Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION:
Mammalian subtilisin/kexin isozyme SKI-1: a proprotein convertase with a
unique
cleavage specificity
FIELD OF THE INVENTION:
This invention relates to a serine proteinase capable of converting proteic
precursors into mature proteins; particularly a serine proteinase capable of
cleaving
at non-basic amino acid residues.
BACKGROUND OF THE INVENTION:
Limited proteolysis of inactive precursors to produce active peptides and
proteins is an ancient mechanism to generate biologically diverse products
from a finite
set of genes. Most often, such processing occurs at either single or dibasic
residues,
as a result of cleavage by a family of mammalian serine proteinases related to
bacterial subtilisin and yeast kexin(1, 2). These enzymes, known as pro-
protein
convertases (PCs), participate in the tissue-specific intracellular processing
of
precursors at the consensus (RIK)-(X)"-R1 sequence, where X is any amino acid
except Cys and n = 0, 2, 4 or 6 (1-3). PCs have been implicated in the
production of
various bioactive polypeptide hormones, neuropeptides, enzymes, growth
factors,
adhesion molecules, cell surface receptors and surtace glycoproteins of
infectious
agents such as viruses and bacteria (1-3).
Less commonly, bioactive products can also be produced by limited proteolysis
at amino acids such as Leu, Val, Met, Ala, Thr, Ser and combinations thereof
(3). This
type of cellular processing has been implicated in the generation of bioactive
peptides
such as a-and y-endorphin (4), the C-terminal glycopeptide fragment 1-19 of
pro-
vasopressin (5), anti-angiogenic polypeptides such as platelet factor 4 (6)
and
angiostatin (7), the metalloprotease ADAM-10 (8), site 1 cleavage of the
sterol receptor
element binding proteins (9), as well as in the production of the Alzheimer's
amyloidogenic peptides A~40, 42 and 43 (10). Processing of this type occurs in
the
endoplasmic reticulum (ER) (9), or late along the secretory pathway, within
secretory
granules (4, 5}, at the cell surface, or in endosomes (6-8, 10). So far, the
proteinases
responsible for these cleavages have not been unambiguously identified.
Since mammalian convertases process precursors at either single or pairs of
basic residues, we hypothesised that a distinct, but related, enzymes) may
generate
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polypeptides by cleavage at non-basic residues. To test that idea, we employed
an RT-
PCR strategy similar to the one used to identify the PCs (11), except that we
used
degenerate oligonucleotides closer to bacterial subtilisin than to yeast
kexin. This
approach resulted in the isolation of a cDNA fragment encoding a putative
subtilisin-
like enzyme from human cell lines. This partial sequence was identical to a
segment
of a human myeloid cells-derived cDNA reported by Nagase et al. (12). A role
for this
putative subtilase remained undefined up to the present invention.
It was further discovered by Chang, D. et al. (1999) J. Biol. Chem. 274:22805
22812 that an enzyme called S1 p, is capable of cleaving sterol-regulatory
element
binding proteins (SREBPs), which function to control lipid biosynthesis and
uptake in
animal cells. Upon cleavage, SREBPs are released from cell membranes for
translocation to the nucleus, where they activate transcription of genes
involved in the
biosynthesis and uptake of cholesterol and fatty acids. S1 p and the present
enzyme
or the same. Therefore, for diseases involving overexpression of these genes
as well
as any other disease involving SKI-1 activity, it is contemplated that any
inhibitor of
SKI-1 would be useful in their treatment.
SUMMARY OF INVENTION:
We show that the sequences of the rat, mouse and human orthologues of this
putative type-I membrane-bound ~ubtilisin-_kexin-~soenzyme, which we called
SKI-1,
exhibit a high degree of sequence conservation. Tissue distribution analysis
by both
Northern blots and in situ hybridization (ISH) revealed that SKI-1 mRNA is
widely
expressed. A stable transfectant of human SKI-1 in HK293 cells allowed the
analysis
of its biosynthesis and intracellular localization. We present data
demonstrating that
SKI-1 cleaves at a specific Thri residue within the N-terminal segment of
human pro-
brain-derived neurotrophic factor (proBDNF). SKI-1 is the first identified
secretory
mammalian subtilisin/kexin-like enzyme capable of cleaving a proprotein at non-
basic
residues.
Therefore in accordance with the present invention, there is provided a
soluble
proteic fragment of a subtilisin-kexin isoenzyme named SKI-1 which has the
amino
acid sequence defined by amino acids 187 to 996 of any one SEQ ID NOs: 2, 4
and
6, a variant thereof, or an enzymatically active part thereof.
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It is further an object of this invention to provide a proteic fragment of SKI-
1
enzyme, which has the amino acid sequence defined by amino acids 18 to 137 of
any
one of SEQ ID NOs: 2, 4 and 6, a variant thereof, or a part thereof, which is
a pro-
segment capable of binding with amino acids 18 to 1052 of SKI-1 in whole or in
part.
A part of this pro-segment has a molecular weight of about 14 KDa and forms
a tight complex with the soluble fragment of SKI-1.
The pro-segment is an inhibitor of SKI-1 activity.
To improve its inhibitory activity, the pro-segment sequence is modified to
prevent further enzymatic processing in a cell expressing said proteic
fragment.
The mod~cation includes amino acid substitution, deletion or rearrangement.
Nucleic acids encoding any of the above SKI-1 forms are also objects of this
invention.
Recombinant vectors and hosts comprising these nucleic acids are also objects
of this invention.
The recombinant vectors are preferably expression vectors.
The recombinant vectors comprise a promoter expressible in a target cell
wherein expression of said nucleic acid is desirable, be it for a therapeutic
or
manufacturing purposes.
The recombinant vectors may also comprise an inducible promoter.
It is further an object of this invention to provide a method of producing a
proteic
fragment of SKI-1 enzyme, which comprises the steps of:
culturing a recombinant host cell expressing a SKI-1 nucleic acid in a cell
growth and expression-supportive culture medium; and recovering the proteic
fragment
of SKI-1 in the culture medium.
There is also provided a method for cleaving a proteic precursor which is an
enzymatic substrate for SKI-1 enzyme, which comprises the step of:
a) contacting the proteic precursor with a SKI-1 enzyme which as an amino
acid sequence defined by amino acids 18 to 1052 of SEQ ID Nos: 2, 4 or 6, or
a variant thereof, or the soluble form, for a time sufficient and in condition
adequate for such cleavage to occur.
The cleavage may be provoked in vivo or in vitro, e.g. serving a therapeutic
purpose or an industrial protein manufacturing use.
For the purpose of producing a protein or a peptide from a proteic precursor
which is an enzymatic substrate for SKI-1 enzyme, the method would further
comprise
the step of:
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b) recovering and purifying the protein or peptide.
The method may be performed in cell-free assays, or may take place in a cell
or in the presence of a cellular population, and wherein step a) comprises the
step of
transfecting a cell with a nucleic acid expressing a SKI-1 protein.
The cell may express said proteic precursor or 'may be transfected with a
nucleic acid expressing the proteic precursor.
A method of silencing the expression or the activity of SKI-1 enzyme on a
proteic precursor, which comprises the steps of:
contacting the enzyme or a nucleic acid encoding the enzyme with a ligand
molecule which binds to the enzyme or to the nucleic acid, thereby interfering
with the
binding of the enzyme to the proteic precursor or with the expression of the
nucleic
acid encoding the enzyme, is also an object of this invention.
The ligand molecule may comprise an antisense nucleic acid to the nucleic acid
encoding SKI-1, a pro-segment of a precursor protein encoding SKI-1, a SKI-
inhibitor,
a peptide mimicking a proteic precursor SKI-1 binding site, or an antibody
molecule
directed against SKI-1, or one which generates an inactive SKI-1 mutant form.
The pro-segment is a polypeptide extending from amino acids 17 to 137 of SEQ
ID NOs: 2, 4, 6, or a variant thereof or an inhibitory part thereof.
We also provide a peptide of at least 7 amino acids capable of binding to and
of being cleaved by SKI-1 catalytic active site, comprising the following
general
formula:
Arg Xaa, J Xaa2 1 Xaa3 (Z)~ O
wherein Xaa,, 2, 3 and Z are any amino acid
J is an alkyl or aromatic hydrophobic amino acid
n is 1, 2 or 3
O is an acidic amino acid.
Preferably Xaa2 is Lys, Leu, Phe or Thr.
A preferred peptide has the structure:
H2N-Val-Phe-Arg-Ser-Leu-Lys-Tyr-Ala-Glu-Ser-Asp-COOH.
The peptide may be labelled, a fluorogenic label being one of our preferred
embodiments.
A fluorogenic peptide which has the following sequence:
Abz-Val-Phe-Arg-Ser-Leu-Lys-Tyr-Ala-Glu-Ser-Asp-Tyr(NOZ)
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has been synthesized.
These peptides can be used for monitoring SKI-1 activity, for screening
inhibitors of SKI-1 activity or for screening enhancers of SKI-1 activity.
An inhibitor of SKI-1 activity used in the making of a medication for treating
a
disease involving an overexpression of a SKI-1 or a SK1-1 substrate, is also a
further
object of this invention, namely the pro-segment modified or not.
The disease may be associated with any one of hypercholesterolemia, high
levels of fatty acids, lipids or farnesyl pyrophosphate, liver steatosis, Ras-
dependent
cancer, restenosis and amyloid protein formation.
We also provide a method for detecting SKI-1 activity in a sample, which
comprises the steps of contacting the sample with a ligand molecule to SKI-1
protein
or nucleic acid, and detecting the formation of a complex between said ligand
and SKI-
protein or nucleic acid as an indication of the presence of SKI-1 in said
sample. The
ligand includes molecules such as anti-SKI-1-antibodies or a nucleic acid
probes or
primers.
Finally is provided a new use for SKI-1 enzyme in whole or in part which is
for
cleaving substrates not cleaved by other members of the subtilisin-kexin
family.
Variants of SKI-1 are under the scope of this invention, such variants are
encoded by
nucleic acids sharing at least 70% homology with the sequences defined in SEQ
ID
NOs: 1, 3, 5.
DESCRIPTION OF THE INVENTION:
During our search for new members of the subtilisin-kexin family, we obtained
two closely related sequences from mouse and rat tissues. When questioning
gene
data banks to find a match with other known sequences, we found that the human
counterpart has been previously cloned and sequenced. However, no specific
function
for this enzyme was known. We named our new enzyme subtilisin-kexin isoenzyme
1 (SKI-1 ).
We characterized this enzyme and found that SKI-1 has a unique cleavage site
in cognate substrates. One of these substrates is pro-BDNF. Sakai et aG have
found
that another substrate, SREBP-2, which is a sterol-responsive transcription
element,
was cleaved at a first enzyme processing site by an enzyme which they called
site 1
protease (S1 p}. S1 p and SKI-1 appeared to be the same enzyme.
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Since SKI-1 is autocatalytically cleaved, this brings to three the number of
substrates that are known to be recognized and cleaved by SKI-1. One object of
this
invention is therefore the use of SKI-1 as a protein processing enzyme.
SKI-1 is ubiquitously distributed and appears to be very well conserved
amongst mammalian species. Therefore, variants of SKI-1 are within the scope
of this
invention. We have indeed identified two species variants of the human enzyme
disclosed in gene data banks, and per se this is a proof that variants to
screen SKI-1
activity exist.
SKI-1 is first located in the endoplasmic reticulum (ER) membrane. Upon
processing the pro-segment of pro-SKl-1 is removed and SKI-1 is thus
activated. SKI-1
is further processed to remove the transmembrane domain that keeps it
integrated in
the ER membrane, which generates a SKI-1 soluble form that is directed into
the
secretory pathway and which remains active. The soluble active form is indeed
retrievable in culture media as well as the pro-segment. The pro-segment is
itself also
processed into shorter fragments. One of these fragments has an apparent
molecular
weight of about 14 KDa and forms a tight complex with the soluble SKI-1 form.
The
formation of this complex does not hinder the activity of the enzyme. It is
known that
the pro-segment of pro-protein convertases is inhibitory in vitro to the
activity of the
convertases. We demonstrate for the first time hereinbelow that such a
behaviour
occurs in an ex vivo model. SKI-1 pro-segment also has such an inhibitory
activity. We
predict that a SKI-1 pro-segment that would be modified to prevent the pro-
segment
processing wilt be an even better SKI-1 inhibitor. Such a modification is made
by
converting an enzyme recognition and cleavage site into a non-cleavable
sequence.
Such modification is intended to cover amino acid substitutions, deletions or
re-
arrangements to provide a SKI-1 pro-fragment that has an improved inhibitory
activity.
The nucleic acids encoding all the above SKI-1 forms (soluble, pro-segment
and sub-fragments, modified or not) are under the scope of this invention.
Recombinant vectors and hosts comprising these nucleic acids are also objects
of this
invention. More particularly, expression vectors capable of producing the
different SKI
1 forms are preferred. The expression vectors comprise promoter sequences
which
govern the expression of the nucleic acids. The promoter may be compatible
with the
cell wherein the expression of the nucleic acid is sought, be it for a
therapeutic purpose
or for the industrial production of SKI-1. The promoter may also be an
inducible
promoter which needs an exogenous inducing agent to activate the expression.
For
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the production of any SKI-1 form, a recombinant host cell may be used and is
cultured
in a culture medium which supports cell proliferation and the expression of
the nucleic
acids. Under suitable conditions, the SKI-1 form of interest is expressed and
may be
conveniently recovered from the culture medium.
A general method for cleaving a proteic precursor is also an object of this
invention. SKI-1 whole active enzyme or its soluble form or catalytically
active
fragments or variants are added to a proteic precursor which is a SKI-1
substrate, in
conditions adequate for enzymatic precursor processing (cleavage) to occur.
This
method may be performed in vivo for curing a SKI-1 deficiency or in vitro for
the
industrial preparation of active proteins. In the latter case, the processing
may be
performed in a cell-free assay, using purified proteic precursors and SKI-1
whole
enzyme or derived forms. Alternatively, it may be performed using transfected
cells
expressing SKI-1 whole enzyme and derived forms. The transfected cells may
endogenously express the protein precursor or may be co-transfected to express
the
same. The transformed cells therefore become a manufacture of mature proteins
and/or or SKI-1.
Modification of the SKI-1 activity is further an object of this invention. We
have
succeeded in inhibiting SKI-1 activity using the SKI-1 pro-segment.
Alternative ways
to achieve the same results include antisense nucleic acids or
oligonucleotides, SKI-1
inhibitors, peptides mimicking a precursor SKI-1 binding site (cleavable or
not), which
would compete for the binding of SKI-1 to its cognate protein precursor site,
and
antibodies directed against SKI-1 or its cognate proteic precursor binding
site. Another
alternative is a genic therapy replacing the active SKI-1 by an inactive
mutant form. On
the opposite, overexpressing SKI-1 may cure a SKI-1 deficiency. Due to the
ubiquitous
distribution of SKI-1, it may be useful, even necessary, to target the cell
wherein SKI-1
activity is to be modified for such a therapeutic purpose. Such targeting may
include
conjugating or combining molecules capable of modifying or modulating SKI-1
activity
to a ligand capable of targeting the cell of interest. Immunoliposomes are
examples of
targeting vehicles as well as conjugated ligands-oligonucleotides. Even viral
vectors
may be made targeting if they express such a targeting ligand at the membrane
surface. A targetting ligand serves a selection purpose, leaving substantially
intact the
non-targetted cells.
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Peptides of less than 100 amino acids, more preferably of less than 30 amino
acids, mimicking a cognate SKI-1 cleaving site in a proteic precursor have
been
synthesized and are also objects of this invention. Therefore, a peptide of at
least 7
amino acids comprising the following preferred structure is capable of binding
to and
of being cleaved by SKI-1 enzyme catalytic site:
Arg Xaa, J Xaaz 1 Xaa3 (Z)~ O
wherein Xaa,, 2, 3 and Z are any amino acid
J is an alkyl or aromatic hydrophobic amino acid
nis1,2or3
O is an acidic amino acid.
Preferably Xaa2 is Lys, Leu, Phe or Thr.
The preferred peptide has the following sequence:
HZN-Val-Phe-Arg-Ser-Leu-Lys t Tyr-Ala-Glu-Ser-Asp-COOH.
These peptides may be labelled in such a way that labelled fragments produced
upon cleavage are easily detected and identified. Such labelling include any
type of
suitable detectable markers. We have developed a fluorogenic peptide which
shows
a very good affinity for SKI-1. The above preferred peptide has been labelled
at its N-
and C- terminal ends with an orthoaminobenzoic acid and 3-nitrotyrosine
groups,
respectively.
These peptides as well as cell lines expressing SKI-1 will be especially
useful
for monitoring SKI-1 activity and for screening inhibitors or substrates and
enhancers
of SKI-1 activity.
Inhibitors of SKI-1, namely the SKI-1 pro-segment, will be used in the making
of a medication for treating a diseasing involving overexpression of SKI-1 or
of its
substrate.
Conversely, substrates of SKI-1 will be used in the research field to discover
physiological systems involving SKI-1.
Diagnostic methods and kits comprising a ligand to SKI-1 protein or nucleic
acid, which is to be contacted with a sample suspected to express SKI-1, is
also an
object of this invention. Detection of the formation of a ligand-SKI-1 complex
or of a
hybridization complex is an indication of the presence or amount of SKI-1 in
the
sample.
Since we were the first to discover the function of SKI-1 enzyme, the use
thereof for cleaving proteic precursors that are not substrates for the other
members
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of the subtilisin-kexin family is an object of this invention. SKI-1 is
intended in this
broad use to cover the whole enzyme, a catalytic part thereof and its
functional
variants. Variants are encoded by anyone of the nucleic acids depicted in SEQ
ID Nos:
1, 3 or 5, and any other sequences sharing at least 70% homology therewith,
preferably more than 85% homology, under stringent conditions of
hybridization.
Having now defined the general teachings of the present invention, reference
will be made hereinbelow to specific examples and embodiments as well to the
following appended figures, which purpose is to illustrate the invention
rather than to
limit its scope.
BRIEF DESCRIPTION OF FIGURES:
FIG. 1 shows the comparative protein sequences of SKI-1 deduced from rat,
mouse
and human cDNAs (SEQ ID NOs 2, 4, and 6, encoded by nucleic acids SEQ ID NOs:
1, 3, and 5, respectively). The position of the predicted end of the 17 as
signal peptide
is shown by an arrow. The active sites Asp2'°, His249 and Se~"4, as
well as the oxyanion
hole Asn3'e are in bold, shaded and underlined characters. The positions of
the 6
potential N-glycosylation sites are emphasized in bold. The conserved shaded
~DDSHRQK~F~( sequence fits the consensus signature for growth factors and
cytokine receptors family. Each of the two boxed sequences was absent in a
number
of rat clones. The predicted transmembrane segment is in bold and underlined.
FIG. 2 shows a Northern blot analysis of the expression of SKI-1 in adult rat
tissues.
[A] 5 Ng of male rat total RNA were loaded in each lane. Molecular sizes are
based on
the migration of an RNA ladder. The tissues include: adrenal, thyroid,
striatum,
hippocampus, hypothalamus, pineal gland, anterior (AP) and neurointermediate
(NIL)
lobes of the pituitary, submaxillary gland, prostate, ovary and uterus. Notice
the high
level of SKI-1 mRNA in adrenal glands. [B] 2 Ng of poly-A+ of (male + female)
Sprague
Dawley rat adult tissues (Bio/Can Scientific) were loaded, which includes:
liver,
thymus, spleen, kidney, heart and brain. The estimated size of rat SKI-1 mRNA
is
about 3.9 kb.
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F1G. 3 shows in situ hybridization (15 H) of rSKI-1 mRNA in a 2 day old rat.
ISH is
shown at anatomical resolution on X-ray film using an [~5S]-labeled antisense
riboprobe
[A-C] and sense control riboprobe [D]. Abbreviations: Adr - adrenal gland; Cb -
cerebellum; cc - corpus callosum; Cx - cerebral cortex; H - heart; Int -
intestine; K -
kidney; Li - liver; Lu - lungs; M - muscles; Mol - molars; OT - olfactory
turbinates; Pit -
pituitary gland; Rb - ribs; Ret - retina; Sk - skin, SM - submaxillary gland;
Th - thymus.
Magnification x 4; scale bar (in D) = 1cm.
FIG. 4 illustrates the biosynthetic analysis of SKI-1 in HK293 cells. Stable
transfectants
expressing either the pcDNA3 vector alone or one that expresses SKI-1 (clone
9) were
pulse-labeled for 4h with ['SS]Met. Media and cell lysates were
immunoprecipitated with
either a SKI-1 antiserum (Ab: SKI; against as 634-651 ) or a pro-SKI-1
antiserum (Pro).
The stars represent the 4 specific intracellular proteins (Mr 148, 120, 106
and 98 kDa)
immunoprecipitated with the SKI-1 antiserum. In these transfected cells, only
the 148
kDa band is recognized by the Pro-antiserum. A 98 kDa immunoreactive SKI-1 s
protein is also detectable in the medium.
FIG. 5 shows hSKI-1 immunoreactivity in stably transfected HK293 cells.
Representation of the comparative double fluorescence staining using a SKI-1
antiserum (directed against as 634-651 ) [A] and [B] and FITC-labeled WGA [A']
and
[B'] in control [A, A'] and LME-treated [B, B'] cells is shown. Thin arrows
emphasize the
observed punctate staining which is enhanced in the presence of LME. Large
arrows
point to the coincident staining of SKI-1 and WGA. Magnification x 900; bar
(in B') _
10 Nm.
FIG. 6 shows the processing of proBDNF by SKI-1. [A] COS-7 cells were infected
with
w:BDNF and either w:WT (-) or w:SKI-1 in the presence of either w: PIT or
vv:PDX.
The cells were metabolically labeled with [35S]Cys-Met for 4h and the media
(M) and
cell lysates (C) were immunoprecipitated with a BDNF antiserum, prior to SDS-
PAGE
analysis. The autoradiogram shows the migration positions of proBDNF (32 kDa),
the
28 kDa BDNF produced by SKI-1 and the 14 kDa BDNF. [B] Microsequence analysis
of the [35S]Met-labeled 32 kDa proBDNF (maximal scale 1000 cpm) and [ W]Leu-
labeled 28 kDa BDNF (maximal scale 250 cpm), revealing a Met at sequence
position
3 and Leu at positions 2, 13 and 14, respectively.
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FIG. 7 shows the in vitro processing profile of proBDNF by SKI-1. [A] pH
dependence
of the processing of proBDNF by SKI-1. The SKI-1 enzyme preparation was
compared
to that obtained from the media of Schwann cells infected with the wild type
virus (WT)
as control. [B] Inhibitor profile of the processing of proBDNF to the 28 kDa
BDNF by
the same SKI-1 preparation as in [A]. The reaction was performed overnight at
37°C,
pH 6Ø Notice that only PMSF (0.5 mM PMSF+50 NM pAPMSF), o-phenanthroline (5
mM), and EDTA (10 mM) effectively inhibited SKI-1 cleavage of proBDNF.
FIG. 8 shows the in situ hybridization translating SKI-1 mRNA expression in
the
pituitary gland of an adult rat using specific [35S]radiolabeled antisense
(SKI AS) and
control sense (SKI SS} riboprobes. The hybridization signal was detected in
the
anterior (AL), intermediate (IL) and posterior pituitary lobe (PL). Most of
the labeling
was confined to endocrine cells in AL and IL and to some pituicytes in the PL.
Magnification x 5; bar (in b) = 1 mm.
FIG. 9 shows the in situ hybridization translating the presence of SKI-1 mRNA
sites
in the skin of a newborn two days old (p2) rat using antisense (SKI AS) and
control
sense (SKI SS) riboprobes. The hybridization signal was detected in the
stratum
germinativum (sma(I vertical arrows in SGe), in both outer and inner hair
sheath
(medium arrows) and in some cells within the dermis (D). Other abbreviations:
HB -
hair bulb, SC - stratum corneum, SGr - stratum granulosum. Magnification x 80.
Fig. 10 shows the in situ hybridization (ISH) distribution of SKI-1 mRNA in
the rat
central nervous system (CNS). ISH distribution pattern in the CNS of adult rat
demonstrates a higher concentration of SKI-1 mRNA within a grey matter (GM and
all
structures indicated with capital letters) vs the white matter (V1IM)
including corpus
callosum (cc). Representative brain structures are shown in sagittal (a);
horizontal (b)
and coronal plane (c - f) after hybridization with antisense SKI-1 riboprobe
(a - e) and
control sense riboprobe (ssRNA in f). As shown at anatomical level this type
of mRNA
distribution is highly reminiscent to a type of pan-neuronal gene distribution
pattern. As
complementary to this figure a Table 1 demonstrates at cellular level the
predominance
of neuronal SKI-1 mRNA expression over gliai SKI-1 mRNA expression.
Magnification
x 4; bar (in a) = 1 cm. Abbreviations: CA1 - area 1 of corpus Ammonis; CA3 -
area 3
of corpus Ammonis; Cb - cerebellum; cc - corpus callosum; Ch PI - choroid
plexus; Cx
- cerebral cortex; GD - gyrus dentatus; GM - grey matter; Hip - hippocamp; Hy -
hypothalamus; OI - olfactory bulb; Str - striatum; WM - white matter.
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Fig. 11 shows the in situ hybridization (ISH) distribution of SKI-1 mRNA in
the rat
peripheral nervous system (PNS) trigeminal ganglion (TriG). ISH distribution
pattern
in the CNS of adult rat demonstrates a higher concentration of SKI-1 mRNA
within a
region of cell bodies (large arrows) over the region of supportive Schwann
cells (small
arrows). ISH was performed using antisense (SKI-1 as in a) and sense (SKI-1
ss)
riboprobes. Magnification x 12.
Fig. 12 shows the distribution of SKI-1, mRNA and/or protein, in the region of
spinal
cord (SpC) and in the related dorsal root ganglion (DRG) and dorsal root (DR).
Demonstrated are the region of neuronal cell bodies in the DRG (SKI-1 mRNA)
and
the region of nerve terminals in the dorsal horn of the spinal cord (layer i
and II)
characterized by a especial density of SKI-1 protein.
A) Schematic drawing depicting the position of layer I and II in the dorsal
horn as
well as that of the related DRG and DR.
B) SKI-1 mRNA revealed by in situ hybridization labeling (thin arrows) in the
DRG
using antisense riboprobes (SKI-1 AS).
C) Control hybridization in the DRG using sense riboprobes (SKI-1 SS).
D) Immunocytochemical localization of SKI-1 (brown staining) within layer I
and II
of the dorsal hom and in the dorsal root (DR) suggesting the sensory afferents
arriving
from DRG. Neuronal and glial nuclei are stained on blue. Magnification x 300.
E) Immunoreactivity of SKI-1 (thin arrows) detected around neuronal somata
(large
arrows) within layer II of the dorsal horn at high magnification (x 1,500).
Pattern of
immunoreactive spots is reminiscent to that of axo-somatic or axo-dendritic
nerve
terminals.
F) Northern blot revealing the concentrations of 4 kb SKI-1 mRNA in different
tissues including dorsal root ganglia (DRG) and spinal cord (SpC).
Abbreviations: 1
layer I of the dorsal hom; II - layer II of the dorsal horn; Adr - adrenal
gland; Cb
cerebellum; Cx - cerebral cortex; Hip - hippocamp; DH - dorsal horn; DR -
dorsal root;
DRG - dorsal root ganglion; SpC - spinal cord; Stom - stomach and Thyr -
thyroid
gland.
Fig. 13 shows the farnesyl diphosphatase mRNA levels in HK 293 cells treated
with
(+)lipids (cholesterol and 25-hydroxycholesterol) or without lipids (-). 1-2 =
wild type
cells, 3-4 = SREBP-1 overexpressors, 5-6 = a pool of 3 different clones
overexpressing
SREBP-1 and Pro-SKI-1 ; clones 4,6,9.
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Fig. 14 shows the fatty acid synthase mRNA levels in HK 293 cells treated with
(+)
lipids (cholesterol and 25-hydroxycholesterol) or without lipids (-). 1-2 =
wildtype cells,
3-4 = SREBP-1 overexpressors, 5-6 = a pool of 3 different clones
overexpressing
SREBP-1 and Pro-SKI-1; clones 4,6,9.
Fig. 15 shows the HMG CoA reductase mRNA levels in HK 293 cells treated with
lipids
(box A) or without lipids (box B). 1 = wild type cells, 2 = vector only cells,
3 = SREBP-1
overexpressor cells, 4 = SREBP-1 and ProSKI-1 overexpressor cells (high SREBP
expression, clone 6), 5 = SREBP-1 and ProSKI-1 overexpressor cells (low SREBP
expression, clone 9).
Fig. 16 shows the HMG CoA reductase and farnesyl diphosphatase mRNA levels in
Hk 293 cells in different clones overexpressing SREBP-1 (1-5) or SREBP-1 and
ProSki-1 (clone 4, clone 6, clone 9). Cells were treated with fetal calf
serum.
Fig. 17 shows the nuclear SREBP-1 in HK 293 cells in absence of lipids. Mature
SREBP is processed in the ER and translocated into the nucleus. 1 = wild type
cells,
2 = vector only cells, 3 = SREBP-1 overexpressors, 4 = SKI-1 antisense cells,
5 =
ProSki + SREBP-1 overexpressors clone 6, 6 = ProSKI + SREBP-1 overexpressors
clone 9.
Fig. 18 shows the processing of cytoplasmic SREBP-1 in HK 293 cells. 50 Ng of
protein per lane was separated in 6 % (above) and 10 % (below) SDS-PAGE gels.
Uncut SREBP-1 (proSREBP-1 ) and intermediate SREBP-1 (intSREBP-1 ) cleaved by
SKI-1 are indicated with arrows. Cell lines express ProSKI-1 (pSKI), SKI-1
anti-sense
( SKI-1 as), SREBP-1, or ProSKI-1 and SREBP-1 ( pSKI + SRE ), or control
vector
pcDNA3), as indicated. Analysis was pertormed in the presence ( + sterols) or
absence
of sterols ( - sterols).
Fig. 19 [A] is a schematic representation of the structure of FL-SKI-1 and its
truncation
mutant BTMD-SKI-1. The various SKI-1 domains depicted are, respectively, the
signal
peptide, pro-segment, catalytic domain, and the C-terminal region comprising a
cytokine receptor/growth factor motif, a transmembrane domain and a cytosolic
tail.
The positions of polypeptides used to produce SKI-1-specific antisera (Ab: P,
N and
S) are also displayed. Fig. 19 [B] shows the biosynthetic analysis of SKI-1.
W:FL-SKI-
1, BTMD-SKI-1 (bSKI-1) or control W:WT infected LoVo cells were pulse-labeled
with
[35S]CyS for 3h. Media were immunoprecipitated with either Ab:S or Ab:P and
then
resolved by SDS-PAGE on an 8 % gel followed by autoradiography. Arrows point
to
the migration positions of the 100 kDa BTMD-SKI-1 (bSKI-1), the 98 kDa shed
form
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(sSKI-1 ) as well as the 14 kDa prosegment product. Fig. 19 [C] shows a
Western blot
analysis of the overexpressed BTMD-SKI-1. Samples from W:WT or BTMD-SKI-1
infected BSC 40 cells (left and middle panel) were processed as described in
"Experimental Procedures" and run on an 8 % SDS-PAGE reducing gel. Following
electrotransfer to PVDF membranes, protein bands were visualized via ECL
detection
using primary rabbit antisera Ab:S or Ab:N. Purified BTMD-SKI-1 (right panel,
*) was
obtained from a Ni2+ affinity resin as described in "Experimental Procedures",
then
processed as described above. A mixture of Ab:S and Ab:P were used as primary
antisera. Elution buffer was used as a control (CTL}.
Fig. 20 shows the biosynthetic analysis of the rate of zymogen processing and
the fate
of the prosegment of SKI-1. LoVo cells overexpressing W:FL-SKI-1 were pulse-
labeled with [3H]Leu for 15 min and then chased for 2h (P15C2h), or pulsed for
2h in
the presence or absence of BFA (P2h). Cell lysates were immunoprecipitated
with
Ab:P, resolved by SDS-PAGE on a 14 % gel and autoradiographed. The migration
positions of the major ~26, 24, 14, 10 and 8 kDa prosegments are emphasized.
Fig. 21 illustrates the purification and identification of secreted
recombinant pro-SKI-1.
[A] Media obtained from HK293 cells stably expressing FL-SKI-1 were
concentrated
and sequentially applied to C4 semi-preparative column (not shown) followed by
a C4
analytical RP-HPLC columns, and then eluted by the indicated linear CH3CN
gradient.
[B] The fractions labeled I-IV were collected and analyzed by Western blotting
using
the primary antiserum Ab:P. (C,D~ Proteins contained in fraction IV were
separated on
a 10 % SDS-PAGE reducing gel. Following electrotransfer, the proteins were
stained
with Ponceau Red. The immunoreactive 14 kDa and non-immunoreactive but colored
4.5 kDa [D] polypeptides were excised and submitted to N-terminal sequencing
(X
represents an undefined residue). [E] Mass spectrometric analysis by MALDI-TOF
spectrometry of fraction IV. The C-terminal residues sites believed to
corresponding
to the three ~14 kDa polypeptides are underlined, whereas the expected
(potential)
cleavage sites are indicated by dashed arrows.
Fig 22 shows the processing of proSKl-1 autocatalytic prosegement candidate
sequences by purified, shed SKI-1. The proposed prosegment C-terminal mutant
17
as peptide IV [A~ and 15 as peptide IX [B~ were digested for 18 h with metal
chelation
chromatography-purified BTMD-SKI-1. The cleavage products were separated by RP
HPLC using a 5 Nm analytical Ultrasphere C18 column (Beckman) as described
under
"Experimental Procedures". The peptides contained in all but two peaks were
identifed
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by mass spectrometry. The unidentified peaks are attributable to contaminating
activities seen in WT/empty vector controls.
Fig. 23 shows the processing of proBDNF and SREBP-2 peptides by BTMD-SKI-1.
The 14 as peptide I [A] and 27 as peptide II [B] were digested with BTMD-SKI-1
for
150 and 60 min, respectively. The cleavage products were separated by RP-HPLC
using a 5 Nm analytical Ultrasphere C18 column (Beckman) as described under
"Experimental Procedures". The peptides contained in the major peaks were
identified
by mass spectrometry and amino acid analysis (nof shown).
Fig. 24 shows the pH and Ca2' activation profile of BTMD-SKI-1. BTMD-SKI-1
from
W-infected BSC40 cells was assayed as described under "Experimental
Procedures"
using a binary buffer system consisting of MES and HEPES, along with peptides
I or
II for the pH profile [A], and peptide II for the Ca2+ profile [B]. The
results represent the.
average t SD (indicated as error bars) of three separate determinations.
Fig. 25 is a X-ray film autoradiography showing in situ hybridization pattern
for SKI-1
mRNA (A) and APP mRNA (B) at the anatomical plane in sagital section from a 4-
day
mouse. Note similarity of distribution of SKI-1 and APP. A significant
concentration of
both SKI-1 and APP mRNA is revealed in the brain (Br), apinal cord (SpC),
dorsal root
ganglia (DRG), kidney (Ki), skin (Sk) submaxillary gland (SM) and bone tissue
(B).
Fig. 26 shows the comparative distribution of SKI-1 and APP in different
regions of
lacrimal gland of adult male mouse shown by immunocytochemistry. Peripherally
located lobes display immunoreaction for both SKI-1 {A) and APP (B) in acinar
cells.
fn the centrally located lobes the immunoreaction for SKI-1 (C) and APP (D) is
confined to single cells distributed through the acini (medium arrows) and to
intralobular ducts (long arrows).
Fig. 27 illustrates the inhibition of proNGF processing. Rat Schwann cells
were infected
with either W:POMC (antigen control), or co-infected with W:NGF and either
W:POMC (control), W:PDX, W:ppFurin or W:ppPC7. The cells were then pulse-
labeled with [35S]Met for 4h and the media immunoprecipitated with an NGF
antiserum.
The migration positions of the 35 kDa proNGF and the 13.5 kDa NGF are shown.
Fig. 28 illustrates the inhibition of proBDNF processing by furin. Western
blot analysis
of non-transfected (NT) COS-1 or cells transfected with pcDNA3 recombinants of
proBDNF as control (BDNF) or together with recombinants expressing sense (S)
or
antisense (AS) ppPC7 or ppFurin. The secreted products resolved by SDS-PAGE
were
analyzed with a BDNF-specific antiserum [Santa Cruz ].
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Fig. 29 shows the biosynthetic analysis of the fate of the prosegment of SKI-
1.
(A) Zymogen processing of [3H] Leu SKI-1 in LoVo cells. LoVo cells
overexpressing
vaccinia virus full length SKI-1 were pulse-labeled for 15 min with [3H] Leu
and then
chased for 2h (P15C2h). Cell lysates were immunoprecipitated with antibody to
the
prosegment, resolved by SDS-PAGE on a 14% gel and the dried gel
autoradiographed. The migration positions of the major 26, 24, 14, 10 and 8
kDa
prosegments are emphasized.
(B) Zymogen processing of [3H] Leu SKI-1 in BSC40 cells. BSC40 cells
overexpressing
vaccinia virus SKI-1 prosegment were pulse-labeled for 30 min with [3H] Leu
and then
chased for 2h (P30C2h). Cell lysates were immunoprecipitated with antibody to
the
prosegment, resolved by SDS-PAGE on a 14% gel and the dried gel
autoradiographed. The migration positions of the 24 and 14 kDa prosegments are
emphasized.
Fig. 30 shows the inhibition of ha, processing in stable transfectants of
Jurkat T cells
expressing the mPC5 prodomain mutated at Arg~" to Ala. The cell surface
proteins of
X 10g cells were biotinylated and immunoprecipitated with monoclonal ha4
antibody
( HP 2/1). Following SDS gel electrophoresis under reducing conditions and
blotting
to nitrocellulose the 80 kDa cleavage product was revealed by the
chemiluminescence
detection of anti-biotin streptavidin horse radish peroxidase.
EXAMPLE 1:
MATERIALS AND METHODS
Polymerase Chain Reaction and Sequencing. Most reverse transcriptase
polymerase chain reactions (RT-PCR) were performed using a Titan One Tube RT
PCR system (Boehringer Mannheim) on 1 Ng of total RNA isolated from either a
human neuronal cell line (IMR-32), mouse corticotrophic cells (AtT20), or rat
adrenal
glands using a TRlzol reagent kit (Life Technologies). The active site
degenerate
primers were: ~ (sense) 5'-GGICA(C,T)GGIACI(C,T)(A,T)(C,T)(G,T){T,G)IGCIGG-3'
and~(an6sense) 5'-
CCIG(C,T)IACI(T,A)(G,C)IGGI(G,C)(f,A)IGCIACI(G,C)(A,T)GTICG3'
based on the sequences GHGT(H,F)(V,C)AG and GTS(V,M)A(T,S)P(H,V)V(A,T)G,
respectively. The amplified 525 by products were sequenced on an ALF DNA
sequencer (Pharmacia). To obtain the full length of rat and mouse SKI-1, we
used
PCR primers based on the human (12) and mouse sequences, in addition to 5'
(13)
and 3' (14) RACE amplifications. To avoid errors, at least three clones of the
amplified
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cDNAs were fully sequenced. The GenBank accession numbers of the 3788 by mouse
mSKI-1 cDNA and 3895 by rat rSKI-1 are AF094820 and AF094821, respectively.
Transfection and Metabolic Labeling. Human SKI-1 (nt 1-4338) (12) in
Bluescript (a generous gift from Dr. N. Nomura, Kazusa DNA Research Institute,
Chiba, Japan; gene name KIAA0091, accession No. D42053) was digested with
Sacll
(nt 122-4338) and inserted into the vector PMJ602. The construct was digested
with
5' Kpnll3' Nhel, cloned into the Kpnl/Xbal sites of pcDNA3 (Invitrogen), and
the cDNA
transfected into HK293 cells with a DOSPER liposomal transfection reagent
(Boehringer Mannheim). A number of stable transfectants resistant to 6418 and
positive on western blots using a SKI-1 antiserum (see below) were isolated,
and one
of them (clone 9), was further investigated. Cells were pulsed for 4h with
[35S]Met and
the media and cell lysates immunoprecipitated with SKI-1 antisera directed
against
either amino acids (aa) 634-651, or as 217-233, or a pro-SKI-1 antiserum
directed
against the pro-segment comprising as 18-188 (Fig. 1). Immune complexes were
resolved by SDS-PAGE on a 6% polyacrylamidelTricine gel (15).
Northern Blots, in situ Hybridizations and Immunocytochemistry. Northern
blot analyses (16) were done on total RNA from adult male rat tissues using
either a
' TRlzol reagent kit (Life Technologies) or a Quick Prep RNA-kit
(Pharmacia)and on
polyA+ RNA of (male + female) rat adult tissues (BioICan Scientific). The
blots were
hybridized overnight at 68°C in the presence of [~2P]UTP SKI-1 cRNA
probes,
consisting of the antisense of nucleotides 655-1249 of rat SKI-1 (accession
No.
AF094821 ). For ISH, the same rat sense and antisense cRNA probes were doubly
labeled with uridine and cytosine 5'-{J~-[35S]thio}triphosphate (16). The
distribution of
SKI-1 mRNA in different tissues of adult and newborn rat (P1 ) after emulsion
autoradiography was investigated. Relative densities of specific SKI-1 mRNA
labeling
per celi in selected organs have been measured upon counting of silver grains
produced by antisense SKI-1 riboprobes and subtraction of non-specific
background
produced with sense SKI-1 riboprobes. Countings were made under 1000-fold
microscopical magnification in the similar regions of adjacent sections
stained with
hematoxylin and eosin. Results are the mean ( S.E.D. of 10 -16 readings / cell
type.
Newborn rats were frozen at - 35°C in isopentane and then cut into 14-
Nm sagital
cryostat sections (1, 16). After hybridization, all tissue slides were exposed
for 4 or 30
days to X-Ray film or emulsion autoradiography, respectively. For
immunofluorescence
staining we used a rabbit anti-SKI-1 antiserum at a 1:100 dilution and
rhodamine-
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labeled goat anti-rabbit IgGs diluted 1:20 (16). Red SKI-1 immunostaining was
compared with green staining patterns of both fluorescein-labeled concavalin A
(ConA;
Molecular Probes, OR), an ER marker, or fluorescein-conjugated wheat germ
agglutinin (WGA; Molecular Probes, OR), a Golgi marker (17).
Ex vivo and in vitro proBDNF Processing. A vaccinia virus recombinant of
human SKI-1 (vv:SKl-1) was isolated as previously described for human proBDNF
(w:BDNF) (15). The vaccinia virus recombinants of the serpins a1-antitrypsin
Pittsburgh (a1-PIT; w:PIT) and a1-antitrypsin Portland (a1-PDX; w:PDX) (18)
were
generous gifts from Dr. G. Thomas (Vollum Institute, Portland, OR). For
analysis of the
cleavage specificity of hSKI-1, 4 x1 Og COS-7 cells were co-infected with 1
pfu/cell of
w:BDNF and either the wild type virus (vv:W'T7 alone at 2 pfu/cell or with 1
pfu/cell of
each virus in the combinations: [w:SKI-1+w:WT], [w:SKI-1+w:PIT] and [w:SKI-
1+w:PDX]. At 10h post infection, cells were pulse labeled for4h with 0.2 mCi
[~5S]Cys-
Met (Dupont). Media and cell extracts were immunoprecipitated with a BDNF
antiserum (19; kindly provided by Amgen) at a concentration of 0.5 Ng/ml. The
precipitates were resolved on polyacrytamide gradient gels (13-22%) and the
autoradiograms obtained as described (15). Microsequencing analysis was
performed
on the [~5S]Met-labeled 32 kDa proBDNF and [ PI]Leu-labeled 28 kDa BDNF, as
described (20). For in vitro analysis, the 32 kDa proBDNF obtained from the
media of
LoVo cells infected with w:BDNF was incubated overnight with the shed form of
SKI-1
obtained from rat Schwann cells (16) co-infected with w:SKI-1 and w:PDX,
either at
different pHs or at pH 6.0 in the presence of selected inhibitors: pepstatin
(1 NM),
antipain (50 NM), cystatin (5 NM), E64 (5 NM), soya bean trypsin inhibitor
(SBTI, 5 NM),
0.5 M phenylmethylsulfonyl fluoride (PMSF) + 50 NM para-
aminophenylmethylsulfonyl
fluoride (pAPMSF), o-phenanthroline (5 mM) and EDTA (10 mM). The products were
resolved by SDS-PAGE on a 15% polyacrylamide gel, transferred to a PVDF
membrane and then probed with a BDNF antiserum (Santa Cruz) at a dilution of
1:1000.
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RESULTS
Protein Sequence Analysis of SKl-1. We first aligned the protein sequences
within the catalytic domain of PC7 (21 ), yeast subtilases and bacterial
subtilisins,
together with that of a novel subtilisin-like enzyme from Plasmodium
falciparum (J-C.
Barale et al., submitted). This led to the following choice of conserved amino
acids
around the active sites His and Ser: GHGT(HIF)(V/C)AG and
GTS(M/V)A(T/S)P(HIV)V(A!T)G, respectively. Thus, using degenerate
oligonucleotides
coding for the sense His and antisense Ser consensus sequences we initiated a
series
of RT-PCR reactions on total RNA (see Materials and Methods} and isotated a
525 by
cDNA fragment from the human neuronal cell line IMR-32. This sequence was
found
to be 100% identical to that reported for a human cDNA called KIAA0091
(Accession
No. D42053) obtained from a myeloid KG-1 cell line (12) and 88 % identical to
that of
a 324 by EST sequence (Accession No. H31838) from rat PC12 cells. We next
completed the rat and mouse cDNA sequences following RT-PCR amplifications of
total RNA isolated from rat adrenal glands and PC12 cells, and from mouse
AtT20
cells. Starting from the equivalent rat and mouse 525 by fragments, the
complete
sequences were determined using a series of RT-PCR reactions with human-based
oligonucleotides in addition to 5' (13} and 3' (14) RACE protocols. As shown
in Fig. 1,
alignment of the protein sequence deduced from the cDNAs of rat, mouse and
human
SKI-1 revealed a high degree of conservation. Rat and mouse SKI-1 share 98%
sequence identity and a 96% identity to human SKI-1. Interestingly, within the
catalytic
domain (Asp2'e to Ser"4) the sequence similarity between the three species is
100%.
Analysis of the predicted amino acid sequence suggests a 17 as signal peptide,
followed by a putative pro-segment beginning at Lys'8 and extending for some
160-180
amino acids. The proposed catalytic domain encompasses the typical active
sites
Asp2'e, His2°9 and Ser4'4 and the oxyanion hole Asn'38. This domain is
followed by an
extended C-terminal sequence characterized by the presence of a conserved
growth
factor / cytokine receptor family motif C_~'9,~DDSHRQKDCF~'. This sequence is
then
followed by a potential 24 as hydrophobic transmembrane segment and a less
conserved 31 as cytosolic tail that remarkably consists of 35% basic residues.
Some
of the clones isolated from rat adrenal glands suggested the existence of
alternatively
spliced rSKI-1 mRNAs in which the segments coding for as 430-4.83 or 858-901
are
absent. Finally, the phylogenetic tree derived from the alignment of the
catalytic
domain of SKI-1 with subtilases (22) suggests that it is an ancestral protein
that is
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closer to plant and bacterial subtilases than to either yeast or mammalian
homologues
(not shown).
Tissue Distribution of SKI-1 mRNA. Northern blot analyses of SKI-1 mRNA
in adult male rat tissues reveal that rSKI-1 mRNA is widely expressed and is
particularly rich in anterior pituitary, thyroid and adrenal glands (Figs. 2A
and 8). A
Northern blot of polyA+ RNA obtained from mixed adult male and female rat
tissues
also showed a wide distribution and a particular enrichment in liver (Fig.
2B). Similarly,
analysis of 24 different cell lines (23) revealed a ubiquitous expression of
SKI-1 mRNA
(not shown).
In situ hybridization data obtained in a day 2 postnatal rat also provided
evidence of a widespread, if not ubiquitous distribution of rSKI-1 mRNA.
Figure 3
shows at the anatomical level the presence of SKI-1 mRNA in developing skin
(see
also Figure 9), striated muscles, cardiac muscles, bones and teeth as welt as
brain and
many internal organs. Strong hybridization signals were detectable in the
retina,
cerebellum, pituitary, submaxillary, thyroid and adrenal glands, molars,
thymus, kidney
and intestine. Evidence for the cellular expression of rSKI-1 mRNA was
obtained from
analysis of the relative labeling densities per cell in selected tissues,
based on a
semiquantitative analysis of emulsion autoradiographies (not shown). In the
central
nervous system (CNS) rSKI-1 mRNA labeling was mostly confined to neurons,
whereas ependymal cells; supportive glial cells, such as presumed astrocytes,
oligodendrocytes, and microglia, exhibited 5-30 fold less labelinglcell (see
Table 1 and
Figure 10). In addition, within the peripheral nervous system (PNS) trigeminal
ganglia
reveal a 5-10 fold greater expression in neurons as compared to presumptive
Schwann
cells (Figures 11 and 12 and Table 1). Labeling was observed in most of the
glandular
cells in the anterior and intermediate lobes of the pituitary as well as in
the pituicytes
of .the pars nervosa. A semiquantitative comparison in the adult and newborn
rat
pituitary gland, submaxillary gland, thymus and kidney demonstrated an overall
2-fold
decreased labeling of rSKI-1 mRNA with age (not shown).
Biosynthesis of hSKI-1. To define the molecular forms of human SKI-1 and
their biosynthesis, we generated both a vaccinia virus recombinant (w:SKI-1 )
and a
stable transfectant in HK293 cells. Three antisera were produced against as 18-
188
(prosegment), 217-233 and 634-651 of SKI-1. Expression of vv:SKl-1 in 4
different cell
lines revealed that the enzyme is synthesized as a 148 kDa proSKl-1 a zymogen
which
is processed into 120, 106 and 98 kDa proteins. In this system, both the 148
and 120
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kDa forms are recognized by the Pro-domain antiserum, whereas all 4 forms
react with
the other two antisera. Processing of the 148 kDa proSKl-1 a into the 120 and
106 kDa
forms occurs in the ER based on the presence of these proteins in cells pre-
incubated
with the fungal metabolite brefeldin A (see 24 for refs., not shown). The same
SKI-1-
related forms are also observed in stably transfected HK293 cells following a
4h pulse
labeling with [35S)Met (Fig.4). The results reveal the intracellular formation
of a
secretable 98 kDa form (SKI-1s) recognized by both of the SKI antisera but not
by the
Pro antiserum. These data demonstrate that the 148 kDa proSKl-1 a is N-
terminally
cleaved into an intermediate 120 kDa form containing part of the prosegment
(proSKl-
1 b) which is then further excised to form a non secretable 106 kDa SKI-1.
This
suggests that two cleavages occur within the prosegment prior to the formation
of the
presumably membrane-bound 106 kDa form which is later shed into the medium as
a 98 kDa soluble SKI-1 s.
Intracellular localization of SKI-1. Double staining immunofluorescence was
used to compare the intracellular localization of the stably transfected human
SKI-1 in
HK293 cells and that of either the ER or Golgi markers ConA and WGA (17),
respectively. The data show that SKI-1 exhibits: (i) peripheral nuclear
staining,
colocalizing with ConA fluorescence, presumably corresponding to the ER (not
shown);
(ii) paranuclear staining colocalizing with WGA fluorescence, suggesting the
presence
of SKI-1 in the Golgi (Fig. SA,B) and (iii) punctate staining observed in the
cytoplasm
and within extensions of a few cells (Fig. 5A). Some, but not all of the
punctate
immunostaining matched that observed with WGA. This suggests that SKI-1
localizes
in the Golgi but may sort to other organelles, including lysosomal and/or
endosomal
compartments. Since in HK293 cells we observed scant immunoreaction to either
cathepsin B or cathepsin D (not shown), we could not directly assess the
presence of
SKI-1 within lysosomes. An indication of lysosomal/endosomal localization was
provided by the analysis of SKI-1 immunofluorescence within cells pre-
incubated for
4h with 10 mM leucine-methyl ester (LME), a specific IysosomaUendosomal
protease
inhibitor (25). The results showed a net increase in the proportion of cells
exhibiting
punctate staining (Fig. 5C) as compared to control cells. Thus, SKI-1
immunoreactivity
is enhanced upon LME inhibition of lysosomal/endosomal hydrolases.
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Enrymatic Activity and Cleavage Specificity of SKI-1. To prove that SKI-1
is a proteolytic enzyme we examined its ability to cleave five different
potential
precursor substrates. Our choice was based on the tissue expression pattern of
SKI-1
(Figs. 2, 3), which led us to select pro-opiomelanocortin (pituitary), pro-
atrial natriuretic
factor (heart), HIV gp160 (T-lymphocytes) and based on its neuronal
expression, pro-
nenre growth factor and pro-brain-derived neurotrophic factor (proBDNF).
Cellular co-
expression of vv:SKl-1 with the vaccinia virus recombinants of each of the
above
precursors revealed that only proBDNF could be cleaved intracellularly by SKI-
1. Thus,
upon expression of vv:BDNF alone in COS-7 cells we observed a partial
processing
of proBDNF (32 kDa) into the known major 14 kDa BDNF product (15), and the
minor
production of a previously observed (16; Mowla, S.J. et al., submitted but
still
undefined 28 kDa product (Fig. 6A). Upon co-expression of proBDNF and SKI-1, a
net
increase in the level of the secreted 28 kDa BDNF is evident, without
significant
alteration in the amount of 14 kDa BDNF (Fig. 6A). To examine whether the 28
kDa
product results from cleavage at a basic residue or at an alternative site, we
first co-
expressed proBDNF, SKI-1 and either a1-PIT or a1-PDX which are inhibitors of
thrombin and PC cleavages, respectively (18, 26). The results show that
different from
a1-PIT, the serpin a1-PDX selectively blocks the production of the 14 kDa BDNF
and
that neither a1-PIT nor a1-PDX affect the level of the 28 kDa product. This
demonstrates that a1-PDX effectively inhibits the endogenous furin-like
enzymes)
responsible for the production of the 14 kDa BDNF (15), but does not inhibit
the ability
of SKI-1 to generate the 28 kDa product. Thus, it is likely that the
generation of the 28
kDa BDNF takes place via an alternate cleavage. Incubation of the cells with
the Ca2+
ionophore A23187 abolished the production of both the 14 and 28 kDa products
(not
shov~rn), supporting the notion that similar to the PCs (1-3, 24), SKI-1 is a
Ca2'-
dependent enzyme.
In Fig. 6B, we present the N-terminal microsequence analysis of [35S]Met-
labeled 32 kDa proBDNF and [3H]Leu-labeled 28 kDa BDNF. The sequence of the 32
kDa form revealed the presence of an [35S]Met at position 3 (Fig. 6B), which
is in
agreement with the proposed sequence of human proBDNF (27) resulting from the
removal of an 18 as signal peptide cleaved at GCMLA'eIAPMK site. The N-
terminal
sequence of the 28 kDa product revealed a [~H]Leu at positions 2, 13 and 14
(Fig. 6B).
This result demonstrates the 28 kDa BDNF is generated by a unique cleavage at
Thrs'
in the sequence: $GLIS'iSLADTFEHVIEELL (27).
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To prove that SKI-1 is directly responsible far the production of the 28 kDa
BDNF at the novel Thr-directed cleavage, we performed in vitro studies. Thus,
proBDNF was incubated at various pHs with concentrated media of w:SKI-1-
infected
Schwann cells. A similar preparation obtained from wild type vaccinia virus-
infected
cells served as control. The data show that SKI-1 exhibits a wide pH
dependence
proftle revealing activity at both acidic and neutral pHs between pH 5.5 up to
7.3 (Fig.
7A) but also at pH 4.5 and 8 (not shown). Analysis of the inhibitory profile
of this
reaction revealed that metal chelators such as EDTA and o-phenanthroline, or a
mixture of the serine proteinase inhibitors PMSF + pAPMSF effectively inhibit
the
processing of proBDNF by SKI-1. The inhibition by EDTA is expected since like
all
PCs, SKI-1 is a Ca2'-dependent enzyme. The unexpected inhibition by 5 mM
o-phenanthroline may be due to excess reagent since at 1 mM only 25%
inhibition is
observed (not shown). All other class-specific proteinase inhibitors (aspartyl-
,
cysteinyl-, and serine proteases- of the trypsin- type) proved to be inactive.
Tabte 1
Tissue Adult Newborn (PI)
Silver grainslCell Silver Grains/Cell
t SED tSED
C.N.S.
Cerebal Cortex
Neurons, large 19.7 t 5.8 ND*
Neurons, medium & small5.7 t 2.3
Astrocytes, presumptive0.6 t 0.5
Hig) o~ campus ND
Neurons, pyramidal 15.313.9
Neurons, granules 23.7 t 5.3
Opus callosum ND
Oligodendrocytes, presumptØ6 t 0.6
Spinal cord
ND
Motorneurons 27.8 t 7.1
Circumventricular onaans ND
Plexus choroideux 9.6 t 1.9
Ependyma (Ill ventr.) 2.9 t 0.8
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P.N.S. ND
Trigeminal ganglion
Neurons, large 14.6 t 4
Satellite cells 3.8 t 22
Schwann cells, presumpt.1.3 t 1.9
Pituitar~aland
Anterior lobe cells 4.9 t 3.6 9.3 t 2.1
Intermediate lobe cells4.1 t 0.9 7.2 t 1.4
Posterior lobe pituicytes3.6 t 3.9 6.7 t 4.2
Thvmus
Cortical lymphocytes 4.1 t 0.7 7.1 t 1.0
Medullary reticular 2.7 t 1.0 4.4 t 0.9
cells
Adipocytes 0.3 t 0.6 ND
Fibroblats ~ 0.2 t 0.1 ND
Submaxillary c li and
Epithelial cells 2.1 t 1.0 3.9 t 1.7
Acinar cells 2.4 t 1.2 4.5 t 1.7
Kidnev
Glomerular cells 2.8 t 0.9 4.2 t 0.9
Convoluted tubules 4.1 t 2.7 9.8 t 1.4
*ND = not determined
DISCUSSION
This work provides the first evidence for the existence of a mammalian
secretory Ca2+-dependent serine proteinase of the subtilisin-kexin type that
selectively
cleaves at non-basic residues. Thus, SKI-1 processes the 32 kDa human proBDNF
at
an ~CAGS~tGL~ISL sequence generating a 28 kDa form, which may have its own
biological activity (Mowla, S.J. et aL, submitted. Such a cleavage site is
close to the
consensus site deduced from a large body of work done with the PCs, whereby an
(~C)-(X)~ ~1 X-(UIIIn, [where n=0, 2, 4 or 6] motif is favored.by most PCs (1-
3, 28).
Note that in the SKI-1 site, P1 Arg is replaced by Thr and an aliphatic Leu is
present
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at P2', an amino acid also favored by PCs (1-3, 28). Several proteins are
known to be
cleaved following Thr. These include human anti-angiogenic platelet factor 4
(6;
QCLCVKTT1SQ) and angiostatin (7; KGPWCFTT1DP), the neuroendocrine a-
endorphin (4; KSQTPLVT1 LF), the ADAM-10 metalloprotease (8; LLRKKRTT1 SA), as
well as the amyloidogenic peptide A[343 (10; VGGWIAT1VI).
Interestingly, comparison of the phylogenetically highly conserved sequence of
proBDNF revealed an insertion of hydroxylated amino acids (Thr and Ser) just
after the
identified SKI-1 cleavage site of human proBDNF. Thus, in rat and mouse
proBDNF,
two threonines are inserted (RGL'iZ'f-SL) and in porcine proBDNF flue serines
added
(RGLTSSSSS-SL) (27). These observations raised a number of questions: (i) do
these insertions affect the kinetics of proBDNF cleavage by SKI-1? (ii) does
SKI-1
recognize both single and pairs of Thr and Ser and combinations thereof? (iii)
is the
presence of a basic residue at P4, P6 or P8 important for cleavage? and (iv)
similar to
enzymes cleaving at basic residues (29), does the possible phosphorylation at
specific
Thr or Ser residues affect substrate cleavability by SKI-1? Answers to these
questions
are provided hereinbelow.
Biosynthetic analysis of the zymogen processing of proSKl-1 demonstrated a
two-step ER-associated removal of the pro-segment (Fig. 4). Furthermore,
analysis of
the [35S0~]-labeled SKI-1 demonstrated only the presence of sulfated 106 and
98 kDa
forms but not that of either the 148 or 120 kDa forms recognized by the Pro-
segment
antiserum (not shown). Since sulfation occurs in the traps Golgi network, this
confirms
that the removal of the pro-segment occurs in the ER. Like furin and PC5-B (1-
3, 24)
the membrane bound 106 kDa SKI-1 is transformed into a soluble 98 kDa form
that is
released into the medium by an as yet unknown mechanism. The secreted 98 kDa
SKI-1s is enzymatically active since it processes proBDNF in vitro (Fig. 7).
Numerous
attempts to sequence the SDS-PAGE purified ['H]Leu and Val-labeled 148 kDa and
98 kDa forms, resulted in ambiguous results, suggesting that SKI-1 is
refractory to N-
terminal Edman degradation. Presently, we cannot define the two zymogen
cleavage
sites leading to the sequential formation of the 120 kDa proSKl-1 b and 106
kDa SKI-1
deduced by pulse (Fig. 4) and pulse-chase studies (not shown). Examination of
the
pro-segment sequence (Fig. 1 ), the species-specific proBDNF motif potentially
recognized by SKI-1 (see above), and the alignment of SKI-1 with other
subtilases
(22), suggests two possible conserved sites: $NNP~S951 DY_PS and $H~'BZ i
RRLL.
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Both sites predict a cleavage after pairs of Ser with either a P6 or a P4 Arg,
respectively.
Phylogenetic structural analysis of the predicted amino acid sequence of SKI-1
reveals that this serine proteinase is closer to plant and bacterial
subtilases than it is
to yeast and mammalian PCs. The 100% conservation of the catalytic domain
sequence, although striking and suggestive of an important function, is not
far from the
98% similarity between human and rat PC7 (3, 21). The sequence C-terminal to
the
catalytic domain of SKI-1 is very different from that of any of the known PCs.
In fact,
although PCs have a typical P-domain critical for the folding of these enzymes
(for
reviews see 1-3), we did not find the hallmark sequences (3, 30) of the P-
domain within
the SKI-1 structure. Instead different from the PCs, we find a conserved
growth
factorlcytokine receptor motif of which functional importance will need to be
addressed,
especially since this motif is partly missing in alternatively spliced forms
(Fig. 1 ).
Finally, the highly basic nature of the cytosolic tail of SKI-1 (Fig. 1 ) may
be critical for
its probable cellular localization within endosomal/lysosomal compartments
(Fig. 5),
similar to the importance of basic residues for the accumulation of the a-
amidation
enzyme PAM in endosomal compartments (Milgram, S.L., personal communication).
The wide tissue distribution of SKI-1 mRNA transcripts suggests that this
enzyme processes numerous precursors in various tissues. Furthermore, the
observed
developmental down-regulation of the level of its transcripts also suggests a
functional
importance during embryonic development. The fact that SKI-1 can cleave C-
terminal
to Thr and possibly Ser residues suggests that, like the combination of PCs
and
carboxypeptidases E and D (31), a specific carboxypeptidase may also be
required to
trim out the newly exposed C-terminal hydroxylated residues. Such a hypothesis
may
find credence in a report suggesting that the amyloidogenic A~43 (ending at
Thr) may
be transformed in vitro into Aa42 and A~340 by a brain-specific
carboxypeptidase(s)
(32).
A recent report demonstrated the existence of a soluble subtilisin-like enzyme
exhibiting a 29% sequence identity to SKI-1 in Plasmodium falciparum
merozoites
(PfSUB-1 ). This enzyme localizes to granular-like compartments and presumably
cleaves at a Leu tAsn bond (33). In that context, SKI-1 may represent the
first member
of an as yet undiscovered mammalian family of proteinases implicated in the
limited
proteolysis of proproteins at sites other than basic amino acids that may
differ by their
intracellular localization and cleavage specificity.
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EXAMPLE 2
Genetic and biochemical evidence indicates that SKI-1/S1 p is the protease
that
cleaves sterol-regulatory element-binding proteins (SREBPs) which functions to
control
lipid biosynthesis and uptake in animal cells { Sakai, J. et al. (1998)
Molecular Cell 2,
505-514; Cheng, D. et al. (1999) J. Biol. Chem. 274, 22805-22812; Toure, A. et
al.
(1999) In: Peptides for the Now Millennium: Proceedings of the 16th American
Peptide
symposium }. SKI-1 and SREBPs play critical roles in the feedback pathways by
which
cholesterol suppresses transcription of genes encoding HMG CoA reductase and
other
enzymes of cholesterol biosynthesis as well as the low density lipoprotein (
LDL)
receptor. A SKI-1 inhibitor would be of use under clinical conditions in which
there is
not sufficient down regulation of SREBP dependent transcription by sterols.
For
example, in the Nieman-Pick group of diseases a high sphingomylin content of
cells
leads to an increase in proteolysis of SREBP-2 and a subsequent increase in
cholesterol biosynthesis { Scheek, S. et al. (1997) Proc. Natl. Acad. Sci. USA
94,
11179-11183; Spence, M.W., and Callahan, J.W. (1989) Spingomyelin-cholesterol
lipidoses: The Nieman-Pick Group of Diseases. In The Metabolic Basis of
Inherited
Disease ( Scrnrer, C.R., Beaudet, A.L., Sly, W.S., and Valle, D., editors ),
McGraw-Hill
Publ. Co., 6th edition, chapter 86, 1655-1675; Svirirodov, D. (1999) Histology
&
Histopathology 14 (1): 305-319 }. Perhaps of greater significance, nuctear
SREBP-1c
protein levels were significantly elevated in mouse models for non-insulin
dependent
diabetes, ob/ob and aP2 SREBP-1c mice, which was associated with elevated mRNA
levels for known SREBP target genes involved in the biosynthesis of fatty
acids
(Shimomura, I. et aL J. Biol. Chem.1999; 274:30028-30032).
In addition, the inhibition of the SREBP- dependent transcription of farnesyl
diphosphate synthase, like HMG-CoA reductase and farnesyl-protein transferase
inhibitors, by inhibition of farnesyl pyrophosphate biosynthesis could
potentially be
useful to treat a number of diseases such as Ras-dependant cancers and
restenosis
( Reference - United States Patent 5,925,651). With regard to a potential
treatment
for restenosis, HMG-CA reductase inhibitors decrease smooth muscle (SMC) cell
migration and proliferation, and induce SMC apoptosis { Bellosta, S. et al.
(1998)
Atherosclerosis 137, S101-S109; Guijarro, C. et al. (1998) Circulation
Research 83,
490-500 }.
As mentioned above, inhibition of PC activity seems to offer new therapeutical
targets. Unfortunately, previous attempts using inhibitory peptides have
failed either
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due to cytotoxicity of used agents or poor targeting "~'8. We have focused on
the
inhibitory properties of PC prosegments in order to find a safe and effective
way for
enzyme silencing.
To study the effect of the SKI-1 prosegment (ProSki-1 ) on the SREBP
processing and mediated transcriptional activity we isolated a cDNA fragment
covering
the 188 amino acids that make up the signal peptide and the prosegment of SKI-
1
including the predicted cleavage site RRLL"6. This autocatalytic cleavage site
was
confirmed by mass spectral analysis and amino acid sequencing by other
investigators
'9. We isolated stable cell lines overexpressing SREBP-1 (neo resistance) and
ProSki-
1 plasmid (zeo resistance). A background SREBP-1 overexpression was used in
order
to improve detection of nuclear NH2 terminal segment of SREBP in immunoblot
experiments.
The effect of ProSki-1 on target gene mRNA: mRNA expression in HK293 cells was
studied by Northern blotting as described in the methods section. In wild type
(wt),
vector only, and SREBP overexpressor cells in presence of lipids the mRNA
levels
were low for all studied genes: LDL-receptor, HMG-CoA reductase, farnesyl
diphosphate (FDP) (Fig. 13), and fatty acid synthase (FAS) (Fig. 14). However,
when
these cells were treated with media containing no cholesterol a clear increase
was
observed in mRNA expression for all these genes, as demonstrated in earlier
studies.
Interestingly, corresponding mRNA levels were greatly reduced in both
conditions in
cells overexpressing ProSKI-1 and SREBP-1 suggesting .that SREBP mediated
transcription can be blocked efficiently by the prodomain mediated inhibition
of the SKI-
1 protease (Figs. 13 and 14). The effect was observed in early passages of
previously
frozen cell lines. However, when the same clones were kept in culture for
future
passages, in contrast to earlier findings the target gene mRNA levels were now
normal
or even higher than in control cells. (Figure 15). This finding suggests that
cells can
adapt to new conditions and maintain their lipid homeostasis even without
SREBP
mediated regulation and synthesis. This finding was supported in another
experiment
with several cell lines overexpressing SREBP-1 or SREBP-1 and ProSki-1 (Fig.
16).
While HMG CoA reductase and farnesyl diphosphatase varied markedly between
different cell lines containing only SREBP-1 (Fig. 16, lanes 1-5), mRNA levels
measured from cells overexpressing ProSki-1 and SREBP-1 (Fig. 16, lanes c14,
c16,
and cl9) showed no variation and were higher than in SREBP-1 cells.
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The effect of ProSki-1 on nuclear SREBPs: Western blot experiments were
performed to illustrate the effect of ProSKI-1 on SREBP-1 processing in these
cells.
After staining with an antibody against the NHZ terminal end of SREBP-1 a band
around 60 kDa appeared on blots of nuclear extracts (Fig. 17), as demonstrated
earlier
by other investigators Z'3. As expected, only a weak signal was detected in
presence
of sterols. In absence of sterols a significant increase was observed,
especially in
SREBP-1 cells. Only minute amounts of nuclear SREBPs were detected when ProSKi-
1 was present suggesting that sterol mediated proteolysis of SREBPs is
efficiently
blocked in these cells in presence of ProSki-1 (Figure 17 shows the data from
clones
6 {lane 5~ and 9 {lane 6~).
The inhibitory effect of ProSKI-1 was further demonstrated by studying the
processing of cytoplasmic full length SREBP-1 (proSREBP-1) (Fig. 18). The
processing of proSREBP-1 by SKI-1 / S1 P into intermediate (intSREBP-1 ) forms
shown previously by other investigators'e, was clearly demonstrated in clones
overexpressing SREBP-1. Significantly, in cell lines overexpressing SREBP-1
together
with the inhibitory prodomain of SKI-1 (pSKI + SRE) accumulation of the
proSREBP-1
was observed and formation of the intermediate forms) of SREBP-1 was
abolished.
These results, along with the observed reduction in nuclear SREBP ( Fig. 17),
indicate
that ProSKI-1 efficiently inhibits SKI-1 protease activity and blocks SREBP
processing
in mammalian cells. In addition, the specificity of ProSKI-1 inhibition was
studied by
using a substrate not processed by SKI-1 (neurotrophin-3; NT-3). Both the
level and
furin-derived processing of NT-3 were unaffected by the presence of ProSKI-1
(not
shown). These results suggest that ProSKI-1 is SREBP- and pro-BDNF- specific
and
that it does not affect other secretory proteins which are not substrates for
SKI-1.
In these experiments a pro-domain was successfully used for the first time as
a subtilase inhibitor in vivo. ProSki-1 seems to be a promising therapeutical
tool for
SREBP-mediated pathologies, which may or may not be directly related fo
cholesterol
or fatty acid homeostasis. For instance SREBP-dependent isoprenoids, such as
farnesol and geranylgeraniol, have been shown to associate e.g. with
endothelial nitric
oxide synthetase (eNOS) Zo-2', vascular smooth muscle proliferation and
migration as
well as ras-protein mediated cell proliferation 24-28. Furthermore, links to
PPAR-y
mediated signaling system including adipocyte differentiation and insulin
resistance
have already been reported ~~3'. This novel prosegment approach to inhibit
enzyme
activity will certainly also inspire other investigators in different fields,
since it may be
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possible to specifically inhibit other enzymes with this prosegment technology
leading
to new treatments for a variety of diseases. On the other hand, these results
provide
new data supporting the existence of an SREBP-independent, but lipid dependent
(Fig.
3) control of the lipid homeostasis in human cells, although the alternative
sensor of
lipids under these conditions is currently unknown.
Materials and Methods
Materials:
Cell Culture: HK293 cells were maintained as monolayers in Dulbecco's modified
Eagle's medium containing 100 units I ml penicillin and 100 Ng I ml
streptomycin
sulfate (medium A) supplemented with 10 % fetal calf serum. 24 hours before
RNA and
protein extractions medium A was supplemented with 5 % lipoprotein deficient
serum,
50 NM mevalonate (Sigma), 50 NM compactin (Sigma) and with no sterols or 1
Ng/ml
of 25-hydroxy-cholesterol and 10 Nglml of cholesterol. 4 hours before protein
extraction
25 Ng/ml N-acetyl-leucinyl-leucinyl norleucinal was added. Total RNA was
isolated with
Trizol (Gibco BRL) reagent according to the instructions of the manufacturer.
In order
to extract proteins cells were washed and collected in PBS with protease
inhibitors ().
After addition of buffer A (Triton x 1001 %,50 mM tris maleate, 2 mM CaCl2,
inhibitor
coctail (), and ALLN) cells were mixed with pipette and allowed to swell on
ice for 20
minutes Then the solution was centrifuged for 5 minutes at 15 000 rpm and
supernatants representing membrane proteins were collected and stored until
analyzed
at -70 °C. Remaining pellets were resuspended in Buffer B (20 mM Tris
pH 7.9, 400
mM NaCI, 1 mM EDTA, 1 mM EGTA, and protease inhibitors). Samples were shaken
at 4 C for 1 hour and centrifuged and the supernatant was frozen in aliquots
at -70 °C.
Plasmid constructions: SKI-1 prosegment containing as 1-188 was isolated by
PCR
using following ofigonucleotides: [5' GGA TCC GAA GAA ACA TCT GGG CGA CAGA
3'] and [5' CTC GAG GGC TCT CAG CCG TGT GCT 3'] and cloned into PCR 2.1 TA
cloning vector for sequencing. After that it was subcloned into the
pcDNA3Ze~~~ vector
(Invitrogen) (BamHl / Hindlll sites) for transfections.
SREBP-1 in bluescript IISK (ATCC 79810) subcloned into Sall / BamHl sites
of the pcDNA~"",~,~.
Transfections: HK293 cells were plated at a density of 5x105 I 60 mm dish in
medium
A with 10 % fetal calf serum and were cultured until they were 40-60 %
confluent. The
cells were then transfected with 10 Ng plasmid DNA (pcDNA3~~, pcDNA3~~ SREBP-
1,
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pcDNA3~~ SREBP-1 and pcDNA 3Z~roSKl-1 ) using Lipofectin reagent (Life
Technologies, city, state) according to manufactures instructions. On day two
medium
containing appropriate selection agents (800 Ng/ml Geneticin for pcDNA3"~, x00
~g/ml
Zeocin for pcDNA3=~°) were added. The medium was changed every two
days until
defined colonies were evident. Colonies were isolated and formed stable cell
lines
were analyzed by immunoblotting with ProSKI-1 and SREBP-1 antibodies.
Northern blotting: 20 Ng of total RNA was electroforetically separated in an
1.0
agarose gel, and transferred to Hybond N+ filters (Amersham, city, state) by
capillary
blotting. After transfer filters were crosslinked by UV irradiation in a
Stratalinker
(Stratagene). Filters were prehydridized at 42 °C for 1 hour and
hybridized with
random labeled'2P cDNA probes for 16-20 hours. Ultrahyb buffer (Ambion) was
used.
After hybridization filters were washed and exposed to film for indicated time
and
bands were quantified by densitometry. Following primer pairs were used to
clone
cDNA probes: HMG CoA reductase [5' GAG GAA GAG ACA GGG ATA AAC 3'] [5'
GGG ATA TGC TTA GCA TTG AC 3'], farnesyl diphosphate [5' AGC CCT ATT ACC
TGA ACC TG 3'], [5' GAA TCT GAA AGA ACT CCC CC 3'], Fatty acid synthase [5'
TTC CGA GAT TCC ATC CTA CG 3'], [5' TGC AGC TCA GCA GGT CTA TG 3'],
Acetyl CoA carboxylase [ 5' TCT CCT CCA ACC TCA ACC AC 3'], [5' CCA GCC TGT
CAT CCT CAA TAT C 3'], SREBP-1 [5' GGA GCC ATG GAT TGC ACT TTC 3'], [5'
AGG AGC TCA ATG TGG CAG GA 3'], LDL-receptor [5' 3'], [5' 3']. Amplification
products were cloned into pGEM (Promega) and sequenced. 18S cDNA was
purchased from Ambion.
Immunoblot analysis: 50 Ng of nuclear extract and membrane fractions were
separated in an SDS-PAGE gel. After electrophoresis proteins were transferred
to a
nitrocellulose membrane. Membranes were stained wkh appropriate primary SREBP-
1
(Santa Cruz), ProSki-1 and secondary antibodies. After washing
chemiluminescent
substrate (Santa Cruz) was added, and membranes were exposed to x-ray film for
1-
min. Gels were calibrated with prestained molecular weight markers (New
England
Biolabs).
EXAMPLE 3
The soluble SKI-1 isoform, collected from cell media, was used to study the in
vitro cleavage properties of this enzyme on a number of synthetic substrates.
In
addition, we present data on the in vitro inhibitory character of three
prosegment
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constructs of SKI-1, which we obtained as bacterial recombinant proteins.
Moreover,
we examined the processing of hSKI-1 in LoVo cells infected with a W
recombinant
as well as in a stable transfectant of HK293 cells (10).
EXPERIMENTAL PROCEDURES
Vaccinia Virus Recombinant of BTMD-SKI-1- The preparation of a soluble form
of hSKI-1 involved the initial amplification by polymerase chain reaction
(PCR) of a
1250 base pair (bp) product encompassing nucleotides (nts) 491-1740 of the
hSKI-1
cDNA (12), which includes the initiator methionine. The sense (s) and
antisense (as)
oligonucleotides were 5' GTGACCATG-AAGCTTGTCAACATCTGG 3' and 5'
ACACTGGTCCCTGAGAGGGCCCGGCA 3', respectively. This completely sequenced
fragment, which had been inserted into the PCR2.1 TA cloning vector
(Invitrogen), was
first digested with Notl and Accl. It was then ligated with the similarly
digested full-
length hSKI-1 cDNA 3.5 kb product, resulting in a product called 5' hSKI-1-FL.
In order
to obtain a soluble form of hSKI-1 with a hexa-His sequence just before the
stop
codon, PCR amplification was carried out using the sense and antisense
oligonucleotides: 5' ATTGACCTGGACAAGGTGGTG 3' and 5'
G G A T C C T C T A G A T C A G T G G T G G T G G T G G -
TGGTGGTGCTCCTGGTTGTAGCGGCCAGG 3'. This resulted in a 165 by fragment
encoding the C-terminal sequence PGRYNQE99'-(H6)* (10). Following digestion
with
5' EcoNl and 3' Xbal, the product was ligated to the aforementioned and
similarly
digested 5' hSKI-1-FL. This cDNA, coding for BTMD-SKI-1 ending with a hexa-His
sequence, was then transferred to the BamH1/Xbal site of the (W) transfer
vector
PMJ601. A recombinant was then isolated as previously reported (13). The W
recombinant of full-length hSKI-1 has been described (10).
Biosynthetic Analyses - Seventeen hours following infection with 2 pfu each of
W:SKI-1 and W:BTMD-SKI-1 recombinants, human LoVo cells (3 x 106) were
radiolabeled with 500 pCi of [3H]Leu for 2h or pulsed for 15 min followed by a
chase
of 2h, in the presence or absence of 5 Ng/ml of the fungal metabolite
brefeldin A (BFA)
as described (10,14). Media and cell lysates were immunoprecipitated with SKI-
1
antiserum directed against either as 634-651, or the prosegment comprising as
18-188
(10). Immune complexes were resolved by SDS-PAGE on an 8% or 14%
polyacrylamidelTricine gel (10) and the dried gels autoradiographed (10,14).
All
biosynthesis experiments were performed at least twice.
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Isolation and Purification of Recombinant hSKI-1 Prosegments - Three N-
terminal fragments of hSKI-1 were isolated by PCR using a common (s)
oligonucleotide [5' GGATCCGAAGAAACATCTGGGCGACAGA 3'] and one of three
(as) oligonucleotides [5' CTCGAGGGAGAGGCTGGCTCTTCG 3'], [5'
CTCGAGGGCTCTCAGCCGTGTGCT 3'] or [5'
CTCGAGTGTCTGGGCAACCTGGCGCGGG 3']. These prosegment fragments,
ending at as 169, 188, and 196 (10), were cloned in the PCR 2.1 TA cloning
vector for
sequencing. Then they were transferred into the BamHl / Xhol sites of the
bacterial
expression vector pET 24b (Novagen). These recombinants were transformed into
the
E. Coli strain BL21. Protein expression was induced with 1 mM isopropyl (3-D-
thiogalactoside and the cultures were grown for 3h at 37°C. The cell
pellets were
sonicated on ice in a binding buffer containing 6M guanidine-HCI (Novagen)
until a
clear solution was obtained. The clarified and filtered solution was then
applied to a
nickel affinity column (Novagen) and eluted with 500 mM imidazole. The eluates
were
dialyzed overnight at 4°C against 50 mM sodium acetate (pH 7). The
protein
precipitate was solubilized with glacial acetic acid, filtered through a 0.45
Nm disk and
further purified on a 5 Nm C4 column (0.94 x 25 cm; Chromatographic Sciences
Company Inc; CSC) by reverse-phase high performance liquid chromatography (RP-
HPLC). The purity was assessed by Coomassie staining and the identity of the
products verified by mass spectrometry on a Matrix Assisted Laser Desorption
Time
of Flight (MALDI-TOF) Voyageur DE-Pro instrument (PE PerSeptive Biosystems).
The
amounts of prosegments were determined by quantitative amino acid analysis
(13).
Expression and Purification of Recombinant BTMD-SKI-7 - Following infection
of BSC40 cells (75 x 10B cells) with 2 pfu/cell of recombinant W:BTMD-SKI-1,
the cells
were washed and incubated at 37 °C for 18h in a serum-free minimal
essential
medium (MEM; Life Technologies). Media (45 ml) were then dialyzed,
concentrated
20-fold to 2.2 ml on Centriprep-30's (Amicon) and stored at -20 °C in
40 % glycerol.
For purfication2, the concentrated media were applied to a N~+ affinity resin
(Novagen)
or a Co2'' affinity resin (Clontech Laboratories) as described by the
manufacturer. After
two washes with 5 mM imidazole, the protein was eluted with 200 mM imidazole
and
tested for enzymatic activity and immunoreactivity by Western blot (see
below).
Western Blot Analyses - Aliquots of partially purified BTMD-SKI-1 were
separated by 8 or 12 % SDS-PAGE followed by electro-transfer of the proteins
onto
polyvinylidene fluoride (PVDF) membranes (Schleicher and Schuell). These
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membranes were probed with an antiserum directed against either SKI-1 [aa 217-
233
(Ab:N) or as 634-651 (Ab:S)] or pro-SKI-1 [(aa 18-188 (Ab:P)]. Protein bands
were
visualized by enhanced chemiluminescence (ECL) (Boehringer Mannheim).
Purifrcation, N-terminal Sequencing and Mass Spectrometric Analysis of the
Secreted Recombinant Prosegment(s) of hSKI-9 - Concentrated media obtained
from
either W:BTMD-SKI-1 infected BSC40 cells or from a stable transfectant of full-
length
hSKI-1 in HK293 cells (10) were loaded onto an RP-HPLC 5 Nm C4 column (0.94 x
25
cm) (Vydac). Proteins were eluted at 2 ml/min using a 1 %/min linear gradient
(15-70
%) of 0.1 % aqueous trifluoroacetic acid (TFA)/CH3CN with monitoring at 210
nm. The
products were analyzed by Western blotting, after which the immunoreactive
fractions
were further purified on a CSC 5 Nm C4 column {0.2 x 25 cm). Mass values were
obtained by MALDI-TOF spectrometry using the 'matrix 3,5 dimethoxy-4-
hydroxycinnamic acid (Aldrich Chemical Co). For N-terminal sequencing,
fraction IV
proteins (Fig. 21A) were separated by SDS-PAGE, transferred to Immobilon-P
membranes, and stained with Ponceau Red. The 14 and 5 kDa bands were excised
and sequenced using an Applied Biosystems Model 477 sequenator operating in
the
gas-phase mode (15).
Synthesis of Peptide Substrates - All Fmoc amino acid derivatives (L-form),
the
coupling reagents, and the solvents for peptide synthesis were purchased from
PE
Biosystems Inc. {Framingham, Mass, USA), Calbiochem (San Diego, Ca, USA), or
Richelieu Biotechnologies (Montreal, QC, Canada). The various linear synthetic
peptides and internally quenched fluorogenic (Q-) substrates reported in this
article
are: (I) hproBDNF(50-63): KAGSRGLTSLADTF, (II) hSREBP-2(504-530):
GGAHDSDQHPHSGSGRSVLSFESGSGG, (III) hSKI-1(174-191):
WHATGRHSSRRLLRAIPR, (IV) hSKI-1 (174-188+LE): WHATGRHSSRRLLRALE, (V)
hSKI-1 (182-188+LE): SRRLLRALE, (VI) hSKI-1 (156-172):WQSSRPLRRASLSLGSG,
(Vll) hSKI-1(187-201): RAIPRQVAQTLQADV; (VIII) hSKI-1(128-136): PQRKVFRSL;
(IX) hSKI-1(128-142): PQRKVFRSLKYAESD; (X) Q-hSKI-1(132-142): Abz-
' Although we managed to produce limited quantities of partially purfied SKI-1
using
metal chelating resins, there was insufficient enzyme to carry out full
kinetic analyses.
However, since the medium of WT virus-(or control vector)-expressing cells
produced no
significant peptide hydrolysis (with the exception of peptides VIII and IX),
we mainly used
the concentrated media of BSC40 cells infected with W:BTMD-SKI-1. Thus, the
metal
chelation-purified enzyme served mainly to verify that the enzyme from
concentrated media
behaved similarly to this form. We therefore confirmed all of the peptide
cleavage sites, the
SREBP-2 pH optimum, and the Ca2+ requirement presented below.
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VFRSLKYAESD-Y(N02}-A; (XI) Q-hSKI-1(134-142): Abz-RSLKYAESD-Y(NO }~.
Except for the first two peptides, which were purchased from the Sheldon
Biotechnology Institute (McGill University, QC, Canada), all other peptides
were
synthesized with the carboxy-terminus in the amide form. Peptides II I-XI were
prepared
on a solid phase peptide synthesizer (Pioneer model, PE Biosystems) using
either 2-
(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluophosphate (HBTU) /
N-
hydroxybenzotriazole (HOST) or HATU (O-[7-azabenzotriazol-1-yl]-N,N,N',N'-
tetramethyluronium hexafluorophosphate) / diisopropyl ethyl amine (DIEA)-
mediated
Fmoc chemistry with PAL-PEG unloaded resin and the standard side chain
protecting
groups (16). For the incorporation of the two unnatural amino acids [Abz and
Y(N02)],
an extended coupling cycle was used instead of either the standard or fast
cycles.
Purification, Analysis, and Digestion of Peptide Substrates - The crude
peptides
were purified by RP-HPLC using a semi-preparative CSC-Exsil C18 column (2.5 x
25
cm). Monitoring at 210 nm, the peptides were eluted with a 1 %/min linear
gradient (5
% to 60 %) of aqueous 0.1 % TFAICH3CN at 2 ml/min and. The peptide purity and
concentration were determined by quantitative amino acid analysis (16). The
identity
of each purified peptide was conflmied by MALDI-TOF spectrometry using the
matrix
a-cyano 4-hydroxycinnamic acid (Aldrich Chemical Co).
For digestions, each peptide was typically reacted at 37 °C with 10 NI
of the
concentrated enzyme preparation in a buffer consisting of 50 mM HEPES (N-2
Hydroxyethyl Piperazine-N'-2 EthaneSulfonic acid) (ICN Biomedicals Inc), 50 mM
MES
(2-[N-Morpholino] EthaneSulfonic acid) (Sigma Chem Co.), and 3 mM Ca2i-acetate
(pH
6.5). The digestion products were separated by RP-HPLC on a Beckman 5 Nm
Ultrasphere C18 column (0.2 x 25 cm) and eluted with a 1 %Imin linear gradient
of
aqueous 0.1 %TFA/CH3CN (5-45 %) at a flow rate of 1 mllmin. The collected
peptides
were characterized by mass spectrometry and amino acid composition, which was
also
used to quantitate the amount of various substrates and products. The
digestions of
the quenched fluorogenic peptides were analyzed by RP-HPLC using a dual UV
(210
nm) and fluorescence (excitation and emission wavelengths of 320 and 420 nm,
respectively) detector (Rainin).
pH Optimum, Calcium-Dependence and Inhibitor Profrle - The protocols used
were essentially the same as reported previously (13). Stocks of the buffer
described
above were adjusted to pH 5.0-8.5 at 0.5 unit increments by addition of either
acetic
acid or sodium hydroxide. In order to investigate the calcium requirement of
SKI-1,
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increasing concentrations of Ca2'-acetate were used ranging from 0 to 10 mM.
For
inhibition studies, the enzyme in the reaction buffer was preincubated with
the desired
agents for 30 min prior to addition of peptide II.
K,"~,~~, Vmex~,,~~ and Ku,~~ determinations - Following digestion reactions
with
increasing substrate concentrations, the products were separated by RP-HPLC.
The
rate of substrate hydrolysis was obtained from the integrated peak areas of
the
chromatograms. K,~e~~ and V"",~~~ values were estimated using nonlinear
regression
analysis (Enzfitter software; Elsevier Biosoft, Cambridge, UK) of plots of the
hydrolysis
rate vs the substrate concentration. For apparent inhibitor constant
[IC~~,Pp~]
determinations, variable inhibitor concentrations within the range of 15-70 %
inhibition
were used at three concentrations of peptide IV ranging from 0.6 to 3.5 times
the K,~app~
value. The K~~P~ values were estimated from Dixon plots as described (16). For
the two
quenched peptides, kinetic parameters were determined as described (17).
RESULTS
SKI-9 Overexpr~ession, Purification, Biosynthesis, and Prosegment Processing
We have previously shown that overexpression of full-length SKI-1 (FL-SKI-1 )
in HK293 cells results in shedding of a 98 kDa form (sSKI-1) of this enzyme
into the
medium (10). Based on this finding, we engineered a soluble form of SKI-1
(BTMD-
SKI-1), ending at residue 997, to which we added a hexa-His sequence at the C-
terminus (Fig. 19A). In a comparative biosynthetic analysis, shown in Fig.
19B, LoVo
cells were infected with the SKI-1 virus constructs W:FL-SKI-1, W:BTMD-SKI-1,
and
wild type virus (W:WT). After labeling the cells for 3h with [35S]Cys,
proteins in the
media were immunoprecipitated with an antiserum directed against either the
prosegment of SKI-1 (Ab:P) or an internal SKI-1 sequence (Ab:S). In both
cases, a
protein of ~14 kDa co-immunoprecipitated with the 98 kDa sSKI-1 or the 100 kDa
BTMD-SKI-1 (bSKI-1, Fig. 19B) that was not seen with W:WT infections. Since
Ab:P
was raised against a recombinant SKI-1 prosegment peptide and has been shown
previously to detect the SKI-1 zymogen (10), we concluded that the ~14 kDa
peptide
is most likely derived from the cleaved prosegment (the full-length prosegment
is ~24
kDa - see below). The fact that it co-immunoprecipitated with the enzyme under
denaturing conditions suggests a strong interaction between SKI-1 and this
region of
its prosegment. The actual stoichiometry of enzyme-to-prosegment is not clear
from
this experiment, since it was carried out using two different antisera and
denaturing
conditions. We also observed that some of the 100 kDa BTMD-SKI-1 is cleaved
into
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a 98 kDa form similar to that found with FL-SKI-1 (Fig. 19B). This conversion
is
presumably carried out by endogenous °shedding enzymes" (10,18) that
can act on
both forms of SKI-1, although C-terminal sequencing would be needed to confirm
this
hypothesis.
Western blot analyses of media now obtained from BSC40 cells infected with
W:BTMD-SKI-1 also revealed a secreted 100 kDa immunoreactive band (Fig. 19C).
The same band was detected using either an antiserum against the N-terminal
region
of the SKI-1 catalytic domain (Ab:N) or one against a more C-terminal region
(Ab:S).
When Ab:P was mixed together with Ab: S and used to probe the metal affinity
column-
purified SKI-1 preparation (indicated by the * in Fig. 19C), we were able to
again detect
the ~14 kDa prosegment fragment, further supporting our hypothesis that it
forms a
strong association with the enzyme. (It should be noted that although a
mixture of Ab:S
and Ab:P was used in order to detect both proSKl-1 and BTMD-SKI-1
simultaneously,
when either Ab:N or Ab:S were used alone, only the 100 kDa or 14 kDa species
were
observed, respectively (nof shown)).
In order to evaluate the rate of zymogen processing and the fate of the
prosegment, LoVo cells overexpressing W:FL-SKI-1 were pulse-labeled with
[~H]Leu
for 15 min and then chased for 2h. Figure 20 shows an SDS-PAGE analysis of the
cell
lysates immunoprecipitated with Ab:P (left panel). At least five
immunoreactive
polypeptides (molecular masses of ~26, 24, 14, 10 and 8 kDa) which were not
present
in controls infected with W:WT, were detected. In order to further define in
which
organelles) this processing occured, LoVo cells infected with W:FL-SKI-1- were
pulse-labeled with [3H]Leu for 2h in the presence or absence of BFA (Fig. 20,
right
panel). In both cases, the same five major, intracellular, immunoreactive
prosegment
forms could still be detected. Since the fungal metabolite BFA is known to
disassemble
the Golgi complex and cause the ER to fuse with the cis, medial and frans
Golgi (but
not the trans Golgi network, TGN) (19), this result strongly implies that the
initial
zymogen processing of proSKl-1 occurs early along the secretory pathway.
Possible
locations include the ER or cis Golgi, as was previously reported (10).
Moreover,
further processing of the prosegment into yet smaller fragments also occurs in
these
organelles.
To further characterize the prosegment of SKI-1, we took advantage of a stable
transfectant of FL-SKI-1 in human HK293 cells that we had made previously
(10). This
system has the added advantage that the possibility of W overexpression
artifacts
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influencing the processing of the prosegment is eliminated. Concentrated
culture
medium from these cells (serum-free) was purified via RP-HPLC using first a
semi-
preparative C4 column (not shown) followed by an analytical C4 column (Fig.
21A).
The eluted fractions were analyzed by Western blot using Ab:P (Fig. 21 B).
Immunoreactive peptides ranging from ~4.5-24 kDa were apparent. N-terminal
sequencing of the very abundant ~14 kDa protein in fraction IV (Fig. 21 C)
revealed a
major sequence starting at Gly'° of pre-proSKl-1 (10,12). This clearly
defines the signal
peptidase cleavage site as LWLLC"1 GKKHLG, which is one as before that
predicted
by signal peptidase cleavage site algorithms (10,11). The N-terminal sequence
of the
~4.5 kDa polypeptide (Fig. 21 D) revealed that it starts at Pro'43, indicating
a cleavage
at the sequence KYAESD'°21 PTVPCNETRWSQK. This fragment is most likely
the
product of cleavage between Asp and Pro that may be caused by the acidic
conditions
encountered in either RP-HPLC, Edman sequencing (20), or sample preparation
for
SDS-PAGE analysis (21). An unexpected benefit of this cleavage was our finding
that
phenylthiohydantoin (PTH)-Asn'48, which occurs in the putative N-glycosyiation
site
~4snGI~Thr was readily detected in this sequence. Thus, the predicted N-
glycosylation
site Asn'48 within the prosegment of SKI-1 is not employed, at least in this
expression
system. This conclusion was also supported by the prosegment's resistance to
endo
H and endo F digestion (not shown). Of the two eukaryotic subtilases known to
contain
a potential N-glycosyiation AsnGIuThr site, i.e. kexin (22) and SKI-1 (10), it
appears
that at least the tatter's prosegment is not N- glycosylated. Finally, the
separation of
the above prosegment fragments from mature SKI-1 using RP-HPLC (Fig. 21A,B)
and
non-reducing SDS-PAGE (not shown), suggests that none of the Cys residues in
the
prosegment (10) are linked by disulfide bridges to the rest of the enzyme.
As a preliminary means of characterizing the SKI-1 prosegment fragments, MALDI-
TOF analysis (Fig. 21 E) of fraction IV from Fig. 21 B was carried out. Three
major
molecular ions of masses 13,351, 13,518, and 13,685 Da were detected, with an
expected error of + 25 Da for this mass range. Combined with the previous N-
terminal
sequencing results of the ~14 kDa peptide (Fig. 21 C), these mass values
indicate that
this peptide has heterogeneous C-termini that are derived from cleavages near
the
sequence RKV_FRSLI~'3', as indicated in Fig. 21 E. In fact this region
contains three
potential SKI-1 cleavage sites (8) with an R or K at the P4 position and
either an F, R
or K at the P1 position. Although the calculated molecular masses of 13,339,
13,496
and 13,696 for the polypeptides G"KK---RKVF'33, G"KK---RKVFR"4 and G"KK---
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RKVFRSL'3g, respectively, match within experimental error (~ 22 Da) the
observed
masses in Fig. 21 E, these assignments should only be taken as a first
indication (see
below). Moreover, the predicted G"KK--RKVFRSL"e fragment does not correspond
to the expected SKI-1 cleavage motif of a basic residue at the P4 position.
Hence, this
secreted peptide could result either from cleavage at G"KK---RKVFRSL"6 or,
more
likely, at G"KK---RKVFRSLK'3' Lys"' followed by basic carboxypeptidase
cleavage
of the C-terminal Lys (23). Since we were unable to obtain consistent mass
spectra of
the ~4.5 kDa polypeptide that was sequenced in Fig. 21 D, we could not use
this
technique to approximate its C-terminus, which presumably corresponds to the C-
terminus of the processed SKI-1 pro-segment. We therefore resorted to
synthetic
peptide cleavage as a tool to accurately define potential prosegment cleavage
sites.
Analysis of Synthetic Prosegment derived Peptide Cleavages -Based on our
detection of ~26and 24 kDa SKI-1 prosegment products (Fig. 20), as well as on
a
mutagenesis study of SREBP-2 cleavage sites (8), we synthesized three SKI-1
prosegment peptides encompassing potential, C-terminal, autocatalytic cleavage
sites
(10,11 ). All contain Arg at P4 and either Leu, Lys, Ala or Phe at P1
(peptides III, VI and
VII shown in Table II-A). Of these peptides containing only native sequences,
the only
one with detectable cleavage by SKI-1-containing concentrated medium (from
either
W:BTMD-SKI-1-infected BSC40 cells or SKI-1 transfected HK293 cells) was
peptide
Ill (WHATGRHSS$RL,~'~1RAIPR) (see Table II-A). No cleavages were observed
when W:WT-infected or empty vector-transfected media were used (not shown).
Metal chelation chromatography-purified enzyme further supported that this
cleavage
is effected by SKI-1 (Fig. 22A; peptide III), and the products were positively
identified
via mass spectrometry.
Similarly, based on the mass spectrometry data in Fig. 21 E, we synthesized
two
peptides (VI II and IX) encompassing the putative internal processing sites)
of the SKI-
1prosegment. Both were cleaved at multiple locations by SKI-1-containing
concentrated medium from HK293 transfectants (not shown). Further analysis
revealed
that one of these cleavages, corresponding to PQRKVF'331$SL, was as prevalent
in
empty vector-transfected HK293 medium as in SKI-1-transfected medium (see.
Table
III-A, peptide VIII). In contrast, the PQRKVF$SLI~'3' 1 YAESD cleavage was
only seen
in SKI-1-containing medium. This cleavage was also confirmed using metal
chelation
chromatography-purified enzyme (Fig. 22B; peptide IX) and mass spectrometry to
identify the products. However, also clearly visible are the
PQRKVF'331$SL~YAESD
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cleavage products. We acknowledge that there could be residual contaminating
proteases in our purified SKI-1 preparations (minor bands were visible on
colloidal
gold-stained membranes of SKI-1 preparations). Thus, while we are confident
that SKI-
1 cleaves its prosegment at the C-terminal WHATGRHSS$RL~,'esl RAIPR site and
at
the internal PQRKVF$SL~'3' 1 YAESD site, our data do not allow us to rule out
SKI-1-
mediated cleavage at the PQRKVF"31$SL~YAESD site.
Comparing the simple cleavage rates of the SKI-1 prosegment internal and C-
terminal sites, we observed that the former was vastly superior to the latter
(not
shown). We also noticed that the peptides best processed by SKI-1 contain an
acidic
residue at the P3' or P4' substrate site, whereas those that did not appeared
to be
cleaved poorly or not at all (Table III-A). Moreover, we had previously
established that
SKI-1 does not cleave the fluorogenic peptides RGLT-MCA, RGLTT-MCA and RSVL-
MCA (10), which lack P' residues. Based on these observations, we asked if
replacing
the Ile and Pro residues at P3' and P4' of the C-terminal prosegment
processing site
would significantly improve the SKI-1-mediated cleavage of peptide III. Thus,
we
synthesized two mutants of this peptide (peptides IV and V, the latter
truncated by 8
as at the N-terminus) in which the Ile and Pro residues at P3' and P4' were
replaced
by Leu and Glu, respectively. As shown in Table II-B, this change
significantly
improved the processing of these peptides, such that we were able to determine
Vmax(app) ~ "m(app) Values. The approximately two-fold difference in these
values for
peptides IV and V further suggests that determinants N-terminal to the P4
position may
also play a role in substrate specificity. The SKI-1 specificity of these
peptide
cleavages was also verified using metal chelation chromatography-purified
enzyme
(when W:WT-infected or empty vector-transfected media were used, no peptide
processing was observed).
In Vitro Kinetic Properties of SKI-1: Comparative Analysis of Synthetic
Peptide
Cleavages -In a previous report (10), sSKI-1 was shown, to cleave the 32 kDa
proBDNF into a 28 kDa form at the $GL~1 SL sequence in vitro with a pH optimum
close to neutrality. Similar to PCs (1-3), we suggested that SKI-1 might be a
Ca2+-
dependent enzyme since the calcium ionophore A23187 inhibited the ex vivo
cleavage
of proBDNF (10). In order obtain kinetic analyses of defined SKI-1 substrates,
we
examined a 14 as peptide spanning the hproBDNF processing site (10),
KS°AGS$GLI1 SLADTFg' (peptide I) and a 27 as hSREBP-2-related
peptide (8),
G~GAHDSDQHPHSGSG$SVL_1 SFESGSGGS'° (peptide II). Concentrated SKI-
1-
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containing medium (from either W:BTMD-SKI-1-infected BSC40 cells or SKI-1
transfected HK293 cells) was reacted with these peptides at pH 6.5, followed
by
MALDI-TOF mass spectrometric analysis of the RP-HPLC-purified products. The
expected cleavages were confirmed and did not occur using WT-/empty vector-
derived
media (Fig. 23). Again, the metal chelation chromatography-purified enzyme
generated
the same products as the concentrated media (not shown). We then demonstrated
that
the optimal pH and calcium concentrations for efficient cleavage of the hSREBP-
2
peptide (II) are pH 6.5 and 2 mM Ca2+, respectively (Fig. 24). Interestingly,
the pH
optimum observed with the the proBDNF peptide (I) is sharper than that
obtained with
peptide II. In the former case, the enzyme still retains about 30% of its
activity at pH
5.0 and 55 % of its activity at pH 8.5 (Fig. 24A). Similar results for the pH
optimum of
peptide II cleavage were obtained with metal chelation-purified BTMD-SKI-1
(not
shown). In contrast, however, the pH optimum of peptide IX with the purified
enzyme
was 8.0, with no activity detectable below pH 5.5.
A summary of the kinetic analyses of the synthetic proBDNF (peptide I) and
SREBP-2
(peptide 11) cleavages by SKI-1 is shown in Table II-B. Both peptides are
cleaved at
comparable kinetic efficiencies with V,"~,~c,pp~ / KmcaPP~ values of 0.002 and
0.004 h-',
respectively. In comparison, the V",~cePp~ / K,"caPp~ value estimated with
peptide IV is 5-
10-fold higher than those obtained with peptides I and If (Table 11-B). The N-
terminal
truncation of peptide IV from 17 to 9 as (peptide V, Table II-A) caused a 4-
fold
reduction in catalytic efficiency (Table I1-B).
Table II I shows the inhibitor profile of SKI-1, in which it is clear that
this enzyme
is quite sensitive to metal chelators such as EDTA and to the calcium chelator
EGTA.
In addition, the transition metals Cu2' and Zn2+, but not Ni2+ or Co2',
inhibit the enzyme
at mM concentrations. As reported using the 32 kDa proBDNF (10), assays with
the
synthetic SREBP-2 peptide demonstrated that the metal chelator o-
phenanthroline
becomes inhibitory at concentrations above 1 mM. The other non-chelator
inhibitors
tested had minimal or no effects on SKI-1 activity.
In order to develop a convenient in vitro assay for SKI-1, we designed a
number of
internally quenched fluorogenic substrates and tested their cleavage efficacy
by SKI-1.
The two best peptides encompassed the processing site RSLK1 within the hSKI-1
prosegment (peptides X and XI, Table II-A). Mass spectrometric analysis
confirmed
that both peptides were cleaved at the $SL~1 site by shed SKI-1 derived from
HK293
cell transfects, but not by medium obtained from HK293 empty vector
transfectants.
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This processing generated the fluorescent N-terminal peptides Abz-VFRSLK or
Abz-
RSLK, and a non-fluorescent C-terminal peptide YAESDY(NOZ)-A (not shown).
Measurements of kinetic parameters demonstrated that peptides X and XI are
about
3- and 16-fold better substrates than the C-terminal prosegment peptide IV
(Tables I-B
and III), suggesting that the shorter peptide XI may be the best SKI-1
substrate tested
to date. This cleavage was completely abolished in the presence of 10 mM EDTA,
in
agreement with the Ca2+-dependence of SKI-1 activity (Fig. 24B).
SKI-1 Inhibition by its Prosegment =One important question remaining is
whether the SKI-1 prosegment functions as an inhibitor of its enzymatic
activity,
analogous to the prosegments of other subtilases (3). We thus prepared
prosegment
constructs, designated ending near the proposed C-terminal processing site
RRLL'es
(Fig. 22A): PS1, extending to Leu's9; PS2, extending'to Ala'88; and PS3,
extending to
Leu'9' To each C-terminus we coupled a hexa-His tag. These prosegment
constructs
were expressed in bacteria and purified by Ni2+-chelation chromatography
followed by
RP-HPLC (see Experimental Procedures). The purity of these prosegments was
confirmed by SDS-PAGE/Coomassie staining and as analysis (notshown). A summary
of the inhibitory potency of each prosegment using peptide IV as a substrate
is shown
in Table V. Kinetic analysis using Dixon plots (15) indicated a competitive
inhibition
mechanism (not shown). Although PS2 exhibits the best apparent inhibitory
constant
(K;~aPP~ = 97 nM), PS3 (KK,~P~ = 127 nM) and PS1(K;~,PP~ = 182 nM) are
similarly potent
SKI-1 inhibitors. When PS2 was digested with carboxypeptidase B to eliminate
the His-
tag, its inhibitory potency was not affected (not shown), confirming that this
tag is not
responsible for the observed inhibition. We also tested the inhibitory
activity of the RP-
HPLC-fractionated native prosegment (see Fig. 21). Only, the material from
fraction
IV, which included the full-length ~24 kDa prosegment, was inhibitory, whereas
that of
the others , including the ~14 kDa peptide alone or in combination with
smatter
fragments, were not inhibitory (not shown).
DISCUSSION
Limited proteolysis of inactive precursor proteins at sites marked by paired
or
multiple basic residues is a widespread process (1,2). Less common is the
recent
finding that bioactive peptides or proteins can also be generated by limited
proteolysis
after either hydrophobic or small residues (3). SKI-1 represents the first
mammalian
member of subtilisin-like processing enzymes with such substrate specificity
(10,11 ).
It is a widely expressed enzyme (10) that may play a crucial role in
cholesterol and
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fatty acid metabolism (11). Due to its very recent discovery, information
regarding its
enzymatic properties, substrate specificity, and the function of its proregion
have only
begun to be addressed.
Many peptidyl hydrolases, including subtilases, possess a prodomain which
acts both as an intramolecular chaperone and a highly potent inhibitor of its
associated
protease (24,25}. Activation of the enzyme typically requires release of the
prosegment
in an organelle-specific manner. For furin (26) the release occurs in the TGN,
whereas
for PC1 and PC2 (27) it occurs in immature secretory granules. The data
presented
in this report demonstrate that SKI-1 is unique among the mammalian
subtilases, since
,r 10 both the C-terminal and internal cleavages of its prosegment occur in
the ER. Hence,
this enzyme does not appear to require an acidic environment for activation,
assuming,
by analogy with other subtilases (3), that prosegment release is the crucial
step leading
to zymogen activation. We propose the following sequence of events presumably
leading to SKI-1 activation: 1) The signal peptide is removed in the ER by a
signal
peptidase cleavage at LWLLC"1GKKHLG (Fig. 21C). 2) The prosegment is
processed into a non-N-glycosylated polypeptide with an apparent molecular
mass of
~24-26 kDa (Fig. 20). 3) This prosegment is further processed into 14, 10 and
8 kDa
intermediates (Fig. 20). While these multiple cleavages may be catalyzed by
SKI-1
itself, the participation of other proteases cannot be excluded. The major
cleavages
leading to the formation of the ~24 and ~14 kDa products occur within 10 min,
and the
other secondary ones within 30 min (not shown). Since treatment of cells with
BFA did
not significantly alter these processing events, they most likely occur in the
ER (Fig.
20). It is possible that the generation of prosegment fragments from the ~24-
26 kDa
pro-form leads to a loss of inhibition in a fashion similar to that of
subtilisin E (24,25).
Indeed, our results demonstrate that while the full-length prosegment is
inhibitory, its
~14 kDa product is not. Surprisingly, some pro-region-derived polypeptides are
found
associated with SKI-1 in cell culture media. Thus, in contrast to furin (26},
the low pH
and high Ca2+ concentrations prevailing in the TGN do not lead to propeptide
dissociation. High ionic concentrations (up to 1M NaCI) such as those used in
immunoprecipitation (Fig. 19B) and metal chelation protein purification (Fig.
19C) also
do not disrupt the complex. It is only during RP-HPLC purification (Fig. 21A),
in the
presence of strong acids and organic solvents, that the prosegment peptides
dissociate from SKI-1. These data suggest that hydrophobic interactions may be
critical, as is the case for subtilisin (24,25).
CA 02349587 2001-05-02
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To distinguish the SKI-1 prosegment autoprocessing sites (C-terminal and
internal)
from several closely situated candidate sites, we employed a combination of
mass
spectrometry and synthetic peptide digestion. In the case of the C-terminal
site, only
one of three candidate peptides (III) was processed by SKI-1 (Table II-A),
indicating
that $RLj~'$giRAIP is the most likely autoprocessing site. For the internal
site,
preliminary mass spectrometric data suggested three distinct cleavages
occuring
within the sequence PQRKVFRSLKYAESD'°2 (Fig. 21 E). Two of the three
possible
sites (PQ$KVF"'1 RSLKYAESD and PQR~VFR'~' 1 SLKYAESD) appeared to satisfy
the proposed SKI-1 recognition motif requiring a P4 basic residue (8). The
third
possibility (PQRKVERSL'~1KYAESD) could be considered by assuming the cleavage
actually occurred at PQRKVF$SLK'3'1YAESD, followed by endogenous, basic
carboxypeptidase removal of the C-terminal Lys residue (23). Assays carried
out in
vifro with synthetic peptides corresponding to this region of the prosegment
(peptides
VIII and IX) produced the same cleavage products (nof shown), but only the
PQRKVF$SLK'3'IYAESD cleavage was unique to SKI-1. Thus, we propose that the
aforementioned site is the most likely internal autoprocessing site, with the
qualification
that PQ$KVF'3' 1 RSLKYAESD may occur to a lesser extent (see Results and Fig.
22).
Other information regarding the substrate preferences of SKI-1 was obtained by
replacing the P3' and P4' lle and Pro residues of the C-terminal cleavage site
peptide
(III) by Leu and Glu (peptides IV and V) to create a very well processed SKI-1
substrate. While it would appear that the presence of an acidic residue at P4'
significantly enhances the rate of substrate hydrolysis, it is also possible
that the
presence of Pro at P4' hinders efficient substrate processing. The presence of
similar
acidic residues at the P3' or P4' position of the two confirmed substrates of
SKl-1
(peptides I and I I) as well as in the prosegment internal cleavage site
RSLK"' 1 YAKS
(Table II-A) lends support to the first argument. In addition to these
residues, others
also appear to play a role in SKI-1 substrate cleavage catalysis. The peptide
pairs IVIV
and X/XI both point to influences of positions N-terminal to the P4 residue.
Interestingly, the efficiency of the truncated C-terminal peptide V is lower
than that of
peptide IV, whereas that of the truncated internal (quenched) peptide XI is
higher.
Taken together, these data indicate the importance of as at both the P and P'
positions
in SKI-1-mediated substrate hydrolysis.
The data presented in Fig. 24 indicate that SKI-1 functions most efficiently
near neutral
pH and at 2-3 mM Caz'. This is in general agreement with the conditions that
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
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reportedly prevail in the ER (28,29). However, closer examination of the data
reveal
that the pH optimum of SREBP-2 cleavage (peptide II, Fig. 24A) is actually
6.5, an
observation that we confirmed using our purified SKI-1 preparation (not
shown). This
suggests that the processing of SREBP might occur outside of the ER, perhaps
in the
Golgi where pH values of ~6.5 have recently been reported (30,31 ). Indeed,
there is
now cellular evidence suggesting that SREBP cleavage may occur in the Golgi
rather
than in the ER (32,33). The pH optimum of SKI-1 appears to be dependent on the
substrate employed; proBDNF (10) and its related peptide (I), appear to be
well
cleaved even at pH 5.5, suggesting that it could cleave this (and possibly
other
substrates) in acidic endosome-like compartments where it was previously
localized
(10). On the other hand, cleavage of the internal, autocatalytic, prosegment
processing
site PQRKVF~SLK"' 1 YAESD (Fig. 22B) is optimal at pH 8 (not shown), implying
that
this event, as we concluded from our biosynthesis assays, takes place most
effectively
in the ER. Overall, the pH and Ca2+ profiles of SKI-1 resemble those of the
constitutively secreted PCs (1,13). The inhibitor profile of SKI-1 (10, Table
III), showing
that enzymatic activity is significantly inhibited by EDTA, EGTA and only high
concentrations of o-phenanthroline, tend to discount the likelihood that SKI-1
is a
transition metal-dependent proteinase. In fact, SKI-1 activity is inhibited by
low
concentrations of certain transition metals, such as Cu2+ and Zn2+.
Directed by the observation that peptides containing the primary processing
site
of the prosegment of PC1 are potent inhibitors of its activity, and that the C-
terminal
basic residues of furin and PC7 are essentiai for enzyme inhibition (34,35),
we
assessed the inhibitory potency of three SKI-1 recombinant propeptides. All of
these
end at sequences near the RRLL'~RA cleavage site. Interestingly, the three
prosegments displayed comparable inhibitory potencies (Table ~. Compared to
proPC1 (34), pro-furin and proPC7 (35), the KKaPP~ values (Table ~ are up to
250 fold
higher. This suggests that the prosegment of SKI-1, although potentially
inhibitory in
vivo, may function more as a chaperone, catalyzing the productive folding of
SKI-1.
Indeed, since SKI-1 may be active in the ER (10,11), whereas the PCs are not
(13,26),
the lower inhibitory potency of the prosegment of SKI-1 may be adapted to the
conditions prevailing in this cellular compartment. In the case of PCs, highly
effective
inhibition by the prosegment may be needed in order to ensure that these
enzymes are
activated only when they reach the TGN or secretory granules (1-3). The 14 kDa
fragment, which represents the major secreted form of the prosegment, is
tightly
CA 02349587 2001-05-02
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associated with SKI-1 (Fig. 19C) yet it is not inhibitory (not shown).
Accordingly, this
segment may serve a chaperonin-like function similar to that reported for the
N-
terminal 150 as of 7B2 towards proPC2 (36,37).
Two articles describing the processing, purification and in vitro activity of
hamster SKI-11S1 P were published (38,39). On most points, our results are in
close
agreement with those recently published. Thus, these authors characterized the
processing of the SKI-11S1 P prosegment, proposing that the ER is the major
site of
autocatalytic activation of SKI-1 at the same cleavage sites as we present
here. They
also went on to purify a soluble form of the enzyme, showing that it correctly
processes
SREBP-2 derived peptides as well as a 16 residue peptide spanning the internal
prosegment cleavage site. In addition, they find that cleavage of fluorogenic
RSLK-
MCA peptide derived from the same sequence is optimal at ~3 mM Ca2+ at
slightly
alkaline pH. Discrepancies such as the lack of detectable shed SKI-1/S1 P,
multiple
secreted prosegment forms, and a different signal peptidase site can most
likely be
attributed to the different cell types and species employed in the two
studies.
In conclusion, the present work firmly establishes that SKI-1 is a Ca2+-
dependent subtilase with a reasonably neutral pH optimum, depending on the
substrate employed.We also demonstrate that SKI-1 can cleave substrates C-
terminal
to Thr, Leu and Lys residues, thus providing direct, in vitro evidence that it
is a
candidate converting enzyme responsible for the generation of 28 kDa proBDNF
(10)
and SREBP-2 processing at site 1 (11 ). For efficient cleavage, it appears
that
substrates should contain a basic residue at P4 and an aliphatic one at P2
(Table ll-A).
Furthermore, as at the P3' and P4' positions seem to exert an important
discriminatory
effect. The best substrate tested so far is the quenched flurorogenic
substrate Abz-
RSLK _ YAESDY(N02), thereby providing a convenient and sensitive assay for SKI-
1
activity. The present data demonstrate that only the full length SKI-1
prosegment is
inhibitory. Thus, overexpression of this prosegment in cell lines may provide
a novel
method for inhibiting the cellular activity of this enzyme in a fashion
similar to the that
of over-expressed profurin and proPC7 (35). Finally, it is anticipated that
precursor
substrates other than the sterol regulating SREBPs (8) and the neurotrophin
proBDNF
(10) will be identified, thereby extending the spectrum of activity of this
unique and
versatile enzyme.
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Table II-A
Synthetic peptide substrates
Peptides were first reacted with approximately equal quantities of BTMD-SKI-1
medium for 2-18 h as described in "Experimental Procedures". When cleavage was
not
detected, a 10-fold concentrated enzyme preparation was tested. Arrow
thickness is
a qualitative estimate of the cleavage efficacy.
I Peptide P16 P12 P8 P4 P1 P4' PS'
1O I K A S R G L S L T F
~ G T _ A
D
II G G A H D S D PH S G R S V S F G S G
Q H G S L _ E G
S
III W H A TG R S R R L R A R
H S L 1 I
P
IV W H A TG R S R R L R A
H S L - L
E
V S R R L R A
L L
8
VI 1 W Q SS R R - S L G
I P L R A S G
L - S
VII 1 R A P R Q V Q T A D V
I A - L
Q
VIII Z P Q R K V R S
F - L
I IX a,3 pQ R F R S L Y A D
K V K _ E
S
X Abz-V F R S L Y A D Y (NOZ)
K E -A
S
2O XI - Y A D Y(NOZ)
~ Abz-R E -A
S L S
K -
' No cleavage detected even with a 10-fold excess of enzyme.
2 Cleavage detected but not attributable to SKI-1.
3 Kinetic determinations of this peptide were not attempted due to the
presence of
multiple cleavages.
Table II-B
Kinetic constants for the hydrolysis of peptide substrates by BTMD-hSKI-1
Increasing concentrations of peptides were reacted with identical quantities
of
BTMD-SKI-1 medium for times chosen to produce 5-30 % substrate hydrolysis.
Data
analysis was carried out as described in "Experimental Procedures". The values
are
averages of duplicate assays.
Peptide Km(app} vmax(app) Vmax(app} ~
Km(app)
( nM*1000) (nmoUh) (h-' L'')
I 169 0.4 0.002
I I 124 0.5 0.004
IV 17 0.4 0.023
V 109 1.1 0.010
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Table III
Effect of selected protease inhibitors on BTMD-hSKI-1 activity
Digestion reactions using BTMD-SKI-1 medium plus peptide II were carried out
as described in "Experimental Procedures". The agents were preincubated with
the
enzyme for 30 min.
Inhibitor Concentration Hydrolysis of
SREBP-2 peptide
(mM) (% of control)
'
Control - 100
APMSF , 95
1.0
PMSF 1.0 85
~
TPCK 1.0 71
TLCK 1.0 100
SBTI 0.5 2 100
Cystatin 0.01 100
Antipain 1.0 100
~
Chymostatin 1.0 100
Leupeptin 1.0 100
Pepstatin 0.1 97
E-64 0.01 100
O-Phenanthroline 0.05 135
~
1.0 90
5.0 0
EDTA 10.0 0
EGTA 10.0 15
Dithiothreitol 10.0 92
~
CuS04 1.0 0
ZnS04 1.0 0
NiSO, 1.0 93
MgCl2 1.0 100
CoCl2 1.0 100
~
' Values represent averages of duplicate assays (variation is + 5 %).
2 Concentration in mglml.
Table IV
Kinetic constants for the hydrolysis of quenched fluorogenic substrates by
shed-hSKI-1
Assays and data analysis were carried out as described in Table II-A. The
values are averages of duplicate assays.
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WO 00/26348 PCT/CA99/01058
- 49 -
Peptide Km(aPP) vmax(app) Vmax(aPP) / Km(aPP)
(NM) (Nmoles/h) (h'' L-')
X 31.3 34.0 1.1
~ XI 8.7 56.9 6.5
Table V
Effect of pro-segment peptide constructs on BTMD-hSKI-1 activity
Digestion reactions using BTMD-SKI-1 medium plus peptide IV were carried out
as described in "Experimental Procedures". The prosegment peptides were
preincubated with the enzyme for 30 min. Values were deduced from the Dixon
plots
obtained from three separate experiments.
Pro-segment construct K~(aPP)
(nM)
PS1 182.0 t 0.5
PS2 97.5 t 4.5
PS3 127.3 t 6.2
EXAMPLE 4
SIMILARITY OF ANATOMICAL DISTRIBUTION OF SKI-1 mRNA TO THAT OF APP
~-amyloid precursor protein ( ~-APP ) is a member of a highly conserved gene
family, which includes amyloid precursor-like protein-1 and amyloid precursor-
like
protein-2 { McNamara, M.J. et al. (1998) Brain Research 804, 45-51;
Rassoulzadegan,
M. et al. (1998) The EMBO Journal 17, 4647-4656 }. Mammalian subtilases,
exempl~ed by SKI-1, may be responsible for limited cleavage at hydrophobic
residues
present in biologically important precursor proteins such as f3-amyloid
precursor
protein ( f3-APP ) ( TabIeVl). SKI-1 has recently been identified as the
enzyme which
cleaves sterol-regulatory element-binding protein (SREBP) in a fashion
analogous to
the f3-secretase cleavage of APP { Sakai, J. et al. (1998) Molecular Cell 2,
505-514 }.
The cleavage of SREBP by SKI-1 ( Site 1 protease ) at a position 20 residues
to the
lumenal side of the first membrane-spanning segment is analogous to the ~i-
secretase
cleavage of (i-APP at a position 28 amino acids from the membrane { Brown,
M.S. and
Goldstein, J.L. (1997} Cell 89, 331-340 }.
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
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Similarity of anatomical distribution of SKI-1 mRNA to that of APP suggests a
functional link between both proteins.
In situ hybridization performed in 4-day-mouse provides evidence of a similar
distribution of mRNA coding for the membrane proteins SKI-1 and APP ( Fig. 25
).
Their spatial distribution was observed to be significantly overlapping within
different
tissues such as brain and spinal cord, cranial and spinal ganglia,
submaxillary gland,
thymus, kidney, bones, skin and many other. Their mRNA distribution was
partially
similar to that of two other proteases, namely the convertase furin and the
peptidase
neprilysin. A much different distribution was observed with convertases PC1,
PC2 and
PCS. It is clearly established that an increase in cellular cholesterol levels
results in
the inhibition of activity of SKI-1 I S1 P { reviewed in Edwards, P.A., and
Ericsson, J.
(1999) Annu. Rev. Biochern. 68, 157-185 }. In a similiar fashion, an increase
in dietary
cholesterol leds to significant reductions in brain levels of secreted APP
derivatives,
including sAPPa, sAPP~, A~1-40 and A~1-42 { Howland, D.S. et al. (1998) J.
Biol.
Chem. 273, 16576-16582 }. The nature of the relationships between cholesterol,
SKI-1
and APP metabolism are complex.
Cellular association between SKI-1 and APP in facrimal gland. Potential use of
shed SKI-1 in tears as diagnostic tool.
Results of immunocytochemistry performed in mouse lacrimal glands provides
evidence for the presence of SKI-1 and APP in the same cells types, including
intralobular duct epithelial cells and some acinar cells ( Fig. 26 ). The
finding of SKI-1
in the lacrinal gland suggests the possibility of developing a diagnostic
assay
analyzing tears; perhaps based on two -dimensional polyacrylamide gel
electrophoresis for disease diagnosis { Molley, M.P. et al. (1997)
Electrophoresis 18,
2811-2815; Glasson, M.J. et al. (1998) Electrophoresis 19, 852-855; Grus,
F.H., and
Augustin, A.J. (1999) Electrophoresis 20, 875-880; Iskeleli, G. et al. (1999)
Electrophoresis 2fl, 875-880 }.
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
-51 -
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o 0 ~IC f w S I
~ ~I ~" r U
" I U
S-INi-I
US-1 S-1 . -12i -I~ c2 ~
P4 S-I U rl~ t:l N V1
U1 J.. N
.
,
U
~
U
V p, ~p, t l I
, W , I
, p,
_ _ N
_
d.
d.
SUBSTITUTE SHEET (RULE 26)
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EXAMPLE 5
Prodomains in general ( for example furin and PC7 prodomains ) function in
trans when expressed in mammalian cells to inhibit their cognate subtilise-
like
convertase
We have recently shown that the prosegment of furin expressed as an
independent domain ( preprofurin, ppfurin ) can specifically inhibit
neurotrophin
processing. In these assays, successful inhibition requires not only that the
prodomain
enter the secretory pathway, but that it remain there long enough to interact
with the
target PC (most likely furin within the ~TGN ). Figures 27 & 28 depict
vaccinia virus
constructs or transient transfections of prosegments preventing the maturation
of the
neurotrophins NGF and BDNF in Schwann or COS-1 cells, respectively. The modest
inhibition with the prodomain of PC7 ( ppPC7) is most likely due to inhibition
of furin,
since PC7 is a poor effector of proNGF and proBDNF maturation in these cells.
The
complementary experiment to demonstrate selectivity by the prosegment of PC7
will
be carried out once we are able to establish unique in vivo PC7 substrates.
Most proteases from the four major classes ( thiol, aspartic, serine, and
metallo)
are synthesized as inactive precursor molecules with N-terminal extensions
(prosegments ) that play critical roles in folding, stability and regulation
of enzymatic
activity { Khan, A.R., and James, M.N. (1998) Protein Sci. 7, 815-836 }. The
proregions
of the PCs have been shown to function as potent inhibitors of their cognate
enzymes
in vitro. We present data for the first time showing that the expression of a
prosegment
as an independent domain in a cell-based ( ex vivo ) assay functions as a PC
inhibitor
( Figs. 27 and 28 ). In these assays, successful inhibition requires not only
that the
prodomain enter the secretory pathway, but that it remain there long enough to
interact
with the target PC ( most likely furin within the TGN ).
We have shown that expression of full length SKI-1 prosegment ( 22-24 kDa
with sequence ending at the secondary cleavage sequence RHSSRRLL ) inhibits
SKI-
1 activity in stable HK 293 cell lines (Example 2). However, since the
prodomain of
SKI-1 is processed at an internal primary cleavage site RKVFRSLK to give a 14
kDa
N-terminal fragment ( Fig. 29A&B ) we predict that mutation of this site will
generate
an even more effective SKI-1 inhibitor. In fact, in the case of the mouse PC5
prodomain we have shown that mutation of the internal prosegment cleavage site
does
in fact generate a inhibitor of integrin ha, 150 kDa processing to 80kDa and
70kDa
species ( Fig. 15 ).
CA 02349587 2001-05-02
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EXAMPLE 6
SKI-1 Peptide Substrates for fluorescence resonance energy transfer ( FRET )
- Based Proteolysis Assays
A large number of synthetic peptides based on potential cleavage sites in the
hSKI-1 prodomain, proBDNF and the loop region of SREBP-2 were synthesized.
These are:
(i) hSKI-1 (156-172)
Trp-Gln-Ser-Ser-Arg-Pro-Leu-Arg-Arg-Ala-Ser-LeulSer-Leu-Gly Ser-Gly
(ii) hSKI-1 (174-191 )
Trp-His-Ala-Thr-Gly Arg-His-Ser-Ser-Arg-Arg-Leu-Leu 1 Arg-Ala-Ile-Pro-Arg
(iii) hSKI-1 (174-188+Leu+Glu)
Trp-His-Ala-Thr-Gly-Arg-His-Ser-Ser Arg-Arg-Leu-Leu 1 Arg Ala-~eu
(iv) hSKI-1 (181-188+Glu)
Ser-Ser-Arg-Arg-Leu-Leu 1 Arg-Ala-Ile-~l
(v) hSKI-1 (187-201)
Arg-Ala-lle-Pro-Arg-Gln-Val-Ala 1 Gln-Thr Leu-Gln-Ala Asp-Val
(vi) hSKI-1 (128-136)
Pro-Gln-Arg-Lys-Val Phe-Arg-Ser-Leu
(vii) hSKI-1 (128-142)
Pro-Gln Arg-Lys-Val Phe-Arg-Ser-Leu-Lys 1 Tyr-Ala-Glu-Ser-Asp
(viii) hProBDNF (50-63)
Lys-Ala-Gly Ser-Arg-Gly-Leu-Thrl Ser-Leu-Ala-Asp-Thr-Phe
(ix) SREBP-2 27 mer
Gly-Gly-Ala-His-Asp-Ser-Asp-Gln-His-Pro-His-Ser-Gly-Ser-Gly Arg-Ser-Val-
LeulSer-Phe-Glu-Ser-Gly Ser-Gly Gly
(x) SREBP-210 mer
Ser-Gly Ser-Gly-Arg-Ser-Val LeulSer-Phe-Glu-Ser
These peptides were examined as possible substrates of SKI-1. Our data
indicate
that only the peptides (iii), (iv), (vii), (viii) (ix) and (x) are efficiently
cleaved by
the recombinant SKI-1.
NOVEL FLUOROGENIC SUBSTRATE BASED ASSAY OF SKI-1 ACTIVITY:
CA 02349587 2001-05-02
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Based on the results reported above with various synthetic peptides we
designed
a number of internally quenched fluorogenic substrates of SKI-1. Our main goal
vvas to develop a rapid and a sensitive method for the assay of SKI-1
enzymatic
activity. SKI-1 activity was monitored by following the cleavage of suitable
peptide
substrates with HPLC that is often extremely slow and cumbersome. The
following
internally quenched fluorogenic peptides were synthesized and tested as
substrates for SKI-1:
(a) QSKI (132-142):
bz-Val-Phe-Arg-Ser-Leu-Lys lTyr-Ala-Glu-Ser-Asp-T_yrr(NO~) Ala
(b) QSKI (134-142):
-Arg-Ser-Leu-Lys 1 Tyr-Ala-Glu-Ser-Asp-T r )-Aia
(c) QSKI (178-188)
I~z-Arg-His-Ser-Ser-Arg-Arg-Leu-Leu t Arg-Ala-Ile-T_yJfNOz)-Ala
(d) QSKI (181-187+Leu+Glu)
gbz-Ser-Arg-Arg-Leu-Leu 1 Arg Ala-Leu-Glu-T_y r(i NO~)-Ala
(e) QBDNF (4?-58)
A~-Asn-Gly-Pro-Lys-Ala-Gly-Ser-Arg-Gly-Leu-Thr 1 Ser-~vr~N02)-Ala
The main feature of these peptides is the incorporation of finro special amino
acids namely Abz [Ortho amino benzoic acid also known as anthranaiic acid]
and Tyr(N02 ) [3-vitro Tyrosin] at the amino (N-) and carboxy (C-} terminal
end
of the peptide chain respectively. Abz, an electron donor, is a powerful
fluorescent moiety whereas Tyr(NOZ ), an electron acceptor, acts as a
fluorescence quench group. All the above peptides exhibit weak fluorescence
background values (at >~ = 320 nm and )gym = 420 nm). It is expected that upon
cleavage by the proteolytic action of SKI, these peptides will release two
peptide
CA 02349587 2001-05-02
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fragments of which the Abz-containing N terminal part should display a very
high
degree of fluorescence. The net result will be the increase of fluorescence
intensity
that can be measured very accurately with a fluorimeter instrument. This
technique
of measurement of enzymatic activity has been applied to a number of enzymes
{ F. Jean, A. Boudreault, A. Basak, N. G. Seidah and C. Lazure.,., J. Biol.
Chem.,
1995, 270, 19225-19231 }
RESULTS
Our data indicates that among the above quenched fluorogenic peptides,
l~el tide (a) is most effective as a substrate for SKI-1. In fact the
measurement of
kinetic parameters (Vma,~Km) indicted that this peptide is 6-fold more
efficient that
the nearest candidate g ed betide ~k~. HPLC analysis using both UV and
fluorescence detector systems clearly revealed a single site of cleavage in
peptides (a) and (b) (as indicated above by a vertical arrow 1 ), again
reenforcing
the notion that the preferred sequence motif for SKI-1 is characterized by the
presence of an Arg residue at P4 , an alkyl hydrophobic residue at P2 and
possibly
an aromatic hydrophobic residue at P1'. Therefore, peptide (a) is a highly
specific fluorogenic substrate for monitoring the activity of SKI 9
This invention has been described in details hereinabove, and it will be
readily apparent to the skilled artisan that modifications can be made thereto
without departing form the teachings of the present disclosure. These
modifications
are considered within the scope of the present invention, as defined in the
appended claims.
CA 02349587 2001-05-02
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56
Human SKI-1
cagggcacgctgggtcggcggagctgaggctcccagctgtgggcctcgctggcccggtcg
gtcccgtgcgacccagccgcctcgactccgagggtcgacacccggagcgaccgggccagc
1 _________+_________+_________+_________+_______._+_________+60
cccagtctcgcgagagttgggagtaaacagccccgaatggagtgcccaggcgtgttcgcc
gggtcagagcgctctcaaccctcatttgtcggggcttacctcacgggtccgcacaagcgg
61 _________+_________+_________+_________+_________+_________+120
gcggaggcgccgttatcccgggcccgccggccctgagctcccggcggcgcagattggctc
cgcctccgcggcaatagggcccgggcggccgggactcgagggccgccgcgtctaaccgag
121_________+_________+_________f_________+_________+_________+180
acagtggttgattgatcaaccccattggacgttggttctgtggtacaaatggagtacagg
tgtcaccaactaactagttggggtaacctgcaaccaagacaccatgtttacctcatgtcc
181_________+_________+_________+_________+_________+_________+240
actcagtcgtcacggcctgagtgagagaagccttatttccaagatggagaagaagcggag
tgagtcagcagtgccggactcactctcttcggaataaaggttctacctcttcttcgcctc
241_________+_________+_________+_________+_________+_________+300
aaagaaatgaaagcctctcttcaggctgaaccacaaaaggccatgggatttaacttttat
tttctttactttcggagagaagtccgacttggtgttttccggtaccctaaattgaaaata
301_________+_________+_________+_________+_________+_________+360
ttatgttgggcaagactgtaagatggctgatcagtaatgttgcagcttttagctgaaaca
aatacaacccgttctgacattctaccgactagtcattacaacgtcgaaaatcgactttgt
361-________+_________+_________+_________+_________+_________+420
.aaaattcacttttaatcaagaagaaaaaagtgtgatttgaatatatgcaattttatgatc
ttttaagtgaaaattagttcttcttttttcacactaaacttatatacgttaaaatactag
421_________+_________+_________+_________+_________+_________+480
1 M K L V N I W L L L L V V L L 15
atattcgcttgtgaccatgaagcttgtcaacatctggctgcttctgctcgtggttttgct
tataagcgaacactggtacttcgaacagttgtagaccgacgaagacgagcaccaaaacga
981_________+_________+_________+_________+_________+_________+540
16 C G K K H L G D R L E K K S F E K A P C 35
ctgtgggaagaaacatctgggcgacagactggaaaagaaatcttttgaaaaggccccatg
gacacccttctttgtagacccgctgtctgaccttttctttagaaaacttttccggggtac
541_________+_________+_________+_________+_________+_________.~600
36 P G C S H L T L K V E F S S T V V E Y E 55
ccctggctgttcccacctgactttgaaggtggaattctcatcaacagttgtggaatatga
gggaccgacaagggtggactgaaacttccaccttaagagtagttgtcaacaccttatact
601_________+_________+_________+_________+_________+_________+660
56 Y I V A F N G Y F T A K A R N S F I S S 75
atatattgtggctttcaatggatactttacagccaaagctagaaattcatttatttcaag
tatataacaccgaaagttacctatgaaatgtcggtttcgatctttaagtaaataaagttc
661_________+_________+_________+_________+_____.___+_________+720
76 A L K S S E V D N W R I I P R N N P S S 95
tgccctgaagagcagtgaagtagacaattggagaattatacctcgaaacaatccatccag
acgggacttctcgtcacttcatctgttaacctcttaatatggagctttgttaggtaggtc
721_________+_________+_________+_________+_________+_________+780
96 D Y P S D F E V I Q I K E K Q K A G L L 115
tgactaccctagtgattttgaggtgattcagataaaagaaaaacagaaagcggggctgct
actgatgggatcactaaaactccactaagtctattttctttttgtctttcgccccgacga
781-________+_________+_________f_________+_________+_________+890
116T L E D H P N I K R V T P Q R K V F R S 135
aacacttgaagatcatccaaacatcaaacgggtcacgccccaacgaaaagtctttcgttc
ttgtgaacttctagtaggtttgtagtttgcccagtgcggggttgcttttcagaaagcaag
841-________+_________+_________+_________+_________+_________+900
136L K Y A E S D P T V P C N E T R W S Q K 155
cctcaagtatgctgaatctgaccccacagtaccctgcaatgaaacccggtggagccagaa
ggagttcatacgacttagactggggtgtcatgggacgttactttgggccacctcggtctt
901-________+_________+_________+_________+_________+_________+960
156W Q S S R P L R R A S L S L G S G F W H 175
gtggcaatcatcacgtcccctgcgaagagccagcctctccctgggctctggcttctggca
caccgttagtagtgcaggggacgcttctcggtcggagagggacccgagaccgaagaccgt
961_________+_________+_________+_________+_________+_________+1020
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
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176 A T G R H S S R R L L R A I P R Q V A Q 195
tgctacgggaaggcattcgagcagacggctgctgagagccatcccgcgccaggttgccca
acgatgcccttccgtaagctcgtctgccgacgactctcggtagggcgcggtccaacgggt
1021 _________+_________+_________+_________+_________+_________+ 1080
196 T L Q A D V L W Q M G Y T G A N V R V A 215
gacactgcaggcagatgtgctctggcagatgggatatacaggtgctaatgtaagagttgc
ctgtgacgtccgtctacacgagaccgtctaccctatatgtccacgattacattctcaacg
1081 _________+_________+_________+_________+_________+_________+ 1190
216 V F D T G L S E K H P H F K N V K E R T 235
tgtttttgacactgggctgagcgagaagcatccccacttcaaaaatgtgaaggagagaac
acaaaaactgtgacccgactcgctcttcgtaggggtgaagtttttacacttcctctcttg
1141 _-_______+_________+_________+_________+_________+_________+ 1200
236 N W T N E R T L D . D G L G H G T F V A G 255
caactggaccaacgagcgaacgctggacgatgggttgggccatggcacattcgtggcagg
gttgacctggttgctcgcttgcgacctgctacccaacccggtaccgtgtaagcaccgtcc
1201 -________+_________+_________+_________+_________+_________+ 1260
256 V I A S M R E C Q G F A P D A E L H I F 275
tgtgatagccagcatgagggagtgccaaggatttgctccagatgcagaacttcacatttt
acactatcggtcgtactccctcacggttcctaaacgaggtctacgtcttgaagtgtaaaa
1261 -________+_________+_________+_________+_________+_________+ 1320
276 R V F T N N Q V S Y T S W F L D A F N Y 295
cagggtctttaccaataatcaggtatcttacacatcttggtttttggacgccttcaacta
gtcccagaaatggttattagtccatagaatgtgtagaaccaaaaacctgcggaagttgat
1321 _________+_________+_________+_________+_________+_________+ 1380
296 A I L K K I D V L N L S I G G P D F M D 315
tgccattttaaagaagatcgacgtgttaaacctcagcatcggcggcccggacttcatgga
acggtaaaatttcttctagctgcacaatttggagtcgtagccgccgggcctgaagtacct
1381 _________f_________+_________+_________+_________+_________+ 1490
316 H P F V D K V W E L T A N N V I M V S A 335
tcatccgtttgttgacaaggtgtgggaattaacagctaacaatgtaatcatggtttctgc
agtaggcaaacaactgttccacacccttaattgtcgattgttacattagtaccaaagacg
1441 _________+_________+_________+_________+_________+_________+ 1500
336 I G N D G P L Y G T L N N P A D Q M D V 355
tattggcaatgacggacctctttatggcactctgaataaccctgctgatcaaatggatgt
ataaccgttactgcctggagaaataccgtgagacttattgggacgactagtttacctaca
1501 _________+_________+_________+_________+_________+_________+ 1560
356 I G V G G I D F E D N I A R F S S R G M 375
gattggagtaggcggcattgactttgaagataacatcgcccgcttttcttcaaggggaat
ctaacctcatccgccgtaactgaaacttctattgtagcgggcgaaaagaagttcccctta
1561 _________+_________+_________+_________+_________+_________+ 1620
376 T T W E L P G G Y G R M K P D I V T Y G 395
gactacctgggagctaccaggaggctacggtcgcatgaaacctgacattgtcacctatgg
ctgatggaccctcgatggtcctccgatgccagcgtactttggactgtaacagtggatacc
1621 -________+_________+_________+_________+_________+_________+ 1680
396 A G V R G S G V K G G C R A L S G T S V 415
tgctggcgtgcggggttctggcgtgaaaggggggtgccgggccctctcagggaccagtgt
acgaccgcacgccccaagaccgcactttccccccacggcccgggagagtccctggtcaca
1681 -________+_________+_________+_________+_________+_________+ 1740
416 A S P V V A G A V T L' L V S T V Q K R E 435
tgcttctccagtggttgcaggtgctgtcaccttgttagtgagcacagtccagaagcgtga
acgaagaggtcaccaacgtccacgacagtggaacaatcactcgtgtcaggtcttcgcact
1741 _________+_________+_________+_________+_________+_________+ 1800
936 L V N P A S M K Q A L I A S A R R L P G 455
gctggtgaatcccgccagtatgaagcaggccctgatcgcgtcagcccggaggctccccgg
cgaccacttagggcggtcatacttcgtccgggactagcgcagtcgggcctccgaggggcc
1801 _________+_________+_________+_________+_________+_________+ 1860
956 V N M F E Q G H G K L D L L R A Y Q I L 475
ggtcaacatgtttgagcaaggccacggcaagctcgatctgctcagagcctatcagatcct
ccagttgtacaaactcgttccggtgccgttcgagctagacgagtctcggatagtctagga
1861 -________+_________+_________+_________+_________+_________+ 1920
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
wo oon63as rcTicAmo~oss
58
476 N S Y K P Q A S L 5 P S Y I D L T E C P 995
caacagctacaagccacaggcaagtttgagccccagctacatagatctgactgagtgtcc
gttgtcgatgttcggtgtccgttcaaactcggggtcgatgtatctagactgactcacagg
1921-________+_________+_________+_________+_________+_________+1980
496 Y M W P Y C S Q P I Y Y G G M P T V V N 515
ctacatgtggccctactgctcccagcccatctactatggaggaatgccgacagttgttaa
gatgtacaccgggatgacgagggtcgggtagatgatacctccttacggctgtcaacaatt
1981_________+_________+_________+_________+_________+_________+2090
516 V T I L N G M G V T G R I V D K P D W Q 535
tgtcaccatcctcaacggcatgggagtcacaggaagaattgtagataagcctgactggca
acagtggtaggagttgccgtaccctcagtgtccttcttaacatctattcggactgaccgt
2041_________+_________+_________+_________+_________+_________+2100
536 P Y L P Q N G D N . I E V A F S Y S S V L 555
gccctatttgccacagaacggagacaacattgaagttgccttctcctactcctcggtctt
cgggataaacggtgtcttgcctctgttgtaacttcaacggaagaggatgaggagccagaa
2101_________+_________+_________+_________+_________+_________+2160
556 W P W S G Y L A I S I S V T K K A A S W 575
atggccttggtcgggctacctggccatctccatttctgtgaccaagaaagcggcttcctg
taccggaaccagcccgatggaccggtagaggtaaagacactggttctttcgccgaaggac
2161-________+_________+_________+_________+_________+_________+2220
576 E G I A Q G H V M I T V A S P A E T E S 595
ggaaggcattgctcagggccatgtcatgatcactgtggcttccccagcagagacagagtc
ccttccgtaacgagtcccggtacagtactagtgacaccgaaggggtcgtctctgtctcag
2221_________+_________+_________+_________+_________+_________+2280
596 K N G A E Q T S T V K L P I K V K I I P 615
aaaaaatggtgcagaacagacttcaacagtaaagctccccattaaggtgaagataattcc
ttttttaccacgtcttgtctgaagttgtcatttcgaggggtaattccacttctattaagg
2281_________+_________+_________+_________+_________+_________+2390
616 T P P R S K R V L W D Q Y H N L R Y P P 635
tactcccccgcgaagcaagagagttctctgggatcagtaccacaacctccgctatccacc
atgagggggcgcttcgttctctcaagagaccctagtcatggtgttggaggcgataggtgg
2391_________+_________+_________+_________+_________+_________+2400
636 G Y F P R D N L R M K N D P L D W N G D 655
tggctatttccccagggataatttaaggatgaagaatgaccctttagactggaatggtga
accgataaaggggtccctattaaattcctacttcttactgggaaatctgaccttaccact
2401_________+_________+_________+_________+_________+_________+2960
656 H I H T N F R D M Y Q H L R S M G Y F V 675
tcacatccacaccaatttcagggatatgtaccagcatctgagaagcatgggctactttgt
agtgtaggtgtggttaaagtccctatacatggtcgtagactcttcgtacccgatgaaaca
2461_________+_________+_________+_________+_________+_________+2520
676 E V L G A P F T C F D A S Q Y G T L L M 695
agaggtcctcggggcccccttcacgtgttttgatgccagtcagtatggcactttgctgat
tctccaggagccccgggggaagtgcacaaaactacggtcagtcataccgtgaaacgacta
2521_________+_________+_________+_________+_________+_________+2580
696 V D S E E E Y F P E E I A K L R R D V D 715
ggtggacagtgaggaggagtacttccctgaagagatcgccaagctccggagggacgtgga
ccacctgtcactcctcctcatgaagggacttctctagcggttcgaggcctccctgcacct
2581
_______ _____ 2690
__+__ __+_________+_________+_________+_________+
716 N G L S L V I F S D W Y N T S V M R K V 735
caacggcctctcgctcgtcatcttcagtgactggtacaacacttctgttatgagaaaagt
gttgccggagagcgagcagtagaagtcactgaccatgttgtgaagacaatactcttttca
2641_________+_________+_________+_________+_________+_________+2700
736 K F Y D E N T R Q W W M P D T G G A N I 755
gaagttttatgatgaaaacacaaggcagtggtggatgccggataccggaggagctaacat
cttcaaaatactacttttgtgttccgtcaccacctacggcctatggcctcctcgattgta
2701_________+_________+_________+_________+_________+_________+2760
756 P A L N E L L S V W N M G F S D G L Y E 775
cccagctctgaatgagctgctgtctgtgtggaacatggggttcagcgatggcctgtatga
gggtcgagacttactcgacgacagacacaccttgtaccccaagtcgctaccggacatact
2761_________+_________+_________+_________+_________+_________+2820
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
59
776G E F T L A N H D M Y Y A S G C S I A K 795
aggggagttcaccctggccaaccatgacatgtattatgcgtcagggtgcagcatcgcgaa
tcccctcaagtgggaccggttggtactgtacataatacgcagtcccacgtcgtagcgctt
2821_________+_________+_________+_________+_________+_________+2880
796F P E D G V V I T Q T F K D Q G L E V L 815
gtttccagaagatggcgtcgtgataacacagactttcaaggaccaaggattggaggtttt
caaaggtcttctaccgcagcactattgtgtctgaaagttcctggttcctaacctccaaaa
2881_________+_________+_________+_________+_________+_________+2940
816K Q E T A V V E N V P I L G L Y Q I P A 835
aaagcaggaaacagcagttgttgaaaacgtccccattttgggactttatcagattccagc
tttcgtcctttgtcgtcaacaacttttgcaggggtaaaaccctgaaatagtctaaggtcg
2941_________+_________+_________+_________+_________+_________+3000
836E G G G R I V L Y G D S N C L D D S H R 855
tgagggtggaggccggattgtactgtatggggactccaattgcttggatgacagtcaccg
actcccacctccggcctaacatgacatacccctgaggttaacgaacctactgtcagtggc
3001_________+_________+_________+_________+_________+_________+3060
856Q K D C F W L L D A L L Q Y T S Y G V T 875
acagaaggactgcttttggcttctggatgccctcctccagtacacatcgtatggggtgac
tgtcttcctgacgaaaaccgaagacctacgggaggaggtcatgtgtagcataccccactg
3061_________+_________+_________+_________+_________+_________+3120
876P P S L S H S G N R Q R P P S G A G S V 895
accgcctagcctcagtcactctgggaaccgccagcgccctcccagtgqagcaggctcagt
tggcggatcggagtcagtgagacccttggcggtcgcgggagggtcacctcgtccgagtca
3121_________+_________+_________+_________+_________+_________+3180
896T P E R M E G N H L H R Y S K V L E A H 915
cactccagagaggatggaaggaaaccatcttcatcggtactccaaggttctggaggccca
gtgaggtctctcctaccttcctttggtagaagtagccatgaggttccaagacctccgggt
3181_________+_________+_________+_________+_________+_________+3240
916L G D P K P R P L P A C P R L S W A K P 935
tttgggagacccaaaacctcggcctctaccagcctgtccacgcttgtcttgggccaagcc
aaaccctctgggttttggagccggagatggtcggacaggtgcgaacagaacccggttcgg
3241_________+_________+_________+_________+_________+_________+3300
936Q P L N E T A P S N L W K H Q K L L S I 955
acagcctttaaacgagacggcgcccagtaacctttggaaacatcagaagctactctccat
tgtcggaaatttgctctgccgcgggtcattggaaacctttgtagtcttcgatgagaggta
3301_________+_________+_________+_________+_________+_________+3360
956D L D K V V L P N F R S N R P Q V R P L 975
tgacctggacaaggtggtgttacccaactttcgatcgaatcgccctcaagtgaggccctt
actggacctgttccaccacaatgggttgaaagctagcttagcgggagttcactccgggaa
3361_________+_________+_________+_________+_________+_________+3420
976S P G E S G A W D I P G G I M P G R Y N 995
gtcccctggagagagcggcgcctgggacattcctggagggatcatgcctggccgctacaa
caggggacctctctcgccgcggaccctgtaaggacctccctagtacggaccggcgatgtt
3421_________+_________+_________+_________+_________+_________+3480
996Q E V G Q T I P V F A F L G A M V V L A 1015
ccaggaggtgggccagaccattcctgtctttgccttcctgggagccatggtggtcctggc
ggtcctccacccggtctggtaaggacagaaacggaaggaccctcggtaccaccaggaccg
3481_________+_________+_________+_________+_________+_________+3540
1016F F V V Q I N K A K S R P K R R K P R V 1035
cttctttgtggtacaaatcaacaaggccaagagcaggccgaagcggaggaagcccagggt
gaagaaacaccatgtttagttgttccggttctcgtccggcttcgcctccttcgggtccca
3591_________+_________+_________+_________+_________+_________+3600
1036K R P Q L M Q Q V H P P K T P S V * 1053
gaagcgcccgcagctcatgcagcaggttcacccgccaaagaccccttcggtgtgaccggc
cttcgcgggcgtcgagtacgtcgtccaagtgggcggtttctggggaagccacactggccg
3601_________+_________+_________+_________+_________+_________+3660
agcctggctgaccgtgagggccagagagagccttcacggacggcgctggtgggtgagccg
tcggaccgactggcactcccggtctctctcggaagtgcctgccgcgaccacccactcggc
3661_________+_________+_________+_________+_________+_________+3720
agctgtggtggcggctggtttaaaagggatccagtttccagctgcaggtttgttagagtc
tcgacaccaccgccgaccaaattttccctaggtcaaaggtcgacgtccaaacaatctcag
3721_________+_________+_________+_________+_________+_________+3780
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO OO/Z6348 PCT/CA99/01058
60
tgttctacatgggcctgccctcctgtgatgggcagaggctcctggtacatcgagaagatt
acaagatgtacccggacgggaggacactacccgtctccgaggaccatgtagctcttctaa
3781_________+_________+_________+__ ____+________-+_________+3840
cctgtggatcccgtcaggagggacttagtggctctgccgccagtgagacttcccgccggc
ggacacctagggcagtcctccctgaatcaccgagacggcggtcactctgaagggcggccg
3841-_____-__+-________+_________+_________+_________+_________+3900
agctgtgcgcaccaaagactcgggagaactggaaaggctgtctggggtcttctgactgca
tcgacacgcgtggtttctgagccctcttgacctttccgacagaccccagaagactgacgt
3901-________+_________+_________+_________+____-____+_________+3960
ggggaaggatgtactttccaaacaaatgatacaaccctgaccaagctaaaagacgcttgt
ccccttcctacatgaaaggtttgtttactatgttgggactggttcgattttctgcgaaca
3961-________+_________+_________+_________+_________+_________+9020
taaaggctattttctatatttattgttgggaaaagtcactttaaagacttgtgctatttg
atttccgataaaagatataaataacaacccttttcagtgaaatttctgaacacgataaac
9021-________+_________+_________+_________+_________+________.+9080
gaagcaaagctattttttttgtcagtggaatgcagtttttttactattccatcatgagga
cttcgtttcgataaaaaaaacagtcaccttacgtcaaaaaaatgataaggtagtactcct
9081_________+__-______+_________+_________+_________+_________+9190
acaacatagattccatgatctttttaatgacagtacagactgagatttgaaggaaacatg
tgttgtatctaaggtactagaaaaattactgtcatgtctgactctaaacttcctttgtac
4141_________+_________+_________+_________+_________+_________+4200
cacaaatctgtaaaacatagaccttcgctttatttttgtaagtatcacctgccaccatgt
gtgtttagacattttgtatctggaagcgaaataaaaacattcatagtggacggtggtaca
4201_________+_________+_________+_________+_________+_________+4260
tttgtaatttgaggtcttgatttcaccattgtcggtgaagaaaattttcaataaatatgt
aaacattaaactccagaactaaagtggtaacagccacttcttttaaaagttatttataca
4261-________+_________+_________+_________+_________+_________+4320
attacccgtctgaagctt
taatgggcagacttcgaa
4321 ---------+-------- 9338
SUBSTITUTE SI3EET (RULE 26)
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
61
Rat SKI-1
1 GCGAGTAAACATCCCCCGAATGGATACCCGAGGCGTGTTCGCGGCGGAGGCCCCGTTTTC 60
CGCTCATTTGTAGGGGGCTTACCTATGGGCTCCGCACAAGCGCCGCCTCCGGGGCAAAAG
61 CCGGGTCCGCCGATCCCGAGCCTGAGGCGACGCAGATCGGCTCAGAGCGGTGGCTTGGGC 120
GGCCCAGGCGGCTAGGGCTCGGACTCCGCTGCGTCTAGCCGAGTCTCGCCACCGAACCCG
121 TCCTGCTAGATTTGGGTCTGTGGTACAAATGGAGTTTAGGACTCAGTGGACTCGGCCCTA 180
AGGACGATCTAAACCCAGACACCATGTTTACCTCAAATCCTGAGTCACCTGAGCCGGGAT
181 ATGAGAGAAGCCCCCTGTCCAAGATGGAGAAGAAGCGGAGAAAGAAATGAAAGCCTCTTT 240
TACTCTCTTCGGGGGACAGGTTCTACCTCTTCTTCGCCTCTTTCTTTACTTTCGGAGAAA
241 TTGGGCCAAGCTGTGGGTGACCATGGGACTGAGGTTTTCTTTACGTTGGACAAGTCTGTA 300
AACCCGGTTCGACACCCACTGGTACCCTGACTCCAAAAGAAATGCAACCTGTTCAGACAT
301 GGATGGCTGATCAGTAAGGTTGCAGCTTTTAGCGAAAACAGAAATCCACTTCTGATCAAG 360
CCTACCGACTAGTCATTCCAACGTCGAAAATCGCTTTTGTCTTTAGGTGAAGACTAGTTC
1 M 1
361 GAAGAGCCTAGTGCAATTTGAATTTATGCAATTTTATGACCATATTCACTTAGGACCATG 920
CTTCTCGGATCACGTTAAACTTAAATACGTTAAAATACTGGTATAAGTGAATCCTGGTAC
2 K L V N I W L L L L V V L L C G K K H L 21
421 AAGCTCGTCAACATCTGGCTTCTTCTGCTGGTGGTTTTGCTCTGTGGGAAAAAGCATCTG 480
TTCGAGCAGTTGTAGACCGAAGAAGACGACCACCAAAACGAGACACCCTTTTTCGTAGAC
22 G D R L G K K A F E K A P C P S C S H L 91
481 GGTGACAGGCTGGGGAAGAAAGCTTTTGAAAAGGCCCCATGCCCCAGCTGTTCCCACCTG 540
CCACTGTCCGACCCCTTCTTTCGAAAACTTTTCCGGGGTACGGGGTCGACAAGGGTGGAC
42 T L K V E F S S T V V E Y E Y I V A F N 61
541 ACTTTGAAGGTGGAATTCTCCTCAACTGTGGTGGAATATGAATATATTGTGGCTTTCAAC 600
TGAAACTTCCACCTTAAGAGGAGTTGACACCACCTTATACTTATATAACACCGAAAGTTG
62 G Y F T A K A R N S E I S S A L K S S E 81
601 GGATACTTCACAGCCAAAGCTAGAAACTCATTTATTTCAAGTGCTCTAAAAAGCAGTGAA 660
CCTATGAAGTGTCGGTTTCGATCTTTGAGTAAATAAAGTTCACGAGATTTTTCGTCACTT
82 V D N W R I I P R N N P S S D Y P S D F 101
661 GTGGACAACTGGAGAATAATACCTCGGAACAACCCATCTAGTGACTACCCTAGTGATTTT 720
CACCTGTTGACCTCTTATTATGGAGCCTTGTTGGGTAGATCACTGATGGGATCACTAAAA
102 E V I Q I K E K Q K A G L L T L E D H P 121
721 GAGGTGATTCAGATAAAAGAGAAGCAGAAGGCGGGGCTGCTCACACTTGAAGATCACCCA 780
CTCCACTAAGTCTATTTTCTCTTCGTCTTCCGCCCCGACGAGTGTGAACTTCTAGTGGGT
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
62
122 N I K R V T P Q R K V F R S L K F A E S 191
781 AACATCAAGCGGGTGACACCCCAGCGGAAAGTCTTTCGTTCCCTGAAGTTTGCTGAATCC 840
TTGTAGTTCGCCCACTGTGGGGTCGCCTTTCAGAAAGCAAGGGACTTCAAACGACTTAGG
192 D P I V P C N E T R W S Q K W Q S S R P 161
841 GACCCCATTGTGCCCTGTAATGAGACCCGGTGGAGCCAGAAGTGGCAGTCATCACGTCCC 900
CTGGGGTAACACGGGACATTACTCTGGGCCACCTCGGTCTTCACCGTCAGTAGTGCAGGG
162 L K R A S L S L G S G F W H A T G R H S 181
901 CTGAAAAGAGCCAGTCTCTCCCTGGGCTCTGGATTCTGGCATGCAACAGGAAGGCATTCA 960
GACTTTTCTCGGTCAGAGAGGGACCCGAGACCTAAGACCGTACGTTGTCCTTCCGTAAGT
182 S R R L L R A I P R Q V A Q T L Q A D V 201
961 AGTCGACGATTGCTGAGAGCCATTCCTCGCCAGGTTGCCCAGACATTGCAGGCAGATGTG 1020
TCAGCTGCTAACGACTCTCGGTAAGGAGCGGTCCAACGGGTCTGTAACGTCCGTCTACAC
202 L W Q M G Y T G A N V R V A V F D T G L 221
1021 CTTTGGCAGATGGGATACACAGGTGCTAATGTCAGGGTTGCCGTTTTTGATACTGGGCTC 1080
GAAACCGTCTACCCTATGTGTCCACGATTACAGTCCCAACGGCAAAAACTATGACCCGAG
222 S E K H P H F K N V K E R T N W T N E R 291
1081 AGTGAGAAGCATCCACATTTCAAGAATGTGAAGGAAAGAACCAACTGGACCAATGAGCGG 1140
TCACTCTTCGTAGGTGTAAAGTTCTTACACTTCCTTTCTTGGTTGACCTGGTTACTCGCC
242 T L D D G L G H G T F V A G V I A S M R 261
1141 ACCCTGGACGATGGGCTGGGCCATGGCACATTCGTTGCAGGTGTGATTGCCAGCATGAGA I200
TGGGACCTGCTACCCGACCCGGTACCGTGTAAGCAACGTCCACACTAACGGTCGTACTCT
262 E C Q G F A P D A E L H I F R V F T N N 281
1201 GAGTGCCAAGGATTTGCCCCAGATGCAGAGCTGCACATCTTCAGGGTCTTTACCAACAAT 1260
CTCACGGTTCCTAAACGGGGTCTACGTCTCGACGTGTAGAAGTCCCAGAAATGGTTGTTA
282 Q V S Y T S W F L D A F N Y A I L K K M 301
1261 CAGGTGTCTTACACGTCTTGGTTTTTGGATGCCTTCAACTATGCCATCCTAAAGAAGATG 1320
GTCCACAGAATGTGCAGAACCAAAAACCTACGGAAGTTGATACGGTAGGATTTCTTCTAC
302 D V L N L S I G G P D F M D H P F V D K 321
1321 GACGTTCTGAACCTTAGCATCGGTGGGCCTGACTTCATGGATCACCCCTTTGTTGACAAG 1380
CTGCAAGACTTGGAATCGTAGCCACCCGGACTGAAGTACCTAGTGGGGAAACAACTGTTC
322 V W E L T A N N V I M V S A I G N D G P 341
1381 GTATGGGAATTAACAGCGAACAATGTAATCATGGTTTCTGCTATTGGCAATGATGGACCT 1440
CATACCCTTAATTGTCGCTTGTTACATTAGTACCAAAGACGATAACCGTTACTACCTGGA
342 L Y G T L N N P A D Q M D V I G V G G I 361
1441 CTCTATGGCACTCTGAATAACCCTGCTGATCAGATGGATGTGATTGGAGTGGGTGGCATT 1500
GAGATACCGTGAGACTTATTGGGACGACTAGTCTACCTACACTAACCTCACCCACCGTAA
362 D F E D N I A R F S S R G M T T W E L P 381
1501 GACTTTGAAGACAACATCGCCCGCTTCTCTTCCAGGGGAATGACTACCTGGGAACTACCG 1560
CTGAAACTTCTGTTGTAGCGGGCGAAGAGAAGGTCCCCTTACTGATGGACCCTTGATGGC
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
63
382 G G Y G R V K P D I V T Y G A G V R G S 401
1561 GGAGGCTATGGTCGTGTGAAGCCTGACATTGTCACCTATGGTGCTGGAGTGCGGGGTTCT 1620
CCTCCGATACCAGCACACTTCGGACTGTAACAGTGGATACCACGACCTCACGCCCCAAGA
402 G V K G G C R A L S G T S V A S P V V A 421
1621 GGTGTGAAAGGGGGCTGCCGTGCACTCTCAGGGACCAGTGTCGCCTCCCCAGTGGTTGCT 1680
CCACACTTTCCCCCGACGGCACGTGAGAGTCCCTGGTCACAGCGGAGGGGTCACCAACGA
422 G A V T L L V S T V Q K R E L V N P A S 991
1681 GGGGCTGTCACCTTGTTAGTAAGCACAGTACAGAAGCGGGAGCTAGTGAATCCTGCCAGT 1740
CCCCGACAGTGGAACAATCATTCGTGTCATGTCTTCGCCCTCGATCACTTAGGACGGTCA
442 V K Q A L I A S A R R L P G V N M F E Q 461
1741 GTGAAGCAAGCTTTGATAGCATCAGCCCGGAGACTTCCTGGTGTCAACATGTTTGAGCAA 1800
CACTTCGTTCGAAACTATCGTAGTCGGGCCTCTGAAGGACCACAGTTGTACAAACTCGTT
462 G H G K L D L L R A Y Q I L S S Y K P Q 481
1801 GGCCATGGCAAGTTGGATCTACTGCGAGCCTATCAGATCCTCAGCAGCTATAAACCGCAG 1860
CCGGTACCGTTCAACCTAGATGACGCTCGGATAGTCTAGGAGTCGTCGATATTTGGCGTC
982 A S L S P S Y I D L T E C P Y M W P Y C 501
1861 GCGAGCCTGAGTCCTAGCTACATCGACCTGACTGAGTGTCCCTACATGTGGCCCTACTGC 1920
CGCTCGGACTCAGGATCGATGTAGCTGGACTGACTCACAGGGATGTACACCGGGATGACG
502 S Q P I Y Y G G M P T I V N V T Z L N G 521
1921 TCCCAGCCCATCTACTATGGAGGAATGCCAACAATTGTTAATGTCACCATCCTCAATGGC 1980
AGGGTCGGGTAGATGATACCTCCTTACGGTTGTTAACAATTACAGTGGTAGGAGTTACCG
522 M G V T G R I V D K P E W R P Y L P Q N 541
1981 ATGGGAGTTACAGGAAGAATTGTGGATAAGCCTGAGTGGCGACCCTATTTACCACAGAAT 2090
TACCCTCAATGTCCTTCTTAACACCTATTCGGACTCACCGCTGGGATAAATGGTGTCTTA
542 G D N I E V A F S Y S S V L W P W S G Y 561
2041 GGAGACAACATTGAAGTGGCCTTCTCCTACTCCTCAGTGTTGTGGCCTTGGTCAGGTTAC 2100
CCTCTGTTGTAACTTCACCGGAAGAGGATGAGGAGTCACAACACCGGAACCAGTCCAATG
562 L A I S I S V T K K A A S W E G I A Q G 581
2101 CTTGCCATCTCCATTTCTGTGACCAAGAAGGCAGCTTCCTGGGAAGGCATCGCGCAGGGC 2160
GAACGGTAGAGGTAAAGACACTGGTTCTTCCGTCGAAGGACCCTTCCGTAGCGCGTCCCG
582 H I M I T V A S P A E T E L K N G A E H 601
2161 CACATCATGATCACAGTGGCTTCCCCAGCAGAGACGGAATTAAAAAATGGTGCCGAGCAT 2220
GTGTAGTACTAGTGTCACCGAAGGGGTCGTCTCTGCCTTAATTTTTTACCACGGCTCGTA
602 T S T V K L P I K V K I I P T P P R S K 621
2221 ACTTCCACAGTGAAGCTGCCCATCAAGGTGAAGATCATTCCCACCCCTCCTCGGAGCAAG 2280
TGAAGGTGTCACTTCGACGGGTAGTTCCACTTCTAGTAAGGGTGGGGAGGAGCCTCGTTC
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
64
622 R V L W D Q Y H N L R Y P P G Y F P R D 691
2281 AGAGTCCTCTGGGACCAGTACCACAACCTCCGCTACCCACCCGGCTACTTCCCCAGGGAC 2340
TCTCAGGAGACCCTGGTCATGGTGTTGGAGGCGATGGGTGGGCCGATGAAGGGGTCCCTG
642 N L R M K N D P L D W N G D H V H T N F 661
2341 AACTTGCGGATGAAGAATGATCCTTTAGACTGGAATGGCGACCACGTCCACACCAACTTC 2900
TTGAACGCCTACTTCTTACTAGGAAATCTGACCTTACCGCTGGTGCAGGTGTGGTTGAAG
662 R D M Y Q H L R S M G Y F V E V L G A P 681
2901 AGGGACATGTACCAGCATCTGCGCAGCATGGGCTACTTTGTGGAGGTGCTTGGTGCCCCA 2460.
TCCCTGTACATGGTCGTAGACGCGTCGTACCCGATGAAACACCTCCACGAACCACGGGGT
682 F T C F D A T Q Y G T L L M V D S E' E E 701
2461 TTCACATGCTTTGACGCCACGCAGTACGGCACTCTGCTTATGGTGGACAGTGAGGAAGAG 2520
AAGTGTACGAAACTGCGGTGCGTCATGCCGTGAGACGAATACCACCTGTCACTCCTTCTC
702 Y F P E E I A K L R R D V D N G L S L V 721
2521 TACTTCCCTGAGGAGATTGCTAAGCTGAGGAGGGACGTGGACAATGGCCTTTCCCTTGTC 2580
ATGAAGGGACTCCTCTAACGATTCGACTCCTCCCTGCACCTGTTACCGGAAAGGGAACAG
722 V F S D W Y N T S V M R K V K F Y D E N 791
2581 GTCTTCAGTGACTGGTACAACACTTCTGTTATGAGAAAAGTGAAGTTTTACGATGAAAAC 2640
CAGAAGTCACTGACCATGTTGTGAAGACAATACTCTTTTCACTTCAAAATGCTACTTTTG
742 T R Q W W M P D T G G A N V P A L N E L 761
2641 ACAAGGCAGTGGTGGATGCCAGATACTGGAGGAGCCAACGTCCCAGCTCTAAACGAGCTG 2700
TGTTCCGTCACCACCTACGGTCTATGACCTCCTCGGTTGCAGGGTCGAGATTTGCTCGAC
762 L S V W N M G F S D G L Y E G E F A L A 781
2701 CTGTCTGTGTGGAACATGGGGTTCAGTGACGGCCTGTATGAAGGGGAGTTTGCCCTGGCA 2760
GACAGACACACCTTGTACCCCAAGTCACTGCCGGACATACTTCCCCTCAAACGGGACCGT
782 N H D M Y Y A S G C S I A R F P E D G V 801
2761 AACCACGACATGTACTATGCATCGGGGTGCAGCATTGCCAGGTTTCCAGAAGATGGTGTG 2820
TTGGTGCTGTACATGATACGTAGCCCCACGTCGTAACGGTCCAAAGGTCTTCTACCACAC
802 V I T Q T F K D Q G L E V L K Q E T A V 821
2821 GTGATCACACAGACTTTCAAGGACCAAGGATTGGAAGTCTTAAAACAAGAGACAGCAGTT 2880
CACTAGTGTGTCTGAAAGTTCCTGGTTCCTAACCTTCAGAATTTTGTTCTCTGTCGTCAA
822 V D N V P I L G L Y Q I P A E G G G R I 841
2881 GTCGACAATGTCCCCATTCTGGGGCTATATCAGATTCCAGCTGAAGGTGGAGGCCGGATT 2940
CAGCTGTTACAGGGGTAAGACCCCGATATAGTCTAAGGTCGACTTCCACCTCCGGCCTAA
842 V L Y G D S N C L D D S H R Q K D C F W 861
2941 GTGCTGTATGGAGACTCCAACTGCTTGGATGACAGTCACAGACAGAAGGACTGCTTTTGG 3000
CACGACATACCTCTGAGGTTGACGAACCTACTGTCAGTGTCTGTCTTCCTGACGAAAACC
SUBSTITUTE SI3EET (RULE 26)
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
65
862 L L D A L L Q Y T S Y G V T P P S L S H 881
3001 CTTCTGGATGCACTCCTTCAGTACACATCCTATGGTGTGACCCCTCCCAGCCTCAGCCAT 3060
GAAGACCTACGTGAGGAAGTCATGTGTAGGATACCACACTGGGGAGGGTCGGAGTCGGTA
882 S G N R Q R P P S G A G L A P P E R M E 901
3061 TCAGGGAACCGGCAGCGCCCACCCAGCGGGGCTGGCTTGGCCCCTCCTGAAAGGATGGAA 3120
AGTCCCTTGGCCGTCGCGGGTGGGTCGCCCCGACCGAACCGGGGAGGACTTTCCTACCTT
902 G N H L H R Y S K V L E A H L G D P K P 921
3121 GGAAACCACCTTCATCGCTACTCGAAAGTTCTTGAGGCCCACTTGGGAGACCCGAAACCT 3180
CCTTTGGTGGAAGTAGCGATGAGGTTTCAAGAACTCCGGGTGAACCCTCTGGGCTTTGGA
922 R P L P A C P H L S W A K P Q P L N E T 991
3181 CGGCCCCTTCCAGCCTGTCCACACTTGTCGTGGGCCAAGCCACAGCCTTTGAATGAGACG 3290
GCCGGGGAAGGTCGGACAGGTGTGAACAGCACCCGGTTCGGTGTCGGAAACTTACTCTGC
942 A P S N L W K H Q K L L S I D L D K V V 961
3291 GCACCCAGTAATCTTTGGAAACACCAGAAGCTGCTCTCCATTGACCTGGACAAAGTAGTG 3300
CGTGGGTCATTAGAAACCTTTGTGGTCTTCGACGAGAGGTAACTGGACCTGTTTCATCAC
962 L P N F R S N R P Q V R P L S P G E S G 981
3301 TTACCCAACTTTCGCTCAAATCGCCCTCAAGTGAGACCTTTGTCCCCTGGAGAAAGTGGT 3360
AATGGGTTGAAAGCGAGTTTAGCGGGAGTTCACTCTGGAAACAGGGGACCTCTTTCACCA
982 A W D I P G G I M P G R Y N Q E V G Q T 1001
3361 GCCTGGGACATTCCTGGAGGGATCATGCCTGGCCGCTACAACCAGGAAGTAGGCCAGACC 3920
CGGACCCTGTAAGGACCTCCCTAGTACGGACCGGCGATGTTGGTCCTTCATCCGGTCTGG
1002 I P V F A F L G A M V A L A F F V V Q I 1021
3421 ATCCCTGTTTTTGCCTTCCTTGGAGCCATGGTGGCCCTGGCCTTCTTCGTGGTACAGATC 3980
TAGGGACAAAAACGGAAGGAACCTCGGTACCACCGGGACCGGAAGAAGCACCATGTCTAG
1022 S K A K S R P K R R R P R A K R P Q L A 1041
3981 AGTAAGGCCAAGAGCCGGCCGAAGCGGAGGAGGCCCAGGGCAAAGCGTCCACAACTTGCA 3540
TCATTCCGGTTCTCGGCCGGCTTCGCCTCCTCCGGGTCCCGTTTCGCAGGTGTTGAACGT
1042 Q Q A H P A R T P S V 1052
3541 CAGCAGGCCCACCCTGCAAGGACCCCGTCAGTGTGATCATCACAGTGGCCAGACACAGAA 3600
GTCGTCCGGGTGGGACGTTCCTGGGGCAGTCACACTAGTAGTGTCACCGGTCTGTGTCTT
3601 GCTGACAAGCTTTGAACCCCTCTGGTGGCCACACAGCATCAGAGAGCATCCTGGGAAGTG 3660
CGACTGTTCGAAACTTGGGGAGACCACCGGTGTGTCGTAGTCTCTCGTAGGACCCTTCAC
3661 CCTGTTTCCAAGGAGCCCTATCTCTGGATTGTGGCTGGCTTAGTGTGTTCTGCCCAGACG 3720
GGACAAAGGTTCCTCGGGATAGAGACCTAACACCGACCGAATCACACAAGACGGGTCTGC
3721 TCTATGAGGTACATCCTGCAGTGCCTCACTGTGTTTGGCTCTGGCCGAAGGTGCCCAGTA 3780
AGATACTCCATGTAGGACGTCACGGAGTGACACAAACCGAGACCGGCTTCCACGGGTCAT
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO 00126348
66
PCT/CA99/01058
3781 GCTCAGCCTCCGGTGGCATCAGGCCCAGTGACAGTGCACCAAAGACACAGAGCCTGGAAG 3840
CGAGTCGGAGGCCACCGTAGTCCGGGTCACTGTCACGTGGTTTCTGTGTCTCGGACCTTC
3841 GGCTGTCGGGACATACTTTCTACATAATGCTACAACCCTGACCAAGCGAAGACAT 3895
CCGACAGCCCTGTATGAAAGATGTATTACGATGTTGGGACTGGTTCGCTTCTGTA
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO 00/26348
67
Mouse SKI-1
PCT/CA99/01058
1 , M K L V S T W L L V L V V L L C G K 18
GCATTCCATGAAGCTCGTCAGCACCTGGCTTCTTGTGCTGGTGGTTTTGCTCTGTGGGAA
1 CGTAAGGTACTTCGAGCAGTCGTGGACCGAAGAACACGACCACCAAAACGAGACACCCTT 60
19 R H L G D R L G T R A L E K A P C P S C 38
ACGGCACCTGGGCGACAGGCTGGGGACGAGAGCTTTGGAAAAGGCCCCGTGCCCCAGCTG
61 TGCCGTGGACCCGCTGTCCGACCCCTGCTCTCGAAACCTTTTCCGGGGCACGGGGTCGAC 120
39 S H L T L K V E F S S T V V E Y E Y I V 58
CTCCCACCTGACTTTGAAGGTGGAATTCTCTTCAACTGTGGTGGAGTACGAATATATTGT
121 GAGGGTGGACTGAAACTTCCACCTTAAGAGAAGTTGACACCACCTCATGCTTATATAACA 180
59 A F N G Y F T A K A R N S F I S S A L K 78
GGCTTTCAACGGATACTTCACAGCCAAAGCTAGAAACTCATTTATTTCAAGTGCGCTGAA
181 CCGAAAGTTGCCTATGAAGTGTCGGTTTCGATCTTTGAGTAAATAAAGTTCACGCGACTT 290
79 S S E V E N W R I I P R N N P S S D Y P 98
AAGCAGTGAAGTGGAAAACTGGAGAATAATACCTCGGAACAACCCATCCAGTGACTACCC
291 TTCGTCACTTCACCTTTTGACCTCTTATTATGGAGCCTTGTTGGGTAGGTCACTGATGGG 300
99 S D F E V I Q I K E K Q K A G L L T L E 118
TAGTGATTTTGAGGTGATTCAGATAAAAGAGAAGCAGAAGGCGGGGCTGCTCACACTTGA
301 ATCACTAAAACTCCACTAAGTCTATTTTCTCTTCGTCTTCCGCCCCGACGAGTGTGAACT 360
119 D H P N - I K R V T P Q R K V F R S L K F 138
AGATCACCCCAACATCAAGCGGGTGACACCCCAGCGGAAAGTCTTTCGTTCCCTCAAGTT
361 TCTAGTGGGGTTGTAGTTCGCCCACTGTGGGGTCGCCTTTCAGAAAGCAAGGGAGTTCAA 420
139 A E S N P I V P C N E T R W S Q K W Q S 158
TGCTGAATCCAACCCCATCGTGCCCTGTAATGAAACCCGGTGGAGCCAGAAGTGGCAGTC
921 ACGACTTAGGTTGGGGTAGCACGGGACATTACTTTGGGCCACCTCGGTCTTCACCGTCAG 980
159 S R P L K R A S L S L G S G F W H A T G 178
ATCACGTCCCCTGAAAAGAGCCAGTCTCTCCCTGGGCTCTGGATTCTGGCATGCAACAGG
48I TAGTGCAGGGGACTTTTCTCGGTCAGAGAGGGACCCGAGACCTAAGACCGTACGTTGTCC 590
179 R H S S R R L L R A I P R Q V A Q T L Q 198
AAGACATTCAAGTCGGCGATTGCTGAGAGCCATTCCTCGCCAGGTCGCCCAGACACTGCA
541 TTCTGTAAGTTCAGCCGCTAACGACTCTCGGTAAGGAGCGGTCCAGCGGGTCTGTGACGT 600
lg9 A D V L W Q M G Y T G A N V R V A V F D 218
GGCAGATGTGCTGTGGCAGATGGGATACACAGGTGCTAATGTCAGAGTTGCTGTTTTTGA
601 CCGTCTACACGACACCGTCTACCCTATGTGTCCACGATTACAGTCTCAACGACAAAAACT 660
219 T G L S E K H P H F K N V K E R T N W T 238
TACTGGGCTCAGTGAGAAGCATCCGCATTTTAAGAATGTGAAGGAGAGAACCAACTGGAC
661 ATGACCCGAGTCACTCTTCGTAGGCGTAAAATTCTTACACTTCCTCTCTTGGTTGACCTG 720
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO 00126348 PCT/CA99/01058
68
239 N E R T L D D G L G H G T F V A G V I A 258
CAATGAGCGGACCCTGGATGATGGGCTAGGCCATGGCACATTCGTTGCAGGTGTGATTGC
721 GTTACTCGCCTGGGACCTACTACCCGATCCGGTACCGTGTAAGCAACGTCCACACTAACG 780
259 S M R E C Q G F A P D A E L H I F R V F 278
CAGCATGAGGGAGTGCCAAGGATTTGCTCCAGATGCAGAGCTGCACATCTTCAGGGTCTT
781 GTCGTACTCCCTCACGGTTCCTAAACGAGGTCTACGTCTCGACGTGTAGAAGTCCCAGAA 890
279 T N N Q V S Y T S W F L D A F N Y A I L 298
TACCAACAATCAGGTGTCTTACACATCTTGGTTTCTGGATGCCTTCAACTATGCCATCCT
891 ATGGTTGTTAGTCCACAGAATGTGTAGAACCAAAGACCTACGGAAGTTGATACGGTAGGA 900
299 K K M D V L N L S I G G P D F M D H P F 318
AAAGAAGATGGACGTTCTCAACCTTAGCATCGGTGGGCCCGACTTCATGGATCATCCGTT
901 TTTCTTCTACCTGCAAGAGTTGGAATCGTAGCCACCCGGGCTGAAGTACCTAGTAGGCAA 960
319 V D K V W E L T A N N V I M V S A I G N 338
TGTTGACAAGGTGTGGGAATTAACAGCTAACAATGTAATTATGGTTTCTGCTATTGGCAA
961 ACAACTGTTCCACACCCTTAATTGTCGATTGTTACATTAATACCAAAGACGATAACCGTT 1020
339 D G P L Y G T L N N P A D Q M D V I G V 358
TGATGGACCTCTCTATGGCACTCTGAATAACCCTGCTGATCAGATGGATGTGATTGGAGT
1021 ACTACCTGGAGAGATACCGTGAGACTTATTGGGACGACTAGTCTACCTACACTAACCTCA 1080
359 G G I D F E D N I A R F S S R G M T T W 378
GGGTGGCATTGACTTTGAAGATAACATCGCTCGCTTTTCTTCCAGGGGAATGACTACCTG
1081 CCCACCGTAACTGAAACTTCTATTGTAGCGAGCGAAAAGAAGGTCCCCTTACTGATGGAC 1140
379 E L P G G Y G R V K P D I V T Y G A G V 398
GGAATTACCAGGAGGCTATGGTCGTGTGAAGCCTGACATTGTCACCTATGGTGCTGGAGT
1141 CCTTAATGGTCCTCCGATACCAGCACACTTCGGACTGTAACAGTGGATACCACGACCTCA 1200
399 R G S G V K G G C R A L S G T S V A S P 918
GCGGGGTTCCGGTGTGAAAGGGGGCTGCCGTGCACTCTCAGGGACCAGTGTCGCTTCCCC
1201 CGCCCCAAGGCCACACTTTCCCCCGACGGCACGTGAGAGTCCCTGGTCACAGCGAAGGGG 1260
419 V V A G A V T L L V S . T V Q K R E L V N 438
AGTGGTCGCTGGGGCCGTCACCTTGTTAGTAAGCACAGTACAGAAGCGGGAGCTGGTGAA
1261 TCACCAGCGACCCCGGCAGTGGAACAATCATTCGTGTCATGTCTTCGCCCTCGACCACTT 1320
939 P A S V K Q A L I A S A R R L P G V N M 95B
TCCTGCCAGTGTGAAGCAAGCTTTGATAGCGTCAGCCCGGAGACTTCCTGGGGTCAACAT
1321 AGGACGGTCACACTTCGTTCGAAACTATCGCAGTCGGGCCTCTGAAGGACCCCAGTTGTA 1380
459 F E Q G H G K L D L L R A Y Q I L S S Y 478
GTTCGAGCAAGGTCATGGCAAGTTGGATCTGCTGCGAGCTTATCAGATCCTCAGCAGCTA
1381 CAAGCTCGTTCCAGTACCGTTCAACCTAGACGACGCTCGAATAGTCTAGGAGTCGTCGAT 1490
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
69
479 K P Q A S L S P S Y I D L T E C P Y M W 998
TAAACCGCAGGCAAGCCTGAGTCCTAGCTACATCGACCTGACTGAGTGTCCCTACATGTG
1991 ATTTGGCGTCCGTTCGGACTCAGGATCGATGTAGCTGGACTGACTCACAGGGATGTACAC 1500
499 P Y C S Q P I Y Y G G M P T I V N V T I 518
GCCCTACTGCTCCCAGCCTATCTACTATGGAGGAATGCCAACAATCGTTAATGTCACCAT
1501 CGGGATGACGAGGGTCGGATAGATGATACCTCCTTACGGTTGTTAGCAATTACAGTGGTA 1560
519 L N G M G V T G R I V D K P E W R P Y L 538
CCTCAATGGCATGGGCGTCACAGGAAGAATTGTGGATAAGCCTGAGTGGCGACCCTATTT
1561 GGAGTTACCGTACCCGCAGTGTCCTTCTTAACACCTATTCGGACTCACCGCTGGGATAAA 1620
539 P Q N G D N I E V A F S Y S S V L W P W 558
ACCACAGAATGGAGACAACATTGAAGTGGCCTTCTCCTACTCCTCAGTGTTGTGGCCCTG
1621 TGGTGTCTTACCTCTGTTGTAACTTCACCGGAAGAGGATGAGGAGTCACAACACCGGGAC 1680
559 S G Y L A I S I S V T K K A A S W E G I 578
GTCAGGTTACCTTGCCATCTCCATTTCTGTGACCAAGAAGGCAGCTTCCTGGGAAGGCAT
1681 CAGTCCAATGGAACGGTAGAGGTAAAGACACTGGTTCTTCCGTCGAAGGACCCTTCCGTA 1740
579 A Q G H I M I T V A S P A E T E L H S G 598
CGCTCAGGGCCACATCATGATCACAGTGGCGTCCCCAGCAGAGACAGAGTTACACAGTGG
1741 GCGAGTCCCGGTGTAGTACTAGTGTCACCGCAGGGGTCGTCTCTGTCTCAATGTGTCACC 1800
599 A E H T S T V K L P I K V K I I P T P P 618
TGCGGAGCACACTTCCACCGTGAAGCTGCCCATCAAGGTGAAGATCATTCCCACCCCTCC
1801 ACGCCTCGTGTGAAGGTGGCACTTCGACGGGTAGTTCCACTTCTAGTAAGGGTGGGGAGG 1860
619 R S K R V L W D Q Y H N L R Y P P G Y F 638
TCGGAGCAAGAGAGTCCTCTGGGACCAGTACCACAACCTCCGCTACCCACCTGGCTACTT
1861 AGCCTCGTTCTCTCAGGAGACCCTGGTCATGGTGTTGGAGGCGATGGGTGGACCGATGAA 1920
639 P R D N L R M K N D P L D W N G D H V H 658
CCCCAGGGACAACTTGCGGATGAAGAATGACCCTTTAGACTGGAATGGCGACCACGTCCA
1921 GGGGTCCCTGTTGAACGCCTACTTCTTACTGGGAAATCTGACCTTACCGCTGGTGCAGGT 1980
659 T N F R D M Y Q H L R S M G Y F V E V L 678
CACCAACTTCAGGGACATGTACCAGCATCTGCGCAGCATGGGCTACTTCGTGGAGGTGCT
1981 GTGGTTGAAGTCCCTGTACATGGTCGTAGACGCGTCGTACCCGATGAAGCACCTCCACGA 2090
67 9 G A P F T C F D A T Q Y G T L L L V D S 698
CGGCGCCCCATTCACATGTTTTGACGCCACACAGTATGGCACTTTGCTGCTGGTGGACAG
2041 GCCGCGGGGTAAGTGTACAAAACTGCGGTGTGTCATACCGTGAAACGACGACCACCTGTC 2100
699 E E E Y F P E E I A K L R R D V D N G L 718
TGAGGAAGAGTACTTCCCTGAGGAGATTGCTAAGCTGAGGAGGGATGTGGACAATGGCCT
2101 ACTCCTTCTCATGAAGGGACTCCTCTAACGATTCGACTCCTCCCTACACCTGTTACCGGA 2160
719 S L V I F S D W Y N T S V M R K V K F Y 738
TTCCCTCGTCATCTTCAGTGACTGGTACAACACTTCTGTTATGAGAAAAGTGAAGTTTTA
2161 AAGGGAGCAGTAGAAGTCACTGACCATGTTGTGAAGACAATACTCTTTTCACTTCAAAAT 2220
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
70
739 D E N T R Q W W M P D T G G A N I P A L 758
TGATGAAAACACCAGGCAGTGGTGGATGCCAGACACCGGAGGAGCGAACATCCCAGCTCT
2221 ACTACTTTTGTGGTCCGTCACCACCTACGGTCTGTGGCCTCCTCGCTTGTAGGGTCGAGA 2280
759 N E L L S V W N M G F S D G L Y E G E F 778
GAATGAGCTGCTGTCTGTGTGGAACATGGGGTTCAGTGACGGCCTATATGAAGGGGAGTT
2281 CTTACTCGACGACAGACACACCTTGTACCCCAAGTCACTGCCGGATATACTTCCCCTCAA 2340
779 V L A N H D M Y Y A S G C S I A K F P E 798
TGTCCTGGCAAACCATGACATGTACTATGCGTCGGGGTGCAGCATCGCCAAGTTTCCAGA
2391 ACAGGACCGTTTGGTACTGTACATGATACGCAGCCCCACGTCGTAGCGGTTCAAAGGTCT 2400
?99 D G V V I T Q T F K D Q G L E V L K Q E 818
AGATGGCGTCGTGATCACACAGACTTTCAAGGACCAAGGATTGGAGGTCTTAAAACAAGA
2401 TCTACCGCAGCACTAGTGTGTCTGAAAGTTCCTGGTTCCTAACCTCCAGAATTTTGTTCT 2460
819 T A V V E N V P I L G L Y Q I P S E G G 838
GACAGCAGTTGTGGAAAATGTTCCCATTTTGGGGCTTTATCAGATTCCATCTGAAGGTGG
2461 CTGTCGTCAACACCTTTTACAAGGGTAAAACCCCGAAATAGTCTAAGGTAGACTTCCACC 2520
839 G R I V L Y G D S N C L D D S H R Q K D 858
AGGCCGGATCGTGCTGTATGGAGACTCCAACTGCTTGGATGACAGTCACAGACAGAAGGA
2521 TCCGGCCTAGCACGACATACCTCTGAGGTTGACGAACCTACTGTCAGTGTCTGTCTTCCT 2580
859 C F W L L D A L L Q Y T S Y G V T P P S 878
CTGCTTTTGGCTTCTGGATGCGCTCCTTCAGTACACATCCTATGGCGTGACCCCTCCCAG
2581 GACGAAAACCGAAGACCTACGCGAGGAAGTCATGTGTAGGATACCGCACTGGGGAGGGTC 2690
879 L S H S G N R Q R P P S G A G L A P P E 898
CCTCAGCCATTCAGGGAACCGGCAGCGCCCACCTAGCGGAGCCGGCTTGGCCCCTCCTGA
2641 GGAGTCGGTAAGTCCCTTGGCCGTCGCGGGTGGATCGCCTCGGCCGAACCGGGGAGGACT 2700
899 R M E G N H L H R Y S K V L E A H L G D 918
AAGGATGGAAGGAAACCACCTCCATCGGTACTCCAAAGTTCTTGAAGCCCACTTGGGAGA
2701 TTCCTACCTTCCTTTGGTGGAGGTAGCCATGAGGTTTCAAGAACTTCGGGTGAACCCTCT 2760
919 P K P R P L P A C P H ~ L S W A K P Q P L 938
CCCGAAACCTCGGCCCCTGCCAGCCTGTCCACATTTGTCATGGGGCAAGCCACAGCCTTT
2761 GGGCTTTGGAGCCGGGGACGGTCGGACAGGTGTAAACAGTACCCGGTTCGGTGTCGGAAA 2820
939 N E T A P S N L W K H Q K L L S I D L D 958
GAATGAGACGGCACCCAGTAATCTTTGGAAACATCAGAAGCTGCTCTCCATTGACCTGGA
2821 CTTACTCTGCCGTGGGTCATTAGAAACCTTTGTAGTCTTCGACGAGAGGTAACTGGACCT 2880
959 K V V L P N F R S N R P Q V R P L S P G 978
CAAAGTAGTGTTACCCAACTTTCGATCCAATCGCCCTCAAGTGAGACCTTTGTCCCCTGG
2881 GTTTCATCACAATGGGTTGAAAGCTAGGTTAGCGGGAGTTCACTCTGGAAACAGGGGACC 2940
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
71
979 E S G A W D I P G G I M P G R Y N Q E V 998
AGAGAGTGGTGCCTGGGACATTCCTGGAGGGATCATGCCTGGCCGCTACAACCAGGAGGT
2941 TCTCTCACCACGGACCCTGTAAGGACCTCCCTAGTACGGACCGGCGATGTTGGTCCTCCA 3000
999 G Q T I P V F A F L G A M V A L A F F V 1018
GGGACAGACCATCCCCGTCTTCGCCTTCCTCGGAGCCATGGTGGCCCTGGCCTTCTTTGT
3001 CCCTGTCTGGTAGGGGCAGAAGCGGAAGGAGCCTCGGTACCACCGGGACCGGAAGAAACA 3060
1019 V Q I S K A K S R P K R R R P R A K R P 1038
GGTACAGATCAGCAAGGCCAAGAGCCGGCCGAAGCGGAGGAGGCCCAGGGCAAAGCGTCC
3061 CCATGTCTAGTCGTTCCGGTTCTCGGCCGGCTTCGCCTCCTCCGGGTCCCGTTTCGCAGG 3120
1039 Q L A Q Q A H P A R T P S V 1052
ACAACTTGCACAGCAGGCCCACCCTGCAAGGACCCCATCAGTGTGAGCATCGCAGTAGCC
3121 TGTTGAACGTGTCGTCCGGGTGGGACGTTCCTGGGGTAGTCACACTCGTAGCGTCATCGG 3180
AGCCACAGAAGCTAACAAGCCTTGAACCACTCTGGTGGCCACACAGCGCCTCAGAGAGCA
3181 TCGGTGTCTTCGATTGTTCGGAACTTGGTGAGACCACCGGTGTGTCGCGGAGTCTCTCGT 3240
TTCTGGGAAGTGCCTGTTTCCGAGGACCCTGTCTCCAGCTTGTGGCTATCTTACTGTGTT
3241 AAGACCCTTCACGGACAAAGGCTCCTGGGACAGAGGTCGAACACCGATAGAATGACACAA 3300
CTGCCCAGGCACCTGATGAGGTACATCCTGCAGTGCCTCTCTGTGCTTGGCTCTGGCAGA
3301 GACGGGTCCGTGGACTACTCCATGTAGGACGTCACGGAGAGACACGAACCGAGACCGTCT 3360
AGGCACCCAGTGACATCAGGCATCAGGCCCAGTGACAGTGCACCAAAGACACAGAGCCTG
3361 TCCGTGGGTCACTGTAGTCCGTAGTCCGGGTCACTGTCACGTGGTTTCTGTGTCTCGGAC 3420
GAAGGGCTGTCGGGACATACTTTCTACATAACGCTACAACCCTGACCAAGCAAAGACATG
3421 CTTCCCGACAGCCCTGTATGAAAGATGTATTGCGATGTTGGGACTGGTTCGTTTCTGTAC 3480
CTTGTTACAGGCTATTTTCTATATTTATTGTGGGAGAGTCACTTTAAAGACTGTGCTAGT
3481 GAACAATGTCCGATAAAAGATATAAATAACACCCTCTCAGTGAAATTTCTGACACGATCA 3540
TGGAAACAGAGCTGTTGCTGTTGTCAGTCGAGTGCAGTTTTCTGCAGCGATGTCATAAGG
3541 ACCTTTGTCTCGACAACGACAACAGTCAGCTCACGTCAAAAGACGTCGCTACAGTATTCC 3600
AGTCAGATTCCGTGACCTCCTCTTTGATGGAGGACACACTGAACTGAAGGGGACTTGCGC
3601 TCAGTCTAAGGCACTGGAGGAGAAACTACCTCCTGTGTGACTTGACTTCCCCTGAACGCG 3660
GGATGTGGGAGATGCAAGCCTTCGCTTTATTTTTTTATAACTATCAACTGCCATCATGTT
3661 CCTACACCCTCTACGTTCGGAAGCGAAATAAAAAAATATTGATAGTTGACGGTAGTACAA 3720
TTGTAATTTGGGGATCTTGATTTCACCGTTGTTGGTGAAGGAAATTTTCAATAAATATGC
3721 AACATTAAACCCCTAGAACTAAAGTGGCAACAACCACTTCCTTTAAAAGTTATTTATACG 3780
ATAACCTT
3781 TATTGGAA 3788
SUBSTITUTE SHEET (RULE 26)
CA 02349587 2001-05-02
WO 00/26348 PCT/CA99/01058
-72-
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