAF-AH isolated from plasma, recombinant PAF-
AH, PAF-AH variants or cells expressing such products. Binding proteins are
useful,
in turn, in compositions for immunization as well as for purifying PAF-AH, and
are
useful for detection or quantification of PAF-AH in fluid and tissue samples
by
known immunologicai procedures. Anti-idiotypic antibodies specific for PAF-AH-
specific antibody substances are also contemplated.
The scientific value of the information contributed through the
disclosures of DNA and amino acid sequences of the present invention is
manifest.
As one series of examples, knowledge of the sequence of a cDNA for PAF-AH
makes possible the isolation by DNA/DNA hybridization of genomic DNA sequences
encoding PAF-AH and specifying PAF-AH expression control regulatory sequences
such as promoters, operators and the like. DNA/DNA hybridization procedures
carned out with DNA sequences of the invention under conditions of stringency
standard in the art are likewise expected to allow the isolation of DNAs
encoding
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allelic variants of PAF-AH, other structurally related proteins sharing one or
more
of the biochemical and/or immunological properties of PAF-AH, and non-human
species proteins homologous to PAF-AH. The DNA sequence information provided
by the present invention also makes possible the development, by homologous
recombination or "knockout" strategies [see, e.g., Kapecchi, Science, 244:
1288-1292
(1989)], of rodents that fail to express a functional PAF-AH enzyme or that
express
a variant PAF-AH enzyme. Polynucleotides of the invention when suitably
labelled
are useful in hybridization assays to detect the capacity of cells to
synthesize PAF-
AH. Polynucleotides of the invention may also be the basis for diagnostic
methods
useful for identifying a genetic alterations) in the PAF-AH locus that
underlies a
disease state or states. Also made available by the invention are anti-sense
polynucleotides relevant to regulating expression of PAF-AH by those cells
which
ordinarily express the same.
Administration of PAF-AH preparations of the invention to mammalian
subjects, especially humans, for the purpose of ameliorating pathological
inflammatory conditions is contemplated. Based on implication of the
involvement
of PAF in pathological inflammatory conditions, the administration of PAF-AH
is
indicated, for example, in treatment of asthma [Miwa et al., J. Clin. Invest.,
82:
1983-1991 (1988); Hsieh et al., J. Allergy Clin. Immunol., 91: 650-657 (1993);
and
Yamashita et al., Allergy, 49: 60-63 (1994)], anaphylaxis [Venable et al.,
supra],
shock [Venable et al., supra], reperfusion injury and central nervous system
ischemia
[Lindsberg et al. (1991), supra], antigen-induced arthritis [Zarco et al.,
Clin. Exp.
Immunol. , 88: 318-323 (1992)], atherogenesis [Handley et al. , Drug Dev. Res.
, 7:
361-375 (1986)], Crohn's disease [Denizot et al., Digestive Diseases and
Sciences,
37(3): 432-437 (1992)], ischemic bowel necrosis/necrotizing enterocolitis
[Denizot et
al., supra and Caplan et al., Acta Paediatr., Suppl. 396: 11-17 (1994)],
ulcerative
colitis (Denizot et al., supra), ischemic stroke [Satoh et al., Stroke, 23:
1090-1092
(1992)], ischemic brain injury [Lindsberg et al., Stroke, 21: 1452-1457 (1990)
and
Lindsberg et al. (1991), supra], systemic lupus erythematosus [Matsuzaki et
al.,
Clinica Chimica Acta, 210: 139-144 (1992)], acute pancreatitis [Kald et al.,
Pancreas, 8(4): 440-442 (1993)], septicemia (Kald et al., supra), acute post
streptococcal glomerulonephritis [Mezzano et al. , J. Am. Soc. Nephrol. , 4:
235-242
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(1993)], pulmonary edema resulting from IL-2 therapy [Rabinovici et al., J.
Clin.
Invest., 89: 1669-1673 (1992)], allergic inflammation [Watanabe et al., Br. J.
Pharmacol., 111: 123-130 (1994)], ischemic renal failure [Grino et al., Annals
of
Internal Medicine, 121(5): 345-347 (1994); preterm labor [Hoffman et al., Am.
J.
S Obstet. Gynecol. , 162(2): 525-528 ( 1990) and Maki et al. , Proc. ll~atl.
Acad. Sci.
USA, 85: 728-732 (1988)]; adult respiratory distress syndrome [Rabinovici et
al., J.
Appl. Physiol., 74(4): 1791-1802 (1993); Matsumoto et al., Clin. Fxp.
Pharmacol.
Physiol., 19 509-SIS (1992); and Rodriguez-Roisin et al., J. Clin. Invest.,
93: 188-
194 (1994)]. Also contemplated is the use of PAF-AH preparations to treat
human
immunodeficiency virus (HIV) infection of the central nervous system.
"Treatment"
as used herein includes both prophylactic and therapeutic treatment.
Animal models for many of the foregoing pathological conditions have
been described in the art. For example, a mouse model for asthma and rhinitis
is
described in Example 16 herein; a rabbit model for arthritis is described in
Zarco et
at. , supra; rat models for ischemic bowel necrosislnecrotizing enterocolitis
are
described in Furukawa et al., Ped. Res., 34,(2): 237-241 (1993) and Caplan et
al.,
supra; a rabbit model far stroke is described in Lindsberg et al., (1990),
supra; a
mouse model for lupus is described in Matsuzaki et al., supra; a rat model for
acute
pancreatitis is described in Kald et al., supra: a rat model for pulmonary
edema
resulting from IL-2 therapy is described in Rabinovici et al., supra; a rat
model of
allergic inflammation is described in Watanabe et al. , supra); a canine model
of renal
allograft is described in Watson etal., Transplantation, 56(4): 1047-1049
(1993); and
rat and guinea pig models of adult respiratory distress syndrome are
respectively
described in Rabinovici et al., supra. and Lellouch-Tubiana, Am. Rev. Respir.
Dis.,
137: 948-954 (1988).
Specifically contemplated by the invention are PAF-AH compositions
for use in methods for treating a mammal susceptible to or suffering from PAF-
mediated pathological conditions comprising administering PAF-AH to the mammal
in an amount sufficient to supplement endogenous PAF-AH activity and to
inactivate
pathological amounts of PAF in the mammal.
Therapeutic/pharmaceutical compositions contemplated by the invention
include PAF-AH products and a physiologically acceptable diluent or Garner and
may
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also include other agents having anti-inflammatory effects. Dosage amounts
indicated
would be sufficient to supplement endogenous PAF-AH activity and to inactivate
pathological amounts of PAF. For general dosage considerations see
Remmington's
Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, PA (1990).
Dosages will vary between about 0.1 to about 1000 tcg PAF-AHlkg body weight.
Therapeutic compositions of the invention may be administered by various
routes
depending on the pathological condition to be treated. For example,
administration
may be by intraveneous, subcutaneous, oral, suppository, and/or pulmonary
routes.
For pathological conditions of the lung, administration of PAF-AH by
the pulmonary route is particularly indicated. Contemplated for use in
pulmonary
administration are a wide range of delivery devices including, for example,
nebulizers, metered dose inhalers, and powder inhalers, which are standard in
the art.
Delivery of various proteins to the lungs and circulatory system by inhalation
of
aerosol formulations has been described in Adjei et al., Pharm. Res., 7(6):
565-569
(1990) (leuprolide acetate); Braquet et al., J. Cardio. Pharm., 13(Supp. 5):
s. 143-
146 (1989) (endothelin-1); Hubbard et al., Annals of Internal Medicine,
Ill(3), 206-
212 (1989) (al-antitrypsin); Smith et al., J. Clin. Invest., 84: 1145-1146
(1989) (a-1-
proteinase inhibitor); Debs er al. , J. Immunol., 140: 3482-3488 (1933)
(recombinant
gamma interferon and tumor necrosis factor alpha); Patent Cooperation Treaty
(PCT)
International Publication No. WO 94/20069 published September 15, 1994
(recombinant pegylated granulocyte colony stimulating factor).
BRIEF DESCRIPTION OF THE DRAWING
Numerous other aspects and advantages of the present invention will
be apparent upon consideration of the following detailed description thereof,
reference
being made to the drawing wherein:
FIGURE 1 is a photograph of a PVDF membrane containing PAF-AH
purified from human plasma;
FIGURE 2 is a graph showing the enzymatic activity of recombinant
human plasma PAF-AH;
FIGURE 3 is a schematic drawing depicting recombinant PAF-AH
fragments and their catalytic activity;
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FIGURE 4 depicts mass spectroscopy results for a recombinant PAF-
AH product, rPH.2.
FIGURE 5 depicts mass spectroscopy results for a recombinant PAF-
AH product, rPH.9.
FIGURE 6 is a bar graph illustrating blockage of PAF-induced rat foot
edema by locally administered recombinant PAF-AH of the invention;
FIGURE 7 is a bar graph illustrating blockage of PAF-induced rat foot
edema by intravenously administered PAF-AH;
FIGURE 8 is a bar graph showing that PAF-AH blocks PAF-induced
edema but not zymosan A-induced edema;
FIGURES 9A and 9B present dose response results of PAF-AH anti-
inflammatory activity in rat food edema;
FIGURES l0A and lOB present results indicating the in vivo efficacy
of a single dose of PAF-AH over time;
FIGURE 11 is a line graph representing the pharmacokinetics of PAF-
AH in rat circulation; and
FIGURE i2 is a bar graph showing the anti-inflammatory effects of
PAF-AH in comparison to the lesser effects of PAF antagonists in rat foot
edema.
FIGURE 13 presents results indicating that PAF-AH neutralizes the
apoptotic effects of conditioned media from HIV-I-infected and activated
monocytes.
DETAILED DESCRIPTION
The following examples illustrate the invention. Example i presents
a novel method for the purification of PAF-AH from human plasma. Example 2
describes amino acid microsequencing of the purified human plasma PAF-AH. The
cloning of a full length cDNA encoding human plasma PAF-AH is described in
Example 3. Identification of a putative splice variant of the human plasma PAF-
AH
gene is described in Example 4. The cloning of genomic sequences encoding
human
plasma PAF-AH is described in Example 5. Example 6 desribes the cloning of
canine, murine, bovine, chicken, rodent and macaque cDNAs homologous to the
human plasma PAF-AH cDNA. Example 7 presents the results of an assay
evidencing the enzymatic activity of recombinant PAF-AH transiently expressed
in
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COS 7 cells. Example 8 describes the expression of full length, truncated and
chimeric human PAF-AH DNAs in E. coli, S. cerevisiae and mammalian cells.
Example 9 presents protocols for purification of recombinant PAF-AH from E.
coli
and assays confirming its enzymatic activity. Example 10 describes various
recombinant PAF-AH products including amino acid substitution analogs and
amino
and carboxy-truncated products, and describes experiments demonstrating that
native
PAF-AIi isolated from plasma is glycosylated. Results of a Northern blot assay
for
expression of human plasma PAF-AH RNA in various tissues and cell lines are
presented in Example I1 while results of in situ hybridization are presented
in
Example 12. Example 13 describes the development of monoclonal and polyclonal
antibodies specific for human plasma PAF-AH. Examples 14, 15, 16, 17, 18 and
19
respectively describe the in vivo therapeutic effect of administration of
recombinant
PAF-AH products of the invention on acute inflammation, pleurisy, asthma,
necrotizing enterocolitis, adult respiratory distress syndrome and
pancreatitis in
animal models. Example 20 describes the in vitro effect of recombinant PAF-AH
product on neurotoxicity associated with HIV infection. Example 21 presents
the
results of immunoassays of serum of human patients exhibiting a deficiency in
PAF-
AH activity and describes the identification of a genetic lesion in the
patients which
is apparently responsible for the deficiency.
Example 1
PAF-AH was purified from human plasma in order to provide material
for amino acid sequencing.
A. Optimization of Purification Conditions
Initially, low density lipoprotein (LDL) particles were precipitated from
plasma with phosphotungstate and solubilized in 0.1 % Tween 20 and subjected
to
chromatography on a DEAF column (Pharmacia, Uppsala, Sweden) according to the
method of Stafforini et al. (1987), supra, but inconsistent elution of PAF-AH
activity
from the DEAE column required reevaluation of the solubilization and
subsequent
puriflcatian conditions.
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Tween 20, CHAPS (Pierce Chemical Co., Rocl~ord, IL) and octyl
glucoside were evaluated by centrifugation and gel filtration chromatography
for their
ability to solubilize LDL particles. CHAPS provided 25 ~b greater recovery of
solubilized activity than Tween 20 and 300 ~ greater recovery than ocxyl
glucoside.
LDL precipitate solubilized with lOmM CHAPS was then fractionated on a DBAE
Sepharose Fast Flow column (an anion exchange column; Pharmacia) with buffer
containing 1 mM CHAPS to provide a large pool of partially purified PAF-AH
("the
DEAF pool") for evaluation of additional columns. .
The DEAF pool was used as starting material to test a variety of
chromatography columns for utility in further purifying the PAF-AH activity.
The
columns tested included: Blue Sepharose Fast FlovV (Pharmacia), a dye ligand
affinity column; S-Sepharose Fast Flov~i (Pharmacia), a ration exchange
column; Cu
Chelating Sepharose (Pharmacia), a metal ligand affinity column; Practogel S
(EM
Separations, Gibbstown, N~, a ration exchange column; and Sephacryl-20Q
(Pharmacia), a gel filtration column. These chromatographic procedures all
yielded
low, unsatisfactory levels of purification when operated in 1mM CHAPS.
Subsequent
gel filtration chromatography on Sephacryl S-200 in 1mM CHAPS generated an
enzymatically active fraction which eluted over a broad size range rather than
the
expected 44 kDa approximate size. Taken together, these results indicated that
the
LDL proteins were aggregating in solution.
Different LDL samples were therefore evaluated by analytical gel
filtration chromatography for~aggregation of the PAF-AH activity. Samples from
the
DEAF pool and of freshly solubilized LDL precipitate were analyzed on Superose
12
(Pharmacia) equilibrated in buffer with 1mM CHAPS. Both samples eluted over a
very broad range of molecular weights with most of the activity eluting above
150
kDa. When the samples were then analyzed on Superose 12 equilibrated with lOmM
CHAPS, the bulls of the activity eluted near 44 kDa as expected for PAF-AH
activity.
However, the samples contained some PAF-AH activity in the high molecular
weight
region corresponding to aggregates.
Other samples eluted PAF-AH activity exclusively in the approximately
44 kDa range when they were subsequently tested by gel filtration. These
samples
were an LDL precipitate solubilized in IOmM CHAPS in the presence of O.SM NaCI
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and a fresh DEAF pool that was adjusted to lOmM CHAPS after elution from the
DEAF column. These data indicate that at least lOmM CHAPS is required to
maintain non-aggregated PAF-AH. Increase of the CHAPS concentration from 1mM
to lOmM after chromatography on DEAF but prior to subsequent chromatographic
steps resulted in dramatic differences in purification. For example, the
degree of
PAF-AH purification on S-Sepharose Fast Flow was increased from 2-fold to 10-
fold.
PAF-AH activity bound the Blue Sepharose Fast Flow column irreversibly in 1mM
CHAPS, but the column provided the highest level of purification in lOmM
CHAPS.
The DEAF chromatography was not improved with prior addition of lOmM CHAPS.
Chromatography on Cu Chelating Sepharose after the Blue Sepharose
Fast Flow column concentrated PAF-AH activity 15-fold. It was also determined
that
PAF-AH activity could be recovered from a reduced SDS-polyacrylamide gel, as
long
as samples were not boiled. The activity of material eluted from the Cu
Chelating
Sepharose column when subjected to SDS-polyacrylamide gel electrophoresis
coincided with a major protein band when the gel was silver stained.
B. PAF-AH Purification Protocol
The novel protocol utilized to purify PAF-AH for amino acid
sequencing therefore comprised the following steps which were performed at
4°C.
Human plasma was divided into 900 ml aliquots in 1 liter Nalgene bottles and
adjusted to pH 8.6. LDL particles were then precipitated by adding 90 mI of 3.
85 %
sodium phosphotungstate followed by 23 ml of 2M MgCl2. The plasma was then
centrifuged for 15 minutes at 3600 g. Pellets were resuspended in 800 ml of
0.2
sodium citrate. LDL was precipitated again by adding 10 g NaCI and 24 ml of 2M
MgCl2. LDL particles were pelleted by centrifugation for 15 minutes at 3600 g.
This wash was repeated twice. Pellets were then frozen at -20°C. LDL
particles
from SL of plasma were resuspended in 5 L of buffer A (25mM Tris-HCI, IOmM
CHAPS, pH 7.5) and stirred overnight. Solubilized LDL particles were
centrifuged
at 3600 g for 1.5 hours. Supernatants were combined and filtered with Whatman
113
filter paper to remove any remaining solids. Solubilized LDL supernatant was
loaded
on a DEAF Sepharose Fast Flow column (11 cm x 10 cm; 1 L resin volume; 80
ml/minute) equilibrated in buffer B (25mM Tris-HCI, ImM CHAPS, pH 7.5). The
I I~ I
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column was washed with buffer B until absorbance returned to baseline. Protein
was
eluted with an 8 L, 0 - 0.5M NaCl gradient and 480 ml fractions were
collected.
This step was necessary to obtain binding to the Blue Sepharose Fast Flow
column
below. Fractions were assayed for acetylhydrolase activity essentially by the
method
described in Example 4.
Active fractions were pooled and sufficient CHAPS was added to make
the pool about 1 OmM CHAPS . The DEAF pool was loaded overnight at 4 mUminute
onto a Blue Sepharose Fast Flow column (5 cm x 10 cm; 200 ml bed volume)
equilibrated in buffer A containing 0.5M NaCI. The column was washed with the
equilibration buffer at 16 ml/minute until absorbance returned to baseline.
PAF-AH
activity was step eluted with buffer A containing 0.5M KSCN (a chaotmpic salt)
at
16 ml/minute and collected in 50 ml fractions. This step resulted in greater
than
1000-fold purification. Active fractions were pooled, and the pool was
adjusted to pH
8.0 with I M Tris-HCl pH 8Ø The active pool from Blue Sepharose Fast Flow
chromatography was loaded onto a Cu Chelating Sepharose column (2.5 cm x 2 cm;
10 ml bed volume; 4 ml/minute) equilibrated in buffer C [25mM Tris-HCI, lOmM
CHAPS, 0.5M NaCI, pH 8.0 (pH 7.5 also worked)], and the column was washed
with 50 ml buffer C. PAF-AH activity was eluted with 100 ml 50mM imidazole in
buffer C and collected in 10 ml fractions. Fractions containing PAF-AH
activity were
pooled and dialyzed against buffer A. In addition to providing a 15-fold
concentration of PAF-AH activity, the Cu Chelating Sepharose column gave a
small
purification. The Cu Chelating Sepharose pool was reduced in 50 mM DTT for 15
minutes at 37°C and loaded onto a 0.75 mm, 7.5 R~ polyacrylamide gel.
Gel slices
were cut every 0.5 cm and placed in disposable microfuge tubes containing 200
~cl
'25 25mM Tris-HCl, lOmM CHAPS, 150mM NaCI. Slices were ground up and allowed
to incubate overnight at 4°C. The supernatant of each gel slice was
then assayed for
PAF-AH activity to determine which protein band on SDS-PAGE contained PAF-AH
activity. PAF-AH activity was found in an approximately 44 kDa band. Protein
from a duplicate gel was electrotransferred to a PVDF membrane (Immobilon-P,
Millipore) and stained with Coomassie Blue. A photograph of the PVDF membrane
is presented in FIGURE 1.
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As presented in Table 1 below, approximately 200 ~g PAF-AH was
purified 2 x 106-fold from 5 L human plasma. In comparison, a 3 x 104-fold
purification of PAF-AH activity is described in Stafforini et al. (1987),
supra.
Table 1
J~ Sample Vol.ActivityTotal Prot. Specific% RecoveryFold
Purification
ml (cpm ActivityConc. Activityof ActivityStep Cum.
x
1~ (cpm (mg/ (cpm Sten Cum.
x x
1~ ml~ 1~
Plasma 500023 116 62 0.37 100 100 1 I
LDL 450022 97 1.76 12 84 84 33 33
DEAF 420049 207 1.08 46 212 178 3.7 124
Blue 165 881 14 0.02 54200 70 126 1190 1.5
x
105
Cu 12 12700 152 0.15 82200 104 I31 1.5 2.2
x
105
SDS-PAGE --- --- --- --- --- --- --- ~ 2.2
10 x
106
In summary, the following steps were unique and critical for successful
purification of plasma PAF-AH for microsequencing: {1) solubilization and
chromotography in IOmM CHAPS, (2) chromatography on a blue ligand affinity
column such as Blue Sepharose Fast Flow, (3) chromatography on a Cu ligand
affinity column such as Cu Chelating Sepharose, and (4) elution of PAF-AH from
SDS-PAGE.
Example 2
For amino acid sequencing, the approximately 44 kDa protein band
from the PAF-AH- containing PVDF membrane described in Example 1 was excised
and sequenced using an Applied Biosystems 473A Protein sequencer. N-terminal
sequence analysis of the approximately 44 kDa protein band corresponding to
the
PAF-AH activity indicated that the band contained two major sequences and two
minor sequences. The ratio of the two major sequences was 1:1 and it was
therefore
difficult to interpret the sequence data.
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To distinguish the sequences of the two major proteins which had been
resolved on the SDS gel, a duplicate PVDF membrane containing the
approximately
44 kDa band was cut in half such that the upper part and the lower part of the
membrane were separately subjected to sequencing.
The N-terminal sequence obtained for the lower half of the membrane
was:
SEQ ID NO: 1
FKDLGEENFKALVLIAF
A search of protein databases revealed this sequence to be a fragment of human
serum albumin. The upper half of the same PVDF membrane was also sequenced
and the N-terminal amino acid sequence determined was:
SEQ ID NO: 2
IQVLMAAASFGQTKIP
This sequence did not match any protein in the databases searched and was
different
from the N-terminal amino acid sequence:
SEQ ID NO: 3
MKPLVVFVLGG
which was reported for erythrocyte cytoplasmic PAF-AH in Stafforini et al.
(1993),
supra. The novel sequence (SEQ ID NO: 2) was utilized for cDNA cloning of
human plasma PAF-AIi as described below in Example 3.
Example 3
A full length clone encoding human plasma PAF-AH was isolated from
a macrophage cDNA library.
A. Construction of a Macrophage cDNA LibrarX
Poly A+ RNA was harvested from peripheral blood monocyte-derived
macrophages. Double-stranded, blunt-ended cDNA was generated using the
Invitrogen Copy Kit (San Diego, CA) and BstXI adapters were ligated to the
cDNA
prior to insertion into the mammalian expression vector, pRcICMV (Invitrogen).
The
resulting plasmids were introduced into E. coli strain XL-1 Blue by
electroporation.
Transformed bacteria were plated at a density of approximately 3000 colonies
per
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agarose plate on a total of 978 plates. Plasmid DNA prepared separately from
each
plate was retained in individual pools and was also combined into larger pools
representing 300,000 clones each.
B. Library Screenin~by PCR
The macrophage library was screened by the polymerise chain reaction
utilizing a degenerate antisense oligonucleotide PCR primer based on the novel
N-
terminal amino acid sequence described in Example 2. The sequence of the
primer
is set out below in IUPAC nomenclature and where "I" is an inosine.
SEQ ID NO: 4
5' ACATGAATTCGGIATCYTTIGTYTGICCRAA 3'
The colon choice tables of Wada et al., Nuc. Acids Res., 195: 1981-1986 (1991)
were used to select nucleotides at the third position of each colon of the
primer. The
primer was used in combination with a primer specific for either the SP6 or T7
promoter sequences, both of which flank the cloning site of pRc/CMV, to screen
the
macrophage library pools of 300,000 clones. All PCR reactions contained 100 ng
of
template cDNA, 1 ~cg of each primer, 0.125mM of each dNTP, lOmM Tris-HCl pH
8.4, SOmM MgCl2 and 2.5 units of Taq polymerise. An initial denaturation step
of
94°C for four minutes was followed by 30 cycles of amplification of 1
minute at
94°C, 1 minute at 60°C and 2 minutes at 72°C. The
resulting PCR product was
cloned into pBluescript SK- (Stratagene, La Jolla, CA) and its nucleotide
sequence
determined by the dideoxy chain termination method. The PCR product contained
the sequence predicted by the novel peptide sequence and corresponds to
nucleotides
1 to 331 of SEQ ID NO: 7.
The PCR primers set out below, which are specific for the cloned PCR
fragment described above, were then designed for identifying a full length
clone.
Sense Primer (SEQ ID NO: 5)
5' TATTTCTAGAAGTGTGGTGGAACTCGCTGG 3'
Antisense Primer (SEQ ID NO: 6)
5' CGATGAATTCAGCTTGCAGCAGCCATCAGTAC 3'
PCR reactions utilizing the primers were performed as described above to first
screen
the cDNA pools of 300,000 clones and then the appropriate subset of the
smaller
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pools of 3000 clones. Three pools of 3000 clones which produced a PCR product
of
the expected size were then used to transform bacteria.
C. Liblanr Screenin~by Hybridization
DNA from the transformed bacteria was subsequently screened by
hybridization using the original cloned PCR fragment as a probe. Colonies were
blotted onto nitrocellulose and prehybridized and hybridized in 50 %
formamide,
0.75M sodium chloride, 0.075M sodium citrate, O.OSM sodium phosphate pH 6.5,
1 % polyvinyl pyrolidine, 1 % Ficoll, 1 % bovine serum albumin and 50 ng/ml
sonicated salmon sperm DNA. The hybridization probe was labeled by random
hexamer priming. After overnight hybridization at 42°C, blots were
washed
extensively in 0.03M sodium chloride, 3mM sodium citrate, 0.1 % SDS at
42°C. The
nucleotide sequence of 10 hybridizing clones was determined. One of the
clones,
clone sAH 406-3, contained the sequence predicted by the original peptide
sequence
of the PAF-AH activity purified from human plasma. The DNA and deduced amino
acid sequences of the human plasma PAF-AH are set out in SEQ ID NOs: 7 and 8,
respectively.
Clone sAH 406-3 contains a 1.52 kb insert with an open reading frame
that encodes a predicted protein of 441 amino acids. At the amino terminus, a
relatively hydrophobic segment of 4I residues precedes the N-terminal amino
acid
(the isoleucine at position 42 of SEQ ID NO: 8) identified by protein
microsequencing. The encoded protein may thus have either a long signal
sequence
or a signal sequence plus an additional peptide that is cleaved to yield the
mature
functional enzyme. The presence of a signal sequence is one characteristic of
secreted proteins. In addition, the protein encoded by clone sAH 406-3
includes the
consensus GxSxG motif (amino acids 271-275 of SEQ ID NO: 8) that is believed
to
contain the active site serine of all known mammalian lipases, microbial
lipases and
serine proteases. See Chapus et al., Biochimie, 70: 1223-1224 (1988) and
Brenner,
Nature, 334: 528-530 (1988).
Table 2 below is a comparison of the amino acid composition of the
human plasma PAF-AH of the invention as predicted from SEQ ID NO: 8 and the
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amino acid composition of the purportedly purified material described by
Stafforini
et al. (1987), supra.
Table 2
Clone sAH 406-3 Stafforini et
al.
Ala 26 24
Asp & Asn 48 37
Cys 5 14
Glu & Gln 36 42
Phe 22 12
Gly 29 58
His 13 24
Ile 31 17
Lys 26 50
Leu 40 26
Met 10 7
Pro 15 11
Arg 18 16
Ser 27 36
Thr 20 15
Val 13 14
Trp 7 Not determined
Tyr 14 13
The amino acid composition of the mature form of the human plasma PAF-AH of
the
invention and the amino acid composition of the previously purified material
that was
purportedly the human plasma PAF-AH are clearly distinct.
When alignment of the Hattori et al., supra nucleotide and deduced
amino acid sequences of bovine brain cytoplasmic PAF-AH with the nucleotide
and
amino acid sequences of the human plasma PAF-AH of the invention was
attempted,
no significant structural similarity in the sequences was observed.
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Example 4
A putative splice variant of the human PAF-AH gene was detected
when PCR was performed on macrophage and stimulated PBMC cDNA using primers
that hybridized to the 5' untranslated region (nucleotides 31 to 52 of SEQ ID
NO: 7)
and the region spanning the translation termination codon at the 3' end of the
PAF-
AH cDNA (nucleotides 1465 to 1487 of SEQ ID NO: 7). The PCR reactions yielded
two bands on a gel, one corresponding to the expected size of the PAF-AH cDNA
of Example 3 and the other was about 100 by shorter. Sequencing of both bands
revealed that the larger band was the PAF-AH cDNA of Example 3 while the
shorter
band lacked exon 2 (Example 5 below) of the PAF-AH sequence which encodes the
putative signal and pro-peptide sequences of plasma PAF-AH. The predicted
catalytic
triad and all cysteines were present in the shorter clone, therefore the
biochemical
activity of the protein encoded by the clone is likely to match that of the
plasma
enzyme.
To begin to assess the biological relevance of the PAF-AH splice
variant that is predicted to encode a cytoplasmically active enzyme, the
relative
abundance of the two forms in blood monocyte-derived macrophages was assayed
by
RNase protection. Neither message was present in freshly isolated monocytes
but
both messages were found at day 2 of in vitro differentiation of the monocytes
into
macrophages and persisted through 6 days of culture. The quantity of the two
messages was approximately equivalent throughout the differentiation period.
In
contrast, similar analyses of neural tissues revealed that only full length
message
predicted to encode the full length extracellular form of PAF-AH is expressed.
Example 5
Genomic human plasma PAF-AIi sequences were also isolated. The
structure of the PAF-AH gene was determined by isolating lambda and P1 phage
clones containing human genomic DNA by DNA hybridization under conditions of
high stringency. Fragments of the phage clones were subcloned and sequenced
using
primers designed to anneal at regular intervals throughout the cDNA clone sAH
406-
3. In addition, new sequencing primers designed to anneal to the intron
regions
flanking the exons were used to sequence back across the exon-intron
boundaries to
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confirm the sequences. Exon/intron boundaries were defined as the points where
the
genomic and cDNA sequences diverged. These analyses revealed that the human
PAF-AH gene is comprised of 12 exons.
Exons 1, 2, 3, 4, 5, 6, and part of 7 were isolated from a male fetal
placental library constructed in lamda FIX (Stratagene). Phage plaques were
blotted
onto nitrocellulose and prehybridized and hybridized in 50 % formamide, 0.75M
sodium chloride, 75mM sodium citrate, 50mM sodium phosphate (pH 6.5), 1
polyvinyl pyrolidine, 1 % Ficoll, 1 % bovine serum albumin, and 50 ng/ml
sonicated
salmon sperm DNA. The hybridization probe used to identify a phage clone
containing exons 2-6 and part of 7 consisted of the entire cDNA clone sAH 406-
3.
A clone containing exon 1 was identified using a fragment derived from the 5'
end
of the cDNA clone (nucleotides 1 to 312 of SEQ ID NO: 7). Both probes were
labelled with 32P by hexamer random priming. After overnight hybridization at
42 ° C, blots were washed extensively in 30mM sodium chloride, 3mM
sodium citrate,
0.1 % SDS at 42 ° C. The DNA sequences of exons 1, 2, 3, 4, 5, and 6
along with
partial surrounding intron sequences are set out in SEQ ID NOs: 9, 10, 11, 12,
13,
and 14, respectively.
The remainder of exon 7 as well as exons 8, 9, 10, 11, and 12 were
subcloned from a Pl clone isolated from a human P1 genomic library. P1 phage
plaques were blotted onto nitrocellulose and prehybridized and hybridized in
0.75M
sodium chloride, 50mM sodium phosphate (pH 7.4), 5mM EDTA, 1 % polyvinyl
pyrolidine, 1 % Ficoll, 1 % bovine serum albumin, 0.5 % SDS, and 0.1 mg/ml
total
human DNA. The hybridization probe, labeled with 32P by hexamer random
priming, consisted of a 2.6 kb EcoRl fragment of genomic DNA derived from the
3' end of a lambda clone isolated above. This fragment contained exon 6 and
the part
of exon 7 present on the phage clone. After overnight hybridization at 65
° C, blots
were washed as described above. The DNA sequences of exons 7, 8, 9, 10, 11,
and
12 along with partial surrounding intron sequences are set out in SEQ ID NOs:
15,
16, 17, 18, 19, and 20, respectively.
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Example 6
Full length plasma PAF-AH cDNA clones were isolated from mouse,
canine, bovine and chicken spleen cDNA libraries and a partial rodent clone
was
isolated from a rat thymus cDNA library. The clones were identified by low
stringency hybridization to the human cDNA (hybridization conditions were the
same
as described for exons 1 through 6 in Example 5 above except that 20 %
formamide
instead of 50 % formamide was used). A 1 kb HindIll fragment of the human PAF-
AH sAH 406-3 cDNA clone (nucleotides 309 to 1322 of SEQ ID NO: 7) was used
as a probe. In addition, a partial monkey clone was isolated from macaque
brain
cDNA by PCR using primers based on nucleotides 285 to 303 and 851 to 867 of
SEQ
ID NO: 7. The nucleotide and deduced amino acid sequences of the mouse,
canine,
bovine, chicken, rat, and macaque cDNA clones are set out in SEQ ID NOs: 21,
22,
23, 24, 25, and 26, respectively.
A comparison of the deduced amino acid sequences of the cDNA
clones with the human cDNA clone results in the amino acid percentage identity
values set out in Table 3 below.
Table 3
Human Dog Mouse Bovine Chicken
Dog 80 100 64 82 50
Mouse 66 64 100 64 47
Monkey 92 82 69 80 52
Rat 74 69 82 69 55
Bovine 82 82 64 100 50
Chicken 50 50 47 50 100
About 38 % of the residues are completely conserved in all the
sequences. The most divergent regions are at the amino terminal end
(containing the
signal sequence) and the carboxyl terminal end which are shown in Example 10
as
not critical for enzymatic activity. The Gly-Xaa-Ser-Xaa-GIy motif (SEQ ID NO:
27)
found in neutral lipases and other esterases was conserved in the bovine,
canine,
mouse, rat and chicken PAF-AH. The central serine of this motif serves as the
active
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site nucleophile for these enzymes. The predicted aspartate and histidine
components
of the active site (Example l0A) were also conserved. The human plasma PAF-AH
of the invention therefore appears to utilize a catalytic triad and may assume
the alai
hydrolase conformation of the neutral lipases even though it does not exhibit
other
sequence homology to the lipases.
Moreover, human plasma PAF-AH is expected to have a region that
mediates its specific interaction with the low density and high density
lipoprotein
particles of plasma. Interaction with these particles may be mediated by the N-
terminal half of the molecule which has large stretches of amino acids highly
conserved among species but does not contain the catalytic triad of the
enzyme.
Example 7
To determine whether human plasma PAF-AH cDNA clone sAH 406-3
(Example 3) encodes a protein having PAF-AH activity, the pRcICMV expression
construct was transiently expressed in COS 7 cells. Three days following
transfection
by a DEAF Dextran method, COS cell media was assayed for PAF-AH activity.
Cells were seeded at a density of 300,000 cells per 60 mm tissue
culture dish. The following day, the cells were incubated in DMEM containing
0.5
mg/ml DEAF dextran, O.1mM chloroquine and 5-10 ~cg of plasmid DNA for 2 hours.
Cells were then treated with 10% DMSO in phosphate-buffered saline for 1
minute,
washed with media and incubated in DMEM containing 10 % fetal calf serum
previously treated with diisopropyl fluorophosphate (DFP} to inactivate
endogenous
bovine serum PAF-AH. After 3 days of incubation, media from transfected cells
were assayed for PAF-AH activity. Assays were conducted in the presence and
absence of either 10 mM EDTA or 1 mM DFP to determine whether the recombinant
enzyme was calcium-independent and inhibited by the serine esterase inhibitor
DFP
as previously described for plasma PAF-AH by Stafforini et al. (1987), supra.
Negative controls included cells transfected with pRc/CMV either lacking an
insert
or having the sAH 406-3 insert in reverse orientation.
PAF-AH activity in transfectant supernatants was determined by the
method of Stafforini et al. (1990), supra, with the following modifications.
Briefly,
PAF-AH activity was determined by measuring the hydrolysis of 3H-acetate from
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[acetyl-3H] PAF (New England Nuclear, Boston, MA). The aqueous free 3H-acetate
was separated from labeled substrate by reversed-phase column chromatography
over
octadecylsilica gel cartridges (Baker Research Products, Phillipsburg, PA).
Assays
were carned out using 10 ~cl transfectant supernatant in O.1M Hepes buffer, pH
7.2,
in a reaction volume of 50 ~I. A total of 50 pmoles of substrate were used per
reaction with a ratio of 1:5 labeled: cold PAF. Reactions were incubated for
30
minutes at 37°C and stopped by the addition of 40 ~,1 of 10M acetic
acid. The
solution was then washed through the octadecylsilica gel cartridges which were
then
rinsed with O.1M sodium acetate. The aqueous eluate from each sample was
collected and counted in a liquid scintillation counter for one minute. Enzyme
activity was expressed in counts per minute.
As shown in FIGURE 2, media from cells transfected with sAH 406-3
contained PAF-AH activity at levels 4-fold greater than background. This
activity
was unaffected by the presence of EDTA but was abolished by 1mM DFP. These
observations demonstrate that clone sAH 406-3 encodes an activity consistent
with the
human plasma enzyme PAF-AH.
Example 8
Full length and various truncated human plasma PAF-AH DNAs and
a chimeric mouse-human PAF-AH DNA were expressed in E. coli and yeast and
stably expressed in mammalian cells by recombinant methods.
A. Expression in E. coli
PCR was used to generate a protein coding fragment of human plasma
PAF-AH cDNA from clone sAH 406-3 which was readily amenable to subcloning
into an E. coli expression vector. The subcloned segment began at the 5' end
of the
human gene with the codon that encodes I1e42 (SEQ ID NO: 8), the N-terminal
residue of the enzyme purified from human plasma. The remainder of the gene
through the native termination codon was included in the construct. The 5'
sense
PCR primer utilized was:
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SEQ ID NO: 28
TATTCTAGAATTATGATACAAGTATTAATGGCTGCTGCAAG
3' and contained an XbaI cloning site as well as a translation initiation
codon
(underscored). The 3' antisense primer utilized was:
5 SEQ ID NO: 29
5' ATTGATATCCTAATTGTATTTCTCTATTCCTG 3'
and encompassed the termination codon of sAH 406-3 and contained an EcoRV
cloning site. PCR reactions were performed essentially as described in Example
3.
The resulting PCR product was digested with XbaI and EcoRV and subcloned into
a
pBR322 vector containing the Trp promoter [deBoer et al., PNAS, 80:21-25
(1983)]
immediately upstream of the cloning site. E. coli strain XL-1 Blue was
transformed
with the expression construct, and cultured in L broth containing 100 tcg/ml
of
carbenicillin. Transformants from overnight cultures were pelleted and
resuspended
in lysis buffer containing 50mM Tris-HCl pH 7.5, 50mM NaCI, lOmM CHAPS,
1mM EDTA, 100 wg/ml lysozyme, and 0.05 trypsin-inhibiting units (TILnlml
Aprotinin. Following a 1 hour incubation on ice and sonication for 2 minutes,
the
lysates were assayed for PAF-AH activity by the method described in Example 4.
E. coli transformed with the expression construct (designated trp AH)
generated a
product with PAF-AH activity. See Table 6 in Example 9.
Constructs including three additional promoters, the tacll promoter
(deBoer, supra), the arabinose (ara) B promoter from Salmonella typhimurium
[Horwitz et al., Gene, 14: 309-319 (1981)], and the bacteriophage T7 promoter,
were
also utilized to drive expression of human PAF-AH sequences in E. coli.
Constructs
comprising the Trp promoter {pUC trp AH), the tacll promoter (pUC tac AH), and
the araB promoter {pUC ara AH) were assembled in plasmid pUCl9 (New England
Biolabs, MA) while the construct comprising the T7 promoter (pET AH) was
assembled in plasmid pETlSB (Novagen, Madison, WI). A construct containing a
hybrid promoter, pHAB/PH, consisting of the araB promoter fused to the
ribosome
binding sites of the T7 promoter region was also assembled in pETlSB. AlI E.
coli
constructs produced PAF-AH activity within a range of 20 to 50 U/mIlOD6(y(1.
This
activity corresponded to a total recombinant protein mass of ~ 1 % of the
total cell
protein.
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Several E. coli expression constructs were also evaluated which
produce PAF-AH with extended amino termini. The N-terminus of natural plasma
PAF-AH was identified as I1e42 by amino acid sequencing (Example 2). However,
the sequence immediately upstream of I1e42 does not conform to amino acids
found
at signal sequence cleavage sites [i.e., the "-3-1-rule" is not followed, as
lysine is not
found at position -1; see von Heijne, Nuc. Acids Res., 14:4683-4690 (1986)].
Presumably a more classical signal sequence (M 1-A 17 or M 1-P21 ) is
recognized by
the cellular secretion system, followed by endoproteolytic cleavage. The
entire
coding sequence for PAF-AH beginning at the initiating methionine {nucleotides
162
to 1487 of SEQ ID NO: 7) was engineered for expression in E. coli using the
trp
promoter. As shown in Table 4, this construct made active PAF-AH, but
expression
was at about one fiftieth of the level of the original construct beginning at
I1e42.
Another expression construct, beginning at Va118 (nucleotides 213 to 1487 of
SEQ
ID NO: 7), produced active PAF-AH at about one third the level of the original
construct. These results suggest that amino terminal end extensions are not
critical
or necessary for activity of recombinant PAF-AH produced in E. coli.
Table 4
PAF-AH activitX (Ulml/OD6~
Construct L_,ysate Media
pUC trp AH (I1e42 N-terminus) 177.7 0.030
pUC trp AH Metl 3.1 0.003
pUC trp AH Va118 54.6 0.033
Truncated recombinant human PAF-AH products were also produced
in E. coli using a low copy number plasmid and a promoter that can be induced
by
the addition of arabinose to the culture. One such N-terminally truncated PAF-
AH
product is the recombinant expression product of DNA encoding amino acid
residues
Met46 through Asn~l of the polypeptide encoded by full length PAF-AH cDNA
(SEQ ID NO: 8), and is designated rPH.2. The plasmid used for production of
rPH.2 in bacterial cells was pBAR2lPH.2, a pBR322-based plasmid that carnes
(1)
nucleotides 297 to 1487 of SEQ ID NO: 7 encoding human PAF-AH beginning with
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the methionine codon at position 46, (2) the araB-C promoters and araC gene
from
the arabinose operon of Salmonella typhimurium, (3) a transcription
termination
sequence from the bacteriophage T7, and (4) a replication origin from
bacteriophage
fl.
Specifically, pBAR2/PH.2 included the following segments of DNA:
(1) from the destroyed AatII site at position 1994 to the EcoRI site at
nucleotide
6274, vector sequence containing an origin of replication and genes encoding
resistance to either ampicillin or tetracycline derived from the bacterial
plasmid
pBR322; (2) from the EcoRI site at position 6274 to the XbaI site at position
I31,
IO DNA from the Salmonella typhimurium arabinose operon {Genbank accession
numbers M11045, M11046, MI 1047, J01797); (3) from the XbaI site at position
131
to the NcoI site at position 170, DNA containing a ribosome binding site from
pET-
21b (Novagen, Madison, WI); (4) from the NcoI site at position I70 to the XhoI
site
at position 1363, human PAF-AH cDNA sequence; and (5) from the XhoI site at
position 1363 to the destroyed Aatli site at position 1993, a DNA fragment
from
pET-21b (Novagen) that contains a transcription termination sequence from
bacteriophae T7 and an origin of replication from bacteriophage fl.
Another PAF-AH product, designated rPH.9, is the recombinant
expression product of DNA encoding amino acid residues Met46 through I1e429 of
the polypeptide encoded by full length PAF-AH cDNA (SEQ ID NO: 8). The DNA
encoding rPH.9 was inserted into the same vector used for production of rPH.2
in
bacterial cells. This plasmid was designated pBAR2/PH.9 and specifically
included
the following segments of DNA: (1) from the destroyed Aatl1 site at position
1958
to the EcoRI site at nucleotide 6239 of the vector sequence containing an
origin of
replication and genes encoding resistance to either ampicillin or tetracycline
derived
from the bacterial plasmid pBR322; (2) from the EcoRI site at position 6239 to
the
Xbal site at position 131, DNA from the Salmonella typhimurtum arabinose
operon
(Genbank accession numbers M11045, MI 1046, M 11047, J01797); (3) from the
Xbal
site at position 131 to the Ncol site at position 170, DNA containing a
ribosome
binding site from pET-21b {Novagen, Madison, WI); (4) from the NcoI site at
position 170 to the XhoI site at position 1328, human PAF-AH DNA sequence; (5)
from the XhoI site at position 1328 to the destroyed Aatll site at position
1958, a
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DNA fragment from pET-21b (Novagen, Madison, WI) that contains a transcription
termination sequence from bacteriophage T7 and a origin of replication from
bacteriophage fl.
Expression of PAF-AH products in pBAR2/PH.2 and pBAR2/PH.9 is
under the control of the araB promoter, which is tightly repressed in the
presence of
glucose and absence of arabinose, but functions as a strong promoter when L-
arabinose is added to cultures depleted of glucose. Selection for cells
containing the
plasmid can be accomplished through the addition of either ampicivin (or
related
antibiotics) or tetracycline to the culture medium. A variety of E. coli
strains can be
used as a host for recombinant expression of PAF-AH products, including but
not
limited to strains prototrophic for arabinose metabolism such as W3110, DHSa,
BL21, C600, JM101 and their derivatives, strains containing mutations reducing
proteolysis such as CAG629, KY1429, and strains defective in their ability to
degrade
arabinose such as SB7219 and MC 1061. The advantage of using a strain that is
unable to break down arabinose is that the inducer (arabinose) for production
of PAF-
AH is not depleted from the medium during the induction period, resulting in
higher
levels of PAF-AH compared to that obtained with strains that are capable of
metabolizing arabinose. Any suitable media and culturing conditions may be
used to
express active PAF-AH products in various E. coli strains. For example, either
rich
media formulations such LB, EDM295 (a M9 based minimum medium supplemented
with yeast extract and acid hydrolysed casein), or "defined" media such as
A675, an
A based minimal medium set at pH 6.75 employing glycerol as a carbon source
and
supplemented with trace elements and vitamins, permit substantial production
of
rPAF-AH products. Tetracycline is included in the media to maintain selection
of the
plasmid.
The plasmid pBAR2/PH.2 was transformed into the E. coli strain
MC 1061 (ATCC 53338), which carnes a deletion of the arabinose operon and
thereby
cannot metabolize arabinose. MC 1061 is also a leucine auxotroph and was
cultivated
by batch-fed process using a defined media containing casamino acids that
complement the leucine mutation.
The E. coli MI061 cells transformed with pBAR2/PH.2 were grown
at 30 ° C in batch media containing 2 gml L glucose. Glucose serves the
dual purpose
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of carbon source for cell growth, and repressor of the arabinose promoter.
When
batch glucose levels were depleted ( < 50 mg/L), a nutrient feed (containing
300 gm/L
glucose) was started. The feed was increased linearly for 16 hours at a rate
which
limited acid bi-product formation. At this point, the nutrient feed was
switched to
media containing glycerol instead of glucose. Simultaneously, 500 gmlL L-
arabinose
was added to a final concentration of 5 gm/L. The glycerol feed was kept at a
constant feed rate for 22 hours. Cells were harvested using hollow-fiber
filtration to
concentrate the suspension approximately 10-fold. Cell paste was stored at -
?0° C.
A final cell mass of about 80 gm/L was obtained (OD600 - 50-60) with a PAF-AH
activity of 65-70 UIOD/ml representing about 10 % of total cell protein. The
final
culture volume of about 75 liters contained 50-60 gm PAF-AH.
High level production of rPAF-AH products can be achieved when
pBAR2lPH.2 or PH.9 is expressed by strains SB72I9 or MC1061. Other strains
deficient in arabinose degradation are suitable for high cell density
production.
Preferably, the cells are cultured under the following conditions.
Exponentially
growing SB7219;pBAR2/PH.2 and SB7219;pBAR2/PH.9 strains are seeded into
fermentors containing batch medium containing 2 g/L glucose. Once glucose is
consumed, the tanks are fed with a glycerol solution containing trace
elements,
vitamins, magnesium and ammonium salt to maintain healthy exponential growth.
The tanks are maintained at 30°C, provided air to supply oxygen and
agitated to
maintain the dissolved oxygen level above about 15 % saturation. When the cell
density of the culture is above I I O g/L (wet cell mass), constant feed rate
is imposed
and a bolus addition of L-arabinose is added to the culture (about 0.5 %
final).
Product formation is observed for I6-22 hours. The cultures typically achieve
40-50
g/L (dry cell weight). Cells are harvested by centrifugation, stored at -
70°C, and
rPAF-AH product purified for analysis. Specific productivities in excess of
150
unitslml/OD600 are routinely obtained.
B. Expression in Yeast Cells
Recombinant human PAF-AH was also expressed in Saccharomyces
cerevisiae. The yeast ADH2 promoter was used to drive rPAF-AH expression and
produced 7 U/mllOD600 (Table 5 below).
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Table 5
Enzyme Activity
Construct Promoter Strain (tJ/ml/OD)
pUC tac AH tac E. coli W3110 30
pUC trp AH trp E. coli W3110 40
S pUC ara AH araB E. coli W3110 20
pET AH T7 E. coli BL21 (DE3)SO
(Novagen)
pHAB/PH araBlT7 E. coli XL-1 34
pBAR2/PH. 2 araB MC 1061 9p
pYep ADH2 AH ADH2 Yeast BJ2.28 7
C. Expression of PAF-AH in mammalian cells
1. Expression of Human PAF-AH cDNA Constlvcts
Plasmids constructed for expression of PAF-AH, with the exception of
pSFN/PAFAH.1, employ a strong viral promoter from cytomegalovirus, a
polyadenylation site from the bovine growth hormone gene, and the SV40 origin
of
replication to permit high copy number replication of the plasmid in COS
cells.
Plasmids were electroporated into cells.
A first set of plasmids was constructed in which the 5' flanking
sequence (pDC 1/PAFAH.1 ) or both the 5' or 3' flanking sequences
(PDC 1 /PAFAH.2) of the human PAF-AH cDNA were replaced with flanking
sequences from other genes known to be expressed at high levels in mammalian
cells.
Transfection of these plasmids into COS, CHO or 293 cells led to production of
PAF-
AH at about the same level (0.01 units/ml or 2-4 fold above background) as
that cited
for clone sAH 406-3 in Example 7 after transient transfection of COS cells.
Another
plasmid was constructed which included a Friend spleen focus-forming virus
promoter
instead of the cytomegalovirus promoter. The human PAF-AH cDNA was inserted
into plasmid pmH-neo [Hahn et al., Gene, 127: 267 (1993)] under control of the
Friend spleen focus-forming virus promoter. Transfection of the myeloma cell
line
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NSO with the plasmid which was designated pSFN/PAFAH. l and screening of
several
hundred clones resulted in the isolation of two transfectants (4B11 and 1C11)
that
made 0.15-0.5 unitslml of PAF-AH activity. Assuming a specific activity of
5000
units/milligram, the productivity of these two NSO transfectants corresponds
to about
0.1 mglliter.
2. Expression of Mouse-Human Chimeric PAF-AH Gene Constructs
A construct (pRc/MS9) containing the cDNA encoding mouse PAF-AH
in the mammalian expression vector pRc/CMV resulted in production of secreted
PAF-AH at the level of 5-10 units/ml (1000 fold above background) after
transfection
into COS cells. Assuming that the specific activity of the mouse PAF-AH is
about
the same as that of the human enzyme, the mouse cDNA is therefore expressed at
a
500-1000 fold higher Level than is the human PAF-AH cDNA.
To examine the difference between the expression levels of human and
mouse PAF-AH in COS cells, two mouse-human chimeric genes were constructed and
tested for expression in COS cells. The first of these constructs,
pRc/PH.MHCI,
contains the coding sequence for the N-terminal 97 amino acids of the mouse
PAF-
AH polypeptide (SEQ ID NO: 21 ) fused to the C-terminal 343 amino acids of
human
PAF-AH in the expression vector pRc/CMV (Invitrogen, San Diego, CA). The
second chimeric gene, in plasmid pRcIPH.MHC2, contains the coding sequence for
the N-terminal 40 amino acids of the mouse PAF-AH polypeptide fused to the C-
terminal 400 residues of human PAF-AH in pRcICMV. Transfection of COS cells
with pRc/PH.MHCI led to accumulation of 1-2 units/m1 of PAF-AH activity in the
media. Conditioned media derived from cells transfected with pRcIPH.MHC2 was
found to contain only 0.01 units/mI of PAF-AH activity. From these
experiments,
it appears that the difference in expression level between mouse and human PAF-
AH
genes is attributable at least in part to the polypeptide segment between the
residues
40 and 97, or the corresponding RNA or DNA segment encoding this region of the
PAF-AH protein.
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3. Recoding of the First 290 by of the PAF-AH Coding Sequence
One hypothesis for the low level of human PAF-AH synthesized in
transfected mammalian cells is that the codons utilized by the natural gene
are
suboptimal for efficient expression. However, it does not seem likely that
codon
usage can account for 500-1000 fold difference in expression levels between
the
mouse and human genes because optimizing codons generally has at most only a
10-
fold effect on expression. A second hypothesis to explain the difference
between the
mouse and human PAF-AH expression levels is that the human PAF-AH mRNA in
the 5' coding region forms a secondary structure that leads to either
relatively rapid
degradation of the mRNA or causes inefficient translation initiation or
elongation.
To test these hypotheses, a synthetic fragment encoding the authentic
human PAF-AH protein from the amino-terminus to residue 96 but in which most
of
the codons have been substituted ("recoded") with a codon of a different
sequence but
encoding the same amino acid was constructed. Changing the second codon from
GTG to GTA resulted in the creation of an Asp718 site, which was at one end of
the
synthetic fragment and which is present in the mouse cDNA. The other end of
the
fragment contained the BamHI site normally found at codon 97 of the human
gene.
The approximately 290 by Asp718/BamHI fragment was derived from a PCR
fragment that was made using the dual asymmetric PCR approach for construction
of
synthetic genes described in Sandhu et al., Biotechniques, 12: 14-16 (1992).
The
synthetic Asp718lBamHI fragment was ligated with DNA fragments encoding the
remainder of the human PAF-AH molecule beginning with nucleotide 453 of SEQ ID
NO: 7 such that a sequence encoding authentic human PAF-AH enzyme was inserted
into the mammalian expression vector pRc/CMV (Invitrogen, San Diego) to create
plasmid pRc/HPH.4. The complete sequence of the recoded gene is set out in SEQ
ID NO: 30. The 5' flanking sequence adjacent to the human PAF-AH coding
sequence in pRc/HPH.4 is from that of a mouse cDNA encoding PAF-AH in
pRc/MS9 (nucleotides 1 to 116 of SEQ ID NO: 21).
To test expression of human PAF-AH from pRc/HPH.4, COS cells
were transiently transfected with pRc/HPH.4 (recoded human gene), pRclMS9
(mouse PAF-AH), or pRc/PH.MHCI (mouse-human hybrid 1). The conditioned
media from the transfected cells were tested for PAF-AH activity and found to
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contain 5.7 units/ml (mouse gene)., 0.9 units/ml (mouse-human hybrid 1), or
2.6
units/ml (recoded human gene). Thus, the strategy of recoding the first 290 by
of
coding sequence of human PAF-AH was successful in boosting expression levels
of
human PAF-AH from a few nanograms/ml to about 0.5 microgramlml in a transient
COS cell transfection. The recoded PAF-AH gene from pRcIHPH.4 will be inserted
into a mammalian expression vector containing the dihydrofolate reductase
{DHFR)
gene and DHFR-negative Chinese hamster ovary cells will be transfected with
the
vector. The transfected cells will be subjected to methotrexate selection to
obtain
clones making high levels of human PAF-AH due to gene amplification.
Example 9
Recombinant human plasma PAF-AH (beginning at I1e42) expressed
in E. coli was purified to a single Coomassie-stained SDS-PAGE band by various
methods and assayed for activities exhibited by the native PAF-AH enzyme.
A. Purification of Recombinant PAF-AH
1 S The first purification procedure utilized is similar to that described in
Example 1 for native PAF-AH. The following steps were performed at 4°C.
Pellets
from 50 ml PAF-AH producing E. coli {transformed with expression construct trp
AH) were lysed as described in Example 8. Solids were removed by
centrifugation
at 10,000 g for 20 minutes. The supernatant was loaded at 0.8 mllminute onto a
Blue
Sepharose Fast Flow column (2.5 cm x 4 cm; 20 ml bed volume) equilibrated in
buffer D (25mM Tris-HCI, lOmM CHAPS, O.SM NaCI, pH 7.5). The column was
washed with 100 ml buffer D and eluted with 100 ml buffer A containing O.SM
KSCN at 3.2 ml/minute. A 15 mi active fraction was loaded onto a 1 ml Cu
Chelating Sepharose column equilibrated in buffer D. The column was washed
with
5 rnl buffer D followed by elution with 5 ml of buffer D containing 100mM
imidazole
with gravity flow. Fractions containing PAF-AH activity were analyzed by SDS-
PAGE.
The results of the purification are shown in Table b wherein a unit
equals ~.mol PAF hydrolysis per hour. The purification product obtained at
4°C
appeared on SDS-PAGE as a single intense band below the 43 kDa marker with
some
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diffuse staining directly above and below it. The recombinant material is
significantly
more pure and exhibits greater specific activity when compared with PAF-AH
preparations from plasma as described in Example 1.
Table 6
Sample Volume Activity Total Prot Conc Specific % Recovery Fold
(units/ Act. m lmL Activity of Activity Purification
ml~ (units (units/ Stev Cum. Step Cum.
x mgl
10~
Lysate 4.5 989 4451 15.6 63 100 100 1 1
Blue 15 64 960 0.07 914 22 22 14.4 14.4
Cu 1 2128 2128 0.55 3869 220 48 4.2 61
When the same purification protocol was performed at ambient temperature, in
addition to the band below the 43 kDa marker, a group of bands below the 29
kDa
marker correlated with PAF-AH activity of assayed gel slices. These lower
molecular weight bands may be proteolytic fragments of PAF-AH that retain
enzymatic activity.
A different purification procedure was also performed at ambient
temperature. Pellets (100 g) of PAF-AH-producing E. coli (transformed with the
expression construct pUC trp AH) were resuspended in 200 mI of lysis buffer
(25mM
Tris, 20mM CHAPS, SOmM NaCI, 1 mM EDTA, SO ~cg/ml benzamidine, pH 7.5)
and lysed by passing three times through a microfluidizer at 15,000 psi.
Solids were
removed by centrifugation at 14,300 x g for 1 hour. The supernatant was
diluted 10
fold in dilution buffer [25mM MES (2-[N-morpholino] ethanesulfonic acid), lOmM
CHAPS, 1mM EDTA, pH 4.9] and loaded at 25 ml/minute onto an S Sepharose Fast
Flow Column (200 ml) (a ration exchange column) equilibrated in Buffer E (25mM
MES, IOmM CHAPS, 1mM EDTA, SOmM NaCI, pH 5.5). The column was washed
with 1 liter of Buffer E, eluted with 1M NaCI, and the eluate was collected in
50 ml
fractions adjusted to pH 7.5 with 0.5 ml of 2M Tris base. Fractions containing
PAF-
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AH activity were pooled and adjusted to 0.5M NaCI. The S pool was loaded at 1
ml/minute onto a Blue Sepharose Fast Flow column (2.5 cm x 4 cm; 20 ml)
equilibrated in Buffer F (25mM Tris, IOmM CHAPS, 0.5M NaCI, 1mM EDTA, pH
7.5). The column was washed with 100 ml Buffer F and eluted with 100 ml Buffer
F containing 3M NaCI at 4 ml/minute. The Blue Sepharose Fast Flow
chromatography step was then repeated to reduce endotoxin levels in the
sample.
Fractions containing PAF-AH activity were pooled and dialyzed against Buffer G
(25mM Tris pH 7.5, 0.5M NaCI, 0.1 % Tween 80, lnlM EDTA).
The results of the purification are shown in Table 7 wherein a unit
equals ~cmol PAF hydrolysis per hour.
Table 7
Sample Volume Activity Total Prot Conc Specific % Recovery Fold
ml (units/ Act. m /mL Activity of Activity Purification
ml~ (units (units/ Sten Cum. Step Cum.
x
1~
Lysate 200 5640 1128 57.46 98 100 100 1 1
S 111 5742 637 3.69 1557 57 56 16 16
Blue 100 3944 394 0.84 4676 35 b2 3 48
The purification product obtained appeared on SDS-PAGE as a single intense
band
below the 43 kDa marker with some diffuse staining directly above and below
it.
The recombinant material is significantly more pure and exhibits greater
specific
activity when compared with PAF-AH preparations from plasma as described in
Example 1.
Yet another purification procedure contemplated by the present
invention involves the following cell lysis, clarification, and first column
steps. Cells
are diluted 1:1 in lysis buffer (25mM Tris pH 7.5, 150mM NaCI, 1 % Tween 80,
2mM EDTA). Lysis is performed in a chilled microfluidizer at 15,000-20,000 psi
with three passes of the material to yield > 99 % cell breakage. The lysate is
diluted
1:20 in dilution buffer (25mM Tris pH 8.5, ImM EDTA) and applied to a column
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packed with Q-Sepharose Big Bead chromatography media (Pharmacia) and
equilibrated in 25mM Tris pH 8.5, 1mM EDTA, 0.015 % Tween 80. The eluate is
diluted 1:10 in 25mM MES pH 5.5, 1.2M Ammonium sulfate, ImM EDTA and
applied to Butyl Sepharose chromography media (Pharmacia) equilibrated in the
same
buffer. PAF-AH activity is eluted in 25mM MES pH 5.5, 0.1 % Tween 80, 1mM
EDTA.
Still another method contemplated by the invention for purifying
enzymatically-active PAF-AH from E. toll includes the steps of: (a) preparing
an
E. toll extract which yields solubilized PAF-AH supernatant after lysis in a
buffer
containing CHAPS; (b) dilution of the said supernatant and application to a
anion
exchange column equilibrated at about pH 8.0; (c) eluting PAF-AH enzyme from
said
anion exchange column; (d) applying said adjusted eluate from said anion
exchange
column to a blue dye ligand affinity column; (e) eluting the said blue dye
ligand
affinity column using a buffer comprising 3.OM salt; (f) dilution of the blue
dye
eiuate into a suitable buffer for performing hydroxylapatite chromatography;
(g)
performing hydroxylapatite chromatography where washing and elution is
accomplished using buffers (with or without CHAPS); (h) diluting said
hydroxylapatite eluate to an appropriate salt concentration for cadon exchange
chromatography; (i) applying said diluted hydroxylapatite eluate to a cation
exchange
column at a pH ranging between approximately 6.0 to 7.0; (j) elution of PAF-AH
from said canon exchange column with a suitable formulation buffer; (k)
performing
cation exchange chromatography in the cold; and (1) formulation of PAF-AH in
liquid
or frozen form in the absence of CHAPS.
Preferably in step (a) above the lysis buffer is 25mM Tris, 100mM
NaCI, 1 mM EDTA, 20mM CHAPS, pH 8.0; in step (b) the dilution of the
supernatant for anion exchange chromatography is 3-4 fold into 25mM Tris, 1mM
EDTA, lOmM CHAPS, pH 8.0 and the column is a Q-Sepharose column equilibrated
with 25mM Tris, 1mM EDTA, 50mM NaCI, lOmM CHAPS, pH 8.0; in step (c) the
anion exchange column is eluted using 25mM Tris, 1 mM EDTA, 350mM NaCI,
IOmM CHAPS, pH 8.0; in step (d) the eluate from step (c) is applied directly
onto
a blue dye affinity column; in step (e) the column is eluted with 3M NaCI,
lOmM
CHAPS, 25mM Tris, pH 8.0 buffer; in step (f) dilution of the blue dye eluate
for
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hydroxylapatite chromatography is accomplished by dilution into IOmM sodium
phosphate, IOOmM NaCI, IOmM CHAPS, pH 6.2; in step (g) hydroxylapatite
chromatography is accomplished using a hydroxylapatite column equilibrated
with
IOmM sodium phosphate, 100mM NaCI, lOmM CHAPS and elution is accomplished
using 50mM sodium phosphate, 100mM NaCI (with or without) lOmM CHAPS, pH
7.5; in step (h) dilution of said hydroxylapatite eluate for ration exchange
chromatography is accomplished by dilution into a buffer ranging in pH from
approximately 6.0 to 7.0 comprising sodium phosphate (with or without CHAPS);
in
step (i) a S Sepharose column is equilibrated with 50mM sodium phosphate,
(with or
without) IOmM CHAPS, pH 6.8; in step (j) elution is accomplished with a
suitable
formulation buffer such as potassium phosphate 50mM, 12.5mM aspartic acid,
I25mM NaCI, pH 7.5 containing 0.01 % Tween-80; and in step (k) ration exchange
chromatrography is accomplished at 2-8 ° C . Examples of suitable
formulation buffers
for use in step (1) which stabilize PAF-AH include 54mM potassium phosphate,
12.5mM Aspartic acid, 125mM NaCl pH 7.4 (approximately, with and without the
addition of Tween-80 and or Pluronic F68) or 25mM potassium phosphate buffer
containing (at least) 125mM NaCI, 25mM arginine and 0.01 ~ Tween-80 (with or
without Pluronic F68 at approximately 0.1 and 0.5 % ).
B. Activit~r of Recombinant PAF-AH
The most remarkable property of the PAF acetylhydrolase is its marked
specificity for substrates with a short residue at the sn-2 position of the
substrate.
This strict specificity distinguishes PAF acetylhydrolase from other forms of
PLA2.
Thus, to determine if recombinant PAF-AH degrades phospholipids with long-
chain
fatty acids at the sn-2 position, hydrolysis of 1-palmitoyl-2-arachidonoyl-sn-
glycero-3-
phosphocholine (arachidonoylPC) was assayed since this is the preferred
substrate for
a well-characterized form of PLA2. As predicted from previous studies with
native
PAF-AH, this phospholipid was not hydrolyzed when incubated with recombinant
PAF-AH. In additional experiments, arachidonoylPC was included in a standard
PAF
hydrolysis assay at concentrations ranging from 0 to 125 ~.M to determine
whether
it inhibited the hydrolysis of PAF by recombinant PAF-AH. There was no
inhibition
of PAF hydrolysis even at the highest concentration of PAF-AH, which was 5-
fold
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greater than the concentration of PAF. Thus, recombinant PAF-AH exhibits the
same
substrate selectivity as the native enzyme; long chain substrates are not
recognized.
Moreover, recombinant PAF-AH enzyme rapidly degraded an oxidized phospholipid
(glutaroylPC) which had undergone oxidative cleavage of the Sn-2 fatty acid.
Native
plasma PAF-AH has several other properties that distinguish it from other
phospholipases including calcium-independence and resistance to compounds that
modify sulfhydryl groups or disrupt disulfides.
Both the native and recombinant plasma PAF-AH enzymes are sensitive
to DFP, indicating that a serine comprises part of their active sites. An
unusual
feature of the native plasma PAF acetylhydrolase is that it is tightly
associated with
lipoproteins in circulation, and its catalytic efficiency is influenced by the
lipoprotein
environment. When recombinant PAF-AH of the invention was incubated with
human plasma (previously treated with DFP to abolish the endogenous enzyme
activity), it associated with low and high density lipoproteins in the same
manner as
the native activity. This result is significant because there is substantial
evidence that
modification of low density lipoproteins is essential for the cholesterol
deposition
observed in atheromas, and that oxidation of lipids is an initiating factor in
this
process. PAF-AH protects low density lipoproteins from modification under
oxidizing conditions in vitro and may have such a role in vivo. Administration
of
PAF-AH is thus indicated for the suppression of the oxidation of lipoproteins
in
atherosclerotic plaques as well as to resolve inflammation.
These results all confirm that the cDNA clone sAH 4.06-3 encodes a
protein with the activities of the the human plasma PAF acetylhydrolase.
Example 10
Various other recombinant PAF-AH products were expressed in E.
coli. The products included PAF-AH analogs having single amino acid mutations
and
PAF-AH fragments.
A. PAF-AH Amino Acid Substitution Products
PAF-AH is a lipase because it hydrolyses the phospholipid PAF.
While no obvious overall similarity exists between PAF-AH and other
characterized
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lipases, there are conserved residues found in comparisons of structurally
characterized lipases. A serine has been identified as a member of the active
site.
The serine, along with an aspartate residue and a histidine residue, form a
catalytic
triad which represents the active site of the lipase. The three residues are
not
adjacent in the primary protein sequence, but structural studies have
demonstrated that
the three residues are adjacent in three dimensional space. Comparisons of
structures
of mammalian lipases suggest that the aspartate residue is generally twenty-
four amino
acids C-terminal to the active site serine. In addition, the histidine is
generally 109
to 111 amino acids C-terminal to the active site serine.
By site-directed mutagenesis and PCR, individual codons of the human
PAF-AH coding sequence were modified to encode alanine residues and were
expressed in E. coli. As shown in Table 8 below wherein, for example, the
abbreviation "S 108A" indicates that the serine residue at position 108 was
changed
to all alanine, point mutations of Ser273, Asp296, or His351 completely
destroy PAF-
AH activity. The distances between active site residues is similar for PAF-AH
(Ser
to Asp, 23 amino acids; Ser to His, 78 amino acids) and other iipases. These
experiments demonstrate that Ser273, Asp296, and His351 are critical residues
for
activity and are therefore likely candidates for catalytic triad residues.
Cysteines are
often critical for the functional integrity of proteins because of their
capacity to form
disulfide bonds. The plasma PAF-AH enzyme contains five cysteines. To
determine
whether any of the five is critical for enzyme actvity, each cysteine was
mutated
individually to a serine and the resulting mutants were expressed in E. coli.
Preliminary activity results using partially purified preparations of these
recombinantly produced mutants are shown below in the second column of Table
8,
while results using more purled preparations are shown below in the third
column
of Table 8. The data show that all of the cysteine mutants had largely
equivalent
activity, so that none of the cysteines appears to be necessary for PAF-AH
activity.
Other point mutations also had little or no effect on PAF-AH catalytic
activity. In
Table 8, "++++" represents wild type PAF-AH activity of about 40-b0
UImI/OD600, "+++" represents about 20-40 U/mUOD600 activity, "++"
represents about 10-20 U/ml/OD600 activity, "+" represents 1-10 UImUOD600
activity, and "-" indicates < 1 U/ml/OD600 activity.
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Table 8
Mutation PAF-AH activitySpecific PAF-AH activity
of purified preparations
Wild type + + + + 6.9 mmollmg/hr
S108A ++++
S273A -
D286A -
D286N + +
D296A -
D304A ++++
D338A ++++
H351A -
H395A, H399A + + + +
C67S + + + 5.7 mmol/mglhr
C229S + 6.5 mmol/mglhr
I5 C291 S + 5.9 mmollmglhr
C334S + + + + 6. 8 mmol/mg/hr
C407S + + + 6.4 mmoUmg/hr
C67S, C334S, C407S 6.8 mmol/mg/hr
B. PAF-AH Frag_ment Products
C-terminal deletions were prepared by digesting the 3' end of the PAF-
AH coding sequence with exonuclease 13I for various amounts of time and then
ligadng the shortened coding sequence to plasmid DNA encoding stop codons in
all
three reading frames. Ten different deletion constructs were characterized by
DNA
sequence analysis, protein expression, and PAF-AH activity. Removal of twenty-
one
to thirty C-terminal amino acids greatly reduced catalytic activity and
removal of
fifty-two residues completely destroyed activity. See FIGURE 3.
Similar deletions were made at the amino terminal end of PAF-AH.
Fusions of PAF-AH with E. coli thioredoxin at the N-terminus were prepared to
facilitate consistent high level expression PAF-AH activity [LaVallie et al.,
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Bioltechnology, 11:187-193 (1993)]. Removal of nineteen amino acids from the
naturally processed N-terminus (I1e42) reduced activity by 99 % while removal
of
twenty-six amino acids completely destroyed enzymatic activity in the fusion
protein.
See FIGURE 3. Deletion of twelve amino acids appeared to enhance enzyme
activity
about four fold.
In subsequent purifications of PAF-AH from fresh human plasma by
a method similar to that described in Example 1 (Microcon 30 filter from
Amicon
were utilized to concentrate Blue sepharose eluate instead of a Cu column),
two N-
termini in addition to I1e42 were identified, Ser35 and Lys55. The
heterogeneity may
be the natural state of the enzyme in plasma or may occur during purification.
The purified material described above was also subject to analysis for
glycosylation. Purified native PAF-AH was incubated in the presence or absence
of
N-Glycanase, an enzyme that removes N-linked carbohydrates from glycoproteins.
The treated PAF-AH samples were electrophoresed through a 12 % SDS
polyacrylamide gel then visualized by Western blotting using rabbit polyclonal
antisera. Protein not treated with N-Glycanase migrated as a diffuse band of
45-50
kDa whereas the protein treated with the glycanase migrated as a tight band of
about
44 kDa, demonstrating that native PAF-AH is glycosylated.
N-terminal heterogeneity was also observed in purified preparations of
recombinant PAF-AH (I1e42 N-terminus) . These preparations were a mixture of
polypeptides with N-termini beginning at A1a47, I1e42, or the artificial
initiating
Met-1 adjacent to I1e42.
1. Preliminay comparison of PAF-AH fragments with PAF-AH
In view of the observed heterogeneity of recombinantly produced PAF-
AH, other recombinant products were prepared and tested for homogeneity after
recombinant expression and purification. The composition of the recombinant
expression products of pBAR2/PH.2 and pBAR2/PH.9 in E. coli strain MC1061 was
analyzed at different time points during the production phase of cell
fermentation.
Partially purified samples of the recombinant PH.2 and PH.9 from cells
collected at
time points ranging between 5 and 22 hours after induction of protein
expression were
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analyzed by matrix assisted laser desorption ionization mass spectrometry
(MALDI-
MS).
When the PH.2 expression vector was utilized, two peaks were
observed in the spectrum of the partially purified protein at a mass value
expected for
S rPAF-AH protein. Two peaks were observed at all time points, with greater
heterogeneity being observed at time points when fermentation is stressed as
indicated
by an accumulation of acetate and/or a depletion of oxygen in the media. The
accuracy of the MALDI-MS technique in this mass range was approximately t
0.3 % , about the mass of one amino acid. The higher mass peak observed was
consistent with the presence of the expected full length translation product
for the
PH.2 vector, minus the translation initiating methionine which is expected to
be post-
translationally removed. The lower mass peak was approximately 1200 atomic
mass
units less.
When the PH.9 expression vector was utilized, a single peak
predominated in the spectrum of the partially purified protein at a mass value
expected for rPAF-AH protein. This single peak was observed at all time
points,
with no increase in heterogeneity seen at different time points. The observed
mass
of this protein was consistent with the presence of the expected full length
translation
product for the PH.9 vector, minus the initiating methionine.
2. Purification of PAF-AH fragments
Recombinantly expressed rPH.2 (the expression product of DNA
encoding Met46-Asn~l) and rPH.9 (the expression product of DNA encoding
Met46-I1e429) p~~~ons were purified for further comparison with purified rPAF-
AH (expression product of DNA encoding I1e42-Asn~l). rPH.9 was produced by
E. toll strain SB7219 and purified generally according to the zinc chelate
purification
procedure described above, while rPH.2 was produced by E. toll strain MC106I
and
purified as described below. The transformed cells were lysed by dilution of
the cell
paste with lysis buffer (100 mM succinate, 100 mM NaCI, 20 mM CHAPS, pH 6.0).
The slurry was mixed and lysed by high pressure dismption. The lysed cells
were
centrifuged and the supernatant containing rPH.2 was retained. The clarified
supernatant was diluted 5-fold in 25 mM sodium phosphate buffer containing, 1
mM
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EDTA, 10 mM CHAPS, pH 7Ø The diluted supernatant was then applied to the Q
Sepharose column. The column was washed first with 3 column volumes of 25 mM
sodium phosphate buffer containing 1 mM EDTA, 50 mM NaCI, 10 mM CHAPS,
pH 7.0 (Wash I), then washed with 10 column volumes of 25 mM Tris buffer
containing 1 mM EDTA, IO mM CHAPS, pH 8.0 (Wash 2) and with 10 column
volumes of 25 mM Tris buffer containing 1 mM EDTA, 100 mM NaCI, 10 mM
CHAPS, pH 8.0 (Wash 3). Elution was accomplished with 25 mM Tris buffer
containing 1 mM EDTA, 350 mM NaCI, 10 mM CHAPS, pH 8Ø The Q Sepharose
eluate was diluted 3-fold in 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0 then
applied to a Blue Sepharose column. The column was washed first with 10 column
volumes of 25 mM Tris, I mM EDTA, 10 mM CHAPS, pH 8Ø The column was
then washed with 3 column volumes of 25 mM Tris, 0.5 M NaCI, 10 mM CHAPS,
pH 8Ø Elution was accomplished with 25 mM Tris, 3.0 M NaCI, 10 mM CHAPs,
pH 8Ø The Blue Sepharose eluate was diluted 5-fold in 10 mM sodium
phosphate,
10 mM CHAPS, pH 6.2 then applied to the chromatography column. The column
was washed with 10 column volumes of 10 mM sodium phosphate, 100 mM NaCI,
0.1 % Pluronic F68, pH 6.2. rPH.2 was eluted with 120 mM sodium phosphate, 100
mM NaCI, 0.1 % Pluronic F-68, pH 7.5. The hydroxyapatite eluate was diluted 6-
fold with 10 mM sodium phosphate, 0.1 % Pluronic F68, pH 6.8. The diluted
hydroxyapatite eluate was adjusted to pH 6.8 using 0.5 N succinic acid and
then
applied to a SP Sepharose column. The SP Sepharose column was washed with 10
column volumes 50 mM sodium phosphate, 0.1 % Fluronic F68, pH 6.8 and eluted
with 50 mM sodium phosphate, I25 mM NaCI, 0.1 % Pluronic F68, pH 7.5. The
eluted rPH.2 was formulated by diluting to a final concentration of 4 mg/ml in
50
mM sodium phosphate, 125 mM NaCI, 0.15 % Pluronic F68, pH 7.5, and Tween 80
was added to a final concentration of 0.02 % Tween 80. The formulated product
was
then filtered through a 0.2~u membrane and stored prior to use.
3. Comparison of PAF-AH fragments with PAF-AH bv, seauen
The purified rPH.2 and rPH.9 preparations were compared with
purified rPAF-AH preparations by N-terminal sequencing using an Applied
Biosystems Model 473A Protein Sequencer (Applied Biosystems, Foster City, CA)
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and by C-terminal sequencing using a Hewlett-Packard Model GI009A C-terminal
Protein Sequencer. The rPH.2 preparation had less N-terminal heterogeneity
compared to rPAF-AH. The N-terminus analysis of the rPH.9 preparation was
similar to that of rPH.2, but less C-terminal heterogeneity was observed for
the
rPH.9 preparation relative to rPH.2.
The purified rPH.2 preparation contained a major sequence with an N-
terminus of A1a47 (about 86-89 %) and a minor sequence with an N-terminus of
A1a48
(about 11-14%), with the ratio of the two N-termini being fairly consistent
under
different fermentation conditions. The purified rPH.9 preparation also
contained a
major sequence with an N-terminus of Ata47 (about 83-90%) and a minor sequence
with an N-terminus of A1a48 (about 10-17 % ). In contrast, attempts to produce
in
bacteria the polypeptide beginning at I1e42 (rPAF-AH) resulted in a varying
mixture
of polypeptides with N-termini beginning at A1a47 (20-53 %}, I1e42 (8-10%), or
at the
artificial initiating Met_I methionine (37-72 % ) adjacent to I1e42. For rPH.2
and
rPH.9, the initiating methionine is efficiently removed by an amino-terminal
peptidase
after bacterial synthesis of the polypeptide, leaving the alanine at position
47 (or the
alanine at position 48) as the N-terminal residue.
C-terminal sequencing was carried out on one lot of rPH.2, which was
observed to have a C-terminus of HOOC-Asn-Tyr as the major sequence (about
80%), consistent with the predicted HOOC-Asn~I-Tyr~O C-terminus of the
translation product, while about 20 % was HOOC-Leu. After the rPH.2
preparation
had been fractionated by SDS-PAGE, additional sequencing of the primary and
secondary bands yielded a C-terminal sequence of HOOC-Leu-Met from a lower
secondary band (AHL, described below in section B.S.) consistent with a
product that
is 10 amino acids shorter than the full length translation product, as well as
low levels
of HOOC-His. Further peptide mapping has shown that additional C-termini are
present in some lots of PH.2 protein. The C-terminus of rPH.9 was primarily
HOOC-Ile-His (about ?8 to 91 %, depending on the lot) by direct sequencing,
consistent with the predicted HOOC-Ile42g-His428 C-terminus of the translation
product. There appears to be some background ("noise") in this technique, so
low
levels of other sequences could not be ruled out.
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4. Comparison of PAF-AH fragments with PAF-AH by MALDI-MS
MALDI-MS was performed on purified rPH.2 and rPH.9 preparations.
The rPH.2 spectrum exhibited two peaks in the spectrum at a mass value
expected
for the rPAF-AH product (see FIGURE 4), similar to the pattern observed with
the
partially purified protein in section B.1. above. The secondary, lower
molecular
weight peak was typically present at approximately 20 % to 30 ~ of the total.
The
rPH.9 spectrum showed a predominant peak at a mass consistent with that
expected
for the full length translation product for the PH.9 vector, minus the
translation
initiating methionine (see FIGURE 5). A small slightly lower molecular weight
shoulder peak was also observed for rPH.9 that represented approximately 5 %
of the
total.
5. Comparison of PAF-AH fragments with PAF-AH by SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE)
was performed vn purified rPAF-AH, rPH.2 and rPH.9 preparations. A complicated
banding pattern was observed for rPH.2 around the electrophoretic migration
range
expected for the rPAF-AH product, based on protein molecular weight standards.
One, or in some gels, two predominant bands were seen, with readily observed
secondary bands above and below the primary band. These upper secondary,
middle
primary and lower secondary bands, respectively, were termed AHU, AHM and
AHL. All of these bands reacted with an anti-rPAF-AH monoclonal antibody on
Western blot and have thus been identified as rPAF-AH related products. The
upper
secondary band AHU increased in intensity over time with storage of the
protein and
presumably represents a modified form of the rPAF-AH product. The SDS-PAGE
of the rPAF-AH preparation is similar to that of rPH.2. There are two major
bands
that migrate near the expected molecular weight for rPAF-AH, as well as a
minor
band above and a shadow below the major bands. In contrast, rPH.9 displayed a
single predominant band on SDS-PAGE with no apparent splitting. Faint bands at
a slightly lower molecular weight and at an expected dimer position were also
seen.
No AHU-like band was observed.
The composition of the purified rPH.2 and rPH.9 preparations was also
analyzed on 2D gels (isoelectric focusing (IEF) in urea followed by SDS-PAGE
in the
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second dimension). For rPH.9, the 2D gels showed five main spots separated in
the
IEF direction. The charge heterogeneity appeared consistent between lots of
rPH.9.
In contrast, the 2D gel pattern of rPH.2 was more complicated as it contained
approximately 15 spots separated in the IEF and SDS-PAGE dimensions.
6. Comparison of activity of PAF-AH fragments with PAF-AH
Purified rPH.2 and rPH.9 have enzymatic activity indistin4guishable
from that of endogenous PAF-AH purified from senior, and rPH.2 and rPH.9 bind
to lipropotein in a similar manner as purified endogenous PAF-AH.
Example 11
A preliminary analysis of expression patterns of human plasma PAF
AH mRNA in human tissues was conducted by Northern blot hybridization.
RNA was prepared from human cerebral cortex, heart, kidney,
placenta, thymus and tonsil using RNA Stat 60 (Tel-Test "B", ~iiendswood, TX).
Additionally, RNA was prepared from the human hematopoietic precursor-like
cell
IS line, THP-1 (ATCC TIB 202), which was induced to differentiate to a
macrophage-
like phenotype using the phorbol ester phorbolmyristylacetate (PMA). Tissue
RNA
and RNA prepared from the premyelocytic THP-1 cell line prior to and 1 to 3
days
after induction were electrophoresed through a 1.2 °~ agarose
formaldehyde gel and
subsequently transferred to a nitrocellulose membrane. The full length human
plasma
PAF-AH cDNA, sAH 406-3, was labelled by random priming and hybridized to the
membrane under conditions identical to those described in Example 3 for
library
screening. Initial results indicate that the PAF-AH probe hybridized to a 1.8
kb band
in the thymus, tonsil, and to a lesser extent, the placental RNA.
PAF is synthesized in the brain under normal physiological as weU as
pathophysiological conditions. Given the known pro-infla.mmatory and potential
neurotoxic properties of the molecule, a mechanism for localization of PAF
synthesis
or for its rapid catabolism would be expected to be critical for the health of
neural
tissue. The presence of PAF acetylhydrolase in neural tissues is consistent
with it
playing such a protective role. Interestingly, both a bovine heterotrimeric
intracellular PAF-AH [the cloning of which is described in Hattori et al., J.
Biol.
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Chem., 269(37): 23150-23155 (1994)] and PAF-AH of the invention have been
identified in the brain. To determine whether the two enzymes are expressed in
similar or different compartments of the brain, the human homologue of the
bovine
brain intracellular PAF-AH cDNA was cloned, and its mRNA expression pattern in
the brain was compared by Northern blotting to the mRNA expression pattern of
the
PAF-AH of the invention by essentially the same methods as described in the
foregoing paragraph. The regions of the brain examined by Northern blotting
were
the cerebellum, medulla, spinal cord, putamen, amygdala, caudate nucleus,
thalamus,
and the occipital pole, frontal lobe and temporal lobe of the cerebral cortex.
Message
of both enzymes was detected in each of these tissues although the
heterotrimeric
intracellular form appeared in greater abundance than the secreted form.
Northern
blot analysis of additional tissues further revealed that the heterotrimeric
intracellular
form is expressed in a broad variety of tissues and cells, including thymus,
prostate,
testis, ovary, small intestine, colon, peripheral blood leukocytes,
macrophages, brain,
liver, skeletal muscle, kidney, pancreas and adrenal gland. This ubiquitous
expression suggests that the heterotrimeric intracellular PAF-AH has a general
housekeeping function within cells.
The expression of PAF-AH RNA in monocytes isolated from human
blood and during their spontaneous differentiation into macrophages in culture
was
also examined. Little or no RNA was detected in fresh monocytes, but
expression
was induced and maintained during differentiation into macrophages. There was
a
concomitant accumulation of PAF-AH activity in the culture medium of the
differentiating cells. Expression of the human plasma PAF-AH transcript was
also
observed in the THP-1 cell RNA at 1 day but not 3 days following induction.
THP-1
cells did not express mRNA for PAF-AH in the basal state.
Example 12
PAF-AH expression in human and mouse tissues was examined by in
situ hybridization.
Human tissues were obtained from National Disease Research
Interchange and the Cooperative Human Tissue Network. Normal mouse brain and
spinal cord, and EAE stage 3 mouse spinal cords were harvested from S/JLJ
mice.
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Normal S/JIJ mouse embryos were harvested from eleven to eighteen days after
fertilization.
The tissue sections were placed in Tissue Tek II cryomolds {Miles
Laboratories, Inc., Naperville, IL) with a small amount of OCT compound
{Miles,
Inc., Elkhart, IN). They were centered in the cryomold, the cryomold filled
with
OCT compound, then placed in a container with 2-methylbutane [C2H5CH(CH3)2,
Aldrich Chemical Company, Inc. , Milwaukee, WI] and the container placed in
liquid
nitrogen. Once the tissue and OCT compound in the cryomold were frozen, the
blocks were stored at -80 ° C until sectioning. The tissue blocks were
sectioned at 6
,um thickness and adhered to Vectabond (Vector Laboratories, Inc., Burlingame,
CA)
coated slides and stored at -70 ° C and placed at 50 ° C for
approximately 5 minutes to
warm them and remove condensation and were then fixed in 4 % paraformaldehyde
for 20 minutes at 4 ° C, dehydrated (70 % , 95 % , 100 % ethanol) for 1
minute at 4 ° C
in each grade, then allowed to air dry for 30 minutes at room temperature.
Sections
were denatured for 2 minutes at 70 ° C in 70 % formamide/2X SSC, rinsed
twice in
2X SSC, dehydrated and then air dried for 30 minutes. The tissues were
hybridized
in situ with radiolabeled single-stranded mRNA generated from DNA derived from
an internal 1 Kb HindllI fragment of the PAF-AH gene (nucleotides 308 to 1323
of
SEQ m NO: 7) by in vitro RNA transcription incorporation 35S-UTP (Amersham)
or from DNA derived from the heterotrimeric intracellular PAF-AH cDNA
identified
by Hattori et al. The probes were used at varying lengths from 250-500 bp.
Hybridization was carried out overnight (12-I6 hours) at 50 ° C; the
35S-labeled
riboprobes (6 x 105 cpm/section), tRNA (0.5 ~cg/section) and
diethylpyrocarbonate
{depc)-treated water were added to hybridization buffer to bring it a final
concentration of 50% formamide, 0.3M NaCI, 20 mM Tris pH 7.5, 10% dextran
sulfate, 1 X Denhardt's solution, 100 mM dithiothretol (DTT) and 5 mM EDTA.
After hybridization, sections were washed for 1 hour at room temperature in 4X
SSCI10 mM DTT, then for 40 minutes at 60°C in 50% formamide/1X SSCI10
mM
DTT, 30 minutes at room temperature in 2X SSC, and 30 minutes at room
temperature in 0.1X SSC. The sections were dehydrated, air dried for 2 hours,
coated with Kodak NTB2 photographic emulsion, air dried for 2 hours, developed
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(after storage at 4 ° C in complete darkness) and counterstained with
hematoxylinleosin.
A. Brain
Cerebellum. In both the mouse and the human brains, strong signal
was seen in the Purkinje cell layer of the cerebellum, in basket cells, and
individual
neuronal cell bodies in the dentate nucleus (one of the four deep nuclei in
the
cerebellum). Message for the heterotrimeric intracellular PAF-AH was also
observed
in these cell types. Additionally, plasma PAF-AH signal was seen on individual
cells
in the granular and molecular layers of the grey matter.
Hippocampus. In the human hippocampus section, individual cells
throughout the section, which appear to be neuronal cell bodies, showed strong
signal. These were identified as polymorphic cell bodies and granule cells.
Message
for the heterotrimeric intracellular PAF-AH was also observed in hippocampus.
Brain stem. On both human and mouse brain stem sections, there was
strong signal on individual cells in the grey matter.
Cortex. On human cortex sections taken from the cerebral, occipital,
and temporal cortexes, and on mouse whole brain sections, individual cells
throughout
the cortex showed strong signal. These cells were identified as pyramidal,
stellate
and polymorphic cell bodies. There does not appear to be differentiation in
the
expression pattern in the different layers of the cortex. These in situ
hybridization
results are different from the results for cerebral cortex obtained by
Northern blotting.
The difference is likely to result from the greater sensitivity of in situ
hybridization
compared to that of Northern blotting. As in the cerebellum and hippocampus, a
similar pattern of expression of the heterotrimeric intracellular PAF-AH was
observed.
Pituitary. Somewhat weak signal was seen on scattered individual cells
in the pars distalis of the human tissue section.
B. Human colon
Both normal and Crohn's disease colons displayed signal in the
lymphatic aggregations present in the mucosa of the sections, with the level
of signal
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being slightly higher in the section from the Crohn's disease patient. The
Crohn's
disease colon also had strong signal in the lamina propria. Similarly, a high
level of
signal was observed in a diseased appendix section while the normal appendix
exhibited a lower but still detectable signal. The sections from the
ulcerative colitis
patient showed no evident signal in either the lymphatic aggregations or the
lamina
propna.
C. Human tonsil and th,
Strong signal was seen on scattered groups of individual cells within
the germinal centers of the tonsil and within the thymus.
D. Human lymph node
Strong signal was observed on the lymph node section taken from a
normal donor, while somewhat weak signal was observed in the lymph nodules of
the
section from a donor with septic shock.
E. Human small intestine
Both normal and Crohn's disease small intestine had weak signal in the
Peyer's patches and lamina propria in the sections, with the signal on the
diseased
tissue slightly higher.
F. Human spleen and lung
Signal was not observed on any of the spleen (normal and splenic
abcess sections) or lung (normal and emphysema sections) tissues.
G. Mouse spinal cord
In both the normal and EAE stage 3 spinal cords, there was strong
signal in the grey matter of the spinal cord, with the expression being
slightly higher
in the EAE stage 3 spinal cord. In the EAE stage 3 spinal cord, cells in the
white
matter and perivascular cuffs, probably infiltrating macrophages and/or other
leukocytes, showed signal which was absent in the normal spinal cord.
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F. Mouse embryos
In the day 11 embryo signal was apparent in the central nervous system
in the fourth ventricle, which remained constant throughout the embryo time
course
as it developed into the cerebellum and brain stem. As the embryos matured,
signal
became apparent in central nervous system in the spinal cord (day 12), primary
cortex
and ganglion Gasseri (day 14), and hypophysis (day 16). Signal was observed in
the
peripheral nervous system (beginning on day 14 or 15) on nerves leaving the
spinal
cord, and, on day 17, strong signal appeared around the whiskers of the
embryo.
Expression was also seen in the liver and lung at day 14, the gut (beginning
on day
15), and in the posterior portion of the mouth/throat (beginning on day 16).
By day
18, the expression pattern had differentiated into signal in the cortex,
hindbrain
(cerebellum and brain stem), nerves leaving the lumbar region of the spinal
cord, the
posterior portion of the mouth/throat, the liver, the kidney, and possible
weak signal
in the lung and gut.
G. Summary
PAF-AH mRNA expression in the tonsil, thymus, lymph node, Peyer's
patches, appendix, and colon lymphatic aggregates is consistent with the
conclusions
that the probable predominant in vivo source of PAF-AH is the macrophage
because
these tisues all are populated with tissue macrophages that serve as
phagocytic and
antigen-processing cells.
Expression of PAF-AH in inflamed tissues would be consistent with
the hypothesis that a role of monocyte-derived macrophages is to resolve
inflammation. PAF-AH would be expected to inactivate PAF and the pro-
inflammatory phospholipids, thus down-regulating the inflammatory cascade of
events
initiated by these mediators.
PAF has been detected in whole brain tissue and is secreted by rat
cerebellar granule cells in culture. In vitro and in vivo experiments have
demonstrated that PAF binds a specific receptor in neural tissues and induces
functional and phenotypic changes such as calcium mobilization, upregulation
of
transcription activating genes, and differentiation of the neural precursor
cell line,
PC12. These observations suggested a physiologic role for PAF in the brain,
and
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consistent with this, recent experiments using hippocampal tissue section
cultures and
PAF analogs and antagonists have implicated PAF as an important retrograde
messenger in hippocampal long term potendation. Therefore, in addition to its
pathological effect in inflammation, PAF appears to participate in routine
neuronal
signalling processes. Expression of the extracellular PAF-AH. in the brain may
seine
to regulate the duration and magnitude of PAF-mediated signalling. ~~
Example 13
Monoclonal antibodies specific for recombinant human plasma PAF-AH
were generated using E. coti produced PAF AH as an immunogen.
Mouse X1342 was injected on day 0, day 19, and day 40 with
recombinant PAF-AH. For the prefusion boost, the mouse was injected with the
immunogen in PBS, four days later the mouse was sacrificed and its spleen
removed
sterilely and placed in lOml serum free RPMI 1640. A single-cell suspension
was
formed by grinding the spleen between the frosted ends of two glass microscope
slides submerged in serum free RPMI 1640, supplemented with 2 mM L-glutamine;
1 mM sodium pyruvate, 100 units/ml penicillin, and 100 p,gl ml streptomycin
(RPMI)
,.*
(Gibco, Canada). The cell suspension was filtered thmugh sterile 70-mesh Nitex
cell
strainer (Becton Dickinson, Parsippany, New Jersey), and washed twice by
centrifuging at 200 g for 5 minutes and resuspending the pellet in 20 ml serum
free
ItPMI. Thymocytes taken from 3 naive Balb/c mice were prepared in a similar
manner. NS-1 myeloma cells, kept in log phase in RPMI with 1196 fetal bovine
serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three days prior to
fusion, were centrifuged at 200 g for 5 minutes, and the pellet was washed
twice as
described in the foregoing paragraph.
One x 108 spleen cells were combined with 2.0 x 107 NS-i cells,
centrifuged and the supernatant was aspirated. The cell pellet was dislodged
by
tapping the tube and 1 ml of 37' C PEG 1500 (50 ~ in 75mM Hepes, pH 8.0)
(Boehringer Mannheim) was added with stirring over the course of 1 minute,
followed by adding 7 ml of serum free ItPMI over 7 minutes. An additional 8 ml
RPMI was added and the cells were centrifuged at 200 g for 10 minutes. After
discarding the supernatant, the pellet 'was resuspended in 200 ml ItPMI
containing
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15 % FES, 100 ~cM sodium hypoxanthine, 0.4 ~uM aminopterin, 16 ~M thymidine
(HAT) (Gibco), 25 unitslml IL-6 (Boehringer Mannheim) and 1.5 x 106
thymocyteslml and plated into 10 Corning flat bottom 96 well tissue culture
plates
(Corning, Corning New York).
On days 2, 4, and 6, after the fusion, 100 ~cl of medium was removed
from the wells of the fusion plates and replaced with fresh medium. On day 8,
the
fusion was screened by ELISA, testing for the presence of mouse IgG binding to
recombinant PAF-AH. Immulon 4 plates (Dynatech, Cambridge, MA) were coated
for 2 hours at 37 ° C with 100 ng/well recombinant PAF-AH diluted in
25mM TRIS,
pH 7.5. The coating solution was aspirated and 200u1/well of blocking solution
[0.5 % fish skin gelatin (Sigma) diluted in CMF-PBS] was added and incubated
for
30 minutes at 37 ° C. Plates were washed three times with PBS with 0.05
% Tween
(PBST) and 50 wl culture supernatant was added. After incubation at 37
° C for 30
minutes, and washing as above, 50 ~,I of horseradish peroxidase conjugated
goat anti-
15 mouse IgG(fc) (Jackson ImmunoResearch, West Grove, Pennsylvania) diluted
1:3500
in PBST was added. Plates were incubated as above, washed four times with PBST
and 100 ~cL substrate, consisting of 1 mglml o-phenylene diamine (Sigma) and
0.1
~I/ml 30% H202 in 100 mM Citrate, pH 4.5, was added. The color reaction was
stopped in 5 minutes with the addition of 50 ~.l of 15 % H2S04. A490 was read
onn
20 a plate reader (Dynatech).
Selected fusion wells were cloned twice by dilution into 96 well plates
and visually scoring the number of colonies/well after 5 days. Hybridomas
cloned
were 90D1E, 90E3A, 90E6C, 90G11D (ATCC HB 11724), and 90F2D (ATCC HB
1 /725).
The monoclonal antibodies produced by hybridomas were isotyped
using the Isostrip system {Boehringer Mannheim, Indianapolis, II~. Results
showed
that the monoclonal antibodies produced by hybridomas from fusion 90 were all
IgG 1.
All of the monoclonal antibodies produced by hybridomas from fusion
90 functioned well in ELISA assays but were unable to bind PAF-AH on Western
blots. To generate antibodies that could recognize PAF-AH by Western, mouse
#1958 was immunized with recombinant enzyme. Hybridomas were generated as
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described for fusion 90 but were screened by Western blotting rather than
ELISA to
identify Western-competent clones.
For Western analyses, recombinant PAF-AH was mixed with an equal
volume of sample buffer containing 125mM Tris, pH 6. 8, 4 % SDS, 100mM
dithiothreitol and 0.05 % bromphenol blue and boiled for five minutes prior to
loading
onto a 12% SDS polyacrylamide gel (Novex). Following electrophoresis at 40
mAmps, proteins were electrotransferred onto a polyvinylidene fluoride
membrane
(Pierce) for 1 hour at 125 V in 192mM glycine, 25mM Tris base, 20 % methanol,
and
0.41 % SDS. The membrane was incubated in 20mM Tris, 100mM NaCI (TBS)
containing 5 % bovine senlm albumin (BSA, Sigma) overnight at 4°C. The
blot was
incubated 1 hour at room temperature with rabbit polyclonal antisera diluted
i/8000
in TBS containing 5 % BSA, and then washed with TBS and incubated with
alkaline
phosphatase-conjugated goat anti-mouse IgG in TBS containing 5 % BSA for 1
hour
at room temperature. The blot was again washed with TBS then incubated with
1 S 0.02 % S-bromo-4-chloro-3-indolyl phosphate and 0.03 % nitroblue
tetrazolium in
100mM Tris-HCI, pH 9.5, 100mM NaCI, and SmM MgCl2. The reaction was
stopped with repeated water rinses.
Selected fusion wells, the supernatants of which were positive in
Western analyses, were processed as described above. Hybridoma 143A reacted
with
PAF-AH in Western blots and was cloned (ATCC HB 11900).
Polyclonal antisera specific for human plasma PAF-AH was raised in
rabbits by three monthly immunizations with 100 ~.g of purified recombinant
enzyme
in Fmend's adjuvant.
Example 14
Experimental studies were performed to evaluate the in vivo therapeutic
effects of recombinant PAF-AH of the invention on acute inflammation using a
rat
foot edema model [Henriques et al., Br. J. Pharmacol., 106: 579-582 (1992)].
The
results of these studies demonstrated that rPAF-AH blocks PAF-induced edema.
Parallel studies were done to compare the effectiveness of PAF-AH with two
commercially available PAF antagonists.
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A. Preparation of PAF-AH
E. coli transformed with the PAF-AH expression vector puc trp AH
were lysed in a microfluidizer, solids were centrifuged out and the cell
supernatants
were loaded onto a S-Sepharose column (Pharmacia). The column was washed
extensively with buffer consisting of 50mM NaCI, lOmM CHAPS, 25mM MES and
1mM EDTA, pH 5.5. PAF-AH was eluted by increasing the NaCI concentration of
the buffer to 1M. Affinity chromatography using a Blue Sepharose column
(Pharmacia) was then used as an additional purification step. Prior to loading
the
PAF-AH preparation on the Blue Sepharose column, the sample was diluted 1:2 to
reduce the NaCI concentration to 0.5M and the pH was adjusted to 7.5. After
washing the Blue Sepharose column extensively with buffer consisting of 0.5M
NaCl,
25mM tris, IOmM CHAPS and 1mM EDTA, pH 7.5 the PAF-AH was eluted by
increasing the NaCI concentration to 3.OM.
Purity of PAF-AH isolated in this manner was generally 95 % as
assessed by SDS-PAGE with activity in the range of 5000-10,000 Ulml.
Additional
quality controls done on each PAF-AH preparation included determining
endotoxin
levels and hemolysis activity on freshly obtained rat erythrocytes. A buffer
containing 25mM Tris, lOmM CHAPS, O.SM NaCI, pH 7.5 functioned as storage
media of the enzyme as well as Garner for administration. Dosages used in
experiments were based on enzyme activity assays conducted immediately prior
to
experiments.
B. Induction of Edema
Six to eight-week-old female Long Evans rats (Charles River,
Wilmington, MA), weighing 180-200 grams, were used for all experiments. Prior
to experimental manipulations, animals were anesthetized with a mixture of the
anesthetics Ketaset (Fort Dodge Laboratories, Fort Dodge, IA), Rompun (Miles,
Shawnee Mission, KS), and Ace Promazine (Aveco, Fort Dodge, IA) administered
subcutaneously at approximately 2.5 mg Ketaset, 1.6 mg Rompun, 0.2 mg Ace
Promazine per animal per dose. Edema was induced in the foot by administration
of
either PAF or zymosan as follows. PAF (Sigma #P-1402) was freshly prepared for
each experiment from a 19.1 mM stock solution stored in chloroform/methanol
(9:1 )
a;i;
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at -20' C. Required volumes were dried down under N2, diluted 1:1000 in a
buffer
containing 150mM NaCI, lOmM Tris pH 7.5, and 0.25 ~ BSA, and sonicated for
five
minutes. Animals received 50 ~cl PAF (final dose of 0.96 nmoles)
subcutaneously
between the hind foot pads, and edema was assessed after 1 hour and again
after 2
hours in some experiments. Zymosan A (Sigma flA-8800) was freshly prepared for
each experiment as a suspension of 10 mg/ml in PBS. Animals nxeived 50 ~d of
zymosan (final dose of 500 ~.g) subcutaneously between the hind foot pads and
edema
was assessed after 2 hours.
Edema was quantitated by measuring the foot volume immediately prior
to administration of PAF or zymosan and at indicated time point post-challenge
with
PAF or zymosan. Edema is expressed as the increase in foot volume in
milliliters.
Volume displacement measurements were made on anesthetized animals using a
plethysmometer (UGO Basile, model x!'7150) which measures the displaced water
volume of the immersed foot. In order to insure that foot immersion was
comparable
from one time point to the next, the hind feet were marked in indelible ink
where the
hairline meets the heel. Repeated measurements of the same foot using this
technique
indicate the precision to be within 5 ~ .
C. PAF-AH Administration Routes and Dosaees
PAF-AH was injected locally between the foot pads, or systematically
by IV injection in the tail vein. For local administration rats received 100
~1 PAF-
AH (4000-6000 U/ml) delivered subcutaneously between the right hind foot pads.
Left feet served as controls by administration of 100 ~d carrier (buffered
salt
solution). ~ For systemic administration of PAF-AH, rats received the
indicated units
of PAF-AH in 300 ~.1 of carrier administered IV in the tail vein. Controls
received
the appropriate volume of carrier IV in the tail vein.
D. Local Administration of PAF-AH
Rats (N=4) were injected with 100 ~cl of PAF-AH (4000-6000 U/ml)
subcutaneously between the right foot pads. Left feet were injected with 100
gel
carrier (buffered salt solution). Four other rats were injected only with
carrier. All
rats were immediately challenged with PAF via subcutaneous foot injection and
foot
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volumes assessed 1 hour post-challenge. FIGURE 6, wherein edema is expressed
as
average increase in foot volume (m1) ~ SEM for each treatment group,
illustrates that
PAF-induced foot edema is blocked by local administration of PAF-AH. The group
which received local PAF-AH treatment prior to PAF challenge showed reduced
inflammation compared to the control injected group. An increase in foot
volume of
0.08 ml ~ 0.08 (SEM) was seen in the PAF-AH group as compared to O.b3 ~ 0.14
(SEM) for the carrier treated controls. The increase in foot volume was a
direct
result of PAF injection as animals injected in the foot only with carrier did
not exhibit
an increase in foot volume.
E. Intravenous Administration of PAF-AH
Rats (N=4 per group) were pretreated IV with either PAF-AH (2000
U in 300 ~.1 Garner) or Garner alone, 15 minutes prior to PAF challenge. Edema
was
assessed 1 and 2 hours after PAF challenge. FIGURE 7, wherein edema is
expressed
as average increase in volume (ml) ~ SEM for each treatment group, illustrates
that
IV administration of PAF-AH blocked PAF induced foot edema at one and two
hours
post challenge. The group which received 2000 U of PAF-AH given by the IV
route
showed a reduction in inflammation over the two hour time course. Mean volume
increase for the PAF-AH treated group at two hours was 0.10 ml ~ 0.08 (SEM),
versus 0.56 ml ~ 0.11 far carrier treated controls.
F. Comparison of PAF-AH Protection in Edema Induced by PAF or Zymosan
Rats (N=4 per group) were pretreated IV with either PAF-AH (2000
U in 300 ~.l Garner) or carrier alone. Fifteen minutes after pretreatment,
groups
received either PAF or zymosan A, and foot volume was assessed after 1 and 2
hours, respectively. As shown in FIGURE 8, wherein edema is expressed as
average
increase in volume (ml) ~ SEM for each treatment group, systemic
administration
of PAF-AH (2000 U) was effective in reducing PAF-induced foot edema, but
failed
to block zymosan induced edema. A mean increase in volume of 0.08 + 0.02 was
seen in the PAF-AH treated group versus 0.49 ~ 0.03 for the control group.
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G. Effective Dose Titration of PAF-AH Protection
In two separate experiments, groups of rats (N=3 to 4 per group) were
pretreated IV with either serial dilutions of PAF-AH or carrier control in a
300 ~,1
volume, 15 minutes prior to PAF challenge. Both feet were challenged with PAF
(as
described above) and edema was assessed after 1 hour. FIGURE 9 wherein edema
is expressed as average increase in volume (ml) ~ SEM for each treatment
group,
illustrates the increase in protection from PAF-induced edema in rats injected
with
increasing dosages of PAF-AH. In the experiments, the ID50 of PAF-AH given by
the IV route was found to be between 40 and 80 U per rat.
H. I_n Vivo Efficacv of PAF-AH as a Function of Time After Administration
In two separate experiments, two groups of rats (N=3 to 4 per group)
were pretreated IV with either PAF-AH (2000 U in 300 ~cl carrier) or carrier
alone.
After administration, groups received PAF at time points ranging from 15
minutes
to 47 hours post PAF-AH administration. Edema was then assessed 1 hour after
PAF
challenge. As shown in FIGURE 10, wherein edema is expressed as average
increase
in volume (ml) ~ SEM for each treatment group, administration of 2000 U of PAF-
AH protects rats from PAF induced edema for at least 24 hours.
I. Pharmacokinetics of PAF-AH
Four rats received 2000 U of PAF-AH by IV injection in a 300 ~cl
volume. Plasma was collected at various time points and stored at 4°C
and plasma
concentrations of PAF-AH were determined by ELISA using a double mAb capture
assay. In brief, monoclonal antibody 90G1ID (Example 13) was diluted in SOmM
carbonate buffer pH 9.6 at 100 ng/ml and immobilized on Immulon 4 EI,ISA
plates
overnight at 4 ° C. After extensive washing with PBS containing 0.05 %
Tween 20,
the plates were blocked for 1 hour at room temperature with 0.5 % fish skin
gelatin
(Sigma) diluted in PBS. Serum samples diluted in PBS with lSmM CHAPS were
added in duplicate to the washed ELISA plate and incubated for 1 hour at room
temperature. After washing, a biotin conjugate of monoclonal antibody 90F2D
(Example 13) was added to the wells at a concentration of 5 ~.g/ml diluted in
PBS and
incubated for 1 hour at room temperature. After washing, SO wl of a 1:1000
dilution
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of ExtraAvidin (Sigma) was added to the wells and incubated for 1 hour at room
temperature. After washing, wells were developed using OPD as a substrate and
quantitated. Enzyme activity was then calculated from a standard curve. FIGURE
11, wherein data points represent means ~ SEM, shows that at one hour plasma
enzyme levels approached the predicted concentration based on a 5-6 ml plasma
volume for 180-200 gram rats, mean = 374 U/ml ~ 58.2. Beyond one hour plasma
levels steadily declined, reaching a mean plasma concentration of 19.3 U/ml ~
3.4
at 24 hours, which is still considerably higher than endogenous rat PAF-AH
levels
which have been found to be approximately 4 U/mI by enzymatic assays.
J. Effectiveness of PAF-AIi Versus PAF Antagonists
Groups of rats (N=4 per group) were pretreated with one of three
potential antiinflammatories: the PAF antagonist CV3988 (Biomol #L-103)
administered IP (2 mg in 200 ~.1 EtOH), the PAF antagonist Alprazolam (Sigma
#A-
8800) administered IP (2 mg in 200 ~,l EtOH), or PAF-AH (2000 U) administered
IV. Control rats were injected IV with a 300 ~,l volume of carrier. The PAF
antagonists were administered IP because they are solubilized in ethanol. Rats
injected with either CV3988 or Alprazolam were challenged with PAF 30 minutes
after administration of the PAF antagonist to allow the PAF antagonist to
enter
circulation, while PAF-AH and Garner-treated rats were challenged 15 minutes
after
enzyme administration. Rats injected with PAF-AH exhibited a reduction in PAF-
induced edema beyond that afforded by the established PAF antagonists CV3988
and
Alprazolam. See FIGURE 12 wherein edema is expressed as average increase in
volume (ml) t SEM for each treatment group.
In summary, rPAF-AH is effective in blocking edema mediated by
PAF in vivo. Administration of PAF-AH products can be either local or systemic
by
IV injection. In dosing studies, IV injections in the range of 160-2000 U/rat
were
found to dramatically reduce PAF mediated inflammation, while the ID50 dosage
appears to be in the range of 40-80 Ulrat. Calculations based on the plasma
volume
for 180-200 gram rats predicts that a plasma concentration in the range of 25-
40 U/ml
should block PAF-elicited edema. These predictions are supported by
preliminary
pharmacokinetic studies. A dosage of 2000 U of PAF-AH was found to be
effective
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in blocking PAF mediated edema for at least 24 hours. At 24 hours following
administration of PAF-AH plasma concentrations of the enzyme were found to be
approximately 25 U/ml. PAF-AH was found to block PAF-induced edema more
effectively than the two known PAF antagonists tested.
Collectively, these results demonstrate that PAF-AH effectively blocks
PAF induced inflammation and may be of therapeutic value in diseases where PAF
is the primary mediator.
Example IS
Recombinant PAF-AH of the invention was tested in a second in vivo
model, PAF-induced pleurisy. PAF has previously been shown to induce vascular
leakage when introduced into the pleural space [Henriques et al. , supra] .
Female rats
(Charles River, 180-200 g) were injected in the tail vein with 200 ~cl of 1 %
Evans
blue dye in 0.9 % with 300 ~,1 recombinant PAF-AH (1500 ~mol/ml/hour, prepared
as described in Example I4) or with an equivalent volume of control buffer.
Fifteen
minutes later the rats received an 100 ~cl injection of PAF (2.0 nmol) into
the pleural
space. One hour following PAF challenge, rats were sacrificed and the pleural
fluid
was collected by rinsing the cavity with 3 ml heparinized phosphate buffered
saline.
The degree of vascular leak was determined by the quantity of Evans blue dye
in the
pleural space which was quantitated by absorbance at 620 nm. Rats pretreated
with
PAF-AH were found to have much less vascular leakage than control animals
(representing more than an 80% reduction in inflammation).
The foregoing results support the treatment of subjects suffering from
pleurisy with recombinant PAF-AH enzyme of the invention.
Example 16
Recombinant PAF-AH enzyme of the invention was also tested for
efficacy in a model of antigen-induced eosinophil recruitment. The
accumulation of
eosinophils in the airway is a characteristic feature of late phase immune
responses
which occur in asthma, rhinitis and eczema. BALB/c mice (Charles River) were
sensitized by two intraperitoneal injections consisting of 1 ~cg of ovalbumin
(OVA)
in 4 mg of aluminum hydroxide (Imject alum, Pierce Laboratories, Rockford, IL)
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given at a 2 week interval. Fourteen days following the second immunization,
the
sensitized mice were challenged with either aerosolized OVA or saline as a
control.
Prior to challenge mice were randomly placed into four groups, with
four micelgroup. Mice in groups 1 and 3 were pretreated with 140 ~1 of control
buffer consisting of 25mM tris, 0.5M NaCI, 1mM EDTA and 0.1 % Tween 80 given
by intravenous injection. Mice in groups 2 and 4 were pretreated with 750
units of
PAF-AH {activity of 5,500 units/mI given in 140 ~1 of PAF-AH buffer). Thirty
minutes following administration of PAF-AH or buffer, mice in groups 1 and 2
were
exposed to aerosolized PBS as described below, while mice in groups 3 and 4
were
exposed to aerosolized OVA. Twenty-four hours later mice were treated a second
time with either 140 ~cl of buffer (groups 1 and 3) or 750 units of PAF-AH in
140 ~cl
of buffer (groups 2 and 4) given by intravenous injection.
Eosinophil infiltration of the trachea was induced in the sensitized mice
by exposing the animals to aerosolized OVA. Sensitized mice were placed in 50
ml
conical centrifuge tubes (Corning) and forced to breath aerosolized OVA (50
mg/ml)
dissolved in 0.9 % saline for 20 minutes using a nebulizer (Model 646,
DeVilbiss
Corp., Somerset, PA). Control mice were treated in a similar manner with the
exception that 0.9 % saline was used in the nebulizer. Forty-eight hours
following the
exposure to aerosolized OVA or saline, mice were sacrificed and the tracheas
were
excised. Tracheas from each group were inbeded in OCT and stored at -70
° until
sections were cut.
To evaluate eosinophil infiltration of the trachea, tissue sections from
the four groups of mice were stained with either Luna solution and hematoxylin-
eosin
solution or with peroxidase. Twelve 6 ~,m thick sections were cut from each
group
of mice and numbered accordingly. Odd numbered sections were stained with Luna
stain as follows. Sections were fixed in formal-alcohol for 5 minutes at room
temperature, rinsed across three changes of tap water for 2 minutes at room
temperature then rinsed in two changed of dH20 for I minute at room
temperature.
Tissue sections were stained with Luna stain 5 minutes at room temperature
(Lung
stain consisting of 90 ml Weigert's Iron hematoxylin and 10 ml of 1 % Biebrich
Scarlet). Stained slides were dipped in 1 % acid alcohol six times, rinsed in
tap water
for 1 minute at room temperature. dipped in 0.5 % lithium carbonate solution
five
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times and rinsed in running tap water for 2 minutes at room temperature.
Slides were
dehydrated across 70 %-95 %-100 % ethanol 1 minute each, at room temperature,
then
cleared in two changes of xylene for 1 minute at room temperature and mounted
in
Cytoseal 60.
For the peroxidase stain, even numbered sections were fixed in 4 °
C
acetone for 10 minutes and allowed to air dry. Two hundred ~.1 of DAB solution
was
added to each section and allowed to sit 5 minutes at room temperature. Slides
were
rinsed in tap water for 5 minutes at room temperature and 2 drops of 1 % osmic
acid
was applied to each section for 3-5 seconds. Slides were rinsed in tap water
for 5
minutes at room temperature and counterstained with Mayers hematoxylin at 25
° C
at room temperature. Slides were then rinsed in running tap water for S
minutes and
dehydrated across 70 %-95 % -100 % ethanol 1 minute each at room temperature.
Slides were cleared through two changes of xylene for 1 minute each at room
temperature and mounted in Cytoseal 60.
The number of eosinophils in the submucosal tissue of the trachea was
evaluated. Trachea from mice from groups 1 and 2 were found to have very few
eosinophils scattered throughout the submucosal tissue. As expected tracheas
from
mice in group 3, which were pretreated with buffer and exposed to nebulized
OVA,
were found to have large numbers of eosinophils throughout the submucosal
tissue.
In contrast, the tracheas from mice in group 4, which were pretreated with PAF-
AH
and exposed to nebuiized OVA were found to have very few eosinophils in the
submucosal tissue comparable to what was seen in the two control groups,
groups 1
and 2.
Thus, therapeutic treatment with PAF-AH of subjects exhibiting a late
phase immune response involving the accumulation of eosinophils in the airway,
such
as that which occurs in asthma and rhinitis is indicated.
Example 17
A PAF-AH product of the invention was also tested in two different
rat models for treatment of necrotizing enterocolitis (NEC), an acute
hemorrhagic
necrosis of the bowel which occurs in low birth weight infants and causes a
significant morbidity and mortality. Previous experiments have demonstrated
that
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treatment with glucocorticoids decreases the incidence of NEC in animals and
in
premature infants, and the activity of glucocorticoids has been suggested to
occur via
an increase in the activity of plasma PAF-AH.
A. Activity in Rats With NEC Induced by PAF Challenge
1. Prevention of NEC
A recombinant PAF-AH product, rPH.2 (25,500 units in 0.3 ml,
groups 2 and 4), or vehicle/buffer alone (25mM tris, O.SM NaCI, 1mM EDTA and
0.1 % Tween 80) (groups 1 and 3) was administered into the tail veins of
female
Wistar rats (n =3) weighing 180-220 grams. Either BSA (0.25 % )-saline (groups
1
and 2) or PAF (0.2 ~ug/100 gm) suspended in BSA saline (groups 3 and 4) was
injected into the abdominal aorta at the level of the superior mesenteric
artery 15
minutes after rPH.2 or vehicle injection as previously described by Furukawa,
et al.
(J.~'ediatr.Res. 34:237-241 (1993)). The small intestines were removed after 2
hours
from the ligament of Trietz to the cecum, thoroughly washed with cold saline
and
examined grossly. Samples were obtained from microscopic examination from the
upper, middle and lower portions of the small intestine. The tissues were
fixed in
buffered formalin and the sample processed for microscopic examination by
staining
with hematoxylin and eosin. The experiment was repeated three times.
Gross findings indicated a normal appearing bowel in groups treated
with the vehicle of BSA saline. Similarly, rPH.2 injected in the absence of
PAF had
no effect on the gross findings. In contrast, the injection of PAF into the
descending
aorta resulted in rapid, severe discoloration and hemorrhage of the serosal
surface of
the bowel. A similar hemorrhage was noted when a section of the small bowel
was
examined on the mucosal side and the intestine appeared to be quite necrotic.
When
rPH.2 was injected via the tail vein 15 minutes prior to the administration of
PAF
into the aorta, the bowel appeared to be normal.
Upon microscopic examination, the intestine obtained from groups l,
2 and 4 demonstrated a normal villous architecture and a normal population of
cells
within the lamina propria. In contrast, the group treated with PAF alone
showed a
full thickness necrosis and hemorrhage throughout the entire mucosa.
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The plasma PAF-AH activities were also determined in the rats utilized
in the experiment described above. PAF-AH activity was determined as follows.
Prior to the tail vein injection, blood samples were obtained. Subsequently
blood
samples were obtained from the vena cava just prior to the injection of PAF
and at
the time of sacrifice. Approximately 50 ~.1 of blood was collected in
heparinized
capillary tubes. The plasma was obtained following centrifugation (980 x g for
5
minutes). The enzyme was assayed as previously described by Yasuda and
Johnston,
Endocrinology, 130:708-716 (1992).
The mean plasma PAF-AH activity of all rats prior to injection was
found to be 75.5 ~ 2.5 units (1 unit equals I nmoles x min-1 x mi-1 plasma).
The
mean plasma PAF-AH activities 15 minutes following the injection of the
vehicle
were 75.2 ~ 2.6 units for group 1 and 76.7 ~ 3.5 units for group 3. After 15
minutes, the plasma PAF-AH activity of the animals injected with 25,500 units
rPH.2
was 2249 ~ 341 units for group 2 and 2494 ~ 623 units for group 4. The
activity
of groups 2 and 4 remained elevated (1855 ~ 257 units) until the time of
sacrifice
(2 114 hours after rPH.2 injection) (Group 2 = 1771 ~ 308; Group 4 = 1939 ~
478). These results indicate that plasma PAF-AH activity of the rats which
were
injected with the vehicle alone (groups I and 3) did not change during the
course of
the experiment. All the animals receiving the PAF injection alone developed
NEC
while all rats that were injected with rPH.2 followed by PAF injection were
completely protected.
2. Dose-Dependency of Prevention of NEC
In order to determine if the protection against NEC in rats was dose
dependent, animals were treated with increasing doses of rPH.2 15 minutes
prior to
PAF administration. Initially, rPH.2, ranging from 25.5 to 25,500 units were
administered into the tail vein of rats. PAF (0.4 ~.g in 0.2 ml of BSA-saline)
was
subsequently injected into the abdominal aorta 15 minutes after the
administration of
rPH.2. The small intestine was removed and examined for NEC development 2
hours after PAF administration. Plasma PAF-AH activity was determined prior to
the exogenous administration of the enzyme, and 15 minutes and 2 1/4 hours
after
rPH.2 administration. The resutts are the mean of 2-5 animals in each group.
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Gross findings indicated that all rats receiving less than 2,000 units of
the enzyme developed NEC. Plasma PAF-AH activity in animals receiving the
lowest protective amount of enzyme (2040 units) was 363 units per ml of plasma
after
15 minutes, representing a five-fold increase over basal levels. When rPH.2
was
administered at less than 1,020 total units, resultant plasma enzyme activity
averaged
approximately 160 or less, and all animals developed NEC.
3. Duration of Protection Against- NEC
In order to determine the length of time exogenous PAF-AH product
afforded protection against development of NEC, rats were injected once with a
fixed
amount of the enzyme via the tail vein and subsequently challenged with PAF at
various time points. rPH.2 (8,500 units in 0.3 ml) or vehicle alone was
administered
into the tail vein of rats, and PAF (0.36 dug in 0.2 ml of BSA-saline) was
injected into
the abdominal aorta at various times after the enzyme administration. The
small
intestines were removed 2 hours after the PAF injection for gross and
histological
examinations in order to evaluate for NEC development. Plasma PAF-AH
activities
were determined at various times after enzyme administration and two hours
after
PAF administration. The mean value ~ standard error for enzyme activity was
determined for each group.
Results indicated that none of the rats developed NEC within the first
eight hours after injection of rPH.2, however 100 % of the animals challenged
with
PAF at 24 and 48 hours following injection of the enzyme developed NEC.
4. Reversal of NEC
In order to determine if administration of PAF-AH product was capable
of reversing development of NEC induced by PAF injection, 25,500 units of
enzyme
was administered via injection into the vena cava two minutes following PAF
administration (0.4 ~cg). None of the animals developed NEC. However, when
rPH.2 was administered via this route 15 minutes after the PAF injection, all
animals
developed NEC, consistent with the rapid time course of NEC development as
induced by the administration of PAF previously reported Furukawa et al.
[supra].
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The sum of these observations indicate that a relatively small (five-fold)
increase in the plasma PAF-AH activity is capable of preventing NEC. These
observations combined with previous reports that plasma PAF-AH activity in
fetal
rabbits [Maki, et al., Proc.Natl.Acad.Sci. (USA) 85:728-732 (1988)] and
premature
infants [Caplan, et al. , J. Pediatr. 116:908-964 ( 1990)] has been
demonstrated to be
relatively low suggests that prophylactic administration of human recombinant
PAF-
AH products to Iow birth weight infants may be useful in treatment of NEC.
B. Activity in a Neonatal Model of NEC
The efficacy of a PAF-AH product, rPH.2, was evaluated as follows
in an NEC model in which newborn rats are stressed by formula feeding and
asphyxia, two Gammon risk factors for the disease in humans. In this model,
approximately 70-80% of the animals develop gross and microscopic intestinal
injury
similar to neonatal NEC by the third day of life. Newborn rats were obtained
from
pregnant Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, III that
were
anesthetized with C02 and delivered via abdominal incision. Newborn animals
were
collected, dried, and maintained in a neonatal incubator during the entire
experiment.
First, separate groups of animals were used to assess the dosing and
absorption characteristics of rPH.2. Normal newborn rat pups were given one of
three different enteral or intraperitoneal doses of rPH.2 {3~, 15~, or 75~) at
time 0,
and blood was collected at 1 hour, 6 hours, or 24 hours later for assessment
of
plasma PAF-AH activity. PAF-AH activity was measured using a substrate
incubation assay [Gray et al., Nature, 374:549 (1995)] and an ELISA utilizing
an
anti-human rPAF-AH monoclonal antibody for each sample (90F2D and 90G11D,
described in Example i3). For selected samples, immunohistochemical analysis
was
performed using two different monoclonal antibodies developed against human
rPAF-
AH (90F2D and 90GlID, described in Example 13). Immunohistochemistry was
done with standard techniques using a 1:100 dilution of the antibody and
overnight
incubations.
Following enteral dosing of rPH.2 in normal newborn rats, there was
no measurable plasma PAF-AH activity at any time point using either the
substrate
incubation assay or the ELISA technique. With intraperitoneal administration
of
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rPl<i.2, significant circulating PAF-AH activity was measurable using both
methods
by 1 hour after dosing, and this activity peaked at 6 hours. Higher doses of
rPH.2
(from 3 to 75~, 10 to 250 In resulted in higher plasma PAF-AH activity.
Immunohistochemical analysis revealed the presence of rPAF-AH product in the
epithelial cells of the intestinal mucosa following enteral administration.
The
reactivity clustered mostly in the intestinal villi with minimal staining
present in the
crypt cells. There was more staining in the ileum than jejunum, and some rPAF-
AH
product was immunochemically identified in portions of colon. There was no
demonstrable staining in any control samples or in specimens recovered from
animals
dosed via the intraperitoneal route. Thus, enteral administration of rPAF-AH
product
resulted in local mucosal epithelial accumulation of the enzyme without any
measurable systemic absorption, while, in contrast, intraperitoneal
administration of
rPAF-AH product resulted in high circulating enzyme levels but no local
mucosal
accumulation.
In the NEC model, NEC was induced in newborn rats according to
Capian et al., Pediatr. Pathol., 14:1017-1028 (1994). Briefly, animals were
fed with
newborn puppy formula reconstituted from powder (Esbiliac, Borden Inc) every
three
hours via a feeding tube. The feeding volume began at 0.1 ml/feed initially
and
advanced as tolerated to 0.4 ml/feed by the 4th day of the protocol. All
animals were
challenged with asphyxial insults twice daily by breathing 100 % nitrogen for
50
seconds in a closed plastic chamber followed by exposure to cold (4°C)
for 10
minutes. Bowel and bladder function was stimulated with gentle manipulation
after
every feeding. Animals were maintained for 96 hours or until they showed signs
of
distress. Morbid animals had abdominal distention, bloody stools, respiratory
distress, cyanosis, and lethargy, and were euthanized via decapitation. After
sacrifice, the intestine of each rat was examined grossly for signs of
necrosis and then
formalin-fixed for later histological analysis. Specimens were paraffin-
embedded,
sectioned with a microtome, stained with hematoxylin and eosin, and examined
in a
blinded fashion by two observers. Intestinal injury was scored as 1 + for
epithelial
cell lifting or separation, 2+ for sloughing of epithelial cells to mid
villous level, 3+
for necrosis of entire villi, and 4+ for transmural necrosis.
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To assess the efficacy of rPH.2, three different groups of rats were
treated with the compound via enteral delivery, intraperitoneal delivery or
both. The
rPH.2 preparation had 0.8 mg/ml protein and approximately 4000 Units/mg PAF-AH
activity, with a < 0.5 EU/mg endotoxin/protein ratio. Enterally dosed animals
were
given 25~ (80 U) of rPH.2 via the orogastric tube diluted into each feeding
(every
three hours). Intraperitoneally dosed animals were given 75~ by
intraperitoneal
injection twice daily. Control animals received appropriate volumes of buffer
(20
mM NaP04, pH 7.4) without the rPH.2 and were studied simultaneously with each
experimental group. Mortality and signs of NEC were evaluated for each
treatment
group, and differences were analyzed statistically using Fischer's Exact test.
A p-
value of < 0.05 was considered significant. Results are shown in Table 9
below.
Table 9
NEC Death
Control (i.p. admin.) 7110 8/10
rPH.2 (240 U i.p. twice daily) 6/11 8/11
IS Control (enteral admin.) 19/26 21126
rPH.2 (80 U enterally every 3 hours) 6/26 7/26
Control (i.p.+enteral admin.) 10/i7 12/17
rPH.2 (240 U i.p. twice daily and 3/14 7114
80 U enteraIly every 3 hours)
Data represent cumulative results from four different experiments for i.p.
dosing, four
experiments for enteral dosing, and three experiments for i.p. +enteral
dosing.
Enteral rPH.2 administration significantly reduced the incidence of both
NEC and death compared to control animals. Results from four different
enterally-
dosed experiments showed that pretreatment with rPH.2 decreased NEC from 19/26
(control) to 6/26 (p < 0.001). Intestinal injury was variable among treated
and control
animals, but in most cases was characterized by midvillous necrosis in some
segments. total villous necrosis in other areas, occasional areas of
transmural
necrosis, and remaining portions of normal intestinal histology. The worst
degree of
NEC in treated animals and control animals with intestinal injury was similar
(median
score 2.8 in controls vs. 2.4 in rPH.2-treated rats, p > 0.05).
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Intlaperitoneal dosing with rPH.2 had no significant impact on NEC
or death in this model. 'The onset of symptoms was similar between this group
and
controls (40 t 5 hours in controls vs 36 ~ 7 hours in rPH.2-treated rats) and
the
degree of NEC in both groups was similar (median score 2.6 in controls vs. 2.5
in
rPH.2-treated rats).
Additional experiments were done in which rats were dosed both
enterally and intraperitoneally with rPH.2 at the same doses as the single
treatment
groups (25~ of rPH.2 in each feeding every three hours, plus 75a by
intraperitoneal
injection twice daily). Results are shown above in Table 9. Although there
were no
significant differences between treated and control groups in the incidence of
death,
the rPH.2 treatment significantly reduced the incidence of NEC (10/17 in
controls vs.
3/14 in rPH.2-treated rats, p = 0.04). Of note, 6 out of the 7 animals who
died in
the rPH.2-treated group had positive blood cultures for E. coli obtained just
prior to
death.
These results further support the protective role of PAF-AH products
in a neonatal model of non-PAF-induced NEC. Enteral treatment with rPAF-AH
product prevented NEC while intraperitoneal treatment at these doses had no
demonstrable effect. These findings suggest that PAF-AH product
supplementation
for formula-fed premature newborns at risk for NEC may reduce the incidence of
this
disease.
Example 18
The efficacy of PAF-AH product in a guinea pig model of acute
respiratory distress syndrome CARDS) was examined.
Platelet-activating factor (PAF) injected intravenously into guinea pigs
produces a profound lung inflammation reminiscent of early ARDS in humans.
Within minutes after intravenous administration of PAF, the lung parenchyma
becomes congested with constricted bronchi and bronchioles [Lellouch-Tubiana
et al. ,
supra. Platelets and polymorphonuclear neutrophils begin to marginate and
cellular
aggregates are easily identified along arterioles of the lung [Lellouch-
Tubiana, Br. J.
Exp Path., 66:345-355 (1985)]. PAF infusion also damages bronchial epithelial
cells
which dissociate from the airway walls and accumulate in the airway lumens.
This
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damage to airway epithelial cells is consistent with hyaline membrane
formation that
occurs in humans during the development of ALRDS. Margination of the
neutrophils
and platelets is quickly followed by diapedesis of these cells into the
alveolar septa
and alveolar spaces of the lung. Cellular infiltrates elicited by PAF are
accompanied
by significant vascular leakage resulting in airway edema [Kirsch, Exp. Lung
Res. ,
18:447-459 (1992)J. Evidence of edema is further supported by in vitro studies
where
PAF induces a dose-dependent (10-1000 ng/ml) extravasation of 1251 labeled
fibrinogen in perfused guinea pig lungs [Basran, Br. J. Pharmacol., 77:437
(1982)].
Based on the above observations, an ARDS model in guinea pigs was
developed. A cannula is placed into the jugular vein of anaesthetized male
Hartly
guinea pigs (approximately 350-400 grams) and PAF diluted in a 500 ~ul volume
of
phosphate buffered saline with 0.25 % bovine serum albumin as a carrier (PBS-
BSA)
is infused over a 15 minute period of time at a total dosage ranging from 100-
400
ng/kg. At various intervals following PAF infusion, animals are sacrificed and
lung
tissue is collected. In guinea pigs infused with PAF, dose dependent lung
damage
and inflammation is clearly evident by 15 minutes and continues to be present
at 60
minutes. Neutrophils and red blood cells are present in the alveolar spaces of
PAF
treated guinea pigs but absent in control or sham infused animals. Evidence of
epithelial cell damage is also evident and reminiscent of hyaline membrane
formation
in human ALZDS patients. Protein determinations done on bronchoalveolar lavage
(BAL) samples taken from guinea pigs infused with PAF shows a dramatic
accumulation of protein in the inflamed lung, clear evidence of vascular
Leakage.
rPH.2 was found to completely protect against PAF mediated lung
injury in the guinea pig model of ARDS. Groups of guinea pigs were pretreated
with
either rPH.2 (2000 units in 500 ~,l) or 500 ~,I of the PAF-AH buffer only.
Fifteen
minutes later these guinea pigs were infused with 400 ng/kg PAF in a 500 ~,l
volume,
infused over a 15 minute period. In addition, a sham group of guinea pigs was
infused with 500 ~,1 of PBS-BSA. At the completion of the PAF infusion the
animals
were sacrificed and BAL fluid was collected by lavaging the lungs 2X with lOml
of
saline containing 2 ~./ml heparin to prevent clotting. To determine protein
concentration in the BAL, samples were diluted 1:10 in saline and the OD 280
was
determined. BAL fluid from sham guinea pigs was found to have a protein
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concentration of 2.10 t 1.3 mglml. In sharp contrast, BAL fluid from animals
infused with PAF was found to have a protein concentration of 12.55 ~ 1.65
mg/ml.
In guinea pigs pretreated with rPH.2, BAL fluid was found to have a protein
concentration of 1.13 t 0.25 mg/ml which is comparable to the sham controls
and
demonstrates that PAF-AH product completely blocks lung edema in response to
PAF.
Example 19
The efficacy of a PAF-AH product, rPH.2, was evaluated in two
different models of acute pancreatitis.
A. Activity in a Rat Pancreatitis Model
Male Wistar rats (200-250 g) were purchased from Charles River
Laboratories (Wilmington, MA). They were housed in a climate controlled room
at
23 t 2 ° C with a 12 hour light/dark cycle and fed standard laboratory
chow with
water ad libitum. Animals were randomly assigned to either control or
experimental
groups. Rats were anesthetized with 50 mglkg pentobarbital sodium
intraperitoneally,
and a polyvinyl catheter (size V3, Biolab products, Lake Havasu, AZ) was
placed by
cutdown into the jugular vein. The catheter was tunneled subcutaneously to
exit in
the dorsal cervical area, and the animals were allowed to recover from
anesthesia.
The rats were given free access to water but were fasted overnight.
Experiments
were performed the next day on conscious animals. During the interim, catheter
patency was maintained by constant infusion of saline (0.2 ml/h). On the day
of the
experiment, the animals were intravenously injected with rPH.2 or vehicle
control,
followed by an infusion of either (1) 5 ~,g/kg per hour of caerulein for 3.5
hours, or
(2) 10 ~.g/kg per hour of caerulein for 5 hours, (Research Plus, Bayonne, NJ}.
Immediately after completion of the infusion, the animals were anesthetized
with
pentobarbital sodium, their abdomens were opened, and 5 ml of blood aspirated
from
the inferior versa cava for subsequent assays. They were then sacrificed by
exsanguination. Serum amylase, serum lipase and serum bilirubin were measured,
and the pancreas was harvested. Pieces of pancreas were either fixed in a 4%
phosphate buffered formaldehyde solution for histological examination or
immediately
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deep frozen at -80°C for measurements of myeloperoxidase activity.
Additional
pieces of pancreas were assessed for pancreatic water content and pancreatic
amylase
and trypsin as described below. Myeloperoxidase activity, a measure of
neutrophil
sequestration, was assessed in the pancreas and lung as described below.
Pulmonary
vascular permeability was also assessed as described below. Statistical
analysis of the
data was accomplished using unpaired Student's t-test. The data reported
represent
means + S.E.M. of at least three different experiments. Differences in the
results
were considered significant when p < 0.05.
1. Pancreatic water content
Pancreas pieces were blotted dry and weighed (wet weight), and were
then desiccated for 34 hrs at 120°C and reweighed (dry weight).
Pancreatic water
content was calculated as the difference between wet and dry weight and
expressed
as a percentage of the pancreatic wet weight. A rise in pancreatic water
content was
considered to indicate the development of edema.
2. Serum and Pancreatic Am, lY ase
Amylase activity in serum was measured using 4,6-ethylidene (G7)-p
nitrophenyl (G1)-a1D-maltoplaside (ET-G7PNP) (Sigma Chemical Co., St. Louis,
MO) as substrate according to Pierre et al., Clin. Chem., 22:1219 (1976).
Amylase
activity in pancreatic tissue homogenized in 10 mM phosphate buffer, pH 7.4,
was
measured using the same method.
3. Pancreatic T sin
Trypsin activity was measured fluorimetrically using Boc-Gin-Ala-Arg-
MCA as the substrate. Briefly, 200 ~cl of the sample and 2.7m1 of 50 mM Tris-
buffer
(pH 8.0) containing 150 mM NaCI, 1mM CaCl2 and 0.1 % bovine serum albumin
were mixed in a cuvette. One hundred ~l of substrate was added to the sample
after
20 seconds of preincubation to start the reaction. The fluorescence reading
was taken
(excitation 380 nm, emission 440 nm) and expressed as slope. To allow pooling
of
data from different experiments trypsin activity in the fractions was
expressed as
percent of total trypsin activity.
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4. Histology and Moiphometry
For light microscopy, complete random cross-sections of the head,
body and tail of the pancreas were fixed in 10 % neutral phosphate-buffered
formalin.
Paraffin embedded-5 ~,m sections were stained with hematoxylin-eosin (H&E) and
examined in a blinded fashion by an experienced morphologist. Acinar cell
injury/necrosis was defined as either (a) the presence of acinar cell ghosts
or (b)
vacuolization and swelling of acinar cells and destruction of the histo-
architecture of
whole or parts of the acini, both of which had to be associated with an
inflammatory
reaction. The amount of acinar cell injury/necrosis and the total area
occupied by
acinar tissue were each quantitated morphometrically using computerized
planimetric
image analysis video unit (model CCD-72, Dage-MTl, Michigan city, III equipped
with NIH-1200 image analysis software. Ten randomly chosen microscopic fields
(125x) were examined for each tissue sample. The extent of acinar cell
injury/necrosis was expressed as the percent of total acinar tissue which was
occupied
by areas which met the criteria for injury/necrosis.
5. Pancreas and Lune Myleoperoxidase (MPO) Activity Measurement
Neutrophil sequestration in pancreas and lung was evaluated by
measurement of tissue myeloperoxidase activity. Tissue samples harvested at
the time
of sacrifice were stored at -70 °C until the time of assay. Samples (50
mg) were
thawed and homogenized in I mL of 20 mM phosphate buffer (pH 7.4) and
centrifuged (10,000 x g, 10 min 4 °C). The resulting pellet was
resuspended in 50
mM phosphate buffer (pH 6.0) containing 0.5 % hexadecyltrimethylammonium
bromide (Sigma, St. Louis, MO) and subjected to four cycles of freezing-
thawing.
The suspension was then further disrupted by sonication for 40 sec. and
centrifuged
{10,000 x g, 5 min. at 4 °C). A reaction mixture consisting of the
extracted enzyme,
1.6 mM tetramethylbenzidine (Sigma Chemical Co., St. Louis, MO), 80 mM sodium
phosphate buffer (pH 5.4) and 0.3 mM hydrogen peroxide was incubated at
37°C
for I IO sec, and the absorbance was measured at 655 nm in a CobasBio
autoanalyzer.
This absorbance was then corrected for the fraction dry weight of the tissue
sample.
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6. Measurement of Pulmonary Vascular Permeabilitx
Obstruction of the common biliopancreatic duct also typically results
in severe pancreatids-associated lung injury quantifiable by lung vascular
permeability
and histological examination.
Two hours before the animals were killed, an intravenous bolus
injection of 5 mg/kg fluorescein isothiocyanate albumin (FITC-albumin, Sigma
Chemical Co., St. Louis, MO) was given. Pulmonary microvascular permeability
was evaluated by quantifying the leakage of FITC-albumin from the vascular
compartment into the bronchoalveolar space. Briefly, just after sacrifice, the
right
bronchus was blocked using a clamp and the trachea exposed. Subsequently, the
right
lung was lavaged by using a cannula inserted into the trachea. Three washes of
saline
(60 ml lavage) were pooled and the FITC fluorescence in serum and lavage was
measured at excitation 494 nm and emission 520 nm. The fluorescence ratio of
lavage fluid to blood was calculated and taken as a measure of microvascular
1 S permeability in the lung. The lung was also stained with H&E and examined
histologically.
7. Effect of Caerulein and rPH.2 administration
Infusion of caerulein alone at 5 ~cg/kg/h for 3.5 hours resulted in a
typical mild secretagogue-induced pancreatitis in the rats, which was
characterized by
hyperamylasemia, pancreatic edema as measured by pancreatic water content, and
histological changes including marked acinar cell vacuolization and pancreatic
edema.
Saline infusion in control animals did not result in any of these biochemical
or
histological changes. Administration of rPH.2 intravenously at doses of 5, 10
or 20
mg/kg 30 min. before the start of caerulein infusion did not significantly
alter the
magnitude of the changes in pancreatic edema (water content) and histology
that were
induced by infusion of caerulein alone. Administration of rPH.2 also had no
effect
on caerulein-induced activation of pancreatic trypsinogen or amylase content.
Infusion of a higher dose of caerulein, 10 ,uglkg/h for 5 hours, to rats
resulted in a more severe pancreatitis, characterized relative to the controls
by a more
pronounced increase in serum amylase activity and pancreatic edema, a marked
increase in pancreatic MPO activity, and a significant increase in trypsinogen
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activation and amylase activity in the pancreas. Pancreatic histology
indicated not
only pancreatic edema and acinar cell vacuolization but also some patchy
necrosis and
a few infiltrating cells.
Administration of rPH.2 (5 or 10 mglkg intravenously) 30 min. before
the start of caerulein (10 ~cg/kg/h) infusion ameliorated the magnitude of
many of the
pancreatic changes induced by the infusion of caerulein alone. Results are
shown in
Table 10 below. rPH.2 treatment at a dose of 5mglkg resulted in decrease of
serum
amylase activity (from 10984 t 1412 to 6763 t 1256). The higher 10 mg/kg dose
of
rPH.2 did not result in further improvement of hyperamylasemia. Treatment with
either 5 or 10 mg/kg rPH.2 also resulted in some decrease in caerulein-induced
development of pancreatic edema as measured by water content (90.6110.27 for
caerulein alone vs. 88.21 X0.61 for caerulein + 5 mglkg rPH.2). The 5 mg/kg
dose
of rPH.2 provided a significant amelioration of pancreatic MPO activity
(2.92+0.32
fold increase over controls for caerulein alone vs. 1.19+0.21 for caerulein
with
rPH.2, p < 0.05). Higher doses of rPH.2 did not result in further improvement
of
MPO activity. Neither dose of rPH.2 significantly altered the extent of
trypsinogen
activation or the amylase content in the pancreas. Pancreatic histology
indicated some
improvement in microscopic necrosis and infiltration after rPH.2 pretreatment.
Pancreatitis associated lung injury has been observed both clinically and
in several models of pancreatitis. Infusion of caerulein at 5 ~cg/kg/h for 3.5
h, which
resulted in a mild form of pancreatitis, did not result in significant injury
to the lungs.
However, infusion of caerulein at 10 ~,g/kg/h for 5 hours, which resulted in
more
severe pancreatitis, also resulted in lung injury quantified by increased lung
vascular
permeability (0.3110.04 to 0.7910.09), lung MPO activity (indicating
neutrophil
sequestration) and neutrophil infiltration on histological examination.
Administration of rPH.2 at a dose of 5 mg/kg 30 min prior to caerulein
infusion significantly ameliorated the rise in lung MPO activity induced by
the
infusion of caerulein alone (3.55 ~ 0.93 for caerulein alone vs. 1.51 t 0.26
for
caerulein with rPH.2). rPH.2 treatment significantly decreased the severity of
microscopic changes observed in the lung tissue after caerulein infusion. The
caerulein-induced increase in lung vascular permeability was reduced by rPH.2
treatment, although not statistically significant. The higher 10 mg/kg dose of
rPH.2
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was no more effective than the lower dose in decreasing the severity of
caerulein-
induced lung injury.
Table 10
Caerulein CER + CER +
Control (CER) 5 mg/kg 10 mg/kg
(no CER) l0~cg/kg/h rPH.2 rPH.2
Serum
Amylase 961 t 174 10984 t 14126763 t 1256 8576 t 1024
(U/I)
Pancreas
Water
Content 72.7110.64 90.610.27 88.2110.61 89.0010.94
(%wet
weight)
Pancreas
MPO (fold 1.0 2.920.32 1.1910.21 1.420.19
increase
over control)
Pancreas
Trypsin
Activity 0.12 t 0.069.70 t 2.50 8. 33 ~ 1. 9.15 t 1.28
75
( 1000xslope/
~.g DNA
Pancreas
Amylase 0.280.06 0.420.07 0.450.04 0.4610.044
Content
(U/~.g DNA)
Lung
Vascular
Permeability 0.3110.04 0.790.09 0.700.09 0.700.07
(Lavage/
Serum % )
Lung MPO
(fold 1.0 3.550.93 1.51 0.26 1.640.22
increase
over control)
B. Activity
in an Opossum
Pancreatitis
Model
Healthy, randomly
trapped American
opossums (DidelpJ~is
virginiana)
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of either sex (2.0 kg to 4.0 kg) were obtained from Scott-Haas and housed in
climate
controlled rooms at 23 t2°C with a 12 hour lightldark cycle and fed a
standard
laboratory chow with water ad libitum. After an overnight fast, the animals
were
anesthetized with 50mg/kg sodium-pentobarbital i.p. (Veterinary Laboratories
Inc.,
Lenexa, KS). A celiotomy was performed through a midline incision under
sterile
conditions and the common bile pancreatic duct was ligated in all animals to
induce
acute necrotizing pancreatitis. Additionally, the cystic duct was ligated to
prevent the
gallbladder from serving as a bile reservoir. The animals were randomly
assigned
to either control or experimental groups. Starting at Day 2 after ligation of
the
pancreatic duct, the experimental group received 5 mg/kg body weight per day
of
rPH.2 (supplied in a 4mg/ml solution) intravenously via the tail vein, while
the
control group received an intravenous injection of the same volume of placebo
vehicle
only. After 1 and 2 days of treatment (at Day 3 and Day 4 after ligation of
the
pancreatic duct) the animals were euthanized by a sodium-pentobarbital
overdose.
Blood samples were drawn from the heart for measurements of serum amylase,
serum
lipase and serum bilirubin, and the pancreas was harvested. Pieces of pancreas
were
either fixed in a 4 % phosphate buffered formaldehyde solution for
histological
examination or immediately deep frozen at -80°C for measurements of
myeloperoxidase activity. Additional pieces of pancreas were assessed for
pancreatic
water content and pancreatic amylase as described above in section A of this
example.
Myeloperoxidase activity, a measure of neutrophil sequestration, was assessed
in the
pancreas as described above. Pulmonary vascular permeability was also assessed
as
described above.
The results reported represent mean ~ standard error of the mean
(SEM) values obtained from multiple determinations in 3 or more separate
experiments. The significance of changes was evaluated using Student's t-test
when
the data consisted of only two groups or by analysis of variance (ANOVA) when
comparing three or more groups. If ANOVA indicated a significant difference,
the
data were analyzed using Tukey's method as a post hoc test for the difference
between groups. A p-value of < 0.05 was considered to indicate a significant
difference.
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Results are shown in Table 11. Obstruction of the common
biliopancreatic duct resulted in severe necrotizing pancreatitis characterized
by
hyperamylasemia, hyperlipasemia and extensive necrosis of the pancreas.
Furthermore, obstruction of the common biliopancreatic duct was associated
with an
S marked increase in serum bilirubin levels. Intravenous administration of
rPH.2 (5
mg/kg/day) starting at Day 2 after ligation of the pancreatic duct ameliorated
the
magnitude of many of the pancreatic changes induced by duct obstruction and
placebo
treatment alone. One day of rPH.2 treatment reduced senim amylase levels in
comparison to placebo treated animals, although the difference was not
statistically
significant, and two days of rPH.2 treatrnent (at Day 4 after ligation of the
pancreatic
duct) significantly reduced serum amylase levels compared to placebo. One or
two
days of rPH.2 treatment reduced serum lipase levels relative to controls,
although the
difference was not statistically significant. Two days of rPH.2 treatment
reduced
pancreatic amylase content relative to controls, although one day of treatment
resulted
in an increase in pancreatic amylase. Treatment with rPH.2 was not observed to
affect serum bilirubin levels, pancreas myeloperoxidase activity or pancreas
water
content.
The major characteristic histological changes induced by obstruction
of the biliopancreatic duct included marked necrosis, infiltration of
inflammatory
cells, acinar cell vacuolization, and marked distention of the acinar lumina.
Morphometrical examination of the pancreas for acinar cell injury showed a
major
protective effect of rPH.2 on the pancreas after one and two days of rPH.2
treatment.
After one day of rPH.2 treatment, the acinar cell injury was reduced to about
23 %
of total acinar cell tissue, compared to 48 % injury for the placebo-treated
animals.
This reduction of acinar cell injury was even more pronounced after two days
of
treatment, at which time rPH.2 treatment resulted in about 35 % injury of the
total
acinar cell tissue, compared to about 60 % injury for the placebo-treated
animals.
Lung vascular permeability, quantified by FITC injection showed a
highly significant difference after one and two days of rPH.2 treatment
compared to
placebo group. Histological examination of the lung showed severe lung injury
in all
placebo-treated animals. Lung injury was characterized by an extensive
inflammatory
response with interstitial and intraalveolar infiltration of mainly
macrophages,
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lymphocytes and neutrophils, and by a patchy but marked interstitial edema and
thickening of the alveolar membranes. Administration of rPH.2 resulted in a
marked
decrease of infiltration of inflammatory cells and a reduction of interstitial
edema at
all times.
In summary, these results showed that administration of rPH.2
intravenously at a dose of 5 mglkg/day beginning at 48 hours after ligation of
the
pancreatic duct resulted in significant amelioration of the increase in blood
levels of
amylase and lipase and acinar cell injury as quandtated by morphometric
analysis of
H&E stained sections, and a significant decrease in the severity of
pancreatitis-
induced lung injury. Administration of rPAF-AH product in this clinically
relevant
model of pancreatitis showed beneficial effects in decreasing the severity of
pancreatitis.
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Table 11
After 1 After 2
day of days of
treatment treatment
(Sacrifice (Sacrifice
at Day at Day
3) 4)
rPH.2 5mg/kg rPH.2
Placebo Placebo 5mglkg
Serum bilirubin 5.490.96 7.I0~0.60 6.540.55 4.9110.79
(mg/dl)
Serum amylase 5618899 4288675 653811355 3106467*
(U/l)
Serum lipase 2226 t 554 1241 t 263 1424257 1023 295
(U/l)
Pancreas 'Water 81.1010.56 81.520.79 80.0511.07 79.320.49
Content {%)
Pancreas MPO 1345 286 1142 t 83 I 149 t 1033 t
232 130
(OD/fraction
dry weight)
Pancreatic
Amylase 70692 1101 105 95085 712 t 131
(UI ~cg DNA)
Lung Vascular
Permeability 0.760.09 0.21 X0.04**0.5710.13 0.230.04*
(FITC Lavagel
Serum % )
Acinar Cell
Injury ( % of 48 % 23 % 60 % 35
Total Acinar
Tissue)
*p=0.02 vs. placebo
**p < 0.001 vs.
placebo
Example 20
A study was conducted to evaluate the effect of a PAF-AH product,
rPH.2, on neurotoxicity associated with HIV infection. Human immunodeficiency
virus type 1 (HIV-I) infection of the central nervous system results in
neuronal loss
by apoptosis. HIV-1-infected monocytes activated by a variety of antigenic
stimuli,
including contact with neural cells, secrete high levels of neurotoxic pro-
inflammatory
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cytokines, including PAF. The effect of rPH.2 on the neurotoxicity of
conditioned
media from HIV-infected and activated monocytes was assessed.
Monocytes were infected with HIV and activated as follows.
Monocytes were recovered from peripheral bone marrow cells (PBMC) of HIV- and
hepatitis B-seronegative donors after leukopheresis and purified ( > 98 ~ ) by
countercurrent centrifugal elutriation as described in Genis et al. , J. Exp.
Med. ,
176:1703-1718 (1992). Cells were cultured as adherent monolayers (1 x 104
cells/ml
in T-75 culture flasks) in DMEM (Sigma, St. Louis, MO) with recombinant human
macrophage colony stimulatory factor (MSCF) (Genetics Institute, Inc.
Cambridge,
MA). Under these conditions, monocytes differentiate into macrophages. After 7-
10
days of culture, macrophages were exposed to HIV-1~A (accession number
M60472) at a multiplicity of infection (MOI) of 0.01 infectious virions/target
cell.
Under these conditions, 20-50 ~ of the monocytes were infected at 7 days after
HIV-1
inoculation, as determined by immunofluorescent and in situ hybridization
techniques
[Kalter et al., J. Immunol., 146:298-306 (1991)]. All cultures were refed with
fresh
medium every 2 to 3 days. Five to seven days after HIV-I infection and during
the
peak of reverse transcriptase activity (107 cpm/ml), assessed according to
Kalter et
al., supra, cultures of HIV-I-infected and parallel cultures of uninfected
monocytes
were stimulated with LPS (10 nglml) or vehicle for 30 min. at 37°C,
then snap-
frozen at -80°C until used in the neurotoxicity assay.
Human cerebral cortical neuron cell cultures were established as
follows. Human fetal brain tissue was obtained from the telencephalon of
second
trimester (13-16 weeks gestation) human fetal brain tissue according to a
modified
procedure of Banker and Cowan, Brain Res., 126:397-425 (1977). Briefly, brain
tissue was collected, washed in 30 ml of cold Hank's BSS (containing Ca+2 and
Mg+2 + 25 mM HEPES, and 5X gentamicin), separated from adherent meninges
and blood, and cut into 2 mm3 pieces. The tissue was forced through a 230 uM
Nitex bag and gently triturated through a flame-polished Pasteur pipet 10-15
times.
The tissue was centrifuged at 550 rpm, 5 minutes, 4°C, and the
pellet was
. resuspended in 5-10 ml of MEM-hipp (D-glucose, 5 grams/liter; L-glutamine, 2
mM;
HEPES, 10 mM; Na pyruvate, 1 mM; KCI, 20 mM) containing Nl components
(insulin, 5 mg/l; transferrin, 5 mg/l; selenite, 5 ~cg/1, progesterone 20 nM;
putrescine,
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100 ,uM), as well as 10 % fetal calf serum (FCS), PSN antibiotic mix
(penicillin, SO
mgll; streptomycin, SO mg/1; neomycin, 100 mgll), and fungizone {2.S mg/1).
The
cell count and viability were determined by diluting Hank's BSS with 0.4 %
trypan
blue (1:1 v/v) and counting with a hemocytometer. Cells were tently triturated
S
S times with a 10 ml pipet and plated at a density of 105 cells/12 mm glass
coverslip
pre-coated with poly-L-lysine (70K-1SOK MW, Sigma, St. Louis, MO) placed in 24
well culture dishes. One ml of media was pipetted into each culture well.
Cells were
cultured for 10-28 days at 37°C in a humidified atmosphere of S %
C02/9S % air,
changing media every 3 days. Under these conditions, cultures were > 60-70 %
homogeneous for neurons, with 20-30 % astrocytes, < 1 % microglia and ~ 10 %
macrophage and microglia staining. After 14-28 days of culture, neuronal
cultures
express sufficient levels of N-methyl-D-aspartate (NMDA) or non-NMDA receptors
to die after excitotoxic doses of NMDA or alpha-amino-3-hydroxy-S-methyl-4
isoxazole proprionic acid (AMPA).
1S The neurotoxicity assay was conducted as follows. The test samples,
which were (a) conditioned media from LPS-stimulated HIV-1 infected monocytes,
(b) control media, (c) conditioned media with added rPH.2 at SI ~cg/ml or (d)
conditioned media with added vehicle for rPH.2, were applied to the neuronal
cell
cultures at a 1:10 v/v concentration for 24 hours. Neurotoxicity was measured
by
identifying apoptotic nuclei in situ on neuronal coverslips fixed in 4
paraformaldehyde, employing a commercial kit {Apop Tag; ONCOR, Gaithersburg,
MD) that uses terminal deoxynucleotidyl transferase (TdT) to bind digoxigenin-
dUPT
to free 3'-OH ends of newly cleaved DNA (TiJNEL staining). Digitized images of
TUNEL-stained neurons in > 1S randomly selected microscopic fields were
analyzed
2S for number of TUNEL-stained nucleilnumber of total neurons per SOX field
using
computerized morphometry (MCID, Imaging Research, St. Catherine, Ontario,
Canada). Data were expressed at % neuronal nuclei positive for TUNEL staining
~
SEM and are shown in FIGURE 13. Tests of statistical significance between
control
and experimental treatments were determined by ANOVA or paired t-tests, with
significance at p < O.OS. Quantitation of these cultures confirmed that
conditioned
media from HIV-infected and activated monocytes induced neuronal cell death in
nearly 2S % of the total population of cerebral cortical neurons, and rPH.2
was able
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to reduce this toxicity to less than 5 9~ of the total neumris. The rPH.2 by
itself was
not neurotoxic, since 50 ~cg/ml rPH.2 had no effect on neuronal cell death
relative to
cultures treated with control media. These results clearly indicate that a
major
component of the neurotoxicity induced by application of conditioned media
from
activated HIV-1 infected rnonocytes must be due to PAF, since neurotoxity can
be
almost completely abrogated by co-incubation with PAF-AH product, the enzyme
responsible for metabolism of PAF in the central nervous system. These
findings
suggest potential therapeutic interventions in the treatment of the CNS -
neurologic
disease associated with HIV-1 infection.
Example 21
Nearly four percent of the 3apanese population has low or undetectable
levels of PAF-AH activity in their plasma. This deficiency has been correlated
with
severe respiratory symptoms in asthmatic children [Miwa et al., J. Clin.
Invert,. 82:
1983-1991 (1988)] who appear to have inherited the deficiency ~in an autosomal
recessive manner.
To determine if the deficiency arises from an inactive but present
enzyme or from an inability to synthesize -PAF-AH, plasma from multiple
patients
deficient in PAF-AH activity was assayed both for PAF-AH activity (by the
method
described in Example 10 for transfectants) and for the presence of PAF-AH
using the
monoclonal antibodies 90G11D and 90F2D (Example 13) in a sandwich ELISA a~
follows. Immulon 4 flat bottom plates (Dynatech, Chantilly, VA) were coated
with
100 ng/well of monoclonal antibody 9061 I D and stored overnight. The plates
were
blocked for 1 hour at room temperature with 0.5 9~ fish skin gelatin (Sigma)
diluted
in CMF-PBS and then washed three times. Patient plasma was diluted in PBS
containing lSmM CHAPS and added to each well of the plates (50 Icl/well). The
plates were incubated for 1 hour at room temperature and washed four times.
Fifty
~cl of 5 ~g/ml monoclonal antibody 90F2D, which was biotinylated by standard
methods and diluted in PBST, was added to each well, and the plates were
incubaxed
for 1 hour at room temperature and then washed three times. Fifty ~cl of
ExtraAvidin-~'
(Sigma) diluted 1 / 1000 in CMF-PEST was subsequently added to each well and
plates
were incubated for 1 hour at room temperature before development.
*Trade-mark
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A direct correlation between PAF-AH activity and enzyme levels was
observed. An absence of activity in a patient's serum was reflected by an
absence
of detectable enzyme. Similarly, plasma samples with half the normal activity
contained half the normal levels of PAF-AH. These observations suggested that
the
deficiency of PAF-AH activity was due to an inability to synthesize the enzyme
or
due to an inactive enzyme which the monoclonal antibodies did not recognize.
Further experiments revealed that the deficiency was due to a genetic
lesion in the human plasma PAF-AH gene. Genomic DNA from PAF-AH deficient
individuals was isolated and used as template for PCR reactions with PAF-AH
gene
specific primers. Each of the coding sequence exons were initially amplified
and
sequenced from one individual. A single nucleotide change within exon 9 was
observed (a G to T at position 996 of SEQ ID NO: 7). The nucleotide change
results
in an amino acid substitution of a phenylalanine for a valise at position 279
of the
PAF-AH sequence (V279F). Exon 9 was amplified from genomic DNA from an
additional eleven PAF-AH deficient individuals who were found to have the same
point mutation.
To test whether this mutation crippled the enzyme, an E. coli
expression construct containing the mutation was generated by methods similar
to that
described in Example 10. When introduced into E. toll, the expression
construct
generated no PAF-AH activity while a control construct lacking the mutation
was
fully active. This amino acid substitution presumably results in a structural
modification which causes the observed deficiency of activity and lack of
immunoreactivity with the PAF-AH antibodies of the invention.
PAF-AH specific antibodies of the invention may thus be used in
diagnostic methods to detect abnormal levels of PAF-AH in serum (normal levels
are
about 1 to 5 U/ml) and to follow the progression of treatment of pathological
conditions with PAF-AIi. Moreover, identification of a genetic lesion in the
PAF
AH gene allows for genetic screening for the PAF-AH deficiency exhibited by
the
Japanese patients. The mutation causes the gain of a restriction endonuclease
site
(Mae II) and thus allows for the simple method of Restriction Fragment Length
Polymorphism (RFLP) analysis to differentiate between active and mutant
alleles.
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See Lewin, pp. 136-141 in Genes V, Oxford University Press, New York, New York
(1994).
Screening of genomic DNA from twelve PAF-AH deficient patients
was carried out by digestion of the DNA with MaeII, Southern blotting, and
hybridization with an exon 9 probe (nucleotides 1-396 of SEQ ID NO: 17). All
patients were found to nave RFLPs consistent with the mutant allele.
While the present invention has been described in terms of specific
embodiments, it is understood that variations and modifications will occur to
those
skilled in the art. Accordingly, only such limitations as appear in the
appended
claims should be placed on the invention.
CA 02267994 1999-04-09
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: ICOS CORPORATION
(ii) TITLE OF INVENTION: TRUNCATED PLATELET-ACTIVATING FACTOR
ACETYLHYDROLASE
(iii) NUMBER OF SEQUENCES: 30
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE : ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATION DATA:
2 0 (A) APPLICATION NUMBER: CA
(B) FILING DATE: 13-AUG-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 64267-976
3 O (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486
(B) TELEFAX: (613)-232-8440
64267-976
CA 02267994 1999-04-09
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(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
64267-976
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Aia
1 5 10 15
Phe
(2) INFORMATION FOR SEQ ID N0:2:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gly Gln Thr Lys Ile Pro
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Met Lys Pro Leu Vai Val Phe Val Leu GIy Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: group(13, 21, 27)
(C) OTHER INFORMATION: /note= "The nucleotide at each of
these positions is an inosine."
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
ACATGAATTC GGNATCYTTG NGTYTGNCCR AA 32
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
TATTTCTAGA AGTGTGGTGG AACTCGCTGG 30
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CGATGAATTC AGCTTGCAGC AGCCATCAGT AC 32
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1520 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
( ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: 162..1484
(xi) SEQUENCE DESCRIPTION: 5EQ ID N0:7:
GCTGGTCGGAGGCTCGCAGT GGGTTTGGAG CGCTTGGGTC60
GCTGTCGGCG
AGAAGCAGTC
GCGTTGGTGCGCGGTGGAAC CCGCGAGCAG CTCCGCGCCG120
GCGCCCAGGG
ACCCCAGTTC
CGCCTGAGAGACTAAGCTGA G CA CCC 173
AACTGCTGCT ATG
CAGCTCCCAA GTG
C
Met ro Pro
Val
P
1
AAA TTG GTGCTT TGC TGCGGC TGCCTGGCT GTT TAT 221
CAT TTC CTC GTG
Lys Leu ValLeu Cys CysGly CysLeuAla Val Tyr
His Phe Leu Val
10 15 20
CCT TTT TGGCAA ATA CCTGTT GCCCATATG TCA TCA 269
GAC TAC AAT AAA
Pro Phe TrpGln Ile ProVal AlaHisMet Ser Ser
Asp Tyr Asn Lys
25 30 35
GCA TGG AACAAA CAA CTGATG GCTGCTGCA TTT GGC 317
GTC ATA GTA AGC
Ala Trp AsnLys Gln LeuMet AlaAlaAla Phe Gly
Val Ile Val Ser
40 45 50
CAA ACT ATCCCC GGA GGGCCT TATTCCGTT TGT ACA 365
AAA CGG AAT GGT
Gln Thr IlePro Gly GlyPro TyrSerVal Cys Thr
Lys Arg Asn Gly
55 60 65
GAC TTA TTTGAT ACT AAGGGC ACCTTCTTG TTA TAT 413
ATG CAC AAT CGT
Asp Leu PheAsp Thr LysGly ThrPheLeu Leu Tyr
Met His Asn Arg
70 75 80
TAT CCA CAAGAT GAT CTTGAC ACCCTTTGG CCA AAT 461
TCC AAT CGC ATC
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TyrProSer Arg Leu ThrLeuTrp IlePro
Gln Asp Asn
Asp
Asn
Asp
85 90 95 100
AAAGAATAT TGG GGTCTTAGC AAATTT CTTGGAACA CACTGGCTT 509
TTT
LysGluTyr Trp GlyLeuSer LysPhe LeuGlyThr HisTrpLeu
Phe
105 110 lI5
ATGGGCAAC TTG AGGTTACTC TTTGGT TCAATGACA ACTCCTGCA 557
ATT
MetGlyAsn Leu ArgLeuLeu PheGly SerMetThr ThrProAla
Ile
120 125 130
AACTGGAAT CCT CTGAGGCCT GGTGAA AAATATCCA CTTGTTGTT 605
TCC
AsnTrpAsn Pro LeuArgPro GlyGlu LysTyrPro LeuValVa1
Ser
135 140 145
TTTTCTCAT CTT GGGGCATTC AGGACA CTTTATTCT GCTATTGGC 653
GGT
PheSerHis Leu GlyAlaPhe ArgThr LeuTyrSer AlaIleGly
Gly
150 155 160
ATTGACCTG TCT CATGGGTTT ATAGTT GCTGCTGTA GAACACAGA 701
GCA
IleAspLeu Ser HisGlyPhe IleVal AlaAlaVal GluHisArg
Ala
165 170 175 180
GATAGATCT TCT GCAACTTAC TATTTC AAGGACCAA TCTGCTGCA 749
GCA
AspArg5er Ser AlaThrTyr TyrPhe LysAspGln SerAlaAla
Ala
185 190 195
GAAATAGGG AAG TCTTGGCTC TACCTT AGAACCCTG AAACAAGAG 797
GAC
GluIleGly Lys SerTrpLeu TyrLeu ArgThrLeu LysGlnGlu
Asp
200 205 210
GAGGAGACA ATA CGAAATGAG CAGGTA CGGCAAAGA GCAAAAGAA 845
CAT
GluGluThr Ile ArgAsnGlu Glnval ArgGlnArg AlaLysGlu
His
215 220 225
TGTTCCCAA CTC AGTCTGATT CTTGAC ATTGATCAT GGAAAGCCA 893
GCT
CysSerGln Leu SerLeuIle LeuAsp IleAspHis GlyLysPro
Ala
230 235 240
GTGAAGAAT TTA GATTTAAAG TTTGAT ATGGAACAA CTGAAGGAC 941
GCA
ValLysAsn Leu AspLeuLys PheAsp MetGluGln LeuLysAsp
Ala
245 250 255 260
TCTATTGAT GAA AAAATAGCA GTAATT GGACATTCT TTTGGTGGA 989
AGG
SerIleAsp Glu LysIleAla ValIle GlyHisSer PheGlyGly
Arg
265 270 275
GCAACGGTT CAG ACTCTTAGT GAAGAT CAGAGATTC AGATGTGGT 1037
ATT
AlaThrVal Gln ThrLeuSer GluAsp GlnArgPhe ArgCysGly
Ile
280 285 290
ATTGCCCTG GCA TGGATGTTT CCACTG GGTGATGAA GTATATTCC 1085
GAT
IleAlaLeu Ala TzpMetPhe ProLeu GlyAspGlu ValTyrSer
Asp
295 300 305
AGAATTCCT CCC CTCTTTTTT ATCAAC TCTGAATAT TTCCAATAT 1133
CAG
ArgIlePro Pro LeuPhePhe IleAsn SerGluTyr PheGlnTyr
Gln
310 315 320
CCTGCTAAT ATA AAAATGAAA AAATGC TACTCACCT GATAAAGAA 1181
ATC
ProAlaAsn Ile LysMetLys LysCys TyrSerPro AspLysGlu
Ile
325 330 335 340
AGAAAGATG ACA ATCAGGGGT TCAGTC CACCAGAAT TTTGCTGAC 1229
ATT
ArgLysMet Thr IleArgGly SerVal HisGlnAsn PheAlaAsp
Ile
345 350 355
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TTC GCA ACT GGC AAA ATA CAC ATG AAG 1277
ACT ATT GGA CTC AAA
TTT TTA
Phe Phe Ala Thr Gly Lys Ile His Met Lys Lys
Thr Ile Gly Leu Leu
360 365 370
GGA ATA GAT TCA AAT GTA GCT CTT AGC AAA TCA 1325
GAC ATT GAT AAC GCT
Gly Ile Asp Ser Asn Val Ala Leu Ser Lys Ser
Asp Ile Asp Asn Ala
375 380 385
TTA TTC TTA CAA AAG CAT TTA CAT AAA TTT CAG 1373
GCA GGA CTT GAT GAT
Leu Phe Leu Gln Lys His Leu His Lys Phe Gln
Ala Gly Leu Asp Asp
390 395 400
TGG TGC TTG ATT GAA GGA GAT AAT CTT CCA ACC 1421
GAC GAT GAG ATT GGG
Trp Cys Leu Ile Glu Gly Asp Asn Leu Pro Thr
Asp Asp Glu Ile Gly
405 410 415 420
AAC AAC ACA ACC AAT CAA CAC TTA CAG TCT GGA 1469
ATT ATC ATG AAC TCA
Asn Asn Thr Thr Asn Gln His Leu Gln Ser Gly
Ile Ile Met Asn Ser
425 430 435
ATA AAA TAC AAT TAGGATTAAA ATAGGTTTTT A AAAAAA 1520
GAG TAAAAAAAA
Ile Lys Tyr Asn
Glu
440
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 441 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Val Pro Pro Lys Leu His Val Leu Phe Cys Leu Cys Gly Cys Leu
1 5 10 15
Ala Val Val Tyr Pro Phe Asp Trp Gln Tyr Ile Asn Pro Val Ala His
20 25 30
Met Lys Ser Ser Ala Trp Val Asn Lys Ile Gln Val Leu Met Ala Ala
35 40 45
Ala Ser Phe Gly Gln Thr Lys Ile Pro Arg Gly Asn Gly Pro Tyr Ser
50 55 60
Val Gly Cys Thr Asp Leu Met Phe Asp His Thr Asn Lys Gly Thr Phe
65 70 75 80
Leu Arg Leu Tyr Tyr Pro Ser Gln Asp Asn Asp Arg Leu Asp Thr Leu
85 90 g5
Trp Ile Pro Asn Lys Glu Tyr Phe Tzp Gly Leu Ser Lys Phe Leu Gly
100 105 lI0
Thr His Trp Leu Met Gly Asn Ile Leu Arg Leu Leu Phe Gly Ser Met
115 120 125
Thr Thr Pro Ala Asn Trp Asn Ser Pro Leu Arg Pro Gly Glu Lys Tyr
130 135 140
Pro Leu Val Val Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr
145 150 155 160
Ser Ala Ile Gly Ile Asp Leu Ala Ser His Gly Phe Ile Val Ala Ala
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165 170 175
Val Glu His Arg Asp Arg Ser Ala Ser Ala Thr Tyr Tyr Phe Lys Asp
180 185 190
Gln Ser Ala Ala Glu Ile Gly Asp Lys Ser Trp Leu Tyr Leu Arg Thr
195 200 205
Leu Lys Gln Glu Glu Glu Thr His Ile Arg Asn Glu Gln Val Arg Gln
210 215 220
Arg Ala Lys Glu Cys Ser Gln Ala Leu Ser Leu Ile Leu Asp Ile Asp
225 230 235 240
His Gly Lys Pro Val Lys Asn Ala Leu Asp Leu Lys Phe Asp Met Glu
245 250 255
Gln Leu Lys Asp Ser Ile Asp Arg Glu Lys Ile Ala Val Ile Gly His
260 265 270
Ser Phe Gly Gly Ala Thr Val Ile Gln Thr Leu Ser Glu Asp Gln Arg
275 280 285
Phe Arg Cys Gly Ile Ala Leu Asp Ala Trp Met Phe Pro Leu Gly Asp
290 295 300
Glu Val Tyr Ser Arg Ile Pro Gln Pro Leu Phe Phe Ile Asn Ser Glu
305 310 315 320
Tyr Phe Gln Tyr Pro Ala Asn Ile IIe Lys Met Lys Lys Cys Tyr Ser
325 330 335
Pro Asp Lys Glu Arg Lys Met Ile Thr Ile Arg Gly Ser Val His Gln
340 345 350
Asn Phe Ala Asp Phe Thr Phe Ala Thr Gly Lys Ile Ile Gly His Met
355 360 365
Leu Lys Leu Lys Gly Asp Ile Asp Ser Asn Val Ala Ile Asp Leu Ser
370 375 3g0
Asn Lys Ala Ser Leu Ala Phe Leu Gln Lys His Leu Gly Leu His Lys
385 390 395 400
Asp Phe Asp Gln Trp Asp Cys Leu Ile Glu Gly Asp Asp Glu Asn Leu
405 410 415
Ile Pro Gly Thr Asn Ile Asn Thr Thr Asn Gln His Ile Met Leu Gln
420 425 430
Asn Ser Ser Gly Ile Glu Lys Tyr Asn
435 440
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1123 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: Not Determined
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(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:9:
AAATATAAAT TTTAATAACA CCACACATAA ATTTCAAACTACTTTCCCTA AGTTTCTAGC60
TGAAGTTTTA AATGAGTGTG TTTTTAATTT ATTAGAAAGTGGATTGAAGA GAAAACATTG120
GAAGATGAAG GAAGGCGTTT CAGTTAAACC CCAAATAACTCTGTGTTACA CTGAGCTATG180
AAACGGCTCC TTCTAGCTCC ATTTCTCCTC AGACCTAAGTGCTATTCCTG ATTGTCCTTC240
ATTGTCATTT CCAGGGAGAA ATGACACCAG CACAGTGGCAGGCCTTCCAA TCTGGAGCAC300
GGTCCACACA ACTTCCGAAT TGGTGTTCAG TGTAAAGTGTATCGGAGTGC GGAAAATGCG360
CAGGGCATTG CCAACTATAG ATGCTCGGAG TAATTCAGTGTATTCAGAGA ACACGGTGAA420
ACAAGGAAAA CCGGCCTGAC TGGGGGGTGA ATTCAGCAGGGAGTAAATCT GATCGGCATC480
AGGTCTGCGG AAAGGAGCTG GTGAGCACGA CACCACCAGGCATTGCCTGG CTCTCTCCGC540
GGCGGGCTAA GTTAACCTCG GGTCCAGGTG CGGGCCATGGTCTTGGGGAG GGTGCTGGGT600
GCGCTCGAGC AGGCTACGTC GGGAGCCGCC GCTGCTAGTGAGAGCCGGGC CACACACGCT660
CCTCCCCGGT ACCTCCTCCA GCATCACCAG GGGAGGAGAGGGTCGGGCAC AAGGCGCGCT720
AGGCGGACCC AGACACAGCC GCGCGCAGCC CACCCGCCCGCCGCCTGCCA GAGCTGCTCG780
GCCCGCAGCC AGGGGGACAG CGGCTGGTCG GAGGCTCGCAGTGCTGTCGG CGAGAAGCAG840
TCGGGTTTGG AGCGCTTGGG TCGCGTTGGT GCGCGGTGGAACCCCCCAGG GACCCCAGTT900
CCCGCGAGCA GCTCCGCGCC GCGCCTGAGT GAGGAGGGGCCCCGGGGGCG AGGCGGGAGT960
GGGAGGAAGG GCACGGTCGC CGCGCTGGAG GTCGGGACCCCGGAGCGGCG ACCGGCCGGG1020
GTGGGCTCGC TGAGTCGCAC CCGCTCTGCT GGCCGGTCCTGGGCTCACAG TCCCTGCAGC1080
CCTCGGAAAC AGCGCTAGGA TCCTTCGGGA GAGGAGAGATGAC 1123
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 417 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
( i.x) FEATURE
(A) NAME/KEY : exon
(B) LOCATION: 145..287
(xi) SEQUENCE DESCRIPTION: SEQ ID
NO:10:
GTACCAATCT AAAACCCAGC ACAGAAAAAT ACATGTTTTATTTTTTCCAA GTGTTACTAG60
TACCTCAGCC TTTCTTGATT TGTCAGCTTA TTTAAGGCCTCTTCATTGCA TACTTCTTTT120
TTCTTTTAAT CATCTGCTTC GAAGGAGACT AAGCTGAAACTGCTGCTCAG CTCCCAAGAT180
GGTGCCACCC AAATTGCATG TGCTTTTCTG CCTCTGCGGCTGCCTGGCTG TGGTTTATCC240
TTTTGACTGG CAATACATAA ATCCTGTTGC CCATATGAAATCATCAGGTA AGAGGTGTAT300
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TTGTTCAAGG TCTTGAGCAA CTGATCTGTC GCCATACTTC AAGTGGGCCC CAAGAAGTTG 360
CACATCTGCA CATCTAAACA AGTCCTATTT AAAGGCTTAT GGAGATCCTG TATTCTC 417
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 498 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 251..372
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: I1:
CATTAGGAGG TAACAGTCCA AGGCAGCTGA GAGAAAGGCT ATGTCTACTT TCATCTCTTT 60
ACCCTCCAAA ACCCCTACAC AGTGTTTCAA ACAGAGAGAC CCTCAATAAT TGCATATCTT 120
ACTTGTTAGG TTGAGAAAGA AAGAAGGCCA GAAACTATGG GAAGTAACTT GATTCCGTTG 18 0
GAATTCTTTT GCATAATAAA ATCTGATATG TAATGGATGA CAAATGAGAT AATATTTACC 240
TGTTTTTCAG CATGGGTCAA CAAAATACAA GTACTGATGG CTGCTGCAAC GTTTGGCCAA 300
ACTAAAATCC CCCGGGGAAA TGGGCCTTAT TCCGTTGGTT GTACAGACTT AATGTTTGAT 360
CACACTAATA AGGTAATGCT TTGATTTATA CAACTTATCC TGATACTCTA ATATTGTCTG 420
TCGCTATGGA CCACTAGAAG GTGTTCAAAT GTGACCTTGC CCTCACCTGA GAATGACTCA 480
TTTTCGAATT TGTATTGT 4 g 8
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 433 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 130..274
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
CAGCAGCCTA AAGTCTTAGA CTTTGTGAAC ACAGAGGTAT TGAGTCCCAC TAATTAATAT 60
CGAAAATAGC TGCTGGAATA TGTTTGAGAC ACAACTTCTC TAAAAGTGCA TTAATTTCTT 120
TCTTAACAGG GCACCTTCTT GCGTTTATAT TATCCATCCC AAGATAATGA TCACCTTGAC 180
ACCCTTTGGA TCCCAAATAA AGAATATTTT TGGGGTCTTA GCAAATTTCT TGGAACACAC 240
TGGCTTATGG GCAACATTTT GAGGTTACTC TTTGGTAAGA TTTCTGTTGA TCCTTCTTTG 300
TAGGCTCTTG CATGTATGAA AACCTTGAAA ACAACAAGAA CTTCAAGTAG TTAAGACCAA 360
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AGTAGATTTT TCTTCAGTCC AAATAGCTCC TAAAATGATA AGGAAAGTAT TTCTTTAAAG 420
CCCAGGCAAC TAC 433
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 486 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 164..257
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
TTGGTGGGTA TCTAGTAGCA GTCTTTTTAA TGAATCTACT ATTCATCCAT AAAAAAGTAG 60
ATATAAATCA GATGGGTCTG CATTTTATGC TAATGAGATA TGAATTAAAT TCACTAGCAA 120
CACTCAGAGA AAACCTTAAC TATAACCTTC CATTGTTGTC TAGGTTCAAT GACAACTCCT 180
GCAAACTGGA ATTCCCCTCT GAGGCCTGGT GAAAAATATC CACTTGTTGT TTTTTCTCAT 240
GGTCTTGGGG CATTCAGGTA ATGTTTGAGA GGTTGAACAA TTTTGGCTTC CAGGAATAAA 3 0 0
TGACAATTTT TTTATTCAAG AAAGAAATAG CAGAGTTTGG AATGTCATGC AGGCCCTTGT 360
CTGGAGGAGT TGGGGTTCCT CAATAATTGG CTGTGGGTCT ATTGATCAGT CCTAGACCTG 420
TCTGGTCAAG TAGTTTTTTC CCTACTATCA GCTCATTGGG ATTAGCCTCA CAGCAGAGAA 480
GAAAGG 486
(2) INFORMATION FOR 5EQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 base pairs
(B) TYPE: nucleic acid
(C) STRANDBDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 113..181
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
CCCCAGGCTC TACTACAGGG TGTAATGGCC TCCATGTTCC CAGTTTTATT AGTGACTCAG 60
CCTTGTAATT CATGACTGGT AGTTGTAATT CTTCCCTCTT TTTGTTTTGA AGGACACTTT 120
ATTCTGCTAT TGGCATTGAC CTGGCATCTC ATGGGTTTAT AGTTGCTGCT GTAGAACACA 180
GGTATGTTAC CTGATATAAT TGGGCTCTTT GGCCAACTAC AGGGAATGTC AATGCTCATA 240
ACTATGTTTC TAATTTTCAT AAAAGTTTAT TTAAAATGTT GATGGAACTT TCAAGTATGG 3 0 0
TAACATCATG AGCAAAAAAG GAGATTGAGT TTTATCGACT TAAAAGACTT AAAAGCACCT 360
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363
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 441 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 68..191
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
GAACTGAGAA ACATGGTCAG ATGAGGAAGG GAAGGAGCAT GCATAAATAA TTTTGCTTGT 60
ATTATAGAGA TAGATCTGCA TCTGCAACTT ACTATTTCAA GGACCAATCT GCTGCAGAAA 120
TAGGGGACAA GTCTTGGCTC TACCTTAGAA CCCTGAAACA AGAGGAGGAG ACACATATAC 180
GAAATGAGCA GGTACATTGC AGTGAAAGGA GAGGTGGTTG GTGACCTAAA AGCATGTACA 240
AAAGGATGAC ATTTGTTAAT TTAATTTTAC ACCTGGCAAG TTATGCTCCT AGCTCTCCTA 300
TTTCCCATTC CCAAAAGATC TGTCAATAGA TTCCTGGAGC AGTAAAATTC CCTTAATGGA 360
ATATCTAGTT CATAGTAAAA ACAAAGGCAA ATACAAAAAT TTGGGAGATG ACAGTGAATA 420
TTCAGAATTC CTCGAGCCGG G 441
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 577 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 245..358
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
GGTTAAGTAA ATCGTCTGAA GTCACATAGT AGGTAAGGCA AAACAGAGCC AGGATTTGGA 60
CTAAGGCTAT ACCTATGTGC AAAGCTGGGG CCTGTGTCAT TATGGTAGCA AGTAATAGTC 120
ACTAATCAGA TTTCCAGTTT ATAACTGACC AACGATTTTT CCCAAATACA GCTTCTACCT 180
AAACTTTAAA ATAAGTGTTA TAACTTTTTA CTTTGTCATT TCCTTCTTCT AATAATTATA 240
TTAGGTACGG CAAAGAGCAA AAGAATGTTC CCAAGCTCTC AGTCTGATTC TTGACATTGA 300
TCATGGAAAG CCAGTGAAGA ATGCATTAGA TTTAAAGTTT GATATGGAAC AACTGAAGGT 360
AAGCTATAAA AAGTAATTTT TCTCTTGTCC TACAGTTCTT TATTGTTTTT TGTCATTTAA 4 2 0
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TTTTCTGCCT ATATTGCAAG GTACAATATG ATAAAGGGCT GCAACCAGCC CCTCCCCAAT 480
GCGCACACAC AGACACACAA AGCAGTACAG GTAAAGTATT GCAGCAATGA AGAATGCATT 540
ATCTTGGACT AGATATGAAT GCAAAGTTAG TCAGTTT 577
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 396 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B} LOCATION: 108..199
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
ATCAATGTAT TTACCATCCC CATGAAATGA ACAATTATAT GATTGACAAA 60
TCATTTCTTC
TAACACCACG AAATAGCTAT AAATTTATAT CATGCTTTTT CAAATAGGAC 120
TCTATTGATA
GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC AACGGTTATT 180
CAGACTCTTA
GTGAAGATCA GAGATTCAGG TAAGAAAATA AGATAGTAAA GCAAGAGAAT 240
AGTAAATTAT
TGGAAGAAAT TATATTGTGA GATATAATTT TTATTCAAAT TCTTAGTGAA 300
GGAAGGGGAT
CTCTTGGAGT TTATAAGGCT ATTCTTTTGC CCCCATAAAA TACTCTATAT 360
ACATTTTCCT
AGGCTAAAAC ATCTCCTCTC CTGCTATTAA AATCTC 396
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 519 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 181..351
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
CTTACAAAGT TAATCATATC CCTTTCCCAC ATTGAAGTAT GATACCTCTT TATTCCAATC 60
AGATAACCCA TAATAAACTG GTATGGTGCG TGTCCACCAA TCCTAGCATT ATTAGGATGT 120
CCTCAATGTT GGCTAGTATG TAACCAGTTT AATTTCATCA TTGTCAACAA ATATCTACAG 180
ATGTGGTATT GCCCTGGATG CATGGATGTT TCCACTGGGT GATGAAGTAT ATTCCAGAAT 240
TCCTCAGCCC CTCTTTTTTA TCAACTCTGA ATATTTCCAA TATCCTGCTA ATATCATAAA 300
AATGAAAAAA TGCTACTCAC CTGATAAAGA AAGAAAGATG ATTACAATCA GGTAAGTATT 360
AGTGACTTAT TTCATTATGT GAAACAAACT TGAAGCTTGG GTAAATATCA ATCGATATCA 420
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TTTGGTAACT ATTAAAGAAT TGCTGAATTG GTTGTTTAGA CTTTCAATAA GGAGAGAATT 480
AGATAATCTC AGTTTCTAAG TACATTTAGT CTACTCTTT 519
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 569 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix} FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 156..304
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:19:
TGAAACACAT CTAAGTAGATCAAATTACAA GTTTTATTTCTTCTTTGGTT TTCAGTAAAC60
AGACCAACAA GACCAGTACCTTTCCTTACA CTCTAACTAAAAAAATAATA ATTTTATCAA120
ACAATGTGAC TTTTAAATGTCTTGTTCTCT TTTAGGGGTTCAGTCCACCA GAATTTTGCT180
GACTTCACTT TTGCAACTGGCAAAATAATT GGACACATGCTCAAATTAAA GGGAGACATA240
GATTCAAATG TAGCTATTGATCTTAGCAAC AAAGCTTCATTAGCATTCTT ACAAAAGCAT300
TTAGGTAAGA AACTATTTTTTTCATGACCT AAACCGAGATGAATCTCGAG GACAAAGCTG360
TCTATCTTAA TACAGCTTTAGTACTATTTA AACTATTTCCAGTTGGTTTA CAATGGAACA420
AAGCAGTATA TCAATTTGAAAACAGAAATT TGAGAAAGTCAATTTTGCTG CTTTACATCT480
CTATATCATA GAAAGCAAATCAACTGTTAA AGGTAATATTCTTTGTATGA GCTAGAGTGA540
CTCATGTGAG GATATCGAACGACGGTGCT 569
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 469 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY : exon
(B) LOCATION: 137..253
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:20:
GATACAGAGG CACATCGTCT CTACCATCCT AACGGAACTT GTGTAATTTG TAAATCTTTA 60
TTGCCACCTAGGGGCATCCA AACTGTTTAA TGCTCTCAAA AGTTTAATATGTTGATTAAC 120
ACTTTATATTTTATAGGACT TCATAAAGAT TTTGATCAGT GGGACTGCTTGATTGAAGGA 180
GATGATGAGAATCTTATTCC AGGGACCAAC ATTAACACAA CCAATCAACACATCATGTTA 240
CAGAACTCTTCAGGAATAGA GAAATACAAT TAGGATTAAA ATAGGTTTTTTAAAAGTCTT 300
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GTTTCAAAAC TGTCTAAAAT TATGTGTGTG TGTGTGTGTG TGTGTGTGTG AGAGAGAGAG 360
AGAGAGAGAG AGAGAGAATT TTAATGTATT TTCCCAAAGG ACTCATATTT TAAAATGTAG 420
GCTATACTGT AATCGTGATT GAAGCTTGGA CTAAGAATTT TTTCCCTTT 469
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1494 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 117..1436
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
GGCACGAGCT CGCTCAGGGT TCTGGGTATC
60
AGGATCTGAC
TCGCTCTGGT
GGCATTGCTG
CGGGAGTCAG TGCAGTGACC CTGCTCAGCT CCTAAG
116
AGAACATCAA
ACTGAAGCCA
ATGGTACCACTC AAA CAGGCGCTT TTCTGC CTCCTCTGCTGC CTC 164
CTG
MetValProLeu Lys GlnAlaLeu PheCys LeuLeuCysCys Leu
Leu
1 5 10 15
CCATGGGTCCAT CCT CACTGGCAA GACACA TCTTCTTTTGAC TTC 212
TTT
ProTrpValHis Pro HisTxpGln AspThr SerSerPheAsp Phe
Phe
20 25 30
AGGCCGTCAGTA ATG CACAAGCTC CAATCG GTGATGTCTGCT GCC 260
TTT
ArgProSerVal Met HisLysLeu GlnSer ValMetSerAla Ala
Phe
35 40 45
GGCTCTGGCCAT AGT ATCCCCAAA GGAAAT GGATCGTACCCC GTC 308
AAA
GlySerGlyHis Ser IleProLys GlyAsn GlySerTyrPro Val
Lys
50 55 60
GGTTGTACAGAT CTG TTCGGTTAT GGGAAT GAGAGCGTCTTC GTG 356
ATG
GlyCysThrAsp Leu PheGlyTyr GlyAsn GluSerValPhe Val
Met
65 70 75 80
CGTTTGTACTAC CCA CAAGATCAA GGTCGC CTCGACACTGTT TGG 404
GCT
ArgLeuTyrTyr Pro GlnAspGln GlyArg LeuAspThrVal Trp
Ala
85 90 95
ATCCCAAACAAA GAA TTTTTGGGT CTTAGT ATATTTCTTGGA ACA 452
TAT
IleProAsnLys Glu PheLeuGly LeuSer IlePheLeuGly Thr
Tyr
100 105 110
CCCAGTATTGTA GGC ATTTTACAC CTCTTA TATGGTTCTCTG ACA 500
AAT
ProSerIleVaI Gly IleLeuHis LeuLeu TyrGIySerLeu Thr
Asn
115 I20 125
ACTCCTGCAAGC TGG TCTCCTTTA AGGACT GGAGAAAAATAC CCG 548
AAT
ThrProAlaSer Trp SerProLeu ArgThr GlyGluLysTyr Pro
Asn
130 135 140
CTCATTGTCTTT TCT GGTCTCGGA GCCTTC AGGACGATTTAT TCT 596
CAT
LeuIleValPhe Ser GlyLeuGly AlaPhe ArgThrIleTyr Ser
His
145 150 155 160
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GCT GGC GGC TTGGCATCT AAT GGG GCC GTC 644
ATT ATT TTT ATA ACT
GTG
AlaIleGly Gly LeuAlaSer Aen Gly IleVal AlaThrVal
Ile Phe
165 170 175
GAACACAGA AGA TCTGCATCG GCA ACT TTTTTT GAAGACCAG 692
GAC TAC
GluHisArg Arg SerAlaSer Ala Thr PhePhe GluAspGln
Asp Tyr
180 185 190
GTGGCTGCA GTG GAAAACAGG TCT TGG TACCTG AGAAAAGTA 740
AAA CTT
ValAlaAla Val GluAsnArg Ser Trp TyrLeu ArgLyeVal
Lys Leu
195 200 205
AAACAAGAG TCG GAAAGTGTC CGG AAA CAGGTT CAGCAAAGA 788
GAG GAA
LysGlnGlu Ser GluSerVal Arg Lys GlnVal GlnGlnArg
Glu Glu
210 215 220
GCAATAGAA TCC CGGGCTCTC AGT GCG CTTGAC ATTGAACAT 836
TGT ATT
AlaIleGlu Ser ArgAlaLeu Ser Ala LeuAsp IleGluHis
Cys Ile
225 230 235 240
GGAGACCCA GAG AATGTACTA GGT TCA TTTGAC ATGAAACAG 884
AAA GCT
GlyAspPro Glu AsnValLeu Gly Ser PheAsp MetLysGln
Lys Ala
245 250 255
CTGAAGGAT ATT GATGAGACT AAA ATA TTGATG GGACATTCT 932
GCT GCT
LeuLysAsp Ile AspGluThr Lys Ile LeuMet GlyHisSer
Ala Ala
260 265 270
TTTGGAGGA ACA GTTCTTCAA GCC CTT GAGGAC CAGAGATTC 980
GCA AGT
PheGlyGly Thr ValLeuGln Ala Leu GluAsp GlnArgPhe
Ala Ser
275 280 285
AGATGTGGA GCT CTTGATCCA TGG ATG CCGGTG AACGAAGAG 1028
GTT TAT
ArgCysGly Ala LeuAspPro Trp Met ProVal AsnGluGlu
Val Tyr
290 295 300
CTGTACTCC ACC CTCCAGCCT CTC CTC ATCAAC TCTGCCAAA 1076
AGA TTT
LeuTyrSer Thr LeuGlnPro Leu Leu IleAsn SerAlaLys
Arg Phe
305 310 315 320
TTCCAGACT AAG GACATCGCA AAA ATG AAGTTC TACCAGCCT 1124
CCA AAA
PheGlnThr Lys AspIleAla Lys Met LysPhe TyrGlnPro
Pro Lys
325 330 335
GACAAGGAA AAA AATGATTAC AAT CAA CTCAGG CACCAGAAC 1172
AGG GGG
AspLysGlu Lys AsnAspTyr Asn Gln LeuArg HisGlnAsn
Arg Gly
340 345 350
TTTGACGAC ACT TTTGTAACT GGC AAA ATTGGA AACAAGCTG 1220
TTT ATA
PheAspAsp Thr PheValThr Gly Lys IleGly AsnLysLeu
Phe Ile
355 360 365
ACACTGAAA GAA ATCGATTCC AGA GTA ATCGAC CTCACCAAC 1268
GGA GCC
ThrLeuLys Glu IleAspSer Arg Val IleAsp LeuThrAsn
Gly Ala
370 375 380
AAAGCTTCG GCT TTCTTACAA AAG CAT GGGCTT CAGAAAGAC 1316
ATG TTA
LysAlaSer Ala PheLeuGln Lys His GlyLeu GlnLysAsp
Met Leu
385 390 395 400
TTTGATCAG GAC CCTCTGGTG GAA GGA GATGAG AACCTGATT 1364
TGG GAT
PheAspGln Asp ProLeuVal Glu Gly AspGlu AsnLeuIle
Trp Asp
405 410 415
CCTGGGTCA TTT GACGCAGTC ACC CAG CCGGCT CAGCAACAC 1412
CCC GCC
ProGlySer Phe AspAlaVal Thr Gln ProAla GlnGlnHis
Pro Ala
420 425 430
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TCT CCA GGA TCA CAG ACC CAG AAT TAGAAGAACT TGCTTGTTAC ACAGTTGCCT 1466
Ser Pro Gly Ser Gln Thr Gln Asn
435 440
TTTAAAAGTA GAGTGACATG AGAGAGAG 1494
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2191 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 92..1423
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
CCGCGCGCTC CGGCCGGGGG CTCAGCGCGGCGCCCGGAAG60
ACCCTGGTTC
CGGCGAGCGG
TTTAAGCTGA CT CAGCTTCCAA TTG CCA CCC CTG CAT 112
AACCACTG G ATG AAA
Met Leu Pro Pro Leu His
Lys
1 5
GCGCTT TGC TGC AGC CTC CTG GTT CAT ATT GAC 160
TTC CTC TGC ACA CCT
AlaLeu Cys Cys Ser Leu Leu Val His Ile Asp
Phe Leu Cys Thr Pro
15 20
TGGCAA CTA CCT GTT CAT AGA TCA TCA TGG GCC 208
GAC AAT GCC ATT GCA
TrpGln Leu Pro Val His Arg Ser Ser Trp Ala
Asp Asn Ala Ile Ala
25 30 35
AATAAA CAA CTG ATG GCT AGT ATT AGG AGT AGA 256
ATA GCT GCT GCA CAA
AsnLys Gln Leu Met Ala Ser Ile Arg Ser Arg
Ile Ala Ala Ala Gln
40 45 50 55
ATTCCC GGA GGA TCT TCT GGT TGT ACA TTG ATG 304
AAA AAT TAT GTC GAT
IlePro Gly Gly Ser Ser Gly Cys Thr Leu Met
Lys Asn Tyr Val Asp
60 65 70
TTTGAT ACT AAG GGC TTT CGT TTG TAT CCA TCG 352
TAT AAT ACC TTG TAT
PheAsp Thr Lys Gly Phe Arg Leu Tyr Pro Ser
Tyr Asn Thr Leu Tyr
75 80 85
CAAGAG GAC TCT GAC CTT ATC CCA AAC GAA TAT 400
GAT CAC ACG TGG AAA
GlnGlu Asp Ser Asp Leu Ile Pro Asn Glu Tyr
Asp His Thr Trp Lys
90 95 100
TTTTTT CTT AAA TAT GGA CCC TGG CTT GGC AAA 448
GGT AGT CTT ACA ATG
PhePhe Leu Lys Tyr Gly Pro Trp Leu Gly Lys
Gly Ser Leu Thr Met
105 lI0 115
ATATTG TTC TTT GGT GTG ACT CCT GCG TGG AAT 496
AGC TTT TCA ACA AAC
IleLeu Phe Phe Gly Val Thr Pro Ala Trp Asn
Ser Phe Ser Thr Asn
120 125 130 135
TCCCCT AGG GGT GAA TAT CTG ATT GTT TCT CAT 544
CTG ACT AAA CCA TTT
SerPro Arg Gly Glu Tyr Leu Ile Val Ser His
Leu Thr Lys Pro Phe
140 145 150
GGTCTT GCA CGG ACA TAT GCT ATT GGC GAT CTA 592
GGA TTC ATT TCT ATT
GlyLeu Ala Arg Thr Tyr Ala Ile Gly Asp Leu
Gly Phe Ile Ser Ile
CA 02267994 1999-04-09
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155 i60 165
GCA CAT GGGTTCATC GTTGCT GCTATAGAA CACAGA GGATCC 640
TCA GAT
Ala His GlyPheIle ValAla AlaIleGlu HisArg GlySer
Ser Asp
170 175 180
GCC GCG ACTTACTAT TTCAAG GACCAGTCT GCTGCA ATAGGG 688
TCT GAA
Ala Ala ThrTyrTyr PheLys AspGlnSer AlaAla IleGly
Ser Glu
185 190 195
AAC TCT TGGTCTTAT CTTCAA GAACTAAAA CCAGGG GAGGAG 736
AAA GAT
Asn Ser TrpSerTyr LeuGln GluLeuLys ProGly GluGlu
Lys Asp
200 205 210 215
ATA GTT CGAAATGAG CAGGTA CAGAAAAGG GCAAAG TGCTCC 784
CAT GAG
Ile Val ArgAsnGlu GlnVal GlnLysArg AlaLys CysSer
His Glu
220 225 230
CAA CTC AACTTGATT CTGGAC ATTGATCAT GGAAGG ATTAAG 832
GCT CCA
Gln Leu AsnLeuIle LeuAsp IleAspHis GlyArg IleLys
Ala Pro
235 240 245
AAT CTA GACTTAGAG TTTGAT GTGGAACAA CTGAAG TCTATT 880
GTA GAC
Asn Leu AspLeuGlu PheAsp ValGluGln LeuLys SerIle
Val Asp
250 255 260
GAC GAT AAAATAGCA GTAATT GGACATTCT TTTGGT GCCACA 928
AGG GGA
Asp Asp LysIleAla ValIle GlyHisSer PheGly AlaThr
Arg Gly
265 270 275
GTT CAG GCTCTTAGT GAAGAC CAGAGATTT AGGTGC ATTGCC 976
CTT GGG
Val Gln AlaLeuSer GluAsp GlnArgPhe ArgCys IleAla
Leu Gly
280 285 290 295
TTG GCA TGGATGCTT CCACTG GATGATGCA ATATAT AGAATC 1024
GAT TCC
Leu Ala TrpMetLeu ProLeu AspAspAla IleTyr ArgIle
Asp Ser
300 305 310
CCT CCC CTCTTTTTT ATTAAC TCGGAACGG TTCCAA CCTGAG 1072
CAG TTT
Pro Pro LeuPhePhe IleAsn SerGluArg PheGln ProGlu
Gln Phe
3i5 320 325
AAT AAA AAAATGAAA AAATGC TACTCACCT GACAAA AGAAAA 1120
ATC GAA
Asn Lys LysMetLys LysCys TyrSerPro AspLys ArgLys
Ile Glu
330 335 340
ATG ACA ATCAGGGGT TCAGTC CATCAGAAC TTTGCT TTCACT 1168
ATT GAT
Met Thr IleArgGly SerVal HisGlnAsn PheAla PheThr
Ile Asp
345 350 355
TTT ACT GGCAAAATA GTTGGA TACATATTC ACATTA GGAGAT 1216
ACA AAA
Phe Thr GlyLysIle ValGly TyrIlePhe ThrLeu GlyAsp
Thr Lys
360 365 370 375
ATA TCA AATGTAGCA ATTGAT CTTTGCAAC AAAGCT TTGGCA 1264
GAT TCA
Ile Ser AsnValAla IleAsp LeuCysAsn LysAla LeuAla
Asp Ser
380 385 390
TTT CAA AAGCATTTA GGACTG CGGAAAGAT TTTGAT TGGGAT 1312
TTA CAG
Phe Gln LysHisLeu GlyLeu ArgLysAsp PheAsp TrpAsp
Leu Gln
395 400 405
TCT ATT GAAGGAAAA GACGAA AATCTTATG CCAGGG AACATT 1360
TTG ACC
Ser Ile GluGlyLys AspGlu AsnLeuMet ProGly AsnIle
Leu Thr
410 415 420
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AAC ATC ACC AAC GAA CAT GAC ACT CTA TCT CGA GAA GCA GAG 1408
CAG AAC
Asn Ile Thr Asn Glu His Asp Thr Leu Ser Pro Glu Ala Glu
Gln Asn
425 430 435
AAA TCG AAT TTA GAT TAAAAGCACT TTTTTAAAGA 1463
TCTTGTTTAA AAACTGTCAA
Lys Ser Asn Leu Asp
440
AAAATGTGTG TATGACTTTT AATATATTTT CTCAAATAACTCATATTGGA AAATGTAGGC1523
TATCCCATAA AAGTGATTGA AGCTTGGACT AGGAGGTTTTTTTCTTTAAA GAAAGATTGG1583
TGTCTATCGA AATCATGCCA GCCTAAATTT TAATTTTACTAAAATGATGC TGTGTCAAAA1643
TTAATAACTA CTTTTACATT CTTTAATGGA CAAGTATAACAGGCACAAGG CTAATGAAAA1703
CGTGTTGCAA TGACATAACA ATCCCTAAAA ATACAGATGTTCTTGCCTCT TTTTTCTATT1763
ATAATTGAGT TTTAGCAACA TGTTATGCTA GGTAGAATTTGGAAGCACTT CCCTTTGACT1823
TTTGGTCATG ATAAGAAAAA TTAGATCAAG CAAATGATAAAAGCAGTGTT TTACCAAGGA1883
TTAGGGATAC TGAACAATTT CACTATGGTA ACTGAATGGGGAGTGACCAA GGGTAAAAAT1943
ATTAAAGCCA AGGCAAAGGC AGCAGATTAG AATGGATTAAAGAGAGTTTA TAATTTGTTT2003
GCATTTACTT GATGGTTTAT CTCATGGATT CATGAGTCAAGAAAGGTGCG TAGGACAGGC2063
CAGGGATTCC AGTTATAACA CATTATTCAC CCAAAGGGTTCTTTAATTCT GTATGAGTAT2123
TGGGAGTGGA TTAGCACAAT AGAGGCATAT GTTGCTTTAAAAAAAAAAAA 2183
AAAAAAAA 2191
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1533 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 62..1394
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
CCGCGAGCAG TTCACCGCGG CGTCCGGAAG GTTAAGCTGA AACGGCAGCT CAGCTTCGGA 60
G ATG TTA CCG TCC AAA TTG CAT GCG CTT TTC TGC CTC TGC ACC TGC 106
Met Leu Pro Ser Lys Leu His Ala Leu Phe Cys Leu Cys Thr Cys
1 5 10 15
CTT GCA CTG GTT TAT CCT TTT GAC TGG CAA GAC CTG AAT CCA GTT GCC 154
Leu Ala Leu Val Tyr Pro Phe Asp Trp Gln Asp Leu Asn Pro Val Ala
20 25 30
TAT ATT GAA TCA CCA GCA TGG GTC AGT AAG ATA CAA GCT CTG ATG GCT 202
Tyr Ile Glu Ser Pro Ala Trp Val Ser Lys Ile Gln Ala Leu Met Ala
35 40 45
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GCT AAC GGTCAA CCC AATGGA TAT 250
GCA ATT TCT AGA TCT
AAA GGA
ATC
AlaAlaAsn IleGlyGln SerLys IleProArg AsnGly SerTyr
Gly
50 55 60
TCCGTCGGT TGTACAGAC TTGATG TTTGATTAC AATAAG GGCACC 298
ACT
SerValGly CysThrAsp LeuMet PheAspTyr AsnLys GlyThr
Thr
65 70 75
TTCTTGCGT TTGTATTAT CCATCT CAAGATGAT CACTCC GACACC 346
GAT
PheLeuArg LeuTyrTyr ProSer GlnAspAsp HisSer AspThr
Asp
80 85 90 95
CTTTGGATC CCAAACAAA GAATAT TTTTTGGGT AGTAAA TTTCTT 394
CTT
LeuTrpIle ProAsnLys GluTyr PheLeuGly SerLye PheLeu
Leu
100 105 110
GGAACACAC TGGCTTGTG GGCAAA ATTATGGGC TTCTTC GGTTCA 442
TTA
GlyThrHis TrpLeuVal GlyLys IleMetGly PhePhe GlySer
Leu
115 120 125
ATGACAACT CCTGCAGCC TGGAAT GCACATCTG ACTGGG GAAAAA 490
AGG
MetThrThr ProAlaAla TrpAsn AlaHisLeu ThrGly GluLys
Arg
130 135 140
TACCCACTA ATTATTTTT TCTCAT GGTCTTGGA TTCAGG ACGATT 538
GCA
TyrProLeu IleIlePhe SerHis GlyLeuGly PheArg ThrIle
Ala
145 150 155
TATTCTGCT ATTGGCATT GATCTG GCATCCCAC TTTATA GTTGCT 586
GGG
TyrSerAla IleGlyIle AspLeu AlaSerHis PheIle ValAla
Gly
160 165 170 175
GCTGTAGAA CACAGGGAT GGCTCT GCATCCTCG TACTAT TTCAAG 634
ACA
AlaValGlu HisArgAsp GlySer AlaSerSer TyrTyr PheLys
Thr
180 185 190
GACCAGTCT GCTGTAGAA ATAGGC AACAAGTCT CTCTAT CTCAGA 682
TGG
AspGlnSer AlaValGlu IleGly AsnLysSer LeuTyr LeuArg
Trp
195 200 205
ACCCTGAAG CGAGGAGAG GAGGAG TTTCCTTTA AATGAG CAGTTA 730
CGA
ThrLeuLys ArgGlyGlu GluGlu PheProLeu AsnGlu GlnLeu
Arg
210 215 220
CGGCAACGA GCAAAGGAA TGTTCT CAAGCTCTC TTGATT CTGGAC 778
AGT
ArgGlnArg AlaLysGlu CysSer GlnAlaLeu LeuIle LeuAsp
Ser
225 230 235
ATTGATCAC GGGAGGCCA GTGACG AATGTACTA TTAGAG TTTGAT 826
GAT
IleAspHis GlyArgPro ValThr AsnValLeu LeuGlu PheAsp
Asp
240 245 250 255
GTGGAACAG CTGAAGGAC TCTATT GATAGGGAT ATAGCC ATTATT 874
AAA
ValGluGln LeuLysAsp SerIle AspArgAsp IleAla IleIle
Lys
260 265 270
GGACATTCT TTTGGTGGA GCCACA GTTATTCAG CTTAGT GAAGAC 922
ACT
GlyHisSer PheGlyGly AlaThr ValIleGln LeuSer GluAsp
Thr
275 280 285
CAGAGATTC AGGTGTGGC ATTGCT CTGGATGCA ATGTTT CCCGTG 970
TGG
GlnArgPhe ArgCysGly IleAla LeuAspAla MetPhe ProVal
Trp
290 295 300
GGTGATGAA GTATATTCC AGAATT CCTCAACCC TTTTTT ATCAAC 1018
CTC
GlyAspGlu ValTyrSer ArgIle ProGlnPro PhePhe IleAsn
Leu
305 310 315
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TCGGAA TTC CAA CCT AAT 1066
CGA TAC TCT ATC
ATA
AGA
ATG
AAA
AAA
TGC
SerGlu Phe GlnTyrPro Asn Ile Arg Lys Cys
Arg Ser Ile Met Lys
320 325 330 335
TTCTTA GAT AGAGAACGA ATG ATT ATC GGT GTC 1114
CCT AAA ACA AGG TCG
PheLeu Asp ArgGluArg Met Ile Ile Gly Val
Pro Lys Thr Arg Ser
340 345 350
CATCAG TTT GTTGACTTC TTT GCC AGC ATA GGC 1162
AAT ACT ACT AAA ATT
HisGln Phe ValAspPhe Phe Ala Ser Ile Gly
Asn Thr Thr Lys Ile
355 360 365
TACCTA ACA CTGAAAGGA ATC GAT AAT GCC AGC 1210
TTC GAC TCC GTA ATC
TyrLeu Thr LeuLysGly Ile Asp Asn Ala Ser
Phe Asp Ser Val Ile
370 375 380
CTTAGC AAA GCTTCCTTA TTC TTA AAA TTA CTT 12
AAC GCG CAA CAT GGA 5
8
LeuSer Lys AlaSerLeu Phe Leu Lys Leu Leu
Asn Ala Gln His Gly
385 390 395
CAGAAA TTT GATCAGTGG TCT TTA GAA GAA CAC 1306
GAT GAT GTT GGC GAT
GlnLys Phe AspGlnTrp Ser Leu Glu Glu His
Asp Asp VaI Gly Asp
400 405 410 415
AATCTT CCA GGGACCAAC AAC ACA AAC CAA ATT 1354
ATT ATT ACC CAC GCC
AsnLeu Pro GlyThrAsn Asn Thr Asn Gln Ile
Ile Ile Thr His AIa
420 425 430
CTGCAG TCC ACAGGAATA AGA CCA TTA T AAAAGAGCTT 1404
AAC GAG AAT GAT
LeuGln Ser ThrGlyIle Arg Pro Leu
Asn Glu Asn Asp
435 440
TTTAAAAAGT TTTGTTTACG AACTTGTCTA AAAGTGTGTG TGTGTATGAT TTAAATGTAT 1464
TTTCTCAAAT AGCTCATATT AAAAAATGTA GGCTATAGCA C;AAAAAAAAA AAAAAAAAAA 1524
1533
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1876 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 468..1734
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:24:
CGGCGGGCTGCTGGCCCTTCCCGGCTGTTCGTAGAGCCGGATCCTGCAGCGCCCCTGAGA 60
CGAACCGCCCCGATGCGGTGCTCCTCAGCGCCACGGGACGCAGCCGGGGCCGGCCGTGTT 120
GGCGCAGCTCCCACGACGTACGCTTCCTTTCCAGGCTCGAGGAAAGCCTCTCCCACAAAC 180
ACCGTCCCAGCTGGGAAGTGAGGCGGAGTTTTGGTCCCTCCCCTCCGGCAGCGCCCGGCA 240
TTCCGTCCGTCCGTCCGTCCGTCCGTGCGGCGCACGGCGCCCTGCAGAGCCGGGACACCG 300
CA 02267994 1999-04-09
WO 99!09147 PCTlUS97/14212
-110-
CAGCAGGGTA GTTTCCATCC 360
GGAGGACCCG TGCCCCCACC
GAGGTGGTGT
GCAGCCACAG
TCCCGGGGAG CAGCCCTGTG AGAGCACTGA
420
CTATACCCAA GCCGGCTGCT
CCCCCCGCAC
GCCTGCCTGC ACAAGTG ATG GCATCG 476
ACCCCGCCGT
GGGACCTTCT
GCTCTTCCCA
Met AlaSer
1
CTGTGGGTG AGAGCC AGGAGGGTG TTCATGAAA AGTCGTGCT TCAGGT 524
LeuTrpVal ArgAla ArgArgVal PheMetLys SerArgAla SerGly
10 15
TTCTCGGCG AAGGCG GCGACGGAG ATGGGGAGC GGCGGCGCG GAGAAG 572
PheSerAla LysAla AlaThrGlu MetGlySer GlyGlyAla GluLys
20 25 30 35
GGCTATCGG ATCCCC GCCGGGAAG GGCCCGCAC GCCGTGGGC TGCACG 620
GlyTyrArg IlePro AlaGlyLys GlyProHis AlaValGly CysThr
40 45 50
GATCTGATG ACCGGC GACGCGGCC GAGGGAAGC TTTTTGCGC CTGTAT 668
AspLeuMet ThrGly AspAlaAla GluGlySer PheLeuArg LeuTyr
55 60 65
TACCTATCG TGTGAC GACACAGAT ACTGAAGAG ACACCCTGG ATTCCA 716
TyrLeuSer CysAsp AspThrAsp ThrGluGIu ThrProTrp IlePro
70 75 80
GATAAAGAG TACTAC CAGGGGCTG TCTGACTTC CTCAACGTG TACCGG 764
AspLysGlu TyrTyr GlnGlyLeu SerAspPhe LeuAsnVaI TyrArg
85 90 95
GCCCTGGGA GAAAGG CTTTTCCAG TACTACGTT GGCTCAGTG ACCTGT 812
AlaLeuGly GluArg LeuPheGln TyrTyrVal GlySerVal ThrCys
100 105 110 115
CCTGCAAAA TCAAAC GCTGCTTTT AAGCCAGGA GAGAAATAC CCACTG 860
ProAlaLys SerAsn AlaAlaPhe LysProGly GluLysTyr ProLeu
120 125 130
CTCGTTTTT TCCCAT GGACTTGGA GCTTTTCGG ACCATCTAT TCTGCT 908
LeuValPhe SerHis GlyLeuGly AlaPheArg ThrIleTyr SerAla
135 140 145
ATCTGCATA GAGATG GCTTCTCAA GGCTTTCTA GTGGCAGCT GTGGAG 956
IleCysIle GluMet AlaSerGln GlyPheLeu ValAlaAla ValGlu
150 155 160
CACAGAGAT GAATCG GCTTCAGCA ACGTATTTC TGTAAAAAG AAGGCT 1004
HisArgAsp GluSer AlaSerAla ThrTyrPhe CysLysLys LysAla
165 170 175
GATTCTGAG CCAGAG GAGGATCAA ACATCAGGC GTGGAGAAG GAGTGG 1052
AspSerGlu ProGlu GluAspGln ThrSerGly ValGluLys GluTrp
180 185 190 195
ATCTACTAC AGGAAG CTCAGAGCA GGAGAGGAG GAGCGCTGT CTGCGT 1100
IleTyrTyr ArgLys LeuArgAla GlyGluGlu GluArgCys LeuArg
200 205 210
CACAAGCAG GTACAG CAGAGAGCA CAGGAGTGC ATCAAAGCG CTCAAC 1148
HisLysGln ValGln GlnArgAla GlnGluCys IleLysAla LeuAsn
215 220 225
CTCATTCTT AAGATC AGTTCAGGA GAGGAAGTG ATGAATGTG CTGAAC 1196
LeuIleLeu LysIle SerSerGiy GluGluVal MetAsnVal LeuAsn
230 235 240
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-111-
TCA GAC TTT TGGAAC CAC AAGGAT GTT AGC 1244
GAC CTG TCT GAT AGA
ACT
Ser Asp Phe TrpAsn His LysAsp Val Thr SerArg
Asp Leu Ser Asp
245 250 255
ATA GCT GTG GGACAC TCT GGTGGT ACA ATT GAGAGC 1292
ATG TTT GCT GTT
Ile Ala Val GlyHis Ser GlyGly Thr Ile GluSer
Met Phe Ala Val
260 265 270 275
CTC AGC AAA ATTAGA TTT TGTGGC GCC GAT GCGTGG 1340
GAA AGG ATT CTT
Leu Ser Lys IleArg Phe CysGly Ala Asp AlaTxp
Glu Arg Ile Leu
280 285 290
ATG CTC CCG GGCGAT GAC TACCAA AGT CAG CAACCA 1388
GTA ACT AGC GTG
Met Leu Pra GlyAsp Asp TyrGln Ser Gln GlnPro
Val Thr Ser Val
295 300 305
CTG CTC TTT AATTCC GAA TTCCAG GCT AAT ATCTTA 1436
ATT AAA TGG GCC
Leu Leu Phe AsnSer Glu PheGln Ala Asn IleLeu
Ile Lys Trp Ala
310 315 320
AAG ATG AAG CTTAGC TCC GATACC AAG ATG ATCACC 1484
AAG AAT AAC AAA
Lys Met Lys LeuSer Ser AspThr Lys Met IleThr
Lys Asn Asn Lys
325 330 335
ATC AAA GGA GTACAT CAG TTTCCT TTT TTT GTGAGT 1532
TCG AGC GAT ACT
Ile Lys Gly ValHis Gln PhePro Phe Phe ValSer
Ser Ser Asp Thr
340 345 350 355
GGA GAA ATC GGAAAG TTT AAGTTA GGA ATA GACCCA 1580
ATT TTC AAA GAA
Gly Glu Ile GlyLys Phe LysLeu Gly Ile AspPro
Ile Phe Lys Glu
360 365 370
AAT GAA GCT GATATA TGC CACGCT TTG TTC CTGCAG 1628
ATT AAC TCA GCC
Asn Glu Ala AspIle Cys HisAla Leu Phe LeuGln
Ile Asn Ser Ala
375 380 385
AAA CAT CTG CTTAAG AGA TTTGAT TGG TCA CTCGTG 1676
AGT GAT AAG GAT
Lys His Leu LeuLys Arg PheAsp Trp Ser LeuVal
Ser Asp Lys Asp
390 395 400
GAT GGC ATA CCCAAT GTT TCTGGT AAT GAC TTATCT 1724
GGA ATT ACC ATC
Asp Gly Ile ProAsn Val SerGly Asn Asp LeuSer
Gly Ile Thr Ile
405 410 415
CCA ACT GAG CAAAGGCCAC 1774
T AAGGAGTACA CAGCAGCAGG
AGAAGTACTG
Pro Thr Glu
420
ACACCAACGT CTGAGATAGC ACTGGCCTCC 1834
TGGCCACACA CACACAGCTT
TTGCTTGGAG
TTGGAGTGTG ACAGGGGAGC CG 1876
AAACAACAAA
AAAAAAAp,TC
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 517 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..514
CA 02267994 1999-04-09
WO 99/09147 PCT/US97/14212
-112-
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
G 46
GGG
CAT
TCT
TTT
GGA
GGA
GCA
ACA
GTT
TTT
CAA
GCC
CTA
AGT
GAA
Gly ly Ala
His Thr Val
Ser Phe Gln
Phe Ala Leu
Gly Ser Glu
G
1 s to is
GACCAG AGA TTC AGA GGG ATT CTT GATCCGTGG ATGTTT CCC 94
TGT GCC
AspGln Arg Phe Arg Gly Ile Leu AspProTrp MetPhe Pro
Cys Ala
20 25 30
GTGAGT GAG GAG CTG TCC AGA CCT CAGCCTCTC TTCTTT ATC 142
TAC GTT
ValSer Glu Glu Leu Ser Arg Pro GlnProLeu PhePhe Ile
Tyr Val
35 40 45
AACTCT GCC GAA TTC ACT CCA GAC ATTGCAAAA ATGAAA AAC 190
CAG AAG
AsnSer Ala Glu Phe Thr Pro Asp IleAlaLys MetLys Asn
Gln Lys
50 55 60
TTCTAC CAG CCT GAC GAA AGG ATG ATTACGATC AAGGGC TCA 238
AAG AAA
PheTyr Gln Pro Asp Glu Arg Met IleThrIle LysGly Ser
Lys Lys
65 70 75
GTGCAC CAG AAT TTT GAC GGG TTT GTAACTGGC AAAATA ATT 286
GCT ACT
ValHis Gln Asn Phe Asp Gly Phe ValThrGly LysIle Ile
Ala Thr
80 85 90 95
GGAAAC AAG CTG TCA AAA GGA ATA GACTCCAGA GTTGCC ATA 334
CTG GAC
GlyAsn Lys Leu 5er Lys Gly Ile AspSerArg ValAla Ile
Leu Asp
100 105 lI0
GACCTC ACC AAC AAG TCC TTG TTC TTACAAAAA CATTTA GGA 382
GCT GCT
AspLeu Thr Asn Lys Ser Leu Phe LeuGlnLys HisLeu Gly
Ala Ala
115 120 125
CTTCAT AAA GAC TTT CAG TGG TGT CTGGTGGAG GGAGAG AAC 430
GAT GAC
LeuHis Lys Asp Phe Gln Trp Cys LeuValGlu GlyGlu Asn
Asp Asp
130 135 140
GAGAAC CTC ATC CCG TCA CCC GAT GTAGTCACC CAGTCC CCG 478
GGG TTT
GluAsn Leu Ile Pro Ser Pro Asp ValValThr GlnSer Pro
Gly Phe
145 150 155
GCTCTG CAG AGT TCT GGA TCA AAC CAGAATTAG 517
CCC CAC
AlaLeu Gln Ser Ser Gly Ser Asn GlnAsn
Pro His
160 165 170
(2)INFORMATION ID N0:26:
FOR
SEQ
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:
580 base
pairs
(B) TYPE:
nucleic acid
(C) STRANDEDNESS:
single
(D) TOPOLOGY:linear
(ii)MOLECULE TYPE:cDNA
(ix)FEATURE:
(A) NAME/KEY:CDS
(B) LOCATION:1..580
(xi)SEQUENCE DESCRIPTION: D
SEQ I N0:26:
CAA GTA CTG ATG GCT GCT GCA AGC TTT GGC GAA CGT AAA ATC CCT AAG 48
Gln Val Leu Met Ala Ala Ala Ser Phe Gly Glu Arg Lys Ile Pro Lys
CA 02267994 1999-04-09
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-113-
1 5 10 15
GGAAAT GGGCCT TATTCCGTT TGT GACTTA ATGTTTGAT TAC 96
GGT ACA
GlyAsn GlyPro TyrSerVal Cys AspLeu MetPheAsp Tyr
Gly Thr
20 25 30
ACTAAA AAGGGC ACCTTCTTG TTA TATCCA TCCCAAGAT GAT 144
CGT TAT
ThrLys LysGly ThrPheLeu Leu TyrPro SerGlnAsp Asp
Arg Tyr
35 40 45
GATCGC CTTGAC ACCCTTTGG CCA AAGGAG TATTTTTGG GGT 192
ATC AAT
AspArg LeuAsp ThrLeuTrp Pro LysGlu TyrPheTrp Gly
Ile Asn
50 55 60
CTTAGC AAGTAT CTTGGAAAA TGG ATGGGC AACATTTTG AGT 240
CAC CTT
LeuSer LysTyr LeuGlyLys Trp MetGly AsnIleLeu Ser
His Leu
65 70 75 80
TTACTC TTTGGT TCAGTGACA CCT AACTGG AATTCCCCT CTG 288
ACT GCA
LeuLeu PheGly SerValThr Pro AsnTrp AenSerPro Leu
Thr Ala
85 90 95
AGGCCT GGTGAA AAATACCCA GTT TTTTCT CATGGTCTT GGA 336
CTT GTT
ArgPro GlyGlu LysTyrPro Val PheSer HisGlyLeu Gly
Leu Val
100 105 110
GCATTC AGGACA ATTTATTCT ATT ATTGAC CTGGCATCT CAT 384
GCT GGC
AlaPhe ArgThr IleTyrSer Ile IleAsp LeuAlaSer His
Ala Gly
115 120 125
GGGTTT ATAGTT GCTGCTGTA CAC GATAGA TCTGCATCT GCA 432
GAA AGA
GlyPhe IleVal AlaAlaVal His AspArg SerAlaSer Ala
Glu Arg
130 135 140
ACTTAC TATTTC AAGAACCAA GCT GAAATA GGGAAAAAG TCT 480
TCT GCA
ThrTyr TyrPhe LysAsnGln Ala GluIle GlyLysLys Ser
Ser Ala
145 150 155 160
TGGCTC TACCTT AGAACCCTG GAA GAGGAG ATACATATA CGA 528
AAA GAG
TrpLeu TyrLeu ArgThrLeu Glu GluGlu IleHisIle Arg
Lys Glu
165 170 175
AATAAG CAGGTA CGACAAAGA AAA TGTTCC CAAGCTCTC AGT 576
GCA GAA
AsnLys GlnVal ArgGlnArg Lys CysSer GlnAlaLeu Ser
Ala Glu
180 185 190
CTGA 580
Leu
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Gly Xaa Ser Xaa Gly
1 5
CA 02267994 1999-04-09
WO 99/09147 PCT/US97/14212
-114-
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
TATTCTAGAA TTATGATACA AGTATTAATG GCTGCTGCAA G 41
(2) INFORMATION FOR 5EQ ID N0:29:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
{B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
ATTGATATCC TAATTGTATT TCTCTATTCC TG 32
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1335 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
ATGGTACCCC CAAAGCTGCA CGTCCTGTTT TGTCTGTGTG GATGTCTCGC CGTCGTGTAC 60
CCCTTCGATT GGCAGTATAT CAACCCCGTG GCTCACATGA AGAGCAGCGC CTGGGTGAAT 120
AAGATCCAGG TGCTCATGGC CGCACCAAGC TTCGGTCAGA CCAAGATTCC TAGAGGCAAC 180
GGCCCCTACA GCGTGGGCTG CACCGATCTG ATGTTCGACC ATACCAACAA AGGAACTTTT 240
CTGAGACTGT ACTACCCCAG CCAGGACAAC GACAGACTGG ATACTCTGTG GATCCCAAAT 300
AAAGAATATT TTTGGGGTCT TAGCAAATTT CTTGGAACAC ACTGGCTTAT GGGCAACATT 360
TTGAGGTTAC TCTTTGGTTC AATGACAACT CCTGCAAACT GGAATTCCCC TCTGAGGCCT 420
GGTGAAAAAT ATCCACTTGT TGTTTTTTCT CATGGTCTTG GGGCATTCAG GACACTTTAT 480
TCTGCTATTG GCATTGACCT GGCATCTCAT GGGTTTATAG TTGCTGCTGT AGAACACAGA 540
GATAGATCTG CATCTGCAAC TTACTATTTC AAGGACCAAT CTGCTGCAGA AATAGGGGAC 600
AAGTCTTGGC TCTACCTTAG AACCCTGAAA CAAGAGGAGG AGACACATAT ACGAAATGAG 660
CAGGTACGGC AAAGAGCAAA AGAATGTTCC CAAGCTCTCA GTCTGATTCT TGACATTGAT 720
CATGGAAAGC CAGTGAAGAA TGCATTAGAT TTAAAGTTTG ATATGGAACA ACTGAAGGAC 780
CA 02267994 1999-04-09
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-115-
TCTATTGATA GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC 840
AACGGTTATT
CAGACTCTTA GTGAAGATCA GAGATTCAGA TGTGGTATTG CCCTGGATGC 900
ATGGATGTTT
CCACTGGGTG ATGAAGTATA TTCCAGAATT CCTCAGCCCC TCTTTTTTAT 960
CAACTCTGAA
TATTTCCAAT ATCCTGCTAA TATCATAAAA ATGAAAAAAT GCTACTCACC 1020
TGATAAAGAA
AGAAAGATGA TTACAATCAG GGGTTCAGTC CACCAGAATT TTGCTGACTT 1080
CACTTTTGCA
ACTGGCAAAA TAATTGGACA CATGCTCAAA TTAAAGGGAG ACATAGATTC 1140
AAATGTAGCT
ATTGATCTTA GCAACAAAGC TTCATTAGCA TTCTTACAAA AGCATTTAGG 1200
ACTTCATAAA
GATTTTGATC AGTGGGACTG CTTGATTGAA GGAGATGATG AGAATCTTAT 1260
TCCAGGGACC
AACATTAACA CAACCAATCA ACACATCATG TTACAGAACT CTTCAGGAAT 1320
AGAGAAATAC
AATTAGGATT CTAGA 1335
