Note: Descriptions are shown in the official language in which they were submitted.
WO 93/14220 PCr~US93/00031
2~7 ~32
ANTIBODIES RECOGNI ZING THE FOURTH
I~Ot:;LOBULIN-LIKE_DOMAIN OF VCAMl
FIELD OF THE INVENTIOl~
This invention relates to monoclonal antibodies
(MAbs), specifically monoclonal antibodies that bind to
the domain 4 cf Vascular Cell Adhesion Molecule-1 (VCAM1~.
BACKGROUND OF THE TNV~NTION
VCA~l (also known as INCAM-110) was first
identified as an adhesion molecule induced on endothelial
cells by inflammatory cytokines (TNF and IL-1~ and LPS
(Rice et al., 1989 [1~; Osborn et al., 1~89 [2]). VCAM1
binds tQ cells sxhîbiting the integrin VLA~4 (~4~
including T ànd B l~mphocytes, monocytes/ and-eosinophils,
but not neutrophils, and is thought to participate in
recruitment of these cells from the bloodstream to a~eas
of infection and infla~mation (Elices et al, 1990 [3];
Osborn, lg90 [4~. The VCAMl/~LA-4 adhesion pathway has
been associated with a number of physiolQgic.al and
pathological processes. Although VL~-4 is normally
restricted to hematopoietic lineages, it is found cn
melanoma cell lines, and thus it has been suggested that
VCAM1 may participate in metastasis of such tumors (Rice
st al., 1989 ~1]~.
W093/14220 PCT/US93/00031
, 2
In vivo, VCAMl is found on areas of arterial
endothelium representing early atherosclerotic plaques in
a rabbit model system (Cybulsky and Gimbrone, 1991 [5]).
VCAM1 is also found on fo~llicular dendritic cells in human
5 lymph nodes (Freedman et al., 1990 ~6]). It is also
present on bone marrow stromal cells in the mouse (Miyake
et al., 1991 ~7]), thus VCAMl appears to play a role in B-
cell development.
VCAMl belongs to the immunoglobulin (Ig)
superfamily. The major form on endothelial cells,
referred to herein as VCAM-7D, has seven Ig homology units
or domains; domains 4, 5 and ~ are similar in amino acid
sequence to domains 1, 2 and 3, respectively, suggesting
an intergenic duplication event in the evolutionary
history of the gene (Osborn et al., 1989 ~2]; Polte et al.
lg90 [83; Hession et al., 1991 [9]). There is also a
minor 6-domain form (referred to as VCAM-6D herein) -~
generated by alternative splicing, in which the fourth
domain is deleted (Osborn et al., 1989 ~2]; Hession et al.
1991 ~9], Cybulsky et al., 1991 [10]). The biological
significance of this alternate splicing is not known,
however as shown below VCAM-6D can bind VLA-4-expressing
cells and thus clearly has potential functionality in
ViVQ .
The apparent involvement of the VCAMlIVLA-4
adhesion pathway in infection, inflammation and possibly
atherosclerosis has led to continuing intensive research
to understand the mechanisms of cell-cell adhesion on a
molecular level and has led investigators to propose
intervention in this adhesion pathway as a treatment for
diseases, particularly inflàmmation (Os~orn et al., 1989
[2]).
WO93/14220 PCT/US93/00031
2~7 ~
- 3 - .
Monoclonal antibodies that inhibit VCAMl binding
to VLA-4 are known. For example, anti-YLA-4 MAbs HP2/1
and HPlt3 have been shown to block attachment of VLA-4-
expressing Ramos cells to human umbilical vein cells and
VCAM~-transfected COS cells (Elices et al., 1990 [3]).
Also, anti-VCAN1 antibodies such as the monoclonal
a~tibody 4B9 (Carlos et al., 1990 [11]) have been shown to
inhibit adhesion of Ramos (B-cell-like), Jurkat ~T-cell-
like) and HL60 (granulocyte-like) cells to COS cells
transfected to express VCAM-6D and VCAM-7D (Hession et
al., 1991 ~9]). ::
Although blocking antibodies that might be .-
potential therapeutics are known, new monoclonal
antibodies recognizing epitopes on other, non-binding
domains of VCAM1 would be important research tools to map
the portionts) of VCAM1 a sential for binding to VLA-4.
Antibodies that bind to VCAM1 without competing for the
epitope of a blocking antibody such as 4Bs would also have
import~nt diagnostic uses. For instance, a radiolabeled
anti-V~AM1 antibody recognizing an epitopa not essential
to VLA-4 binding could be used to monitor the course and
location of acute inflammatory events but would not
~nterfere with the action of a 1 ter-administëred
therapeutic that interacts with the VLA-4-binding domain
of ~CA~l.
SUM~ARY OF THE INVENTION
We have now isolated a panel of monoclonal
antibodies which bind to VCAM-7D but not to VCAM-6D, and
therefore presumably recognize an epitope in domain 4 of
VCAM1. We have surprisingly discovered that some of these
domain 4 antibodies inhibit Ramos binding to VC~M-7D-
expressing cells, and through the use of these antibodies
we have demonstrated that domain 4 of VCAN-7D contains a
2 1 2 7 5 3 2 PCT/US93/00031
site involved in VLA-4 binding, which is in addition to
the site recognized and blocked by MAb 4B9 (domain l).
Thus, VCAMl is identified as the first known adhesion
molecule to have two binding sites for a single receptor ;~
5 (VLA-4); and the domain 4 monoclonal antibodies according ~-
to this invention are useful not only in mapping the -
molecular functionality of VCAMl but al o in at least
partially inhibiting VCAMl recognition by VLA-4.
Accordingly, it is an object of this invention
to provide monoclonal antibodies recogni2ing VCAM-7D but
not VCAM-6~. ;
It is a further object of this invention to
provide antibodies recognizing an epitope dependent on the
fourth immunoglobulin-like domain ~"domain 4") of VCAM-7D
("domain 4 antibodies"), and ~articularly domain 4
antibodies that do not cross-block the anti-VCAMl MAb 4B9.
It is a further object of the present invention
to provide domain 4 antibodies capable of blocking
adhesion of VLA-4-expressing cells to VCAMl.
These and other objects are accomplished by the
monoclonal antibodies described and claimed herein.
RIEF DESCRIPTION OF THE DRAWIN~S
Figure 1 is a schematic diagram of VCAM-7D,
showing the linear arrangemPnt of functional domains from
the amino terminus to the carboxyl terminus. These are
the signal peptide ('Isig")~ immunoglobulin-like domains 1
through 7, the transmembrane sequence ("tm"), and the
cytoplasmic domain or "tail" ("cyt")~ Also indicated are
the relative positions of several restriction enzyme
recognition sites, which were used to prepare additional
VCAMl constructs having one or more altered domains
(described infra~. In the examples set forth below, cDNA
WO93/14220 PCT/US93/00031
~4~ ~f itif2
- 5 -
encoding VCAM-7D having the diagrammed conformation was
used to transfect CoS7 cells to produce CoS7 cells
exhibiting VC~M-7D on their surfaces.
Figure 2 is a schematic diagram of VCAM-6D,
5 showing the linear arrangement of functional domains from :~
the amino terminus to the carboxyl terminus --the signal
peptide ("sig"), immunoglobulin-like domains l, 2, 3, 5, 6
and 7, the transmembrane sequence ("tm")y and the
cytoplasmic domain or "tail" ("cyt"). In the examples set
forth below, cDNA encoding VCAM-6D having the diagrammed
conformation was used to transfect COS7 cells to produce
COS7 cells exhibiting VCAM-6D on their surfaces.
Figure 3 is a schematic diagram of a chimeric
VCANl, designated VCAM/ICAM-l, showing the linear
arrangement of functional domains. This polypeptide has
the general structure of VCAM-7D, except the cDNA encoding
VCAM-7D was modified so that a segment encoding most of
domain l and part of domain 2 was excised and replaced by
cDNA encoding analogous portions of Intercellular Adhesion
Molecule-l (I~AMl). The modified portion of the VCAM-7D
molecule is indicated by shading.
Figura 4 is a schematic diagram of another
chimeric VCAMl, designated VCAM/ICAM-2, showing the linear
arrangement of functional domains. This polypeptide has
the general structure of VCAM-7D, except as indicated by
the shaded area, the cDNA coding for most of domain l and
a large part of domain 2 was excised and replaced with
ICAM1 cDNA .
Figure 5 is a schematic diagram of another
30 chimeric VCAMl, designated VCAM/ICAM-3, ~howing the linear
arrangement of functional domains. This polypeptide has
the general structure of VCAM-7D, except as indicated by
the shaded area, the cDNA coding for part of domain l, all
W093/14220 PCT/US93/00031
~1 2~r'J32
of domains 2 and 3, and a small N-terminal part of domain
4 was excised and replaced with ICAMl cDNA.
Fîgure 6 is a graph showing relative le~els of
expression in COS7 cells transfected with either cDNA
encoding VCAM-6D or cDNA encoding VCAM-7D. Expression of
either VCAM-6D or VCAM-7D was analyzed by flow
cytofluorometry following indireot immunofluorescent
staining with MAb 4B9. The number of cells with
fluorescent intensity greater than that shown by g9% of
control transfectants (pCDM8 vector only) waæ det:ermined.
The data depicted combines the results of two experiments;
standard deviation is indicated by error bars.
Figure 7 is a graph showing the extent of Ramos
cell binding to COS cells separately tranfected with cDNA
encoding VC~M-7D, VCAM-6D, VCAM/ICAM-l, VCAM/ICAM-2, and
VCAM/ICAM-3. In addition, the effects on Xamos binding of
preincubation of the transfected COS cells with 20 ~g/ml
of M~b 4B~ (anti-VCAMl) and lO ~g/ml of MAb HPl/2 (anti- :~
~LA-4) are shown. In addition, the negli~ible Ramos
binding to cells transfected with pCDM8 (expression vector
only; negative control) and cells transfected to exhibit
surface ICAMl is shown. Binding :is expressed as Ramos
cells bound per square millimeter ("Ramos cel~s bound/mm"
in the figures).
Figure 8 is a schematic diagram of another
chimeric VCAMl, designated VCAM6D/IC~M-l, showing the
linear arrangement of functional domains. This
polypeptide has the general structure of VC~M-6D (no`
domain 4), however, as indicated by the shaded area, the
cDNA coding for most of domain l and a small part of
domain 2 was excised and replaced with ICAMl cDNA.
Figure 9 is a schematic d~agram of another
chimeric VCAMl, designated VCAM6D/ICAM-2, showing the :
W093/l4220 PCT/US93/00031
3 ~
linear arrangement of functional domains. This
polypeptide has the general structure of VCAM-6D ~no
domain 4), however, as indicated by the sh~ed area, the
cDNA coding for most of domain 1 and a large part of
S domain 2 was excised and replaced with ICAMl cDNA.
Figure 10 is a graph depicting the extent of
Ramos cell binding to COS cells separately tranfected with
cDNA encoding ~CAM/ICAM-l, VCAM~D/ICAM-l, VCAM/ICAM-2, and
VCAM6D/ICAM-2. In addition, the negligible Ramos binding
to cells transfected with pCDM8 ~expression vector only;
negative control) and cells transfected to exhibit surface
ICAMl is shown.
Figure 11 is a schematic diagram of another
chimeric VC~M1, designated VCAM6D/V~M4-1, showing the ;
linear arrangement of functional dom.~ins. This
polypeptide has the general structure of VCAM-6D (no
domain 4), however, as indicated by the shaded area, ths
cDNA coding for domain 1 and a small part of domain 2 was
excised and replaced with cDNA encoding domain 4 ~and one
amino acid of domain 5) of VCAM-7D.
Figure 12 i5 a graph depicting the extent of
Ramos cell binding of COS cells separately tranfected with
cDNA encoding VCAM/ICAM-l, VC~M~D/ICAM-l, an~- .
VC~M6D/VCAM4-1. Negligible Ramos binding to ICAM1 and
negative (pCDM83 controls is also shown.
Figure 13 is a graph depicting the ~xtent of
Ramos cell binding to transfectants expressing various
chimeric VCAMl constructs in the absence of any
interfering antibody or in the presence of MAb 4B9 or MAb
GH12 (this invention).
Figure 14 is a graph depicting the extent of
Ramos cell binding to transfectants expressing various
chimeric VCAMl constructs in the absence of any
WO 93/14220 PCrtUS93/00031
'~ 1'"~ ~ r~"~ .z
-- 8
interfering antibody or in the presence of MAb 4B9 or MAb
ED11 (this invention).
Figure 15 is a ~raph depicting the extent of
Ramos cell binding to transfectants expressing various
chimeric VCAM1 constructs in the absence of any
interfering antibody or in the presence of MAb 4~9 or MAb
GE4 (this invention).
Figures 16, 17, 18 and 19 are graphs showing
Ramos cell binding to cultured H W ECs treated with 20
ng/ml of recombinant human TNF for 4 hours tFig. 16), 24
hours (Fig. 17), 48 hours (Fig. 18), and 72 hours (Fig.
19). Binding of Ramos cells to uninduced HUVECs
(control), induced ~TNF-treated) HUVECs alone, and in the
presence of HPl/2 (anti-VLA-4 ~4 MAb), 4B9 (anti-VCAM1 --
MAb), EDll (anti-VCAMl NAb, this invention), and GH12
(anti-VCAMl MAb, this invention) is compared.
DE~AILED DESCRIP~ L~OF THE INVENTION
Th~ technology for producing monoclonal
antibodies is well known. Briefly, an immortal cell line
(typically murine myeloma cells) is fused to lymphocytes
(typically splenocytes) from a ma~mal immunized with whole `~
cells expressing a given antigen, e.g., VCAMl, and/or with
purified antigen, and the culture supernatants of ~he
resulting hybridoma cells are screened for antibodiPs
against the antigen. See, generally, Kohler and Milstein,
lg75 [12]~
Immunization may be accomplished using standard
procedures. The unit dose and immunization regimen depend
on the species of mammal immunized, its immune status, the
body weight of the mammal, et~. Typically, the immunized
mammals are bled and the serum from each blood cample is
assayed for particular antibodies using appropriate
WO93/14220 PCT/US93/00031
212 ~ 2
g
screening assays. For example, anti-VCAMl antibodies
according to the present invention were identified by
testing the ability of the immune serum to block Ramos
binding to plates coated with a recombinant soluble form
of VCAM-7D (rsVCAM-7D). The lymphocytes used in the
production of hybridoma cells typically are isolated from ;
immunized mammals whose sera have already tested positive
for the presence of anti-VCAM1 antibodies using such
screening assays.
10Typically, the immortal cell line (e.g., a
myeloma cell line~ is derived from the same mammalian
species as the lymphocytes. Preferred immortal cell lines
are mouse myeloma cell lines that are sensitive to culture
medium containing hypoxanthine, aminopterin and thymidine
("HAT medium").
Typically, HAT-sensitive mouse myeloma cells are
fused to mouse splenocytes using polyethylene glycol -
(e.g., PEG ~350). Hybridoma cells resulting from the
fusion are then selected using HAT medium, which kills
unfused and unproductively fused myeloma cells (unfused
splenoGytes die after several days because they are not
transformed). Hybxidomas producing a desired antibody are
detected by screening the hybridoma culture s~pernatants
using assays which detect MAbs having the desired
specificity. For example, hybridomas according to the
invention were identified by their ability to bind to
VCAM-7D-expressing cells but not to VCA~-6D-expressing
cells~ (See Example 1, suPra.)
To produce anti-VCAMl antibodies, hybridoma
cells that tested positive in ~uch screening assays were
cultured in a nutrient medium under conditions and for a
time suffici~nt to allow the hybridoma cells to secrete
the monoclonal antibodies into the culture medium. Tissue
WO93/14220 PCT~US93/00031
532 ' 10-
culture techniques and culture media suitable for
~ybridoma cells are well known. The conditioned hybridoma
culture supernatant may be collected and the anti-VCAM1
antibodies optionally further purified by well-known
methods.
Alternatively, the desired antibody may be
produced by injecting the hybridoma cells into the
peritoneal cavity of a mouse primed with 2,6,10,14-
tetramethylpentadecane (PRISTANE; Sigma Chemical Co., St.
Louis MO). The hybridoma cells proliferate in the
peritoneal cavity, secreting the antibody which
accu~ulates as ascites fluid. The antibody may be
harvested by withdrawing the ascites fluid from the -~
peritoneal cavity with a syringe. -
The monoclonal antibodies of the pr~sent -
învention recognize epitopes dependent on the fourth Ig
domain of the seven-Ig-domain form of VCAMl, i.e., VCAM-
7D. That is, the MAbs of the present invention bind to
epitopes that either are contained within the fourth Ig
domain of VCAM-7D or are partly contained within the
fourth Ig domain, such that elimination of the fourth Ig
domain eliminates their ability ~o recognize the VCAMl
structure. These M~bs are referred to as "domain 4 MAbs"
herein.
The domain 4 MAbs bind to VCAM-7D but do not
bind to VCAM-6D. All of the antibodies of this invention
are distinct from known VCAMl/VL~-4 blocking antibodies
such as 4B9. We show herein that 4B9 recognizes an
epitope in domain 1 of VCAM1, and thus recognizes both
VCAM-7D and VCAM-6D. The monoclonal antibodies of the
present invention recognize VCAM-7D but do not cross-block
MAb 4B9. Some of the monoclonal antibodies described
herein are effective to inhibit adhesion of Ramos and
WO93/14220 PCT/US93/00031
2 i . ~
-- 11 -
other VLA-4-expressing cells to VCAM1-expressing cells.
Some of the domain 4 monoclonal antibodies described
herein bind to epitopes of VCAMl which are not involved in
the adhesion pathway between mononuclear leukocytes and
VCAM1-expressing cells that is mediated by VCAM1. This
latter type of antibody may be useful, for example, to
detect the presence of VCAM1, in particular VCAM-7D, in
vitro or in vivo without interference with the binding of
VCAM1.
Using the monoclonal antibodies of the present
invention and other anti-VCAMl MAbs obtained from other
investigators, we have discovered that binding of VLA-4 to
VCAM1 can occur via two separate sites, one requiring
domain 1, and the other requiring domain 4. The fact that
some of the anti-VCAM1 domain 4 MAbs of this invention can
inhibit domain 4-dependent cell binding and can inhibit
cell binding to YCAM1 on cultured endothelial cells,
indicates that binding through domain 4 is a ~ignificant
functional component of the VCAMl/VLA-4 interaction n
vivo.
The monoclonal antibodies of the present
invention may be produced naturally as outlined above or
may be synthesized using recombinant DNA techniques.
Suitable recombinant antibodies include antibodies
produced, e.g., by transforming a host cell with a
suitable expression vector containing ~NA encoding the
light and heavy immunoglobulin chains of the desired
antibody, and recombinant chimeric antibodies, wherein
some or all of the hinge, constant and/or variable regions
of the heavy andlor the light chains of the anti-VCAM1
antibody have been substituted with corresponding regions
of an immunoglobulin light or heavy chain of a different
species. This includes so-called "humanized" antibodies.
WOg3/a4220 PCT/US93/00031
2~i 2~S~3~ ~
- 12 ~
(See, e.g., ~ones et al., ~986 [13], Ward et al., 1989
~14], and U.S. Patent 4,816,397 (Boss et al.~ tl5], all
incorporated herein by reference.) `~
Furthermore, domain 4-binding fragments of anti-
VCAMl antibodies, such as Fab, Fab', F(ab)2, and F(v)
fragments; heavy chain monomers or dimers; light chain
monomers or dimers; and dimers consisting of one heavy
chain and one light chain are also contemplated herein.
Such antibody fragments may be produced by chemical
methods, e.g., by cleaving an intact antibody with a
protease, such as papain or pepsin, or via recombinant DNA
techniques, e.g., by using host cells transformed with
truncated heavy and/or light chain genes. Heavy and light `
chain monemers may similarly be produced by treating an
intact antibody with a reducing agent such as
dithiothreitol or ~-mercaptoethanol or by using host cells
transformed with DNA encoding either the desired heavy ~-
chain or light chain or both. As an alternative to
hybxidoma technology, antibody fragments having similar
specificities may be isolated by phage cloning methods.
~See, e.g., Clackson et al. (ls9l) ~16].)
Monoclonal antibodies of the present invention
may be used in any application where antibody~recognition
of domain 4 of VCAMl is advantageous, including
applications where inhibiting binding between VCAM1 and
its ligand, VLA-4, is desired. For example, monoclonal
antibodies of the present invention may be used in the
treatment of inflammation or diseases characterized by
leukocyte binding to endothelium, such as post-reperfusion
injury, microbial infections or other cell migration
(e g., metastasis) dependent on a VCAMl-mediated binding
pathway, vasculitis, etc.
WO93/14220 PCT/VS93/~31
- 13 -
The monoclonal antibodies of the present
invention may also be u~ed in combination with other
antibodies, bioactive agents or materials for various
purposes. For example, the present monoclonal antibodies
S may be used in combination with 4B9 or other anti-VCAM1
antibodies in the treatment of disorder~ characterized by
VCAMl expression in endothelium. Alternatively, the
present monoclonal antibodies may be used in combination
with antibodies recognizing other endothelial cell
receptors identified in inflammatory events (e.g., ELAMl,
ICAM1, etc.) in a combination therapy designed to suppress
a range of leukocyte-endothelial cell binding pathways.
Alæo, the antibodies of the present invention, or VCAM1-
recognizing fragments thereof, may be combined or linked
to cytotoxic molecules such as TNF, ricin or the A chain
of diphtheria toxin, in order to provide antibody/toxin
conjugates capable of targeting cytotoxic elements to
VCAMl-expressing cells. The antibodies of the present
invention may also be i~mobi~i2ed on a chromatographic
~ubstrate (e.g., Protein A-sepharose) to provide an
affinity chromatography resin useful, for example, for
separating or purifying VCAM~7D preferentially over VCAM-
6D.
Detectably ~abeled anti~odies according to the
present invention may also be used in screening methods or
diagnostic methods for detecting VCAMl protein in a sample
or VCAMl-expre~sing endothelial cells ln_yi~Q or in vivo.
For example, samples may be screened for the presence of
VCAN1 or VCAM1-expressing cells by contacting the sample
with a labeled antibody according to the present invention
and detecting whether a VCAMl/anti-VCAM1 antibody complex
iæ formed.
W093/14220 PCT/US93/~31
2~ 32 - 14 - :.
Suitable labels can be radioactive, enzymatic,
fluorescent, maqnetic or chemiluminescent. Radiolabeled
antibodies are prepared in known ways by coupling a ~
radioactive isotope such as ~, 32p~ ~S, ~Fe, '~I~ which can ~:
5 then be detected by gamma counter, scintillation counter
or by autoradiography. Antibodies of this invention may
be suitably labeled with enzymes such as yeast alcohol
dehydrogenase, horseradish peroxidase, alkaline
phosphatase, and the like, then developed and detected
spectrophotometrically or visually. Suitable fluorescent
labels include fluorescein isothiocyanate, fluorescamine,
rhodamine, and the like Suitable chemiluminescent labels
include luminol, imidazole, oxalate aster, luciferin, and
the like.
For therapeutic uses, the monoclonal antibodies
of the present invention may be formulated as a
pharmaceutical composition comprising an effective amount
of the antibody admixed with a pharmaceutically acceptable
carrier. Typically, the antibodies of ~he present
invention will be suspended in a sterile saline solution
for therapeutic uses. The pharmaceutical compositions may
alternatively be formulated to control release of the
actiYe ingredients or to prolong their prese~ce in a
patient's system. Numerous suitable drug delivery systems
are known and include, e.g., hydrogels,
hydroxymethylcellulose, microcapsules, liposomes,
microemulsions, microspheres, and the like.
The pharmaceutical compositions contemplated
herein may be administered by any suitable means such as
orally, intrana~ally, subcutaneously, intramuscularly,
intravenously, intra-arterially, or parenterally.
Ordinarily, intravenous (i.v.) or parenteral
administration will be preferred.
WO 93/14220 PCI`~US93/00031
~ ~ ~ 7 -, ~ 9
' J
~ pl~ 1
MAbs Recoan zinq VCAM-7D But Not VCAM-6D
Monoclonal antibodies according to the invention
that recognize domain 4 of VCAM-7D were prepared in the
following manner. We injected four BALB/c mice with whole
CHO cells that express VCAM-6D on their surfaces ("VCAM-
6D/CHO" cells). The CHO culture was a Chin~se Hamster
Ovary cell line stably transfected with cDNA encoding
VCAM-6D and was obtained from Biogen, Inc. (Cambridge,
MA). Initially, each mouse received approximately 107
cells in P8S intraperitoneally (i.p.). We in3ected
complete Freund's adjuvant i.p. at a different site 2-24
hours later. We boosted the mice twice i.p. with VCAM-
6D/CHO cells, without adjuvant. We then immu~ zed each of
the mice with 55 ~g of a recombinant soluble ~rm of VCAM-
7D (rsVCAM-7D, consisting of the N-terminal 674 amino -
acids of mature VCAM-7D and lacking the transmembrane and
cytoplasmic regions; obtained from Biogen, Inc.,
Cambridge, MA)` injected intraperitoneally with incomplete
Freund's adjuvant We boosted the mice twice with rsVCAM-
7D i.p. without adjuvant. Four days after the rsVCAM1
injections, we removed the spleen from one mouse and fused
the splenocytes with P3/X63-Ag8.653 murine m~loma cells
according to the method described by Lerner t17].
Supernatants of cultures of individual clones of
fused cells were screened for their ability to bind to
rsVCAM-7D-coated plates. ELISA plates (Corning) were
coated with rsVCAM-7D (0.1 ~g/ml, 50 ~l/well) and
incubated overnight at 4 C. The coating solution was
aspirated and blocking buffer was added (phosphate
buffered saline (PBS) with 5% fetal calf ~erum (FCS), pH
7.2) to block the remaining sites, and the plates were
incubated for 2 hrs. at room temperature. The blocking
W093/14220 P~T/US93/00031
2 ~ 2 7 .1 3 2
- 16 -
buffer was removed, and 50 ~1 of hybridoma culture
supernatants (or supernatant diluted into blocking buffer)
were added to the plates and then incubated for 1 hr. at
room temperature. Next, the plates were washed with PBS
containing 0.05% detergent (Tween-20) to r2move unbound
antibody and incubated with a horseradish peroxidase-
conjugated goat anti-mouse immunoglobulin for 1 hr. at
room temperature. The plates were washed with the
PBS/Tween-20 solution and developed with the chromogenic
horseradish peroxidase substrate, TMB (3,3',5,5'-
tetramethylbenzidine; ICN, Lisle IL) 42mM in 0.lM sodium
acetate-citric acid, pH 4.9. The reaction was initiated
by addition of hydrogen peroxide (15 ~1, 30% solution) and
quenched by addition of 2N sulfuric acid. The colored
product was quantified at 450 nm using a Thermofax plate
reader (Molecular Devices; Palo Alto CA). ~-~
The cells in positive culture wells were -
subcloned by limiting dilution and re-evaluated using the
screening assay described above. We then evaluated the
hybridoma supernatants for their ability to stain, by FACS
analysis, COS7 cells transiently expressing VCAM-6D or
~CAM-7D. In these experiments cDNA encoding VCAM-6D or
VCAM-7D was inserted into an expression vector (pCDM8) and
subsequently transfected into C~S7 cells by
electroporation, as described in Osborn et al. 19S9 [2
and Hession et al. 1991 [9~.
The COS7 cells, transfected with either VCAM-6D
or VCAM-7D cDNA, were washed and suspendad in suspension
buffer (PBS/5mM EDTA/2% ~CS/o.05% sodium azide) and
incubated 45 min. at 4 C with supernatant from hybridoma
cultures. The transfected cells were then washed with
suspension buffer and incubated with a fluoresceinated
goat anti-mouse Ig (Jackson Immunoresearch, West Grove PA)
W093~14220 PCT/US~3~31
~ ~ 7~
~ t ~
- 17 -
for 45 min. at 4O C. The cells weire washed, fixed with
o.2% paraformaldehyde in PBS and evaluated using a FACStar
(Becton Dickinson, Mountain View CA).
FACS analysis showed that the isolated anti-
VCAM1 monoclonal antibodies bound to VCAM-7D but not to
VCAM-6D. The binding data for these antibodies are
~ummarized in Table I (infra p. 28).
E~mpl~ 2
Ramos Cell Bindinq_Assay
Ramos is a B-lymphoblastoid cell line t.hat
expre~ses VLA-4 and thus binds to VCAM1 but does not bind
to other induced endothelial cell molecules such as ICAM-l
or ELAM-l (Osborn et al., 1989 [2]). Ramos is therefore a
convenient model cell line with which to assay VLA-4- ~:
15 dependent bindinq. Ramos cells bind to both recombinant ~
VCAM-6D and recombinant VCAM-7~ when these molecules are ~--
expressed on COS cells. We had previously noted that ~`
binding to VCAM-7D was somewhat (less than 2 fold) greater
than to VCAM-6D in transient transfection assays of a
single preparation of each plasmid (Hession et al., 1991
[9~). To determine if this ~uantitative difference in
binding activity was reproducible, several preparations of ~.
each plasmid were made and electroporated in parallel into
COS cells. Reipeated transfections revealed that although
expression of the two plasmids is similar, VCAM-7D on
average binds significantly more cells per square ..
millimeter than does VCAM-6D. (Cf. Figure 6 and Figure
7.~ Binding to both six- and seven-domain forms of VC~Ml
was inhibited by the anti-VLA-4 M~b HP1~2 or by anti-VCAM
MA~ 4B9 (Figurei 7).
In all of our procedures, the binding of Ramos
cells was measured as follows:
: `
WO93/14220 PCT/US93/00031
~2~ ~t~32
- 18 -
Confluent monolayers of the target cells (e.g., COS
transfectants or induced H W ECs) in 48-well plastic tissue
culture plates were washed with RPMI/10% fetal calf serum
(FCS), and an amount (e.g~ g/ml, varying per MAb and
5 per experiment) of the test MAb (e.g., 4B9, HPl/2, GH12, ~;
etc.) in a volume of 0.15 ml was added. In the case of
H W ECs, the cells were preincubated 4, 24, 48 or 72 hours
at 37 C with 20 ng/ml recombinant human TNF (Biogen,
Inc.) to induce expression of VCAM1. Ramos cells were
fluorescently labeled by adding 2',7'-bis(2-carboxyethyl)-
5(and 6)-carboxy-fluorescein ("BCECF", Molecular Probes,
Eugene OR) according to the manufacturer's instructions.
The cells were first centrifuged and then resuspended in
RPMI/10% fetal calf serum (FCS) to a concentration of S x
106 cells ml. The BCECF was added to 2 ~M and the
suspension incubated for 30 min. at 37 C~ The cells were
washed twice in RPMI/10% FCS and then resuspended in the
same solution at 3 x 10~ cells/ml.
4 . 5 X 105 labeled Ramos cells were added to each :
well of the 48-well tissue culture plates (final volume,
0.3 ml) and incubated 10 minutes at 37 C. The plates
were then emptied by inversion with gentle shaking and
washed 3X with HANKS Buffered Salt Solution (HBSS) with
Ca'+ and Mg++. Adherent cells were lysed by addition of
0.12 ml of a 1% detergent solution (Nonidet P-40),
followed by incubation for 5 min. at 37 C. The lysate
was transferred to 96-well microtiter plates by
multichannel micropipette, and the number of adherent
Ramos cells was calculated by reading the plate in a
Titertek Fluorskan II ELISA reader (Flow Laboratories).
(See, also, Gimbrone et al., 1989 ~18].)
W093~14220 PCT/US93/~031
~ 1~7 ` 32
-- 19 --
Antibody staininq Assay
The antibodies were tested for their ability to
bind to VCAM-7D, VCAM-6D and various VCAM/ICAM constructs
exhibiting domain 4-dependent cell binding (see Figures 1-
13 and Example 3, infra). COS7 cells transfected with aparticular VCAM, ICAM (control~ or chimeric VCAM/ICAM (or
VCAM/VCAM, see Fig. 11) cDNA construct were removed from
100 mm culture dishes by incubation in HBSS/5 mM EDTA for ;-`
15 min. at 37 C, followed by vigorous pipetting.
Aliquots of 3 5 x 105 cells were incubated indiviclually
with 2 ~g/ml of the MAb being tested, or with rabbit
polyclonal antiserum raised against rsVCAM-7D (diluted
1:500), in PNF tPBS/0.1% sodium azide/2% FCS) for 20 min.
at room temperature. After incubation the cells were
washed twice with PNF and incubated 20 min. at room
temperature with 0.2 ~g/ml of a secondary fluoresceinated
antibody (rabbit anti-mouse immunoglobulin or goat anti-
rabbit immunoglobulin) in PNF. Cells were then washed 3X
with PBS, suspended in PBS/1% formaldehyde, stored dark at
4 C, and analyzed using a FACStar (Becton Dickinson;
Mountain View CA). The number of cells versus fluorescent
intensity was plotted, and the number of positive cells in
each sample was determined from the FACS hist~grams.
Fluorescence peaks for each transfectant and for negative
controls (pCDM8 vector only) run in parallel were compared
and the peaks marked to exclude 99% of the negative cells.
The number of positive cells (to the right of the marker)
was determined for each transfectant. This method results
in a slight underestimate of the number of positive cells
in each ~ase, since weakly positive cells that overlap the
negative control fluorescence peak are excluded.
WO93/14~20 PCT/US93/~031
,~ ~7 1 ~2
- 20 -
~ pl~ 3
Construction and Expression of
Chimeric VCAM/ICAM PolyDeptides
In order to identify regions of VCAMl involved
in recognition of VLA-4-expressing cells, recombinant
genes encoding VCAM/ICAM chimeras were constru~ted by
excising portions of the VCAM-7D and VCAM-6D cDNAs and
replacing them with analogous regions of ICAM-l, using
unique restriction endonuclease sites in the VCAMl cDNA
tsee Fig. l) and polymerase chain reaction tPCR)-generated
fragments of ICAMl. Substitution of the excised VCAMl
regions was done to eliminate as much as possible the
~tructural distortions that would accompany deletion of
one or more domains of the VCAMl molecules. ICAMl was `~
selected as a source of substituant structures because it
is also a member of the Ig superfamily and has the highest
degree of amino acid homology to VCAMl among Ig
superfamily members (Osborn et al., 1989 ~2]).
- Schematic diagrams of the VCAMl/ICAMl chimeric
polypeptides are shown in Figures 1-5, 8,~ 9 and ll. The
cDNA constructs coding for each of these were prepared as
follows: ~
Aliquots of the parent plasmids VCAMlEll/CDM8 (encoding
VCAM-7D; see, Hession et al., l99l [~]~ or VCAM4ltCDM8
(encod~ng VCAM-6D; Osborn et al., 1989 [2]) were diqested
using restriction endonucleases at the appropriate sites
indicated in Figure 1 and agarose gel purified. ICAMl
inserts were made by PCR from an ICAMl/CDN8 plasmid
isolated from an endothelial cDNA library previously
described (Osborn et al., l989 [2]), using oligonucleotide
probes based on the published sequence of ICAMl (Simmons
et al~, 1988 [l9~; Staunton et al., 1988 [20]). The PCR
primers were designed as follows:
WO~3/14220 PCT/US93/~031
21~7 vJ~ `
Primer P-1 (SE0 ID N0:1) ;.
EcoRV
5'-TCTAGATATC TTCTGCCCCG GGAGGCTCCG TGCTG-3'
The first twelve nucleotides correspond to the coding
5 region for VCAMl amino acid rssidues 9-12; the following .
twenty-three nucleotides correspond to the coding region
for ICAMl amino acid residues 11-18.
Primer P-2 ISE0 ID N0:2)
Bgl2
5'-GGTGGAGATC TACTGGACTC CAGAACGGGT GGAA-3'
The first thirteen nucleotides correspond to the coding
region for VCAMl amino acid residues 86-89; the following
twenty-one nucleotides correspond to the coding region for
ICAMl amino acid residues 84-90.
Primer P-3 (SE0 ID N0:3)
Hind3
5'-TCTCAA CTT TTACTGTTGA GATCTCCCCT GG-3'
The first twelve nucleotides correspond to the coding
region for VCAMl amino acid residues -3-1; the following
twenty nucleotides correspond to the coding region for
ICAM1 amino acid residues 300-30~.
PrinLer P-4 (SEQ ID N0:4)
Bsu3 6 1
5 '--CTGGAT CT~ AGGAGTCCAG TACACGGTGA GGAAGG-3'
The first fourteen nucleotides correspond to the coding
region for VCAMl amino a~id resi~ues 92-9~; the following
twenty-two nucleotides correspond to the coding region for
ICAMl amino acid residues 79-85.
Primer P-5 fSEQ ID N0:5)
BspHl
5'-CTCTTCATGA GCTTCTCCC C~CGGAGCAG CACCAC-3'
The first eleven nucleotides correspond to the coding
region for VCAMl amino acid residues 134-137; the
` -,`,
WO93~14220 PCT/US93/~31 ~
5 J ~ ;~
-- 22 --
following twenty-four nucleotides correspond to the coding
region for ICAMl amino acid residues 121-128.
Primer P-6 ~SE0 ID NO:6~ -
Bgl2
5'-GGGGAGATCT CGGGCGCCGG AAAGCTGTAG ATGGT-3'
The first eleven nucleotides correspond to the coding
region for VCAMl amino acid residues 302-305; the
following twenty-four nuclsotides correspond to the coding
region for ICAM1 amino acid residues 279-2~6.
Primer P-7 (SE0 ID N0:7
Bsu361
5'-CTGGATCCTT AGGGAATGAG TAGAGCTCCA C-3'
The first thirteen nucleotides correspond to the coding
region ~or VCAMl amino acid residues 92-96; the following
eighteen nucleotides correspond to the coding region for
VCAM1 amino acid residues 385 394.
PCR synthesis for inserts for construct
VCAM/ICAM-1 (Fig. 3) was primad with P-l and P~4; for
construct V~A~/ICAM-2 (Fig. 4), P-l and P-5; for construct
VCAM/ICAM-3 (Fig. 5), P-2 and P-6; for construct
VCAM6D/V~AM4-l (Fig. 11), P-3 and P-7. VCAM6D/ICAM-l and
~CAM6D/ICAM-2 ~Figs. 8 and 9~ were made using the same
inserts as for VCAM/I~AM-1 and VC~M/ICAM-2, respectively,
clonad into the VCAM-6D expressic)n vector (VCAM41/CDM8)
25 instead of the VCAM-7D expression vector (VCAM~Ell/CDM8).
~fter PCR synthesis, performed as described in Hession at
al. (1991) [9], the insert fragments were digested with
the appropriate r~striction endonuclaases and gel
purified. Expression vectors and insert fragments were
ligated using NEB ligase and buffer, ethanol precipitated,
then electroporatad into bacterial host strain MCl061/p3
using a Biorad Gene Pulser.
WO93/14220 PCT/US93/00031 ~
2 ~ 3~ J
- 23 - ~.
Clones were screened by PCR and restriction
digests, and at least one isolate of each clone was
sequenced across cloning junctions and insert using a
Sequenase kit (United Stàtes Biochemical Corp~). The
complete cDNA sequences for VCAM-7D, VCAM-6D, and each of
the chimeric constructs VCAN/ICAM-l, VCAM/ICAM-2,
VCAMtICAM-3, VCAM6DtICAM-1, VCAM6D/ICAM-2, and
VCAM6D/VCAM4-l are set forth in the Sequence Listing below
(in order, SEQ ID NOS: 8 through 15~.
The diagrammed constructs were transfected into
COS7 cells and found to be well expresæed when tested by
FACS analysis after staining with polyclonal anticerum to
VCAMl.
In constructs VCAM/ICAM-l (Fig. 3) and
15 VCAM/ICAM-2 (Fig. 4), most of VCAM domain l, or most of `:
domain l and the first half of domain 2, were replaced by
ICAMl sequence (see shaded areas, Figs. 3 and 4,
respectively). In construct VCAM/ICAM-3 (Fig. 5), VCAM :~
domains 2 and 3, and small portions of domains l and 4,
20 were replaced by ICAMl sequence (see shaded area, Fig. 5). :~:
Ex~mple 4
MAb 4B9 Bindinq to VCAMl domain l.
It has been previously shown that the N-terminal
three domains of VCAMl support adhesion to VLA-4 (Taichman
et al., l99l [2l]) and that the anti-VCA`Ml MAb 4B9 ::
significantly inhibits binding of VLA-4-bearing cells to
in~uced HUVECs or VCAMl transfectants (Carlos et al., l990
[22]). These results suggested that the binding site of
VCAMl recognized by VLA-4 and obstructed by 4B9 is i~ the
N-terminal portion of the VCAMl molecule.
Using the MAb binding assay described above
(Example 2), we confirmed that MAb 4B9 recognizes an
`~:
' ''
WO93/14220 PCT/US93/00031
2127~2 - 24 -
epitope in VCAMl domain l. It was found that 4B9 bound -
readily to COS7 transfectants expressing both seven~ and
six-domain forms of VCAMl. Furthermore, chimeric
constructs VCAM/ICAM-l and VCAM/ICAM-2 ~Figs. 3 and 4,
respectively), wherein most of domain l was replaced by
ICAMl amino acid residues, did not bind to 4B9 (see, Table
I, nf~a). Th~se results indicated that the epitope to
which 4Bg binds requires domain l for recognition.
Finally, construct VCAM/ICAM-3 (see, Fig. 5, domain 1
mostly intact) did bind 4B9, confirming that domain l
contains the 4B9 epitope. The binding and ~amos blocking ~
characteristics of 4B~ are summarized in Table I, infra. -
Although constructs VCAM/ICAM 1 and VCAM/ICAM-2 -
did not bind MAb 4B9, both bound Ramos cells at about 30%
of the level seen with intact VCAM-7D (Figure 7). This
binding was mediated by VLA-4/VCAMl, since anti-VLA-4 mAb
HPl/2 completely inhibited the interaction, while a
blocking antibody to the ICAM-l pathway (MAb 60.3) did not
affect binding (data not shown). Construct VCAM/ICAM-3
bound Ramos cells weakly but measurably, at about 10% of
the level seen with intact VCAM-7D (Figure 7).
Ramos Cells Bind At_Two Reions On VCA~l
To determine whether Ramos cell binding to
constructs VCAM/ICAM-l and VC~M/ICAM-2 was due to residual
2S portions of the domain l-dependent binding site, or to
domain 4 (which is structurally similar to domain l~,
constructs analogous to VCAM/ICAM-l and VCAM/ICAM-2 were
made,~except that cDNA coding for VCAM-6D (no domain 4)
instead of VCAM-7D was used as the substrate or backbone
for the substituted ICAMl inserts (see, Figs. 8 and 9).
These constructs, designated VCAM6D/ICAM-l and
VCAM6~/ICAM-2, respectively, were wèll expressed in COS7
cells as measured by anti-VCAMl polyclonal antiserum
WO93/14220 PCT/US93/00031
g .~
- 25 -
staining, but were unable to bind ~amos cells (Figure lO).
These results indicated that the binding of Ramos cells to
constructs VCAM~ICAM-l and VCAM/ICAM-~ appears to be due
to the presence of domain 4.
~e next prepared a construct designated
VCAM6D/VCAM4-1, in which domain 4 of VCAM-7D was
substituted for domain 1 of VCAM-6D (Fig. 11). Referring
to the results depicted in Figures 7 and 12, Ramos binding ~ ;
of VCAM6D/VCAM4-1 was comparable to that of VCAM-6D (see
10 Fig. 2, intact domain 1) and VCAM/ICAM-l ~see Fig. 3, :~
intact domain 4), indicating that domain 4 can incleed bind
cells. This result was somewhat surprising in light of
the fact that MAb 4B9, which binds to domain 1 but not to ~
domain 4, can completely inhibit binding of Ramos cells to -:
VCAM-7D. Stearic hindrance is one possible explanation;
it is also possible that binding of 4B9 to domain 1
perturbs the structure of the molecule so that domain 4 is
no longer in the proper conformation to bind cells.
~x~mple S -
Nonoclonal Antibodies That Block
Domain 4-deendent Cell Adhçsion
According to the procedure set forth in Ex~mple
l, three monoclonal antibodies were produced.that .
recognize VCAM-7D but not VCAM-6D, indicating that they
bind domain 4, or a domain 4-dependent epitope. These
MAbs were designated GE4, ED11 and GH12. All three MAbs
were of IgG1 isotype. The binding and Ramos blocking
characteristics of each antibody were determined using the
antibody and Ramos binding assays described above (Example
2).
MAb GH12 bound to COS7 transfectants expressing
VCAM-7D, VCAM/ICAM-l and 2, and VCAM6D/VCAM4-1 but did not
bind to transfectants expressing VCAM-6D or VCAM/ICAM-3.
`:
WO93/14220 PCT/US93/~03~
2 i27i.i32
- 26 -
Referring to Figure 13, Ramos binding experiments showed
that MAb GH~2 at 10 ~g/ml inhibited Ramos binding to COS7
transfectants expressing the VCAM/ICAM-l, VCAM/ICAM-2 and
VCAM6D/VCAM4-1 constructs but did not bind to the
S VCAM/ICAM-3-expressing tranfectants, indicating that the
~mall portion of domain 4 eliminated from the VCAM/ICAM-3
construct was important to GH12 binding. MAb GH12 did not
block Ramos cell binding to VCAM-6D or VCAM 7D.
Binding experiments showed that MAb EDll bound
to COS7 cells expressing VCAM-7~, VCAM/ICAM-1 and
VCAM/ICAM-2 but not to cells expressing VCAM-6D,
VCAM/ICAM-3 or VCAM6D/VCAM4-1. Referring to Figure 14,
Ramos binding experiments showed that MAb ED11 at 5 ~g/ml
inhibited Ramos binding to COS7 transfectants expressing
th~ VCA~'/ICAM-1 and VCAM/ICAM-2 constructs but did not
inhibit VCAM-6D-expressing, VCA~/ICAM-3-expressing or
VCAM6D/VCAM4-1-expressing tranfectants. Surprisingly, MAb
EDll did partially block Ramos cell binding to VCAM-7D-
expressing COS7 cells.
Bi~ding experiments showed that MAb GE4 bound to
COS7 cells expressing VCAM-7D, VCAM/ICAM-l, VC~M/ICAM-2
and VC~M/ICAM-3 but not to cells expressing VCAM-6D or
VCAM6D/VCAM4-1. Referring to Figure 15, Ramos binding
experiments showed that MAk GE4 at 10 ~glml did not
inhibit Ramos binding to COS7 transfectants expressing
VCAM/ICAM-1 and VC~M/ICAM-2. MAb GE4 did not block Ramos
adhesion to transfectants expressing VCAM6~/VCAM4-1,
VCAMlICAM-3 or VCAM-6D, and also did not inhibit Ramos
binding to VCAM-7D-expressing COS7 ~ells at the single
concentration testad.
The binding and blocking data are summarized
below in Table I. From these results it can be concluded
that each of the three domain 4 antibodies recognize
WO93/14220 PCT/US93/~031
2~27.i.~
- 27 -
distinct epitopes, and two are capable of blocking domain
4-dependent adhesion of VL~-4 to VCAM1. These antibodies
represent a new rlass of anti-VCAMl blocking antibody.
Table I
constructs* .
_~a~ VCAM-7D VCAM-6D A B C D
GH12
binding + - ~ +
blocking - - + ~ - +
10 EDll .. :
binding + - + ~ - -
blocking ~ +
GE4
binding + - + ~ + - -::
15 blocking
489 ~control) --~
binding ~ + - ::
blockîng + +
20 * A - VCAM/ICAM-l C = VCAM/ICAM-3
B = VCAM/ICAM-2 D - VCAM6D/VCAM4-1 .
E~ample 6
Domain 4 MAb Inhibition of Ramos Binding
to Cvtokine-activated EndQthelial Cells
To determine whether the domain-4-dependent MAbs
ED11 or GH12 were capable of blockin~ adhesion to VCAMl on
e~dothelial cells (which is predominantly VCAM-7D), human `~
umbilical vein endothelial cells (H W ECs) were treated
with 20 ng/ml human recombinant TNF for 4, 24, 48, or 72
hours, and binding of Ramos cells and t~e ability of the
antibodies to block binding was measured. All MAbs were
added at a concentration of 10 ~g/ml. The results are
shown for each activation period in Figures 16-19.
WO93/14220 pcT/us93/noo3l
~1275~
- 28 -
The results indicate overall that MAb GH12 did
not block HUVEC/Ramos binding reliably at the cingle
concentration tested (10 ~glml), but MAb EDll blocked more
than 80% of Ramos cell binding to induced H W ECs at all
time points tested. MAb 4B9 blocked HUVEC/Ramos binding
by greater than 90%. These results suggest that although
the two cell binding sites of VCAMl (domain 1- and domain-
4-dependent) can be separated and remain functional,
antibodies to each can interfere with Ramos cell binding
to the other. This is so even though the antibodies do
not cross-react with each other's binding sites (shown by
the antibody binding data to constructs, above~.
In contrast to stimulated endothelial cells, as
in this Example, where VCAM-7D is the predominantly
expressed (RNA and protein) form of VCAM-l, VCAM-6D tRNA)
may be the predominant species of VCAM-l in bone marrow
stromal cells and tonsillar follicular dendritic cells.
This suggests a potential therapeutic advantage of domain
4 spe~ific anti-VCAMl antibodies over domain 1 specific
anti-VCAMl antibodies, in that domain 4 antibodies
according to the present invention might potentially block
the recruitment of leukocytes via stimulated endothelial
cells into sites of infla~mation, without blocking
lymphopoiesis, which is the role that VCAM-l plays in bone
marrow (Miyake et al., 1977 [7]). Such an inhibition of
B-cell lymphopoiesis may be undesirable in longer term
therapy with anti-VCAM1 antibodies.
The foregoing examples are intended as an
illustration of the present invention and are not
presented as a limitation of the invention as claimed
hereinafter. From the foregoing disclosure, numerous
modifications and additional embodiments of the invention
will be apparent to and easily practiced by those
WO93/14220 PCT/US93~31
2127 -;3 ~ ~
- 29 -
experienced in this art. All such modifications and .`
additional embodiments are within the contemplation of - :
t~is application and within the scope of the appended
claims. :
We deposited subclones of hybridomas producing -~
EDll, GE4 and GH12 monoclonal antibodies under the
Budapest Treaty with the American Type Culture Collection ~
(ATCC), 12301 Parklawn Dr~, Rockville, MD (US) on January ~;
9~ 1992. The cultures were identified as follows: .
desiq__tion ATCC_accession no. `:
MAb CB.EDll.AG3 HB 10962
MAb CB.GE4.BG5 HB 10961 ~ -
MAb CB.GH12.AAl2 HB 10963 ~
W093/14220 PCT/U~93/~031
3 ~
- 30 -
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cytokine-activated cultured human endothelial cells,"
Blood, 76(5), 965-970.
The foregoing publications are incorporated
herein by reference.
r 3 7 . ~ ;)
~ .J ~
SEQUENCE LISTING
(1) GENERAL INFO~ATION:
(i) APPLICANT:
(A) NAME: BIOGEN, Inc.
(B) STREET: 14 Cambridge Center
(C) CITY: Cambridge
(D) STATE: Massachusetts
(E~ COUNTRY: USA
(F) POSTAL CODE (ZIP): 02142
(A) NAME: OSBORN, Laurelee
(B) STREET: 19 Harding Street, ~2
(C) CITY: Cambridge
(D) STATE: Massachusetts
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 02141
(A) NAME: BENJAMIN, Christopher D.
(B) STREET: 2 Oak Hill Lane
(C) CITY: Beverly
(D) STATE: Massachu;ettes
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 01915
(ii) TITLE OF INVENTION: ANTIBODIES RECOGNIZING THE FOURTH
IMMVNOGLOBULIN-LIKE DOMAIN OF VCAM-l
(iii) NUMBER OF SEQUENCES: 23
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATINC SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: WQ PCT/US93/00031
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/821,712
(B) FILING DATE: 13-JAN-1992
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single :
(D) TOPOLOGY: linear
~UeSTl~ E S~IEET
212~ ~ 2~ 2 '
-34-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
TCTAGATATC TTCTGCGCCG GGAGGCTCCG TGCTG 35
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C~ STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GGTGGAGATC TACTGGACTC CAGAACGGGT GG M 34
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TCTC M GCTT TTACTGTTGA GATCTCCCCT GG 32 . ~.
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: ~ucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - -
CTGGATCCTT AGGAGTCCAG TACACGGTGA GG MGG 36
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~;116S~ IJ~E S~EE~T
~ 1 2 ~ ;3 3 ~
-35- :~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CTCTTCATGA GCTTCTCCCC ACGGAGCAGC ACCAC 35
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs ~. -
(B) TYPE: nucleic acid .
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GGGGAGATCT CGGGCGCCGG AAAGCTGTAG ATGGT 35
(2) INFORMATION FOR SEQ ID NO:7:
(i) ~EQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base. pairs
(B) TYPE: nucleic acid .
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear :: :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ;
CTGGATCCTT AGGGAATGAG TAGAGCTCCA C ~ 31
'.''
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2217 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
tA) NAME/KEY: CDS
(B) LOCATION: 1..2217
(ix) FEATURE:
(A) NAME/KEY: mat peptide
(B) LOCATION: 73..2217
(ix) FEATURE:
tA) NAME/KEY: sig_peptide
(B) LOCATION: 1..72
81~3S~3~E SffE~ ~
2 1 ~ ~ . i ~ 2
.... .
-36-
(~i) SEQUENCE DESCRIPTION: SEQ ID No:8:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp
--24 --20 --1 5 --10
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96 .
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu
_5 1 5 '.
TCT AGA TAT CTT GCT CAG ATT GGT GAC TCC GTC TCA TTG ACT TGC AGC 144 ~; ~
Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser .:~:
10 15 20 ~:
ACC ACA GGC TGT GAG TCC CCA TTT TTC TCT TGG AGA ACC CAG ATA GAT 192
Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln Ile Asp :~ :
25 30 35 40 :~
AGT CCA CTG M T GGG M G GTG ACG AAT GAG GGG ACC ACA TCT ACG CTG 240
Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu : ~
45 50 55 - :
ACA ATG AAT CCT GTT AGT TTT GGG M C G M CAC TCT TAC CTG TGC ACA 288
Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr .~
60 65 70 .
GCA ACT TGT GAA TCT AGG AAA TTG GAA AAA GGA ATC CAG GTG GAG ATC 336 ::-
Ala Thr Cys Glu Ser Arg Lys Leu Glu Lyc Gly Ile Gln Val Glu Ile -~;
75 80 85 ,
TAC TCT TTT CCT M G GAT CCA GAG ATT CAT TTG AGT GGC CCT CTG GAG 384
Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu
90 95 100
GCT GGG M G CCG ATC ACA GTC AAG TGT TCA GTT GCT GAT GTA TAC CCA 432
Ala Gly Lys Pro Ile Thr Val Lys Gys Ser Val Ala Asp Val Tyr Pro
105 110 115 120
TTT GAC AGG CTG GAG ATA GAC TTA CTG AAA GGA GAT CAT CTC ATG AAG 480
Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys
125 130 135
AGT CAG GAA TTT CTG GAG GAT GCA GAC AGG AAG TCC CTG GAA ACC AAG 52B
Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
140 145 150
AGT TTG GAA GTA ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT 576
Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val
155 160 165
elJBSTlTllTE SHE~ ` ` `
.' `,``'
~ 1 27'~ ~2
--37--
CTT GTT TGC CGA GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC 624
Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro
170 175 180
ACA GTA AGG CAG GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC AAG 6 7 2
Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pr~ Lys
185 190 195 200
AAT ACA GTT ATT TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GGT GGG 720
Asn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly
205 210 215
TCT GTG ACC ATG ACC TGT TCC AGC GAG GGT CTA CCA GCT CCA GAG ATT 7 6 8
Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile
220 225 230
TTC TGG AGT AAG AAA TTA GAT MT GGG AAT CTA CAG CAC CTT TCT GGA 816 . :
Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly -`:
235 240 245 `~
AAT GCA ACT CTC ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT 864
Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile
250 2S5 260
TAT GTG TGT GAA GGA GTT AAT TTG ATT GGG AAA AAC AGA AAA GAG GTG 912
Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val
265 270 27S 280
GAA TTA ATT GTT CAA GAG AAA CCA TTT ACT GTT GAG ATC TCC CCT GGA 960
I:lu Leu Ile Val Gln Glu Lys Pro Phe Thr Val Glu Ile Ser Pro Gly .
285 290 295
CCC CGG ATT GCT GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TGT AGT 1008
Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Ser
300 305 310
GTC ATG GGC TGT GM TCC CCA TCT TTC TCC TGG AGA AC,C CAG ATA GAC 1055
Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp
315 320 325
AGC CCT CTG AGC GGG AAG GTG AGG AGT GAG GGG ACC AAT TCC ACG CTG 1104
Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr LPU
330 335 340
ACC CTG AGC CCT GTG AGT TTT GAG MC GAA CAC TCT TAT CTG TGC ACA 115 2
Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr
345 350 355 360
GTG ACT TGT GGA CAT MG AAA CTG GM MG GGA ATC CAG GTG GAG CTC 12 00
Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu
365 370 375
~U13ST~ E S~
2~ 7'.3f32 '~
--38--
TAC TCA TTC CCT AGA GAT CCA GAA ATC GAG ATG AGT GGT GGC CTC GTG 1248
Tyr Ser Phe Pro Arg Asp Pro Glu lle Glu Met Ser Gly Gly Leu Val
380 385 39~ -
AAT GGG AGC TCT GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC CCC 1296
Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro :~
395 400 405 --
CTT GAC CGG CTG GAG ATT GAA TTA CTT MG GGG GAG ACT ATT CTG GAG1344
Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu
410 415 420 -
AAT ATA GAG TTT TTG GAG GAT ACG GAT ATG AAA TCT CTA GAG AAC AAA 1392
Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys
425 430 435 440 .
AGT TTG GAA ATG ACC TTC ATC CCT ACC ATT GM GAT ACT GGA AAA GCT1440
Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala -
445 450 455
CTT GTT TGT CAG GCT AAG TTA CAT ATT GAT GAC ATG GAA TTC GAA CCC 1488
Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro
460 465 470
AAA CAA AGG CAG AGT ACG CAA ACA CTT TAT GTC AAT GTT GCC CCC AGA 1536
Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg
475 480 485
GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG GAG GAA GGC AGT 1584
Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser
490 495 500
TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT CCG AAA ATC 1632
Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile
505 510 515 520
CTG TGG AGG AGG CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TGT GAG 1680
L,eu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu
525 530 535
AAT GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GAA GAT TCT GGG GTT 1728
Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val
540 545 SS0
TAT TTA TGT GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA AAG GM GTG1776
Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val
555 560 56S
GAA TTA ATT ATC C M GTT ACT CCA AAA GAC ATA AAA CTT ACA GCT TTT 1824
Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe ~:
570 57S S80
2 127 t~ 32
-39-
CCT TCT GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT ACA 1872
Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr
585 590 595 600 ~ ~
TGT GGA AAT GTT CCA GAA ACA TGG ATA ATC CTG AAG AAA AAA GCG GAG 1920 `-. :
Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu
605 610 615
ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC CGA 1968
Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg ~ :
620 ~25 630
AAG GCC CAG TTG M G GAT GCG GGA GTA TAT GAA TGT GAA TCT AAA M C 2016
Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn
635 640 645
AAA GTT GGC TCA CAA TTA AGA AGT TTA ACA CTT GAT GTT C M GGA AGA 2064
Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg
650 65S 660
GAA AAC AAC AAA GAC TAT TTT TCT CCT GAG CTT CTC GTG CTC TAT TTT 2112 :Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe
665 670 675 680
GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT GCA 2160
Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala
685 690 695
AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CTT GTA GAA GCA CAG AAA 2208
Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys
700 705 710
TCA AAA GTG 2217
Ser Lys Val
715
(2) INFORMATION FOR SEQ ID NO:9:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 739 amino acids
(B) l~PE: amino acid -~;
(~) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
SU~S~ T
212'7`132 ' `
-40-
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn ~le Leu Trp
-24 -20 -15 -10
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu
-5 1 5
Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser
Thr Thr Gly Cys Glu Ser Pro Phe Phe Se. Trp Ar~ Thr Gln Ile Asp
er Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
hr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr
la Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile
~5
yr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu
100
la Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro
105 110 115 120
he Asp Arg Leu Glu Ile~ Asp Leu Leu Lys Gly Asp His Leu Met Lys
125 130 135
er Gln Glu Phie Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
140 145 150
Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val
155 160 165
Leu Val Cys Arg A~a Lys Leu His Ile Asp Glu Met Asp Ser Val Pro
~.70 175 180
Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys
185 190 195 200
sn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly
205 210 215
er Val Thr Met ~r Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile
220 225 ~30
Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln Hi.q Leu Ser Gly
235 240 245
Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile
250 255 260
T~ E ~
21 27~-j32
-41-
Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Va~
265 270 275 280
Glu Leu Ile Val Gln Glu Lys Pro Pha Thr Val Glu Ile Ser Pro Gly
285 290 295
Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Ser
300 305 310
Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp
315 320 325
Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu
330 335 340
-~:
Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr
345 350 355 360
,~:
Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu
365 370 375
Tyr Ser Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val
380 385 390
Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro
395 400 4~5
Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu
410 415 420
Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys
425 430 435 440
Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala
445 450 455
Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro
460 465 470
Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg
475 480 485
Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser
490 495 50Q
Ser Val Asn Met Thr Cys Leu Ser 51n Gly Phe Pro Ala Pro Lys Ile
505 51Q 515 520
SllBST~7 UTE S~
.
` 2~7~3~ -
-42-
eu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu
525 530 535
sn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val
540 545 550
Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val
555 560 565
Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe
570 575 580 - `
Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr
585 590 595 600 : ~:
ys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu
605 610 615
Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg ~ .
620 625 630 .
Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn ~:
635 640 645
ys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg
65~ 655 660
lu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe
665 670 675 680 `
Ala Ser Ser Leu Ile Ile Pro Ala Il~ Gly Met Ile Ile Tyr Phe Ala ~ .-
685 690 ~5 ~:
rg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys
700 70S 710
Ser Lys Val -
715
(2) INFO~ATION FOR SEQ ID NQ:10:
( i) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 1941 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: 1inear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..19
Sll~3ST~T~ $~
( i~c) FEATURE:
(A) NAME/KEY: mat peptide
(B) LOCATION: 73. .1941
( i:c ) FEATURE:
(A) NAME/KEY: sig peptide
(B) LOCATION: 1. .72 : -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: .
ATG CCT GGG MG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp
--24 --20 --15 --10
ATA ATG TTT GCA GCT TCT CM GCT TTT AAA ATC GAG ACC ACC CCA GAA 96
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu -
-5 1 5 : .
TCT AGA TAT CTT GCT CAG ATT GGT GAC TCC GTC TCA TTG ACT TGC AGC 144
Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser
10 15 20
ACC ACA GGC TGT GAG TCC CCA TTT TTC TCT TGG AGA ACC CAG ATA GAT 192
Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln Ile Asp
25 30 35 40
AGT CCA CTG MT GGG MG GTG ACG MT GAG GGG ACC ACA TCT ACG CTG 240
Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
45 50 55
ACA ATG MT CCT GTT AGT TTT GGG MC GM CAC TCT TAC CTG TGC ACA 288
Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr
60 65 70
GCA ACT TGT GAA TCT AGG AAA TTG GM AAA GGA ATC CAG GTG GAG ATC 336
Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile
75 80 85
TAC TCT TTT CCT MG GAT CCA GAG ATT CAT TTG AGT GGC CCT CTG GAG 384
Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu
90 95 100
GCT GGG MG CCG ATC ACA GTC MG TGT TCA GTT GCT GAT GTA TAC CCA 432
Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro
105 110 115 120
TTT GAC AGG CTG GAG ATA GAC TTA CTG AAA GGA GAT CAT CTC ATG MG 480
Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys
125 130 135
SUBSTI~V~E S~ET
2 I 2~
--44--
AGT CAG GAA TTT CTG GAG GAT GCA GAC AGG AAG TCC CTG GAA ACC AAG 528 :
Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
140 145 150
AGT TTG GAA GTA ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT 576 .`~
Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val -~ ~
155 160 165 .- .
CTT GTT TGC CGA GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC 624
Leu Val Cys Ar~ Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro
170 175 180 .
ACA GTA AGG CAG GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC MG 672
Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys
185 190 195 200
AAT ACA GTT ATT TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GGT GGC 720
Asn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly .
205 210 215
TCT GTG ACC ATG ACC TGT TCC AGC GAG GGT CTA CCA GCT CCA GAG ATT 768 ~:
Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile
220 225 230 :~.
TTC TGG AGT ~AG AAA TTA GAT AAT ÇGG MT CTA CAG CAC CTT TCT GGA 816 :
Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly
235 240 245 -
AAT GCA ACT CTC ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT 864
Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile .
250 255 260 - ~-
TAT GTG TGT GM. GGA GTT AAT TTG ATT GGG AAA AAC AGA AAA GAG GTG 912
Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val
265 270 275 280
GM TTA ATT GTT CM GCA TTC CCT AGA GAT CCA GAA ATC GAG ATG AGT . 960
Glu Leu Ile Val Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser
285 290 295
GGT GGC CTC GTG AAT GGG AGC TCT GTC ACT GTA AGC TGC AAG GTT CCT 1008
Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro
300 305 310
AGC GTG TAC (::CC CTT GAC CGG CTG GAG ATT GAA TTA CTT AAG GGG GAG 1056
Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu
315 320 325
ACT ATT CTG GAG AAT ATA GAG TTT TTG GAG GAT ACG GAT ATG AAA TCT 1104
Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser
330 335 340
SIJ~ T
2.-i 2 rl ~ 3 ~
--45--
CTA GAG MC AAA AGT TTG GAA ATG ACC TTC ATC CCT ACC ATT GAA GAT 1152
Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp
345 350 355 360
ACT GGA AAA GCT CTT GTT TGT CAG GCT MG TTA CAT ATT GAT GAC ATG 1200
Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met
365 370 375
GAA TTC GAA CCC AAA CAA AGG CAG AGT ACG CAA ACA CTT TAT GTC MT 1248
Glu Phe Glu Pro Lys Gln Ar~s Gln Ser Thr Gln Thr Leu Tyr Val Asn
380 385 390
GTT GCC CCC AGA GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG 1296
Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu
395 400 405
GAG GAA GGC AGT TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT 1344
Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro
410 415 420
GCT CCG AAA ATC CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG 1392
Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln
425 430 435 440
CCT CTT TCT GAG AAT GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GAA 1440
Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu
`~' 445 450 455
GAT TCT GGG GTT TAT TTA TGT GAA GGA ATT AAC CAG GCT GGA AGA AGC 1488
Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser
460 465 470
AGA AAG GM GTG GM TTA ATT ATC CM GTT ACT CCA AAA GAC ATA AAA 1536
Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys
475 480 485
CTT ACA GCT TTT CCT TCT GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC- 1584
Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile
490 495 500
ATC TCT TGT ACA TGT GGA AAT GTT CCA GAA ACA TGG ATA ATC CTG AAG 1632
Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys
505 510 515 520
AAA AAA GCG GAG ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC 1680
Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys ~er Ile Asp Gly Ala
525 530 535
TAT ACC ATC CGA AAG GCC CAG TTG AAG GAT GCG GGA GTA TAT GM TGT 1728
Tyr Thr Ile Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys
540 545 550
SU~
~ ~ 2i2~5r~2 r
- 46 -
' ,
GAA TCT AAA AAC AAA GTT GGC TCA CAA TTA AGA AGT TTA ACA CTT GAT 1776
Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp ~:
555 560 565
GTT CAA GGA AGA GAA AAC AAC AAA GAC TAT TTT TCT CCT GAG CTT CTC 1824
Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu
570 575 580
GTG CTC TAT TTT GCA TCC TCC TTA ATA ATA CCT ~CC ATT GGA ATG ATA 1872
Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile -~
585 590 595 600
ATT TAC TTT GCA AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CTT GTA 1920
Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val
605 610 615
GAA GCA CAG AAA TCA M A GTG 1941
Glu Ala Gln Lys Ser Lys Val
620
(2) INFORMATION FOR SEQ ID NO~
~i~ SEQUENCE CHARACTERISTICS: .
(A) LENGT~: 647 amino acids ~
(B) TYPE: amino acid - .
(D) TOPOLOGY: linear -~
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: :~
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp
-24 -20 -15 -10 ;
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu
-5 l 5
Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser
Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln Ile Asp
Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
hr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr
6~ 65 70
Ala Thr Cys ~lu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile
8UB~ T
212~7 ~232
-4~-
Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu
100
Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro
105 110 115 120
he Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys
125 130 135
er Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
140 145 150
Ser Leu Glu V~l Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val
155 160 1~5
Leu~Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro
170 175 180
Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys
185 190 195 200
sn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly
205 210 215
er Val Thr Met Thr Cys Ser Ser Gl~ Gly Leu Pro Ala Pro Glu Ile
220 225 230
Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly
235 240 245
Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile
250 255 260
Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val
265 270 275 280
lu Leu Ile Val Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser
285 290 2~5
ly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro
300 305 310
Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu
315 320 325
Thr lle Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser
330 335 3~0
Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp
345 350 35S- 360
SUE~ EET ~;
' ' '. ?J'~ J~J~
-48- :
Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Mee ~: .
365 370 375
lu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn
380 385 390
Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu ~:
395 400 405
Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro
410 415 420
Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln
425 430 435 440 :
Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu
445 450 455
sp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser
460 465 470
Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys : -
475 480 485
Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile -
490 4g5 500
le Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys
505 510 515 520 .
ys Lys Ala S:lu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala
525 530 535 .
yr Thr Ile Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys
540 545 550
Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp
555 560 565
Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu
570 575 5~0
Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile
585 590 595 600
le Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val
605 610 615
Glu Ala Gln Lys Ser Lys Val
620
su~ s~
2127 t~32
-49-
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2205 base pairs
~B~ TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: 1 inear
( ix ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..2205
( ix ) FEATURE:
(A) NAME/KEY: mat peptide
- (B) LOCATION: 73.. 2205
( ix) FEATURE:
(A) NAME/KEY: sig peptide
(B) LOCATION: 1..72
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48
Me~ Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp
--24 --20 --15 --10
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96 :.
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu .
--5 1 5
TCT AGA TAT CTT CTG CCC CGG GGA GGC TCC GTG CTG GTG ACA TGC AGC 144
Ser ArE~ Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser
10 15 20 , ~:;
ACC TCC TGT GAC CAG CCC AAG TTG TTG GGC ATA GAG ACC CCG TTG CCT . 192
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro
25 30 35 40
AAA AAG GAG TTG CTC CTG CCT GGG AAC AAC CGG AAG GTG TAT GAA CTG 240
Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu .
45 50 55 -:
AGC MT GTG C M GAA GAT AGC CAA CCA ATG TGC TAT TCA AAG TGC CCT 288
Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 70
GAT GGG CAG TCA ACA GCT AM ACC TTC CTC ACC GTG TAC TGG ACT CCT 3 3 6
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
75 80 85
~VB~T~
` 2ri 2 7 ~ 3 2
-so~
AAG GAT CCA GAG ATT CAT TTG AGT GGC CCT CTG GAG GCT GGG AAG CCG 384
Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro
90 95 100
ATC ACA GTC AAG TGT TCA GTT GCT GAT GTA TAC CCA TTT GAC AGG CTG 432
Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu
105 110 115 120
GAG ATA GAC TTA CTG AAA GGA GAT CAT CTC ATG MG AGT CAG GAA TTT 4 8 0Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe
125 130 135
CTG GAG GAT GCA GAC AGG MG TCC CTG GM ACC AAG AGT TTG GAA GTA 528
Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val
140 145 150
ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT CTT GTT TGC CGA 5 7 6Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Arg
155 160 165
GCT MA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC ACA GTA AGG CAG 624
Ala Lys Lèu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln
170 175 180
GCT. GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC MG AAT ACA GTT ATT 672
Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val Ile
l~S 190 l9S 200
TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GGT GGC TCT GTG ACC ATG 720
Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met .
`205 210 215
ACC TGT TCC AGC GAG GGT CTA CCA GGT CCA GAG ATT TTC TGG AGT AAG 7 6 8 .
Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser Lys
220 225 230
AAA TTA GAT MT GGG AAT CTA CAG CAC CTT TCT GGA AAT GCA ACT CTC . 816Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu
235 240 245
ACC TTA ATT GCT ATG AGG ATG GM GAT TCT GGA ATT TAT GTG TGT GM 864 -~
Thr Leu Ile Ala Met Arg Me~ Glu Asp Ser Gly Ile Tyr Val Cys Glu :
250 255 260 ~ :
GGA GTT A.AT TTG ATT GGG AAA AAC AGA AAA GAG GTG GAA TTA ATT GTT912
Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Gl.u Leu Ile Val
265 270 275 280
CAA GAG AAA CCA TTT ACT GTT GAG ATC TCC CCT GGA CCC CGG ATT GCT960
Gln Glu Lys Pro Phe Thr Val Glu Ile Ser Pro Gly Pro Arg Ile Ala
285 290 295
81JE~ E ~;H~Fr
~ ~ ~7~ ~
~J ' ~
--51--
GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TGï AGT GTC ATG GGC TGT 1008
Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Sar Val Met Gly Cys
300 305 310
GAA TCC CCA TCT TTC TCC TGG AGA ACC CAG ATA GAC AGC CCT CTG AGC 1056
Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu S~r
315 320 325
GGG AAG GTG AGG AGT GAG GGG ACC AAT TCC ACG CTG ACC CTG AGC CCT 1104
Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser Pro
330 335 340
GTG AGT TTT GAG AAC GAA CAC TCT TAT CTG TGC ACA GTG ACT TGT GGA 1152
Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys Gly
345 350 355 360
CAT AAG AAA CTG GAA AAG GGA ATC CAG GTG GAG CTC TAC TCA TTC CCT :L200
His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu Tyr Ser Phe Pro
365 370 375
AGA GAT CCA GAA ATC GAG ATG AGT GGT GGC CTC GTG AAT ~GG AGC TCT 1248
Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser Ser :;
380 385 390
GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC CCC CTT GAC CGG CTG 1296
Val Thr ~al Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu
395 400 405
GAG ATT GAA TTA CTT AAG GGG GAG ACT ATT CTG GAG AAT ATA GAG TTT 1344
Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe
410 415 4~0 `:
TTG GAG GAT ACG GAT ATG AAA TCT CTA GAG AAC AAA AGT TTG GAA ATG 1392 :.
Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met - ~
425 430 435 440 . ~ :`
ACC TTC ATC CCT ACC ATT GM GAT ACT GGA AAA GCT CTT GTT TGT CAG 1440
Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala Leu Val Cys Gln
445 450 455
GCT AAG TTA CAT ATT GAT GAC ATG GAA TTC GAA CCC AAA CAA AGG CAG 1488
Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln
460 465 470
AGT ACG CAA ACA CTT TAT GTC AAT GTT GCC CCC AGA GAT ACA ACC GTC 1536
Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val
475 480 485
TTG GTC AGC CCT TCC TCC ATC CTG GAG GAA GGC AGT TCT GTG MT ATG 1584
Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met
490 495 500
SIIE~STITUTE ~gtÆET
52- ~127~.?~
ACA TGC TTG AGC CAG GGC TTT CCT GCT CCG AM ATC CTG TGG AGC AGG 1632
Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg
505 S10 515 520
CAG CTC CCT MC GGG GAG CTA CAG CCT CTT TCT GAG AAT GCA ACT CTC 1680
Gln Leu Pro Asn Giy Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu
525 53~ 535
ACC TTA ATT TGT ACA AAA ATG GAA GAT TCT GGG GTT TAT TTA TGT GAA1728
Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu
540 545 550
GGA ATT MC CAG GCT GGA AGA AGC AGA MG GAA GTG GAA TTA ATT ATC 1776
Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile
555 560 565
CAA GTT ACT CCA AAA GAC ATA AM CTT ACA GCT TTT CCT TCT GAG AGT 1824
Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu Ser
570 575 580
GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT ACA TGT GGA AAT GTT1872 ~
Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cy.g Gly Asn Val `
585 590 595 600 -~ ~-
CCA GAA ACA TGG ATA ATC CTG AAG AAA AAA GCG GAG ACA GGA GAC ACA1920
Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr
605 610 615
GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC CGA AAG GCC CAG TTG1968 : :
Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln Leu
620 625 630 ,:
MG GAT GCG GGA GTA TAT GAA TGT GAA TCT AAA AAC AAA GTT GGC TCA 2016
Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser :: :
635 640 645
CAA TTA AGA AGT TTA ACA CTT GAT GTT CM GGA AGA GAA AAC AAC AAA 2064
Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys
650 655 660
GAC TAT TTT TCT CCT GAG CTT CTC GTG CTC TAT TTT GCA TCC TCC TTA 2112 . :
Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser Leu
665 670 675 680
ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT GCA AGA AAA GCC AAC 2160
Ile I1Q PrO AIa Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala Asn
685 690 695
ATG AAG GGG TCA TAT AGT CTl' GTA GAA GCA CAG AM TCA AAA GTG 2205
Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln~Lys Ser Lys Val
700 705 710
S~ E Sff~E~
2127332
-53- ,
(2) INF0RMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 735 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu ~rp
-24 -20 -15 -10
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu ~;
_5 1 5 `:-.
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser
l0 15 20 ~
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro ~ :
25 30 35 40 ~ :
~ .......................................................................... ..
Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu -~
45 50 55 ~ :
Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro : .~
60 65 70 ~ -`
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro
9Q 95 100
Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu
105 110 115 120-
lu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe
125 130 135
eu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val
140 145 150
Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Ar~
155 160 165
Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln
170 175 180
SU~STIT~IESlLi!~
3~ 5 3 ~
ds
D N0:13:
.y Ala Ser Asn Ile Leu Trp
.5 -10
s Ile Glu Thr Thr Pro Glu
s
r Val Leu Val Thr Cys Ser
.y Ile Glu Thr Pro Leu Pro
~;n Arg Lys Val Tyr Glu Leu
O 55
~t Cys Tyr Ser Asn Cys Pro
~u Thr Val Tyr Trp Thr Pro
~-o Leu Glu Ala Gly Lys Pro
100
Tyr Pro Phe Asp Arg Leu
llS 120
~u Met Lys Ser Cln Glu Phe
135
u Thr Lys Ser Leu Glu Val
150
y Lys Val Leu Val Cys Arg
165
~r Val Pro Thr Val Arg Gln
180
T~
:
~ r r r ~ ~ r
2~2~ 2
--55-
la Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln
460 465 470
Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val
475 480 485
Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met
490 495 500
Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg
505 510 515 520
ln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu
525 530 535
hr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu
540 545 550
Gly Ile Asn Gln Ala Gly Ar~s Ser Arg Lys Glu Val Glu Leu Ile Ile
555 560 565
Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu Ser
570 575 580
Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn Val
585 590 595 600
ro Glu ll~r Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr
605 610 515
al Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln Leu
620 625 630
Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser
6~5 640 645
Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys
650 655 660
Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser Leu
665 670 675 680
le Ile ~ro Ala Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala Asn
685 690 695
et Lys Gly Ser Tyr Ser Leu Val Gl.u Ala Gln Lys Ser Lys Val
700 705 710
SUI~STITU~ EEr
2 ~ ` 3 ~
-56-
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2208 base pairs
(B) TYPE: nucleic acid -~
(C) STRANDEDNESS: double
(D~ TOPOLOGY: linear
(ix) FEATURE: :
(A) NAME/KEY: CDS
(B) LOCATION: 1..2208
(i~) FEATURE:
(A) NAME~KEY: mat_peptide
- (B) LOCATION: 73.. 2208
~ix) FEATURE:
(A) NAME/KEY: sig_peptide ~:
(B) LOCATION: 1..72
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GAA 96
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile GIu Thr Thr Pro Glu
-5 l 5
TCT AGA TAT CTT CTG CCC CGG GGA GGC TCC GTG CTG GTG ACA TGC AGC 144
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser
10 15 20
ACC TCC TGT GAC CAG CCC AAG TTG TTG GGC ATA GAG ACC CCG TTG CCT 192
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu l'hr Pro Leu Pro
25 30 35 40
AAA AAG GAG TTG CTC CTG CCT GGG AAC AAC CGG AAG GTG TAT GAA CTG 240
L~s Lys Glu Leu Leu Le~ Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
45 50 55
AGC AAT GTG CAA GAA GAT AGC C M CCA ATG TGC TAT TCA M C TGC CCT 288
Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 70
GAT GGG CAG TCA ACA GCT AAA ACC TTC CTC ACC GTG TAC TGG ACT CCA 336
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
75 80 85
SU135~Tl~TE ~
c ' '2127 ,,'3?,'
--~7-- -
GAA CGG GTG GAA CTG GCA CCC CTC CCC TCT TG(' CAG CCA GTG GGC AAG 384
Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys
90 9 5 100
AAC CTT ACC CTA CGC TGC CAG GTG GAG GGT GGG GCA CCC CGG GCC AAC 432
Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn
105 110 115 120
CTC ACC GTG GTG CTG CTC CGT GGG GAG AAG CTC ATG MG AGT CAG GAA 480
Leu Thr Val Val Leu Leu Arg Gly Glu Lys Leu Met Lys Ser Gln Glu
125 130 135
TTT CTG GAG GAT GCA GAC AGG AAG TCC CTG GM ACC AAG AGT TTG GAA 5 2 8
Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu
140 145 150
GTA ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT CTT GTT TGC 576
Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys
155 160 165
CGA GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC ACA GTA AGG 6 24
Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg
170 175 180
CAG GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC AAG AAT ACA GTT 6 7 2
Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val
185 190 195 200
ATT TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GGT GGC TCT CTG ACC 7 2 0
Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr
205 210 215
ATG ACC TGT TCC AGC GAG GGT CTA CCA GCT CCA GAG ATT TTC TGG AGT 7 6 8
Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser
220 225 230
AAG AAA TTA GAT MT GGG MT CTA CAG CAC CTT TCT GGA AAT GCA ACT . 816
T~y5 Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr
235 240 245
CTC ACC TTA ATT GCT ATG AGG AT& GM GAT TCT GGA ATT TAT GTG TGT 864
Leu lhr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys
250 255 260
GAA GGA GTT AAT TTG ATT GGG MA AAC AGA AAA GAG GTG GAA TTA ATT 912
Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile
265 270 27S ~0
GTT CAA GAG AAA CCA TTT ACT GTT GAG ATC TCC CCT GGA CCC CGG ATT 960
Val Gln Glu Lys Pro Phe Thr Val Glu Ile Ser Pro Gly Pro Ar~g Ile
285 290 295 -
SU~ EE~ ~
~127~33~
-58-
GCT GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TGT AGT G,TC ATG GGC 1008
Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Ser Val Met Gly
300 305 310
TGT GAA TCC CCA TCT TTC TCC TGG AGA ACC CAG ATA GAC AGC CCT CTG 1056
Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu ~::
315 320 325
AGC GGG AAG GTG AGG AGT GAG GGG ACC AAT TCC ACG CTG ACC CTG AGC 1104
Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser
330 335 340
CCT GTG AGT TTT GAG AAC GAA CAC TCT TAT CTG TGC ACA GTG ACT TGT 1152
Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys
345 350 355 360
GGA CAT MG AAA CTG GAA AAG GGA ATC CAG GTG GAG CTC TAC TCA TTC 1200
Gly His Lys Lys L~u Glu Lys Gly Ile Gln Val Glu Leu Tyr Ser Phe
365 370 375
CCT AGA GAT CCA GAA ATC GAG ATG AGT GGT GGC CTC GTG AAT GGG AGC 1248
Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser
380 385 390
TCT GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC CCC CTT GAC CGG 1296
Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg : ~-
395 . 400 405
CTG GAG ATT GM TTA CTT AAG GGG GAG ACT ATT CTG GAG AAT ATA GAG 1344
Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu
410 415 420 -
TTT TTG GAG GAT ACG GAT ATG AAA TCT CTA GAG MC AAA AGT TTG GAA 1392
Phe Leu Glu Asp Thr Asp Met l,ys Ser Leu Glu Asn Lys Ser Leu Glu
425 430 435 440
ATG ACC TTC ATC CCT ACC ATT GM GAT ACT GGA AAA GCT CTT GTT TGT 1440
Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala Leu Yal Cys
445 450 455
CAG GCT MG TTA CAT ATT GAT GAC ATG GAA TTC GAA CCC AAA CAA AGG 1488
Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg
460 465 470
CAG AGT ACG CM ACA CTT TAT GTC AAT GTT GCC CCC AGA GAT ACA ACC 1536
Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr
~75 480 485
GTC TTG 5TC AGC CCT TCC TCC ATC CTG GAG GM GGC AGT TCT GTG AAT 1584
Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn
490 495 S00
su~ r~ ET
2 ~ 2 7 ~ 3 2
_59_
ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT CCG AAA ATC CTG TGG AGC 1632
Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser
505 510 515 520
AGG CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TCT GAG AAT GCA ACT 1680
Arg Gln Leu Pro Asn Gly Glu Leu Gl~ Pro Leu Ser Glu Asn Ala Thr
~25 530 535
CTC ACC TTA ATT TCT ACA AAA ATG GAA GAT TCT GGG GTT TAT TTA TGT 1728
Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys
540 545 550
GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA AAG GAA GTG GAA TTA ATT 1776
Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile
S55 560 565
ATC C M GTT ACT CCA AAA GAC ATA AAA CTT ACA GCT TTT CCT TCT GAG 1824
Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu
570 575 580
AGT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT ACA TGT GGA AAT 1872 ~-
Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn
585 S90 595 600
GTT CCA GAA ACA TGG ATA ATC CTG AAG AAA AAA GCG GAG ACA GGA GAC 1920
Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp
605 610 615 .~.
ACA GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC CGA AAG GCC CAG 1968
Thr Val Leu Lys Ser lle Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln
620 625 630 ~-
TTG AAG GAT GCG GGA GTA TAT G M TGT GAA TCT AAA AAC AAA GTT GGC 2016
Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly `
635 640 645
TCA CAA TTA AGA AGT TTA ACA CTT GAT GTT C M GGA AGA GAA .~AC AAC- 2064Ser Gln Leu Arg Ser Leu Thx Leu Asp Val Gln Gly Arg Glu Asn Asn
6S0 655 660
AAA GAC TAT TTT TCT CCT GAG CTT CTC GTG CTC TAT TTT GCA TCC TCC 2112
Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser -
S65 670 675 680
TTA ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT GCA AGA AAA GCC ~160
Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala
685 690 695
AAC ATG M G GGG TCA TAT AGT CTT GTA G M GCA CAG AAA TCA AAA GTG 2208
Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val
700 705 710
81JBST~T~TE ~EEl'
2 } 27~3 ~
-60-
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 736 a~ino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Met Pro Gly Lys Met Val Val Ila Leu Gly Ala Ser Asn Ile Leu Trp
-24 -20 15 -10
le Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu
_5 1 5
er Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cy5 Ser
10 15 20 :
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro
ys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
' 45 50 55
er Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys
100
Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn
105 11.0 115 12
eu Thr Val Val Leu Leu Arg Gly Glu Lys Leu Met Lys Ser Gln Glu
125 130 135
he Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu
140 145 150
Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys
155 160 165
Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg
170 175 180
Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val
185 190 195 200
SU~ ET
; 2~7S~- 2
-61-
Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr
205 210 215
et Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser
220 225 230
ys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr
235 240 245
Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys
250 255 260
Glu Gly Val Asn Leu Ile Gly Lys Asn Ar~ Lys Glu Val Glu Leu Ile :~
265 270 275 280
Val Gln Glu Lys Pro Phe Thr Val Glu Ile Ser Pro Gly Pro Arg Ile
285 290 295 :
la Ala Gln I18 Gly Asp Ser Val Met Leu Thr Cys Ser Val Met Gly
300 305 310
ys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu
315 320 325 ;
Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser
330 335 340
Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys ;~
345 350 355 360 ..
Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu Tyr Ser Phe `
365 370 375 `:
ro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser
380 385 390
er Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg
3g5 400 ~OS
Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu
410 415 420
Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu
425 430 435 440
Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala Leu Val Cys
445 4S0 455
ln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg
460 465 470
SlJB~ S~
~ r ~ r ~
--62--
Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr
475 480 485 .
Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn
490 495 500
Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser
505 510 515 520
rg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr
525 530 535
eu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys
540 545 550
lu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile
555 560 565
Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu
570 575 580
Ser Val I.ys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn
585 5~0 595 600
al Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp
605 610 61 s
hr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln
Ç20 625 630
eu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly
635 640 645
Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn
650 655 660
Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser
665 670 675 680
eu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala
685 690 695
sn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val
700 705 710
-
~JBS~ TF~
~1"7~2
-63-
(2) INFORMATION FOR SEQ ID NO:16- -:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2220 base pairs ~-
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear ~;
(ix) FEATURE:
(A) NAME/KEY: CDS .
(B) LOCATION: 1.. 2220 -~ .
(i~) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 73..2220
(ix) FEATURE:
(A) NAME/KEY: sig peptide :
(B) LOCATION: 1.. 72 :~.
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:16:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TG~ 48
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT C M GCT TTT AAA ATC GAG ACC ACC CCA GAA 96
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu
--5 1 5
TCT AGA TAT CTT GCT CAG ATT GGT GAC TCC GTC TCA TTG ACT TGC AGC 144
Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser
lQ 15 20
ACC ACA GGC TGT GAG TCC CCIA TTT TTC TCT TGG AGA ACC CAG ATA GAT 192
Thr Thr Gly Cys Clu Ser Pro Phe Phe Ser Trp Arg Thr Gln Ile Asp
25 30 35 40
AGT CCA CTG AAT GGG AAG GTG ACG AAT GAG GGG ACC ACA TCT ACG CTG 240
Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
45 50 55
ACA ATG AAT CCT GTT AGT TTT GGG AAC GAA CAC TCT TAC CTG TGC ACA 288
Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr
60 65 70
GCA ACT TGT GAA TCT AGG AAA TTG GAA AAA GGA ATC CAG GTG GAG ATC 336
Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile
75 80 85
SlJB~T~TU~E ~
2127 ~32
-64- -
TAC TGG ACT CCA GAA CGG GTG GAA CTG GCA CCC CTC CCC TCT TGG CAG 384
Tyr Trp Thr Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln
~0 95 100
CCA GTG GGC AAG AAC CTT ACC CTA CGC TGC CAG GTG GAG GGT GGG GCA 432
Pro Val Gly Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala
105 110 115 120
CCC CGG GCC M C CTC ACC GTG GTG CTG CTC CGT GGG GAG AAG GAG CTG 480
Pro Arg Ala Asn Leu Thr Val Val Leu Leu Arg Gly Glu Lys Glu Leu
125 130 135
AAA CGG GAG CCA GCT GTG GGG GAG CCC GCT GAG GTC ACG ACC ACG GTG 528
Lys Arg Glu Pro Ala Val Gly Glu Pro Ala Glu Val Thr Thr Thr Val
140 145 150
CTG GTG AGG AGA GAT CAC CAT GGA GCC AAT TTC TCG TGC CGC ACT GAA 576
Leu Val Arg Arg Asp His His Gly Ala Asn Phe Ser Cys Arg Thr Glu
155 .~.160 165
CTG GAC CTG CGG CCC C M GGG CTG GAG CTG TTT GAG AAC ACC TCG GCC 624
Leu Asp Leu Arg Pro Gln Gly Leu Glu Leu Phe Glu Asn Thr Ser Ala
170 175 180
CCC TAC CAG CTC CAG ACC TTT GTC CTG CCA GCG ACT CCC CCA CAA CTT 672
Pro Tyr Gln Leu Gln Thr Phe Val Leu Pro Ala Thr Pro Pro Gln Leu
185 190 195 200
GTC AGC CCC CGG GTC CTA GAG GTG GAC ACG CAG GGG ACC GTG GTC TGT 720
Val Ser Pro Arg Val Leu Glu Val Asp Thr Gln Gly Thr Val Val Cys
205 210 215
TCC CTG GAC GGG CTG TTC CCA GTC TCG GAG GCC CAG GTC CAC CTG GCA 768
Ser Leu Asp Gly Leu Phe Pro Val Ser Glu Ala Gln Val His Leu Ala
220 225 230
CTG GGG GAC CAG AGG TTG AAC CCC ACA GTC ACC TAT GGC AAC GAC TCC- 816
Leu Gly Asp Gln Axg Leu~Asn Pro Thr Val Thr Tyr Gly Asn Asp Ser
~35 240 245
TTC TCG GCC AAG GCC TCA GTC AGT GTG ACC GCA GAG GAC GAG GGC ACC 864
Phe Sex Ala Lys Ala Ser Val Ser Val Thr Ala Glu Asp Glu Gly Thr
250 255 260
CAG CGG CTG ACG TGT GCA GTA ATA CTG GGG AAC CAG AGC CAG GAG ACA 912
Gln Arg Leu Thr Cys Ala Val Ile Leu Gly Asn Gln Ser Gln Glu Thr
265 270 275 280
CTG CAG ACA GTG ACC ATC TAC AGC TTT CCG GCG CCC GAG ATC TCC CCT 960
I.eu Gln Thr Val Thr Ile Tyr Ser Phe Pro Ala Pro Glu Ile Ser Pro
285 290 295
~;~.IB5~ ; t ~ T
2 7 ~ 3 2
-65-
GGA CCC CGG ATT GCT GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TGT 1008
Gly Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys
300 305 310 .
AGT GTC ATG GGC TGT GAA TCC CCA TCT TTC TCC TGG AGA ACC CAG ATA 1056
Ser Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile ::~
315 320 325
GAC AGC CCT CTG AGC GGG AAG GTG AGG AGT GAG GGG ACC AAT TCC ACG 1104
Asp Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr
330 335 340 :
CTG ACC CTG AGC CCT GTG AGT TTT GAG AAC GM~CAC TCT TAT CTG TGC 1152 :
Leu Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys
345 350 355 360
ACA GTG ACT TGT GGA CAT AAG AAA CTG GAA AAG GGA ATC CAG GTG GAG 1200
Thr Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu
365 370 375
CTC TAC TCA TTC CCT AGA GAT CCA GAA ATC GAG ATG AGT GGT GGC CTC 1~48
Leu Tyr Ser Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu
380 385 390
GTG AAT GGG AGC TCT GTC ACT GTA AGC TG5 AAG GTT CCT AGC GTG TAC 1296
Val Asn Gly Ser Ser Val-Thr Val Ser Cys Lys Val Pro Ser Val Tyr
3gS 400 405
CCC CTT GAC CGG CTG GAG ATT GAA TTA CTT A.9G G(;G GAG ACT ATT CTG 1344
Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu
410 415 420
GAG AAT ATA GAG TTT TTG GAG GAT ACG GAT ATG MA TCT CTA GAG AAC 1392
Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn
425 430 435 440
AAA AGT TTG GAA ATG ACC TTC ATC CCT ACC ATT GM GAT ACT GGA AAA 1440
Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys
4~5 450 455
GCT CTT GTT TGT CAG GCT AAG TTA CAT ATT GAT GAC ATG GAA TTC GAA 1488
Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu
460 465 470
CCC AAA CM AGG C:AG AGT ACG CAA ACA CTT TAT GTC AAT GTT GCC CCC 1536
Pro Lys Gln Ar~s Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro
475 480 48S
AGA GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG GAG GM GGC 1584
Arg Asp Thr Thr Val Leu ~lal Ser Pro Ser Ser Ile Leu Glu Glu Gly
490 495 500
r ~ ~
J~
-66-
AGT TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT CCG AAA 1632Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys
505 510 515 5~0
ATC CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TCT 1680Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser
525 530 535
GAG M T GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GAA GAT TCT GGG 1728Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly
540 545 550
GTT TAT TTA TGT GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA M G GAA 1776Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu
555 560 565
GTG GAA TTA ATT ATC CAA GTT ACT CCA AAA GAC ATA AAA CTT ACA GCT 1824Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala
570 575 580
TTT CCT TCT GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT 1872Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys
585 590 595 600
ACA TGT GGA AAT GTT CCA G M ACA TGG ATA ATC CTG AAG AAA AAA GCG 1920Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala
605 610 615
GAG ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC 1968Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile
620 625 630 ~ :-
CGA AAG GCC CAG TTG ~AG GAT GCG GGA GTA TAT GAA TGT GAA TCT AAA 2016Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys
635 640 645
M C AAA GTT GGC TCA CM TTA AGA AGT TTA ACA CTT GAT GT~ C~A GGA 2064Asn Lys ~al Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly
650 655 6~0
AGA GAA M C AAC AAA GAC TAT TTT TCT CCT GAG CTT CTG GTG CTC TAT 2112Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr
665 670 675 680
TTT GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT 2160
Phe Ala Ser Ser Leu Ile Ile Pro ~la Ile Gly Met Ile Ile Tyr Phe
685 690 6~5
GCA AGA AAA GCC M C ATG M G GGG TCA TAT AGT CTT GTA GAA GCA CAG 2208
Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln
700 705 710
`,
:.
8UE~STIT~E ~
: 212~2 -:
--67--
AAA TCA AAA GTG 2 2 2 0
Lys S e r Lys Val
715
(2) INFORMATION FOR SEQ ID NO:17:
( i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 740 amino acids
(B) TYPE: amino acid -
( D ) TOPOLOGY: 1 ine ar
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO :17:
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp
--24 --20 --15 --lO
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu
-5 1 5
Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser
Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln Ile Asp
er Pro Leu Asn Gly ~ys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
5~
hr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr
Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile
Tyr Trp Thr Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln
100
Pro Val Gly Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala
105 110 115 120
ro Arg Ala Asn Leu Thr Val Val Leu Leu Arg Gly Glu Lys Glu Leu
125 130 135
ys Arg Glu Pro Ala Val Gly Glu Pro Ala Glu Val Thr Thr Thr Val
140 145 150
Leu Val Arg Arg Asp His His Gly Ala Asn Phe Ser Cys Arg Thr Glu
155 160 165
~;U8~;1 IT~)TE S~EE~
2 1 ~ 7 ~5 3 ~
-68-
Leu Asp Leu Arg Pro Gln Gly Leu Glu Leu Phe Glu Asn Thr Ser Ala
170 175 lB0
Pro Tyr Gln Leu Gln Thr Phe Val Leu Pro Ala Thr Pro Pro Gln Leu
185 190 195 200
Val Ser Pro Arg Val Leu Glu Val Asp Thr Gln Gly Thr Val Val Cys
205 210 215
er Leu Asp Gly Leu Phe Pro Val Ser Glu Ala Gln Val His Leu Ala
220 225 230
Leu Gly Asp Gln Arg Leu Asn Pro Thr Val Thr Tyr Gly Asn Asp Ser
235 240 245
Phe Ser Ala Lys Ala Ser Val Ser Val Thr Ala Glu Asp Glu Gly Thr
250 255 260
Gln Arg Leu Thr Cys Ala Val Ile Leu Gly Asn Gln Ser Gln Glu Thr
265 270 275 280
eu Gln Thr Val Thr Ile Tyr Ser Phe Pro Ala Pro Glu Ile Ser Pro
285 290 295
ly Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys
30~ 305 310
Ser Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile
315 320 325
Asp Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr
330 335 340
Leu Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys
345 350 355 360
hr Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu
365 370 375
eu Tyr Ser Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu
380 385 390
Val Asn Gly Ser- Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr
395 400 405
Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu
410 415 420
Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met. Lys Ser Leu Glu Asn
425 430 435 440
811BST~Tl~E S~
2 1 2 7 S 3 2
-69-
Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys
445 450 455
Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu
460 465 470
Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro
475 480 485
Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly
490 495 500
Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys
505 510 515 520
Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser
525 530 535
Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly
540 545 550
Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu
555 560 565
Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala
570 575 580
Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys
585 590 595 600
Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala
- 605 610 615
Glu Thr Gly As~ Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile
S2(~ 625 630
Arg Lys Ala Gln Leu l.ys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys
635 640 645
Asn Lys Val Gly Ser Gln Leu Arg Ser Leu lhr l,eu Asp Val Gln Gly
650 655 660
Ar~ Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr
665 670 675 680
Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe
685 690 695
Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln
700 705 ~ 710
~;UB~;T~Tl~E ~Ef
2 ~ 2 7 !J 3 ~
--70--
Lys Ser Lys Val
715
(2) INFORMATION FOR SE~ ID NO:18:
(i) SEQUENCE CHAXACTERISTICS:
(A) LENGTH: 1929 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A~ NAME/KEY: GDS
(B) LOCATION: 1..1929
( ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 73..1929
( ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B~ LOCATION: l . . 72
(xi) SEQUENCE DESCRIPTION: SE~ ID N0:18:
ATG CCT GGG AAG ~TG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TGG 48 :~
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA GM 96
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu
-5 1 5
TCT AGA TAT CTT GTG CCC CGG GGA GGC TCC GTG CTG GTG ACA TGC AGC . 144
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val. Thr Cys Ser
10 15 20
ACC TCC TGT GAC CAG CCC AAG TTG TTG GGC ATA GAG ACC CCG TTG CCT 192
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro
25 30 35 40
AAA AAG GAG TTG CTC CTG CCT GGG AAC MC CGG AAG GTG TAT GAA CTG 240
Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
45 50 55
AGC MT GTG CM GM GAT AGC CM CCA ATG TGC TAT TCA AAC TGC CCT 288
Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 ` 70
SUE~ U~ LE~
~; ~ ?, 7 ~ 3 2
--71--
GAT GGG CAG TCA ACA GCT AAA ACC TTC CTC ACC GTG TAC TGG ACT CCT 336
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro
7S 80 ~5
AAG GAT CCA GAG ATT CAT TTG AGT GGC CCT CTG GAG GCT GGG AAG CCG 3 84
Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro
90 95 100
ATC ACA GTC MG TGT TCA GTT GCT GAT GTA TAC CCA TTT GAC AGG CTG 432
Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu
105 110 115 120
GAG ATA GAC TTA CTG AAA GGA GAT CAT CTC ATG MG AGT CAG GAA TTT 480
Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe
125 130 135
CTG GAG GAT GCA GAC AGG AAG TCC CTG GM ACC AAG AGT TTG GAA GTA 528
Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val
140 145 150 ~:~
ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA MA GTT CTT GTT TGC CGA 5 7 6
Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Arg
155 160 165
GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC ACA GTA AGG CAG 624 : .
Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln
170 175 1~0
GCT GTA AAA GAA TTG CM GTC TAC ATA TCA CCC AAG AAT ACA GTT ATT 6 7 2
Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val Ile
185 190 l9S 200
TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GGT GGC TCT GTG ACC ATG 720 ~-
Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met
205 210 215
ACC TGT TCC AGC GAG GGT CTA CCA (:CT CCA GAG ATT TIC TGG AGT MG 7 6 8
Thr Cys Ser Ser Gl~ Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser Lys
220 225 230
AAA TTA GAT AAT GGG AAT CTA CAG CAC CTT TCT GGA MT GCA ACT CTC 816
Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu -
~35 240 245
ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT TAT GTG TGT GAA 8 6 4
Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly lle Tyr Val Cys Glu
250 255 260
GGA GTT MT TTG ATT GGG AM MC AGA AAA GAG GTG GM TTA ATT GTT 912
Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile Val
265 270 275 280
I;U~ 7a~E ~3~.r~r
2 ~ ~ 7 ~ 3 2
CAA GCA TTC CCT AGA GAT CCA GM ATC GAG ATG AGT GGT GGC CTC GTG 960
Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val
285 290 295
AAT GGG AGC TCT GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC CCC 1008
Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro
300 305 310
CTT GAC CGG CTG GAG ATT GAA TTA CTT AAG GGG GAG ACT ATT CTG GAG 1056
Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu
315 320 325
AAT ATA GAG TTT TTG GAG GAT ACG GAT ATG AM TCT CTA GAG AAC AAA 1104
Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys ~ .
330 335 340
AGT TTG GM ATG ACC TTC ATC CCT ACC ATT GAA GAT ACT GGA AAA GCT 1152
Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala
345 350 355 360 :
CTT GTT TGT CAG GCT MG TTA CAT ATT GAT GAC ATG GAA TTC GAA CCC 1200
Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro :
365 370 375 ;-
AAA CAA AGG CAG AGT ACG CM ACA CTT TAT GTC AAT GTT GCC CCC AGA 1248 -'
Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg -:
380 385 390
GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG GAG GAA GGC AGT 1296
Asp Thr Thr Val Leu Val Ser Pro Sar Ser Ile Leu Glu Glu Gly Ser
395 400 405
TCT GTG MT ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT CCG AAA ATC 1344
Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile
410 415 420
CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TCT GAG 1392
Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu
425 430 435 440
MT GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GM GAT TCT GGG GTT 1440
Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val
445 450 455
TAT TTA TGT GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA AAG GM GTG 14~8
Tyr Leu Cys &lu Gly Ile Asn Gln Ala Gly Ar~ Ser Arg Lys Glu Val
460 465 470
GM TTA ATT ATC CM GTT.ACT CCA MA GAC ATA AM CTT ACA GCT TTT 1536
Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile~Lys Leu Thr Ala Phe
475 480 485
SUE~S~lTtlTE ~fflEf
- 21~75~
--73-- .
CCT TCT GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT ACA 1584
Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr
490 495 500
TGT GGA AAT GTT CCA GM ACA TGG ATA ATC CTG AAG MA AAA GCG GAG 1632
Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu
505 510 515 520
ACA GGA GAC ACA GTA CTA AAA TCT ATA :;AT GGC GCC TAT ACC ATC CGA 1680
Thr Gly Asp Thr ~.'al Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg
525 530 535 .
MG GCC CAG TTG MG GAT GCG GGA GTA TAT GAA TGT GM TCT MA AAC 1728 -
Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn
540 545 550 :
AAA GTT GGC TCA CM TTA AGA AGT TTA ACA CTT GAT GTT CAA GGA AGA 1776
Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg
555 560 565 ~
GAA AAC MC AAA GAC TAT TTT TCT CCT GAG CTT CTC GTG CTC TAT TTT 1824 :~.
Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Lell Val Leu Tyr Phe
570 575 580 .
GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT GCA 1872
Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala ;
585 590 595 600
AGA AM GCC AAC ATG AAG GGG TCA TAT AGT CTT GTA GAA GCA CAG AAA 1920
Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys
605 610 615
TCA AAA GTG 1~29
Ser Lys Val
(2) INFORMATION FOR SEQ ID NO: 19:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 643 anlino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECUI,E TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp
--24 --20 --15 --10
8UBSTIT~T~
lle Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu
-5 1 5
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser ~-
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro
Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu - ~ -- 45 50 55 ~.
er Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro .
Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu Ala Gly I~s Pro
g5 100
Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu
105 110 115 120
Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe
125 130 135 :
Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val
140 145 150 `
Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Arg
155 - 150 165
Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln
170 - 175 180
Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val Ile
185 190 195 200
Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met
205 210 ~15
Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser Lys :~
220 225 230
Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu
235 240 245
Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys Glu
250 255 ~ 260
SUE3ST~T~E S~
~ 1 2 7 i~ 3 ~
-75-
Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile Val
265 270 275 280 :~
ln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val
285 2gO 295
sn Gly Ser Ser Val Thr Val Ser Cy5 Lys Val Pro Ser Val Tyr Pro
300 305 310
eu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu
315 320 325
Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys
330 335 340
Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala
345 350 355 360
eu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro
365 ~370 375
ys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg
380 385 390
sp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser
395 400 405
Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile
410 415 420
Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu
425 ~30 435 440
sn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val
445 450 455
yr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val
460 465 470
Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe
475 480 485
Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr
490 495 S00
Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu
505 510 515 s2n
hr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg
525 530 535
~u~ r
~ ~ 12 rl ~ 3 ~ ;:
-76-
ys Ala Gln Leu Lys Asp Ala Gly Val Tyr Gl~l Cys Glu Ser Lys Asn
540 545 550
Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg
555 560 565
Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe
570 575 580
Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala
585 590 595 600
Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys
605 610 615
Ser Lys Val
(2) I~FORMATION FOR SEQ ID NO:20:
(i) SEQUENCE C~ ACTERISTICS:
(A) LENGTH: 1932 base pairs
(B) TYPE: nucleic acid
(C~ STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1932
(ix) FEAT~RE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 73..1932
(ix) FEATURE:
(A~ NAME/KEY: sig_peptide
(B) LOCATION: 1.. 72 ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: :
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA .~Ar ATA CTT TGG 48
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp
-24 -20 -15 -lO
ATA ATG TTT GCA GCT TCT CAA GCT TTT AAA ATC GAG ACC ACC CCA G.~ 96
Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu
_5 1 5
TCT AGA TAT CTT CTG CCC CGG GGA GGC TCC GTG~CTG GTG ACA TGC AGC 144
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser
lO 15 20 `
81JB~TIT~T~
-b~ 2~32
ACC TCG TGT GAC CAG CCC AAG TTG TTG GGC ATA GAG ACC CCG TTG CCT 192
Thr Ser Cys Asp Gln Pr~ Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro
25 30 3 S 40
AAA AAG GAG TTG CTC CTG CCT GGG AAC AAC CGG AAG GTG TAT GM CTG 240
Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
45 50 5 5
AGC AAT GTG CAA GAA GAT AGC CAA CCA ATG TGC TAT TCA AAC TGC CCT 2 8 8
Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 65 70
GAT GGG CAG TCA ACA GCT AAA ACC TTC CTC ACC GTG TAC TGG ACT CCA 336
Asp Gly Gln Ser Thr Ala Lys Thr Phe l.eu Thr Val Tyr Trp Thr Pro
75 80 85
GAA CGG GTG GAA CTG GCA CCC CTC CCC TCT TGG CAG CCA GTG GGC AAG 3 84
Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys
90 95 lC0
AAC CTT ACC CTA CGC TGC CAG GTG GAG GGT GGG GCA CCC CGG GCC MC 432
Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn ~ .
105 110 - 115 120 ~,
CTC ACC GTG GTG CTG CTC CGT GGG GAG MG CTC ATG AAG AGT CAG GAA 480
Leu Thr Val Val Leu Leu Arg Gly Glu Lys Leu Met Lys Ser Gln Glu
125 130 135 ~-
TTT CTG GAG GAT GCA GAC AGG AAG TCC CTG GAA ACC AAG AGT TTG GAA 5 2 8
Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu
140 145 150
GTA ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT CTT GTT TGC 576
Val lrhr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys
155 160 165
CGA GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG ('CC ACA GTA AGG 6 24
Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg
170 175 lB0
CAG GCT GTA AAA GAA TTG CM GTC TAC ATA TCA CCC AAG AAT ACA GTT 6 7 2
Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val
185 190 195 200
ATT TCT GTG AAT CCA TCC ACA AAG CTG CAA GAA GGT GGC TCT GTG ACC 7 2 0
Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr
205 210 215
ATG ACC TGT TCC AGC GAG .GGT CTA CCA GCT CCA GAG ATT TTC TGG AGT 7 6 8
Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser
220 225 230
Sll~S~3Tt~TE S~~
2~ 2~3~
-78-
AAG AAA TTA GAT AAT GGG AAT CTA CAG CAC CTT TCT GGA AAT GCA ACT 816
Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr
235 240 245
CTC ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT TAT GTG TGT 864
Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys
250 255 260
GAA GGA GTT MT TTG ATT GGG AAA MC AGA MA GAG GTG GAA TTA ATT 912
Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile
265 270 275 280
GTT CAA GCA TTC CCT AGA GAT CCA GAA ATC GAG ATG AGT GGT GGC CTC 960
Val Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu :
285 290 2~5
GTG MT GGG RGC TCT GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC 1008 :~
Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr
300 305 310 `:
CCC CTT GAC CGG CTG GAG ATT GM TTA CTT AAG GGG GAG ACT ATT CTG 1056
Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu
3~5 320 325
GAG MT ATA GAG TTT TTG GAG GAT ACG GAT ATG AAA TCT CTA GAG MC 1104
Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser I.eu Glu Asn
33~ 335 340 :
AAA AGT TTG GAA ATG ACC TTC ATC CCT ACC ATT GAA GAT ACT GGA AAA 1152
Lys Ser Leu Glu Met Thr Phe lle Pro Thr Ile Glu Asp Thr Gly Lys.
345 350 355 ~ 360
GCT CTT GTT TGT CAG GCT AAG TTA CAT ATT GAT GAC ATG GAA TTC GAA 1200
Ala Le.u Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu , :
365 370 375 :~
CCC AAA CAA AGG CAG AGT ACG CAA ACA CTT TAT GTC AAT GTT GCC CCC 1248
Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro
380 385 390
AGA GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG GA& G~ GGC 1296
Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Çlu Gly
395 400 405
AGT TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT GCT C:CG AAA 1344
Ser Sex Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys
410 415 420
ATC CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG CCT CTT TCT 1392
Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser
425 430 435 440
~3U!13STIT~
7 ~ <J h
--79--
GAG AAT GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GAA GAT TCT GGG 1440
Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly
445 450 455
GTT TAT TTA TGT GAA GGA ATT AAC CAG GCT GGA AGA AGC AGA AAG GAA 1488
Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu
460 465 470
GTG GAA TTA ATT ATC CAA GTT ACT CCA AAA GAC ATA AAA CTT ACA GCT 1536
Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala
475 480 485
TTT CCl` TCT GAG AGT GTC AAA GAA GGA GAC ACT GTC ATC ATC TCT TGT 1584
Phe Prc Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys
490 495 500 :
ACA TGT GGA AAT GTT CCA GAA ACA TGG ATA ATC CTG AAG AAA AAA GCG 1632
Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala
505 510 515 520
GAG ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC 1680
Glu Thr Gly Asp,Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile
525 530 535
CGA AAG GCC CAG TTG M G GAT GCG GGA GTA TAT GM TGT G M TCT AAA 1728
Arg Lys Ala Gin Leu Lys A.~p Ala Gly Val Tyr Glu Cys Glu Ser Lys
540 545 550
M C AAA GTT GGC TCA C M TTA AGA AGT TTA ACA CTT GAT GTT CAA GGA 1776
Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly
555 S60 565
AGA GAA AAC AAC AAA GAC TAT TTT TCT CCT GAG CTT CTC GTG CTC TAT 1824
Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr
570 575 580
TTT GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA ATG ATA ATT TAC TTT 1872
Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe
585 590 59~ 603
GCA AGA AAA GCC M C ATG AAG GGG TCA TAT AGT CTT GTA GAA GCA CAG 1920
Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln
. 605 610 615
AAA TCA AAA GTG 1932
Lys Ser Lys ~al
620
S~
: 2 ~ 2 7 ~ `, 2
-80-
(2) I~FORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 644 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp :.
-24 -20 -15 -10 -
Ile Me~ Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu
-5 1 5 ~:
Ser Arg Tyr Leu Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser
Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu Pro
ys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu
er Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys Pro
60 ~5 70 ::
Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro -
Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly Lys
100
Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala E'ro Arg Ala Asn
105 110 115 120
eu Thr Val Val Leu Leu Arg Gly Glu Lys Leu Met Lys Ser Gln Glu
125 130 135
he Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu
140 145 150
Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly I.ys V~l Leu Val Cys
155 160 165
Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg
170 175 180
Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val
185 190 195 ~00
S~ TlTl~
. -81- 2127~32
Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr
205 210 215
et Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser
220 ~25 230
Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr
235 240 245
Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys
250 255 260
Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile
265 270 275 280
al Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly l.eu
285 290 295
al Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr
300 305 310
Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu
315 320 325
Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn
330 335 340
Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys
345 350 355 360
la Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu
365 370 375
ro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro
380 385 390
Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly
395 400 405
Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys
410 415 420
Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser
425 430 435 440
lu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly
445 450 455
al Tyr Leu Cys Glu &ly Ile Asn Gln Ala Gly Ar~ Ser Arg Lys Glu
460 465 ~ 470
SU~3T~
r ~ ~ .
--8 2-- 2 1 2 7 J r3
Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala
475 480 485 .:
Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys
490 495 500
~hr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu ~ys Lys Lys Ala
505 510 515 520
Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile ..
525 530 535
Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys :
540 545 550 :
Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly
555 560 565
Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr
570 575 580 .
Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe
585 590 595 600
Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln :~605 610 615 -~.
Lys Ser Lys Val
620
(2) INFORMATION FOR SEQ ID NO:22: ~ `
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 1941 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix~ FEATURE:
(A) NAME/KEY: CDS
~B) LOCATION: 1..1941
(ix) FEATURE:
(A) NAME/KEY:`mat_peptide
(B) LOCATION: 73..1941
(ix~ FEATURE:
~A) NAME/KEY: sig peptide
(B) LOCATION: 1..72
SUB~ F~
2127~32
-83-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
ATG CCT GGG AAG ATG GTC GTG ATC CTT GGA GCC TCA AAT ATA CTT TCG 48
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp
-24 -20 -15 -10
ATA ATG TTT GCA GCT TCT CAA GCT TTT ACT GTT GAG ATC TCC CCT GGA 96
Ile Met Phe Ala Ala Ser Gln Ala Phe Thr Val Glu Ile Ser Pro Gly
_5 1 5
CCC CGG ATT GCT GCT CAG ATT GGA GAC TCA GTC ATG TTG ACA TGT AGT 144
Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Ser
10 15 20
GTC ATG GGC TGT GAA TCC CCA TCT TTC TCC TGG AGA ACC CAG ATA GAC 192
Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp
25 30 35 40
AGC CCT CTG AGC GGG M G GTG AGG AGT GAG GGG ACC AAT TCC ACG CTG 240
Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu
45 50 S5
ACC CTG AGC CCT GTG AGT TTT GAG AAC GAA CAC TCT TAT CTG TGC ACA 288
Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr
60 65 70
GTG ACT TGT GGA CAT AAG AAA CTG GAA M G GGA ATC CAG GTG GAG CTC 336
Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu
75 80 85
TAC TCA TTC CCT M G GAT CCA GAG ATT CAT TTG AGT GGC CCT CTG GAG 384
Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu
90 95 100
GCT GGG AAG CCG ATC ACA GTC AAG TGT TCA GTT GCT GAT GTA TAC CCA 432
Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro
105 110 115 12~
TTT GAC AGG CTG GAG ATA GAC TTA CTG AAA GGA GAT CAT CTC ATG AAG 480
Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys
125 130 135
AGT CAG GAA TTT CTG GAG GAT GCA GAC AGG M G TCC CTG GAA ACC AAG 528
Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys
140 145 150
AGT TTG GAA GTA ACC TTT ACT CCT GTC ATT GAG GAT ATT GGA AAA GTT 576
Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val
155 160 165
SUE~S~UTE~
r r r
2 1 2 7 5 ~3 2
-84- :
CTT GTT TGC CGA GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC 624
Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro :
170 175 180
ACA GTA AGG CAG GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC AAG 672 ~:
Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys
185 190 195 200
MT ACA GTT ATT TCT GTG AAT CCA TCC ACA MG CTG CAA GAA GGT GGC 720
Asn Thr Val Ile Sar Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly
205 210 215
TCT GTG ACC ATG ACC TGT TCC AGC GAG GGT CTA CCA GCT CCA GAG ATT 768 :~
Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile
220 225 230 :~
TTC TGG AGT AAG AAA TTA GAT MT GGG AAT CTA CAG CAC CTT TCT GGA 816
Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly
235 240 245
AAT GCA ACT CTC ACC TTA ATT GCT ATG AGG ATG GAA GAT TCT GGA ATT 864 ` :
Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile ~:
250 255 260 ~`
TAT GTG TGT GAA GGA GTT AAT TTG ATT GGG AAA AAC AGA AAA GAG GTG 912 -~
Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val
265 270 275 280
GM TTA ATT GTT CM GCA TTC CCT AGA GAT CCA GAA ATC GAG ATG AGT 960
Glu Leu Ile Val Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser
285 290 295 :
GGT GGC CTC GTG AAT GGC: AGC TCT GTC ACT GTA AGC TGC MG GTT CCT 1008
Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro -
300 305 310
AGC GTG TAC CCC CTT GAC CGG CTG GAG ATT GAA TTA CTT AAG GGG GAG 1056
Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu
315 320 325
ACT ATT CTG GAG AAT ATA GAG TTT TTG GAG GAT ACG GAT ATG AM TCT 1104
Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser
330 335 340
CTA GAG MC AAA AGT TTG GAA ATG ACC TTC ATC CCT ACC ATT GM GAT 1152
Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp
345 350 355 360
ACT GGA AM GCT CTT GTT TGT CAG GCT MG TTA CAT ATT GAT GAC ATG 1200
Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu-His Ile Asp Asp Mec
365 370 375
SU~
2 1 2 7 ~ 3 2
GAA TTC GAA CCC AAA CM AGG CAG AGT ACG CAA ACA CTT TAT GTC MT 1248
Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn
380 385 390
GTT GCC CCC AGA GAT ACA ACC GTC TTG GTC AGC CCT TCC TCC ATC CTG 1296
Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu
395 400 405
GAG GAA GGC AGT TCT GTG AAT ATG ACA TGC TTG AGC CAG GGC TTT CCT 1344
Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro
410 415 420
GCT CCG AAA ATC CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG 1392
Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln
425 430 435 440
CCT CTT TCT GAG AAT GCA ACT CTC ACC TTA ATT TCT ACA AAA ATG GAA 1440
Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu
445 450 455
GAT TCT GGG GTT TAT TTA TGT GM GGA ATT AAC CAG GCT GGA AGA AGC 1488
Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser
460 465 470
AGA AAG GAA GTG GM TTA ATT ATC CM GTT ACT CCA AAA GAC ATA AAA 1536
Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys
475 480 485
CTT ACA GCT TTT CCT TCT GAG AGT GTC AAA GM GGA GAC ACT. GTC ATC 1584
Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile
490 495 500
ATC TCT TGT ACA TGT GGA MT GTT CCA GM ACA TGG ATA ATC CTG AAG 1632
Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys
505 510 515 520
AAA AAA GCG GAG ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GC~ 1680
Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala
525 530 535
TAT ACC ATC CGA AAG GCC CAG TTG AAG GAT GCG GGA GTA TAT GAA TGT 1728
Tyr Thr Ile Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys
540 545 550
GAA TCT AAA AAC AAA GTT GGC TCA CM TTA AGA AGT TTA ACA CTT GAT 1776
Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp
555 560 565
GTT CM GGA AGA GM AAC AAC MM GAC TAT TTT TCT CCT GAG CTT CTC 1824
Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu
570 575 580
SU~
~ 1 2 7 '.~
-86-
GTG CTC TAT TTT GCA TCC TCC TTA ATA ATA CCT GCC ATT GGA ATG ATA 1872
Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile
585 590 595 600
ATT TAC TTT GCA AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CTT GTA 1920
Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val
605 610 61S
GAA GCA CAG AAA TCA AAA GTG 1941
Glu Ala Gln Lys Ser Lys Val
620
(2) INFO~MATION FOR SE~ ID NO:23: :~
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 647 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: :.
Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp :
-24 -2~ -15 -10
Ile Met Phe Ala Ala Ser Gln Ala Phe Thr Val Glu Ile Ser Pro Gly
-5 1 5
Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Ser
Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp
er Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu
hr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr
Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu
7S 80 85
Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu
100
Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro
iO5 110 llS 120
SUI3!~T~9~E ~tEES
2 7 ~
-87-
Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met l.ys
125 130 135
er Gln Glu Phe Leu Glu Asp Ala Asp Ar~ Lys Ser Leu Glu Thr Lys
140 145 150
Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val
155 160 165
Leu Val Cys Ar~ Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro
170 175 180
Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys
185 190 195 200
sn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly
205 210 215
er Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile
220 225 230
Phe Trp SPr Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly
235 240 245
Rsn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile
250 255 260
Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val
~65 270 275 280
lu Leu Ile Val Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser
285 290 295
ly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro
300 305 .~.. 310
Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu-
315 320 325
Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser
330 335 340
Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp
345 350 355 360
hr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met
3~5 370 375
lu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn
380 385 ` 390
8)Jg3ST~TJ~ ~
~ ~ 2 7 ~ .~ 2 :~
-88-
Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu
395 400 405
Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro
410 415 420
Ala Pro Lys Ile Leu Trp Ser Arg 51n Leu Pro Asn Gly Glu Leu Gln
425 430 435 440
Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu
445 450 455
Asp Ser ~ly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser
. 460 465 470
Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys
475 480 485
Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile
490 4~5 500
Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys
505 S10 515 520
Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala
525 530 535 ~:
Tyr Thr Ile Arg Lys Ala Gln Leu Lyc Asp Ala Gly Val Tyr Glu Cys
540 54S 550
Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp
555 56Q S65 .
Val Gln Gly Arg Glu Asn Asn Lys Asp-Tyr Phe Ser Pro Glu Leu Leu ~;-
570 575 580
Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile
585 590 595 600
Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val
605 610 615 .
Glu Ala Gln Lys Ser Lys Val
620
... . ~
8UBÇT3TU~E~ S9~EE~
