Note: Descriptions are shown in the official language in which they were submitted.
W094/06829 ~ 1 4 2 2 0 3 PCT/US93/08511
BASOPHIL GRANULE PROTEINS
Technical Field
This invention i~ related to no~el proteins,
pharmaceutical compositions containing such, therapeutics
and human immunology. More specifically, it relates to
proteins found in the cytoplasmic granules of human
- basophils, to the gene~ which encode them, to the
antibodies which recognize them, and to the use of these
lS proteins, oligonucleotide~, and antibodies in the
diagnosis and treatment of disease.
Back~round Art
The~basophil,~along-with the mast cell,
20~ contains~cytoplasmic~ granules with an affinity for basic
s~ The basophil~is produced by the bone~marrow and
9~ circulates~in the blood. 9asophils are associated with
he~iminthic parasiti~c infections and allergic reactions
and~they~possess a high affinity receptor for IgE
25~antibod~les.~ Little is known howe~er about the proteins
which~comprise the~granùle,~ in part because, under normal
co~ditlo~s, basophils constitute le8s than l~ of
peripheral blood cells and it is therefore difficult to
obtain~an adequate ~amount of purified material for study.
While some researchers have proposed that
ba~ophils-areitheipre~ursors of mast cells, recent data
suggests that basophils represent terminally
differentiated leukocyte8, pos~ibly more closely related
to eo8inophils~(Galli, S~J. and Lichtenstein, ~.M., in
., :
,"~.
~ " ,~
WO94/06829 ~ O3 PCI/US93/01~5ll-~ ~
All~rqy:Princi~les and Practice, Middleton et al (Ed~.), -
3rd Ed, Vol. 1 (1988), pp 106-134).
Ba~ophil~ appear to participate in many
inflar~unatory, irr~nunological and pathological react~ions.
5 For a general review ~ee ~alli et al, Proq_Allergy (1984)
34:1. The most ~triking tissue infiltrates of ba~ophils
occur in cutaneou~ ba~ophil hypersensitivity reaction~
(Galli and A~kenase, in The Reticuloendothelial Sy~tem:A
Com~rehensi~re Treatise, Abramof~ et al (Eds.) pg 321,
10 Plenum Press 19~6). Recent studies suggest that
basophils are es~ential for expression of i~nunity to the
feeding of larval Amblyo~ma americanum ticks. Here,
basc)phils rn~y collaborate with eosinophils in the
expression of inmunity by acting to attract eosinophils
15 into tissues where the eosinophils subsequently release
toxic cationic proteins (Brown, S.J. et ai, J Immunol
(1982) 129:790). ~asophils are also elevated during
` helminthic infections, suggesting that they might
participate in host defense to these parasites (Ogilvie,
3.M. et al, I~nunol (19aO) 39:3~5; Lindor, h.J., Para~ite
I~nuno} (1983) 4:13; Juhlin, h. and Michaelsson, G.,
Lance~ (1977) 1:1233). E~ridence also exists that
basophils function in hypersensitivity reaction
(Schwartz, L.B. and Austen, K.F. in Immunological
~; 25 Disease~, Samter et al (Eds) Little E~rown & Co 4th Ed, pg
157 (1988); Mitchell, E.B. Clin Re~ ~ller~ sa3)
427), and in IgE mediated cutaneous late phase
reactions (Solley, G.O. et al, J Clin Invest (1976)
~1.:408; Charlesworth, E.N. et al, J Clin Invest (1989)
~.:1519).
Studies (of human basophil granule proteins ha~re
been limited by the difficulty of obtaining sufficient
- numbers of basophils because they constitute o~ly about
0.5~ of the total leukocyte population. Prior studies f
proteins isolated from the basophil3 of guinea pig8 i~
W094/06B29 ~14 2 ~ 0 3 PCT/US93tO8511
3-
reFeatedly immunized with sheep blood revealed a mixture
of neutral esterases-pro~ea~es and both trypYin and
chymotrypsin-like serine hydrolase~, Dvorak, H.F. et al,
J Immunol ~1974) ll3:l69; J Im~munQl (1977) 119:38J~
Studies of the glycosaminoglycans (GAG) of these proteins
showed a mixture of GAGs including chondroitin sulfate,
dermatin sulfate, and small amount~ of heparin sulfate
(Orenstein, N.S. et al, J Immunol (1978) l2l:586).
Several proteins have been localized to the
human ba30phil granule including the eosinophil major
basic protein (Ackerman, S.J. et al, J Ex~ Med (1983)
158:946) and the Charcot-Leyden crystal protein
(Ackerman, S.J. et al, J Ex~ Med (1982) l55:l597). In
addition, mast cell tryptase can be identified in human
ba~ophils at about 40 pg/cell, a level roughly 500-fold
lower than in human mast cellq (Castells, M.C. et al, J
Imm~aQl (1987) l38:2l84). In addition, bradykinin
~generating aceivity has been ascribed to basophils by
virtuè;of the release of this enzyme from peripheral
~whlte~blood cel1s by IgE dependent stimulation (Newball,
H.H. et al, J Clin Invest (1979) 64:466),
The present invention was facilitated by a
patient that presented with basophilic leukemia.
~ Leukocyte counts were over 105 cell/~l and contained 78%
r,~ 25 basophils. On two~occasions this patient underwent
~ cytophoresis for removal of leukocytes and a total of
-~ l.SxlOll basophi1s were obtained. Examination of the
~-~ granule proteins of these ba~ophils have revealed a
number of novel pro~eins with unique N-tenminal amino
acid sequences.
DisclQ~ure of the Invention
Several newly identified polypeptides (basophil
;~
granule proteins, nBGP9 n ) are de8cribed which constitute
somè of the proteins found in the cytopla~mic gra~ules of
' ~
:~
,:~
W094/06829 PCT/US93/08~
2 ~ 03 - 4-
human basophils. These polypeptides, the DNA which
encodes them and antibodies which recognize them, are
critical for diagnostics for, and treatments for,
pathologies involving inflammatory and IgE-mediated
responses, parasitic and.helminthic infections,
hypersensitivity reactions and certain types of
leukocytic leukemias.
One aspect of the invention is directed to
BGPs, which include proteins found in the granules of
basophils, and fragments, mutations and modifications of
these natural proteins which retain their respective BGP
biological characteristics. The polypeptides can be
recombinantly produced by cell~ in culture, produced by
chemical synthesis or isolated and purified from
basophils.
Other aspects of the invention'are an
: : expression system comprising DNA~ which encode these
. BGPs; host cells transfonmed with these expression
systems; and methods to produce BGPs~which utilize host
~ 20 cells transformed with said expression systems.
;~ ~ Sti}l other aspects include antibodies, both
monoclonal and polyclonal, which are specific for BGPs.
Additional aspects include methods of diagnosis
and treatment of diseases characterized by abnormal
.25 expression or re}ea~e of BGPs by basophils or other
cells, or by genetic abnormalities within genes encoding
BGPs. Further aspects include methods of treating
`~: disease by the~ administration of the BGPs, antibodies,
and DNA described herein.
Brief Descript~onlof!the Drawin~s
Figure 1 shows a HPLC chromatogram of human
~ basophil granule proteins obtained per Example l;
-~ 35
, ~
~, - ,,
,, ~
~, ,
W094/06X29 ~ 1 4 2 2 ~ 3 PCT/US93~08511
Figure 2 include~ HPLC chromatograms 2-1 to 2-8
of human basophil granule protein~ obtained per
Example 2; and
Figure 3 include~ HPLC chromatograms 3-l to 3-3
of human basophil granule proteins obtained per
Example 3.
Modes of Carryinq Out the In~ention
Unless defined otherwi~e, all technical and
scientific terms used herein have the same meaning as
- commonly understood by one of ordinary skill in the art
to which this invention belongs. Although any methods
and materials similar or equi~alent to those described
herein may be used in the practice or testing of the
present in~ention, the preferred methods and materials
are described. All publications mentioned herein are
incorporated by reference for the purpose;of disclosing
and~describing the~specific aspects of the invention to
whih those publications relate.
~ As used herein, "basophil granule protein" or
"BGPn;refers~to purified forms of any and/or all of the
novel proteins that may be purified from basophil
~; granules as disclosed here as well as fragments and
variantq thereof, which retain a useful biological
cha~acteristic of natural BGP~which BGP may be obtained
by extraction, synthetically produced or produced using
recombinant DNA methodology. It is recognized that
basophil granules contain certain proteins that have been
demonstrated from other 30urces. The in~ention allow~
the description of additional novel proteins that are
discoverable f~omlbasophil granules.
A fragment of a BGP is a polypeptide having a
primary amino acid se~uence identical to any part of a
naturally occurring BGP and retaining a u9eful biological
characteristic of the BGP.
,"~, :
~':
,~
WO 94/06829 ~ L ~ h 2 0 3 PCT/US93/08511~
-- 6 -
A ~ariant of a ~GP i9 any naturally occurring
(allelic variant), recom~inantly engineered or chemically
synthesized peptide or protein resulting from changes in
the primary amino acid ~equence or posttranslatio~nal
p.ocessing of the BGP described, but retaining useful
~iological characteristic~ of the BGP isolated from the
cytoplasmic granule of human basophils. The variant
forms of natural BGP include tho~e wherein one or more
instances of amino acid deletions, substitutions or
;0 insertions occur. The variant forms of natural BGP also
include those wherein aItered patterns of glycosylation
or lipidation occur. Variants also include ~GP made
~ynthetically wherein substitutions by amino acids which
are not encoded by the gene are made. Examples of such
amino acids include but are not limited to norleucine,
citrulline, ornithine, hydroxyproline, and cysteic acid.
- The biological l'characteri~tics" refer to the
structural and/or biochemical properties of a BGP and
include i~ts specific antigenicity or immunogenicity and
its abl}ity to mediate inflammatory and immunological
- ~ ~ respon9e9 n vL~Q. ~;
A "mutated" protein i8 a protein with an
altered primary structure (relative to the commonly
: : :
- - occurring protein) resulting from changes in the
nucleotide-sequence of the DNA which encodes it. These
mutations~include allelic variant~. A "modified" protein
differ~s from the commonly occurring protein a9 a result
~ ; of post-translational e~ent~ which change the
- glycosylation or lipidation pattern, or the primary,
3econdary, or tertiary structure of the protein. Changes
in ~the9primaryl'structure!of a protein can also result
from deletions, additions or sub~titutions. A "deletionl~
-~ - i9 defined~as a polypeptide in which one or more internal
~-- amino acid residues are absent. An ~addition" i9 defined
as a polypeptide which has one or more additional
. , ,
,
W094/068~9 ~1~ 2 2 0 3 PCT/US93/08~11
internal amino acid residue~ as compared to the wild
type. A '~sub~titution" result~ from the replacement of
one or more amino acid residue~ by other residues. A
protein "fragment" i~ a polypeptide consisting of a
primary amino acid sequen~e which i~ identical to a
portion of the primary sequence of the protein to which
the polypeptide is related.
Preferred altered forms of "natural" BGP
described above are those which have at least 80~
homology with natural BGP. At least 90~ homology is more
preferred, especially those including conservati~e
substitution~.
Homology is calculated by standard methods
which involve aligning two sequences to be compared 90
that maximum matching occurs; and then calculating the
percentage of matches. The altered forms of natural ~GP
include those wherein one or more of the residues of the
native 3eque~ce i8 deIeted, substituted for, or inserted
by a different amino acid or acids.
~ Preferred substitutions are those which are
; conservati~e, i.e., wherein a residue i8 replaced by
another of the same general type. As is well understood,
naturally occurring amino acids can be subclassified a~
~ acidic, basic, neutral and polar, or neutral and
- ~ 25 nonpolar. Furthermore, three of the encoded amino acids
are aromatic. It is generaIly preferred that peptides
~- ~ differing from the natural BGP contain substitutions
,
which are from the same group as that of the amino acid
~-1 replaced. Thus, in general, the basic amlno acids ~y9,
Arg, and Hi~ are interchangeable; the acidic amlno acids
aspartic and gl~tamic'are interchangeable; the neut~ai
;~ polar amino acids Ser, Thr, Cys, Gln, and Asn are
interchangeable; the nonpolar aliphatic acids Gly, Ala,
Val, Ile, and Leu are conservative with respect to each
other (but because of size, Gly and Ala are more closely
. ~ ~
, ~
`',~:
~ ~ .
W094/06829 214 2 2 0 3 PCT/US93/~8~
related and Val, Ile and Leu are more clo~ely related),
and the aromatic amino acids Phe, Trp, and Tyr are
interchangeable. While proline i8 a nonpolar neutral
amino acid, it presents difficulties becau~e of ~ts
effects on confonmation, and substitutions by or for
proline are not preferred~ except when the same or
similar conformational result~ can be obtained. Polar
amino acid~ which represent conservative charge include
Ser, Thr, Gln, Asn; and to a le~ser extent, Met. In
addition, althoùgh classified in different categories,
Ala, Gly, and Ser seem to be interchangeable, and Cys
additionally fits into this group, or may be classified
with the polar neutral amino acids. Some substitutions
by amino acid~ from different classes may also be useful
to produce altered 3GP.
It should further be noted that if the BGP i~
- made synthetically, substitutions by amino acids which
cannot be encoded by genes~may also be made. Alternative
~residues include, for example, the omega amino acids of
the fonmula N2N(CH2)nCOOH wherein n i9 2-6. These are
neutral, nonpolar amino acids, as are sarco3ine ~Sar, t-
butyl alanine (t-BuA), t-butyl glycine (t-BuG), N-methyl
Ile (N-MeIle), and norleucine (Nle)~ Phenyl glycine, for
example, can be substituted for Trp, Tyr, or Phe as
aromatic neutral amino acids; citrulline (Cit) and
- . methionine sulfoxide (MSO) are polar but neutral,
.-~, .
;;~; cyclohexyl alanine (Cha) is neutral and nonpolar, cysteic
~- acid ~(Cya) is~acid~;c, and ornithine (Orn) i9 basic. The
~ confonmation conferring properties of the pxoline
; 30 residues may be retained if one or more of these i9
substituted by hydroxyproline (Hyp).
It should be further noted that if the ~GP is
" ~ ..
produced recombinantly as an intracellular protein, an N-
terminal methionine residue may be retained in the
fini~hed product. Cleavage of the N-terminal methionine
' , '
~;
~ ; WO 94t06829 2 i 4 2 ~ ~ 3 PCT/US93/08~11
to liberate the native sequence may or may not be
complete.
The biological "characteristic~ll of a protein
refer to the structural or biochemical function of the
protein in the normal biological processes of the
organism in which the protein naturally occurs. Examples
of biological characteristics of a BGP include its
specific antigenicity or immunogenicity, its anti-
helminthic activity when this is associated with a
particular protein, and/or it~ ability to mediate
inflammatory and immunological respon~eR in vivo.
A host cell "expresse~" a gene or DN~ when the
gene or DNA i~ transcribed. A protein or polypeptide is
"expressed" when the protein or polypeptide has been
produced.
"Recombinant host cell n means a procaryotic or
eucaryotic~cell which contains an expression vector
comprising heterologous structural DNA and is capable of
expressing~the polypeptides encoded by the heterologou~
^ ~ - 20 DNA.
`
-~ A. Isolation of Baso~hil G~anules
ClinicaI hematology laboratories are monitored
to identify patients with chronic myelogenous leukemias
~with~greater ~han~2-3x104 leukocytes/~l of blood and lO-
20~ basophils. The basophils are purified by
, ~
,7',` centrifugation over a cushion of Ficoll-Hypaque from
~- ~ which 95% are recovered from the inter~ace with greater
than 90~ purity.
30 ~ , Purified basophi1s are lysed u8ing
modifications of the procedures de8cribed by Dvorak et
al, J Immunol (1977) 119:38 (5~ ), for purification of
~-~ guinea pig basophil granule proteins. Briefly, and in a
-;~ typical and illustrative procedure, purified basophils
~ 35 are washed with PBS and contaminating erythrocyte~ are
-;
,~,,
W094/06829 PCT/US93/08~11r--
` 21~2203 `
- 10 -
ly~ed by e~posure to Tris-ammonium chloride for 5
minutes. The cell suspension is centrifuged at about
400g, washed with Hank's ~SA-EDTA and su~pended in cold
0.25M sucrose containing DNAase and heparin using~a
volume of l5 ml for 8x108 basophils. The cell suspension
i~ next centrifuged at 400g for lO minutes and the
sediment i5 again suspended in 0.25M ~ucrose containing
2mg DNAase per 15ml cell suspen~ion. After 1-2 minutes,
heparin (20 IU) dissolved in 2ml 0.24M sucrose i5 added
and the preparation is subjected to a shearing force by
repeated passage (15 times) through a 20 gauge needle.
The suspen~ion is centri~uged a~ 400g to remo~e any
remaining intact cells, and the granules are then
purified by centrifugation through a cushion of 40~
sucrose Finally, the proteins of the isolated basophil
granules are solubilized by expo~ure to O.C5M borate
buffer at pH 9 in the presence of 5mM
diisopropylfluorophosphate, lx10-7M pepstatin A, and lOmM
~ .
~ EDTA~to inhibit protease acti~ity.
~ - .
~- 20
B. Protein Fractionation
The solubilized proteins of human basophilic
granu1es, in the same sol~ent described su~ra, are
separated by reverse phase HPLC using a Brownlee BU-300
25~ C4 column. ~The mobile phase i9 0.1~ trifluoroacetic acid
~TFA) containing 0-70~ acetonitrile. Fractions are
collect~ed across the acetonitrile gradient as shown in
Figure l, where absorbance at 214 nm is shown on the
ordinate. The relative homogeneity of each fraction i8
determined by SDS-PAGE electrophoresis.
~ ~ Although re~erse phase HPLC is an extremely
;~ powerful technique, not all human basophil gra~ule
- protein~ can be purified by thi technique alone. Thus,
~ ize exclusion chromatography can also be employed as a
-~ 35
~-
'''"~:'
~ :- W094/06829 2 ~ 4 2 2 ~ 3 PCT/US93/08511
preliminary fractionation (e.g. Bio Sel TSK 125 in 50mM
phosphate pH 6.9) prior to HP~C.
If additional purification of size exclusion
chromatography fraction~ i~ necessa~y prior to HPLC, ion
exchange chromatography can also be employed. A Mono-Q
column (Pharmacia) is used under conditions as would be
understood in the art whereby mo~t typical proteins would
bind to the column (e.g. 20 mM Tris, pH9.0). The
protein~ are then eluted in a gradient from 0 to 2 M
NaCl.
Purified proteins recovered from re~erse phase
HPLC are sequenced by subjecting up to 100 pmole~
(estimated ~rom chromatographic peak height and ~taining
intensity on acrylamide gels) to automated Edman
degradation.
For some granule proteins N-terminal sequencing
may not be adequate to support efforts to clone the
-~ cDNAs. ~For instance, some granule proteins~may be
blocked~or modified at the N-terminal or alternatively,
the~-N-~terminal sequences may not show fa~orable regions
for generation of oligonucleotide probes. Such proteins
are~digested with trypsin and the tryptic peptides are
purified and sequenced in order to generate additional
information. The protein i9 concentrated to a 5~1 volume
by vacuum centrifugation, and i9 then digested by
i~cu~atian (4 hours, 37 C) with 1/40 (w:w) TPC~-trypsin
in~1 ml: of 50mM ammonium bicarbonate, pH 8Ø Tryptic
fragments are puri~fied for sequencing by reverse phase
-~ -
HP~C using a ~rownlee RP 18 narrow bore column and an
Applied Biosystems 130A liquid chromatograph or other
suitable de~ice for HPLC - designed specifically for
~; ~ purification of pmole samples.
-~ Sequence data thus obtained are compared to
krown protein ~equences by computerized searches of the
Protein Identification Resource of the N~RF, and/or of
,
,, ~
',~
~,
W094/06829 PCT/US93/08511f
2142~03
i ` -12-
th~ Swi~9 protein databa~e, in order to determine their
novelty or relationship to other protein sequences.
C. Protein Sequencing.
Protein sequen~ing i5 performed by automated
Edman degradation utilizing a Applied ~i 09y8 t ems 477A
Protein Sequenator. Those skilled in the art will
recognize that sequence assignments based on a ~ingle
analysi~ are subject to a degree of uncertainty.
[Hunkapiller, M.W. et al. in Method~ of Protein
Microcharacterization ed. Shively, J.E. (19~6) The Humana
Press, Clifton, N.J., pp. 223-247; Hunkapiller M.W.,
Applied Biosystems Protein Sequence~ Users Bulletin #14
(1985)]. Particularly, when assignments at a given cycle
lS are based on analysis of low quantities of the
deri~atized amino acid~, a certain le~el of error i~
expected. These error~ are most frequently
misidentification~ of amino acids which are more
difficult to identify, namely Serj Thr, His, Arg and Trp.
~ Po~itive identification of Cys is a special
problem. Without prior modification of the pxotein~, Cys
is not detectable but its presence may be inferred by the
a~aence of an appropriate signal in that cycle.
Lag i~ a well known phenomenon in sequencing,
where signal deri~ing from cycle n i9 also present in
~ cycle n~+l. Lag present3 a special problem when Cys is
- ~ present at cycle n+1 since the amino acid present at
~ cycle n may mistakenly be as3igned at cycle n+1 due to
-~ the lag phenomenon.
Becau3e of such characterigtic uncertainties,
data from an analysis may support more than one
intexpretation. Nevertheless, examination of the
~- sequence data allows one skilled in the art to judge
whether two qequences derive from the same protein, e~en
when ~ome discrepancies exist.
~: :
,~ ,
,
W094/06X29 PCT/US93/0~
2 0 3
In accordance with the considerations, the
sequences determined are submitted as a description
rather than a definition of the proteins which have been
isolated.
D. Screenin~ of cDNA Libraries and the Molecular Cloninq
of Unique Basophil Granule Protein Encodinq PNA
Basophils mature in culture~ of human umbilical
cord blood cell~. Thus these cultures can be used to
prepare a cDNA library which is then screened for
particular DNA sequences that encode proteins unique to
human basophil granules (BGP) (Saito, H. et al, Proc Nat
Acad Sci (1988) 85:2288).
Other candidate cDNA libraries which may
express BGP~ include unstimulated HL-60 cells, or Hh-60
cells driven to basophilic differentiation by culturing
in a;protein free medium (Muroi, K. et a}, heukemia Res
(1989) l3:l~57) or in the pre3ence of sodium butyrate
(~ut~t-~Taylor, S.R. et al., ~l~Ç~ (1988) 71:209).
20 ~ Although~basophils and mast cel}s appear to be distinct
n~eheir lineageq, granules of both cells contain mast
cell~tryptase (Castells, M.C. et al, ~_Immunol (1987)
13~:21843 and these cells may therefore share other
common pxoteins. Therefore rDNA libraries made from
human mast cel}s (~e.g. HMC-l) are another source of BGP
encoding~sequences.~The preparation of these cDNA
libraries i9 described in detail in Maniatis, T. et al,
Molecular~ClQ~lng, (1982) CSHL Press, and i8 well known
to those skilled in the art. A convenient approach is
~ 30~ the insertion of cDNA fragments into a lambda phage
: vector e.g. lambda gtiO or lambda gtll as`described by
M~niatiq, .su~ra.
i- Methods of screening cDNA libraries are also
- well known to those skilled in the art. The amino acid
sequence of the BGPs is analyzed for example utilizing
i, , ,:: -
, ,
'''
-
~ '
W094/06829 ~ PCT/US93/08511;
. -14-
programs from DNAstar (Madi30n WI) in order to identify
optimal regions for construction of oligonucleotide
probe~. Redundant oligonucleotide probes are Qynthe~ized
with a DNA synthesizer (380A: Applied Bio~ystem~ Inc.
Fo~ter City CA) by the phosphoramidite method.
Oligonucleotides are purified on Sephadex G-50 columns
and ~tored at -20C. The redundant probe~ are 5'-labeled
with T- [32P)ATP (E.I. du Pont de Nemours & Co., Inc.,
Boston, MA) using T4 polynucleotide kinaqe. ~ibraries
are ~creened u~ing up to 1o6 individual plaques per
library, with the redundant oligonucleotide probes.
Duplicate nylon membranes containing phage are prepared
and prehybridized in 5x SSPE ~0.9M Nail, 50mM NH2P04, 5mM
- EDTA, pH7.4), 0.2~ SDS, and 0.005% denatured salmon ~perm
DNA for 2 hours at 50C with 8 filters per 50 ml
prehybridization fluid per bag. Membranes are hybridized
with approximately 1 ng of labeled probe per ml, in fresh
hybridization fluid, overnight at the appropriate
temperature for the redundant probe mixture. Membranes
are then wa~hed at room temperature for 45 minutes in 1
liter of 5x SSPE per 40 filters, followed by a 1 minute
wash in fre~h buffer at 50C, slightly air-dried, a~d
exposed to Rodak XAR-5 film, with intensifying screens,
for 72 hours at -70C.
.~ .
After analysis, filters are stripped of
hy~ridized label by incubation in 5x SSPB at 70C for 10
minutes and sub~equently hybridized with a second probe
-~ under the same conditions. This procedure is repeated
, .~
for each probe. Recombinant clones which hybridize ~ith
probes will be ~elected from the library and plaque
pur~fied.
Recombinant phage DNA i9 then purified and
digested with an appropriate reqtriction endonuclea~e to
-~ yield the amplified cDNA insert. Inserts are then
~ 35 ligated into M13mp series phage and sequenced u~ing the
,
~ W094/~6829 ~1422a3 PCT/~S93/0851i
di~eoxy method described by Sanger ~igginl M.D. et al,
Proc Nat Acad Scl ~l983) 80:3963). Depending on the ~ize
of the cDNA, it may be necessary to restrict the clone,
and subclone the fragments into ~13. If the cDNA clones
are not complete, a repeat ~creen of the library with the
partial cDNA would be required. The complete ~equence of
the ~GP cDNA i then compared again~t known sequences in
the GenBank databa~e. DNAstar is used for nucleotide and
polypeptide analy~es and sequence comparison~.
Selected cDNA insert~ which encode a BGP can
then be incorporated into an expression ~ystem. The cDNA
is operably linked to heterologous control sequences to
form an expression ~ector. The control sequences are
chosen to be functionally compatible with the recombinant
host cell into which the expressio~ vector is introduced.
These procedures are known to tho9e skilled in the art
and described in Maniatis, supra.
Expression can be in procaryotic or eucaryotic
systems. ~Procaryotes most frequently are represented by
varioùs~strains of E~coli. Howe~er, other microbial
strains~ay also be used, ~uch a9 bacilli (e.g. Bacillus
subtilis), various species of Pseudomonas, or other
bacterial strains. In such procaryotic systems, pla3mid
" ~
~ectors which contain replication sites and control
25~ sequences~derived from a species compatible with the host
are~;us~ed.~ For example, E coli is typically transformed
using deri~atives of pBR322, a pla~mid derived from an E.
li species by Bolivar et al., Gene (l977~ 2:95.
Commonly used procaryotic control sequences, which are
defined herein to include operons with promoters for
transcriptional i1nitiatlon, optionally w~th an operator,
:~:
along with ribosome binding site sequences, include such
commonly used promoter9 as the beta-lactamase
(penicillina9e) promoter, lacto9e ~lac) promoter 9y9tems
(Chang et al., Nature (1977) l98:1056), the tryptophan
,,".~
,, :
:,
:
WO 94/06829 2 1 ~ 2 2 0 3 PCT/US93/0851~
,
-16-
(trp) promoter system (Goeddel et al., Nucleic Acids Res
(1980) 8:4057), the lambda-deri~red PL promoter and N-gene
ribosom~ binding site (Shimatake et al., Nature (1981)
292:128). Any available promoter sy~tem compatiple with
S procaryotes can be u~ed.
The expression systems useful in eucar}rotic
hosts comprise promoters derived from appropriate ~`
eucaryotic genes. A class of promoters useful in yea~t,
for example, includes promoters for synthesis of
10 glycolytic enzyme~, including tho~e for 3-
phosphoglycerate kinase (Hitzeman et al., J Biol Chem
(1980) 255:207). Other promoters include those from the
enolase gene (Holland, M.J., et al. J Biol Chçm (1981)
~56:1385) or the ~eu2 gene obtained from YEpl3 (Broacht,
J.~ et al., ~aÇ (1978) ~:121).
Suitable ma~lian promoters include
metallothionein, the early and late promoter~ from SV40
-~ (Fiers et al.~, Nature (1978) 273:113), or other Yiral
promote~rs~such a those derived from polyoma, adeno~rirus
;20 II, bo~ine papilloma virus or retroviruses. Suitable
- ~;
`In~ Yiral and~mammalian enhancers may also be used. In the
event~plant cells are used as an expression system, the
nopaline synthesis promoter is appropriate (Depicker, A.,
: ~ ~ et al~., J Mol A~?l Gen (1982) 1:561).
~ ~ The expreqsion system is constr~cted from the
foresoing control elements~ which are operably linked to
the BGP sequences by employing standard ligation and
restriction techniques which are well understood in the
~- ~ art. Isolated pla mid~, DNA sequences, or synthe~ized
~; 30 oligonucleotides are cleaved, tailored, and relegated in
the fo`rn~s desi~ed.
;~
~:~
'
; w094/06829 ~ 2 2 0 ~ PCT/US93/08511
-17-
E. Productlon_of AntisenSe~ and Trlple Helix
01iqonucleo~cldes
Antisense oligonucleo~ides can be designed and
produced ba~ed upon the nucleotide sequence of cDN~A
encoding a BGP. The anti$ense oligonucleotide can be
designed and used to regulate the translation within the
cell of the specific mRNA to which it i9 complementary
according to methods known in the art (Green et al., Ann
Rev Biochem (1986) 55:569; Rossi et al., Pharmacol Ther
(1991) 50:245).
The antisense reagent is made complementary to
some portion of the mRNA encoding the BGP, preferably
including a portion of the mRNA at or near the
translation initiation site in the 5' region. In one
approach, the antisense reagent may be ~NA or preferably
a modified RNA wherein the sugar phosphatq backbone has
been~modified to increase resistance to RNase acti~ity or
otherwise impro~e pharmacokinetic or pharmacodynamic
properties. In another approach the antisense
oligonucleotide may be a DNA, which has been ligated to a
promoter in an antisense orientation, such that
transcription of the DNA produces a mRNA complementary to
the mRNA encoding a BGP. The antisense DNA may be
incorporated into an expression ~ector for introduction
into the body.
-~ Triple helix oligonucleotides can also be
de~igned and prod~ced based upon the nucleotide sequence
of cDNA encoding a BGP. The triple helix oligonucleotide
can be used to-regulate the transcription within the cell
30 of the specific DNA to which it is taryeted and -~
particularly to'inhibit expression of a specific gene in
individuals having diseases associated with expres~ion of
the gene. ~omopurine and homopyrimidine sequenceq which
are appropriate for triple helix formation can be
designed by methods k~nown in the art (Griffen et al.
. -~
W094/06~29 ~1 A 2 2 ~ 3 PCTtUS93/08
-18-
ScIe ce (19a9~ 245:967). The effectivenes~ of triple
helix oligonucleotide can be improved by synthe~izing the
reagents using the unnatural ~-anomeric nucleotides to
improve their nuclease resistance properties or b~
derivitizing the oligonucleotides with an intercalating
agent such as ethidium bromide to 3tabilize the triple
helix, once it has been formed.
Anti~ense molecules are introduced into the
body in one method by injecting the oligonucleotide
either alone, encapsulated in a liposome or incorporated
into a viral particle.
~ .
F. Analysis of the Genomic Seq~nce of B~P DNA
~GP-encoding genes are obtained from the
genomic library of human fetal liver DNA in Charon 4A
phage (ATCC 37333). The library contains 106 independent
recombinants with an insert size of 15-20 kb and it is
~ screened wieh cDNA essentially as previously described.
-~ Phage are seguentially adsorbed onto duplicate 8x8 cm
nylon membrane filters. Filters are prehybridized in 5x
~-- SSPE, 50~ formamide, 5x Denhardt's solution, 0.5% SDS and 0.005~ denatured salmon qperm DN~ for 2 hours at 42C
with 8 filters per 50 ml of prehybridization fluid.
Fi1ters are hybridized with approximately 1.0 ng of
25~ lab~eled ba ophil protein cDNA per ml of fre~h
- prehybridization fluid, containing 10~ dextran sulphate
and 2x Denhardt's solution, overnight at 42C. BGP cDN~
~- is labeléd with ~32p dCTP and purified by Sephadex G-50
chromatography. Filters are then washed twice at room
temperature for 15 minutes in 1 liter 2x SSPE and 0.2~
SDS per!40 filt~rs', followed by two 15 minute 50C washes
in O.lx SSPE and 0.2~ SDS, ~lightly air-dried,and expoQed
to Kodak XAR-5 film, with intensifying screens, for 48
hour~ at -70C.
~ 35
'~:
~'
:'
;~- W094/06829 ~1 ~ 2 2 0 3 PCT/US93/08511
- 19 -
Po~itive clone~ are selected from the library
and plaque purified. Various probes derived from the
cDNA are utilized to determine whether or not a complete
copy of the gene is contained within the genomic clone.
Recombinant phage DNA is next extracted, purified, and
subjected to restriction digestion - all processes which
are well known to those skilled in the art. Soùthern
blots of the restriction fragments are hybridized with
BGP cDNA to identify fragments containin~ the BGP gene.
10 These f-agments are then isolated and sequenced. From "`
this inf-ormation a restriction map is con~tructed and the
inerons of the gene are identified.
. .
G. Preparaeion of Antibodies to BGP3 `.
Two approaches are utilized to raise antibodies
to BGP and both approaches can be used to generate either
poly~clonal~or monoclonal antibodies. In ~ne approach, as
`dena~ured~pro~ein from~ehe~reverse~phase HPLC separation
is-~0=btàlned in~quantities up~to 75 ~g, this~denatured
20~ proeein~can~-be~used to immunlze mice;using standard
protocols;~abQut 25 ~g is adequaee for immu~ization. For
c~reening~hybridomas, the denatured protein, which is
soluble in 0.1% TFA~and acetonitrile, can'be
radioiodinated;and used to s~creen murine ~-cell
25~ hybrid~mas'~for those which produce antibody. This
proc~dure~r~equire9 only small quantitie~ of protein such
ehat 20~g would be~sufficient for labeling and screening
of~several~thou:~and~clones.
~`-9~ In the sec~ond approach, the amino acid sequence
of BGPs as deduced from the gene is analyzed to determine
' regions of high' i~mu~o'genicity. The corresponding I '
polypeptides are synthesized and are used in suitable
immunizat~ion~prQtocols to rai9e antibodie9. Analy9is to
se1ect appropriate~epitopes is described by, for example,
Au-qubel,~ F.~M. et al, in ~rent Protocols in Molecular
, ~ ~
W094/06X29 ~ 1 4 2 ~ 0 3 PCT/US93/08511
-20-
~i~loqy, John Wiley ~ Sons, Vol. 2, Sec. IV, ppll.14.l,
1989). The optimal selections are usually the C
terminus, the N terminus and internal regions of the
polypeptide, which are likely to be exposed to the
external environment when the protein is in its natural
conformation (this determination is based on the
hydrophilicity of the sites). Typically, selected
peptides, about 15 residues in length, are synthesized
usi$g an Applied ~iosystems Peptide Synthesizer Model
431A using fmoc-chemistry and coupled to keyhole limpet
hemocyanin (KLH; Sigma) by reaction with m-
maleimidobenzoyl-N-hydroxysuccinimide e~ter (MBS) (See
Ausubel et al, supra at pp ll.15.l). A cysteine is
introduced at the N-terminus of the peptide to permit
coupling to KLH. Rabbits are lmmunized with the peptide-
KLH complex in complete Freund' 9 adjuvant and the
resulting antisera tested for antipeptide acti~ity, for
example, by blnding the peptide tQ plastic, blocking with
-~ 0.1%~ SA, reacting with antisera, washing and reacting
w1th radioiodinated affinity purified specific goat
antirabbit IgG.
; Hybridomas may be also be prepared and screened
- using standard techniques. Hybrids are scrèened using
radioiodinated 9GP to identify those producing monoclonal
antibody. In a typical protocol, prongs of plates (FAST,
9ecton-Dickinson, Palo Alto, CA), are coated with
- affinity purified specific rabbit-antimouse (or suitable
anti species Ig) antibodies at lO ~g/ml. The coated
prongs are blocked with O.l~ ~SA, washed and exposed to
supernatants from hybridoma~. After incubation the
prongslare exposed td radiolabeled protein, 1 ng/ml.
Clone~ producing antibodies will bind a quantity of
radioactivity which is detectable abo~e background. Such
clones are expanded and 3ubjected to 2 cycles of cloning
at 0.3 cell/well. Cloned hybridomas are injected into
~":
"- ~
, ~,
~ W094/06829 ~1 ~ 2 2 0 3 Pcr/usg3/o85l,
-21-
pristine treated mice ~o produce ascite~q, and monoclonal
antibody i9 purified from the ascitic fluid by affinity
chromatography on protein A.
H. Use of Anti-BGPs in Diagnosls
Anti-~GPs are useful for the diagnoqis of
prepathologic conditionq and aq well as chronic and acute
di~ea~es which are characterized by abnormalities in the
amount or distribution of BGPs. For example, the ~GPs
disclosed herein can be used to generate polyclonal and
preferably monoclonal antibodies. The3e antibodies can
be used to detect the BGPs in a sample such as a blood
sample and determine the presence of and level of the
BGPs in the ~ample. The type and/or amount of BGPs
detected can be compared to a known standard -- an
average for healthy individuals. Readings out~ide of the
standard would be useful information in diagnosing
abnormalities such as basophilic leukemia.
A variety of protocols for the conduct of
immunoassays, using either polyclonal or monoclonal
antibodies specific for BGPs, known in the art and
include competiti~e binding assays and immNnoradiometric
assays. A two-site monoclonal-based immunoassay
utilizing monoclonal antibodies reactive to two
noninterfering epitopes on a specific BGP is preferred,
but a competitive binding a~say can also be employed.
The~e assays are deYcribed in the following publications,
--~ hereby incorporated by reference: Maddox, D.E. et al, J
Ex~ Med (1983) 1$8:1211; Gleich, G.J. et al, J Lab Clin
~ (1971) 77:690; Gleich, G.J. et al, J ~llergy Clin
Immun (~974) ~ 15~; Gleich, G.~. et al, J Allergy Clin
Immun (1977) 60:188; Dunnette, S.h. et al, J ImmunQl
- (1977) 11~:1727; Wa~som, D.~. et al, J Clin Invest (19
67:651.
:
W094/06829 ~ 2 ~ O ~ PCT/US93/08511
-22-
Immunoassay procedures are utilized to mea~ure
several major parameters in immunopathologic and
prepathologic condition~ which are characterized by ~GP
abnormalitie~ - e.g. the increased or decrea~ed
production of BGPs by basophils, the aberrant production
o~ BGPs by cells other than basophils, and the change in
intracellular or extracellular distribution o~ BGPs
during the genesis of disea~e. In order to determine the
normal distribution of ~GP in leukocytes, peripheral
blood mononuclear cells from normal individual~ are
prepared and analy~ed a~ described by Ackerman et al for
the localization of eosinophil granules ~3P and Charcot-
Leyden crystal protein to human basophils. (J Exp Med
(19~3) 158:946; J Exp Med (1982) 155:1597). To determine
the ~uantity of ~GPs in basophils, freeze-thawed
detergent extracts of cell ~uspensions enriched for
basophils are analyzed by immunoassay, and the slope of
the b~nding curves are then compared to comparable
binding curves generated by the purified protein.
2~
I. Pharmaceutical Compositions
BGPs are also useful to remedy deficiencies in
these proteins or to amplify immune-responses which are
stimulated by these proteins. BGPs can be administered
to subjects exhibiting such conditions using standard
formulations such as those set forth in Reminaton's
~ Pharmaceutical Sciences, Mac~ Publishing Co., Easton PA,
-~ Latest Ed.
Thus the present in~ention also provides
compositions containing an effective amount of compounds
of the;present invent;lon,`including the nontoxic add!ition
f salts, amides and esters thereof, which may alone
ser~e to pro~ide the abo~e-recited therapeutic benefits.
.
Such compositions can also be pro~ided together with
:
.
W094/06829 ~ 4 2 2 0 3 PCT/US93/~8511
-23-
phy~iologically tolerable liquid, gel o~ solid diluents, `~
adjuvant~ and excipients.
These compounds and composition~ can be
administered tO mammal5 for veterinary use, such a5 with
domestic animals, and cllnical use in humanc in a manner
similar to other therapeutic agents. In general, the
dosage required for therapeutic efficacy will range from
about 0.01 to 10,000 mcg/kg, more u~ually 0.1 to 1000
mcg/kg of the host body weight. Alternatively, dosages
within these ranges can be administered by constant
infusion o~er an extended period of time, usually
exceeding 24 hours, until the desired therapeutic
benefits have been obtained.
Typically, such compo~itions are prepared as
injectibles, either as liquid solutions or suspensions;
solid form suitable for solution in, or suspended in,
liquid prior to injection may also be prepared. The
prepara~ion may al80 be emulsified. The active
ingredient is often mixed with diluents or excipients
~-~ 20 which are physiologically tolerable and compatible with
the acti~e ingredient. Suitable diluents or excipients
-~ are/ for example, water, saline, dextrose, glycerol, or
the like, and combinations thereof~ In addition, if
desired, the compositions may contain minor amounts of
auxiliary 3ubs~ances such as wetting or emulsifying
agents, stabilizing or pH-buffering agents, and the like.
The compositions are conventionally
-~ administered parenterally, by injec~ion, for example,
either 3ubcutaneously or intra~enously. Additional
formulations which are suitable for other modes of
-~ - administrationiinclude 3uppositories, intranasal
aeroso1s, and in some cases, oral formulations. For
suppositories, traditional binders and excipients may
include, for example, polyalkylene glycols or
;~ 35 triglycerides; such suppositories may be formed from
:,
W094/068~9 PCT/US93/085l1;~;
-24-
mi~ures containing the active ingredient in the range of
0.5~ to 10~, preferably 1%-2~. Oral formulations include
such normally employed excipients a~, for example,
phar~ceutical grades of mannitol, lactose, starch~,
S magnesium stearate, sodiu~ saccharin, cellulose,
magne~ium carbonate, and the like. These compo~itions
take the fonm of solutions, suspensions, tablets, pills,
capsule~, su~tained-release formulations, or powders, and
contain 10~-95% of active ingredient, preferably 25~-70~.
The peptide compound~ may be formulated into
the compositions as neutral or salt forms.
Pharmaceutically acceptable nontoxic salt~ include the
acid addition salts (formed with the free amino groups)
and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or ~uch
organic acids as acetic, oxalic, tartaric, mandelic, and
the like. Salts formed with the free carboxyl groups may
be derived from inorganic bases such as, for example,
sodium, potassium, ammonium, calcium, or ferric
20~ hydroxides, and such organic bases a~ isopropylamine,
~; ~ trimethylamine, 2-ethylamino ethanol, histidine,
procaine, and the like.
Based on the above, it can be seen that the
core of the present in~ention relates to the novel
; 25 proteins which are present in the cytoplasmic granules of
human~basophils, including a subset of those basophil
granule proteins which are disclosed and described
herein. In addition, the proteins can be applied to
~-~; various procedures for the diagnosis of disease3 related
to the abnormal (e.g., o~er or under) expres~ion of the
protein. The p~ot!eins can be formulated into
pharmaceutical compositions of ~arious types and used for
various types of treatments. In addition, the genetic
material which encodes the proteins is useful in
producing the proteins. The genetic material can be
:
~ .
~ '
, , .
;; W094/06829 ~1 4 2 2 0 3 PCT/US93/08511
-25-
pl~ced in plasmids and the plasmid~ used to transfect
ho~ts each of which are part of the present in~ention.
The proteins can be used to produce monoclonal and
polyclonal antibodies and these antibodies can be~used in
detection ~ssays of the t~pe described above.
EXAMPL~S
The following examples are put forth so as to
provide those of ordinary skill in the art with a
complete disclosure and description of how~to make the
proteins of the invention~and are not inte~ded to limit
the scope of what the inventors regard as their
invention Efforts ha~e been made to ensure accuracy
with respect to numbers used (e.g., amount~, temperature,
etc.) but some experimental errors and deviation should
-~ be accounted for. Unless indicated~otherwi~e, parts are
parts~by~weight, molecule weight is weight average of
molec~ular~weight, temperature is in degrees Centigrade,
and~pressure~is~at or near atmo~pheric.
The following procedures are used to purify
h ~ n ba~ophil3, isolate the basophil granules, extract
basophil granule proteins, fractionate and purify those
proteins and determine~their N-terminal amino acid
; 25 ~ sequence.
Pre~ara~ion~l~f Purifled Huma~ ~asophil Granules
A patient with a form of chronic myelogenous
leukemia (basophil~leukemia with leukocyte counts o~er
105 cell~/~l and 78% basophils) underwent two treatment3
of cyt~phorèsis f~om which 1.5xlO11 baqophils were
~:
~ - reco~ered. The basophils were purified by centrifugation
-~ o~er a cu~hion of Ficoll-Hypaque from which 95% were
;, -
recovered from the interface with greater than 90
` 35 purity.
", ~ .
,~:
W094/068~9 ~ ~ g~ PCT/US93/~8511
-26-
Subsequently, these basophils were lysed using
modifications of the procedures described by
Dvorak et al. Purified basophil~ were washed with PBS
and erythrocytes were lysed by exposure to Tris-ammonium
5 chloride for 5 minutes. The cell su~penqion wa~ ;
centrifuged at 400 x g, washed with Hank'~ ~SS-EDTA and
suspended in cold 0.25M sucrose containing DNAase and
heparin u~ing a ~olume of 15 ml for 8 x 108 basophil~.
The cell suspension was centrifuged at 400 x g for 10
minute~ and the sediment was again ~uspended in 0.25M
sucrose containing DNAase (2mg/15ml solution). After 1-2
minutes, heparin (20 IU) dissolved in 2ml 0.24M sucrose
- was added and the preparation was subjected to a shearing
force by repeated passage ~15 times) through a 20 gauge
needle. The suspension was centrifuged at 400 x g to
remove intact cells and granules were purified by
centrifugation through a cushion of 40~ sucrose. The
granules were resuspended and stored in 30 aliquots at
70C.
, :
Exam~le 1
~-~ Extraction of Baso~hil ~xanule Proteins.
Four consecutive freeze/thaw cycle~ were
carried out on a single aliquot of purified ba30phil
granules in;order to lyse the granule~. Proteins were
extracted at 4C by addition of 1 ml of SOmM ~odium
borate pH 9.0 to the lysed granules. The extraction mix
was ~ortexed frequently over a 1 hour period to aid
extraction. The insoluble material was removed by
centrifugation for 15 minutes at 20000 x g, and the
soluble extract was takén for chromatographic ! '`
~ fractionation.
,~
. ~
" .
~ 35
-
W O 94/06829 ~ ~ /US93/08511
-27-
Re~rse Phase HPLC.
The borate extract was acidified by adjustment
to 0.05-0.1~ trifluoroacetic acid and wa~ fractionated by
reverse phase chromatography on a ~rownlee ~U300 column
(2.1mm x 30mm) eluted with a 0-70~ gradient of
acetonitrile in 0.1~ trifluoroacetic acid. Fractions
judged to be appropriate for analysis were concentrated
in a SpeedVac concentrator and sequenced by automated
Edman degradation on the ABI 477 Protein Sequenator.
The results are shown in Figure 1 and de~cribed
as follows:
Analyses of ba~ophil granule extracts by HPLC
chromatogram. Basophil granules were solubilized in
0.05M borate buffer, pH 9, and separated-by re~er~e phase
HPLC using a Brownlee BU-300 C4 column. The mobile phase
was 0.1~ trifluororacetic acid (TFA) conta-ning 0-70~
` ~ acetonitrile. The ~ B (0.1~ TFA and 70~ acetonitrile) is
indlcated on the graph. The fractions are indicated on
the absclssa. The ordinate shows absorbance at 214 nm.
TA~LE 1. N-terminal amino acid ~equences of proteins
i ~ purified from basophil granule extract~ X" represents
,- ~
an unidentified residue.
Each of the sequences li3ted in Examples 1-3 i~
25~ given a number designation at the left referred to as the
"Praction No. n which is provided only as a matter of
re~erence convenience.
,~ ~
~,
W094/06829 PCT/US93/0~511~;`
-28-
Fraction
No.
9 - Asp-Ile-Gly-Pro-Asp-Gln-His-Thr-Ser-Arg-Pro-Trp-Gly
-Gln-Thr
11 - Asp-Val-Lys-Lys-Asp-Met-Glu-Val-Ser- Cy9 - Pro-Asp-Gly
-Tyr-Thr
12 - Val-Met-X-Pro-Asp-Ala-Arg-Ser-X-Arg-Pro-Asp-Gly-X
-Thr
15 - Ala-Ile-Tyr-X-Arg-Ile-Pro-X-X-Ile-Ala-Gly-Glu-Phe
-Arg-Tyr-Gly-Thr-Val-Tyr-Tyr-Gln-Gly-Ser-Leu
20 - Asp-Ile-Pro-Glu-Val-X-Val-X-Leu-Ala-Ala-A~p-Glu-Ser
-Leu-Ala-Pro-Lys
,
30 - Tyr-Pro-Gln-Leu-Al -Ile-Asn
43~- Ser-Ile-Gly-Phe-Val-Glu-Val-X-~eu-Val-Leu
, ., ~
Exam~le 2
Extraction of Baso~hil Granule Proteins.
A~cocktail of protease inhibitors was prepared
25~ containing~;diisopropylfluorophosphate,
e~th~ylenediaminetetraacetic acid and pepstatin A, and
0.~50 ml~of~the mix was added to each of 2 aliquots of
; frozen~granules. F~our consecutive freezejthaw cycles
were`carried out in order to lyse the ~ranules. Protein~
~ 30 were extracted at 4C by addition of 3 ml of 50mM sodium
-~ borate pH 9.0 to the pooled lysed granul~s. The
~ extraction mix was vortexed occa~ionally over 1 hour to
q~ aid extraction. The insoluble material was removed by
c-n~rlfugaelon for lS minute~ at 20000 x g, and the
, ~
:.
-: .
` W094/06829 PCT/~S93/08511 -.
-29-
soP~ble extract was taken for chromatographic
fractionation.
Size Exclusion HPLC.
~orate extract (4 ml) was concentrated to 0.6
ml and the pH was adjusted to neutral pH wi~h 0.5M
NaH2PO4. The neutralized extract was fractionated by
size exclusion chromatography on a Bio-Sil TS~ 250 HPLC -~
column (7.5mm x 600mm). The A280 was monitored and the
fractions containing protein~ were combined into 7
separate pools based on silver ~taining of Laemmli SDS
polyacrylamide gels.
Reverse Pha~e HPLC.
Pools of fractions from size exclusion HPLC
were acidified by adjustment to 0.05-O.l~ trifluoroacetic
. acid and each pool was separately fractionated further by
reverse~phase chromatography on a Brownlèe BU300 column
(2.1mm x 30mm) eluted with a 0-70~ gradient of
~ 20 acetonitr1le in O.l~ tri~luoroacetic acid. Fractions
:~ :judged to be appropriate for analysis were concentrated
in a SpeedVAc and sequenced by automated Edman
degradation on the A~I 477 Protein Sequenator.
The following se~uences were obtained:
25 l.18 Ala-Cys-Tyr-Cys-Arg-Ile-Pro-Ala- Cy9 -Ile-Ala-
~ Gly-G1u-Arg-Arg-Tyr-Thr-Cys-Ile
: 2.17b Ala-Pro-Ala-Leu-Thr-Ile-Ser-Asn-Gln
4.10a Asp-Ile-G1y-Pro-Asp-Gln-His-Thr-Ser-Xl-Pro-X2-
Gly-Gln~Thr-Arg-X-Pro-Gln-Leu-Thr-Gly-Gly-Glu-
Ala-X-Val
. ~ " ,
where -l is Ser or Arg and X2 is Val or Trp
4.10b Arg-Asp-Val-Pro-Pro-Asp-X-Val-Val-Ser-X-Pro-
Ser-Ser-Asp-Thr
4.12 Gly-Asp-Val-Lys-X-Asp-Met-Glu-Val-Ser-X-Pro-
Asp-Gly-Tyr-Thr-X-X-Arg-Leu-Gln-Ser-Gly-Ala
' ~':
: ~ ,
,~ .
, ,
W094/06829 PCT/US93/08511'~
~ ~14~2~ 30
A~p-Val-Lys-X-A9p-Met-Glu-Val-Ser-X-Pro-Asp- '~
Gly-Tyr-Thr-X-X-Arg-Leu-Gln-Ser~Gly-Ala
4.38 Gly-Pro-Pro-Thr-Ph2-A~n-Lys-Ile-Thr-Pro-~sn-
Asp-Ala-Asp-Phe
:~
It is pointed out that the above-listed
sequence 1.18 is homologous to a family of peptides
previously described (Selsted, M.E., Harwig, S.L., Ganæ,
T., Schilling, J.W., and Lehrer, R.I. (1985) J. Clin.
Invest. 76,1436.), but differs from them by virtue of a
- deletion of Gly found in position 17 of those peptides.
The above-listed sequence 4.12 is nearly
identical to t~he ~equence BGP 11 of U.S. parent
application serial number 07/55~,263 filed July 10, lg90,
which i~ in turn very similar or identical to granulin A
described by Bateman et al. ~Bateman, A., Belcourt, D.,
Bennètt~ H., Lazure,~ C. and Solomon, S. (1990) Biochem.
Biopkys. Res;.~Comm~ 173, 1161), and bears sequence
simi~l~a~ity~to~rat pept~ides termed epithelins,
;20~ par~ticularly~epithelin 1, described by Shoyab et al.
Shoyab~,~M.~ McDonald, V.L., 3yles, C., Todaro, G.J. and
Pl~owman,~G.D. ~1990j Proc. Nat. Acad. Sci. 87, 7912).
Other members of a fami}y o~human and rat
granulin~/epithelins have been described (Bhandari, V.,
~Pal~f~ree,~ R.G.~E. and Bateman, A. ~1992j Proc. Nat. Acad.
S~ci.~89, ~1715~; Plowman, G.D., Green, J.M., Neubauer,
M.G.~, Buckley, S.D., McDona}d! V.L., Todaro, G.J. and
Shoyab~, M~. ~1992)~J. 3iol. Chem. 267, 13073.) Except for
a Gly in~position l, the peptide of seguence 4.12 may be
3~0j ;identlcal to,peptlide i1~ of parent application 07/551,263.
The above-listed 4.38 i9 homologous to human
antitrypsin (Long, G.L., Chandra, T., Woo, S.L.C.,
Davie,~.W.-and Kurachi, K. ~1984) ~iochemistry, ~l,
4828) and may represent an allelic variant of al-
antitrypsin or may represent another member of a family
,
."
, ~
:
W094/06829 ~14 2 ~ ~ 3 PCT/US93/0~511
of related molecule~, the serpins, of which ~1-
antitrypsin is one.
Example 3
S Extraction of Basophil ~ranule Prot~ins.
A coc~tail of protease inhibitors was prepared
containing diisopropylfluorophosphate, ,~
ethylenediaminetetraacetic acid and pepstatin A, and
O.25 ml of the mix was added to each of 10 aliquots of
frozen granules. Four consecutive freeze/thaw cycle~
were carried out in order to lyse the granules. Proteins
were extracted at 4C by addition of 5 ml of 50mM sodium
.
borate pH 9.0 to the pooled lysed granules. The
extraction mix was vortexed occasionally over a 1 hour
period to aid extraction. The insoluble material was
removed by centrifugation for 15 minutes at 20000 x g,
and~the soluble extract was~taken for chromatographic
fractionation.
20~ Size~Exc~usion-~HPLC.
Borate extract ~7.2 ml) was concentrated to
1.6 ml and~was adjusted to neutral pH with 0.5M NaH~PQ4.
~r,,''~ The neutralized extract was fractionated by size
exclusion chromatography in 3 identical runs on a Bio-Sil
25~ TSK~125 HP~C column (7~5mm~x 600mm). The A2~0 was
nitored and the fractions containing proteins were
combined~into 4 sepàrate pools based on sil~er staining
of~àemmli SDS polyacrylamide gels.
3a Revers Phase HPLC.
e
Pools of fractions from size exclusion HP~C
were acidi~ied by adjustment to 0.05-0.1~ trifluoroacetic
ac~id~and ea~ch~pool was ~eparately fractionated further by
reverse phase~chromatography on a Vydac C4 column
~- 35 ~(4.6mm x 25cm) eluted with a 0-100% gradient of
,,~,.
, i ~ .
" " ",
W094/06829 PCT/USg3/08511~ .
~1~22~3
ace~onitrile in 0.1% trifluoroacetic acid. Fraction~ .
judged to be appropriate for analy~is were concentra~ed
in a SpeedVac and sequenced by automated Edman
degradation on the ABI 477 Protein Sequenator.
Proteins in ~ome fractions were further
fractionated by an additional reverse phase HPLC step
performed as described in Example 2. Proteins present in
other reverse phase fractions were prepared for
sequencing of component proteins by Laemmli SDS
polyacrylamide gel electrophoresis followed by
electrophoretic transfer (2h at 500mA in lOmM CAPS
ph 11.0, 10% methanol, 0.05~ SDS) of proteins from the
gel onto a polyvinylidineaifluoride membrane (ProBlott, :;
ABI). Such electroblotted bands were excised from the
membrane and loaded directly onto the ABI 477 Protein
Sequenator.
The following sequences were obtained:
19 Ala-~Ile-Gln-Cys-Pro-X3-Ser-Gln-Phe-X4-X5-Pro-X6-Phe-
:~ Leu-Ala-Thr-Gly-Val-Met
where 83 is Leu or Asp, _4 is Met or Glu, X5 is Lys,
Ile, or Cys, and X6 i8 Pro or Leu.
26 Asp-Ile-Pro-Glu-Val-Cys-Phe-A~n
29 Asp-Pro-Gly-Glu-Val-Lys-Ala-Leu-Pro-Met-Gln
Lys-Pro-Gln-Met-Phe-Thr-Ile-X-Gln-Asn-X-Ala-Thr-Trp-
: ~ 25 Met
31 Lys-Ile-Gly-Gly-Phe-Glu-Val-Thr-Asp-Val-Phe-Ala-Pro-
~ ` Val-Met-Ala
- ~ ~ 31rp Ile-Leu-Gly-Val-Phe-X-Val-Glu-Gln-X-Phe-Ser-Phe-X-
`~ Leu
37 Asp-Pro-Pro-Thr-Phe-Asn-Lys-Ile-Thr-Pro-Asn-Leu-Leu-
Glu-Phe-Ala-Asp-Gly-Leu-Tyr-Lys-Gln-Glu
~- : 26bbl Ser-Glu-Leu-Thr-Lys-Met-Asn-Gln-Arg-Ser-Phe
rp indicates that the sequence was obtained
~:~ 35 ~ollowing repurification of Fraction 31 of this example.
.
,
, ~
~::
--- W0~4J06829 hl 4 2 Z 0 3 PCTIUS93/08511
-33-
bbl indicates that the sequence was o~tained
following electroblotting of Fraction 26 of this example.
The above-listed sequence 37 has homology wit~
re~pect to human ~l-antitrypsin (Long, G.L., Chandra, T.,
Woo, S.L.C., Davie, E.W. and Kurachi, K. (1984) ;
Biochemi~try, 23,4828) and may represent an allelic
variant of ~l-antitrypsin or may represent another member
of a family of related molecules, the serpins, of which
~l-antitrypsin i~ one.
Each of the 29 remaining vials of basophil cell~
contains an estimated 200 ~g of extractable protein.
Individual proteins recovered had yields ranging from 250
pmoles for peak 21 down to 2S-50 pmoles for peak3 9 and
37 (Figure l). Since 25 pmoles i9 usually sufficient for
sequencing 20 or more residues at the N-terminus, the
expenditure of~more vials will enable rarer specieq of
proteins~to be sequenced and will a1so e able more
residues to be sequenced from all proteins.
The instant invention has been shown and
described herein and was considered to be the most
praatical, and preferred embodiments. It i9 recognized,
however, that departures may be made therefrom which are
within the scope of the in~entionj and that obvious
modifications wilI occur to one ~killed in the art upon
- ~ reading this disclosure.
, :
,. . ..
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