Note: Descriptions are shown in the official language in which they were submitted.
21170.99
-
FILE, I~THI`~
~F TRANSLArlC)N ~ :
RECEPTOR DERIVATIVES HAVING BINDING SITES
FOR HUMAN RHINOVIRUSES
The present invention describes receptor derivatives
having binding sites for human rhinoviruses of the
"small rhinovirus receptor group", the use thereof and
DNA coding for the receptor derivatives.
Human rhinoviruses represent a large genus within the
family of the picorna viruses and include approximately
115 different serotypes (Melnick, J.L. (1980) Prog. Med.
Virol. 26, 214-232). These RNA viruses attack the
respiratory tract in humans and cause acute infections
which lead to colds.
The human rhinoviruses can be subdivided into two groups
if the criterion used for categorisation is their
competition for binding sites on the surface of human
cell culture cells such as HeLa cells. Competitive
experiments show that, apart from one single exception
(serotype 87), there are two different receptors on the
cell surface. Hitherto, 91 serotypes have been
allocated to the "large rhinovirus receptor group" and
10 serotypes to the "small rhinovirus receptor group"
(Abraham and Colonno R.J. (1984) J. Virol. 51, 340-345;
Uncapher et al. (1991) Virology 180, 814 - 817). The
receptor of "large rhinovirus receptor group" was
purified and identified as ICAM-1, a protein belonging
to the immunoglobulin superfamily, which acts as a cell
adhesion molecule (Tomassini et al. (1989) Proc. Natl.
Acad. Sci. USA 86, 4907-4911; Staunton et al. (1989)
Cell 56, 849-853; Greve et al. (1989) Cell 56, 839-847)-
~ 2117099
-- 2
By demonstrating specific binding of the purified ICAM-l
to the virus and because of the possibility of
transferring the rhinovirus binding activity, by gene
transfer, to cells which had no such activity before the
transfer, it was possible to show clearly that ICAM-l is
the receptor for the majority of rhinoviruses (Greve et
al. (1989) loc. cit.; Staunton et al. (1989) loc. cit).
Moreover, it has been shown that monoclonal antibodies
against ICAM-1 prevent the binding and infection of HeLa
cells by rhinoviruses (Staunton et al. (1989), loc.
cit.). Furthermore, monoclonal antibodies which inhibit
the binding of ICAM-l to leukocytes via LFA-l
("lymphocyte function associated antigen-l") - another
natural ligand of ICAM-l - are also able to block the
binding of the rhinovirus to the receptor. Thus, the
¦ LFA-l and rhinovirus binding sites must be at least
I adjacent. Tests with chimeric and mutated ICAM-l
molecules additionally showed that the binding site for
1 the rhinovirus-ICAM-l interaction does not coincide with
the binding site for LFA-l (Staunton et al. (1990) Cell
61, 243-254).
The receptor binding site of the human rhinovirus
serotype 14, an example of the "large rhinovirus
receptor group", lies in a so-called "canyon", a
depression in the surface of the virus (Rossmann et al.
(1985) Nature 317, 145-153). The amino acids which are
located in this canyon are conserved to a relatively
great extent, whilst the amino acids in the surrounding
area are variable and constitute binding sites for
antibodies with a neutralising effect. According to
this "canyon hypothesis", viruses can accept mutations
in the hypervariable antibody binding sites and thus
escape the natural immune response. In this way a
constant receptor binding site is maintained which is
not accessible for antibodies (Rossmann and Palmenberg
(1988) Virology 164, 373-382).
.~
~ :
;~ ~ :~` ~ff ~
4~
2ll7ass
-- 3
As far as is known at present, the receptor of the
"small rhinovirus receptor group" permits the uptake of
about 10 serotypes of human rhinoviruses into the
corresponding host cells. This receptor on the membrane
has been isolated by various purification steps, whilst
the binding activity in the various frac~ions has been
demonstrated by a filter binding assay (Mischak et al.
(1988) J. Gen. Virol., 69, 2653-2656). The apparent
molecular weight of the native receptor in the presence
of nonionic detergents (determined by gel
chromatography) corresponds to about 450 kD, that of the
denatured form corresponds to about 120 kD, although a
number of other forms were found (Mischak (1988) loc.
cit.). It has also been found that a protein isolated
from the cell culture supernatant from HeLa cells has
the capacity to bind rhinoviruses of the "small rhino-
virus receptor group" (Hofer et al. (1992) J. gen.
Virol. 73, 627 - 632). ;
The natural receptor is less suitable for inhibiting the
uptake of rhinoviruses of the "small rhinovirus receptor
group" on the basis of the low solubility of this
membrane protein in polar, e.g. aqueous solution systems
such as aqueous buffer solutions.
Surprisingly, it has now been found that the members of
LDL ("low density lipoprotein") receptor family act as
receptors for rhinoviruses of the "small rhinovirus
receptor group".
~ . :
The identical nature of the receptors of the LDL-
receptor family and the receptors of rhinoviruses of the
"small rhinovirus receptor group" now surprisingly makes
it possible to prepare polypeptides, particularly
soluble polypeptides, which have at least one bindinq -
site for rhinoviruses of the "small rhinovirus receptor
group".
21170~9
The polypeptides according to the invention are
hereinafter referred to as ~functional derivatives" of
the receptor proteins. A func~ional derivative is
therefore a component with the biological activity which
corresponds essentially to the biological activity of
the native receptor of the "small rhinovirus receptor
group". This biological activity relates to the binding
capacity of the receptor for rhinoviruses of the "small
rhinovirus receptor group". The expression "functional
derivatives" is intended to include "variants" and
"chemical derivatives". The term derivative refers to
any polypeptide which is small in size, compared with
the native receptor protein, and has at least one
binding site for rhinoviruses of the "small rhinovirus
receptor group". A "variant" comprises the molecules
which are essentially derived from the native receptor
molecule in function and structure, such as the allelic
forms, for example. Accordingly, the term "variant"
includes molecules which are capable of binding
rhinoviruses of the "small rhinovirus receptor group"
but have a different amino acid sequence, for example.
A "chemical derivative" includes additional chemical
groups which are not normally part of this molecule.
These groups may improve the molecule solubility, the
absorption, the biological half-life etc. or
alternatively may reduce the toxicity of undesirable
side effects. Groups having such effects are known
(Remington's Pharmaceutical Sciences (1980)).
~ .
The biological activity of the receptor derivatives
according to the invention or the chemical derivatives
obtained after modification can be tested using methods
known from the prior art, e.g. the filter binding assay
described by Mischak et al. (Mischak et al. (1988) J.
Gen. Virol. 69, 2653-2656 and Mischak et al. (1988)
Virology 163, 19-25): the polypeptide is applied to a
~ 2~170~9
-- 5
,
suitable membrane, such as nitrocellulose. Then, in
order to block any non~specific binding, it is saturated
with a detergent mixture. The membrane pretreated in
this way is then incubated with labelled rhinovirus,
e.g. with HRV2 labelled with 35S-methionine, in order to
check the specific binding. After washing and drying of
the membrane specific binding can then be visualised by
autoradiography.
One aspect of the invention relates to the receptor
derivatives which are present in the form of
extracellular, soluble polypeptides and are released
into the medium, for example, by receptor-carrying
cells. These receptor derivatives are exceptionally
well suited to inhibiting the binding of rhinoviruses to
their receptors. Thus, they can be used for the
therapeutic or prophylactic treatment of the human body
or for producing pharmaceutical preparations. In
particular, their use as antiviral and preferably
antirhinoviral agents may be considered. The phenomenon
of releasing a soluble receptor derivative has been ;~;
described for numerous receptor proteins, e.g. for the
interleukin-4- and interleukin-7-receptor (Mosley et al.
(1989) Cell 59, 335-348; Goodwin et al. (1990) Cell. 60,
941-951). ;
Naturally, soluble receptor derivatives may also be
formed by enzymatic, especially proteolytic or chemical
cleaving. Receptor-carrying cell lines may be used for
this purpose, which are reacted with enzymes such-as
papain, trypsin etc. If the amino acid sequence of the
receptor molecule is known, the person skilled in the
art can of course deliberately prepare extracellular
derivatives by a suitable choice of proteases. The
binding capacity of such derivatives can be checked
using the filter binding assay described above, thus
making it possible to prepare deliberately smaller
~ 21~709~
-- 6
receptor derivatives which are capable of binding
rhinoviruses of the "small rhinovirus receptor group".
In addition to enzymatic cleaving it is also possible to
cleave extracellular receptor regions by chemical
methods, e.g. by cleaving with cyanogen bromide.
A further aspect of this invention consists of the
formation of soluble derivatives by enzymatic or
chemical cleaving of native receptor molecules. After a
native receptor protein has been isolated, for example,
the native receptor protein can be cleaved by reaction
with proteases or by chemical cleaving (as described
above) and the reduced in size, rhinovirus-binding
region can be identified by the filter binding assay,
for example, and isolated. Suitable proteases can be
derived from the particular amino acid sequence of the
receptor protein. Chemical cleaving reactions can also
be carried out using cyanogen bromide or cleaving the
receptor protein by a reductive treatment, e.g. with
dithiothreitol.
More specifically, the present invention comprises the
following aspects:
It has been found, surprisingly, that proteins of the
LDL-receptor family are capable of binding and
internalising rhinoviruses of the "small rhinovirus
receptor group". Consequently, all the members of the
LDL-receptor family can now be used to prepare
functional derivatives capable of binding the
rhinoviruses of the "small rhinovirus receptor group".
The LDL-receptor family is formed from three
structurally related cell surface receptors which bring
about the endocytosis of lipoproteins and other plasma
proteins (Brown et al. (1991) Curr. Opin. Lipidology 2,
65-72). The receptors have the following common
21170~3
- 7 -
features: cysteine-rich repeats, which are responsible
for ligand binding, cysteine-rich repeats of the EGF
("epidermal growth factor")-type, Y-W-T-D-repeats, a
single region spanning the membrane and at least one
NPXY-internalising signal (Willnow et al. (1992) J.
Biol. Chem. 267, 26172-21180).
Surprisingly, it has been shown that all three ~embers
of this family - the LDL-receptor, the ~2MR/LRP (~2-
macroglobulin/LDL-receptor-related protein) and also the
gp330 (Heymann nephritis antigen gp330) - are capable of
binding and internalising rhinoviruses of the "small
rhinovirus receptor group" (Examples 1 to 2). All
members of this receptor family can thus be used to form
functional derivatives with binding properties for ~1
rhinoviruses of the "small rhinovirus receptor group".
For example, in order to isolate soluble LDL-receptor
derivatives released into the medium, the method
included in Example 3 can be followed. This describes
the purification of a binding protein released into the
cell culture supernatant. Surprisingly, it was found
that this is an LDL-receptor derivative (Example 4).
For the purpose of isolation, the receptor derivative is
purified by ion exchange chromatography (anionic),
affinity chromatography (Lens culinaris lectin and
Jacalin agarose) and ammonium sulphate precipitation.
The binding activity was checked using the filter
binding assay (Mischak et al. (1988) 163, 19-25). This
method of production can also be applied to the other
two proteins of the LDL-receptor family.
Isolation of the native receptor proteins is known and
I is described by Yamamoto et al. (1984) Cell 39, 27-38;
I Goldstein et al. (1985) Annu. Rev. Cell Biol. 1, 1-39;
I Mischak et al. (1988) Virology 163, 19-25; Kowal et al.
j (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5810-5815 and
Willnow et al. (1992) loc. cit.). The native proteins
~ 2117~g.~ '
-- 8
can then be converted by enzymatic and chemical cleaving
into the functional soluble derivatives. Since the
amino acid sequence of the LDL-receptor (Fig. 1), the
MR/LRP (Fig. 2) and, at least partially, the gp330
(Fig. 3) are known, proteolytically active enzymes or
chemicals can be deliberately selected in order to
release, in particular, the particular extracellular
receptor region. The present invention therefore also
relates to polypeptides which are derived from the amino
acid sequences of the LDL-receptor, ~zMR/LRP and gp330
and, in particular, in their soluble form are capable of
binding rhinoviruses of the "small rhinovirus receptor
group". Preferably, these polypeptides are derived from
the amino acid sequences which correspond to the human
proteins of the LDL-receptor family, although, as
explained in Examples 1 and 2, corresponding receptors -
from mammals and amphibia are also suitable.
Receptor derivatives may be used in the form in which
they are released into the cell supernatant from
eukaryotic cells. The receptor derivatives of the
present invention may also, however, correspond to the
membrane-bound members of the LDL-receptor family in
which that part of the protein which is responsible for ~ -
binding the protein to the membrane is missing or has
lost its function.
Particularly preferred are receptor derivatives which
consist essentially of domains 1, 2 and 3 of the
receptor protein, or domains 1 and 2 or only domain 1
according to Figure 4. According to this, domain 1
comprises the N-terminal cysteine-rich receptor portion
which binds the various ligands, domain 2 comprises a
region having higher homology to the EGF-precursor
protein, domain 3 comprises a relatively short, O-
glycosylated peptide region, domain 4 comprises the
transmembrane region and domain 5 the cytoplasmic part
. - : : '
21170~9
_ 9 -
of the receptor ~olecule. Polypeptides consisting
essentially of domains l, l and 2 and 1, 2 and 3 m~y be
obtained from the culture supernatant of eukaryotic
cells (Example 3) or by recombinant DNA techniques known '
se, such as that described by Davis et a],. (1987)
Nature 326, 760-765 for the LDL-receptor. Of the
proteins of the LDL-receptor family the human,LDL- :,
receptor is the preferred starting compound. In
particular, the invention include~ functional receptor
derivatives which essentially comprise amino acids 1 to
750 tdomains l and 2) and 1-322 (domain 1) (Fig. 1). , ,
The C-terminus of these polypeptides can be shortened,
provided that the ~inding capacity for rhinoviruses of
the small rhinovirus receptor group remains intact.
The preferred receptor derivatives have essentially the
following amino acid sequences:
Domains l and 2 (amino acids 1 to 750, SEQ.ID.NO.l):
1 MGPWGWKLRW TVALLLAAAG TAVGDRCERN EFQCQDGKCI SYKWVCDGSA
51 ECQDGSDESQ ETCLSVTCKS GDFSCGGRVN RCIPQFWRCD GQVDCDNGSD
101 EQGCPPKTCS QDEFRCHDGK CISRQFVCDS DRDCLDGSDE ASCPVLTCGP
151 ASFQCNSSTC IPQLWACDND PDCEDGSDEW PQRCRGLYVF QGDSSPCSAF
201 EFHCLSGECI HSSWRCDGGP DCKDKSDEEN CAVATCRPDE FQCSDGNCIH
251 GSRQCDREYD CKDMSDEVGC VNVTLCEGPN KFKCHSGECI TLDKVCNMAR
301 DCRDWSDEPI KECGTNECLD NNGGCSHVCN DLKIGYECLC PDGFQLVAQR
351 RCEDIDECQD PDTCSQLCVN LEGGYKCQCE EGFQLDPHTK ACKAVGSLAY
401 LFFTNRHEVR KMTLDRSEYT SLIPNLRN W ALDTEVASNR IYWSDLSQRM '
451 ICSTQLDRAH GVSSYDTVIS RDIQAPDGLA VDWIHSNIYW TDsvLGTvsv
501 ADTKGVKRKT l,FRENGSKPR AI W DPVHGF MYWTDWGTPA KIKKGGLNGv
551 DIYSLVTENI QWPNGITLDL LSGRLYWVDS KLHSISSIDV NGGNRKTILE
601 DEKRLAHPFS LAVFEDKVFW TDIINEAIFS ANRLTGSDVN LLAENLLspE
651 DMVLFHNLTQ PRGVNWCERT TLSNGGCQYL CLPAPQINPH SpKFTcAcpD
701 GMLLARDMRS CLTEAEAAVA TQETSTVRLK VSSTAVRTQH TTTRpvpDTs
'
-
~ 211709~
-- 10 --
Domain 1 (amino acid 1 to 322, SEQ.ID.NO.2):
1 MGPWGWKLRW TVALLLAAAG TAVGDRCERN EFQCQDGKCI SYKWVCDGSA
51 ECQDGSDESQ ETCLSVTCKS GDFSCGGRVN RCIPQFWRCD GQVDCDNGSD
101 EQGCPPKTCS QDEFRCHDGK CISRQFVCDS DRDCLDGSDE ASCPVLTCGP
151 ASFQCNSSTC IPQLWACDND PDCEDGSDEW PQRCRGLYVF QGDSSPCSAF
201 EFHCLSGECI HSSWRCDGGP DCKDKSDEEN CAVATCRPDE FQCSDGNCIH
251 GSRQCDREYD CKDMSDEVGC VNVTLCEGPN KFKCHSGECI TLDKVCNMAR
301 DCRDWSDEPI KECGTNECLD NN
The polypeptides according to the invention may occur as
dimers, trimers, tetramers or multimers. The processes
for preparing the receptor derivative, enzymatic or
chemical treatment of the native receptor molecules,
isolation of the derivatives released by cells and
processes for recombinant preparation are also part of
the invention.
A further aspect of the invention concerns DNA molecules
which code for the polypeptides according to the
invention.
:'
The starting molecules can be obtained by the person
skilled in the art using known methods. The cloning of
the correspondind cDNA is described for all three
members (Yamamoto et al. (1984) loc. cit.; Goldstein et
al. (1985) loc. cit.; Pietromonaco et al. (1990) Proc.
Natl. Acad. Sci. U.S.A. 87, 1811-1815; Herz et al.
(1988) loc. cit.). Moreover, the DNA molecules, where ;
the amino acid sequence is known, may also be produced
synthetically (e.g. according to Edge et al. (1981) 292,
756-762) or by the PCR method (Sambrook et al., loc.
cit.).
The invention relates to DNA sequences which have
modifications obtained simply by methods known to those
skilled in the art, by mutation, deletion, transposition
~: .
:~ ", ~ : :.,"~;-. :~: .: ~ ~ :
-- 21170~9
or addition. All DNA sequences which code for a
polypeptide according to the invention and the
correspondingly degenerate forms of the DNA sequences
are included.
In addition, the invention relates to DNA vectors which
contain the DNA sequences described above. In
particular, these may be vectors in which the DNA
molecules described are functionally linked to a control
sequence which allows expression of the corresponding
polypeptides. These are preferably plasmids which can
be replicated and/or expressed in prokaryotes such as
E. coli and/or in eukaryotic systems such as yeasts or
mammalian cell lines.
The invention also relates to correspondingly
transformed host organisms.
Expression in prokaryotes may be carried out using other
organisms known from the prior art, especially E. coli.
The DNA sequences according to the invention may be
expressed as fusion polypeptides or as intact, native
polypeptides. .
Fusion proteins may advantageously be produced in large
quantities. They are generally more stable than the
native polypeptide and are easy to purify. The
expression of these fusion proteins can be controlled by
normal E. coli DNA sequences.
I
For example, the DNA sequences according to the
invention can be cloned and expressed as lacZ fusion
genes. The person skilled in the art has a variety of
vector systems available for this purpose, e.g. the pUR-
vector series (Ruther, U. and Muller-Hill, B. (1983),
EMB0 J. 2, 1791). The bacteriophage promoter ~PR may
also be used, in the form of the vectors pEX-1 to -3,
21170~9
- 12 -
for expressing large amounts of Cro-~-galactosidase
fusion protein (Stanley, K.K. and Luzio, J.P. (1984)
EMBO J. 3, 1429). Analogously, the tac promoter which
can be induced with IPTG can also be used, for example
in the form of the pROK-vector series (CLONTECH
Laboratories).
The prerequisite for producing intact native
polypeptides using E. coli is the use of a strong,
regulatable promoter and an effective ribosome binding
site. Promoters which may be used for this purpose
include the temperature sensitive bacteriophage ~pL-
promoter, the tac-promoter inducible with IPTG or the
T7-promoter. Numerous plasmids with suitable promoter
structures and efficient ribosome binding sites have
been described, such as for example pKC30 (~pL; Shimatake
and Rosenberg (1981) Nature 292, 128, pKK173-3 (tac,
Amann and Brosius (1985) Gene 40, 183) or pET-3 ~T7-
promoter (Studier and Moffat (1986) J. Mol. Biol. 189,
113).
A number of other vector systems for expressing the DNA -~
according to the invention in E. coli are known from the
prior art and are described for example in Sambrook et
al. (1989) "A Laboratory Manual", Cold Spring Harbor
Laboratory Press).
Suitable E. coli strains which are specifically tailored
to the expression vector in question are known to those
skilled in the art (Sambrook et al. (1989), loc. cit.).
The experimental performance of the cloning experiments,
the expression of the polypeptides in E. coli and the
working up and purification of the polypeptides are
known and are described for example in Sambrook et al.
(1989, loc. cit.). In addition to prokaryotes,
eukaryotic microorganisms such as yeast may also be
used.
-- 2117~
- 13 -
For expression in yeast, the plasmid YRp7 (StinchcoMb et
al. Nature 282, 39 (1979); Kings~an et al., Gene 7, 141
(1979); Tschumper et al., Gene 10, 157 (1980)) and the
plasmid YEpl3 (Bwach et al., Gene 8, 121-133 (1979)) are
used, for example. The plasmid YP~p7 contains the TRPl-
gene which provides a selection marker for a yeast
mutant (e.g. ATCC No. 44076) which is incapable of
growing in tryptophan-free medium. The presence of the
TRPl defect as a characteristic of the yeast strain used
then constitutes an effective aid to detecting
transformation when cultivation is carried out without -
tryptophan. The same is true with the plasmid YEpl3, -
which contains the yeast gene LEU-2, which can be used
to complete a LEU-2-minus mutant.
.. .
Other suitable marker genes for yeast include, for
example, the URA3- and HIS3-gene. Preferably, yeast
hybrid vectors also contain a replication start and a
marker gene for a bacterial host, particularly E. coli,
so that the construction and cloning of the hybrid
vectors and their precursors can be carried out in a
bacterial host. Other expression control sequences
suitable for expression in yeast include, for example,
those of PHO3- or PHO5-gene.
Other suitable promoter sequences for yeast vectors
contain the 5'-flanking region of the genes of ADH I
tAmmerer, Methods of Enzymology 101, 192-210 (1983)), 3-
phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem.
255, 2073 (1980)) or other glycolytic enzymes (Kawaski
and Fraenkel, BBRC 108, 1107-1112 (1982)) such as
enolase, glycerinaldehyde-3-phosphate-dehydrogenase,
hexokinase, pyruvate-decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, phosphoglucose-isomerase
and glucokinase. When constructing suitable expression
plasmids, the termination sequences associated with
these genes may also be inserted in the expression
21170~
vector at the 3~-end of the sequence to be expressed, in
order to enable polyadenylation and termination of the
mRNA.
Other promoters are the promoter regions of the genes
for alcohol dehydrogenase-2, isocytochrome C, acid
phosphatase and enzymes which are responsible for the
metabolism of maltose and galactose~ Promoters which
are regulated by the yeast mating type locus, such as
promoters of the genes BARI, MF~1, STE2, STE3, STE5 can
be inserted in temperature regulated systems by the use -
of temperature-dependent sir mutations. (Rhine Ph.D.
Thesis, University of Oregon, Eugene, Oregon (1979), --
Herskowitz and Oshima, The Molecular Biology of the
Yeast Saccharomyces, part I, 181-209 (1981), Cold Spring
Harbor Laboratory). However, generally, any vector
which contains a yeast-compatible promoter and origin
replication and termination sequences is suitable.
Thus, hybrid vectors which contain sequences homologous
to the yeast 2~ plasmid DNA may also be used. Such
hybrid vectors are incorporated by recombination within
the cells of existing 2~-plasmids or replicate
autonomously. ~-
. ~
In addition to yeasts, other eukaryotic systems may, of
course, be used to express the polypeptides according to
the invention. Since post-translational modifications
such as disulphide bridge formation, glycosylation,
phosphorylation and/or oligomerisation are frequently
necessary for the expression of biologically active
eukaryotic proteins by means of recombinant DNA, it may
be desirable to express the DNA according to the
invention not only in mammalian cell lines but also
insect cell lines.
:~:
Functional prerequisites of the corresponding vector
systems comprise, in particular, suitable promoter,
~ A
21170~9
- 15 -
termination and polyadenylation signals as well as
elements which make it possible to carry out replication
and selection in mammalian cell lines. For expression -
of the DNA molecules according to the invention it is
particularly desirable to use vectors which are
replicable both in mammalian cells and also in
prokaryotes such as E. coli.
Vectors derived from viral systems such as SV40,
Epstein-Barr-virus, etc., include, for example, pTK2,
pSV2-dhfv, pRSV-neo, pKO-neo, pHyg, p205, pHEBo, etc.
(Sambrook et al. 1989, loc. cit.).
After transformation in suitable host cells, e.g. CHO-
cells, corresponding transformed cells may be obtalned
with the aid of selectable markers (thymidine-kinase,
dihydrofolate-reductase etc.) and the corresponding
polypeptides are isolated after expression. The host
cells suitable for the vectors are known, as are the
techniques for transformation (micro-injection,
electroporation, calcium phosphate method, etc.) (e.g.
Sambrook et al., 1989).
For cloning corresponding DNA fragments in prokaryotic
or eukaryotic systems, the selected vector is cut, for
example, with a restriction endonuclease and, optionally
after modification of the linearised vector thus formed,
an expression control sequence equipped with
corresponding restriction ends is inserted. At the 3'-
end (in the direction of translation) the expression
control sequence contains the recognition sequence of a
restriction endonuclease, so that the vector already
containing the expression control sequence is digested
with the said restriction enzyme and the DNA molecule
according to the invention, provided with ends which
fit, can be inserted. It is a~vantageous to cleave the
vector which already contains the expression control
21170~9
- 16 -
sequence with a second restriction endonuclease inside
the vector DNA and to insert the DNA molecule provided
with the correct ends into the vector fragment produced.
The techniques required are described for example by
Sambrook et al. (1989, loc. cit.).
Apart from the DNA molecules specified, the invention
also relates to processes for preparing the vectors
described, particularly expression vectors. These
vectors are characterised in that a DNA provided with
corresponding ends and coding for a functional
derivative of the receptor of the "small rhinovirus
receptor group" is inserted into a vector DNA cut with
restriction endonucleases and containing the expression
control sequences described by way of example, in such a
way that the expression control sequences regulate the
expression of the DNA inserted. The polypeptides
according to the invention which are obtained by the
expression of recombinant DNA or from the native
receptor molecule may, of course, also be derivatised by
chemical or enzymatic processes.
The expression of the LDL-receptor is explained in
Example 6. Here, expression takes place in a eukaryotic
system, by way of example. It is clearly shown that the
LDL-receptor expressed brings about a binding of
radioactively labelled human rhinovirus serotype 2
(HRV2) (Fig. 5). The polypeptides according to the
invention may be obtained, for example, by deletion of
DNA sequences in the expression plasmid. This can-be
done, for example, using the method of Davis et al.
(1987) Nature 326, 760-765, which describes the deletion
of the entire EGF domain. Moreover, soluble forms of
the receptor can be formed by inserting a stop codon in
front of the cytoplasmic or transmembrane domain (Yokade
et al. (1992) J. Cell. Biol. 117, 39).
. ~
.'
-' 211709~
- 17 -
The invention further relates to hybrid cell lines which
secrete monoclonal antibodies specifically against one
of the polypeptides according to the invention or
functional derivatives thereof. These monoclonal
antibodies are capable of wholly or partially
neutralising the activity of the polypeptides or
specifically binding to one of the said polypeptides.
The monoclonal antibodies can then be used for
qualitative and/or quantitative measurement or for
purifying the polypeptides according to the invention.
The invention naturally also includes test systems which
contain the monoclonal antibodies mentioned. The
process for preparing the monoclonal antibodies is
characterised in that host animals are immunised with
one of the polypeptides and B-lymphocytes of these host
animals are fused with myeloma cells; the hybrid cell
lines which secrete the corresponding monoclonal
antibodies can then be subcloned and cultivated (Harlow,
G. and Lane, D.: "Antibodies. A Laboratory Manual"
(1988) Cold Spring Harbor Laboratory Press, USA).
A further aspect of the invention is the use of
physioloqical ligands of the LDL-receptor family for
preparing pharmaceutical compositions for inhibiting the
binding of rhinoviruses of the "small rhinovirus
receptor group". The physiological ligands comprise the
substances which are bound and/or internalised by the
LDL-receptor family. For example, LDL (low density
lipoprotein) inhibits the uptake of rhinoviruses of the
"small rhinovirus receptor group" (Example 9). other
natural ligands of the LDL-receptor family are described
for example by Willnow et al. (1992) J. Biol. Chem. 267,
26172-2618~.
Thus, for example, the 39 kDa receptor-associated
protein (RAP) can reduce the yield of rhinoviruses of
the "small rhinovirus receptor group" (Example 7). RAP
~ 2 1 1 7 0 .~ .~
is known ~er se. Its isolation and binding to members
of the LDL-receptor family has been described for
example by Kounnas et al. (1992) J. Biol. Chem. 267,
21162-21166.
: .
It is also, of course, possible to use the native
receptors of the LDL-receptor family, the LDL-receptor,
~2MR/LRP and gp330 as the receptor derivatives according
to the invention for inhibition.
Conversely, it is also, of course, possible to use
rhinovirus material of the "small rhinovirus receptor
group" to inhibit the binding of physiological LDL-
ligands. This rhinovirus may, for example, be derived
from human rhinovirus serotype 2 (HRV2). Preferably,
inactivated rhinovirus, rhinovirus coat material or
rhinovirus peptides with a binding activity to a
receptor of the LDL-receptor family may be used as
rhinovirus material. Rhinoviruses of the "small
rhinovirus receptor group" can be obtained from the
"American Type Culture Collection". Corresponding virus
material may be prepared by known methods (e.g. Putnak
and Phillips (1981) Microbiol. Reviews 45, 287-315 and
Palmenberg (1990) Annu. Rev. Microbiol. 44, 603-623 and
the literature cited therein).
Of course, the invention also includes the
pharmaceutically acceptable salts of the polypeptides
according to the invention and the pharmaceutically
acceptable adducts and covalent compounds between the
polypeptides and an inert carrier for the prophylactic
and/or therapeutic treatment of the human or animal
body. The adducts or covalent compounds may be formed
with polyethyleneglycol, for example. The polypeptides
according to the invention and the native receptor ~-
proteins, the physiological ligands of the LDL-receptor
family such as LDL and RAP, for example, may be used to
~ 2117Q~
-- 19 --
produce pharmaceutical preparations for the therapeutic
and/or prophylactic treatment of the human or animal
body. In particular, the polypeptides can be used as
competitively acting substances for inhibiting the
binding of viruses, particularly rhinoviruses, to the
native receptor and/or physiological LDL-ligands. The
polypeptides and natural ligands, particularly the
extracellular, soluble form of the receptor, may be used
especially as antiviral and preferably antirhinoviral
agents.
For treating viral infections the substances described
may be administered nasally, for example, the quantities
supplied being sufficient to suppress or competitively
interact or inhibit the binding of the rhinovirus to the
natural receptor. The dosage should generally be
between 0.01 pg/kg of the weight of the patient up to
1 mg/kg of the weight of the patient, although larger or
smaller quantities may also be used. The rhinovirus
I material which may be used to inhibit the binding of
physiological LDL-ligands can be used in suitable
pharmaceutical compositions in the ranges of
concentration specified for the polypeptides.
The receptor derivatives according to the invention and
the pharmacologically acceptable salts thereof may be
converted in the usual way into conventional
formulations such as plain or coated tablets, pills,
granules, aerosols, syrups, emulsions, suspensions and
solutions, using inert pharmaceutically acceptable
carriers or solvents. The proportion of the
pharmaceutically active compound or compounds should be
in the range from 0.5 to 90 wt.-% of the total
composition, i.e. in amounts which are sufficient to
achieve the dosage range specified above.
The formulations are prepared, for example, by admixing
.1 ' ~
~ `,:
.~.. ,......... - ~ . . ~ . , . ~ ~ ............................ ..
. - ~ .. . .. ., . .. . . ,.. .,. ..... , ., ., ~ ... . . ... .
211709~ ~ ~
- 20 -
the active substances with solvents and/or carriers,
optionally using emulsifiers and/or dispersants, whilst
if water is used as the diluent, organic solvents may be
used as solubilising agents or auxiliary solvents.
The excipients used include, for example, water,
pharmaceutically acceptable organic solvents such as
paraffins, vegetable oils, mono- or polyfunctional
alcohols, carriers such as natural mineral powders,
synthetic mineral powders, sugars, emulsifiers and
lubricants.
The substances are administered in the usual way,
preferably nasally. In the case of oral administration
the tablets may, of course, contain in addition to the
above-mentioned carriers, additives such as sodium
citrate, calcium carbonate and dicalcium phosphate
together with various additives such as starch,
preferably potato starch, gelatine and the like.
Furthermore, lubricants such as magnesium stearate,
sodium laurylsulphate and talc can be used to form
tablets. In the case of aqueous suspensions, the active
substances may contain, in addition to the above-
mentioned excipients, various flavour improvers or
colourings.
The invention also relates to processes for isolating
substances which inhibit the binding of ligands to the
LDL-receptor. These processes comprise incubating the
LDL-receptor protein or an LDL-receptor derivative with
a potentially inhibiting substance. This process can be
carried out in the presence of labelled rhinovirus
material. The extent of the binding of labelled
rhinovirus material then allows conclusions to be drawn
as to the activity of the test substance. The
preparation of rhinovirus material with various binding
activities is described in Example 9. ~ -~
r~ 21~7099
- 21 --
The invention also relates to processes for detecting
LDL-receptors, in which a substance derived from virus
material of the "small rhinovirus receptor group" with a
binding activity for the LDL-receptor is labelled,
incubated with a suitable sample and the extent of
binding is detected. Another process serves to supply
therapeutically active substances in which virus
material of the "small rhinovirus receptor" group with a
binding activity for the LDL-receptor is coupled with
the therapeutic substance and the said conjugate is
added to cell material carrying LDL-receptor and the
therapeutically actlve substance is inserted into the
j cell by binding and internalisation.
.
'1 :
,~ 21~7~
- 2~ -
LEGENDS T0 THE FIGURES
Fig. 1: Amino acid sequence of the "Low Density
Lipoprotein Receptors" (LDL, Yamamoto et al.
(1984) Cell 31, 27-38).
Fig. 2: Amino acid sequence of the "Low Density
Lipopro-tein Receptor Related Proteins" (LRP,
Herz et al. (1988) EMB0 J. 7, 4119-4127).
Fig. 3: Part of the amino acid sequence of the
"Heymann Nephritis Antigen gp330 (Pietromonaco
et al. (1990) Proc. Natl. Acad. Sci. U.S.A.
87, 1811-1815).
Fig. 4: Diagrammatic representation of a receptor of
the LDL-receptor family (according to Yamamoto
et al. (loc. cit.). The receptor has five
domains: domain 1 comprises the N-terminal
cysteine-rich receptor part which is
presumably responsible for the ligand binding.
Domain 2 with homology for the EGF-precursor
protein adjoins domain 3, the amino acids of
which are partially 0-glycosylated. Domain 4
forms the part of the receptor situated at the
membrane whilst domain 5 forms the cytoplasmic
part of the receptor.
Fig. 5: A) Binding and internalisation of labelled
HRV2 on normal human fibroblast cells and
on LDL-receptor deficient FH cells,
respectively (Example 1).
t: cultivated without the addition of
cholesterol/25-hydroxycholesterol
1: cultivated with the addition of
cholesterol/25-hydroxycholesterol
D,
21~70~9
- 23 -
B) Competition of HRV2 and LDL for the
receptor binding site
+: with the addition of unlabelled HRV2
or LDL
-: without the addition of unlabelled
HRV2 or LDL.
ig. 6: Binding of [35S]-labelled HRV2 to ~2MR/LRP and
gp330. Membrane extracts have been
electrophoretically separated and transferred
to nitrocellulose. Detection was carried out
with [35S]-labelled HRV2 (trace 1 and 2) with
~2MR/LRP antiserum (trace 3) or with gp330
antiserum (trace 4).
Trace 1: LM-extracts,
Trace 2: Rat kidney microvilli-extracts,
Trace 3: Protein extracts as in trace 1,
Trace 4: Protein extracts as in trace 2.
ig. 7: Gel electrophoretic analysis of the purified
HRV2 binding protein.
a) The purified H~V2 binding protein was
subjected to electrophoresis in a 7.5
SDS gel under reducing (trace 1) and non-
reducing conditions ~trace 2) and made -~
visible by silver staining. Under non-
reducing conditions a molecular weight of
about 120 kDa is obtained whilst under
reducing conditions the molecular weight
is 160 kDa.
b) Ligand blots of a gel as described under
a (trace 2), developed with [3sS]-HRV2
(trace 1) according to Mischak et al.
(1988) Virology 163, 19-25. Trace 2
shows the development with an antibody
specific for the human LDL-receptor
(IgG-C7, Beisiegel et al. (1982) J. Biol.
~ 21170~
- 24 -
Chem. 257, 13150-13156).
,, .
Fig. 8: Shows the column chromatographic separation of
the tryptic peptides of the soluble form of
the receptor of the "small rhinovirus receptor
group", obtained from the HeLa cell
supernatant. The peptides were separated on a
~Bondapak C18,250-4 column under the following
conditions:
~ Buffer A: distilled water/0.06% TFA; buffer B:
¦ 80% acetonitrile/0.052~ TFA; flow rate:
0.5 ml/min; gradient: 2% B to 37.5% B from 0
to 60 min, 37.5% B to 75% 8 from 60 to 90 min,
75% B to 98% B from 90 to 105 min; ~-
temperature: ambient temperature; detection:
photometrically at 214 nm, 0.08 AUFS (paper
advance: 0.25 cm/min).
Fig. 9: Chromatographic separation of fractions 23 to
27 under the following conditions:
Column: Merck Superspher 4 ~m, C18, 125-H;
buffer A: distilled water/0.1% TFA; buffer B:
acetonitrile/0.1% TFA; flow rate: 1 ml/min,
linear gradient from 0% B to 70% B in 70 min; -~-
temperature: 30C; detection: photometrically '
at 214 nm, 0.1 AUFS, paper advance 1 cm/min.
Fig. 10: Rechromatography of fraction 29. The
experimental conditions are described in the
legend to Fig. 9.
Fig. 11: Rechromatography of fraction 38. The
experimental conditions are described in the
legend to Fig. 9.
Fig. 12: Sequences of the peptide analysed X = amino
acid not identifiable; subscript = amino acid
.`
' :
~ 5~
~11709~
- 25 -
not clearly identifiable.
*: The sequencing of fraction 33 (Fi~. g)
yielded 2 amino acids for each breakdown step;
however, peptides B and E were able to be
classified because of the different
quantities.
ig. 13: Inhibition of binding of [35S]-labelled HRV2 to
the immobilon-bound LDL-receptor by jacalin.
A) filter binding test in the absence of
jacalin
B) as in ~, but in the presence of 0.1 mg/ml
of jacalin.
ig. 14: Expression of the human LDL-receptor in COS-7
cells (Example 6).
Detection of bound[3sS]-HRV2 on
u: untransfected COS-7 cells
+: transformed with the "sense" (pSVL-LDLR+)-
vector
-: transformed with the "antisense"
(pSVL-LDLR-)-vector
ig. 15: Reduction of the virus yield by RAP, given in
p.f.u./ml (infectious particles per
millilitre).
ig. 16: Inhibition of HRV2-infection of HeLa cells by
human LDL.
ig. 17: Comparison of sequences for determining the
positions in or on the edge of the canyon
which are conserved in rhinoviruses of the
small group.
ig. 18: Binding characteristics of HRV2ll48pG and
HRV23lB2RT to HeLa cells
^` 2117099 ~:
- 26 -
AHRv2 1 148P:G
HRV2-Wild type
_HRV23l8ZR r
Fig. l9: Competition of binding of HRV2~148pG and .
HEV2~zpl by HRVl4 (-) or HRV2 (~
:~;
~ 2117~9~
- 27 -
EXAMPLES
Example 1: sinding and internalisation of [35S]-labelled
HRV2 by human fibroblast cells and
competition between HRV2 and LDL for the
receptor binding site -~
a~ Binding and internalisation of HRV2
Normal human fibroblast cells or LDL receptor-deficient
cells (FH cells; NIH Collection No. GM 00486A) were
grown for 24 hours on 6-well plates (Nunc) in MEM,
containing 10% delipidated foetal calf serum (Gibco),
either with (1) or without (t) the addition of 12 ~g/ml
of cholesterol and 2 ~g/ml of 25-hydroxycholesterol.
Then the cells were washed twice with PBS, 10,000 cpm
[35S]-labelled HRV2 in 0.5 ml of PBS, containing 2% ~SA
and 30 mM MgCl2 were added and the mixture was incubated
for 60 minutes at 34C (Mischak et al. (1988) Virology
163, 19-25). After the removal of superficially bound
HRV2 using 10 ~g/ml of trypsin and 25 mM EDTA in PBS the
cells were washed once more and then the bound
radioactivity was measured. The data provided are the
averages from four experiments in each case. The
radioactivity levels of the cell pellets from normal
fibroblasts (normally about 1900 cpm) grown without
steroids, minus background radioactivity, was set at
100%. The background activity was determined either
with HRV2 which had been heated to 56'C for 30 minutes
(Mischak et al. (1988) loc. cit.) or by incubation with
a 1000-fold excess of unlabelled HRV2. For both methods
it was between 40 and 50 cpm. The data obtained from ~f~
four separate experiments are shown in Fig. Sa. ~
:.
b) Competition of HRV2 and LDL for the receptor bindinq ;;~
site :~
Normal fibroblast cells were grown as described under a)
(without the addition of cholesterol and 25-hydroxy-
. ~
~ 21170~9
- 28 -
cholesterol). The cells were incubated at 37C for 60
minutes with approximately 1.4 x 106 cpm lZsI-labelled LDL
(250 cpm/ng; Huettinger et al. (1992) J. Biol. Chem.,
267, 18551-7) with (+) and without (-) the addition of
100 Pfu ("plaque forming units"; corresponding to about
2400-24000 virus particles; Abraham & Colonno (1984) J.
Virol. 51, 340-345) per cell on purified, unlabelled
HRV2 or with about 10000 cpm of [35S]-labelled HRV2 with
(+) or without (-) 80 ~g/ml of unlabelled LDL. The
cell-associated radioactivity was measured with a y- or
~-counter. The radioactivity levels for the high
affinity binding of l2sI-LDL were determined by
subtracting the radioactivity, obtained in the presence
of a 20-fold excess of unlabelled LDL (approximately
40000 cpm/mg of whole cell protein), from the entire
LDL-bond (150,000 cpm/mg). Without a competitor, a
radioactivity level of 1900 cpm was normally found for
HRV2 binding. The maximum binding levels wère put at
100% in each case. The data obtained (Fig. 5b) show the
results of two separate experiments in each case.
Example 2: Binding of [35S]-labelled HRV2 by ~2MR/LRP
and gp 330
Plasma membrane preparations were tested for HRV2-
binding in order to demonstrate the binding of
rhinoviruses of the small rhinovirus receptor group to
other members of the LDL receptor family. Plasma
membranes were isolated from murine LM fibroblasts and
kidney epithelial microvilli (Malathi et al. (1979)
Biochem. Biophys. Acta, 554, 259-263; Fornistal et al.
(1991) Infect. Immun. 59, 2880-2884 and Kerjaschki and
Farquhar (1982) Proc. Natl. Acad. Sci. U.S.A. 79,
5557-5561). Proteins from the membrane extracts were
separated by SDS-qradient-polyacrylamide-electrophoresis
and transferred on to nitrocellulose.
^` 211709~ ~
29
The incubation of the separated ~-extracts with [35S]-
labelled HRV2 showed a binding to a protein with an
apparent molecular weight of about 500 kDa (Fig. 6,
trace 1). This band comigrates with ~2MR/LRP. This was
demonstrated by an identical blot which was developed
with ~2MR/LRP antiserum (Moestrup and Gliemann (1991) J.
Biol. Chem. 266, 14011-14017) (Fig. 6; trace 3).
A protein with an apparent molecular weight of again
about 500 kDa could be detected with radioactively
labelled HRV2 in extracts from rat kidney microvilli
(Fig. 6; trace 2). After analysis with a gp330
antiserum the band was identified as Heymann Nephritis
' Antigen gp330 (Fig. 6, trace 4~.
:i
l Example 3: Purification of a binding protein for the
i rhinoviruses of the "small rhinovirus
¦ receptor group"
200 1 of HeLa-cell culture supernatant (made by Computer
Cell Culture Center, Mons, Belgium) were concentrated
down to 20 1 by ultrafiltration and dialysed against
`~ 250 1 of distilled water (with 0.02% NaN3). Then the
bùffer concentration was adjusted to 20 mM N-
methylpipe~azine, pH ~.5, the mixture was centrifuged at
4000 rpm in a Beckman J6B centrifuge, filtered through a
0.8 ~m preliminary filter and the filtrate was
transferred on to an anion exchanger column (0.5 1
Makroprep 50 Q; Biorad). Bound material was eluted with
20 mM N-methylpiperazine, pH 4.5, 0.5 M NaCl. The
eluate was adjusted to a pH of 7.2 using 1 M tris-HCl,
pH 8.0, and transferred to a Lens culinaris lectin
column (100 ml; Pharmacia); bound protein was eluted -~
with 0.5 M ~-[D]-methylglucose in PBS, the eluted
protein was precipitated at 50% saturation with ammonium
sulphate, pH 7.2, the precipitate was washed with 50% ~ ;
:~
~ 211709~
- 30 -
saturated ammonium sulphate solution, pH 7.2, and taken
up in 200 ml of PBS. The protein solution was
transferred to a Jacalin agarose column (40 ml; Vector-
Labs) and eluted with 120 ml of 100 mM ~-tD]-methyl-
galactopyranoside in PBS. The eluted protein was
precipitated with ammonium sulphate as described above,
washed, taken up in 20 mM methylpiperazine, pH 4.5, and
desalinated using a PD10-column (Pharmacia). The
desalinated material was added to a Mono Q anionic
exchanger column (HR5/5; Pharmacia) in 5 aliquots per ml
and eluted with a gradient from o to 0.5 M NaCl, 20 mM
methylpiperazine, pH 4.5. 0.5 ml fractions were
collected and tested for binding activity using a filter
binding assay (Mischak et al., Virology (1988) 163,
19-25). The active fractions from all five
chromatographies were concentrated down to 1.5 ml in a
Centricon 30 (Amicon) and resolved by preparative gel
electrophoresis under non-reducing conditions on a 7.5%
polyacrylamide gel (Laemmli, U.K. (1970) Nature 227,
680 - 685). The gel was stained with copper chloride
(Lee et al. (1987) Anal. Biochem. 166, 308 - 312), the
band corresponding to the active protein was located and
excised. The gel fragments were decolorised in 0.25 M
Tris-HCl, 0.25 M EDTA, pH 9.0, and the protein was
eluted in 50 mM N-ethylmorpholinoacetate, pH 8.5, by
electrophoresis. One aliquot was again tested for
activity using the filter binding assay. The protein
was then separated by gel electrophoresis under reducing
conditions, eluted and lyophilised.
Example 4: Tryptic digestion and sequence analysis of
the binding protein for the rhinoviruses of
the "small rhinovirus receptor group"
The purified and lyophilised protein (Example 3) was
taken up in 30 ~1 of 6 M guanidine-HCl, 0.4 M ammonium
'-` 2117~99
- 31 -
hydrogen carbonate, pH 7.6, and mixed with 3 ~1 of 45 mM
dithiothreitol and incubated for 15 minutes at 56C.
After cooling to ambient temperature, 3 ~1 of 100 mM
iodoacetamide were added and the mixture was incubated
for a further 15 minutes at ambient temperature. Then
84 ~1 of water and 80 ~1 of 0.1 M ammonium hydro~en
carbonate, pH 7.6, were added, the protein solution was
mixed with 800 ng of trypsin (in 5 ~1) under the
conditions specified by the manufacturer (Promega) and
incubated for 18 hours at 37~C. The solution was
acidified with 1/10 volume of 10% trifluoroacetic acid
(TFA), centrifuged for 5 minutes and the peptides were
eluted on a C-18 "reversed phase" column (Baker) which
had been equilibrated with a 0.06~ aqueous TFA solution,
with a gradient up to 80~ acetonitrile, 20~ water,
0.052% TFA (Fig. 8). ;~
Fractions 20 and 33 were sequenced directly using a gas
¦ phase sequenator whilst fractions 23 to 27 and 29 and 38
I were re-chromatographed under the conditions specified
j in the Figures (C18 "reversed-phase" column, Merck;
I Figs. 9, 10 and 11). The peptides designated "A", "D" and "F" in the Figures and fractions 33 and 20 were
selected for sequencing in the gas phase sequenator.
The results are assembled in Fig. 12. The sequences
obtained were compared with the protein sequences
available in the "SwissProt" databank. Comparison
showed total agreement with corresponding peptide
sequences of the human LDL-receptor:
The following Table shows the sequences of the isolated
tryptic peptides and the position in the sequence of the
human LDL-receptor (Fig. 1).
~ .~
2~17ass
- 32 -
Peptide Position Sequence
A 165 XLYVFQGDSSPXXAFEFXXLXXXXI
B 373 XFGSIAXLFFTN
C 420 XYWSDLSQR
D 451 DIQAPXGLAVXXIXSNIYXXXXVL
E 500 XI WXPVHGFMYXTXXGTPAK
F 584 XAHPFSLAVFEXK
The sequencing of fraction 33 yielded two amino acids
per breakdown step. Taking as the basis the LDL-
sequence and the ratio of amino acid quantities present
in each breakdown step, from about 40% to 60~, fraction
33 was identified as a mixture of two peptides. The
sequences of these two peptides also correspond to
sequences of the human LDL-receptor.
~ Fig. 1 shows the total sequence of the human LDL-
i receptor (Yamamoto et al. (1984) Cell 31, 27 - 38).
~,
j Example 5: Expression of the human LDL-receptor in
COS-7 cells
The plasmid pTZl, which contains the entire coding
sequence of the human LDL-receptor from the plasmid
pLDLR2 (Yamamoto et al., loc. cit.) was introduced into
competent E. coli SK and amplified using known methods
(Sambrook et al., loc. cit.). After extraction and
purification of the plasmid DNA the latter was digested
with the restriction enzyme HindIII and the fragments
were separated in a 0.8% agarose gel. After elution of
the fragment coding for the LDL-receptor the latter was
precipitated with ethanol, taken up in TE-buffer and
partially filled with Klenow fragment using dATP and
dGTP.
21170~9
- 33 -
The eukaryotic expression vector pSVL (Pharmacia) was
replicated in E. coli 5K, purified and cut with XbaI.
After partial filling with dCTP and dTTP, phenol-
chloroform extraction and ethanol precipitation, the
plasmid was dephosphorylated with alkaline phosphatase.
By partial filling both of the vector and of the LDL-
receptor DNA the restriction cutting sites were made
compatible and LDL-receptor coding and vector DNA were
ligated using T4-ligase. Competent E. coli bacteria
were transformed as described. A number of colonies
were investigated, by restriction digestion with XhoI,
for orientation of the insert with regard to the SV40
"late promoter". One colony with positive (sense,
pSVL-LDLR+) orientation and one colony with negative
(antisense, pSVL-LDLR-) orientation of the insert were
cultivated and plasmids were obtained in large
quantities.
The transfection of COS-7 cells (ATCC CRL 1651) was
carried out by lipofection (lipofectin reagent, BRL) in
9 cm petri dishes in accordance with the manufacturer's
instructions. After transfection the cells were sown in
6-well dishes and cultivated for a further 24 hours in
RPMI/10% HiFCS and 12 ~g/ml of cholesterol as well as -
2 ~g/ml of 25-hydroxycholesterol. The cells were washed
with PBS/2% BSA and then incubated for 1 hour at 34C
with about 10,000 cpm/well of [35S ] -HRV2 in PBS/2~ BSA.
After washing several times, the cells were lysed in
PBS/2% SDS and the quantity of bound [3sS]-HRV2 was
determined by counting in a liquid scintillation
counter. The addition of foetal calf serum, cholesterol
and 25-hydroxycholesterol brings about a suppression of
the endogenous LDL-receptors (Davis et al., 1987, Nature
326, 760), so that in the subsequent binding test only
the LDL-receptors expressed by transfection are
detected. As shown in Fig. 14, the quantity of bound
-' 21~70~9
- 34 -
HRV2, compared with untransfected control cells, is
twice as great if the cells are transfected with the
sense construct pSVL-LDLR+. Transfection with pSVL-
LDLR- shows no difference in binding compared with the
control cells.
Example 6: Inhibition of binding of [35S]-labelled
rhinovirus serotype 2 (HRV2) by Jacalin
Two aliquots of the protein purified by all but Jacalin
agarose chromatography (see Example 3, corresponding to
a starting quantity of cell supernatant of about 50 ml)
were separated on a 7.5% SDS polyacrylamide gel
(Laemmli, U.K. 1970, Nature 227, 680 - 685) under non-
reducing conditions and transferred by electrophoresis
on to an immobilon membrane (Millipore) (Mischak et al.,
1988, loc. cit., Hofer et al., 1992, loc. cit.). One
trace was incubated in the absence (Fig. 13, trace A) or
in the presence (trace B) of 0.1 mg/ml of jacalin
(Vector Labs) with radioactively labelled rhinovirus
(Mischak et al., 1988, loc. cit.), washed, dried and
exposed on X-ray film (Hofer et al.~ 1992, loc.cit.).
As shown in Figure 13 the binding of the virus to the
LDL-receptor is totally inhibited under the conditions
specified.
Example 7: ~eduction of the virus yield by RAP
(receptor associated protein)
FH cells (cf. Example 1) were sown in 24-well dishes
(Nunc) in RPMI with 10% foetal calf serum and cultivated
overnight to a cell density of about sx104 cells per
well. The cells were washed once with PBS and mixed
with RPMI/2% foetal calf serum/30 mM MgC12. Human ~ -
recombinant RAP was obtained as described in Kunnas et ~
~ ' :
21170~
- 35 -
al., loc. cit., then purified and added to the mediu~ in -
concentrations of 0.5 ~g/ml, 5 ~g/ml, 10 ~g/ml and
20 ~g/ml, and the cells were incubated for 2 hours at
4C. HRV2 was added to each test batch in an m.o.i of
lO0 and incubation was continued for a further 2 hours
at 4C. Then the cells were washed 3x with PBS, mixed
with RPMI/2% foetal calf serum/30 mM MgC12 and incubated
overnight at 34C. The next day the cells were broken
up by freezing and thawing three times. Cell fragments
were removed by centrifuging at lO,OOOxg and the number
of infectious virus particles in the supernatant was
determined by the plaque test (Neubauer et al., loc.
cit.). Fig. 15 shows that the yield of HRV2 decreases
as the concentration of RAP rises and at an RAP
concentration of 20 ~g/ml it is reduced to about 5% of
the comparison value without RAP.
xample 8: Inhibition of ~RV2-infection of HeLa cells
by human LDL
HeLa cells were sown in 24-well dishes (Nunc) in MEM
with 10% foetal calf serum and cultivated overnight to a
cell density of about 2xlO5 cells per well. The cells
were washed once with PBS and mixed with RPMI/2% foetal
calf serum/30 mM MgCl2. Purified LDL (Huttinger et al.,
loc. cit.) was added in concentrations of 0.1 mg/ml,
0.3 mg/ml, 0.5 mg/ml and 1 mg/ml and the cells were
incubated for 30 minutes at 34C. HRV2 or HRV14 (a
virus of the large receptor group, used as a control)
were added to each test batch in an m.o.i of 100 and
incubation was continued for 45 minutes at 34C. Then
the cells were washed three times with PBS, mixed with
RPMI/2% foetal calf serum/30 mM M~Cl2 and incubated for
60 hours at 34C. The medium was suction filtered and
intact cells were stained with crystal violet. Fig. 16
shows that in the presence Oe LDL at a concentration Oe
21170~9
- 36 -
1 mg/ml the infection of HeLa cells by HRV2 is prevented
(all the cells are intact). In the case of HRV14, no
effect was observed (cells fully lysed).
Example 9: Mutations of the HRV2-receptor binding site
Different receptor binding sites of the rhinoviruses of
the "small" and "large rhinovirus receptor group" should
also be reflected in the "canyon structure" of the viral
capsids which is responsible for the interaction with
the corresponding receptor. The term "canyon" is used
to denote the 5-numbered axis of symmetry of the viral
capsid with an extent of roughly 30 ~. One hypothesis
regarding the canyon structure claims that the amino
acid side groups in this region are inaccessible to
immunoglobulins and are therefore not exposed to any
immunological pressure (Rossmann et al. (1985) Nature
317, 145-154). The different rhinoviral serotypes of a
receptor group might in this way conserve structures
which are important for the interaction with the
corresponding receptor and at the same time develop a
broad serotypical diversity (Rossmann (1989) Viral
Immunology 2, 143-161). The hypothesis goes on to say
that differences in the canyon structure between the
"large" and the "small rhinovirus receptor group" are
responsible for the use of two different receptors.
This would contain one set of amino acid groups which is
conserved in specific pOsitiolls of the rhinoviruses of
the "large rhinovirus receptor group" and a second set
which is conserved in specific positions of the
rhinoviruses of the "small rhinovirus receptor group".
Fig. 17 shows a sequence comparison for determining the
positions in or on the edge of the canyon which are
preserved in the rhinoviruses of the small group, by
contrast with the rhinoviruses of the large group. They
21 17099
- 37 -
include the basic groups at position 1081 (HRV2
numbering: Blaas et al. (1987) Proteins 2, 263-272) and
3182, Ile or Leu at position 3229 and the sequence
Thr-Glu-Lys (TEK at position 1222-1224).
The following HRV2 mutants were constructed: at position
1081 (1081K:E) and at 1222-1224 (replacement of TEK by
the corresponding sequence derived from HRV14, HRV39,
HRV89) in the VPl-protein and the mutants 3182R:T and
3229L:T in VP3. Another mutant (1148P:G) was
constructed analogously to HRV14 (1155P:G) (Colonno et
al. (1988) Proc. Natl. Acad. Sci~ U.S.A. 85, 5449-5453).
The preparation of cDNA required for mutagenesis, the in
vitro production of corresponding infectious RNA and the
transfection of corresponding cells have been described
Duechler et al. (1989) Virology 168, 159-161; Maniatis
et al. (1982) "Molecular Cloning: A laboratory manual".
I Cold Spring Harbour Laboratory, Cold Spring Harbour, New
York; Taylor et al. (1989) Nucl. Acids. Res. 13,
8764-8785; Ho et al. (1989) Gene 77, 51-59 and Herlitze
& Koenen (1990) Gene 91, 143-147.
i~ .
The transfection with RNAs which corresponded to the
j Wild-type HRV2 and the mutants HRV2l148pG and HRV23182RT, in
HeLa cells gave yields of about 300 Pfu/ml. The plaque
size and morphology of the Wild type and the two mutants
were identical. Fig. 18 also shows that there was no
significant difference in the binding characteristics of
these two viruses to HeLa cells (HRV2wt ( ), HRV21148pG
( ) and HRV23182RT (_); Neubauer et al. (1987) Virology
158, 255-258). No viable mutants were obtained Erom the
1081K:E, 3229L:T or the mutant with the exchange TEK-
motif at position 1222.
To demonstrate whether the mutants are capable of
binding to the receptor of the small rhinovirus group,
::
21170~
- 38 -
competitive experiments were carried out (Fig. 19). As
soon as increasing quantities of unlabelled HRV2 (-)
were present in the incubation of HeLa cells, the
binding of [35S]-labelled virus material of the mutants
was reduced. The addition of HRV14 (O) had no effect on
the binding. Obviously the affinity of the viruses for
the receptor of the small rhinovirus receptor group is
not affected by the mutations. These data show that the
Pro 148 of VPl (equivalent to Pro 155 in HRV14) is not
involved in the interaction of HRV2 with its receptor.
- Unlike HRV14, which indicates an important function of
this amino acid in the interaction of HRV14 with ICAM-1
(Colonno et al. (1988) Proc. Natl. Acad. Sci. USA 85,
5449-5453).
No conserved oligopeptide sequence corresponding to the
TEK element could be detected in rhinoviruses of the
large group.
mutantS HRV2 1081~:~, HRV23229~r and the TEK mutant were
not viable. Analysis of the three-dimensional structure
of HRVlA - a serotype closely related to ~RV2 - provided
no indications of sterical or electrostatic disorders
caused by the exchanged amino acid side groups. All the ~-
side groups are located on the surface and are
accessible for the solvent. For this reason it is
certainly possible that they are involved in the
interaction with the receptor of the small group and
that their change results in a loss of binding capacity
and infectiousness.
.
The change from Pro114~ to Gly in HRV2 had no effect on
the ability of the virus to bind to its receptor. In
HRV14, a corresponding exchange leads to a substantially
firmer bonding to the receptor. Prollss forms a kind of
base at the foot of the canyon and appears to prevent
the receptor from penetrating further into the viral
~ 2~170~
- 39 -
capsid. The increase in the binding affinity caused by
replacing Pro with Gly can therefore be interpreted as a
reduction in the sterical hindrance. Since no such
effect was observed in HRV2 it is probable that the
virus/receptor interaction occurs at a different place
in rhinoviruses of the small group than that used by the
rhinoviruses of the large group.
~ 21170~9
Cas~ IV140, 141, 149-PCT 40
SEQUENCE LISTING
(1) GENERAL INFORMATION:
S (i) APPLICANT:
(A) NAME: Boehringer Ingelheim
(B) STREET: Binger Strasse
(C) CITY: Ingelheim
(D) STATE: Rheinland-Pfalz
(E) COUNTRY: Deutschland
(F) POSTAL CODE: W-6507
(G) TELEPHONE: 06132-77-0
(H) TEL8FAX: 06132-77-3000
- (I) TELEX: 418791-0 bi d
: 15
(ii) TITLE OF INVENTION: RECEPTOR DERIVATIVES HAVING BINDING SITES
WITH RECEPTOR BINDING SITES
(iii) N~MBER OF SEQUENCES: 5
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/NS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPA)
I (2) INFORMATION ZU SEQ ID NO: 1:
3 30
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 750 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLEC~LE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: :
Met Gly Pro Trp Gly Trp Lyg Leu Arg Trp Thr Val Ala Leu Leu Leu
1 5 10 15
Ala Ala Ala Gly Thr Ala Val Gly Asp Arg Cys Glu Axg Asn Glu Phe
20 25 30
Gln Cys Gln Asp Gly Lys Cys Ile Ser Tyr Lya Trp Val Cy8 Asp Gly
35 40 45
:
Ser Ala Glu Cys Gln A8p Gly Ser Asp Glu Ser Gln Glu Thr Cys Leu ~ :
50 S5 60 ~:
Ser Val Thr Cys Lys Ser Gly Asp Phe Ser Cys Gly Gly Arg Val Asn
65 70 75 80
Arg Cys Ile Pro Gln Phe Trp Arg Cys Asp Gly Gln Val Asp Cys Asp
A8n Gly Ser ABP Glu Gln Gly Cys Pro Pro Lys Thr Cys Ser Gln Asp
100 105 110 --
Glu Phe Arg Cys His Asp Gly Lys Cys Ile Ser Arg Gln Phe Val Cys
115 120 125
: Asp Ser Asp Arg Agp Cy8 Leu Asp Gly Ser Asp Glu Ala Ser Cys Pro
130 135 140
2117~
CaseIV140,141,149-PCT 41
Val Leu Thr Cy8 Gly Pro Ala Ser Phe Gln Cy8 Asn Ser Ser Thr Cys
145 150 155 160
Ile Pro Gln Leu Trp Ala Cy5 Asp Asn A6p Pro Asp Cys Glu Asp Gly
165 170 175
Ser Asp Glu Trp Pro Gln Arg Cy5 Arg Gly Leu Tyr Val Phe Gln Gly
laO 185 190
Asp Ser Ser Pro Cys Ser Ala Phe GlU Phe His Cys Leu Ser Gly Glu
195 200 205
Cys Ile His Ser Ser Trp Arg Cys Agp Gly Gly Pro Asp Cys Lys Asp
lS 210 215 220
Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro Asp Glu
225 230 235 240
Phe Gln Cys Ser Asp Gly Asn Cys Ile His Gly Ser Arg Gln Cys Asp
245 250 255
Arg Glu Tyr A~p Cys Lys Asp Met Ser Asp Glu Val Gly Cys Val Asn
260 265 270
Val Thr Leu Cys Glu Gly Pro Asn Lys Phe Lys Cy~ His Ser Gly Glu
275 280 285
Cys Ile Thr Leu Asp Lys Val Cys Asn Met Ala Arg Asp Cys Arg Asp
290 295 300
Trp Ser A~p Glu Pro Ile Lys Glu Cys Gly Thr Asn Glu Cy8 Leu A6p
305 310 315 320
Asn A~n Gly Gly Cys Ser Hig Val Cy~ Asn Asp Leu Lys Ile Gly Tyr
325 330 335
Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys
340 345 350
Glu Asp Ile Asp Glu Cys Gln Asp Pro Asp Thr Cys Ser Gln Leu Cys
355 360 365
Val Asn Leu Glu Gly Gly Tyr Lys Cys Gln Cys Glu 51u Gly Phe Gln
370 375 380
Leu Asp Pro His Thr Ly8 Ala Cys Lys Ala Val Gly Ser Ile Ala Tyr
385 390 395 400
Leu Phe Phe Thr Asn Arg Hi~ Glu Val Arg Lys Met Thr Leu Asp Arg
405 410 415
Ser Glu Tyr Thr Ser Leu Ile Pro Asn Leu Arg Asn Val Val- Ala Leu
420 425 430
Asp Thr Glu Val Ala Ser Asn Arg Ile Tyr Trp Ser Asp Leu Ser Gln
435 440 445
Arg Met Ile Cys Ser Thr Gln Leu Asp Arg Ala His Gly Val Ser Ser
450 455 460
Tyr Asp Thr Val Ile Ser Arg Asp Ile Gln Ala Pro Asp Gly Leu Ala
465 470 475 480
Val Asp Trp Ile His Ser Asn Ile Tyr Trp Thr Asp Ser Val Leu Gly
485 490 495
211709~
Casc IV140,141,149-PCT 42
Thr Val Ser Val Ala Asp Thr Lys Gly Val Lys Arg Lys Thr Leu Phe
500 505 510
Arg Glu Agn Gly Ser Lys Pro Arg Ala Ile Val Val AEP Pro Val His
515 520 525
Gly Phe Met Tyr Trp Thr Asp Trp Gly Thr Pro Ala Lys Ile Lys Lys
530 535 540
Gly Gly Leu Asn Gly Val Asp Ile Tyr Ser Leu Val Thr Glu Asn Ile
545 550 555 560
Gln Trp Pro Asn Gly Ile Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr
565 570 575
Trp Val Asp Ser Lys Leu Hig Ser Ile Ser Ser Ile Asp Val Asn Gly
580 585 590
Gly Asn Arg Lys Thr Ile Leu Glu Asp Glu Lys Arg Leu Ala His Pro
595 600 605
Phe Ser Leu Ala Val Phe Glu A~p Lys Val Phe Trp Thr A&p Ile Ile
610 615 620
Asn Glu Ala Ile Phe Ser Ala Agn Arg Leu Thr Gly Ser Asp Val Asn
625 630 635 640
Leu Leu Ala Glu A8n Leu Leu Ser Pro Glu Asp Met Val Leu Phe His
645 650 655
Asn Leu Thr Gln Pro Arg Gly Val Agn Trp Cys Glu Arg Thr Thr Leu
1 660 665 670 :.
! 35 Ser Asn Gly Gly Cys Gln Tyr Leu Cys Leu Pro Ala Pro Gln Ile Asn - ~-
675 680 685
Pro Hi8 Ser Pro Ly8 Phe Thr Cya Ala Cys Pro Asp Gly Net Leu Leu
1 690 695 700 ; -~
¦ Ala Arg Asp Met Arg Ser Cys Leu Thr Glu Ala Glu Ala.Ala Val Ala
' 705 710 715 720
Thr Gln Glu Thr Ser Thr Val Arg Leu Lys Val Ser Ser Thr Ala Val
725 730 735
Arg Thr Gln His Thr Thr Thr Arg Pro Val Pro Asp Thr Ser
740 7~5 750
(2) INFORMATION ZU SEQ ID NO: 2: -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 322 amino acids
(B) TYPE: amino acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) NOLEC~LE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
¦ Met Gly Pro Trp Gly Trp Lys Leu Arg Trp Thr Val Ala Leu Leu Leu
~ 1 5 10 15
.- 2ll7n~.~
Caso IV140,141,149-PCI 43
Ala Ala Ala Gly Thr Ala Val Gly Asp Arg Cy8 Glu Arg Asn Glu Phe
Gln Cys Gln Asp Gly Lys Cys Ile Ser Tyr Lys Trp Val Cy8 Aap Gly
35 40 45
Ser Ala Glu Cys Gln Asp Gly Ser Asp Glu Ser Gln Glu Thr Cys Leu
50 55 60
Ser Val Thr Cys Lys Ser Gly Agp Phe Ser Cys Gly Gly Arg Val Asn
65 70 75 80
Arg Cys Ile Pro Gln Phe Trp Arg Cys Asp Gly Gln Val Asp Cys Asp
85 90 95
Asn Gly Ser Asp G1U Gln Gly Cys Pro Pro Lys Thr Cys Ser Gln Asp
100 105 110
Glu Phe Arg Cys His Asp Gly Lys Cys Ile Ser Arg Gln Phe Val Cys
115 120 125
Asp Ser Asp Arg Asp Cys Leu Asp Gly Ser Asp Glu Ala Ser Cys Pro
130 135 140
Val Leu Thr Cys Gly Pro Ala Ser Phe Gln Cys Asn Ser Ser Thr Cys
145 150 155 160
Ile Pro Gln Leu Trp Ala Cys Asp Asn Asp Pro Asp Cys Glu Asp Gly
165 170 175
Ser Asp Glu Trp Pro Gln Arg cyg Arg Gly Leu Tyr Val Phe Gln Gly
180 185 190
Asp Ser Ser Pro Cys Ser Ala Phe Glu Phe His Cys Leu Ser Gly Glu
195 200 205
Cys Ile His Ser Ser Trp Arg Cys Asp Gly Gly Pro Asp Cy5 Lys Asp
210 215 220
Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro Asp Glu
225 230 235 240
Phe Gln Cys Ser Asp Gly Asn Cys Ile His Gly Ser Arg Gln Cys Asp
245 250 255
Arg Glu Tyr Asp Cys Lys Asp DIet Ser Asp Glu Val Gly Cys Val Asn
260 265 270
Val Thr Leu Cys Glu Gly Pro Asn Lys Phe Lys Cys ~lis Ser Gly Glu
275 280 285
Cys Ile Thr Leu Asp Lys Val Cy8 Asn Met Ala Arg Asp Cys. Arg Asp
290 295 300
Trp Ser Asp Glu Pro Ile Lys Glu Cys Gly Thr Asn Glu Cys Leu Asp
305 310 315 320
Asn Asn
65 (2) INFORMATION ZU SEQ ID NO: 3:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 860 amino acids
~` ~117~9
Cl~se 12/140,141,149-K~ 44
(B) TYPE: amino ac~id
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear :
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTIO~: SEQ ID NO: 3:
Met Gly Pro Trp Gly Trp Lys Leu Arg Trp Thr Val Ala Leu Leu Leu
5 10 15
Ala Ala Ala Gly Thr Ala Val Gly Asp Arg Cys Glu Arg Asn Glu Phe
20 25 30
Gln Cys Gln Asp Gly Lys Cys Ile Ser Tyr Lys Trp Val Cys Asp Gly
35 40 45 . :
20 Ser Ala Glu Cys Gln Asp Gly Ser Asp Glu Ser Gln Glu Thr Cys Leu
50 55 60
Ser Val Thr Cy8 Lys Ser Gly Asp Phe Ser Cy8 Gly Gly Arg Val Asn
Arg Cys Ile Pro Gln Phe Trp Arg Cys Asp Gly Gln Val Asp Cys Asp
85 90 95
Asn Gly Ser Asp Glu Gln Gly Cys Pro Pro Lys Thr Cys Ser Gln Asp
100 105 110
Glu Phe Arg Cys His Asp Gly Lys Cys Ile Ser Arg Gln Phe Val Cys
115 120 125
35 Asp Ser ABP Arg Asp Cys Leu Asp Gly Ser Agp Glu Ala Ser Cys Pro
130 135 140
Val Leu Thr Cys Gly Pro Ala Ser Phe Gln Cys Asn Ser Ser Thr Cys
145 150 155 1~0
Ile Pro Gln Leu Trp Ala Cyg A~p Asn Asp Pro A~3p Cys Glu Asp Gly
165 170 175 ~;
Ser Asp Glu Trp Pro Gln Arg Cys Arg Gly Leu Tyr Val Phe Gln Gly : -
180 185 190
Asp Ser Ser Pro Cy8 Ser Ala Phe Glu Phe His Cys Leu Ser Gly Glu
195 200 205
50 Cys Ile His Ser Ser Trp Arg Cys Asp Gly Gly Pro Asp Cys Lys Asp
210 215 220
Lys Ser ABP Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro- Asp Glu
225 230 235 240
Phe Gln Cys Ser Asp Gly Asn Cys Ile His Gly Ser Arg Gln Cys Asp
245 250 255 :
Arg Glu Tyr Asp Cys Lys Asp Met Ser A p Glu Val Gly cyg Val Asn
260 265 270
Val Thr Leu Cyg Glu Gly Pro Asn Lys Phe Lys Cys His Ser Gly Glu
275 280 285
65 Cys Ile Thr Leu Asp Lys Val Cys Asn Met Ala Arg A6p cyg Arg Asp
290 295 300
'~
-
~: ::::::
- 21170.~9
Ca~e 12/140,141,149-PCT 45
Trp Ser Asp Glu Pro Ile Lys Glu Cy8 Gly Thr Asn Glu Cys Leu Asp
305 310 315 320
5 Asn Asn Gly Gly Cy8 Ser His Val Cys Asn Asp Leu Lys Ile Gly Tyr
325 330 335
Glu Cy8 Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys
340 345 350
, 10 :: :
Glu Asp Ile Asp Glu Cy8 Gln Asp Pro Asp Thr Cys Ser Gln Leu Cys
355 360 365
Val Asn Leu Glu Gly Gly Tyr Lyg Cys Gln Cys Glu Glu Gly Phe Gln
370 375 380
Leu Asp Pro His Thr Lys Ala Cys Lys Ala Val Gly Ser Ile Ala Tyr
385 390 395 400 ::
' 20
Leu Phe Phe Thr Asn Arg His Glu Val Arg Lys Met mr Leu Asp Arg
405 410 415
25 Ser Glu Tyr Thr Ser Leu Ile Pro Asn Leu Arg Asn Val Val Ala Leu
, 420 425 430
Asp Thr Glu Val Ala Ser A8n Arg Ile ~yr Trp Ser Asp Leu Ser Gln ::
435 440 445
~¦ Arg Met Ile Cys Ser Thr Gln Leu Asp Arg Ala His Gly Val Ser Ser
450 455 460
Tyr Asp Thr Val Ile Ser Arg Asp Ile Gln Ala Pro Asp Gly Leu Ala
35 465 470 475 480
Val Asp Trp Ile Hig Ser Asn Ile Tyr Trp Thr Asp Ser Val Leu Gly
485 490 495
~¦ 40 Thr Val Ser Val Ala A8p Thr 1,y8 Gly Val l.ys Arg Lys Thr Leu Phe
500 505 510
Arg Glu Asn Gly Ser Lys Pro brg Ala Ile Val Val Asp Pro Val His
515 520 525
. 45
Gly Phe Met Tyr Trp Thr A6p Trp Gly Thr Pro Ala Ly8 Ile Ly6 Ly5
530 535 540
Gly Gly Leu Asn Gly Val Asp Ile Tyr Ser Leu Val Thr Glu Asn Ile
50 545 550 SSS 560
Gln Trp Pro Asn Gly Ile Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr
: 565 570 575
. 55 Trp Val A8p Ser Lys Leu His Ser Ile Ser Ser Ile A8p Val A8n Gly
, 580 5a5 590
Gly Asn Arg Lys mr Ile Leu Glu Asp Glu Lys Arg Leu Ala His Pro
595 600 605
Phe Ser Leu Ala Val Phe Glu A6p Ly8 Val Phe Trp Thr Asp Ile Ile
610 615 620
Asn Glu Ala Ile Phe Ser Ala Asn Arg Leu Thr Gly Ser Asp Val Asn
65 625 630 635 640
Leu Leu Ala Glu Asn Leu Leu Ser Pro Glu Asp Me~ Val Leu Phe His
645 650 655 ;.
:
. '
, , ,, ~ ~.. ,... , . . , ,~, , , j. .. . .. ..
~ 2117099
12/140,141,149-PCI' 46
Asn Leu Thr Gln Pro Arg Gly Val Asn Trp Cys Glu Arg Thr Thr Leu
660 ~ 665 670 - :
Ser Asn Gly Gly Cys Gln Tyr Leu Cys Leu Pro Ala Pro Gln Ile Asn
675 680 685
Pro His Ser Pro Lys Phe Thr CYB Ala Cy6 Pro A~p Gly Met Leu Leu
690 695 700
Ala AIg Asp Met Arg Ser Cys Leu Thr Glu Ala Glu Ala Ala Val Ala
705 710 715 720
Thr Gln Glu Thr Ser Thr Val Arg Leu Ly~ Val Ser Ser Thr Ala Val
725 730 735
Arg Thr Gln His Thr Thr Thr Arg Pro Val Pro Asp Thr Ser Arg Leu
740 745 750
Pro Gly Ala Thr Pro Gly Leu Thr Thr Val Glu Ile Val Thr Met Ser
755 760 765
His Gln Ala Leu Gly Asp Val Ala Gly Arg Gly Asn Glu Lys Lys Pro
770 775 780
Ser Ser Val Arg Ala Leu Ser Ile Val Leu Pro Ile Val Leu Leu Val
785 790 795 800 ~:
,Phe Leu Cys Leu Gly Val Phe Leu Leu Trp Lys Asn Trp Arg Leu Lys
805 810 815
Asn Ile A~n Ser Ile Asn Phe Asp A6n Pro Val Tyr Gln Lys Thr Thr
820 825 830
Glu Asp Glu Val His Ile Cy6 His A6n Gln Asp Gly Tyr Ser Tyr Pro
835 840 845
Ser Arg Gln Met Val Ser Leu Glu Asp Asp Val Ala
850 855 860
(2) INFORMATION ZU SEQ ID NO: 4~
(i) SEQUENCE CHARACTERISTICS: - :
(A) LENGTH: 4492 amino acid6
(B) TYPE: amino acid
(C) STRANDEDNESS: ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide . ~:
: ::
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Leu Thr Pro Pro Leu Leu Leu Leu Leu Pro Leu Leu Ser Ala Leu
1 5 10 15
Val Ala Ala Ala Ile Asp Ala Pro Lys Thr Cys Ser Pro Lys Gln Phe
20 25 30 .: ~ :
Ala Cys Arg Asp Gln Ile Thr Cyg Ile Ser Lys Gly Trp Arg Cy6 Asp
r~
2117~
C~e IV140.141,149-PCr 47
.
Gly Glu Arg Asp CYE; Pro Asp Gly Ser Asp Glu Ala Pro Glu Ile Cys
Pro Gln Ser Lys Ala Gln Arg Cys Gln Pro Asn Glu His A~n Cy5 Leu
65 70 75 80
Gly Thr Glu Leu Cys Val Pro Met Ser Arg Leu Cys Asn Gly Val Gln
85 90 95
Asp Cys Met Asp Gly Ser Asp Glu Gly Pro His Cy8 Arg Glu Leu Gln
100 105 110
Gly Asn Cys Ser Arg Leu Gly Cy8 Gln His His Cys Val Pro Thr Leu
115 120 125
Asp Gly Pro Thr Cys Tyr Cy8 Asn Ser Ser Phe Gln Leu Gln Ala Asp
130 135 140
Gly Lys Thr Cy8 Lys Asp Phe Asp Glu Cys Ser Val Tyr Gly Thr Cys
145 150 155 160
Ser Gln Leu Cys Thr Asn Thr Asp Gly Ser Phe Ile Cys Gly Cys Val
165 170 175
Glu Gly Tyr Leu Leu Gln Pro Asp Aan Arg Ser Cys Lys Ala Lys Asn
180 185 190
Glu Pro Val Asp Arg Pro Pro Val Leu Leu Ile Ala Asn Ser Gln Asn
195 200 205
Ile Leu Met Pro Gly Leu Lys Gly Phe Val A~p Glu His Thr Ile Asn
210 215 220
Ile Ser I:-eu Ser Leu His His Val Glu Gln ~et Ala Ile Asp Trp Leu
225 230 235 240
Thr Gly Asn Phe Tyr Phe Val A8p A8p Ile Asp A8p Arg Ile Phe Val
245 250 255
Cys Asn Arg A~n Gly Asp Thr CYB Val Thr Leu Leu Asp Leu Glu Leu
260 265 270
Tyr Asn Pro Lys Gly Ile Ala Leu Asp Pro Ala Met Gly Lys Val Phe
275 280 285
Phe Thr Asp Tyr Gly Gln Ile Pro Lys Val Glu Arg Cys Asp Met Asp
290 295 300
Gly Gln A8n Arg Thr Lys Leu Val Asp Ser Lys Ile Val Phe Pro His
305 310 315 320
Gly Ile Thr Leu Asp Leu Val Ser Arg Leu Val Tyr Trp Ala Asp Ala
325 330 335
Tyr Leu Asp Tyr Ile Glu Val Val A~p Tyr Glu Gly Lys Gly Arg Gln
340 345 350
Thr Ile Ile Gln Gly Ile Leu Ile Glu His Leu Tyr Gly Leu Thr Val
355 360 365
Phe Glu A8n Tyr Leu Tyr Ala Thr Asn Ser A8p Asn Ala Asn Ala Gln
370 375 380
Cln Lys Thr Ser Val Ile Arg Val Asn Arg Phe Asn Ser Thr Glu Tyr
385 390 395 400
2117(1~
Ca~e Ivl40,141,149-PCr 48
Gln Val Val Thr Arg Val Asp Lys Gly Gly Ala Leu Hia Ile Tyr His
405 410 415
Gln Arg Arg Gln Pro Arg Val Arg Ser Hia Ala Cys Glu Asn Asp Gln
420 425 430
Tyr Gly Ly8 Pro Gly Gly Cys Ser Asp Ile Cys Leu Leu Ala Asn Ser
435 440 445
His Lys Ala Arg Thr Cys Arg Cys Arg Ser Gly Phe Ser Leu Gly Ser
450 455 460
Asp Gly Lys Ser Cys Lys LYB Pro Glu His Glu Leu Ph~ Leu Val Tyr
465 470 475 ~80
Gly Lys Gly Arg Pro Gly Ile Ile Arg Gly Met Asp Met Gly Ala Lys
485 490 495
Val Pro Agp Glu His Met Ile Pro Ile Glu Asn Leu Net Asn Pro Arg
500 505 510
Ala Leu Asp Phe His Ala Glu Thr Gly Phe Ile Tyr Phe Ala Asp Thr
515 520 525
Thr Ser Tyr Leu Ile Gly Arg Gln Lys Ile Asp Gly Thr Glu Arg Glu
530 535 540
Thr Ile Leu Lys Asp Gly Ile His Agn Val Glu Gly Val Ala Val Asp :: ~
545 550 555 560 ~ ::
Trp Met Gly Asp Asn Leu Tyr Trp Thr Asp Asp Gly Pro Lys Lys Thr
565 570 575
: -
Ile Ser Val Ala Arg Leu Glu Lys Ala Ala Gln Thr Arg Lys Thr Leu :~
580 5a5 590 .. ::
Ile Glu Gly Lys Met Thr His Pro Arg Ala Ile Val Val Asp Pro Leu ;~
595 600 605 ~:~
Asn Gly Trp Met Tyr Trp Thr Asp Trp Glu Glu Asp Pro Lys Asp Ser
610 615 620 ~ :
Arg Arg Gly Arg Leu Glu Arg Ala Trp Met Asp Gly Ser His Arg Asp p
625 630 635 640
Ile Phe Val Thr Ser Lys Thr Val Leu Trp Pro Asn Gly Leu Ser Leu ~ -~
645 650 655
ABP Ile Pro Ala Gly Arg Leu Tyr Trp Val Asp Ala Phe Tyr Asp Arg .
660 665 670
Ile Glu Thr Ile Leu Leu Asn Gly Thr Asp Arg Lys Ile Val Tyr Glu
675 680 685 :.
Gly Pro Glu Leu Asn Hi8 Ala Phe Gly Leu Cys His His Gly Asn Tyr
690 695 700
Leu Phe Trp Thr Glu Tyr Arg Ser Gly Ser Val Tyr Arg Leu Glu Arg .:
705 710 715 720 -
Gly Val Gly Gly Ala Pro Pro Thr Val Thr Leu Leu Arg Ser Glu Arg
725 730 735 :
Pro Pro Ile Phe Glu Ile Arg Met Tyr Asp Ala Gln Gln Gln Gln Val
740 745 750
:~
: :
2117099
ca~e IVI40 141 149-PCl-
Gly Thr Asn Lys Cy5 Arg Val Asn Asn Gly Gly Cy9 Ser Ser Leu Cy5
7S5 760 765
Leu Ala Thr Pro Gly Ser Arg Gln Cy8 Ala Cys Ala Glu Asp Gln Val
770 775 780
Leu Asp Ala Asp Gly Val Thr Cys Leu Ala Asn Pro Ser Tyr Val Pro
785 790 795 800
Pro Pro Gln Cys Gln Pro Gly Glu Phe Ala cy5 Ala Asn Ser Arg Cy8
805 810 815
Ile Gln Glu Arg Trp Lys Cys A~p Gly Asp Asn Asp Cys Leu Asp Asn
820 825 830
Ser A~p Glu Ala Pro Ala Leu Cys His Gln His Thr Cys Pro Ser Asp
835 840 845
Arg Phe Lys Cy8 Glu Asn Asn Arg Cys Ile Pro Asn Arg Trp Leu Cys
850 855 860 :'
Asp Gly Asp Asn Asp Cys Gly AF:n Ser Glu Asp Glu Ser Asn Ala Thr
865 870 875 880
~.
Cys Ser Ala Arg Thr Cys Pro Pro Asn Gln Phe Ser Cys Ala Ser Gly
885 890 895 .
Arg Cys Ile Pro Ile Ser Trp Thr Cys Asp Leu Asp Asp Asp Cys Gly
900 905 910
Asp Arg Ser Asp Glu Ser Ala Ser Cys Ala Tyr Pro Thr Cys Phe Pro
915 920 925
! 35
Leu Thr Gln Phe Thr Cys Asn Asn Gly Arg Cys Ile Asn Ile Asn Trp
930 935 940
Arg Cys Asp Asn Asp Asn Asp Cys Gly Asp Asn Ser Asp Glu Ala Gly
945 950 955 960
Cys Ser His Ser Cy9 Ser Ser Thr Gln Phe Lys Cys Asn Ser Gly Arg
965 970 975
Cys Ile Pro Glu His Trp Thr Cys Asp Gly ~sp Asn Asp Cys Gly Asp
980 985 990
Tyr Ser Asp Glu Thr His Ala Asn Cys Thr Asn Gln Ala Thr Arg Pro
995 1000 1005
Pro Gly Gly Cys His Thr Asp Glu Phe Gln Cys Arg Leu Asp Gly Leu
1010 1015 1020
Cys Ile Pro Leu Arg Trp Arg Cys Asp Gly Asp Thr Agp Cys Net Asp
1025 1030 1035 1040
Ser Ser Asp Glu Lys Ser Cy9 Glu Gly Val Thr His Val Cys Asp Pro
1045 1050 lOS5
Ser Val Lys Phe Gly Cys Lys Asp Ser Ala Arg Cys Ile Ser Lys Ala
1060 1065 1070
Trp Val Cys Asp Gly Asp Asn Asp Cys Glu Asp Asn Ser Asp Glu Glu
. 1075 1080 1085
Asn Cys Glu Ser Leu Ala Cys Arg Pro Pro Ser His Pro Cys Ala Asn
1090 1095 1100
f' 21~709~
Caso IV140,141,149-PC~ 50
Asn Thr Ser Val Cy8 Leu Pro Pro AE:p Lys Leu Cys A3p Gly Asn Asp
1105 1110 1115 1120
Asp Cys Gly Asp Gly Ser Asp Glu Gly Glu Leu Cys Asp Gln Cy8 Ser
1125 1130 1135
Leu Asn Agn Gly Gly Cy~ Ser His Asn Cys Ser Val Ala Pro Gly Glu
1140 1145 1150
Gly Ile Val Cys Ser Cys Pro Leu Gly Met Glu Leu Gly Pro Asp Asn
1155 1160 1165
His Thr Cys Gln Ile Gln Ser Tyr Cys Ala Lys His Leu Lys Cy8 Ser
1170 1175 1180
Gln LYB Cys Asp Gln Asn Lys Phe Ser Val Lys Cys Ser Cy8 Tyr Glu : ~:
1185 1190 1195 1200 :
Gly Trp Val Leu Glu Pro Asp Gly Glu Ser Cys Arg Ser Leu Asp Pro
1205 1210 1215 -.;
Phe Lys Pro Phe Ile Ile Phe Ser Asn Arg His Glu Ile Arg Arg Ile
1220 1225 1230
Asp Leu His Lys Gly Asp Tyr Ser Val Leu Val Pro Gly Leu Arg Asn
1235 1240 1245
Thr Ile Ala Leu Asp Phe His Leu Ser Gln Ser Ala Leu Tyr Trp Thr
1250 1255 1260
Asp Val Val Glu Asp Lys Ile Tyr Arg Gly Lys Leu Leu Asp Asn Gly
1265 1270 1275 1280 : ~:
Ala Leu Thr Ser Phe Glu Val Val Ile Gln Tyr Gly Leu Ala Thr Pro
1285 1290 1295
;:
Glu Gly Leu Ala Val Asp Trp Ile Ala Gly Asn Ile Tyr Trp Val Glu
1300 1305 1310 ~ :.
Ser Asn Leu Asp Gln Ile Glu Val Ala Lys Leu Asp Gly Thr Leu Arg
1315 1320 1325
Thr Thr Leu Leu Ala Gly Asp Ile Glu His Pro Arg Ala Ile Ala Leu
1330 1335 1340 :
Aap Pro Arg A8p Gly Ile Leu Phe Trp Thr A~p Trp Aap Ala Ser Leu .,
1345 1350 1355 1360
Pro Arg Ile Glu Ala Ala Ser Met Ser Gly Ala Gly Arg Arg Thr Val
1365 1370 1375 ~:
His Arg Glu Thr Gly Ser Gly Gly Trp Pro Aan Gly Leu Thr Val Asp
1380 1385 1390
Tyr Leu Glu Lys Arg Ile Leu Trp Ile Asp Ala Arg Ser Asp Ala Ile
1395 1400 1405
Tyr Ser Ala Arg Tyr Asp Gly Ser Gly His Met Glu Val Leu Arg Gly
1410 1415 1420
His Glu Phe Leu Ser Hia Pro Phe Ala Val Thr Leu Tyr Gly Gly Glu
1425 1430 1435 1~40
Val Tyr Trp Thr Asp Trp P,rg Thr Agn Thr Leu Ala Lys Ala Agn Lys
1445 1450 1455
~ 211709~
C~o 121140,141.149-PCr 5 l
Trp Thr Gly His A~n Val Thr Val Val Gln Arg Thr Asn Thr Gln Pro
lg60 1465 1470
Phe Asp Leu Gln Val Tyr His Pro Ser Arg Gln Pro Met Ala 2ro Asn
1475 1480 1485
Pro Cys Glu Ala Asn Gly Gly Gln Gly Pro Cy~ Ser His Leu Cy8 Leu
1490 1495 1500
Ile Asn Tyr Asn Arg Thr Val Ser Cys Ala Cy5 Pro His Leu Met Lys
1505 1510 1515 1520
Leu Hi~ Lys Asp Asn Thr Thr Cys Tyr Glu Phe Lys Ly8 Phe Leu Leu
1525 1530 1535
Tyr Ala Arg Gln Met Glu Ile Arg Gly Val Asp Leu Asp Ala Pro Tyr
1540 1545 1550
Tyr Asn Tyr Ile Ile Ser Phe Thr Val Pro Asp Ile A~p Asn Val Thr
1555 1560 1565 .
Val Leu Agp Tyr Asp Ala Arg Glu Gln Arg Val Tyr Trp Ser Asp Val
1570 1575 1580
Arg Thr Gln Ala Ile Lys Arg Ala Phe Ile Asn Gly Thr Gly Val Glu
1585 1590 1595 1600
Thr Val Val Ser Ala Asp Leu Pro Asn Ala His Gly Leu Ala Val Asp
1605 1610 1615
Trp Val Ser Arg Asn Leu Phe Trp Thr Ser Tyr Asp Thr Asn Lys Lys
1620 1625 1630
Gln Ile Aan Val Ala Arg Leu Asp Gly Ser Phe Lys Asn Ala Val Val
1635 1640 1645
Gln Gly Leu Glu Gln Pro His Gly Leu Val Val His Pro Leu Arg Gly
1650 1655 1660
Lys Leu Tyr Trp Thr Asp Gly Asp Aan Ile Ser Met Ala Asn Met Asp
1665 1670 1675 1680
Gly Ser Asn Arg Thr Leu Leu Phe Ser Gly Gln Lys Gly Pro Val Gly
1685 1690 1695
Leu Ala Ile Asp Phe Pro Glu Ser Lys Leu Tyr Trp Ile Ser Ser Gly
1700 1705 1710
Asn Hi8 Thr Ile Asn Arg Cys Asn Leu Asp Gly Ser Gly Leu Glu Val
1715 1720 1725
Ile Asp Ala Met Arg Ser Gln Leu Gly Lys Ala Thr Ala Leu Ala Ile
1730 1735 1740
Met Gly Asp Lys Leu Trp Trp Ala Asp Gln Val Ser Glu Lys Met Gly
1745 1750 1755 1760
Thr Cys Ser Lys Ala Asp Gly Ser Gly Ser Val Val Leu Arg Asn Ser
1765 1770 1775
Thr Thr Leu Val Met His Met Lys Val Tyr Asp Glu Ser ~le Gln Leu
1780 1785 1790
Asp His Lys Gly Thr Agn Pro Cyg Ser Val Asn Asn Gly Asp Cys Ser
1795 1800 1805
,~ 211709~
C~se IV140,141,149-PCI` 52
Gln Leu Cy8 Leu Pro Thr Ser Glu Thr Thr Arg Ser Cyæ Met Cys Thr ~:
1810 1815 1820 :
Ala Gly Tyr Ser Leu Arg Ser Gly Gln Gln Ala Cya Glu Gly Val Gly
1825 1830 la35 1840
Ser Phe Leu Leu Tyr Ser Val ~i8 Glu Gly Ile Arg Gly Ile Pro Leu ~:
1845 1850 1855 :
Asp Pro Asn Asp Lys Ser Aap Ala Leu Val Pro Val Ser Gly Thr Ser .
1860 1865 1870
Leu Ala Val Gly Ile Asp Phe His Ala Glu Asn ABP Thr Ile Tyr Trp
1875 1880 1885 . :
Val Asp Met Gly Leu Ser Thr Ile Ser Arg Ala Lys Arg Asp Gln Thr .:
1890 la95 1900
: .
Trp Arg Glu Asp Val Val Thr A8n Gly Ile Gly Arg Val Glu Gly Ile
1905 1910 1915 1920
Ala Val Asp Trp Ile Ala Gly A6n Ile Tyr Trp Thr Asp Gln Gly Phe ~:
1925 1930 1935 ~ :
::
Asp Val Ile Val Ala Arg Leu A8n Gly Ser Phe Arg Tyr Val Val Ile : :
1940 1945 1950 ~.
Ser Gln Gly Leu Asp Lys Pro Arg Ala Ile Thr Val His Pro Glu Lys
1955 1960 1965 .
Gly Tyr Leu Phe Trp Thr Glu Trp Gly Gln Tyr Pro Arg Ile Glu Arg ~ :
1970 1975 1980
Ser Arg Leu Asp Gly Thr Glu Arg Val Val Leu Val A~n Val Ser Ile
1985 1990 1995 2000 - :
Ser Trp Pro Asn Gly Ile Ser Val Asp Tyr Gln ABP Gly Lys Leu Tyr
2005 2010 2015
Trp Cys Asp Ala Arg Thr Asp Lys Ile Glu Arg Ile Asp Leu Glu Thr
2020 2025 2030
Gly Glu Asn Arg Glu Val Val Leu Ser Ser Asn Asn Met Asp Met Phe
2035 2040 2045 ~:
Ser Val Ser Val Phe Glu Asp Phe Ile Tyr Trp Ser Asp Arg Thr His
2050 2055 2060
Ala A8n Gly Ser Ile Ly8 Arg Gly Ser Lys ABP Asn Ala Thr A8p Ser
2065 2070 2075 2080 . :
Val Pro Leu Arg Thr Gly Ile Gly Val Gln Leu Lys Asp Ile Lys Val
2085 2090 2095
Phe Asn Arg Asp Arg Gln Lys Gly Thr Agn Val Cys Ala Val Ala Asn
2100 2105 2110
Gly Gly Cys Gln Gln Leu Cys Leu Tyr Arg Gly Arg Gly Gln Arg Ala
2115 2120 2125
CYB Ala Cys Ala His Gly Net Leu Ala Glu Asp Gly Ala Ser Cys Arg
2130 2135 2140
Glu Tyr Ala Gly Tyr Leu Leu Tyr Ser Glu Arg Thr Ile Leu Lys Ser
2145 2150 2155 2160
~ 21170~ -
C~e 12/140,141,149-P~r 53
Ile His Leu Ser Asp Glu Arg Asn Leu Asn Ala Pro Val Gln Pro Phe
2165 2170 2175
Glu Asp Pro Glu His Met Lys Asn Val Ile Ala Leu Ala Phe Asp Tyr
2180 2185 2190
Arg Ala Gly Thr Ser Pro Gly Thr Pro Asn Arg Ile Phe Phe Ser Asp
2195 2200 2205
Ile His Phe Gly Asn Ile Gln Gln Ile Agn Asp Asp Gly Ser Arg Arg
2210 2215 2220
Ile Thr Ile Val Glu Aan Val Gly Ser Val Glu Gly Leu Ala Tyr His
2225 2230 2235 2240
', Arg Gly Trp Asp Thr Leu Tyr Trp Thr Ser Tyr Thr Thr Ser Thr Ile
2245 2250 2255
Thr Arg Hia Thr Val Asp Gln Thr Arg Pro Gly Ala Phe Glu Arg Glu
2260 2265 2270
I
Thr Val Ile Thr Met Ser Gly Asp Asp His Pro Arg Ala Phe Val Leu
2275 2280 2285
Asp Glu Cys Gln Asn Leu Met Phe Trp Thr Asn Trp Asn Glu Gln Hi s
2290 2295 2300
Pro Ser Ile Met Arg Ala Ala Leu Ser Gly Ala Aan Val Leu Thr Leu
2305 2310 2315 2320
Ile Glu Lys Asp Ile Arg Thr Pro Asn Gly Leu Ala Ile Asp His Arg
2325 2330 2335
Ala Glu Lys Leu Tyr Phe Ser Asp Ala Thr Leu Asp Lys Ile Glu Arg
2340 2345 2350
Cys Glu Tyr Asp Gly Ser His Arg Tyr Val Ile Leu Lys Ser Glu Pro
2355 2360 2365
Val His Pro Phe Gly Leu Ala Val Tyr Gly Glu His Ile Phe Trp Thr
2370 2375 2380
Asp Trp Val Arg Arg Ala Val Gln Arg Ala Asn Lys His Val Gly Ser
2385 2390 2395 2400
Asn Met Ly~ Leu Leu Arg Val Asp Ile Pro Gln Gln Pro Met Gly Ile
2405 2410 2415
Ile Ala Val Ala Asn Asp Thr Asn Ser Cys Glu Leu Ser Pro Cys Arg
2420 2425 2430
Ile Asn Asn Gly Gly Cy8 Gln Asp Leu Cys Leu Leu Thr His Gln Gly
2435 2440 2445
His Val Asn Cys Ser Cys Arg Gly Gly Arg Ile Leu Gln Asp Asp Leu
2450 2455 2460
Thr Cys Arg Ala Val Asn Ser Ser Cys Arg Ala Gln Asp Glu Phe Glu
2465 2470 2475 2480
Cys Ala Asn Gly Glu Cy8 Ile Asn Phe Ser Leu Thr Cys Asp Gly Val
2485 2490 2495
Pro His Cya Lys Asp Lys Ser Agp Glu Lys Pro Ser Tyr Cyg Agn Ser
2500 2505 2510
~ 211709~
Case IV140,141.149-PCT 54
Arg Arg Cy8 Ly8 Lys Thr Phe Arg Gln Cy5 Ser Asn Gly Arg Cys Val
2515 2520 2525
Ser Asn Met Leu Trp Cys Asn Gly Ala Asp Asp Cys Gly Asp Gly Ser
2530 2535 2540
Asp Glu Ile Pro Cyg A~n Ly8 Thr Ala Cys Gly Val Gly Glu Phe Arg
2545 2550 2555 2560
Cys Arg Asp Gly Thr Cy8 Ile Gly Asn Ser Ser Arg Cys Asn Gln Phe ~ ~
2565 2S70 2575 ~: :
Val Asp Cys Glu Agp Ala Ser Asp Glu Met Asn Cyg Ser Ala Thr Asp ::
2580 2585 2590 :
Cys Ser Ser Tyr Phe Arg Leu Gly Val Lys Gly Val Leu Phe Gln Pro
2595 2600 2605
Cys Glu Arg Thr Ser Leu Cys l'yr Ala Pro Ser Trp Val Cys Asp Gly
2610 2615 2620 ~ :
Ala Asn Asp Cys Gly Asp Tyr Ser Asp Glu Arg Asp Cys Pro Gly Val
2625 2630 2635 2640
Lys Arg Pro Arg Cys Pro Leu Asn Tyr Phe Ala Cys Pro Ser Gly Arg
2645 2650 2655
Cys Ile Pro Net Ser Trp ~r Cys Asp Lys Glu Asp Asp Cys Glu His
2660 2665 2670 ~ -
Gly Glu ABP Glu Thr Hi5 Cya Asn Lys Phe Cys Ser Glu Ala Gln Phe ~ -
2675 2680 2685
Glu Cys Gln Asn His Arg Cys Ile Ser Lys Gln Trp Leu Cys Asp Gly
2~90 2695 2700
Ser Asp Asp Cys Gly Asp Gly Ser Asp Glu Ala Ala His Cys Glu Gly
2705 2710 2715 2720
Lys Thr Cy8 Gly Pro Ser Ser Phe Ser Cys Pro Gly Thr His Val Cys
2725 2730 2735
Val Pro Glu Arg Trp Leu Cys Asp Gly Asp Lys Asp Cys Ala Aæp Gly
2740 2745 2750
Ala Asp Glu Ser Ile Ala Ala Gly Cys Leu Tyr Asn Ser Thr Cys Asp
2755 2760 2765
Asp Arg Glu Phe Met Cys Gln Asn Arg Gln Cys Ile Pro Lys His Phe
2770 2775 2780
Val Cys Asp Hi8 Asp Arg Agp Cys Ala Asp Gly Ser Asp Glu Ser Pro
2785 2790 2795 2800
Glu Cys Glu Tyr Pro Thr Cys Gly Pro Ser Glu Phe Arg Cys Ala Asn
2805 2810 2815
Gly Arg Cys Leu Ser Ser Arg Gln Trp Glu Cys ABP Gly Glu Agn Asp
2820 2825 2830
CYB Hi8 Asp Gln Ser Asp Glu Ala Pro Lys Asn Pro His Cys Thr Ser
2835 2840 2845 ~ .
6~ :
Pro Glu HiS Lys Cys Asn Ala Ser Ser Gln Phe Leu Cys Ser Ser Gly
2850 2855 2860
~ ,"-'"',""',:"~
- 21171~9
Casc IV140.141,14!~-PCI 55
Arg Cy8 Val Ala Glu Ala Leu Leu Cys Asn Gly Gln Asp Asp Cys Gly
2865 2870 2875 2880
Asp Ser Ser Asp Glu Arg Gly Cys Hig Ile A8n Glu Cys Leu Ser Arg
2885 2890 2895
Lys Leu Ser Gly Cys Ser Gln Agp Cys Glu A~p Leu Lys Ile Gly Phe
2900 2905 2910
Lys Cys Arg Cyg Arg Pro Gly Phe Arg Leu Lys Asp Asp Gly Arg Thr
2915 2920 2925
Cys Ala Asp Val Asp Glu Cys Ser Thr Thr Phe Pro Cys Ser Gln Arg
2930 2935 2940
Cy8 Ile Asn Thr His Gly Ser Tyr Lys Cy8 Leu Cys Val Glu Gly Tyr
2945 2950 2955 2960
Ala Pro Arg Gly Gly Asp Pro His Ser Cys Lys Ala Val Thr Asp Glu
2965 2970 2975
Glu Pro Phe Leu Ile Phe Ala Asn Arg Tyr Tyr Leu Arg Lys Leu Asn
2980 2985 2990
Leu Asp Gly Ser Asn Tyr Leu Leu Lys Gln Gly Leu Asn Asn Ala Val
2995 3000 3005
Ala Leu Asp Phe A~p Tyr Arg Glu Gln Met Ile Tyr Trp Thr Asp Val
3010 3015 3020
Thr Thr Gln Gly Ser Met Ile Arg Arg Met His Leu Asn Gly Ser Asn
3025 3030 3035 3040
Val Gln Val Leu His Arg Thr Gly Leu Ser Asn Pro Asp Gly Leu Ala
3045 3050 3055
Val Asp Trp Val Gly Gly Asn Leu Tyr Trp Cys Asp Lys Gly Arg Asp
3060 3065 3070
Thr Ile Glu Val Ser Lys Leu A8n Gly Ala Tyr Arg Thr Val Leu Val
3075 3080 3085
Ser Ser Gly Leu Arg Glu Pro Arg Ala Leu Val Val Asp Val Gln Asn
3090 3095 3100
Gly Tyr Leu Tyr Trp Thr Asp Trp Gly Asp Hi8 Ser Leu Ile Gly Arg
3105 3110 3115 3120
Ile Gly Met Asp Gly Ser Ser Arg Ser Val Ile Val Asp Thr Lys Ile
3125 3130 3135
Thr Trp Pro Asn Gly Leu Thr Leu Asp Tyr Val Thr Glu Arg Ile Tyr
3140 3145 3150 .:
Trp Ala Asp Ala Arg Glu Asp Tyr Ile Glu Phe Ala Ser Leu Asp Gly
3155 3160 3165
Ser Asn Arg His Val Val Leu Ser Gln A8p Ile Pro His Ile Phe Ala
3170 3175 3180
Leu Thr Leu Phe Glu Asp Tyr Val Tyr Trp Thr Asp Trp Glu Thr Lys
3185 3190 3195 3200
Ser Ile Aan Arg Ala His Lys Thr Thr Gly Thr Asn Lys Thr Leu Leu
3205 3210 3215
2~ ~ ~ ~ 2 ~
~ 2~17099
C.~s~ 12/140,141.149-PCI' 56
Ile Ser Thr Leu His Arg Pro Met ABP Leu His Val Phe His Ala Leu
3220 3225 3230
Arg Gln Pro Asp Val Pro Asn His Pro Cys Ly~; Val Asn Asn Gly Gly
3235 3240 3245
Cys Ser Asn Leu Cys I.eu Leu Ser Pro Gly Gly Gly His Lys Cys Ala
3250 3255 3260
Cys Pro Thr Asn Phe Tyr Leu Gly Ser Asp Gly Arg Thr Cys Val Ser
3265 3270 3275 3280
Asn Cys Thr Ala Ser Gln Phe Val Cys Lys Asn Asp Lys Cys Ile Pro
32a5 3290 3295
Phe Trp Trp Lys Cys Asp Thr Glu Asp Asp Cys Gly Aap Hi s Ser Asp
3300 3305 3310
Glu Pro Pro A8p Cys Pro Glu Phe Lys Cys Arg Pro Gly Gln Phe Gln
3315 3320 3325
Cys Ser Thr Gly Ile Cys Thr Asn Pro Ala Phe Ile Cys Asp Gly Asp
3330 3335 3340
Asn Asp CYB Gln Asp Asn Ser Asp Glu Ala Asn Cys Asp Ile His Val
3345 3350 3355 3360
Cys Leu Pro Ser Gln Phe Lys Cys Thr Asn Thr Asn Arg Cys Ile Pro
3365 3370 3375
Gly Ile Phe Arg Cys Asn Gly Gln Asp A~n Cy8 Gly Asp Gly Glu Asp
3380 3385 3390
Glu Arg A8p Cys Pro Glu Val Thr Cys Ala Pro Asn Gln Phe Gln Cys
3395 3400 3405
Ser Ile Thr Lys Arg Cys Ile Pro Arg Val Trp Val Cys Asp Arg Asp
3410 3415 3420
Asn Asp Cys Val Asp Gly Ser Asp Glu Pro Ala Asn Cy6 Thr Gln Met
3425 3430 3435 3440 : :
Thr Cy~ Gly Val Asp Glu Phe Arg Cys Lys Asp Ser Gly Arg Cys Ile
3445 3450 3455
Pro Ala Arg Trp Lys Cys Asp Gly Glu Agp Asp Cys Gly Asp Gly Ser
3460 3465 3470
A8p Glu Pro Ly8 Glu G1U Cy~ Agp Glu Arg Thr Cy8 Glu Pro Tyr Gln
3475 3480 3485
Phe Arg Cy~ Lys Asn Asn Arg Cy~ Val Pro Gly Arg Trp Gln.Cys Asp
3490 3495 3500
Tyr Asp Asn Asp Cys Gly Asp Asn Ser Asp Glu Glu Ser Cys Thr Pro
3505 3510 3515 3520 ; :
Arg Pro Cys Ser Glu Ser Glu Phe Ser Cys Ala Asn Gly Arg Cys Ile
3525 3530 3535
Ala Gly Arg Trp Lys Cys Asp Gly Agp Hi8 Agp Cys Ala Agp Gly Ser
3540 3545 3550 - :
~5 ~:
Asp Glu Lys Asp Cys Thr Pro Arg Cys Asp Met Asp Gln Phe Gln Cys
3555 3560 3565 -
, ,, :
- 2~170~.~
Case 12/140,141,149-PC1 57
Lys Ser Gly His Cy8 Ile Pro Leu Arg Trp Arg Cy8 Asp Ala Asp Ala
3570 3575 3580
Asp Cys Net Asp Gly Ser Asp Glu Glu Ala Cys Gly Thr Gly Val Arg
3590 3595 3600
Thr Cys Pro Leu Asp Glu Phe Gln Cys Asn Asn Thr Leu Cys Lys Pro
3605 3610 3615
O Leu Ala Trp Lys Cys Asp Gly Glu Asp Asp Cy8 Gly Asp Asn Ser Asp
3620 3625 3630
Glu Asn Pro Glu Glu Cy8 Ala Arg Phe Val Cys Pro Pr~ Asn Arg Pro
3635 3640 3645
Phe Arg Cys Lys Asn Asp Arg Val Cys Leu Trp Ile Gly Arg Gln Cys
3650 3655 3660
Asp Gly Thr Asp Asn Cys Gly Asp Gly Thr Asp Glu Glu Asp Cys Glu
3665 3670 3675 3680
Pro Pro Thr Ala His Thr Thr His Cy6 Lys Asp Lys Lys Glu Phe Leu
3685 3690 3695
Cys Arg Asn Gln Arg Cys Leu Ser Ser Ser Leu Arg Cys Asn Met Phe
3700 3705 3710
Asp Asp Cys Gly Asp Gly Ser Asp Glu Glu Asp Cys Ser Ile Asp Pro
3715 3720 3725
Lys Leu Thr Ser Cys Ala Thr Asn Ala Ser Ile Cys Gly Asp Glu Ala
3730 3735 3740
Arg Cys Val Arg Thr Glu Lys Ala Ala Tyr Cys Ala Cys Arg Ser Gly : :
3745 3750 3755 3760 :
Phe His Thr Val Pro Gly Gln Pro Gly Cys Gln Asp Ile Asn Glu Cys
3765 3770 3775
Leu Arg Phe Gly Thr Cys Ser Gln Leu Cys Asn Asn Thr Lys Gly Gly
3780 3785 3790
His Leu Cys Ser Cys Ala Arg Asn Phe Met Lys Thr His Asn Thr Cys
3795 3aoo 3805
Lys Ala Glu Gly Ser Glu Tyr Gln Val Leu Tyr Ile Ala Asp Asp Asn
3810 3815 3820
Glu Ile Arg Ser Leu Phe Pro Gly His Pro His Ser Ala Tyr Glu Gln
3825 3830 3835 3840
Ala Phe Gl~ Gly Asp Glu Ser Val Arg Ile Asp Ala Met Asp.Val His
3845 3850 385~5
Val Lys Ala Gly Arg Val Tyr Trp Thr Asn Trp His Thr Gly Thr Ile
3860 3865 3870
: ~
Ser Tyr Arg Ser Leu Pro Pro Ala Ala Pro Pro Thr Thr Ser Asn Arg , ~:
3875 3880 3885
His Arg Arg Gln Ile Asp Arg Gly Val Thr His Leu Asn Ile Ser Gly -
3890 3895 3900
Leu Lys Met Pro Arg Gly Ile Ala Ile Asp Trp Val Ala Gly Asn Val
3905 3910 3915 3920
~-
~ 2~1709~
Case 121140.141,149-PCl' 58
Tyr Trp Thr Asp Ser Gly Arg Asp Val Ile Glu Val Ala Gln Met Lys
3925 3930 3935
Gly Glu A6n Arg Lys Thr Leu Ile Ser Gly 2iet Ile Asp Glu Pro His
3940 3945 3950
Ala Ile Val Val Asp Pro Leu Arg Gly Thr Met Tyr Trp Ser A~p Trp
3955 3960 3965
Gly Asn His Pro Lys Ile Glu l~hr Ala Ala Met Asp Gly Thr Leu Arg
3970 3975 3980
Glu Thr Leu Val Gln A~p Asn Ile Gln Trp Pro Thr Gly Leu Ala Val
39a5 3990 3995 4000
Asp Tyr His Asn Glu Arg Leu Tyr Trp Ala Asp Ala Lys Leu Ser Val
4005 4010 4015
Ile Gly Ser Ile Arg Leu Asn Gly Thr Asp Pro Ile Val Ala Ala Asp
4020 4025 4030
Ser Lys Arg Gly Leu Ser His Pro Phe Ser Ile Asp Val Phe Glu Asp
4035 4040 4045
Tyr Ile Tyr Gly Val Thr Tyr Ile Asn Asn Arg Val Phe Lys Ile His
4050 4055 4060
Lys Phe Gly Hi8 Ser Pro Leu Val Asn Leu Thr Gly Gly Leu Ser His
4065 4070 4075 4080
Ala Ser A8p Val Val Leu Tyr His Gln His Lys Gln Pro Glu Val Thr
4085 4090 409~
Asn Pro Cy~ Asp Arg Lys Lys Cys Glu Trp Leu Cys Leu Leu Ser Pro
4100 4105 4110
Ser Gly Pro Val Cys Thr Cys Pro Asn Gly Lys Arg Leu Asp Asn Gly
4115 4120 4125 :
Thr Cys Val Pro Val Pro Ser Pro Thr Pro Pro Pro Asp Ala Pro Arg
4130 4135 4140
Pro Gly Thr Cys Asn Leu Gln Cys Phe Asn Gly Gly Ser Cys Phe Leu
4145 4150 4155 4160
Asn Ala Arg Arg Gln Pro Lys Cys Arg Cys Gln Pro Arg Tyr Thr Gly
4165 4170 4175 . ~
Asp Ly~ Cys Glu Leu Asp Gln Cys Trp Glu His Cys Arg Asn Gly Gly ~3
4180 41a5 4190 ~:
Thr Cy5 Ala Ala Ser Pro Ser Gly Met Pro Thr Cys Arg Cys . Pro Thr
4195 4200 4205
Gly Phe Thr Gly Pro Lys Cys Thr Gln Gln Val Cys Ala Gly Tyr Cys
4210 4215 4220
Ala Asn Asn Ser Thr Cys Thr Val Asn Gln Gly Asn Gln Pro Gln Cys
4225 4230 4235 4240
Arg Cys Leu Pro Gly Phe Leu Gly Asp Arg Cy8 Gln Tyr Arg Gln Cys ~-
4245 4250 4255
Ser Gly Tyr Cys Glu Asn Phe Gly Thr Cys Gln Met Ala Ala Asp Gly
4260 4265 4270
r~ 2117(3~
Cas; ~V140.141.149.PCI 59
: Ser Arg Gln Cy8 Arg Cys Thr Ala Tyr Phe Glu Gly Ser Arg Cys Glu
4275 4280 4285
Val Asn Lys Cys Ser Arg Cy8 Leu Glu Gly Ala Cys Val Val Aan Lya
1 5 4290 4295 4300
Gln Ser Gly Asp Val Thr Cys Asn Cy9 Thr Asp Gly Arg Val Ala Pro
4305 4310 4315 4320
Ser Cys Leu Thr Cys Val Gly His Cys Ser Asn Gly Gly Ser Cys Thr
4325 4330 4335
' Met Asn Ser Lys Met Met Pro Glu Cy8 Gln Cys Pro Pro His Net Thr
; 4340 4345 4350
IS
Gly Pro Arg Cys Glu Glu His Val Phe Ser Gln Gln Gln Pro Gly His
4355 4360 4365
Ile Ala Ser Ile Leu Ile Pro Leu Leu Leu Leu Leu Leu Leu Val Leu
4370 4375 43ao
Val Ala Gly Val Val Phe Trp Tyr Lys Arg Arg Val Gln Gly Ala Lys
4385 4390 4395 4400
Gly Phe Gln His Gln Arg Met Thr Asn Gly Ala Met Asn Val Glu Ile
4405 4410 4415
Gly Asn Pro Thr Tyr Lys Met Tyr Glu Gly Gly Glu Pro Asp Asp Val
4420 4425 4430
Gly Gly Leu Leu Asp Ala Asp Phe Ala Leu Asp Pro Asp Lys Pro Thr
4435 4440 4445
Asn Phe Thr Asn Pro Val Tyr Ala Thr Leu Tyr Met Gly Gly His Gly
4450 4455 4460
Ser Arg His Ser Leu Ala Ser Thr Asp Glu Lys Arg Glu Leu Leu Gly
4465 4470 4475 4480
Arg Gly Pro Glu Asp Glu Ile Gly Asp Pro Leu Ala
4485 4490
' , '
(2) INFORMATION ZU SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 319 amino acids
(P) TYPE: amino acid
(C) STRANDEDNESS: 8ingle
(D) TOPOLOGY: linear
(ii) MOLEC~LE TYPE: peptide
(xi) SEQ~ENCE DESCRIPTION: SEQ ID NO: 5:
Arg Ser Ala Glu Lys Asn Glu Pro Glu Met Ala Ala Lys Arg Glu Ser
1 5 10 15
Gly Glu Glu Phe Arg Met Glu Lyg Leu ~sn Gln Leu Trp Glu Lys Ala
20 25 30
: :
2 1 1 7 0 ~r`~ 9
CA~C 12/14~),141,149-PC~ 60
Lys Arg Leu His Leu Ser Pro Val Arg Leu Ala Glu Leu His Ser A~p
Leu Lys Ile Gln Glu Arg Asp Glu Leu Asn Trp Lys Ly~ Leu Lys Val
50 55 60
Glu Gly Leu Asp Gly Asp Gly Glu Lys Glu Ala Lys Leu Val His Asn
.:
Leu Asn Val Ile Leu Ala Arg Tyr Gly Leu Asp Gly Arg Lys Asp Thr
85 90 95
Gln Thr Val His Ser Asn Ala Leu Asn Glu Asp Thr Gln Asp Glu Leu
IS 100 105 110
Gly Asp Pro Arg Leu Glu Lys Leu Trp His Lys Ala Lys Thr Ser Gly
115 120 125
Ile Ser Val Arg Leu Thr Ser Cys Ala Arg Val Leu His Tyr Lys Glu
130 135 140
Lys Ile His Glu Tyr Asn Val Leu Leu Asp Thr Leu Ser Arg Ala Glu
145 150 155 160
Glu Gly Tyr Glu Asn Leu Leu Ser Pro Ser Asp Net Thr His Ile Lys . .
165 170 175 ~ .
Ser Asp Thr Leu Ala Ser Lys His Ser Glu Leu Lys Asp Arg Leu Arg
180 185 190
Ser Ile A8n Gln Gly Leu Asp Arg Leu Arg Lys Val Ser His Gln Leu -~
195 200 205 . ~:
Arg Pro Ala Thr Glu Phe Glu Glu Prc Arg Val Ile Asp Leu Trp Asp
210 215 220
Leu Ala Gln Ser Ala Asn Phe Thr Glu Lys Glu Leu Glu Ser Phe Arg
225 230 235 240
:::
Glu Glu Leu Lys His Phe Glu Ala Lys Ile Glu Lys His Asn His Tyr
245 250 2~5 ~.
Gln Lys Gln Leu Glu Ile Ser His Gln Lys Leu Lys His Val Glu Ser
260 265 270
Ile Gly Asp Pro Glu His Ile Ser Arg Asn Lys Glu Lys Tyr Val Leu
275 280 285
Leu Glu Glu Lys Thr Lys Glu Leu Gly Tyr Lys Val Lys Lys His Leu
290 295 300
Gln Asp Leu Ser Ser Arg Val Ser Arg Ala Arg His Asn Glu-Leu
305 310 315
