JP2003212791A - Composition for modulating cell adhesion activity and/or cell motility activity containing laminin-6 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition containing a laminin-6 capable of modulating cell adhesion activity and cell motility activity. <P>SOLUTION: The short-chain type laminin-6 has α3-chain, β1-chain and γ1-chain where G4 and G5 domains are deleted. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a composition containing laminin-6 capable of regulating cell adhesion activity and / or cell motility activity.

[0002]

BACKGROUND OF THE INVENTION Basement membrane components not only support tissue structure but also control a variety of cell functions such as cell adhesion, migration, differentiation, growth and apoptosis. Among them, the laminin molecule is one of the most important components in various epithelial tissues, nerve tissues, basement membranes such as muscles. It is known that in these tissues, laminin directly interacts with cells and plays an important role in the construction and maintenance of normal tissues and the regulation of cell function. Deletion of laminin, which plays a role as an essential scaffold for maintaining the life of cells, makes it impossible to maintain the vital activity of cells and the function of tissues, causing various diseases. The interaction between laminin and the receptor on the cell membrane surface is closely related to such life support, and intracellular signal transduction via integrin as a receptor has been emphasized (Gian).
cotti, F.F. G. , And Ruoslahti,
E. , Science, 285: 1028-1032.
1999; Howe, A .; et al. Curr.
Opin. Cell Biol. , 10: 220-23
1, 1998). However, few examples explain how different ligand-integrin interactions produce different biological effects.

Laminins are a family of extracellular matrix proteins that are primarily localized to the basement membranes of various tissues and play important roles in maintaining tissue structure and controlling cell function (Aumailley, M., and.
Rousselle, P.M. , Matrix Bio
l. , 18: 19-28, 1999; Cologna.
to, H.M. , And Yurchenco, P .; D. ，
Dev. Dyn. , 218: 213-234,200.
0). Among them, laminin-5 (α3β3γ2) is particularly abundant in the basement membrane of epithelial tissues such as skin, digestive organs, kidneys and lungs. Laminin-5 is a protein essential for stable adhesion of the epidermis to the dermis, and deficiency of laminin-5 causes epidermolytic varicella. On the other hand, laminin-6 (α3β1γ
1) has been reported to coexist with laminin-5 in the skin and amniotic membrane, but the details of its physiological role have not been clarified. Studies by the present inventors suggest that laminin-5 is abundant in the villus tip portion and laminin-6 is abundant in the crypt (crypt) where cell division is active in the human small intestinal basement membrane. That is, it is assumed that the balance of laminin-5 and laminin-6 regulates the proliferation and differentiation of epithelial cells forming villi.

Laminin is a heterotrimeric molecule in which α-chain, β-chain and γ-chain are cross-linked by disulfide bonds.
It has a characteristic cross structure. Each chain (or subunit) is composed of multiple domains, and domains I and II form a triple helix. Prior to the filing of the present application, the laminin molecule had five developmentally different α chains (α1
To α5), at least 15 kinds of which are specifically expressed in the embryonic development process and adult tissues by different combinations of three kinds of β chains (β1 to β3) and three kinds of γ chains (γ1 to γ3) It has been identified (Table 1), and it is suggested that there are actually several times as many species (Miyazaki et al., Experimental Medicine Vol. 16 No. 16).
(Special Issue) 1998 114-119; Co
lognato, H .; , And Yurchenco,
P. D. , 2000, supra; Libby, R .; T. e
t al. J. Neurosci. , 20: 6517
-6528, 2000). These laminins have different properties and are considered to play different roles in vivo.

[0005]

[Table 1] Table 1 Laminin molecular species and subunit composition Official name Composition Alias laminin-1 α1β1γ1 EHS laminin laminin-2 α2β1γ1 merosin laminin-3 α1β2γ1 s-laminin laminin-4 α2β2γ1 s-merocin laminin-5 α3β3γ2 / radin Epiligrin / Calinine / Nicein Laminin-6 α3β1γ1 k-Laminin Laminin-7 α3β2γ1 ks-Laminin Laminin-8 α4β1γ1 Laminin-9 α4β2γ1 Laminin-10 α5β3γ2 Laminin α3β2γ1 La2 Laminin ααββγγ2 Laminin-11 α5β2γ3 Laminin 15 α5β2γ3 All laminin α chains share a large globular (G) domain in their carboxyl (C) terminal region, which is often a laminin G-like molecule (LG1). -5) consisting of five homologous tandem aligned small G subunits (G1-5) (Timpl, R. et al., Matrix).
Biol. , 19: 309-317, 2000). Each G domain consists of about 200 amino acids, and this region is a binding site for extracellular matrix proteins (eg, perlecan and fibulin-1) and cellular receptors that include integrins, syndecans, heparins and α-dystroglycans. Including (Clognato,
H. , And Yurchenco, P .; D. , 200
0, supra; Nomiz, M .; et al. J. B
iol. Chem. , 270: 20583-2059.
0, 1995; Aumailley, M .; , And
Krieg, T .; J. Invest. Dermato
l. , 106: 209-214, 1996; Niel.
sen, P.M. K. et al. J. Biol. Che
m. , 275: 14517-14523, 2000;
Talts, J. et al. F. et al. , EMBO J .; ，
18: 863-870, 1999).

Recently, LG5 and LG4 of laminin α2 chain
A crystal structure of -5 was reported, indicating that in each module the seven folded β-strands form a compact double β-sheet structure (Hohenester, E. et. al., Mo
l. Cell, 4: 783-792, 1999; Ti
si, D.D. , EMBO J .; , 19: 1432-144
0, 2000). A similar modular structure is found in Neulexin (Rudenko, G. et al., Cell, 9
9: 93-101, 1999) and sex hormone-binding globulin (Grishkovskaya, I. et a.
l. , EMBO J .; , 19: 504-512, 200
0). It has been postulated that various extracellular and transmembrane proteins have similar LG modules (Timpl, R. et al., 2000,
Above).

Among the α chains, the α that the present inventors have paid particular attention to
The 3 chain has a short arm (short chain) structure in which several domains on the N-terminal side are deleted compared to the α1 chain, and the homology of the amino acid sequence with the α1 chain is 50% or less. In particular, the homology of the globular (G) domain existing in the C-terminal region of the α chain is low (25%), and it is considered that this region is related to the function unique to each laminin molecule.

The α3 chain is classified into α3A having a short arm (N-terminal portion) and α3B having a long arm (Ryan, M. et al.
C. et al. J. Biol. Chem. 269:
22779-22787, 1994). So far α3B
Is known, but laminin with α3B has not been obtained. Therefore, in the present specification, laminin-
The α3 chain of 5, laminin-6, and laminin-7 is preferably α3A. The structure of α3A is included in α3B because the two α3 chains are different due to alternative splicing. It should be noted that α4 is also a short chain type similar to α3A, and the other α1 chain, α2 chain and α5 chain are all long arm types.

Laminin-5 (LN5) contains α3, β3 and γ2 chains and is initially an anchoring filament of keratinocytes.
t) component (kalinin / epiligulin / nicein) (R
ousselle, P.M. et al. J. Cell B
iol. , 114: 567-576, 1991; Car.
ter, W. G. et al. , Cell, 65:59.
9-610, 1991; Verrando, P .; et
al. , Biochem. Biophys. Act
a. , 942: 45-56, 1988), and a cell spreading factor (radosine) secreted by gastric mucosal tumor cells.
(Miyazaki, K. et al., Proc. N.
atl. Acad. Sci. USA, 90: 11767.
-117771, 1993). afterwards,
It was revealed that radsin is the same molecule as laminin-5 (also called epiligulin, kalinin, or neisine), which is a laminin molecule derived from epidermal cells (Miz.
Ushima, H .; et al. J. Biochem.
(Tokyo), 120: 1196-1202, 199.
6). Hereinafter, in the present specification, the term "laminin-5" will be used as a standard.

Laminin-5 is unique in its bioactivity and structure. Laminin-5 strongly stimulates both cell adhesion and cell motility in culture (Miyaza).
ki, K.K. et al. , 1993, supra; Kikk
awa, Y. et al. J. Biochem. , 1
16: 862-829, 1994; Rossell.
e, P. , And Aumailley, M .; J. C
ell Biol. , 125: 205-214, 199.
4). Laminin-5 is currently the only laminin molecule with the amino-terminal region truncated for all three subunits (Colognato, H., and.
Yurchenco, P.M. D. , 2000, supra). That is, the β3 chain and γ2 chain of laminin-5 are the β1 chain and the γ chain.
It has a short-arm (short chain) structure in which several domains on the N-terminal side are deleted as compared with one chain. Known laminin in which all of α-chain, β-chain and γ-chain are short-chain type is laminin-5.
(Colognato, H., and Yu
rchenco, P.R. D. , 2000, supra). When the structure of the constituent subunits of laminin-5 is compared with that of laminin-1, the homology of the amino acid sequence between each subunit is 5
It is 0% or less. Also, the β3 chain and the γ2 chain are found only in LN5, while the α3 chain is laminin-6 (LN5
6; α3β1γ1), laminin-7 (LN7; α3β2
γ1) and laminin-13 (LN13; α3β2γ3)
Found in. Laminin-5 is secreted by epithelial cells of various types in tissues and cultures (Miyaza).
ki, K.K. et al. , 1993, supra; Mizu
Shima, H .; et al. , 1996, supra). i
In n vivo, it accumulates in the basement membrane. The interaction of LN5 with integrin α6β4 in hemidesmosomes is essential for adhesion of basal cells to stable substrates, polarity of epithelial sheets, and stabilization of cellular architecture through intermediate filaments (Roussell, P. et a.
l. Carter, W .; G. et
al. , 1991, supra).

Laminin-5 is a 190 kDa α3 chain,
Synthesized and secreted as a precursor containing a 140 kDa β3 chain and a 150 kDa γ2 chain, the α3 chain and γ2
The chains are proteolytically processed into smaller species of 160 and 105 kDa, respectively (Ma
rinokovich, M .; P. et al. J. B
iol. Chem. 267: 17900-17906.
1992). Recent studies have suggested that post-translational processing of the laminin-5 molecule alters the function of the laminin-5 molecule. Gelatinase A or MT1
-Proteolysis of γ2 chain by MMP is laminin-5
The cell motility activity of G. (Giannelli, G. et al.
et al. , Science, 277: 225-22.
8, 1997; Koshikawa, N .; et a
l. J. Cell Biol. , 148: 615-6
24, 2000) and decrease cell adhesion activity (Gag.
noux-Palacios, L .; et al. J.
Cell Biol. , 153: 835-850, 20.
01). Similarly, 190kD of α3 chain by plasmin
Conversion of the laminin-5 precursor from a migratory ligand to a scaffolding ligand in hemidesmosomes has been reported by processing a to 160 kDa (G
oldfinger, L .; E. et al. J. Ce
ll Biol. , 141: 255-265,199
8). However, mature laminin-5, which has a processed α3 chain, has been shown to potently promote integrin α3β1-mediated cell motility or cell dispersal (Miyazaki, K., et al., 1993,).
Above; Kikkawa, Y .; , Et al. , 199
4, above). Cleavage of the α3 chain occurs between the G3 and G4 domains, releasing the G4-5 fragment (Tsubot
a, Y. , Et al. , Biochem. Bioph
ys. Res. Commun. , 278: 614-62.
0, 2000), and the G3 domain is essential for the potent activity of laminin-5 to promote cell adhesion and cell migration (Hirosaki, T. et al., JB.
iol. Chem. , 275: 22495-2250.
2, 2000) was recently found. It has also been found that the G2 domain contains an integrin α3β1 binding site (Mizushima, H. et al., Cel.
l Growth Differ. , 8: 979-98.
7, 1997). Although these studies show that the G1-3 domain of the α3 chain is the fundamental site for binding cell surface receptors, the γ2 chain and the possible short arm β3 chain influence the physiological activity of laminin-5. It shows that it seems to give.

Laminin-5 and laminin-6 have a common α
Having three chains, these laminins are produced by cells of the same type. Therefore, it is important to characterize laminin-6 in order to clarify the relationship between the structure and function of laminin-5. Laminin-6 was first identified as K-laminin from cultures of human keratinocytes and squamous cell carcinoma cell lines (Marinkovich, K. et.
al. , 1992, supra). In human amniotic membrane, about half of laminin-5 exists as a complex with laminin-6 or laminin-7 (Champliaud,
M. F. et al. J. Cell Biol. , 1
32: 1189-1198, 1996). This complex is subjected to rotation-shadowed image analysis (rotary-shadowed).
image analysis) (Engel, J., M
methods Enzymol. , 245: 469-4
88, 1994) to provide laminin-5
Have been suggested to bind via laminin-6 or laminin-7 short arm interactions. It is postulated that the formation of a complex allows for a stable association between laminin-5 and the amnion basement membrane (Champl).
iaud, M .; F. et al. , 1996, supra).
However, no studies have been conducted so far to investigate the physiological activity of laminin-6 or laminin-7. Probably because it is difficult to isolate laminin-6 and laminin-7 using the laminin-5 educt. Therefore,
For laminin-6, each constituent subunit (α3,
The amino acid sequences of β1 and γ1) have been known, but their physiological activities have not been elucidated.

[0013]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a composition containing laminin-6 capable of regulating cell adhesion activity and cell motility activity.

In one embodiment, the composition of the present invention comprises each subunit of α3 chain having the amino acid sequence of SEQ ID NO: 2, β1 chain having the amino acid sequence of SEQ ID NO: 4, and γ1 chain having the amino acid sequence of SEQ ID NO: 6. Laminine consisting of
6, or has an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in these sequences, and has weak cell adhesion activity, and / or substantially Is characterized by containing laminin-6 having no cell motility activity.

In a preferred embodiment of the present invention, the composition of the present invention comprises α3 having the amino acid sequence of SEQ ID NO: 2.
A laminin-6 comprising a chain, a β1 chain having the amino acid sequence of SEQ ID NO: 4 and a γ1 chain having the amino acid sequence of SEQ ID NO: 6.

The composition of the invention is also in one aspect:
Amino acid residue 132 in the amino acid sequence of SEQ ID NO: 2
Α-17 chain in which all or part of 5-1713 is deleted, β1 chain having the amino acid sequence of SEQ ID NO: 4, and SEQ ID NO: 6
Laminin-6 consisting of a γ1 chain having the amino acid sequence of, or having an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in these sequences, and cell adhesion It is characterized in that it comprises laminin-6 which has activity and / or has weak cell motility activity.

The composition of the present invention comprises epithelial tissue, nerve tissue,
Used to maintain or treat muscle function. The composition of the present invention is also used as an additive for culturing cells for transplantation or as an adhesive device.

A further object of the present invention is to provide a method for regulating cell adhesion activity and / or cell motility activity using the composition.

[0019]

The inventor of the present invention has conducted extensive research to solve the above problems, and as a result, laminin-6
As a single unprocessed protein, and G4-5 or G in the α chain of laminin-6
Truncated form lacking 3-5 domain (LN6
ΔG4-5, LN6ΔG3-5) were isolated. further,
It was found that laminin-6 or laminin-6 in which the G4-5 domain of the α3 chain is deleted exhibits cell adhesion activity and / or cell motility activity different from that of laminin-5, and thus regulates these, thus completing the present invention. Came to do.

Laminin-6 The present invention relates to a composition containing laminin-6 capable of regulating cell adhesion activity and / or cell motility activity. The laminin-6 of the present invention, as long as it has the characteristics described herein,
The origin and manufacturing method are not limited. That is, the laminin-6 of the present invention may be either a recombinant protein expressed from DNA by a genetic engineering method or a chemically synthesized protein. Alternatively, it may be a naturally obtained protein that is not artificially modified as long as it has the characteristics described in the present specification. Alternatively, a naturally-occurring protein may be modified at the protein level.

Laminin-6 in the present invention is typically, but not exclusively, α3 chain (amino acid residue 1-1713) (R residue) having the amino acid sequence of SEQ ID NO: 2.
yan, M. C. et al. , 1994, supra), β1 chain having the amino acid sequence of SEQ ID NO: 4 (amino acid residues 1-1786) (Vuolteenaho, R. eta.
l. J. Biol. Chem. , 265: 15611
-15616, 1990) and the γ1 chain having the amino acid sequence of SEQ ID NO: 6 (amino acid residues 1-1609) (P
ikkaraine, T .; et al. J. Bio
l. Chem. , 263: 6751-6758, 199.
It is a protein consisting of each subunit of 8). α3
The chain has five globular (G) domains (or LG modules) at the C-terminus, and the core sequence of each domain is G1:
Amino acid residues 769-974 of SEQ ID NO: 2, G2: 98
0-1148, G3: 1149-1324, G4: 13
66-1537 and G5: 1538-1713 (the sequence between each domain (amino acid residues 975-97
9 and 1325-1365) correspond to linker sequences). Homology between the domains is low, about 25% or less.

In Example 2, in order to examine the characteristics of laminin-6 (LN6), mat-LN5 (mature laminin-free) lacking the G4 and G5 domains of laminin-5.
5) and other matrix proteins. As a result, in the cell adhesion assay and the cell motility assay using HT1080 cells, laminin-6 was found to be ma.
The cell adhesion activity was weaker than that of t-LN5 and substantially did not promote cell motility activity (and cell dispersion activity) (FIG. 3,
(See FIGS. 5 and 6).

Thus, full length laminin-6 preferably has weak cell adhesion activity and / or substantially no cell motility activity. Although not limited thereto, having a weak cell adhesion activity means that the cell adhesion activity is significantly increased as compared with the case where nothing is added.
Short-chain type LN-6 (LN6ΔG4-
It means that the cell adhesion activity is 2/3 or less, more preferably 1/2 or less, further preferably 2/5 or less, as compared with 5). Although it is not limited to have substantially no cell motility activity, it has a cell adhesion activity of ½ or less, more preferably 2/5 or less, further preferably 1 /, as compared with LN6ΔG4-5. It means about 3 or less. In addition, comparing the cell morphology adhered to each substrate, the cells on mat-LN5 spread, while the cells on LN6 maintained a spherical morphology. Therefore, LN6 can be used as an adhesion device capable of inducing cell division (see FIG. 4).

It should be noted that among natural proteins or polypeptides, one to a plurality of natural proteins or polypeptides may be present due to differences in the species of organisms that produce them or the presence of gene mutations due to differences in ecotype. It is well known that there are muteins or polypeptides with amino acid mutations. Therefore, the LN6 of the present invention has an α3 chain having the amino acid sequence of SEQ ID NO: 2, a β1 chain having the amino acid sequence of SEQ ID NO: 4 and the SEQ ID NO: 6 as long as it has a function of regulating cell adhesion activity and / or cell motility activity. Each subunit of the γ1 chain having the amino acid sequence of may have an amino acid sequence in which one or more amino acid residues are deleted, added or substituted.

The protein of the present invention typically comprises α3 chain having the amino acid sequence of SEQ ID NO: 2, β1 chain having the amino acid sequence of SEQ ID NO: 4 and SEQ ID NO: 6, based on the inference from the nucleotide sequence of a gene. Γ1 having an amino acid sequence
It consists of each subunit in the chain. However, it is not limited to only proteins having that sequence, but is intended to include all homologous proteins as long as they have the properties described herein. 1 to a plurality of amino acid mutations, preferably 1 to 20, more preferably 1
To 10 and most preferably 1 to 5.

Α3 having the amino acid sequence of SEQ ID NO: 2
Chain, the amino acid sequence of each subunit of the β1 chain having the amino acid sequence of SEQ ID NO: 4 and the γ1 chain having the amino acid sequence of SEQ ID NO: 6, at least 50% or more, preferably 60% or more, more preferably 70% or more, It is more preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more.

The percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be calculated according to Needleman,
S. B. And Wunsch, C .; D. (J. Mol. B
iol. , 48: 443-453, 1970) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
It may be determined by comparing the sequence information using the P computer program. The preferred default parameters for the GAP program are: (1) Heniko
ff, S and Henikoff, J .; G. (Proc.
Natl. Acad. Sci. USA, 89: 1091.
5-10919, 1992), scoring matrix, blosum62; (2) 1.
Gap weight of 2; (3) Gap length weight of 4; and (4) No penalty for end gaps.

Other programs used by those skilled in the art of sequence comparison may also be used. The percent identity is
For example, Altschul et al. (Nucl. Acids. R
es. 25. , P. 3389-3402, 1997) and can be determined by comparison with the sequence information. The program is on the Internet at National Center
r for Biotechnology Info
mation (NCBI) or DNA Data
It is available from the Bank of Japan (DDBJ) website. Various conditions (parameters) for homology search by the BLAST program are described in detail on the same site, and some settings can be changed as appropriate, but the search is usually performed using default values.

Generally, substitution of amino acids having similar properties (for example, substitution of one hydrophobic amino acid with another hydrophobic amino acid, substitution of one hydrophilic amino acid with another hydrophilic amino acid, certain acidic amino acid) To another acidic amino acid, or from one basic amino acid to another basic amino acid), the resulting mutant protein often has the same properties as the original protein. Techniques for producing recombinant proteins having such desired mutations using gene recombination techniques are well known to those skilled in the art, and such mutant proteins are also included in the scope of the present invention.

Short-Chain Laminin-6 The present invention also relates to a composition containing short-chain laminin-6 capable of regulating cell adhesion activity and / or cell motility activity.
The short-chain laminin-6 of the present invention is not limited in its origin, production method, etc., as long as it has the characteristics described in the present specification.
That is, the short-chain laminin-6 of the present invention may be either a recombinant protein expressed from DNA by a genetic engineering method or a chemically synthesized protein. Alternatively, it may be a naturally obtained protein that is not artificially modified as long as it has the characteristics described in the present specification. Alternatively, a naturally-occurring protein may be modified at the protein level.

Variants of proteins produced by such a method include mutants, derivatives and oligomers,
Fusion proteins or fragments thereof are included. Although the short-chain laminin-6 in the present invention is not limited, it is typically an α3 chain or sequence in which all or part of amino acid residues 1325-1713 are deleted in the amino acid sequence of SEQ ID NO: 2. Β1 chain having the amino acid sequence of number 4 (amino acid residues 1-1786) (Vuolteenah
o, R. et al. , 1990, supra) and γ1 chain having the amino acid sequence of SEQ ID NO: 6 (amino acid residue 1-1
609) (Pikkaraine, T. et al.,
1998, supra).

In the present specification, LN6ΔG4-5 refers to a deletion mutant in which the entire G4-5 domain (amino acid residues 1366-1713 in the amino acid sequence of SEQ ID NO: 2) of the α3 chain has been deleted. LN6ΔG4-
5, G4-5 domain further including a linker sequence, that is, the entire amino acid residues 1325-1713 may be deleted. The deletion mutant in which the G4-G5 domain containing the linker sequence has been deleted also has the same cell adhesion activity and / or cell motility activity as the deletion mutant in the G4-5 domain alone. Amino acid residue 1299 of G3 domain
It has been revealed that the sequence KRD of -1301 is important for the cell adhesion activity of laminin-5. Therefore, part or all of amino acid residues 1302-1713, including part of the carboxyl-terminal portion of the G3 domain, have been deleted.
N-6 may also have the same cell adhesion activity and / or cell motility activity as LN-6ΔG4-5.

Alternatively, the short-chain laminin-6 of the present invention
Includes, but is not limited to, the G3-5 domain (amino acid residue 1149 in the amino acid sequence of SEQ ID NO: 2).
-1713) in which all or part thereof is deleted, a β1 chain having the amino acid sequence of SEQ ID NO: 4 and a γ1 chain having the amino acid sequence of SEQ ID NO: 6 (amino acid residue 1-1
It may be a protein consisting of each subunit of 609).

In the present specification, the G3-5 domain means amino acid residue 1 in the amino acid sequence of SEQ ID NO: 2.
It refers to the domain corresponding to 149-1713. further,
As used herein, LN6ΔG3-5 refers to a deletion mutant in which the entire G3-5 domain in the α3 chain has been deleted. In the examples described below, amino acid residue 1154
A deletion mutant in which -1173 has been deleted has been prepared and used, which shows substantially the same activity as the deletion mutant in which the entire G3-G5 domain (amino acid residues 1149-1173) has been deleted. .

(1) G4-5 Domain Deletion Mutant In a preferred embodiment of the present invention, although not limited thereto, the entire G4-5 domain of the α3 chain is mutated, preferably deleted. As shown in Example 2 described later, the deletion mutant (LN6ΔG4-5) in which the entire G4-5 domain was deleted was HT1080 cells and BRL.
The cells showed cell adhesion activity equivalent to that of mature laminin-5 (mat-LN5) (see FIG. 3), and cell motility activity higher than LN6 but lower than mat-LN5 (see FIG. 6). Therefore, G4-5 of LN-6 of the present invention
Domain deletion mutants have cell adhesion activity and / or weak cell motility activity.

Specifically, comparing the effective concentrations (ED ₅₀ ) for the half-maximal cell adhesion activity, LN6ΔG4
-5 was about 1.5 times higher than LN6. From the above,
The G4-5 domain is considered to have an important function for the cell adhesion activity of LN6. The G4-5 domain may be deleted entirely or partially. Although not limited, the deletion preferably has a cell adhesion activity of 10 times or more, more preferably 5 times or more, still more preferably 2.5 times or more, still more preferably 2 times or more, and most preferably 1 times that of LN6. .5 times or more. Preferably, the cell adhesion activity is similar to that of mature LN5.

When BRL cells were allowed to adhere to mat-LN5 or when BRL cells and mat-LN5 were mixed and cultured, cell dispersion and cell migration were significantly activated. However, BRL cells and LN6
Cell dispersion did not occur when ΔG4-5 was mixed. On the other hand, when LN6ΔG4-5 was coated as an adhesive substrate and used, a higher dispersion activity was exhibited as compared with LN6. Furthermore, comparing cell motility activity by measuring cell migration rate, LN6ΔG4 used as an adhesion substrate
-5 showed weak cell motility. Specifically, LN
Cells attached to 6ΔG4-5 have approximately half the migration activity of cells attached to mat-LN5, while LN6
It was about 2 times higher than (See Figure 6).

From the above, although not limited, G
The 4-5 domain may be deleted entirely or partially. Although not limited thereto, the deletion preferably has a cell motility activity of 10 times or more, more preferably 5 times or more, still more preferably 3 times or more, still more preferably 2.5 times or more, as compared with LN-6. It is preferably increased by a factor of 2 or more. And, although not limited, the cell motility activity is ½ or less, more preferably 2/5 or less, still more preferably ⅓ or less, as compared with mat-LN5.

In a preferred embodiment of the present invention LN6
ΔG4-5 may be used as a stable adhesive device with substantially no cell migration. LN6Δ of the present invention
G4-5 is preferably, but not limited to, m
at-LN5 and LN6 are mixed, more preferably L
N5 may be mixed and more preferably used alone.

(2) G3-5 domain deletion mutant In a preferred embodiment of the present invention, the entire G3-5 domain of the α3 chain is mutated, preferably deleted, although not limited thereto. As shown in Example 2 described later,
Deletion mutants with the entire G3-5 domain deleted (L
N6ΔG3-5) did not promote cell adhesion activity and cell motility activity in HT1080 cells and BRL cells similarly to fibronectin (FIGS. 3 and 6).

Therefore, the LN6ΔG3-5 of the present invention
Include, but are not limited to, LN5, LN
6 or LN6ΔG4-5 may be used as an adhesion device that can down-regulate cell adhesion activity or cell motility activity. The LN6ΔG3-5 of the present invention may be used preferably as a mixture of mat-LN5, LN6 and LN6ΔG4-5, and more preferably used alone.

The G4-5 domain-deleted LN6 mutant and the G3-5 domain LN6 deleted mutant are also particularly similar to the case of the full-length LN6 in the regions other than the G4-5 or G3-5 domain. Without limitation, as long as it has a function of regulating cell adhesion activity and / or cell motility activity,
Α3 chain having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4
In each subunit of the β1 chain having the amino acid sequence of 1 and the γ1 chain having the amino acid sequence of SEQ ID NO: 6, even if it has an amino acid sequence in which one or more amino acid residues are deleted, added or substituted Good. It is intended to include all homologous proteins as long as they have the properties described herein. "Amino acid mutation" is 1 to multiple,
It is preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5.

Therefore, in the amino acid sequence of SEQ ID NO: 2, α3 is obtained by deleting all or part of amino acid residues 1366-1713 or amino acid residues 1154-1713.
Chain, the amino acid sequence of each subunit of the β1 chain having the amino acid sequence of SEQ ID NO: 4 and the γ1 chain having the amino acid sequence of SEQ ID NO: 6, at least 50% or more, preferably 60% or more, more preferably 70% or more, It is more preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more.

Preparation of laminin-6 and short-chain laminin-6
Production Method Laminin-6 (LN6) and short chain laminin of the present invention
6 (LN6ΔG4-5 and LN6ΔG3-5) expression,
Isolation and purification may be performed using any known technique.
For example, the following method can be applied without limitation.

The short-chain LN6 of the present invention is characterized by having an α3 chain in which all or part of amino acid residues 1366-1713 or amino acid residues 1154-1713 in the amino acid sequence of SEQ ID NO: 2 are deleted. .

Briefly, first, a cDNA clone encoding the laminin α3 chain can be isolated from a cell line cDNA library which expresses laminin having the α3 chain as a constituent, for example, laminin-5. For example, M
izushima, H .; et al. , 1996 (supra)
The cDNA library described in 1. can be used. Furthermore, various primers for polymerase chain reaction (PCR) were prepared, and by combining the primers,
PCR can be performed using the cDNA clone as a template to prepare expression plasmids encoding various recombinant laminin mutants. Here, the antisense primer of the PCR primer can include a stop codon (TGA) followed by a restriction enzyme site.

The cDNA encoding the α3 chain obtained as described above can be added to a known method such as Sambrook.
k, J. Et al., Molecular Cloning, A
Laboratory Manual (3rd ed
edition), Cold Spring Harbor
It is possible to make mutations using Laboratory, 2001. For example, the above G4-5 or G3
When a part or all of -5 is deleted, for example, the laminin-6-encoding nucleic acid should not include the part encoding the desired region of the G4-5 domain or G3-5 domain of the α3 chain. Synthesize appropriate primer and
DNA can be amplified using R to obtain DNA encoding a deletion mutant of the α3 chain. Alternatively,
When substituting one or more specific amino acid residues of G4-5 or G3-5 described above, known point mutation methods (Mullis, KB, In Les Prix) are used.
Nobel (ed.T.Frangsmyr), p. 1
07. Almqvist and Wilsell I
international, Stockholm, 19
93; Smith, M .; , In Les Prix
Nobel (ed.T.Frangsmyr), p. 1
23. Almqvist and Wilsell I
international, Stockholm, 19
93) may be used.

DNA encoding the obtained α3 chain mutant
Is a DNA encoding a β chain, a DNA encoding a γ chain
At the same time, a large amount of laminin protein containing the desired α3 chain mutant can be genetically engineered by introducing and expressing into E. coli, yeast, or insect animal cells using an expression vector that can be amplified in each host. You can

The gene encoding each chain may be incorporated into one expression vector for expression, or may be incorporated into a separate expression vector to express each. However, each subunit of α3 chain, β chain and γ chain is a very large polypeptide having 1000 or more amino acid residues, and the gene encoding each is also composed of 3000 or more nucleic acid residues. Thus, but not limited to, for cells expressing the desired β and γ chains, α
It is preferable to transform with a gene encoding a 3-chain subunit or a deletion substitution product thereof. A laminin heterotrimer containing the mutant α3 chain of the present invention can be obtained by genetically expressing only the α3 chain subunit thus deleted. This is simpler than the case of expressing all subunits by genetic engineering, and is particularly useful in the present invention when it is desired to artificially obtain a mutant.

Disclosed herein are the amino acid sequences of human laminin α3 chain in SEQ ID NOS: 1 and 2, and the DNA sequences encoding them. By using the sequence or a part thereof, hybridization, P
By genetic engineering techniques such as nucleic acid amplification reaction such as CR,
A gene encoding a protein having similar physiological activity can be easily isolated from other organism species. In such a case, those genes and the proteins or polypeptides encoded by the genes can also be used to obtain the deletion substitution product of the α3 chain of the present invention.

Hybridization conditions used for screening homologous genes are not particularly limited, but generally, stringent conditions are preferable, for example, 6 × SSC, 5 × Denhardt's, 0.1.
It is contemplated to use hybridization conditions such as% SDS, 25 ° C to 68 ° C. In this case, the hybridization temperature is more preferably 45 ° C to 68 ° C (without formamide) or 25 ° C.
To 50 ° C. (50% formamide). A DNA containing a base sequence having a certain homology or higher by appropriately setting hybridization conditions such as formamide concentration, salt concentration and temperature.
It is well known to those skilled in the art that H. homologous genes can be cloned, and all homologous genes thus cloned are included in the scope of the present invention.

The nucleic acid amplification reaction is carried out, for example, by a replication chain reaction (PCR) (Saiki, RK et al., Sc).
ience, 230: 1350-1354, 198.
5), ligase chain reaction (LCR) (Wu, D. et al.
Y. , And Wallace, R .; B. , Genom
ics, 4: 560-569, 1989; Barri.
Nger, K .; J. , Gene, 89: 117-12.
2, 1990; Barany, F .; , Proc. Na
tl. Acad. Sci. USA, 88: 189-19.
3, 1991) and transcription-based amplification (Kwoh, D .;
Y. et al. , Proc. Natl. Acad. S
ci. USA, 86: 1173-1177, 1989).
Etc., which require temperature circulation, as well as the strand displacement reaction (S
DA) (Walker, GT et al., Pro
c. Natl. Acad. Sci. USA, 89:39.
2-396, 1992; Walker, G .; T. et
al. , Nuc. Acids Res. 20: 169
1-1696, 1992), self-retaining sequence replication (3S
R) (Guatelli, JC, 1990, Proc.
Natl. Acad. Sci. USA 87, p. 18
74-1878) and the Qβ replicase system (Resaildi et al., BioTechnology, 6: 119.
7-1202, 1988) and the like. Also,
Nucleic acid sequence-based amplification by the competitive amplification of a target nucleic acid and a mutant sequence described in EP 0525882 (N
ucleic Acid Sequence Base
d Amplification: NASABA) reaction and the like can also be used. The PCR method is preferred.

The homologous gene cloned using the above-mentioned hybridization, nucleic acid amplification reaction, etc. has at least 5 nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 except for the regions II and III.
0% or more, preferably 60% or more, more preferably 70%
% Or more, more preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more.

The percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of the two nucleic acid sequences can be calculated as described by Devereux et al. (Nuc
l. Acids Res. , 12: 387 (198
4)) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
It may be determined by comparing the sequence information using the AP computer program, version 6.0. G
Preferred default parameters for the AP program include: (1) unary comparison matrix for nucleotides (including values of 1 for identical and 0 for non-identical), and supervision of Schwartz and Dayhoff, Atlas of Protein Sequence.
ce andStructure, National
Biomedical Research Foun
date, pp. 353-358 (1979), as described in Gribskov and Burges.
s, Nucl. Acids Res. , 14:67
45 (1986) weighted comparison matrix; (2) 3.0 penalty for each gap and an additional 0.10 penalty for each symbol in each gap; and (3).
Includes no penalty for end gaps. Other programs used by those of skill in the art for sequence comparison also
You may use.

The nucleic acid encoding the α3 chain containing the mutation can be ligated to a cloning vector, for example, a T-Easy vector (Promega, Madison, WI, USA) and verified by sequencing.
Various phenotypic selectable markers can also be used for cloning. Furthermore, the insert fragment incorporated into the cloning vector can be incorporated into an expression vector using a corresponding restriction enzyme.

The prepared expression vector is used as a cell line which expresses laminin β1 chain and γ1 chain but not α3 chain, as described above, human fiber cell line HT1080 (Mi.
zushima, H .; et al. , 1996, supra)
Etc. to produce a recombinant laminin containing the α3 chain variant. A large amount of the modified laminin α3 chain can be genetically engineered by introducing and expressing it using an expression vector that can be amplified in each host cell. Cloning and expression vectors suitable for use in bacterial, fungal, yeast, and mammalian cell hosts are described, for example, in Pouwels, P .; H. et a
l. , Cloning Vector; ALabora
tory Manual, Elsevier, NY, 1
986. In addition, the genes encoding each chain may be incorporated into one expression vector for expression, or may be incorporated into separate expression vectors to express each. Expressed recombinant laminin can be purified by a combination of molecular sieve chromatography, gelatin column chromatography and immunoaffinity chromatography using anti-laminin α3 chain antibody. The purified variant of recombinant laminin α3 chain can be analyzed by Western blotting and silver staining using anti-laminin α3 chain antibody, anti-laminin β1 chain antibody and anti-laminin γ1 chain antibody.

More specifically, first, a recombinant vector for incorporating a gene in the present invention to express a protein can be prepared using a known method. Examples of the method for incorporating the DNA fragment of the gene of the present invention into a vector such as a plasmid include, for example, Sambrook,
J. , And Russell, D .; W. , Molec
ural Cloning, A Laborator
y Manual (2nd edition), Col
d Spring Harbor Laborator
y, 1.53 (1989) and the like. For convenience, use a commercially available ligation kit (for example,
Takara Shuzo etc.) can also be used. Recombinant vector (eg, recombinant plasmid) thus obtained
Is a host cell (eg, E. coil TB1, LE
392 or XL-1 Blue etc.).

As a method for introducing a plasmid into a host cell, Sambrook, J. et al. Et al, Molecular
Cloning, A Laboratory Ma
numerical (2nd edition), Cold Sp
ring Harbor Laboratory, 1.
74 (1989), calcium chloride method or calcium chloride / rubidium chloride method, electroporation method, electroinjection method, method by chemical treatment such as PEG, method using gene gun and the like.

The vector can be conveniently prepared by ligating a desired gene to a recombinant vector (for example, plasmid DNA etc.) available in the art by a conventional method. Specific examples of the vector used include plasmids derived from E. coli, such as pB
luescript, pUC18, pUC19, pBR
However, the present invention is not limited to these.

The expression vector is particularly useful for the purpose of producing the desired protein. The type of expression vector is not particularly limited as long as it has a function of expressing a desired gene in various host cells such as prokaryotic cells and / or eukaryotic cells, and producing a desired protein. Expression vector, pQE-30, pQ
E-60, pMAL-c2, pMAL-p2, pSE4
20 or the like is preferable, and pYE is used as an expression vector for yeast.
S2 (Saccharomyces), pPIC3.5K, pP
IC9K, pAO815 (above Pichia), pBacPAK8 / 9, pBK283 as an insect expression vector,
Preferred are pVL1392, pBlueBac4.5 and the like.

Examples of mammalian expression vectors include:
Okayama and Berg (Mol. Cell Bi
ol. , 3: 280, 1983). A system useful for stable, high-level expression of mammalian cDNA in C127 mouse mammary epithelial cells has been essentially constructed by Cosman et al. (Mol.
unol. 23: 935, 1986). Alternatively, in vivo or in
Examples of the vector for expressing in a neuron in vitro include an adenovirus vector or a pEF-BOS vector (Mizushima,
S. et al. , Nucleic Acid Re
s. 18: p. 5322, 1990) modified vector (pEF-CITE-neo: Miyata, S. et al.
et al. , Clin. Exp. Metastasi
s, 16: 613-622, 1998).

The transformant can be prepared by introducing a desired expression vector into a host cell. The host cell used is not particularly limited as long as it is compatible with the expression vector and can be transformed, and a natural cell usually used in the technical field of the present invention, or an artificially established recombinant cell. Various cells such as cells can be used. For example, bacteria (Escherichia, Bacillus), yeast (Saccharomyces, Pichia, etc.), animal cells, insect cells, plant cells and the like can be mentioned. Specifically, E. coli (M15, JM109, BL2
1), yeast (INVSc1 (genus Saccharomyces),
Examples thereof include GS115, KM71 (above Pichia) and insect cells (BmN4, silkworm larva, etc.). ,
Examples of animal cells include mouse-derived cells, Xenopus-derived cells, rat-derived cells, hamster-derived cells, monkey-derived cells, human-derived cells, and cultured cell lines established from these cells. Furthermore, the plant cells are not particularly limited as long as cell culture is possible, and examples thereof include cells derived from tobacco, Arabidopsis, rice, corn, wheat and the like. Preferably, laminin β1
Cell line that expresses a chain and a γ1 chain but not an α3 chain,
For example, the human fiber cell line HT1080 (Mizushi
ma, H.M. et al. , 1996, supra), the human keratinocyte cell line HaCaT (Boukamp, P. e.
t al. J. Cell Biol. , 106: 76
1-771, 1988), etc.

When bacteria, in particular Escherichia coli, are used as host cells, the expression vector is generally composed of at least a promoter / operator region, a start codon, a gene encoding a desired antibacterial protein, a stop codon, a terminator and a replicable unit. .

When yeast, plant cells, animal cells or insect cells are used as host cells, the expression vector generally comprises at least a promoter, a start codon, a gene encoding the desired antibacterial protein, a stop codon and a terminator. Preferably. In addition, it may appropriately contain a DNA encoding a signal peptide, an enhancer sequence, 5 ′ and 3 ′ untranslated regions of a desired gene, a selectable marker region, a replicable unit and the like.

In the vector of the present invention, an example of a suitable initiation codon is methionine codon (ATG). Examples of the stop codon include commonly used stop codons (eg, TAG, TGA, TAA, etc.).

A replication-competent unit is the entire D in a host cell.
It means a DNA capable of replicating the NA sequence, and includes natural plasmids, artificially modified plasmids (plasmids prepared from natural plasmids), synthetic plasmids and the like. Suitable plasmids include E. In E. coli, the plasmids pQE30, pET or pCAL or their artificial modifications (pQE3
0, pET or pCAL) and a plasmid pYES2 or pPIC9K in yeast, and a plasmid pBacPAK8 / 9 in insect cells.

As the enhancer sequence and the terminator sequence, those normally used by those skilled in the art such as those derived from SV40 can be used.

As the selectable marker, a commonly used one can be used by a conventional method. Examples include tetracycline, ampicillin, and antibiotic resistance genes such as kanamycin or neomycin, hygromycin, and spectinomycin.

An expression vector is prepared by linking at least the above promoter, start codon, gene encoding the desired antimicrobial protein, stop codon, and terminator region in a continuous and circular fashion to a suitable replicable unit. can do. At this time, if desired, an appropriate DNA fragment (eg, linker, other restriction enzyme site, etc.) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.

The expression vector can be introduced into a host cell by a conventionally known method. For example, bacteria (E. coli, Bacillus subtilis
Etc.), the method of Cohen et al. (Proc.
Natl. Acad. Sci. USA, 69: 211
0,1972), the protoplast method (Mol. Gen.
Genet. , 168: 111, 1979) and the competent method (J. Mol. Biol., 56: 209, 19).
71) by Saccharomyces cere
In the case of visiae, for example, the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA, 7
5: 1927, 1978) and the lithium method (J. Bact).
eriol. , 153: 163, 1983),
In the case of plant cells, for example, the leaf disk method (Scie)
No., 227: 129, 1985), electroporation method (Nature, 319: 791, 198).
According to 6), in the case of animal cells, for example, Graham
Method (Virology, 52: 456, 197)
3) In the case of insect cells, for example, the method of Summers et al. (Mol. Cell Biol., 3: 2156-2).
165, 1983).

Purification and isolation of the protein or polypeptide of the present invention can be carried out by conventional methods for protein purification and isolation such as ammonium sulfate precipitation, ion exchange chromatography (Mono Q, Q Sepharose or DEAE, etc.). Can be appropriately combined and performed. For example, when laminin containing the modified laminin α3 chain of the present invention accumulates in host cells, the host cells are collected by an operation such as centrifugation or filtration, and this is collected in an appropriate buffer solution (for example, at a concentration of 10 M or A buffer solution such as about 100 mM Tris buffer solution, phosphate buffer solution, HEPES buffer solution, MES buffer solution, etc. After being suspended in a pH range of 5.0 to 9.0, although it depends on the buffer solution to be used) , Destroy the cells by a method suitable for the host cell used,
The contents of the host cell are obtained by centrifugation. On the other hand, when the laminin-5 protein of the present invention is secreted outside the host cell, the host cell and the medium are separated by an operation such as centrifugation or filtration to obtain a culture filtrate. The host cell disruption solution or the culture filtrate can be used for protein purification and isolation as it is, or after ammonium sulfate precipitation and dialysis.
Examples of the purification / isolation method include the following methods. That is, 6x histidine or GS
When a tag such as T or maltose binding protein is attached, a method by affinity chromatography suitable for each commonly used tag can be mentioned. On the other hand, when the protein of the present invention is produced without adding such a tag, for example, a method by antibody affinity chromatography can be mentioned. In addition to this, a method of combining ion exchange chromatography, gel filtration, hydrophobic chromatography, isoelectric focusing chromatography and the like can also be mentioned.

Although a method for expressing laminin-6 containing an α3 chain in which the G4-5 domain has been deleted by genetic engineering has been described, it is artificially modified as long as it has the characteristics described in this specification. It may be a naturally-occurring protein instead of a protein. Alternatively, a naturally-occurring protein may be modified at the protein level.

Cellular Assay The effect of the purified short chain laminin-6 of the present invention on cells may be tested in any suitable assay. Assays include cell adhesion activity, cell dispersal activity or cell motility activity assays.

The assay of cell adhesion activity is performed by the method described previously (Mizushima, H. et al.,
1997, supra). Specifically, as described in Example 2 described later, laminin α3
Laminin-6, including chain deletion mutants or wild type, was coated at varying concentrations on cell culture plates,
Thereafter, a cell culture line, for example, a buffalo rat liver cell line BRL, a human keratinocyte cell line HaCaT,
Alternatively, the human fibrosarcoma cell line HT1080 is seeded, and the cell adhesion activity is measured by comparing the cell adhesion rate after a predetermined time with the case of coating with wild-type laminin.

The assay of cell-dispersing activity was carried out by the method described previously (Hirosaki, T. et al., 2).
000, above). Specifically, as described in Example 2 described later, after seeding the cell suspension on a cell culture plate, the purified laminin α3 chain variant and wild type were added at a predetermined concentration, and after a predetermined time, Measure the degree of cell dispersion.

The cell motility activity can be determined by the method described previously (Hirosaki, T. et al., 2000, supra). Specifically, as described in Examples described later, laminin α3 chain mutants and wild type are coated at various concentrations on a plate for cell culture, and then, a cell culture strain, for example, a buffalo rat. Hepatocyte cell line BRL, human keratinocyte HaCa
T, or human epidermal cell (keratinocyte)
Sow. At this time, as an inhibition assay, monoclonal antibodies against different types of integrins may be pre-incubated with the cells for a predetermined time prior to seeding the cells. After cell seeding, cell movement can be observed under a microscope to quantify the cell migration distance, and the migration speed can be measured from the trajectory of cell migration after a predetermined time using a time-lapse video equipment. The cell dispersal activity is associated with cell motility activity.

Laminin-6 or short-chain laminin-6
In the present invention, a short-chain laminin-6 (LN6ΔG4-
5 and LN6ΔG3-5) are wild-type laminin-6.
It was revealed to have an effect of promoting cell adhesion function and / or cell motility activity as compared with (LN6).

Specifically, as described in Example 2 which will be described later, a short-chain laminin-6 composed of each subunit of α3 chain, β1 chain and γ1 chain in which G4-5 domain is deleted.
When comparing cell adhesion activity with wild type (LN6) using (LN6ΔG4-5), the effective concentration (ED ₅₀ ) for half maximal activity of LN6ΔG4-5 is about 1.
It was 5 times higher. This is because the G4-5 domain of the α3 chain is L
It is shown to contain an important site of cell adhesion activity to N6. Furthermore, LN6ΔG4-5 exhibited cell-dispersing activity at a lower concentration than LN6, and its activity was about 2-fold.

Therefore, the present invention has revealed for the first time that cell adhesion activity and / or cell motility activity can be independently regulated by a deletion mutant of LN6, and a composition containing an effective LN6 or short chain laminin-6. It is to provide things. In one preferred embodiment of the composition of the present invention, preferably short-chain laminin is mixed with other laminin, more preferably short-chain laminin is mixed with LN5, and even more preferably mat-LN5 is mixed. You may include it. Most preferably, it is a composition containing short-chain laminin alone.

LN5 is known and is composed of α3 chain, β3 chain and γ2 chain, and is secreted as a trimer. LN
After secretion, 5 undergoes limited degradation by a protease between the G3 and G4 domains of the α3 chain, and a truncated (or mature) LN5 (mat-L) having no G4-5 domain.
N5) (Colognato, H.,
and Yurchenco, P.M. D. , 2000, supra). The sequence of LN5 is described in Ryan, M .; C. Et al (above)
The LN5 protein can be prepared on the basis of these sequences, as disclosed by Dr.

The composition containing LN6 or short-chain laminin-6 is useful as a pharmaceutical composition. The composition of the present invention can be used for maintaining or treating the functions of epithelial tissue, nerve tissue and muscle. Alternatively, it can be used for skin transplantation, treatment of retinal detachment and epidermolytic varicella, or for dentures. It can also be used to promote. On the other hand, a composition containing a short-chain laminin-6, which is a motion-promoting LN6, can be used in a process requiring cell migration such as repair of injury or wound after surgery, and also for repair of nerves. It can be used.

The composition of the present invention is administered in vivo, in
It may be used for controlling cell adhesion function and / or cell motility activity in vitro or ex vivo, and especially for promoting wound healing based on regulation of epithelial cell function. The composition of the present invention can be used as a transplant device, a tissue or cell culture device for transplant, or an adhesive device for cell transplant. The composition of the present invention can be used for, but not limited to, skin transplantation for treating burns and the like and pancreatic β cell transplantation for treating diabetes. When used for transplantation, the mixing ratio of laminin-6 or laminin-6 deletion mutant contained in the composition may be changed.

The composition of the present invention can be used, for example, by immobilizing it on an incubator such as a plastic plate or a petri dish and bringing it into contact with target cells, as described in Example 2 below. As the incubator, for example, 96-well EL
ISA plate (Costar, Cembridge,
MA, USA) and the like can be used, but the present invention is not limited thereto.

The composition of the present invention is preferably, but not limited to, 0.001 μg / ml to 10 μg when used in vitro or ex vivo.
It can be used at a concentration of / ml.

The composition of the present invention is also useful as a cosmetic composition. The pharmaceutical or cosmetic composition of the present invention comprises a therapeutically effective amount of laminin containing said variant of the α3 chain in a mixture with a pharmaceutically acceptable carrier. The composition of the invention may be administered systemically or locally, preferably intravenously, subcutaneously, intramuscularly and parenterally. Preparation of a laminin solution that can be parenterally administered can be performed within the technical scope of those skilled in the art in consideration of pH, isotonicity, safety and the like.

Dosage regimens of the compositions of the present invention may vary according to the effect of the drug, such as the nature and / or severity of the patient's symptoms, weight, sex, diet, time of administration, and other clinical effects. Factors can be considered and determined by the attending physician. One of ordinary skill in the art can determine the dose of the composition of the present invention based on these factors.

The composition of the present invention can be used by further applying it to a transplant body at the time of skin transplantation and transplanting it in vivo. Specifically, laminin-6 alone or laminin containing a laminin-6 deletion mutant is directly or coated on an appropriate support, or is embedded in a collagen gel or a biodegradable polymer gel. When applied in vivo as a transplant, it is possible to control cell adhesion function and / or cell motility activity.

Alternatively, laminin-6 or laminin-6
By preparing an expression vector incorporating a gene encoding a deletion mutant and expressing it at a target site in vivo, or by transporting cells incorporating the expression vector to the target site, It is possible to control cell adhesion function and / or cell motility activity.

In another embodiment, the composition of the present invention is included,
It can be used as a kit for regulating cell adhesion activity and / or cell motility activity. The kit may optionally include a cell culture container, manufacturer's instructions, and the like. References 1. Aumailley, M .; , And Rous
selle, P.P. : Laminins of the
dermo-epidemal junction.
Matrix Biol. , 18, p. 19-28
(1999) 2. Aumailley, M .; , And Krie
g, T. : Laminins: a family
of diversity multifunctiona
l molecules of base m
embranes. J. Invest. Dermat
ol. , 106, p. 209-214 (1996) 3. Boukamp, P .; , Petrussevs
ka, R.K. T. Breitkreutz, D .; ，
Hornung, J. et al. Markham, A .; , A
nd Fusenig, N.N. E. : Normal k
eratinization in a sponta
neoly immortalized anneup
loid human keratinocyte c
ellline. J. Cell Biol. , 10
6, p. 761-771 (1988) 4. Carter, W.C. G. Ryan, M .;
C. , And Gahr, P .; J. : Epilig
rin, a new cell adhesionli
gand for integrin alpha 3
beta 1 in epithelial base
ment membranes. Cell, 65,
p. 599-610 (1991) 5. Champliaud, M .; F. , Lunst
rum, G.R. P. Roussel, P .; , Ni
shiyama, T .; , Keene, D .; R. , A
nd Burgeson, R.M. E. : Human a
mionconstraints a novel la
minin variant, laminin 7,
while like laminin 6, co
valently associated with
laminin 5 topromote stabl
e epithelial-stromal atta
chemnt. J. Cell Biol. , 132,
p. 1189-1198 (1996) 6. Colognato, H .; , And Yurc
henco, P.H. D. : Form and func
section: the laminin family
of heterotrimers. Dev. Dy
n. 218, p. 213-234 (2000) 7. Engel, J .; : Electron mic
rocopy of extracellular cellar m
atrix components. Methods
Enzymol. 245, p. 469-488 (1
994) 8. Gagnaux-Palacios, L. et al. , A
llegra, M .; , Spirito, F .; , Pom
meret, O.M. , Romero, C .; , Orton
ne, J. P. , And Meneguzzi,
G. : Theshart arm of the l
amin gamma 2 chain play
sa personal roll in the i
n corporation of laminin 5
into the extracellular m
atrix and in cell adhesio
n. J. Cell Biol. , 153, p. 835
-850 (2001) 9. Giancotti, F.M. G. , And Ro
uslahti, E .; : Integrin sign
aling. Science, 285, p. 1028
-1032 (1999) 10. Giannelli, G .; , Falk-Ma
rzillier, J .; Schiraldi, O .; ，
Stettler-Stevenson, W.M. G. , A
nd Quaranta, V.N. : Instruction
of cell migration by mat
rix metalloprotease-2 cle
average of laminin-5. Scien
ce, 277, p. 225-228 (1997) 11. Goldfinger, L .; E. , Stac
k, M. S. , And Jones, J .; C. : P
processing of laminin-5 an
d it's functional conseque
nces: role of plasma and and
tissue-type plasmaminogen
activator. J. Cell Biol. , 1
41, p. 255-265 (1998) 12. Grishkovskaya, I .; , Avv
akumov, G .; V. Sklenar, G .; ，
Dales, D.D. Hammond, G .; L. , A
nd Mullre, Y .; A. : Crystals
structure of human sex hor
mone-binding globulin: st
eroid transport by a lami
ninG-like domain. EMBO
J. , 19, p. 504-512 (2000) 13. Hirosaki, T .; , Mizushima
a, H.A. , Tsubota, Y .; , Moriyam
a, K. , And Miyazaki, K .; : St
realistic requirement of c
arboxyl-terminal global
domains of laminin alpha
3 chain for promotion of
rapid cell adhesion and
migration by laminin-5.
J. Biol. Chem. 275, p. 22495-
22502 (2000) 14. Hohenester, E .; , Tisi,
D. Talts, J .; F. , And Timp
1, R.I. : The crystal structure
re of a laminin G-like mo
dule revs the molecule
r basis of alpha-dystrogl
ycan binding to laminins,
perlecan, and agrin. Mo
l. Cell, 4, p. 783-792 (1999) 15. Howe, A .; Aplin, A .; E. ，
Alahari, S .; K. , And Julian
o, R. L. : Integrin signalin
g and cell growh contro
l. Curr. Opin. Cell Biol. ，
10, p. 220-231 (1998) 16. Kikkawa, Y .; Umeda, M .; ，
and Miyazaki, K .; : Marked
stimulation of cell adhes
ion and mobility by ladsi
n, a laminin-like scatter
factor. J. Biochem. (Tokyo
o), 116, p. 862-869 (1994) 17. Koshikawa, N .; , Giannel
i, G. Cirulli, V .; , Miyazak
i, K. , And Quaranana, V .; : R
ole of cell surface metal
loprotase MT1-MMP in epi
the cell cell migration ov
er laminin-5. J. Cell Bio
l. , 148, p. 615-624 (2000) 18. Koshikawa, N .; , Moriyam
a, K. , Takamura, H .; , Mizush
ima, H .; , Nagashima, Y .; , Yano
ma, S.M. , And Miyazaki, K .; : O
verexpresion of laminin
gamma 2 chain monomer in
invading gastric carcinom
a cells. Cancer Res. , 59,
p. 5596-5601 (1999) 19. Libby, R .; T. , Champliau
d, M. F. Claudepierre, T .; ，
Xu, Y. , Gibbons, E .; P. , Koch,
M. Burgeon, R .; E. , Hunte
r, D. D. , And Brunken, W .; J. :
Laminin expression in adu
lt and developing retina
e: evidence of two novel
CNS laminins. J. Neurosc
i. , 20, p. 6517-6528 (2000) 20. Marinokovich, M .; P. , Lu
nstrum, G.N. P. , And Burgeso
n, R.N. E. : The anchoring fila
ment protein kalinin is s
ynthesized and secreted a
sa high molecular weight
precursor. J. Biol. Chem. , 2
67, p. 17900-17906, 1992 21. Miyata, S .; , Miyagi, Y .; ，
Koshikawa, N .; , Nagashima,
Y. , Kato, Y. , Yasumitsu,
H. Hirahara, F .; , Misugi,
K. , And Miyazaki, K .; : Stim
ulation of cellular growt
h and adhesion to fibrone
ctin and vitronectin in c
ultra and tumorigenicity
in nude mice by overexpr
session of trypsinogen in
human gastric cancer cell
s. Clin. Exp. Metastasis, 1
6, p. 613-622 (1998) 22. Kaori Miyazaki, Hiroto Mizushima, Tomomi Hirosaki "Interaction between cancer cells and extracellular matrix in metastasis-especially laminin 5
Function of ", Experimental Medicine, Vol. 16, No. 1
6 (Supplement), pp. 114-119 (1998) 23. Miyazaki, K .; , Kikkawa,
Y. , Nakamura, A .; , Yasumits
u, H. , And Umeda, M .; : Alarg
e cell-adhesion scatter f
actorprotected by human ga
tric carcinomacells. Pro
c. Natl. Acad. Sci. USA, 90, p.
11767-11771 (1993) 24. Mizushima, H .; , Koshika
wa, N.W. Moriya, K .; , Takam
ura, H .; , Nagashima, Y .; , Hira
hara, F .; , And Miyazaki, K .; :
Wide distribution of lami
nin-5 gamma 2 chain in ba
semen membranes of vario
ushuman diseases. Horm. Re
s. , 50 (Suppl. 2), p. 7-14 (199
8) 25. Mizushima, H .; , Takamur
a, H.A. , Miyagi, Y .; , Kikkawa,
Y. , Yamanaka, N .; , Yasumits
u, H. , Misugi, K .; , And Miya
zaki, K .; : Identification o
f integrin-dependent and
-Independent cell adhesio
n domains in COOH-termina
l global region of lami
nin-5 alpha 3 chain. Cell
Growth Differ. , 8, p. 979-98
7 (1997) 26. Mizushima, H .; , Miyagi,
Y. Kikkawa, Y .; , Yamanaka,
N. , Yasumitsu, H .; , Misugi,
K. , And Miyazaki, K .; : Diff
erential expression of la
minin-5 / ladsin subunits i
n human issues and cancel
r lines and ther induci
on by tumor promoter and
growth factors. J. Bioche
m. (Tokyo), 120, p. 1196-1202
(1996) 27. Nielsen, P.M. K. , Gho, Y.
S. Hoffman, M .; P. , Watanab
e, H .; , Makino, M .; , Nomizu,
M. , And Yamada, Y .; : Identity
ficationof a major hepari
n and cell binding site in
the LG4 module of the la
minin alpah 5 chain. J. Bi
ol. Chem. 275, p. 14517-1452
3 (2000) 28. Nomiz, M .; , Kim. , W. H. ，
Yamamura, K .; , Utami, A .; , S
ong, S.N. Y. , Otaka, A .; , Rolle
r, P. P. Kleinman, H .; K. , An
d Yamada, Y. : Identificati
on of cell bindings i
n the laminin alpha 1 cha
incarboxyl-terminal globu
lar domain bysystematics
creating of synthetic pepper
tides. J. Biol. Chem. , 270,
p. 20583-20590 (1995) 29. Pickkarine, T .; , Kallun
ki, T .; , AndTryggvason, K .; :
Human laminin B2 chain. C
omparison of the complete
aminoacid sequence with
the B1 chain reveals vari
availability in sequence homol
ody between different str
rectual domains. J. Biol. C
hem. , 263, p. 6751-6758 (199
8) 30. Rousselle, P.M. , And Aum
airley, M.A. : Kalinin is more
efficient than laminin i
n promoting admission of p
rimarykeratinocytes and s
ome other epithelial cell
s and has a different req
unity for integran rec
eptors. J. Cell Biol. , 125,
p. 205-214 (1994) 31. Roussel, P.M. , Lunstru
m, G.M. P. , Keene, D .; R. , And B
Urgeson, R .; E. : Kalinin: an
epithelium-specific basem
ent membrane adhesion mol
e that that is a component
of anchoring filaments.
J. Cell Biol. 114, p. 567-57
6 (1991) 32. Rudenko, G .; , Nguyen,
T. , Celiah, Y .; , Sudhof,
T. C. , And Deisenhofer, J .; :
The structure of the lig
and-binding domain of neu
rexin I beta: regulation
of LNS domain function by
alternate splicing. Ce
11, 269, p. 93-101 (1999) 33. Ryan, M .; C. Tizzard, R .; ，
Van Devanter, D.M. R. , And Ca
rter, W.A. G. : Cloning of the
LamA3 gene encoding the a
lpha 3 chain of the adhesi
ve ligand epiglirin. Expr
session in wound report.
J. Biol. Chem. , 269, p. 22779-
22787 (1994) 34. Talts, J. et al. F. , Andac, Z. ，
Gohring, W.M. , Blancaccio,
A. , And Timpl, R .; : Binding
of the G domains of lami
nin alpha 1 and alpha 2 c
heins and perlecanto hepa
rin, sulfatides, alpha-sy
stroglycan and general ex
tracellular matrix protei
ns. EMBO J.M. , 18, p. 863-870
(1999) 35. Timpl, R.A. Tisi, D .; , Ta
Its, J. et al. F. , Andac, Z .; , Sasak
i, T. , And Hohenester, E .; :
Structure and function of
laminin LG modules. Matr
ix Biol. , 19, p. 309-317 (200
0) 36. Tisi, D.M. Talts, J .; F. ，
Timpl, R.A. , And Hohenester,
E. : Structure of the C-ter
minal laminin G-like domain
in pair of the laminin al
pha 2 chain harvesting bi
nding sites for alpha-dys
troglycan and heparin. EM
BO J. , 19, p. 1434-1440 (200
0) 37. Tsubota, Y. , Mizushima
a, H.A. , Hirosaki, T .; , Higash
i, S. , Yasumitsu, H .; , And M
iyazaki, K .; : Isolation and
activity of proteolytic
fragment of laminin-5 alp
ha 3 chain. Biochem. Bioph
ys. Res. Commun. , 278, p. 614-
620 (2000) 38. Verrando, P .; , Pisani,
A. , And Ortonne, J .; P. : The
new basement membrane
igen recognized by the mo
noclonal antibody GB3 is
a large size glycoprotei
n: modulation of its expr
session by retinoic acid.
Biochem. Biophys. Acta. , 94
2, 45-56 (1988) 39. Vuolteenaho, R .; , Chow,
L. T. , AndTryggvason, K .; : S
structure of the human lam
inin B1 chain gene. J. Bio
l. Chem. , 265, 15611-15616 (1
990)

[0090]

EXAMPLES The present invention will be described below with reference to examples.
The examples are illustrative and not limiting of the invention. The scope of the present invention should be determined based on the claims. Furthermore, those skilled in the art can easily make corrections and changes based on the description of the present specification.

Materials and Methods In the examples of the present invention, unless otherwise stated, the following materials and methods were followed.

(1) Materials such as Antibodies Mouse monoclonal antibody against human laminin α3 chain (LSα3c4) and mouse monoclonal antibody against γ2 chain (D4B5) were prepared by the method previously disclosed (Hirosaki, T. et a.
l. J. Biol. Chem. , 275: 22495
-22502, 2000; Mizushima, H .;
et al. , Horm. Res. , 2: 7-14,1
998). Rabbit polyclonal antibodies against the G4 domain of laminin α3 were prepared in our laboratory (Tsubota, Y. et al., Bioc.
hem. Biophys. Res. Commun. , 2
78: 614-620, 2000; Hirosak.
i, T. et al. , 2000, supra). Other antibodies used were obtained from: Human laminin β3 specific mouse monoclonal antibody, Transduct.
Ion Laboratories (Lexingto
n, KY); rat monoclonal antibody against laminin β1 chain (VNR147), mouse monoclonal antibody against human laminin γ1 chain (2E8) and human
Mouse monoclonal antibody (P1D6) against integrin α5 subunit, Chemicon Int
international Inc. (Temecula,
CA); a mouse monoclonal antibody against the integrin α2 subunit (P1D6), an antibody against the α3 subunit (P1B5), and an antibody against the β1 subunit (P4C10), Life Technology.
genes; rat monoclonal antibody against human integrin α6 (G0H3), PharMingen
(San Diego, CA).

Mouse EHS-Laminin (Laminin-1;
LN1), human laminin-10 / 11 (LN10 / 1
1), and human plasma fibronectin is Transd
action Laboratories, and Iwa
Purchased from ki Glass (Tokyo, Japan) respectively.

(2) Cell culture cells and cell culture The human fibrosarcoma cell line HT1080 was prepared using JCRB (Japan).
nese Collection of Rear
ch Bioresources) cell bank (Tokyo,
I got it from Japan. Buffalo rat
The rat-derived epidermal cell line BRL has been used in previous studies (Miyazaki, K. et a.
l. , Proc. Natl. Acad. Sci. U.
S. A. , 90: 11767-11771, 1993;
Hirosaki, T .; et al. , 2000, supra). Spontaneously immortalized human keratinocyte cell line HaCaT (Boukamp, P. et al.,
J. Cell Biol. , 106: 761-771,
1988), N. E. Dr. Fusenig (Deut
sches Krebsforshungszentr
um, Heidelberg, Germany). The integrin expression profile of transduced cells was verified by flow cytometry before the assay.

All cell types were prepared as follows: Dulbecco's modified Eagle medium: Ham's F12 medium 1: 1 mixed medium (DME / F12) (Life Te) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin sulfate.
chnologies, Gaithersburg, M
Maintained in D).

Preparation of conditioned medium HT was added to DME / F12 medium containing 10% FBS.
1080 and transduced HT1080 cells were grown to confluence and their cultures washed with Ca ²⁺ and Mg ²⁺ free phosphate buffered saline (PBS (−)) then serum free medium. Incubated overnight. These media were then replaced with fresh serum-free media and incubated for 2 days. The resulting conditioned medium (C
M) was recovered, clarified by centrifugation and dialyzed against pure water. The dialyzed CM was lyophilized and dissolved in 1/50 volume of PBS (-).

Example 1 Laminin-6 and short chain laminin
Expression and Purification of Nin-6 Laminin-6 has an α3 chain in common with laminin-5, and in laminin-5, it is secreted extracellularly by undergoing proteolytic action between G3 and G4 domains (Tsubot).
a, Y. et al. , 2000, supra), while laminin-6 is secreted with almost no proteolytic action (Fig. 1). So far, laminin-6 having α3 chain
The physiological activity of is not clear. Since it is known that cleavage of G4-5 domain is important for activation of laminin-5, preparation of short-chain laminin-6 in which G4-5 was cleaved in order to investigate physiological activity of laminin-6. Tried.

Full-length cDNA of human laminin α3 chain
(LS / CX WT: nucleotides 1-5 of SEQ ID NO: 1
139) and the following two G4-5 and G3-5 deletion mutants, respectively, were constructed: ΔG4-5 deleted from G4-5 (amino acid residue 1 of SEQ ID NO: 2). -1365); and ΔG lacking G3-5
3-5 (amino acid residues 1-1153 of SEQ ID NO: 2).

(1) Preparation of expression plasmid Specifically, an expression plasmid encoding the short chain laminin-6 of the present invention was prepared as follows.

First, a human gastric cancer cell cDNA library (Mizushima, H. et al., J. B.
iochem. (Tokyo), 120: 1196-1
202, 1996), a cDNA clone LS / CX encoding human laminin α3 chain was isolated. The c
TaKaRa ExTaq using DNA clone as a template
Polymerase chain reaction (PCR) was performed using (Takara Shuzo Co., Ltd., Kyoto, Japan) to prepare expression plasmids encoding various recombinant laminin mutants. PC used
The R primers are as follows (underlines indicate restriction sites; 1) and 3): BamHI, 2) to 4): Xb.
aI):

[0101]

Embedded image 1) G3-5 ′ 5′-CA G GAT CC A GTG GTG TCG TTA GA-3 ′ (SEQ ID NO: 7) 1125-Ser Gly Val Val Arg-1129 (nucleotides 3373-3387 of SEQ ID NO: 1) sense) 2) G3X-3 '5' -GG T CTA GA T CAT CCA TGA TTG GCC TG-3' ( SEQ ID NO: 8) 1365-Gly His Asn Ala Gln-1361 ( SEQ ID NO: 1 nucleotides 4081-4095 , Antisense) 3) G2-5 '5'-CA G GAT CC G TTC TGA GCT TGT AC-3' (SEQ ID NO: 9) 951-Val Leu Ser Leu Tyr-955 (nucleotides 2851 to 2865 of SEQ ID NO: 1) , Sense) 4) G2X-3 ′ 5′-CC T CTA GA T CAG GAG AAT GAG GCA GA-3 ′ (SEQ ID NO: 10) 1153-Ser Phe Ser Ala Ser-1149 (nucleotides 3445-3459 of SEQ ID NO: 1) , Antisense) Each antisense primer has a stop codon (TG
A) followed by XbaI site (TCTAGA, underlined)
Is included. Primers 1 and 2 are LN6ΔG4-
5 and primers 3 and 4 were used to make LN6ΔG3-5. The PCR product was added to the T-Easy vector (Pr
(Omega, Madison, WI, USA) and verified by sequencing.

Then, Cl derived from pGEM-G3X was used.
The aI / SphI fragment and the XhoI / SphI fragment derived from pGEM-G2X were respectively clas of LS / CX.
Ligated to the I / SacI site, and pGEM
PGEM-ΔG4-5 and pGEM-ΔG3-5 were created by inserting into the corresponding restriction sites of -LS / CX.

In addition, the XbaI insert of each deletion mutant was subsequently prepared in order to prepare an expression vector.
Mammalian expression vector pEF-BOS-CITE which is a modified version of pEF-BOS-CITE NEO
NEO2 (Miyata, S. et al., Cli.
n. Exp. Metastasis, 16: 613-6.
22, 1998) at the XbaI site.

(2) Expression of various laminin-6 proteins Wild type (W of human laminin α3 chain prepared in (1))
T), various expression vectors encoding G4-5 domain deletion mutants and G3-5 domain deletion mutants, human fibrosarcoma cell line expressing laminin β1 and γ1 chains but not α3 chains HT1080 (Mizush
ima, H .; et al. , 1996, supra) by the known calcium phosphate method, 500 μg /
Selected with ml G418 (Gibco). Each recombinant laminin-6 protein containing the deletion mutation is then
Purified by a combination of molecular sieve chromatography, gelatin column chromatography and immunoaffinity chromatography using anti-laminin α3 chain antibody. The purified recombinant protein was analyzed by Western blotting using anti-laminin α3, β1 and γ1 chain antibodies and silver staining (FIG. 1).
And Figure 2).

Specifically, first, laminin-6 protein secreted by HT1080 / WT transduced cells was purified from serum-free culture supernatant by essentially the same method as previously reported (Hirosaki, T. ., Et
al. , 2000, supra). Serum-free culture supernatant was transferred to roller bottles (Becton Dickinson, Fr.
was prepared as described above using anklin Lakes, NJ, USA). 80% of collected culture supernatant
Subjected to ammonium sulfate precipitation at saturation. HT
Serum-free conditioned medium of 1080 / WT cells was a Sepharose 4B column (Amersham Pharmacia).
Fractionation by molecular sieve chromatography on Biotech) followed by a heparin-sepharose column. Protein bound to the heparin column was eluted with 0.5M NaCl and then 1.0M
Elute with NaCl. Fibronectin was removed from each fraction by passing through a gelatin-sepharose column,
Then, it was subjected to two types of immunoaffinity chromatography.

To isolate the laminin-6 protein, the 1.0 M NaCl eluate from the heparin column was added to the 1 column.
Dilute 0-fold to reduce NaCl concentration, then
Sepharose HPLC column (Amersham Ph
Armacia Biotech). Those eluted between 0.3 and 0.4 M NaCl were passed through a D4B5-Sepharose column and coexpressed with laminin-.
5 proteins were removed. Finally, the unbound one was passed through a LSα3c4-Sepharose immunoaffinity column. Bound laminin-6 was eluted with 0.05% trifluoroacetic acid and immediately neutralized. Laminin α
LN-6 mutant (LN6Δ4-5) lacking the G4-5 domain of 3 chains and L lacking the G3-5 domain
N-6 mutant (LN6Δ3-5) was HT
Purified from conditioned medium of 1080 / ΔG4-5 and HT1080 / ΔG3-5 cells by essentially the same method as described above for LN-6.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed as previously described (Hirosaki, T. e.
tal. , 2000, supra). Two-dimensional electrophoresis analysis
It was carried out according to the method previously reported (Koshikawa).
a, N.A. et al. , Cancer Res. , 5
9: 5596-5601, 1999). One-dimensional SDS-
PAGE was performed on non-reducing conditions on a 4% polyacrylaminodo gel disc gel (2 mm diameter, 70 mm length). After electrophoresis, the gel was incubated in SDS sample buffer containing 5% 2-mercaptoethanol for 15 minutes at room temperature, placed on a 6% polyacrylamide slab gel (width 85 mm, thickness 1 mm, length 70 mm) and then two-dimensional. SDS-PAGE was started. Subsequently, the proteins separated on the slab gel were subjected to the immunoblotting method.

When human laminin α3 chain cDNA was transduced into human fiber cell line HT1080, exogenous α
The 3 chain assembles with the endogenous β3 and γ2 chains to form α3β3γ2
Were previously found to produce the LN5 heterotrimer of H. hirosaki (Hirosaki, T. et al., 200).
0, above). HT1080 transformant (HT1080 /
The conditioned medium of (WT) is 190 kDa and 160 kDa α
Includes both 3 chains. The 160 kDa α3 chain is associated with the G3 and G4 domains (Tsubota, Y. et al., 2
000, above).
For LN-5, only those with the 160 kDa α3 chain were purified from the conditioned medium. Antibodies to the laminin β3 chain and γ2 chain were isolated from conditioned medium at an α of 160 kDa.
Three chains are precipitated, but one with 190 kDa does not.
This suggests the presence of laminin containing α3 other than LN5.

In the present invention, conditioned medium of HT1080 / WT cells was analyzed by two-dimensional SDS-PAGE and then immunoblotted with antibodies against laminin α3 and γ2 chains. On the first dimension non-reducing SDS-PAGE, the α3 chain has at least four different molecular sizes, namely 1,000 kDa or more (top of gel), 600 k
It was isolated as Da, 450 kDa and 400 kDa (FIG. 1A). The 450 kDa and 400 kDa α3 chains bind to the γ2 chain, which is 150 kDa and 10 respectively.
It was shown to be an LN5 body having γ2 of 5 kDa (FIG. 1B). These results show that in the LN5 molecule,
It shows that the 190 kDa α3 chain was completely converted to the mature form of 160 kDa.

On the other hand, the α3 chain in the 600 kDa complex did not bind to the γ2 chain (FIG. 1B). On the second dimension reduced SDS-PAGE, the α3 chain at this position is 19
It was separated into a large spot of 0 kDa and a small spot of 160 kDa. These results show that the 190 kDa α3
The chain is LN6 (α3β1γ1) or LN7 (α3β2γ)
It was suggested that it could exist as 1). This result also indicates that the proteolytic processing of the laminin α3 chain is H
It means that it occurs preferentially to LN5 in T1080 / WT cells.

To identify a 600 kDa complex containing the laminin α3 chain, the present inventor used HT1080 / WT.
An attempt was made to isolate isomers of laminin from the conditioned medium of. The conditioned medium was fractionated by molecular sieve chromatography followed by heparin affinity chromatography. 190 kDa and 160 kDa α
Both 3 chains bound to the heparin column, but 190 kD
The α3 chain of a was eluted with 1.0 M NaCl, while 16
The 0 kDa α3 chain was eluted mainly with 0.5 M NaCl (data not shown). The difference in affinity for heparin was consistent with our previous observation that the G4-5 fragment of the α3 chain bound tightly to the heparin column (T
subota, Y .; et al. , 2000, supra).
The eluted 190 kDa α3 chain was further subjected to anion exchange H
Purified by PLC, then passed through an anti-laminin γ2 antibody column to remove LN5. Finally, anti-laminin α
It was purified by an immunoaffinity column to which 3 antibody was bound.

The purified laminin-6 was treated with SDS-PAG.
Analysis was performed by immunoblotting using E and 5 kinds of antibodies. The isolated and purified protein is about 60 below the mouse laminin 1 under non-reducing conditions.
It migrated as a single band of 0 kDa (Fig. 2A). Laminin of 600 kDa has a molecular weight of 220, 210 and 190 kDa in reduced SDS-PAGE 3.
It was divided into two bands (Fig. 2B), and these bands were respectively identified as laminin β1, γ1 and α3 chains (Fig. 2C). Neither the anti-β3 antibody nor the anti-γ2 antibody reacted (Fig. 2C). These results indicate that the purified 600 kDa laminin is laminin-6. LN5 purified by immunoaffinity chromatography using anti-γ2 antibody has
It has only the α3 chain of Da, and β3 of 135 kDa.
Chain and a 150/105 kDa γ2 chain (hereinafter sometimes referred to as mature LN5, “mat-LN5”) (FIG. 2B, C).

The present inventor has also determined that heparin column 0.5
A small amount of LN6 with a 160 kDa α3 chain was purified from the M NaCl eluate. The 160 kDa α3 chain did not react with antibodies to the G4 domain (data not shown). The 160 kDa α3 chain is cleaved between the G3 and G4 domains, and the 160 kDa α of mat-LN5.
It is shown to be the same as the 3 chain. Therefore, the present inventor tried to purify the HT1080 / ΔG4-5 cells from the conditioned medium in high yield. A laminin 6 mutant lacking the G4 and G5 domains (LN6ΔG4-5) was purified by the same purification method as described above for LN-6. Furthermore, when analyzed by SDS-PAGE under non-reducing conditions, 220 kDa β1 chain and 210 kDa γ
It was composed of one chain and an α3 chain of 160 kDa (Fig. 2B, C). In addition, the present inventor has found that HT1080 / Δ
L36 lacking G3, G4 and G5 from G3-5 cells
A mutant (LN6ΔG3-5) was prepared. Example 2 Cell Adhesion Activity of Deletion Mutants of Laminin-6
, Cell dispersal activity and cell motility activity LN6, LN6ΔG4-5 and L prepared in Example 1
N6ΔG3-5 at various concentrations in HT1080 cells and B
The cell adhesion activity, cell dispersion activity and cell motility activity on RL cells were examined.

Cell Adhesion Assay The cell adhesion assay was performed as previously described (Hi.
rosaki, T .; et al. , 2000, supra).
Briefly, 96-well microtiter plates (Corning Costar, Cembridge).
e, Acton, MA, USA) 50 μl of each well
Substrate solution (0.05-50 μg / ml LN6, LN
6ΔG4-5, LN6ΔG3-5, mat-LN5, mouse LN1, LN10 / 11, and fibronectin C
a ²⁺ and Mg ²⁺ free phosphate buffered saline (Dulbe
cco's PBS (−); 8 g / l NaCl, 0.
2 g / l KCl, 1.15 g / l Na ₂ HPO ₄ ,
0.2 g / l KH ₂ PO ₄ )) was coated overnight at 4 ° C. Plate these plates at 37 ° C. for 1.5 hours, P
Blocked with 200 μl / well of 1.2% (w / v) bovine serum albumin in BS (−). After treating the cells with trypsin, the cells were washed once with serum-free DMEM / F12 medium,
The cells were suspended in a serum-free medium at a cell density of 2 × 10 ⁵ cells / ml, and 100 μl of the cell suspension was spread on the plate. Three
2.5% of adherent cells after incubation at 7 ° C
Fixed with glutaraldehyde, 0.005% Hoechst 33342-0.001% Triton X-10
Stained at 0 for 1.5 hours. The fluorescence intensity of each well of the plate was measured by CytoFluor 2350 Fluorometer (M
illipore, Bedford, MA, USA). Blank values corresponding to empty wells were automatically subtracted.

FIG. 3 shows HT on plastic cuprates precoated with matrix proteins at different concentrations.
Shows the adhesive activity of 1080 cells (Figure 3A) and BRL cells (Figure 3B). mat-LN5, LN6 and LN6ΔG
4-5 efficiently promoted cell adhesion, but LN6ΔG3
-5 did not promote much. LN6ΔG4-5 and L
A significant difference in cell adhesion activity between N6ΔG3-5 was
The G3 domain indicates that LN6 is essential for high affinity binding to integrins. Comparing the effective concentrations (ED ₅₀ ) for half maximal cell adhesion activity, L
N6ΔG4-5 was about 1.5 times lower than LN6.

The morphology of HT1080 cells adhered on each substrate was observed (FIG. 4). mat-LN5, LN6 and L
Although N6ΔG4-5 promotes high adhesive activity of HT1080 cells as shown above, the morphology of adherent cells was different in LN6 and LN6ΔG4-5. On mat-LN5 as a control, 20 minutes after initiation of cell adhesion, significant membrane ruffling was observed around the cells, and cell surface protrusions were formed even at 60 minutes. Sufficient spreading was also observed in cells on LN6ΔG4-5. In contrast to this, on LN6 substrates, H
T1080 cells barely spread and expressed marked spike formation or small protrusions with little polarity even at 60 minutes. These results indicate that processing of the laminin α3 chain converts LN6 to its active form in cell adhesion activity.

Cell Dispersion Assay Dispersion of BRL cells by purified laminin-6 and laminin-6 deletion mutants was assayed as previously reported (Hirosaki, T. et al., 200.
0, above). Briefly, 500 μl of cell suspension (7,000 cells / well in DME / F12 + 1% FBS) was added to a 24-well plate (Sumibe Medi).
cal, Tokyo, Japan) and then seeded with substrate solution (final concentration 10-200 ng / ml LN6, L
N6ΔG4-5, LN6ΔG3-5 and mat-LN5
Ca ²⁺ and Mg ²⁺ free phosphate buffered saline) was added directly into the culture and incubated at 37 ° C. Two days later, the cells were stained by Giemsa staining and the total number of cells and the number of dispersed single cells were determined in three randomly selected microscopic fields. The degree of cell dispersion was expressed as the percentage of single cells within each field.

Alternatively, a 24-well plate was subjected to various substrate solutions (0.01 to 1 μg / ml) of LN6 and LN6Δ.
Ca of G4-5, LN6ΔG3-5 and mat-LN5
²⁺ and Mg ²⁺ free phosphate buffered saline),
After seeding the cells, the degree of cell dispersion after 2 days of culture was measured in the same manner as above.

LN5 promotes cell dispersal and migration of many cell lines much more potently than other extracellular matrix proteins (Miyazaki, K. et al., 1).
993, supra; Kikkawa, Y .; et al. ，
J. Biochem. , 116: 862-829, 19
94). These activities were confirmed by two LN6 bodies and mat-L.
Assayed for N5. 1% FBS for each laminin
When added directly to a culture of BRL cells in a medium containing C, typical cell dispersal was only observed with mat-LN5 (FIG. 5). mat-LN5 is 10 ng /
It showed cell dispersion at concentrations less than ml, while LN
Both 6 and LN6ΔG4-5 did not show even 200 ng / ml (FIG. 6A).

Cell-dispersion activity was assayed on plastic plates precoated with various concentrations of laminin, and mat-LN5 promoted significant cell-dispersion (FIG. 6B). LN6ΔG4-5 induced a weak cell-dispersing activity, but LN6 hardly induced it (FIG. 6B).

Cell Motility Assay 24-well plates were plated at 500 μl / well of substrate solution (0.01-5 μg / ml, LN6, LN6 as described above).
Phosphate buffered saline of ΔG4-5 and mat-LN5 (Dulbecco's PBS (−); 8 g / l N
aCl, 0.2 g / l KCl, 1.15 g / l Na
₂ HPO ₄ , 0.2 g / l KH ₂ PO ₄ )) and then blocked with 1 ml / well of bovine serum albumin. 500 μl cell suspension (DME / F12 + 1% F
Each well was seeded with 7,000 cells / well in BS) and incubated at 37 ° C. for 1 hour to allow cells to adhere to the substrate. Cell migration, as previously reported (H
irosaki, T .; et al. , 2000, above),
Time-lapse video equipment (Sony, Tokyo, Japan) was used to monitor for 10 hours at 37 ° C. The cell migration rate was quantified by following the migration of nuclei of each cell, and the migration distance was measured with a video micrometer (VM-30, Olympus, Tokyo, Japan). As a result of measuring the migration rate of BRL cells on the plate coated with laminin using a video microscope, mat-LN5 induced the highest cell migration, but LN6 did not induce cell migration (FIG. 6C). LN6ΔG4-5 induced cell migration depending on the dose in the culture medium, but to a lesser extent than mat-LN5. This is because LN6ΔG4-5 is mat-
Despite having a G4-5 deficient α3 subunit like LN5, we show that cell migration by LN6ΔG4-5 is significantly lower than that of mat-LN5. Comparing cell motility, LN6ΔG4-5 showed L
It was about twice as high as N6. Example 3 Laminin-6 Deletion Mutant and Integrin
Interaction with LN5 and two LN6 bodies (LN6 and LN6ΔG4-
The integrin requirement of 5) was examined using a function-shielding anti-integrin antibody. For inhibition assays,
Cell suspensions prepared in the cell adhesion assay were preincubated with function blocking anti-integrin antibody or heparin for 20 minutes at room temperature. 100 μl of cell suspension was added to each well of the plate and then incubated at 37 ° C. for 1 hour. Non-adherent cells were removed by gentle agitation and adherent cells were fixed with 2.5% (v / v) glutaraldehyde. Then 0.0005% Hoechst 33342-0.001% Triton X-100
Was used for staining for 1.5 hours at room temperature. The fluorescence intensity of each well of the plate was measured by CytoFluor 2350 Fluorometer (Millipore, Bedford, MA, US).
It was measured in A). Blank values corresponding to empty wells were automatically subtracted.

As previously reported (Mizushim
a, H.A. et al. , CellGrowth Dif
fer. , 8: 979-987, 1997), mat-
The cell adhesion activity of LN5 was significantly blocked by the anti-integrin α3 antibody and almost completely blocked by the anti-integrin β1 antibody. Although the effect of anti-integrin α6 antibody on cell adhesion activity was small, it effectively blocked cell adhesion in the presence of anti-integrin α3 antibody (FIG. 7A). Almost the same result was observed for LN6 (FIG. 7B) and LN6ΔG4-5 (FIG. 7C). HT1080 cells have integrin β4 on the cell surface
No subunit expression (Carter, WG et al.
al. , Cell, 65: 599-610, 199.
1). Therefore, this result shows that the integrin α3β1
And α6β1 are the first and second (or accessory) receptors for laminin in HT1080 cells, respectively. These results are consistent with the fact that LN5 binds to these integrins via the C-terminal globular region of the α3 chain (Hirosaki, T. et.
al. , 2000, supra; Mizushima,
H. et al. , 1997, supra). It can be said that the presence or absence of the G4 and G5 domains in the α3 chain of LN6 has substantially no effect on the integrin binding specificity of LN6.

[0123]

[Sequence list] SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> Composition comprising Laminin-6 for regulating cellular adhesion activity and / or cell migration activity <130> 012796 <160> 10 <210> 1 <211> 5433 <212> DNA <213> Human <220> <221> CDS <222> (1) ... (5139) <300> <301> Ryan, M.C., Tizard, R., VanDevanter, D.R. And Carter, W.G. <302> Cloning of the LamA3 gene encoding the alpha 3 chain of the       adhesive ligand epiligrin. Expression in wound repair <303> JOURNAL J. Biol. Chem. <304> 269 <305> 36 <306> 22779-22787 <307> 1994 <400> 1 atg gga tgg ctg tgg atc ttt ggg gca gcc ctg ggg cag tgt ctg 45 Met Gly Trp Leu Trp Ile Phe Gly Ala Ala Leu Gly Gln Cys Leu   1 5 10 15 ggc tac agt tca cag cag caa agg gtg cca ttt ctt cag cct ccc 90 Gly Tyr Ser Ser Gln Gln Gln Arg Val Pro Phe Leu Gln Pro Pro                  20 25 30 ggt caa agt caa ctg caa gcg agt tat gtg gag ttt aga ccc agc 135 Gly Gln Ser Gln Leu Gln Ala Ser Tyr Val Glu Phe Arg Pro Ser                  35 40 45 cag ggt tgt agc cct gga tac tat cgg gat cat aaa ggc ttg tat 180 Gln Gly Cys Ser Pro Gly Tyr Tyr Arg Asp His Lys Gly Leu Tyr                  50 55 60 acc gga cgg tgt gtt ccc tgc aat tgc aac gga cat tca aat caa 225 Thr Gly Arg Cys Val Pro Cys Asn Cys Asn Gly His Ser Asn Gln                  65 70 75 tgc cag gat ggc tca ggc ata tgt gtt aac tgt cag cac aac acc 270 Cys Gln Asp Gly Ser Gly Ile Cys Val Asn Cys Gln His Asn Thr                  80 85 90 gcg gga gag cac tgt gaa cgc tgc cag gag ggc tac tat ggc aac 315 Ala Gly Glu His Cys Glu Arg Cys Gln Glu Gly Tyr Tyr Gly Asn                  95 100 105 gcc gtc cac gga tcc tgc agg gcc tgc cca tgt cct cac act aac 360 Ala Val His Gly Ser Cys Arg Ala Cys Pro Cys Pro His Thr Asn                 110 115 120 agc ttt gcc act ggc tgt gtg gtg aat ggg gga gac gtg cgg tgc 405 Ser Phe Ala Thr Gly Cys Val Val Asn Gly Gly Asp Val Arg Cys                 125 130 135 tcc tgc aaa gct ggg tac aca gga aca cag tgt gaa agg tgt gca 450 Ser Cys Lys Ala Gly Tyr Thr Gly Thr Gln Cys Glu Arg Cys Ala                 140 145 150 ccg gga tat ttc ggg aat ccc cag aaa ttc gga ggt agc tgc caa 495 Pro Gly Tyr Phe Gly Asn Pro Gln Lys Phe Gly Gly Ser Cys Gln                 155 160 165 cca tgc agt tgt aac agc aat ggc cag ctg ggc agc tgt cat ccc 540 Pro Cys Ser Cys Asn Ser Asn Gly Gln Leu Gly Ser Cys His Pro                 170 175 180 ctg act gga gac tgc ata aac caa gaa ccc aaa gat agc agc cct 585 Leu Thr Gly Asp Cys Ile Asn Gln Glu Pro Lys Asp Ser Ser Pro                 185 190 195 gca gaa gaa tgt gat gat tgc gac agc tgt gtg atg acc ctc ctg 630 Ala Glu Glu Cys Asp Asp Cys Asp Ser Cys Val Met Thr Leu Leu                 200 205 210 aac gac ctg gcc acc atg ggc gag cag ctc cgc ctg gtc aag tct 675 Asn Asp Leu Ala Thr Met Gly Glu Gln Leu Arg Leu Val Lys Ser                 215 220 225 cag ctg cag ggc ctg agt gcc agc gca ggg ctt ctg gag cag atg 720 Gln Leu Gln Gly Leu Ser Ala Ser Ala Gly Leu Leu Glu Gln Met                 230 235 240 agg cac atg gag acc cag gcc aag gac ctg agg aat cag ttg ctc 765 Arg His Met Glu Thr Gln Ala Lys Asp Leu Arg Asn Gln Leu Leu                 245 250 255 aac tac cgt tct gcc att tca aat cat gga tca aaa ata gaa ggc 810 Asn Tyr Arg Ser Ala Ile Ser Asn His Gly Ser Lys Ile Glu Gly                 260 265 270 ctg gaa aga gaa ctg act gat ttg aat caa gaa ttt gag act ttg 855 Leu Glu Arg Glu Leu Thr Asp Leu Asn Gln Glu Phe Glu Thr Leu                 275 280 285 caa gaa aag gct caa gta aat tcc aga aaa gca caa aca tta aac 900 Gln Glu Lys Ala Gln Val Asn Ser Arg Lys Ala Gln Thr Leu Asn                 290 295 300 aac aat gtt aat cgg gca aca caa agc gca aaa gaa ctg gat gtg 945 Asn Asn Val Asn Arg Ala Thr Gln Ser Ala Lys Glu Leu Asp Val                 305 310 315 aag att aaa aat gtc atc cgg aat gtg cac att ctt tta aag cag 990 Lys Ile Lys Asn Val Ile Arg Asn Val His Ile Leu Leu Lys Gln                 320 325 330 atc tct ggg aca gat gga gag gga aac aac gtg cct tca ggt gac 1035 Ile Ser Gly Thr Asp Gly Glu Gly Asn Asn Val Pro Ser Gly Asp                 335 340 345 ttt tcc aga gag tgg gct gaa gcc cag cgc atg atg agg gaa ctg 1080 Phe Ser Arg Glu Trp Ala Glu Ala Gln Arg Met Met Arg Glu Leu                 350 355 360 cgg aac agg aac ttt gga aag cac ctc aga gaa gca gaa gct gat 1125 Arg Asn Arg Asn Phe Gly Lys His Leu Arg Glu Ala Glu Ala Asp                 365 370 375 aaa agg gag tcg cag ctc ttg ctg aac cgg ata agg acc tgg cag 1170 Lys Arg Glu Ser Gln Leu Leu Leu Asn Arg Ile Arg Thr Trp Gln                 380 385 390 aaa acc cac cag ggg gag aac aat ggg ctt gct aac agt atc cgg 1215 Lys Thr His Gln Gly Glu Asn Asn Gly Leu Ala Asn Ser Ile Arg                 395 400 405 gat tct tta aat gaa tac gaa gcc aaa ctc agt gac ctt cgt gct 1260 Asp Ser Leu Asn Glu Tyr Glu Ala Lys Leu Ser Asp Leu Arg Ala                 410 415 420 cgg ctg cag gag gca gct gcc caa gcc aag cag gca aat ggc ttg 1305 Arg Leu Gln Glu Ala Ala Ala Gln Ala Lys Gln Ala Asn Gly Leu                 425 430 435 aac caa gaa aac gag aga gct ttg gga gcc att cag aga caa gtg 1350 Asn Gln Glu Asn Glu Arg Ala Leu Gly Ala Ile Gln Arg Gln Val                 440 445 450 aaa gaa ata aat tcc ctg cag agt gat ttc acc aag tat cta acc 1395 Lys Glu Ile Asn Ser Leu Gln Ser Asp Phe Thr Lys Tyr Leu Thr                 455 460 465 act gca gac tca tct ttg ttg caa acc aac att gcg ctg cag ctg 1440 Thr Ala Asp Ser Ser Leu Leu Gln Thr Asn Ile Ala Leu Gln Leu                 470 475 480 atg gag aaa agc cag aag gaa tat gaa aaa tta gct gcc agt tta 1485 Met Glu Lys Ser Gln Lys Glu Tyr Glu Lys Leu Ala Ala Ser Leu                 485 490 495 aat gaa gca aga caa gaa cta agt gac aaa gta aga gaa ctt tcc 1530 Asn Glu Ala Arg Gln Glu Leu Ser Asp Lys Val Arg Glu Leu Ser                 500 505 510 aga tct gct ggc aaa aca tcc ctt gtg gag gag gca gaa aag cac 1575 Arg Ser Ala Gly Lys Thr Ser Leu Val Glu Glu Ala Glu Lys His                 515 520 525 gcg cgg tcc tta caa gag ctg gca aag cag ctg gaa gag atc aag 1620 Ala Arg Ser Leu Gln Glu Leu Ala Lys Gln Leu Glu Glu Ile Lys                 530 535 540 aga aac gcc agc ggg gat gag ctg gtg cgc tgt gct gtg gat gcc 1665 Arg Asn Ala Ser Gly Asp Glu Leu Val Arg Cys Ala Val Asp Ala                 545 550 555 gcc acc gcc tac gag aac atc ctc aat gcc atc aaa gcg gcc gag 1710 Ala Thr Ala Tyr Glu Asn Ile Leu Asn Ala Ile Lys Ala Ala Glu                 560 565 570 gac gca gcc aac agg gct gcc agt gca tct gaa tct gcc ctc cag 1755 Asp Ala Ala Asn Arg Ala Ala Ser Ala Ser Glu Ser Ala Leu Gln                 575 580 585 aca gtg ata aag gaa gat ctg cca aga aaa gct aaa acc ctg agt 1800 Thr Val Ile Lys Glu Asp Leu Pro Arg Lys Ala Lys Thr Leu Ser                 590 595 600 tcc aac agt gat aaa ctg tta aat gaa gcc aag atg aca caa aag 1845 Ser Asn Ser Asp Lys Leu Leu Asn Glu Ala Lys Met Thr Gln Lys                 605 610 615 aag cta aag caa gaa gtc agt cca gct ctc aac aac cta cag caa 1890 Lys Leu Lys Gln Glu Val Ser Pro Ala Leu Asn Asn Leu Gln Gln                 620 625 630 acc ctg aat att gtg aca gtt cag aaa gaa gtg ata gac acc aat 1935 Thr Leu Asn Ile Val Thr Val Gln Lys Glu Val Ile Asp Thr Asn                 635 640 645 ctc aca act ctc cga gat ggt ctt cat ggg ata cag aga ggt gat 1980 Leu Thr Thr Leu Arg Asp Gly Leu His Gly Ile Gln Arg Gly Asp                 650 655 660 att gat gct atg atc agt agt gca aag agc atg gtc aga aag gcc 2025 Ile Asp Ala Met Ile Ser Ser Ala Lys Ser Met Val Arg Lys Ala                 665 670 675 aac gac atc aca gat gag gtt ctg gat ggg ctc aac ccc atc cag 2070 Asn Asp Ile Thr Asp Glu Val Leu Asp Gly Leu Asn Pro Ile Gln                 680 685 690 aca gat gtg gaa aga att aag gac acc tat ggg agg aca cag aac 2115 Thr Asp Val Glu Arg Ile Lys Asp Thr Tyr Gly Arg Thr Gln Asn                 695 700 705 gaa gac ttc aaa aag gct ctg act gat gca gat aac tcg gtg aat 2160 Glu Asp Phe Lys Lys Ala Leu Thr Asp Ala Asp Asn Ser Val Asn                 710 715 720 aag tta acc aac aaa cta cct gat ctt tgg cgc aag att gaa agt 2205 Lys Leu Thr Asn Lys Leu Pro Asp Leu Trp Arg Lys Ile Glu Ser                 725 730 735 atc aac caa cag ctg ttg ccc ttg gga aac atc tct gac aac atg 2250 Ile Asn Gln Gln Leu Leu Pro Leu Gly Asn Ile Ser Asp Asn Met                 740 745 750 gac aga ata cga gaa cta att cag cag gcc aga gat gct gcc agt 2295 Asp Arg Ile Arg Glu Leu Ile Gln Gln Ala Arg Asp Ala Ala Ser                 755 760 765 aag gtt gct gtc ccc atg agg ttc aat ggt aaa tct gga gtc gaa 2340 Lys Val Ala Val Pro Met Arg Phe Asn Gly Lys Ser Gly Val Glu                 770 775 780 gtc cga ctg cca aat gac ctg gaa gat ttg aaa gga tat aca tct 2385 Val Arg Leu Pro Asn Asp Leu Glu Asp Leu Lys Gly Tyr Thr Ser                 785 790 795 ctg tcc ttg ttt ctc caa agg ccc aac tca aga gaa aat ggg ggt 2430 Leu Ser Leu Phe Leu Gln Arg Pro Asn Ser Arg Glu Asn Gly Gly                 800 805 810 act gag aat atg ttt gtg atg tac ctt gga aat aaa gat gcc tcc 2475 Thr Glu Asn Met Phe Val Met Tyr Leu Gly Asn Lys Asp Ala Ser                 815 820 825 cgg gac tac atc ggc atg gca gtt gtg gat ggc cag ctc acc tgt 2520 Arg Asp Tyr Ile Gly Met Ala Val Val Asp Gly Gln Leu Thr Cys                 830 835 840 gtc tac aac ctg ggg gac cgt gag gct gaa ctc caa gtg gac cag 2565 Val Tyr Asn Leu Gly Asp Arg Glu Ala Glu Leu Gln Val Asp Gln                 845 850 855 atc ttg acc aag agt gag act aag gag gca gtt atg gat cgg gtg 2610 Ile Leu Thr Lys Ser Glu Thr Lys Glu Ala Val Met Asp Arg Val                 860 865 870 aaa ttt cag aga att tat cag ttt gca agg ctt aat tac acc aaa 2655 Lys Phe Gln Arg Ile Tyr Gln Phe Ala Arg Leu Asn Tyr Thr Lys                 875 880 885 gga gcc aca tcc agt aaa cca gaa aca ccc gga gtc tat gac atg 2700 Gly Ala Thr Ser Ser Lys Pro Glu Thr Pro Gly Val Tyr Asp Met                 890 895 900 gat ggt aga aat agc aat aca ctc ctt aat ttg gat cct gaa aat 2745 Asp Gly Arg Asn Ser Asn Thr Leu Leu Asn Leu Asp Pro Glu Asn                 905 910 915 gtt gta ttt tat gtt gga ggt tac cca cct gat ttt aaa ctt ccc 2790 Val Val Phe Tyr Val Gly Gly Tyr Pro Pro Asp Phe Lys Leu Pro                 920 925 930 agt cga cta agt ttc cct cca tac aaa ggt tgt att gaa tta gat 2835 Ser Arg Leu Ser Phe Pro Pro Tyr Lys Gly Cys Ile Glu Leu Asp                 935 940 945 gac ctc aat gaa aat gtt ctg agc ttg tac aac ttc aaa aaa aca 2880 Asp Leu Asn Glu Asn Val Leu Ser Leu Tyr Asn Phe Lys Lys Thr                 950 955 960 ttc aat ctc aac aca act gaa gtg gag cct tgt aga agg agg aag 2925 Phe Asn Leu Asn Thr Thr Glu Val Glu Pro Cys Arg Arg Arg Lys                 965 970 975 gaa gag tca gac aaa aat tat ttt gaa ggt acg ggc tat gct cga 2970 Glu Glu Ser Asp Lys Asn Tyr Phe Glu Gly Thr Gly Tyr Ala Arg                 980 985 990 gtt cca act caa cca cat gct ccc atc cca acc ttt gga cag aca 3015 Val Pro Thr Gln Pro His Ala Pro Ile Pro Thr Phe Gly Gln Thr                 995 1000 1005 att cag acc acc gtg gat aga ggc ttg ctg ttc ttt gca gaa aac 3060 Ile Gln Thr Thr Val Asp Arg Gly Leu Leu Phe Phe Ala Glu Asn                1010 1015 1020 ggg gat cgc ttc ata tct cta aat ata gaa gat ggc aag ctc atg 3105 Gly Asp Arg Phe Ile Ser Leu Asn Ile Glu Asp Gly Lys Leu Met                1025 1030 1035 gtg aga tac aaa ctg aat tca gag cta cca aaa gag aga gga gtt 3150 Val Arg Tyr Lys Leu Asn Ser Glu Leu Pro Lys Glu Arg Gly Val                1040 1045 1050 gga gac gcc ata aac aac ggc aga gac cat tcg att cag atc aaa 3195 Gly Asp Ala Ile Asn Asn Gly Arg Asp His Ser Ile Gln Ile Lys                1055 1060 1065 att gga aaa ctc caa aag cgt atg tgg ata aat gtg gac gtt caa 3240 Ile Gly Lys Leu Gln Lys Arg Met Trp Ile Asn Val Asp Val Gln                1070 1075 1080 aac act ata att gat ggt gaa gta ttt gat ttc agc aca tat tat 3285 Asn Thr Ile Ile Asp Gly Glu Val Phe Asp Phe Ser Thr Tyr Tyr                1085 1090 1095 ctg gga gga att cca att gca atc agg gaa aga ttt aac att tct 3330 Leu Gly Gly Ile Pro Ile Ala Ile Arg Glu Arg Phe Asn Ile Ser                1100 1105 1110 acg cct gct ttc cga ggc tgc atg aaa aat ttg aag aaa acc agt 3375 Thr Pro Ala Phe Arg Gly Cys Met Lys Asn Leu Lys Lys Thr Ser                1115 1120 1125 ggt gtc gtt aga ttg aat gat act gtg gga gta acc aaa aag tgc 3420 Gly Val Val Arg Leu Asn Asp Thr Val Gly Val Thr Lys Lys Cys                1130 1135 1140 tcg gaa gac tgg aag ctt gtg cga tct gcc tca ttc tcc aga gga 3465 Ser Glu Asp Trp Lys Leu Val Arg Ser Ala Ser Phe Ser Arg Gly                1145 1150 1155 gga caa ttg agt ttc act gat ttg ggc tta cca cct act gac cac 3510 Gly Gln Leu Ser Phe Thr Asp Leu Gly Leu Pro Pro Thr Asp His                1160 1165 1170 ctc cag gcc tca ttt gga ttt cag acc ttt caa ccc agt ggc ata 3555 Leu Gln Ala Ser Phe Gly Phe Gln Thr Phe Gln Pro Ser Gly Ile                1175 1180 1185 tta tta gat cat cag aca tgg aca agg aac ctg cag gtc act ctg 3600 Leu Leu Asp His Gln Thr Trp Thr Arg Asn Leu Gln Val Thr Leu                1190 1195 1200 gaa gat ggt tac att gaa ttg agc acc agc gat agc ggc ggc cca 3645 Glu Asp Gly Tyr Ile Glu Leu Ser Thr Ser Asp Ser Gly Gly Pro                1205 1210 1215 att ttt aaa tct cca cag acg tat atg gat ggt tta ctg cat tat 3690 Ile Phe Lys Ser Pro Gln Thr Tyr Met Asp Gly Leu Leu His Tyr                1220 1225 1230 gta tct gta ata agc gac aac tct gga cta cgg ctt ctc atc gat 3735 Val Ser Val Ile Ser Asp Asn Ser Gly Leu Arg Leu Leu Ile Asp                1235 1240 1245 gac cag ctt ctg aga aat agc aaa agg cta aaa cac att tca agt 3780 Asp Gln Leu Leu Arg Asn Ser Lys Arg Leu Lys His Ile Ser Ser                1250 1255 1260 tcc cgg cag tct ctg cgt ctg ggc ggg agc aat ttt gag ggt tgt 3825 Ser Arg Gln Ser Leu Arg Leu Gly Gly Ser Asn Phe Glu Gly Cys                1265 1270 1275 att agc aat gtt ttt gtc cag agg tta tca ctg agt cct gaa gtc 3870 Ile Ser Asn Val Phe Val Gln Arg Leu Ser Leu Ser Pro Glu Val                1280 1285 1290 cta gat ttg acc agt aac tct ctc aag aga gat gtg tcc ctg gga 3915 Leu Asp Leu Thr Ser Asn Ser Leu Lys Arg Asp Val Ser Leu Gly                1295 1300 1305 ggc tgc agt tta aac aaa cca cct ttt cta atg ttg ctt aaa ggt 3960 Gly Cys Ser Leu Asn Lys Pro Pro Phe Leu Met Leu Leu Lys Gly                1310 1315 1320 tct acc agg ttt aac aag acc aag act ttt cgt atc aac cag ctg 4005 Ser Thr Arg Phe Asn Lys Thr Lys Thr Phe Arg Ile Asn Gln Leu                1325 1330 1335 ttg cag gac aca cca gtg gcc tcc cca agg agc gtg aag gtg tgg 4050 Leu Gln Asp Thr Pro Val Ala Ser Pro Arg Ser Val Lys Val Trp                1340 1345 1350 caa gat gct tgc tca cca ctt ccc aag acc cag gcc aat cat gga 4095 Gln Asp Ala Cys Ser Pro Leu Pro Lys Thr Gln Ala Asn His Gly                1355 1360 1365 gcc ctc cag ttt ggg gac att ccc acc agc cac ttg cta ttc aag 4140 Ala Leu Gln Phe Gly Asp Ile Pro Thr Ser His Leu Leu Phe Lys                1370 1375 1380 ctt cct cag gag ctg ctg aaa ccc agg tca cag ttt gct gtg gac 4185 Leu Pro Gln Glu Leu Leu Lys Pro Arg Ser Gln Phe Ala Val Asp                1385 1390 1395 atg cag aca aca tcc tcc aga gga ctg gtg ttt cac acg ggc act 4230 Met Gln Thr Thr Ser Ser Arg Gly Leu Val Phe His Thr Gly Thr                1400 1405 1410 aag aac tcc ttt atg gct ctt tat ctt tca aaa gga cgt ctg gtc 4275 Lys Asn Ser Phe Met Ala Leu Tyr Leu Ser Lys Gly Arg Leu Val                1415 1420 1425 ttt gca ctg ggg aca gat ggg aaa aaa ttg agg atc aaa agc aag 4320 Phe Ala Leu Gly Thr Asp Gly Lys Lys Leu Arg Ile Lys Ser Lys                1430 1435 1440 gag aaa tgc aat gat ggg aaa tgg cac acg gtg gtg ttt ggc cat 4365 Glu Lys Cys Asn Asp Gly Lys Trp His Thr Val Val Phe Gly His                1445 1450 1455 gat ggg gaa aag ggg cgc ttg gtt gtg gat gga ctg agg gcc cgg 4410 Asp Gly Glu Lys Gly Arg Leu Val Val Asp Gly Leu Arg Ala Arg                1460 1465 1470 gag gga agt ttg cct gga aac tcc acc atc agc atc aga gcg cca 4455 Glu Gly Ser Leu Pro Gly Asn Ser Thr Ile Ser Ile Arg Ala Pro                1475 1480 1485 gtt tac ctg gga tca cct cca tca ggg aaa cca aag agc ctc ccc 4500 Val Tyr Leu Gly Ser Pro Pro Ser Gly Lys Pro Lys Ser Leu Pro                1490 1495 1500 aca aac agc ttt gtg gga tgc ctg aag aac ttt cag ctg gat tca 4545 Thr Asn Ser Phe Val Gly Cys Leu Lys Asn Phe Gln Leu Asp Ser                1505 1510 1515 aaa ccc ttg tat acc cct tct tca agc ttc ggg gtg tct tcc tgc 4590 Lys Pro Leu Tyr Thr Pro Ser Ser Ser Phe Gly Val Ser Ser Cys                1520 1525 1530 ttg ggt ggt cct ttg gag aaa ggc att tat ttc tct gaa gaa gga 4635 Leu Gly Gly Pro Leu Glu Lys Gly Ile Tyr Phe Ser Glu Glu Gly                1535 1540 1545 ggt cat gtc gtc ttg gct cac tct gta ttg ttg ggg cca gaa ttt 4680 Gly His Val Val Leu Ala His Ser Val Leu Leu Gly Pro Glu Phe                1550 1555 1560 aag ctt gtt ttc agc atc cgc cca aga agt ctc act ggg atc cta 4725 Lys Leu Val Phe Ser Ile Arg Pro Arg Ser Leu Thr Gly Ile Leu                1565 1570 1575 ata cac atc gga agt cag ccc ggg aag cac tta tgt gtt tac ctg 4770 Ile His Ile Gly Ser Gln Pro Gly Lys His Leu Cys Val Tyr Leu                1580 1585 1590 gag gca gga aag gtc acg gcc tct atg gac agt ggg gca ggt ggg 4815 Glu Ala Gly Lys Val Thr Ala Ser Met Asp Ser Gly Ala Gly Gly                1595 1600 1605 acc tca acg tcg gtc aca cca aag cag tct ctg tgt gat gga cag 4860 Thr Ser Thr Ser Val Thr Pro Lys Gln Ser Leu Cys Asp Gly Gln                1610 1615 1620 tgg cac tcg gtg gca gtc acc ata aaa caa cac atc ctg cac ctg 4905 Trp His Ser Val Ala Val Thr Ile Lys Gln His Ile Leu His Leu                1625 1630 1635 gaa ctg gac aca gac agt agc tac aca gct gga cag atc ccc ttc 4950 Glu Leu Asp Thr Asp Ser Ser Tyr Thr Ala Gly Gln Ile Pro Phe                1640 1645 1650 cca cct gcc agc act caa gag cca cta cac ctt gga ggt gct cca 4995 Pro Pro Ala Ser Thr Gln Glu Pro Leu His Leu Gly Gly Ala Pro                1655 1660 1665 gcc aat ttg acg aca ctg agg atc cct gtg tgg aaa tca ttc ttt 5040 Ala Asn Leu Thr Thr Leu Arg Ile Pro Val Trp Lys Ser Phe Phe                1670 1675 1680 ggc tgt ctg agg aat att cat gtc aat cac atc cct gtc cct gtc 5085 Gly Cys Leu Arg Asn Ile His Val Asn His Ile Pro Val Pro Val                1685 1690 1695 act gaa gcc ttg gaa gtc cag ggg cct gtc agt ctg aat ggt tgt 5130 Thr Glu Ala Leu Glu Val Gln Gly Pro Val Ser Leu Asn Gly Cys                1700 1705 1710 cct gac cag 5139 Pro Asp Gln        1713 taacccaagc ctatttcaca gcaaggaaat tcaccttcaa aagcactgat 5189 tacccaatgc acctccctcc ccagctcgag atcattcttc aattaggaca 5239 caaaccagac aggtttaata gcgaatctaa ttttgaattc tgaccatgga 5289 tacccatcac tttggcattc agtgctacat gtgtatttta tataaaaatc 5339 ccatttcttg aagataaaaa aattgttatt caaattgtta tgcacagaat 5389 gtttttggta atattaattt ccactaaaaa attaaatgtc tttt 5433 <210> 2 <211> 1713 <212> PRT <213> Human <400> 2 Met Gly Trp Leu Trp Ile Phe Gly Ala Ala Leu Gly Gln Cys Leu   1 5 10 15 Gly Tyr Ser Ser Gln Gln Gln Arg Val Pro Phe Leu Gln Pro Pro                  20 25 30 Gly Gln Ser Gln Leu Gln Ala Ser Tyr Val Glu Phe Arg Pro Ser                  35 40 45 Gln Gly Cys Ser Pro Gly Tyr Tyr Arg Asp His Lys Gly Leu Tyr                  50 55 60 Thr Gly Arg Cys Val Pro Cys Asn Cys Asn Gly His Ser Asn Gln                  65 70 75 Cys Gln Asp Gly Ser Gly Ile Cys Val Asn Cys Gln His Asn Thr                  80 85 90 Ala Gly Glu His Cys Glu Arg Cys Gln Glu Gly Tyr Tyr Gly Asn                  95 100 105 Ala Val His Gly Ser Cys Arg Ala Cys Pro Cys Pro His Thr Asn                 110 115 120 Ser Phe Ala Thr Gly Cys Val Val Asn Gly Gly Asp Val Arg Cys                 125 130 135 Ser Cys Lys Ala Gly Tyr Thr Gly Thr Gln Cys Glu Arg Cys Ala                 140 145 150 Pro Gly Tyr Phe Gly Asn Pro Gln Lys Phe Gly Gly Ser Cys Gln                 155 160 165 Pro Cys Ser Cys Asn Ser Asn Gly Gln Leu Gly Ser Cys His Pro                 170 175 180 Leu Thr Gly Asp Cys Ile Asn Gln Glu Pro Lys Asp Ser Ser Pro                 185 190 195 Ala Glu Glu Cys Asp Asp Cys Asp Ser Cys Val Met Thr Leu Leu                 200 205 210 Asn Asp Leu Ala Thr Met Gly Glu Gln Leu Arg Leu Val Lys Ser                 215 220 225 Gln Leu Gln Gly Leu Ser Ala Ser Ala Gly Leu Leu Glu Gln Met                 230 235 240 Arg His Met Glu Thr Gln Ala Lys Asp Leu Arg Asn Gln Leu Leu                 245 250 255 Asn Tyr Arg Ser Ala Ile Ser Asn His Gly Ser Lys Ile Glu Gly                 260 265 270 Leu Glu Arg Glu Leu Thr Asp Leu Asn Gln Glu Phe Glu Thr Leu                 275 280 285 Gln Glu Lys Ala Gln Val Asn Ser Arg Lys Ala Gln Thr Leu Asn                 290 295 300 Asn Asn Val Asn Arg Ala Thr Gln Ser Ala Lys Glu Leu Asp Val                 305 310 315 Lys Ile Lys Asn Val Ile Arg Asn Val His Ile Leu Leu Lys Gln                 320 325 330 Ile Ser Gly Thr Asp Gly Glu Gly Asn Asn Val Pro Ser Gly Asp                 335 340 345 Phe Ser Arg Glu Trp Ala Glu Ala Gln Arg Met Met Arg Glu Leu                 350 355 360 Arg Asn Arg Asn Phe Gly Lys His Leu Arg Glu Ala Glu Ala Asp                 365 370 375 Lys Arg Glu Ser Gln Leu Leu Leu Asn Arg Ile Arg Thr Trp Gln                 380 385 390 Lys Thr His Gln Gly Glu Asn Asn Gly Leu Ala Asn Ser Ile Arg                 395 400 405 Asp Ser Leu Asn Glu Tyr Glu Ala Lys Leu Ser Asp Leu Arg Ala                 410 415 420 Arg Leu Gln Glu Ala Ala Ala Gln Ala Lys Gln Ala Asn Gly Leu                 425 430 435 Asn Gln Glu Asn Glu Arg Ala Leu Gly Ala Ile Gln Arg Gln Val                 440 445 450 Lys Glu Ile Asn Ser Leu Gln Ser Asp Phe Thr Lys Tyr Leu Thr                 455 460 465 Thr Ala Asp Ser Ser Leu Leu Gln Thr Asn Ile Ala Leu Gln Leu                 470 475 480 Met Glu Lys Ser Gln Lys Glu Tyr Glu Lys Leu Ala Ala Ser Leu                 485 490 495 Asn Glu Ala Arg Gln Glu Leu Ser Asp Lys Val Arg Glu Leu Ser                 500 505 510 Arg Ser Ala Gly Lys Thr Ser Leu Val Glu Glu Ala Glu Lys His                 515 520 525 Ala Arg Ser Leu Gln Glu Leu Ala Lys Gln Leu Glu Glu Ile Lys                 530 535 540 Arg Asn Ala Ser Gly Asp Glu Leu Val Arg Cys Ala Val Asp Ala                 545 550 555 Ala Thr Ala Tyr Glu Asn Ile Leu Asn Ala Ile Lys Ala Ala Glu                 560 565 570 Asp Ala Ala Asn Arg Ala Ala Ser Ala Ser Glu Ser Ala Leu Gln                 575 580 585 Thr Val Ile Lys Glu Asp Leu Pro Arg Lys Ala Lys Thr Leu Ser                 590 595 600 Ser Asn Ser Asp Lys Leu Leu Asn Glu Ala Lys Met Thr Gln Lys                 605 610 615 Lys Leu Lys Gln Glu Val Ser Pro Ala Leu Asn Asn Leu Gln Gln                 620 625 630 Thr Leu Asn Ile Val Thr Val Gln Lys Glu Val Ile Asp Thr Asn                 635 640 645 Leu Thr Thr Leu Arg Asp Gly Leu His Gly Ile Gln Arg Gly Asp                 650 655 660 Ile Asp Ala Met Ile Ser Ser Ala Lys Ser Met Val Arg Lys Ala                 665 670 675 Asn Asp Ile Thr Asp Glu Val Leu Asp Gly Leu Asn Pro Ile Gln                 680 685 690 Thr Asp Val Glu Arg Ile Lys Asp Thr Tyr Gly Arg Thr Gln Asn                 695 700 705 Glu Asp Phe Lys Lys Ala Leu Thr Asp Ala Asp Asn Ser Val Asn                 710 715 720 Lys Leu Thr Asn Lys Leu Pro Asp Leu Trp Arg Lys Ile Glu Ser                 725 730 735 Ile Asn Gln Gln Leu Leu Pro Leu Gly Asn Ile Ser Asp Asn Met                 740 745 750 Asp Arg Ile Arg Glu Leu Ile Gln Gln Ala Arg Asp Ala Ala Ser                 755 760 765 Lys Val Ala Val Pro Met Arg Phe Asn Gly Lys Ser Gly Val Glu                 770 775 780 Val Arg Leu Pro Asn Asp Leu Glu Asp Leu Lys Gly Tyr Thr Ser                 785 790 795 Leu Ser Leu Phe Leu Gln Arg Pro Asn Ser Arg Glu Asn Gly Gly                 800 805 810 Thr Glu Asn Met Phe Val Met Tyr Leu Gly Asn Lys Asp Ala Ser                 815 820 825 Arg Asp Tyr Ile Gly Met Ala Val Val Asp Gly Gln Leu Thr Cys                 830 835 840 Val Tyr Asn Leu Gly Asp Arg Glu Ala Glu Leu Gln Val Asp Gln                 845 850 855 Ile Leu Thr Lys Ser Glu Thr Lys Glu Ala Val Met Asp Arg Val                 860 865 870 Lys Phe Gln Arg Ile Tyr Gln Phe Ala Arg Leu Asn Tyr Thr Lys                 875 880 885 Gly Ala Thr Ser Ser Lys Pro Glu Thr Pro Gly Val Tyr Asp Met                 890 895 900 Asp Gly Arg Asn Ser Asn Thr Leu Leu Asn Leu Asp Pro Glu Asn                 905 910 915 Val Val Phe Tyr Val Gly Gly Tyr Pro Pro Asp Phe Lys Leu Pro                 920 925 930 Ser Arg Leu Ser Phe Pro Pro Tyr Lys Gly Cys Ile Glu Leu Asp                 935 940 945 Asp Leu Asn Glu Asn Val Leu Ser Leu Tyr Asn Phe Lys Lys Thr                 950 955 960 Phe Asn Leu Asn Thr Thr Glu Val Glu Pro Cys Arg Arg Arg Lys                 965 970 975 Glu Glu Ser Asp Lys Asn Tyr Phe Glu Gly Thr Gly Tyr Ala Arg                 980 985 990 Val Pro Thr Gln Pro His Ala Pro Ile Pro Thr Phe Gly Gln Thr                 995 1000 1005 Ile Gln Thr Thr Val Asp Arg Gly Leu Leu Phe Phe Ala Glu Asn                1010 1015 1020 Gly Asp Arg Phe Ile Ser Leu Asn Ile Glu Asp Gly Lys Leu Met                1025 1030 1035 Val Arg Tyr Lys Leu Asn Ser Glu Leu Pro Lys Glu Arg Gly Val                1040 1045 1050 Gly Asp Ala Ile Asn Asn Gly Arg Asp His Ser Ile Gln Ile Lys                1055 1060 1065 Ile Gly Lys Leu Gln Lys Arg Met Trp Ile Asn Val Asp Val Gln                1070 1075 1080 Asn Thr Ile Ile Asp Gly Glu Val Phe Asp Phe Ser Thr Tyr Tyr                1085 1090 1095 Leu Gly Gly Ile Pro Ile Ala Ile Arg Glu Arg Phe Asn Ile Ser                1100 1105 1110 Thr Pro Ala Phe Arg Gly Cys Met Lys Asn Leu Lys Lys Thr Ser                1115 1120 1125 Gly Val Val Arg Leu Asn Asp Thr Val Gly Val Thr Lys Lys Cys                1130 1135 1140 Ser Glu Asp Trp Lys Leu Val Arg Ser Ala Ser Phe Ser Arg Gly                1145 1150 1155 Gly Gln Leu Ser Phe Thr Asp Leu Gly Leu Pro Pro Thr Asp His                1160 1165 1170 Leu Gln Ala Ser Phe Gly Phe Gln Thr Phe Gln Pro Ser Gly Ile                1175 1180 1185 Leu Leu Asp His Gln Thr Trp Thr Arg Asn Leu Gln Val Thr Leu                1190 1195 1200 Glu Asp Gly Tyr Ile Glu Leu Ser Thr Ser Asp Ser Gly Gly Pro                1205 1210 1215 Ile Phe Lys Ser Pro Gln Thr Tyr Met Asp Gly Leu Leu His Tyr                1220 1225 1230 Val Ser Val Ile Ser Asp Asn Ser Gly Leu Arg Leu Leu Ile Asp                1235 1240 1245 Asp Gln Leu Leu Arg Asn Ser Lys Arg Leu Lys His Ile Ser Ser                1250 1255 1260 Ser Arg Gln Ser Leu Arg Leu Gly Gly Ser Asn Phe Glu Gly Cys                1265 1270 1275 Ile Ser Asn Val Phe Val Gln Arg Leu Ser Leu Ser Pro Glu Val                1280 1285 1290 Leu Asp Leu Thr Ser Asn Ser Leu Lys Arg Asp Val Ser Leu Gly                1295 1300 1305 Gly Cys Ser Leu Asn Lys Pro Pro Phe Leu Met Leu Leu Lys Gly                1310 1315 1320 Ser Thr Arg Phe Asn Lys Thr Lys Thr Phe Arg Ile Asn Gln Leu                1325 1330 1335 Leu Gln Asp Thr Pro Val Ala Ser Pro Arg Ser Val Lys Val Trp                1340 1345 1350 Gln Asp Ala Cys Ser Pro Leu Pro Lys Thr Gln Ala Asn His Gly                1355 1360 1365 Ala Leu Gln Phe Gly Asp Ile Pro Thr Ser His Leu Leu Phe Lys                1370 1375 1380 Leu Pro Gln Glu Leu Leu Lys Pro Arg Ser Gln Phe Ala Val Asp                1385 1390 1395 Met Gln Thr Thr Ser Ser Arg Gly Leu Val Phe His Thr Gly Thr                1400 1405 1410 Lys Asn Ser Phe Met Ala Leu Tyr Leu Ser Lys Gly Arg Leu Val                1415 1420 1425 Phe Ala Leu Gly Thr Asp Gly Lys Lys Leu Arg Ile Lys Ser Lys                1430 1435 1440 Glu Lys Cys Asn Asp Gly Lys Trp His Thr Val Val Phe Gly His                1445 1450 1455 Asp Gly Glu Lys Gly Arg Leu Val Val Asp Gly Leu Arg Ala Arg                1460 1465 1470 Glu Gly Ser Leu Pro Gly Asn Ser Thr Ile Ser Ile Arg Ala Pro                1475 1480 1485 Val Tyr Leu Gly Ser Pro Pro Ser Gly Lys Pro Lys Ser Leu Pro                1490 1495 1500 Thr Asn Ser Phe Val Gly Cys Leu Lys Asn Phe Gln Leu Asp Ser                1505 1510 1515 Lys Pro Leu Tyr Thr Pro Ser Ser Ser Phe Gly Val Ser Ser Cys                1520 1525 1530 Leu Gly Gly Pro Leu Glu Lys Gly Ile Tyr Phe Ser Glu Glu Gly                1535 1540 1545 Gly His Val Val Leu Ala His Ser Val Leu Leu Gly Pro Glu Phe                1550 1555 1560 Lys Leu Val Phe Ser Ile Arg Pro Arg Ser Leu Thr Gly Ile Leu                1565 1570 1575 Ile His Ile Gly Ser Gln Pro Gly Lys His Leu Cys Val Tyr Leu                1580 1585 1590 Glu Ala Gly Lys Val Thr Ala Ser Met Asp Ser Gly Ala Gly Gly                1595 1600 1605 Thr Ser Thr Ser Val Thr Pro Lys Gln Ser Leu Cys Asp Gly Gln                1610 1615 1620 Trp His Ser Val Ala Val Thr Ile Lys Gln His Ile Leu His Leu                1625 1630 1635 Glu Leu Asp Thr Asp Ser Ser Tyr Thr Ala Gly Gln Ile Pro Phe                1640 1645 1650 Pro Pro Ala Ser Thr Gln Glu Pro Leu His Leu Gly Gly Ala Pro                1655 1660 1665 Ala Asn Leu Thr Thr Leu Arg Ile Pro Val Trp Lys Ser Phe Phe                1670 1675 1680 Gly Cys Leu Arg Asn Ile His Val Asn His Ile Pro Val Pro Val                1685 1690 1695 Thr Glu Ala Leu Glu Val Gln Gly Pro Val Ser Leu Asn Gly Cys                1700 1705 1710 Pro Asp Gln        1713 <210> 3 <211> 5831 <212> DNA <213> Human <220> <221> CDS <222> (336) ... (5361) <300> <301> Pikkarainen, T., Eddy, R., Fuushima, Y., Byers, M., Shows, T.,       Pihlajaniemi, T., Saraste, M. And Tryggvason, K. <302> Human laminin B1 chain.A multidomain protein with gene (LAMB1)       locus in the q22 region of chromosome 7 <303> JOURNAL J. Biol. Chem. <304> 262 <305> 22 <306> 10454-10462 <307> 1987 <400> gggacctgga agcgccccag ccccgcagcg atcgcagatt cggctttcaa acaaaagagg 60 cgccccgggg ggtgggaccg ggacctcacc cggtcctcgc agagttgcgg ccgcccgccc 120 cttcagcccc ggctctccgt atgcgcatga gcagaggcgc ctccctctgt tcctcccaag 180 gctaaacttt ctaattccct tctttgggct cgggggctcc cggagcaggg cgagagctcg 240 cgtcgccgga aaggaagacg ggaagaaagg gcaggcggct cggcgggcgt cttctccact 300 cctctgccgc gtccccgtgg ctgcagggag ccggcatggg gcttctccag ttgctagctt 360 tcagtttctt agccctgtgc agagcccgag tgcgcgctca ggaacccgag ttcagctacg 420 gctgcgcaga aggcagctgc tatcccgcca cgggcgacct tctcatcggc cgagcacaga 480 agctttcggt gacctcgacg tgcgggctgc acaagcccga accctactgt atcgtcagcc 540 acttgcagga ggacaaaaaa tgcttcatat gcaattccca agatccttat catgagaccc 600 tgaatcctga cagccatctc attgaaaatg tggtcactac atttgctcca aaccgcctta 660 agatttggtg gcaatctgaa aatggtgtgg aaaatgtaac tatccaactg gatttggaag 720 cagaattcca ttttactcat ctcataatga ctttcaagac attccgtcca gctgctatgc 780 tgatagaacg atcgtccgac tttgggaaaa cctggggtgt gtatagatac ttcgcctatg 840 actgtgaggc ctcgtttcca ggcatttcaa ctggccccat gaaaaaagtc gatgacataa 900 tttgtgattc tcgatattct gacattgaac cctcaactga aggagaggtg atatttcgtg 960 ctttagatcc tgctttcaaa atagaagatc cttatagccc aaggatacag aatttattaa 1020 aaattaccaa cttgagaatc aagtttgtga aactgcatac tttgggagat aaccttctgg 1080 attccaggat ggaaatcaga gaaaagtatt attatgcagt ttatgatatg gtggttcgag 1140 gaaattgctt ctgctatggt catgccagcg aatgtgcccc tgtggatgga ttcaatgaag 1200 aagtggaagg aatggttcac ggacactgca tgtgcaggca taacaccaag ggcttaaact 1260 gtgaactctg catggatttc taccatgatt taccttggag acctgctgaa ggccgaaaca 1320 gcaacgcctg taaaaaatgt aactgcaatg aacattccat ctcttgtcac tttgacatgg 1380 ctgtttacct ggccacgggg aacgtcagcg gaggcgtgtg tgatgactgt cagcacaaca 1444 ccatggggcg caactgtgag cagtgcaagc cgttttacta ccagcaccca gagagggaca 1500 tccgagatcc taatttctgt gaacgatgta cgtgtgaccc agctggctct caaaatgagg 1560 gaatttgtga cagctatact gatttttcta ctggtctcat tgctggccag tgtcggtgta 1620 aattaaatgt ggaaggagaa cattgtgatg tttgcaaaga aggcttctat gatttaagca 1680 gtgaagatcc atttggttgt aaatcttgtg cttgcaatcc tctgggaaca attcctggag 1740 ggaatccttg tgattccgag acaggtcact gctactgcaa gcgtctggtg acaggacagc 1800 attgtgacca gtgcctgcca gagcactggg gcttaagcaa tgatttggat ggatgtcgac 1860 catgtgactg tgaccttggg ggagccttaa acaacagttg ctttgcggag tcaggccagt 1920 gctcatgccg gcctcacatg attggacgtc agtgcaacga agtggaacct ggttactact 1980 ttgccaccct ggatcactac ctctatgaag cggaggaagc caacttgggg cctggggtta 2040 gcatagtgga gcggcaatat atccaggacc ggattccctc ctggactgga gccggcttcg 2100 tccgagtgcc tgaaggggct tatttggagt ttttcattga caacatacca tattccatgg 2160 agtacgacat cctaattcgc tacgagccac agctacccga ccactgggaa aaagctgtca 2220 tcacagtgca gcgacctgga aggattccaa ccagcagccg atgtggtaat accatccccg 2280 atgatgacaa ccaggtggtg tcattatcac caggctcaag atatgtcgtc cttcctcggc 2340 cggtgtgctt tgagaaggga acaaactaca cggtgaggtt ggagctgcct cagtacacct 2400 cctctgatag cgacgtggag agcccctaca cgctgatcga ttctcttgtt ctcatgccat 2460 actgtaaatc actggacatc ttcaccgtgg gaggttcagg agatggggtg gtcaccaaca 2520 gtgcctggga aacctttcag agataccgat gtctagagaa cagcagaagc gttgtgaaaa 2580 caccgatgac agatgtttgc agaaacatca tctttagcat ttctgccctg ttacaccaga 2640 caggcctggc ttgtgaatgc gaccctcagg gttcgttaag ttccgtgtgt gatcccaacg 2700 gaggccagtg ccagtgccgg cccaacgtgg ttggaagaac ctgcaacaga tgtgcacctg 2760 gaacttttgg ctttggcccc agtggatgca aaccttgtga gtgccatctg caaggatctg 2820 tcaatgcctt ctgcaatccc gtcactggcc agtgccactg tttccaggga gtgtatgctc 2880 ggcagtgtga tcggtgctta cctgggcact ggggctttcc aagttgccag ccctgccagt 2940 gcaatggcca cgccgatgac tgcgacccag tgactgggga gtgcttgaac tgccaggact 3000 acaccatggg tcataactgt gaaaggtgct tggctggtta ctatggcgac cccatcattg 3060 ggtcaggtga tcactgccgc ccttgccctt gcccagatgg tcccgacagt ggacgccagt 3120 ttgccaggag ctgctaccaa gatcctgtta ctttacagct tgcctgtgtt tgtgatcctg 3180 gatacattgg ttccagatgt gacgactgtg cctcaggata ctttggcaat ccatcagaag 3240 ttggggggtc gtgtcagcct tgccagtgtc acaacaacat tgacacgaca gacccagaag 3300 cctgtgacaa ggagactggg aggtgtctca agtgcctgta ccacacggaa ggggaacact 3360 gtcagttctg ccggtttgga tactatggtg atgccctccg gcaggactgt cgaaagtgtg 3420 tctgtaatta cctgggcacc gtgcaagagc actgtaacgg ctctgactgc cagtgcgaca 3480 aagccactgg tcagtgcttg tgtcttccta atgtgatcgg gcagaactgt gaccgctgtg 3540 cgcccaatac ctggcagctg gccagtggca ctggctgtga cccatgcaac tgcaatgctg 3600 ctcattcctt cgggccatct tgcaatgagt tcacggggca gtgccagtgc atgcctgggt 3660 ttggaggccg cacctgcagc gagtgccagg aactcttctg gggagacccc gacgtggagt 3720 gccgagcctg tgactgtgac cccaggggca ttgagacgcc acagtgtgac cagtccacgg 3780 gccagtgtgt ctgcgttgag ggtgttgagg gtccacgctg tgacaagtgc acgcgagggt 3840 actcgggggt cttccctgac tgcacaccct gccaccagtg ctttgctctc tgggatgtga 3900 tcattgccga gctgaccaac aggacacaca gattcctgga gaaagccaag gccttgaaga 3960 tcagtggtgt gatcgggcct taccgtgaga ctgtggactc ggtggagagg aaagtcagcg 4020 agataaaaga catcctggcg cagagccccg cagcagagcc actgaaaaac attgggaatc 4080 tctttgagga agcagagaaa ctgattaaag atgttacaga aatgatggct caagtagaag 4140 tgaaattatc tgacacaact tcccaaagca acagcacagc caaagaactg gattctctac 4200 agacagaagc cgaaagccta gacaacactg tgaaagaact tgctgaacaa ctggaattta 4260 tcaaaaactc agatattcgg ggtgccttgg atagcattac caagtatttc cagatgtctc 4320 ttgaggcaga ggagagggtg aatgcctcca ccacagaacc caacagcact gtggagcagt 4380 cagccctcat gagagacaga gtagaagacg tgatgatgga gcgagaatcc cagttcaagg 4440 aaaaacaaga ggagcaggct cgcctccttg atgaactggc aggcaagcta caaagcctag 4500 acctttcagc cgctgccgaa atgacctgtg gaacaccccc aggggcctcc tgttccgaga 4560 ctgaatgtgg cgggccaaac tgcagaactg acgaaggaga gaggaagtgt ggggggcctg 4620 gctgtggtgg tctggttact gttgcacaca acgcctggca gaaagccatg gacttggacc 4680 aagatgtcct gagtgccctg gctgaagtgg aacagctctc caagatggtc tctgaagcaa 4740 aactgagggc agatgaggca aaacaaagtg ctgaagacat tctgttgaag acaaatgcta 4800 ccaaagaaaa aatggacaag agcaatgagg agctgagaaa tctaatcaag caaatcagaa 4860 actttttgac ccaggatagt gctgatttgg acagcattga agcagttgct aatgaagtat 4920 tgaaaatgga gatgcctagc accccacagc agttacagaa cttgacagaa gatatacgtg 4980 aacgagttga aagcctttct caagtagagg ttattcttca gcatagtgct gctgacattg 5040 ccagagctga gatgttgtta gaagaagcta aaagagcaag caaaagtgca acagatgtta 5100 aagtcactgc agatatggta aaggaagctc tggaagaagc agaaaaggcc caggtcgcag 5160 cagagaaggc aattaaacaa gcagatgaag acattcaagg aacccagaac ctgttaactt 5220 cgattgagtc tgaaacagca gcttctgagg aaaccttgtt caacgcgtcc cagcgcatca 5280 gcgagttaga gaggaatgtg gaagaactta agcggaaagc tgcccaaaac tccggggagg 5340 cagaatatat tgaaaaagta gtatatactg tgaagcaaag tgcagaagat gttaagaaga 5400 ctttagatgg tgaacttgat gaaaagtata aaaaagtaga aaatttaatt gccaaaaaaa 5460 ctgaagagtc agctgatgcc agaaggaaag ccgaaatgct acaaaatgaa gcaaaaactc 5520 ttttagctca agcaaatagc aagctgcaac tgctcaaaga tttagaaaga aaatatgaag 5580 acaatcaaag atacttagaa gataaagctc aagaattagc aagactggaa ggagaagtcc 5640 gttcactcct aaaggatata agccagaaag ttgctgtgta tagcacatgc ttgtaacaga 5700 ggagaataaa aaatggctga ggtgaacaag gtaaaacaac tacattttaa aaactgactt 5760 aatgctcttc aaaataaaac atcacctatt taatgttttt aatcacattt tgtatgagtt 5820 aaataaagcc c 5831 <210> 4 <211> 1786 <212> PRT <213> Human <400> Met Gly Leu Leu Gln Leu Leu Ala Phe Ser Phe Leu Ala Leu Cys   1 5 10 15 Arg Ala Arg Val Arg Ala Gln Glu Pro Glu Phe Ser Tyr Gly Cys                  20 25 30 Ala Glu Gly Ser Cys Tyr Pro Ala Thr Gly Asp Leu Leu Ile Gly                  35 40 45 Arg Ala Gln Lys Leu Ser Val Thr Ser Thr Cys Gly Leu His Lys                  50 55 60 Pro Glu Pro Tyr Cys Ile Val Ser His Leu Gln Glu Asp Lys Lys                  65 70 75 Cys Phe Ile Cys Asn Ser Gln Asp Pro Tyr His Glu Thr Leu Asn                  80 85 90 Pro Asp Ser His Leu Ile Glu Asn Val Val Thr Thr Phe Ala Pro                  95 100 105 Asn Arg Leu Lys Ile Trp Trp Gln Ser Glu Asn Gly Val Glu Asn                 110 115 120 Val Thr Ile Gln Leu Asp Leu Glu Ala Glu Phe His Phe Thr His                 125 130 135 Leu Ile Met Thr Phe Lys Thr Phe Arg Pro Ala Ala Met Leu Ile                 140 145 150 Glu Arg Ser Ser Asp Phe Gly Lys Thr Trp Gly Val Tyr Arg Tyr                 155 160 165 Phe Ala Tyr Asp Cys Glu Ala Ser Phe Pro Gly Ile Ser Thr Gly                 170 175 180 Pro Met Lys Lys Val Asp Asp Ile Ile Cys Asp Ser Arg Tyr Ser                 185 190 195 Asp Ile Glu Pro Ser Thr Glu Gly Glu Val Ile Phe Arg Ala Leu                 200 205 210 Asp Pro Ala Phe Lys Ile Glu Asp Pro Tyr Ser Pro Arg Ile Gln                 215 220 225 Asn Leu Leu Lys Ile Thr Asn Leu Arg Ile Lys Phe Val Lys Leu                 230 235 240 His Thr Leu Gly Asp Asn Leu Leu Asp Ser Arg Met Glu Ile Arg                 245 250 255 Glu Lys Tyr Tyr Tyr Ala Val Tyr Asp Met Val Val Arg Gly Asn                 260 265 270 Cys Phe Cys Tyr Gly His Ala Ser Glu Cys Ala Pro Val Asp Gly                 275 280 285 Phe Asn Glu Glu Val Glu Gly Met Val His Gly His Cys Met Cys                 290 295 300 Arg His Asn Thr Lys Gly Leu Asn Cys Glu Leu Cys Met Asp Phe                 305 310 315 Tyr His Asp Leu Pro Trp Arg Pro Ala Glu Gly Arg Asn Ser Asn                 320 325 330 Ala Cys Lys Lys Cys Asn Cys Asn Glu His Ser Ile Ser Cys His                 335 340 345 Phe Asp Met Ala Val Tyr Leu Ala Thr Gly Asn Val Ser Gly Gly                 350 355 360 Val Cys Asp Asp Cys Gln His Asn Thr Met Gly Arg Asn Cys Glu                 365 370 375 Gln Cys Lys Pro Phe Tyr Tyr Gln His Pro Glu Arg Asp Ile Arg                 380 385 390 Asp Pro Asn Phe Cys Glu Arg Cys Thr Cys Asp Pro Ala Gly Ser                 395 400 405 Gln Asn Glu Gly Ile Cys Asp Ser Tyr Thr Asp Phe Ser Thr Gly                 410 415 420 Leu Ile Ala Gly Gln Cys Arg Cys Lys Leu Asn Val Glu Gly Glu                 425 430 435 His Cys Asp Val Cys Lys Glu Gly Phe Tyr Asp Leu Ser Ser Glu                 440 445 450 Asp Pro Phe Gly Cys Lys Ser Cys Ala Cys Asn Pro Leu Gly Thr                 455 460 465 Ile Pro Gly Gly Asn Pro Cys Asp Ser Glu Thr Gly His Cys Tyr                 470 475 480 Cys Lys Arg Leu Val Thr Gly Gln His Cys Asp Gln Cys Leu Pro                 485 490 495 Glu His Trp Gly Leu Ser Asn Asp Leu Asp Gly Cys Arg Pro Cys                 500 505 510 Asp Cys Asp Leu Gly Gly Ala Leu Asn Asn Ser Cys Phe Ala Glu                 515 520 525 Ser Gly Gln Cys Ser Cys Arg Pro His Met Ile Gly Arg Gln Cys                 530 535 540 Asn Glu Val Glu Pro Gly Tyr Tyr Phe Ala Thr Leu Asp His Tyr                 545 550 555 Leu Tyr Glu Ala Glu Glu Ala Asn Leu Gly Pro Gly Val Ser Ile                 560 565 570 Val Glu Arg Gln Tyr Ile Gln Asp Arg Ile Pro Ser Trp Thr Gly                 575 580 585 Ala Gly Phe Val Arg Val Pro Glu Gly Ala Tyr Leu Glu Phe Phe                 590 595 600 Ile Asp Asn Ile Pro Tyr Ser Met Glu Tyr Asp Ile Leu Ile Arg                 605 610 615 Tyr Glu Pro Gln Leu Pro Asp His Trp Glu Lys Ala Val Ile Thr                 620 625 630 Val Gln Arg Pro Gly Arg Ile Pro Thr Ser Ser Arg Cys Gly Asn                 635 640 645 Thr Ile Pro Asp Asp Asp Asn Gln Val Val Ser Leu Ser Pro Gly                 650 655 660 Ser Arg Tyr Val Val Leu Pro Arg Pro Val Cys Phe Glu Lys Gly                 665 670 675 Thr Asn Tyr Thr Val Arg Leu Glu Leu Pro Gln Tyr Thr Ser Ser                 680 685 690 Asp Ser Asp Val Glu Ser Pro Tyr Thr Leu Ile Asp Ser Leu Val                 695 700 705 Leu Met Pro Tyr Cys Lys Ser Leu Asp Ile Phe Thr Val Gly Gly                 710 715 720 Ser Gly Asp Gly Val Val Thr Asn Ser Ala Trp Glu Thr Phe Gln                 725 730 735 Arg Tyr Arg Cys Leu Glu Asn Ser Arg Ser Val Val Lys Thr Pro                 740 745 750 Met Thr Asp Val Cys Arg Asn Ile Ile Phe Ser Ile Ser Ala Leu                 755 760 765 Leu His Gln Thr Gly Leu Ala Cys Glu Cys Asp Pro Gln Gly Ser                 770 775 780 Leu Ser Ser Val Cys Asp Pro Asn Gly Gly Gln Cys Gln Cys Arg                 785 790 795 Pro Asn Val Val Gly Arg Thr Cys Asn Arg Cys Ala Pro Gly Thr                 800 805 810 Phe Gly Phe Gly Pro Ser Gly Cys Lys Pro Cys Glu Cys His Leu                 815 820 825 Gln Gly Ser Val Asn Ala Phe Cys Asn Pro Val Thr Gly Gln Cys                 830 835 840 His Cys Phe Gln Gly Val Tyr Ala Arg Gln Cys Asp Arg Cys Leu                 845 850 855 Pro Gly His Trp Gly Phe Pro Ser Cys Gln Pro Cys Gln Cys Asn                 860 865 870 Gly His Ala Asp Asp Cys Asp Pro Val Thr Gly Glu Cys Leu Asn                 875 880 885 Cys Gln Asp Tyr Thr Met Gly His Asn Cys Glu Arg Cys Leu Ala                 890 895 900 Gly Tyr Tyr Gly Asp Pro Ile Ile Gly Ser Gly Asp His Cys Arg                 905 910 915 Pro Cys Pro Cys Pro Asp Gly Pro Asp Ser Gly Arg Gln Phe Ala                 920 925 930 Arg Ser Cys Tyr Gln Asp Pro Val Thr Leu Gln Leu Ala Cys Val                 935 940 945 Cys Asp Pro Gly Tyr Ile Gly Ser Arg Cys Asp Asp Cys Ala Ser                 950 955 960 Gly Tyr Phe Gly Asn Pro Ser Glu Val Gly Gly Ser Cys Gln Pro                 965 970 975 Cys Gln Cys His Asn Asn Ile Asp Thr Thr Asp Pro Glu Ala Cys                 980 985 990 Asp Lys Glu Thr Gly Arg Cys Leu Lys Cys Leu Tyr His Thr Glu                 995 1000 1005 Gly Glu His Cys Gln Phe Cys Arg Phe Gly Tyr Tyr Gly Asp Ala                1010 1015 1020 Leu Arg Gln Asp Cys Arg Lys Cys Val Cys Asn Tyr Leu Gly Thr                1025 1030 1035 Val Gln Glu His Cys Asn Gly Ser Asp Cys Gln Cys Asp Lys Ala                1040 1045 1050 Thr Gly Gln Cys Leu Cys Leu Pro Asn Val Ile Gly Gln Asn Cys                1055 1060 1065 Asp Arg Cys Ala Pro Asn Thr Trp Gln Leu Ala Ser Gly Thr Gly                1070 1075 1080 Cys Asp Pro Cys Asn Cys Asn Ala Ala His Ser Phe Gly Pro Ser                1085 1090 1095 Cys Asn Glu Phe Thr Gly Gln Cys Gln Cys Met Pro Gly Phe Gly                1100 1105 1110 Gly Arg Thr Cys Ser Glu Cys Gln Glu Leu Phe Trp Gly Asp Pro                1115 1120 1125 Asp Val Glu Cys Arg Ala Cys Asp Cys Asp Pro Arg Gly Ile Glu                1130 1135 1140 Thr Pro Gln Cys Asp Gln Ser Thr Gly Gln Cys Val Cys Val Glu                1145 1150 1155 Gly Val Glu Gly Pro Arg Cys Asp Lys Cys Thr Arg Gly Tyr Ser                1160 1165 1170 Gly Val Phe Pro Asp Cys Thr Pro Cys His Gln Cys Phe Ala Leu                1175 1180 1185 Trp Asp Val Ile Ile Ala Glu Leu Thr Asn Arg Thr His Arg Phe                1190 1195 1200 Leu Glu Lys Ala Lys Ala Leu Lys Ile Ser Gly Val Ile Gly Pro                1205 1210 1215 Tyr Arg Glu Thr Val Asp Ser Val Glu Arg Lys Val Ser Glu Ile                1220 1225 1230 Lys Asp Ile Leu Ala Gln Ser Pro Ala Ala Glu Pro Leu Lys Asn                1235 1240 1245 Ile Gly Asn Leu Phe Glu Glu Ala Glu Lys Leu Ile Lys Asp Val                1250 1255 1260 Thr Glu Met Met Ala Gln Val Glu Val Lys Leu Ser Asp Thr Thr                1265 1270 1275 Ser Gln Ser Asn Ser Thr Ala Lys Glu Leu Asp Ser Leu Gln Thr                1280 1285 1290 Glu Ala Glu Ser Leu Asp Asn Thr Val Lys Glu Leu Ala Glu Gln                1295 1300 1305 Leu Glu Phe Ile Lys Asn Ser Asp Ile Arg Gly Ala Leu Asp Ser                1310 1315 1320 Ile Thr Lys Tyr Phe Gln Met Ser Leu Glu Ala Glu Glu Arg Val                1325 1330 1335 Asn Ala Ser Thr Thr Glu Pro Asn Ser Thr Val Glu Gln Ser Ala                1340 1345 1350 Leu Met Arg Asp Arg Val Glu Asp Val Met Met Glu Arg Glu Ser                1355 1360 1365 Gln Phe Lys Glu Lys Gln Glu Glu Gln Ala Arg Leu Leu Asp Glu                1370 1375 1380 Leu Ala Gly Lys Leu Gln Ser Leu Asp Leu Ser Ala Ala Ala Glu                1385 1390 1395 Met Thr Cys Gly Thr Pro Pro Gly Ala Ser Cys Ser Glu Thr Glu                1400 1405 1410 Cys Gly Gly Pro Asn Cys Arg Thr Asp Glu Gly Glu Arg Lys Cys                1415 1420 1425 Gly Gly Pro Gly Cys Gly Gly Leu Val Thr Val Ala His Asn Ala                1430 1435 1440 Trp Gln Lys Ala Met Asp Leu Asp Gln Asp Val Leu Ser Ala Leu                1445 1450 1455 Ala Glu Val Glu Gln Leu Ser Lys Met Val Ser Glu Ala Lys Leu                1460 1465 1470 Arg Ala Asp Glu Ala Lys Gln Ser Ala Glu Asp Ile Leu Leu Lys                1475 1480 1485 Thr Asn Ala Thr Lys Glu Lys Met Asp Lys Ser Asn Glu Glu Leu                1490 1495 1500 Arg Asn Leu Ile Lys Gln Ile Arg Asn Phe Leu Thr Gln Asp Ser                1505 1510 1515 Ala Asp Leu Asp Ser Ile Glu Ala Val Ala Asn Glu Val Leu Lys                1520 1525 1530 Met Glu Met Pro Ser Thr Pro Gln Gln Leu Gln Asn Leu Thr Glu                1535 1540 1545 Asp Ile Arg Glu Arg Val Glu Ser Leu Ser Gln Val Glu Val Ile                1550 1555 1560 Leu Gln His Ser Ala Ala Asp Ile Ala Arg Ala Glu Met Leu Leu                1565 1570 1575 Glu Glu Ala Lys Arg Ala Ser Lys Ser Ala Thr Asp Val Lys Val                1580 1585 1590 Thr Ala Asp Met Val Lys Glu Ala Leu Glu Glu Ala Glu Lys Ala                1595 1600 1605 Gln Val Ala Ala Glu Lys Ala Ile Lys Gln Ala Asp Glu Asp Ile                1610 1615 1620 Gln Gly Thr Gln Asn Leu Leu Thr Ser Ile Glu Ser Glu Thr Ala                1625 1630 1635 Ala Ser Glu Glu Thr Leu Phe Asn Ala Ser Gln Arg Ile Ser Glu                1640 1645 1650 Leu Glu Arg Asn Val Glu Glu Leu Lys Arg Lys Ala Ala Gln Asn                1655 1660 1665 Ser Gly Glu Ala Glu Tyr Ile Glu Lys Val Val Tyr Thr Val Lys                1670 1675 1680 Gln Ser Ala Glu Asp Val Lys Lys Thr Leu Asp Gly Glu Leu Asp                1685 1690 1695 Glu Lys Tyr Lys Lys Val Glu Asn Leu Ile Ala Lys Lys Thr Glu                1700 1705 1710 Glu Ser Ala Asp Ala Arg Arg Lys Ala Glu Met Leu Gln Asn Glu                1715 1720 1725 Ala Lys Thr Leu Leu Ala Gln Ala Asn Ser Lys Leu Gln Leu Leu                1730 1735 1740 Lys Asp Leu Glu Arg Lys Tyr Glu Asp Asn Gln Arg Tyr Leu Glu                1745 1750 1755 Asp Lys Ala Gln Glu Leu Ala Arg Leu Glu Gly Glu Val Arg Ser                1760 1765 1770 Leu Leu Lys Asp Ile Ser Gln Lys Val Ala Val Tyr Ser Thr Cys                1775 1780 1785 Leu 1786 <210> 5 <211> 7923 <212> DNA <213> Human <220> <221> CDS <222> (300) ... (5126) <300> <301> Pikkarainen, T., Kallunki, T. And Tryggvason, K. <302> Human laminin B2 chain. Comparison of the complete amino acid       sequence with the B1 chain reveals variability in sequence       homology between different structural domains <303> JOURNAL J. Biol. Chem. <304> 263 <305> 14 <306> 6751-6758 <307> 1988 <400> 5 gcgcactcgg gcacgcgctc ggaagtcggg ggtcggcgcg gagtgcaggc tgctcccggg 60 gtaggtgagg gaagcgcgga ggcggggcgc gggggcagtg gtcggcgagc agcgcggtcc 120 tcgctagggg cgcccacccg tcagtctctc cggcgcgagc cgccgccacc gcccgcgccg 180 gagtcaggcc cctgggcccc caggctcaag cagcgaagcg gcctccgggg gacgccgcta 240 ggcgagagga acgcgccggt gcccttgcct tcgccgtgac ccagcgtgcg ggcggcggga 300 tgagagggag ccatcgggcc gcgccggccc tgcggccccg ggggcggctc tggcccgtgc 360 tggccgtgct ggcggcggcc gccgcggcgg gctgtgccca ggcagccatg gacgagtgca 420 cggacgaggg cgggcggccg cagcgctgca tgcccgagtt cgtcaacgcc gctttcaacg 480 tgactgtggt ggccaccaac acgtgtggga ctccgcccga ggaatactgt gtgcagaccg 540 gggtgaccgg ggtcaccaag tcctgtcacc tgtgcgacgc cgggcagccc cacctgcagc 600 acggggcagc cttcctgacc gactacaaca accaggccga caccacctgg tggcaaagcc 660 agaccatgct ggccggggtg cagtacccca gctccatcaa cctcacgctg cacctgggaa 720 aagcttttga catcacctat gtgcgtctca agttccacac cagccgcccg gagagctttg 780 ccatttacaa gcgcacacgg gaagacgggc cctggattcc ttaccagtac tacagtggtt 840 cctgcgagaa cacctactcc aaggcaaacc gcggcttcat caggacagga ggggacgagc 900 agcaggcctt gtgtactgat gaattcagtg acatttctcc cctcactggg ggcaacgtgg 960 ccttttctac cctggaagga aggcccagcg cctataactt tgacaatagc cctgtgctgc 1020 aggaatgggt aactgccact gacatcagag taactcttaa tcgcctgaac acttttggag 1080 atgaagtgtt taacgatccc aaagttctca agtcctatta ttatgccatc tctgattttg 1140 ctgtaggtgg cagatgtaaa tgtaatggac acgcaagcga gtgtatgaag aacgaatttg 1200 ataagctggt gtgtaattgc aaacataaca catatggagt agactgtgaa aagtgtcttc 1260 ctttcttcaa tgaccggccg tggaggaggg caactgcgga aagtgccagt gaatgcctgc 1320 cctgtgattg caatggtcga tcccaggaat gctacttcga ccctgaactc tatcgttcca 1380 ctggccatgg gggccactgt accaactgcc aggataacac agatggcgcc cactgtgaga 1440 ggtgccgaga gaacttcttc cgccttggca acaatgaagc ctgctcttca tgccactgta 1500 gtcctgtggg ctctctaagc acacagtgtg atagttacgg cagatgcagc tgtaagccag 1560 gagtgatggg ggacaaatgt gaccgttgcc agcctggatt ccattctctc actgaagcag 1620 gatgcaggcc atgctcttgt gatccctctg gcagcataga tgaatgtaat gttgaaacag 1680 gaagatgtgt ttgcaaagac aatgtcgaag gcttcaattg tgaaagatgc aaacctggat 1740 tttttaatct ggaatcatct aatcctcggg gttgcacacc ctgcttctgc tttgggcatt 1800 cttctgtctg tacaaacgct gttggctaca gtgtttattc tatctcctct acctttcaga 1860 ttgatgagga tgggtggcgt gcggaacaga gagatggctc tgaagcatct ctcgagtggt 1920 cctctgagag gcaagatatc gccgtgatct cagacagcta ctttcctcgg tacttcattg 1980 ctcctgcaaa gttcttgggc aagcaggtgt tgagttatgg tcagaacctc tccttctcct 2040 ttcgagtgga caggcgagat actcgcctct ctgccgaaga ccttgtgctt gagggagctg 2100 gcttaagagt atctgtaccc ttgatcgctc agggcaattc ctatccaagt gagaccactg 2160 tgaagtatgt cttcaggctc catgaagcaa cagattaccc ttggaggcct gctcttaccc 2220 cttttgaatt tcagaagctc ctaaacaact tgacctctat caagatacgt gggacataca 2280 gtgagagaag tgctggatat ttggatgatg tcaccctggc aagtgctcgt cctgggcctg 2340 gagtccctgc aacttgggtg gagtcctgca cctgtcctgt gggatatgga gggcagtttt 2400 gtgagatgtg cctctcaggt tacagaagag aaactcctaa tcttggacca tacagtccat 2460 gtgtgctttg cgcctgcaat ggacacagcg agacctgtga tcctgagaca ggtgtttgta 2520 actgcagaga caatacggct ggcccgcact gtgagaagtg cagtgatggg tactatggag 2580 attcaactgc aggcacctcc tccgattgcc aaccctgtcc gtgtcctgga ggttcaagtt 2640 gtgctgttgt tcccaagaca aaggaggtgg tgtgcaccaa ctgtcctact ggcaccactg 2700 gtaagagatg tgagctctgt gatgatggct actttggaga ccccctgggt agaaacggcc 2760 ctgtgagact ttgccgcctg tgccagtgca gtgacaacat cgatcccaac gcagttggaa 2820 attgcaatcg cttgacggga gaatgcctga agtgcatcta taacactgct ggcttctatt 2880 gtgaccggtg caaagacgga ttttttggaa atcccctggc tcccaatcca gcagacaaat 2940 gcaaagcctg caattgcaat ccgtatggga ccatgaagca gcagagcagc tgtaaccccg 3000 tgacggggca gtgtgaatgt ttgcctcacg tgactggcca ggactgtggt gcttgtgacc 3060 ctggattcta caatctgcag agtgggcaag gctgtgagag gtgtgactgc catgccttgg 3120 gctccaccaa tgggcagtgt gacatccgca ccggccagtg tgagtgccag cccggcatca 3180 ctggtcagca ctgtgagcgc tgtgaggtca accactttgg gtttggacct gaaggctgca 3240 aaccctgtga ctgtcatcct gagggatctc tttcacttca gtgcaaagat gatggtcgct 3300 gtgaatgcag agaaggcttt gtgggaaatc gctgtgacca gtgtgaagaa aactatttct 3360 acaatcggtc ttggcctggc tgccaggaat gtccagcttg ttaccggctg gtaaaggata 3420 aggttgctga tcatagagtg aagctccagg aattagagag tctcatagca aaccttggaa 3480 ctggggatga gatggtgaca gatcaagcct tcgaggatag actaaaggaa gcagagaggg 3540 aagttatgga cctccttcgt gaggcccagg atgtcaaaga tgttgaccag aatttgatgg 3600 atcgcctaca gagagtgaat aacactctgt ccagccaaat tagccgttta cagaatatcc 3660 ggaataccat tgaagagact ggaaacttgg ctgaacaagc gcgtgcccat gtagagaaca 3720 cagagcggtt gattgaaatc gcatccagag aacttgagaa agcaaaagtc gctgctgcca 3780 atgtgtcagt cactcagcca gaatctacag gggacccaaa caacatgact cttttggcag 3840 aagaggctcg aaagcttgct gaacgtcata aacaggaagc tgatgacatt gttcgagtgg 3900 caaagacagc caatgatacg tcaactgagg catacaacct gcttctgagg acactggcag 3960 gagaaaatca aacagcattt gagattgaag agcttaatag gaagtatgaa caagcgaaga 4020 acatctcaca ggatctggaa aaacaagctg cccgagtaca tgaggaggcc aaaagggccg 4080 gtgacaaagc tgtggagatc tatgccagcg tggctcagct gagccctttg gactctgaga 4140 cactggagaa tgaagcaaat aacataaaga tggaagctga gaatctggaa caactgattg 4200 accagaaatt aaaagattat gaggacctca gagaagatat gagagggaag gaacttgaag 4260 tcaagaacct tctggagaaa ggcaagactg aacagcagac cgcagaccaa ctcctagccc 4320 gagctgatgc tgccaaggcc ctcgctgaag aagctgcaaa gaagggacgg gataccttac 4380 aagaagctaa tgacattctc aacaacctga aagattttga taggcgcgtg aacgataaca 4440 agacggccgc agaggaggca ctaaggaaga ttcctgccat caaccagacc atcactgaag 4500 ccaatgaaaa gaccagagaa gcccagcagg ccctgggcag tgctgcggcg gatgccacag 4560 aggccaagaa caaggcccat gaggcggaga ggatcgcaag cgctgtccaa aagaatgcca 4620 ccagcaccaa ggcagaagct gaaagaactt ttgcagaagt tacagatctg gataatgagg 4680 tgaacaatat gttgaagcaa ctgcaggaag cagaaaaaga gctaaagaga aaacaagatg 4740 acgctgacca ggacatgatg atggcaggga tggcttcaca ggctgctcaa gaagccgaga 4800 tcaatgccag aaaagccaaa aactctgtta ctagcctcct cagcattatt aatgacctct 4860 tggagcagct ggggcagctg gatacagtgg acctgaataa gctaaacgag attgaaggca 4920 ccctaaacaa agccaaagat gaaatgaagg tcagcgatct tgataggaaa gtgtctgacc 4980 tggagaatga agccaagaag caggaggctg ccatcatgga ctataaccga gatatcgagg 5040 agatcatgaa ggacattcgc aatctggagg acatcaggaa gaccttacca tctggctgct 5100 tcaacacccc gtccattgaa aagccctagt gtctttaggg ctggaaggca gcatccctct 5160 gacagggggg cagttgtgag gccacagagt gccttgacac aaagattaca tttttcagac 5220 ccccactcct ctgctgctgt ccatcactgt ccttttgaac caggaaaagt cacagagttt 5280 aaagagaagc aaattaaaca tcctgaatcg ggaacaaagg gttttatcta ataaagtgtc 5340 tcttccatca cgttgctacc ttacccacac ttccctctga tttgcgtgag gacgtggcat 5400 cctacttacg tacgtggcat aacacatcgt gtgagcccat gtatgctggg gtagagcaag 5460 tagccctccc ctgtctcatc gatccagcag aacctcctca gtctcagtac tcttgtttct 5520 ataaggaaaa gttttgctac taacagtagc attgtgatgg ccagtatatc cagtccatgg 5580 ataaagaaaa tgcatctgca tctcctgccc ctcttccttc taagcaaaag gaaataaaca 5640 tcctgtgcca aaggtattgg tcatttagaa tgtcggtagc catccatcag tgcttttagc 5700 tattatgagt gtaggacact gagccatccg tgggtcagga tgcaattatt tataaaagtc 5760 cccaggtgaa catggctgaa gatttttcta gtatattaat aattgactag gaagatgaac 5820 tttttttcag atctttgggc agctgataat ttaaatctgg atgggcagct tgcactcacc 5880 aatagaccaa aagacatctt ttgatattct tataaatgga acttacacag aagaaatagg 5940 gatatgataa ccactaaagt tttgttttca aaatcaaact aattcttaca gcttttttat 6000 tagttagtct tggaactagt gttaagtatc tggcagagaa cagttaatcc ctaaggtctt 6060 gacaaaacag aagaaaaaca agcctcctcg tcctagtctt ttctagcaaa gggataaaac 6120 ttagatggca gcttgtactg tcagaatccc gtgtatccat ttgttcttct gttggagaga 6180 tgagacattt gacccttagc tccagttttc ttctgatgtt tccatcttcc agaatccctc 6240 aaaaaacatt gtttgccaaa tcctggtggc aaatacttgc actcagtatt tcacacagct 6300 gccaacgcta tcgagttcct gcactttgtg atttaaatcc actctaaacc ttccctctaa 6360 gtgtagaggg aagaccctta cgtggagttt cctagtgggc ttctcaactt ttgatcctca 6420 gctctgtggt tttaagacca cagtgtgaca gttccctgcc acacaccccc ttcctcctac 6480 caacccacct ttgagattca tatatagcct ttaacactat gcaactttgt actttgcgta 6540 gcaggggctg gggtgggggg aaagaaacct attatcatgg acacactggt gctattaatt 6600 atttcaaatt tatatttttg tgtgaatgtt ttgtgttttg tttatccatg ctatagaaca 6660 aggaatttat gtagatatac ttagtcctat ttctagaatg acactctgtt cactttgctc 6720 aatttttcct cttcactggc acaagtatct gaatacctcc ttccctccct tctagagttc 6780 tttggattgt actccaaaga attgtgcctt gtgtttgcag catctccatt ctctaaatta 6840 atataattgc tttcctccac acccagccac gtaaagaggt aacttgggtc ctcttccatt 6900 gcagtcctga tgatcctaac ctgcagcacg gtggttttac aatgttccag agcaggaacg 6960 ccaggttgac aagctatggt aggattagga aagtttgctg aagaggatct ttgacgccac 7020 agtgggacta gccaggaatg agggagaaat gccctttttg gcaattgttg gagctggata 7080 ggtaagtttt ataagggagt acattttgac tgagcactta gggcatcagg aacagtgcta 7140 cttactggtg ggtagactgg gagaggtggt gtaacttagt tcttgatgat cccacttcct 7200 gtttccatct gcttgggata taccagagtt taccacaagt gttttgacga tatactcctg 7260 agctttcact ctgctggctt ctcccaggcc tcttctacta tggcaggaga tgtggtgtgc 7320 tgttgcaaag ttttcacgtc atcgtttcct ggctagttca tttcattaag tggctacatc 7380 ctaacatatg cattggtcaa ggttgcagca agaggactga agattgactg ccaagctagt 7440 ttgggtgaag ttcactccag caagtctcag gccacaatgg ggtggtttgg tttggtttcc 7500 ttttaacttt ctttttgtta tttgcttttc tcctccacct gtgtggtata ttttttaagc 7560 agaattttat tttttaaaat aaaaggttct ttacaagatg ataccttaat tacactcccg 7620 caacacagcc attattttat tgtctagctc cagttatctg tattttatgt aatgtaattg 7680 acaggatggc tgctgcagaa tgctggttga cacagggatt attatactgc tatttttccc 7740 tgaattcttt tccttggaat tccaactgtg gaccttttat atgtgccttc actttagctg 7800 tttgccttac tctacagcct tgctctccgg ggtggttaat aaaatgcaac acttggcatt 7860 tttatgttat aagaaaaaca gtattttatt tataataaaa tctgaatatt ttgtaaccct 7920 tta 7923 <210> 6 <211> 1609 <212> PRT <213> Human <400> Met Arg Gly Ser His Arg Ala Ala Pro Ala Leu Arg Pro Arg Gly   1 5 10 15 Arg Leu Trp Pro Val Leu Ala Val Leu Ala Ala Ala Ala Ala Ala                  20 25 30 Gly Cys Ala Gln Ala Ala Met Asp Glu Cys Thr Asp Glu Gly Gly                  35 40 45 Arg Pro Gln Arg Cys Met Pro Glu Phe Val Asn Ala Ala Phe Asn                  50 55 60 Val Thr Val Val Ala Thr Asn Thr Cys Gly Thr Pro Pro Glu Glu                  65 70 75 Tyr Cys Val Gln Thr Gly Val Thr Gly Val Thr Lys Ser Cys His                  80 85 90 Leu Cys Asp Ala Gly Gln Pro His Leu Gln His Gly Ala Ala Phe                  95 100 105 Leu Thr Asp Tyr Asn Asn Gln Ala Asp Thr Thr Trp Trp Gln Ser                 110 115 120 Gln Thr Met Leu Ala Gly Val Gln Tyr Pro Ser Ser Ile Asn Leu                 125 130 135 Thr Leu His Leu Gly Lys Ala Phe Asp Ile Thr Tyr Val Arg Leu                 140 145 150 Lys Phe His Thr Ser Arg Pro Glu Ser Phe Ala Ile Tyr Lys Arg                 155 160 165 Thr Arg Glu Asp Gly Pro Trp Ile Pro Tyr Gln Tyr Tyr Ser Gly                 170 175 180 Ser Cys Glu Asn Thr Tyr Ser Lys Ala Asn Arg Gly Phe Ile Arg                 185 190 195 Thr Gly Gly Asp Glu Gln Gln Ala Leu Cys Thr Asp Glu Phe Ser                 200 205 210 Asp Ile Ser Pro Leu Thr Gly Gly Asn Val Ala Phe Ser Thr Leu                 215 220 225 Glu Gly Arg Pro Ser Ala Tyr Asn Phe Asp Asn Ser Pro Val Leu                 230 235 240 Gln Glu Trp Val Thr Ala Thr Asp Ile Arg Val Thr Leu Asn Arg                 245 250 255 Leu Asn Thr Phe Gly Asp Glu Val Phe Asn Asp Pro Lys Val Leu                 260 265 270 Lys Ser Tyr Tyr Tyr Ala Ile Ser Asp Phe Ala Val Gly Gly Arg                 275 280 285 Cys Lys Cys Asn Gly His Ala Ser Glu Cys Met Lys Asn Glu Phe                 290 295 300 Asp Lys Leu Val Cys Asn Cys Lys His Asn Thr Tyr Gly Val Asp                 305 310 315 Cys Glu Lys Cys Leu Pro Phe Phe Asn Asp Arg Pro Trp Arg Arg                 320 325 330 Ala Thr Ala Glu Ser Ala Ser Glu Cys Leu Pro Cys Asp Cys Asn                 335 340 345 Gly Arg Ser Gln Glu Cys Tyr Phe Asp Pro Glu Leu Tyr Arg Ser                 350 355 360 Thr Gly His Gly Gly His Cys Thr Asn Cys Gln Asp Asn Thr Asp                 365 370 375 Gly Ala His Cys Glu Arg Cys Arg Glu Asn Phe Phe Arg Leu Gly                 380 385 390 Asn Asn Glu Ala Cys Ser Ser Cys His Cys Ser Pro Val Gly Ser                 395 400 405 Leu Ser Thr Gln Cys Asp Ser Tyr Gly Arg Cys Ser Cys Lys Pro                 410 415 420 Gly Val Met Gly Asp Lys Cys Asp Arg Cys Gln Pro Gly Phe His                 425 430 435 Ser Leu Thr Glu Ala Gly Cys Arg Pro Cys Ser Cys Asp Pro Ser                 440 445 450 Gly Ser Ile Asp Glu Cys Asn Val Glu Thr Gly Arg Cys Val Cys                 455 460 465 Lys Asp Asn Val Glu Gly Phe Asn Cys Glu Arg Cys Lys Pro Gly                 470 475 480 Phe Phe Asn Leu Glu Ser Ser Asn Pro Arg Gly Cys Thr Pro Cys                 485 490 495 Phe Cys Phe Gly His Ser Ser Val Cys Thr Asn Ala Val Gly Tyr                 500 505 510 Ser Val Tyr Ser Ile Ser Ser Thr Phe Gln Ile Asp Glu Asp Gly                 515 520 525 Trp Arg Ala Glu Gln Arg Asp Gly Ser Glu Ala Ser Leu Glu Trp                 530 535 540 Ser Ser Glu Arg Gln Asp Ile Ala Val Ile Ser Asp Ser Tyr Phe                 545 550 555 Pro Arg Tyr Phe Ile Ala Pro Ala Lys Phe Leu Gly Lys Gln Val                 560 565 570 Leu Ser Tyr Gly Gln Asn Leu Ser Phe Ser Phe Arg Val Asp Arg                 575 580 585 Arg Asp Thr Arg Leu Ser Ala Glu Asp Leu Val Leu Glu Gly Ala                 590 595 600 Gly Leu Arg Val Ser Val Pro Leu Ile Ala Gln Gly Asn Ser Tyr                 605 610 615 Pro Ser Glu Thr Thr Val Lys Tyr Val Phe Arg Leu His Glu Ala                 620 625 630 Thr Asp Tyr Pro Trp Arg Pro Ala Leu Thr Pro Phe Glu Phe Gln                 635 640 645 Lys Leu Leu Asn Asn Leu Thr Ser Ile Lys Ile Arg Gly Thr Tyr                 650 655 660 Ser Glu Arg Ser Ala Gly Tyr Leu Asp Asp Val Thr Leu Ala Ser                 665 670 675 Ala Arg Pro Gly Pro Gly Val Pro Ala Thr Trp Val Glu Ser Cys                 680 685 690 Thr Cys Pro Val Gly Tyr Gly Gly Gln Phe Cys Glu Met Cys Leu                 695 700 705 Ser Gly Tyr Arg Arg Glu Thr Pro Asn Leu Gly Pro Tyr Ser Pro                 710 715 720 Cys Val Leu Cys Ala Cys Asn Gly His Ser Glu Thr Cys Asp Pro                 725 730 735 Glu Thr Gly Val Cys Asn Cys Arg Asp Asn Thr Ala Gly Pro His                 740 745 750 Cys Glu Lys Cys Ser Asp Gly Tyr Tyr Gly Asp Ser Thr Ala Gly                 755 760 765 Thr Ser Ser Asp Cys Gln Pro Cys Pro Cys Pro Gly Gly Ser Ser                 770 775 780 Cys Ala Val Val Pro Lys Thr Lys Glu Val Val Cys Thr Asn Cys                 785 790 795 Pro Thr Gly Thr Thr Gly Lys Arg Cys Glu Leu Cys Asp Asp Gly                 800 805 810 Tyr Phe Gly Asp Pro Leu Gly Arg Asn Gly Pro Val Arg Leu Cys                 815 820 825 Arg Leu Cys Gln Cys Ser Asp Asn Ile Asp Pro Asn Ala Val Gly                 830 835 840 Asn Cys Asn Arg Leu Thr Gly Glu Cys Leu Lys Cys Ile Tyr Asn                 845 850 855 Thr Ala Gly Phe Tyr Cys Asp Arg Cys Lys Asp Gly Phe Phe Gly                 860 865 870 Asn Pro Leu Ala Pro Asn Pro Ala Asp Lys Cys Lys Ala Cys Asn                 875 880 885 Cys Asn Pro Tyr Gly Thr Met Lys Gln Gln Ser Ser Cys Asn Pro                 890 895 900 Val Thr Gly Gln Cys Glu Cys Leu Pro His Val Thr Gly Gln Asp                 905 910 915 Cys Gly Ala Cys Asp Pro Gly Phe Tyr Asn Leu Gln Ser Gly Gln                 920 925 930 Gly Cys Glu Arg Cys Asp Cys His Ala Leu Gly Ser Thr Asn Gly                 935 940 945 Gln Cys Asp Ile Arg Thr Gly Gln Cys Glu Cys Gln Pro Gly Ile                 950 955 960 Thr Gly Gln His Cys Glu Arg Cys Glu Val Asn His Phe Gly Phe                 965 970 975 Gly Pro Glu Gly Cys Lys Pro Cys Asp Cys His Pro Glu Gly Ser                 980 985 990 Leu Ser Leu Gln Cys Lys Asp Asp Gly Arg Cys Glu Cys Arg Glu                 995 1000 1005 Gly Phe Val Gly Asn Arg Cys Asp Gln Cys Glu Glu Asn Tyr Phe                1010 1015 1020 Tyr Asn Arg Ser Trp Pro Gly Cys Gln Glu Cys Pro Ala Cys Tyr                1025 1030 1035 Arg Leu Val Lys Asp Lys Val Ala Asp His Arg Val Lys Leu Gln                1040 1045 1050 Glu Leu Glu Ser Leu Ile Ala Asn Leu Gly Thr Gly Asp Glu Met                1055 1060 1065 Val Thr Asp Gln Ala Phe Glu Asp Arg Leu Lys Glu Ala Glu Arg                1070 1075 1080 Glu Val Met Asp Leu Leu Arg Glu Ala Gln Asp Val Lys Asp Val                1085 1090 1095 Asp Gln Asn Leu Met Asp Arg Leu Gln Arg Val Asn Asn Thr Leu                1100 1105 1110 Ser Ser Gln Ile Ser Arg Leu Gln Asn Ile Arg Asn Thr Ile Glu                1115 1120 1125 Glu Thr Gly Asn Leu Ala Glu Gln Ala Arg Ala His Val Glu Asn                1130 1135 1140 Thr Glu Arg Leu Ile Glu Ile Ala Ser Arg Glu Leu Glu Lys Ala                1145 1150 1155 Lys Val Ala Ala Ala Asn Val Ser Val Thr Gln Pro Glu Ser Thr                1160 1165 1170 Gly Asp Pro Asn Asn Met Thr Leu Leu Ala Glu Glu Ala Arg Lys                1175 1180 1185 Leu Ala Glu Arg His Lys Gln Glu Ala Asp Asp Ile Val Arg Val                1190 1195 1200 Ala Lys Thr Ala Asn Asp Thr Ser Thr Glu Ala Tyr Asn Leu Leu                1205 1210 1215 Leu Arg Thr Leu Ala Gly Glu Asn Gln Thr Ala Phe Glu Ile Glu                1220 1225 1230 Glu Leu Asn Arg Lys Tyr Glu Gln Ala Lys Asn Ile Ser Gln Asp                1235 1240 1245 Leu Glu Lys Gln Ala Ala Arg Val His Glu Glu Ala Lys Arg Ala                1250 1255 1260 Gly Asp Lys Ala Val Glu Ile Tyr Ala Ser Val Ala Gln Leu Ser                1265 1270 1275 Pro Leu Asp Ser Glu Thr Leu Glu Asn Glu Ala Asn Asn Ile Lys                1280 1285 1290 Met Glu Ala Glu Asn Leu Glu Gln Leu Ile Asp Gln Lys Leu Lys                1295 1300 1305 Asp Tyr Glu Asp Leu Arg Glu Asp Met Arg Gly Lys Glu Leu Glu                1310 1315 1320 Val Lys Asn Leu Leu Glu Lys Gly Lys Thr Glu Gln Gln Thr Ala                1325 1330 1335 Asp Gln Leu Leu Ala Arg Ala Asp Ala Ala Lys Ala Leu Ala Glu                1340 1345 1350 Glu Ala Ala Lys Lys Gly Arg Asp Thr Leu Gln Glu Ala Asn Asp                1355 1360 1365 Ile Leu Asn Asn Leu Lys Asp Phe Asp Arg Arg Val Asn Asp Asn                1370 1375 1380 Lys Thr Ala Ala Glu Glu Ala Leu Arg Lys Ile Pro Ala Ile Asn                1385 1390 1395 Gln Thr Ile Thr Glu Ala Asn Glu Lys Thr Arg Glu Ala Gln Gln                1400 1405 1410 Ala Leu Gly Ser Ala Ala Ala Asp Ala Thr Glu Ala Lys Asn Lys                1415 1420 1425 Ala His Glu Ala Glu Arg Ile Ala Ser Ala Val Gln Lys Asn Ala                1430 1435 1440 Thr Ser Thr Lys Ala Glu Ala Glu Arg Thr Phe Ala Glu Val Thr                1445 1450 1455 Asp Leu Asp Asn Glu Val Asn Asn Met Leu Lys Gln Leu Gln Glu                1460 1465 1470 Ala Glu Lys Glu Leu Lys Arg Lys Gln Asp Asp Ala Asp Gln Asp                1475 1480 1485 Met Met Met Ala Gly Met Ala Ser Gln Ala Ala Gln Glu Ala Glu                1490 1495 1500 Ile Asn Ala Arg Lys Ala Lys Asn Ser Val Thr Ser Leu Leu Ser                1505 1510 1515 Ile Ile Asn Asp Leu Leu Glu Gln Leu Gly Gln Leu Asp Thr Val                1520 1525 1530 Asp Leu Asn Lys Leu Asn Glu Ile Glu Gly Thr Leu Asn Lys Ala                1535 1540 1545 Lys Asp Glu Met Lys Val Ser Asp Leu Asp Arg Lys Val Ser Asp                1550 1555 1560 Leu Glu Asn Glu Ala Lys Lys Gln Glu Ala Ala Ile Met Asp Tyr                1565 1570 1575 Asn Arg Asp Ile Glu Glu Ile Met Lys Asp Ile Arg Asn Leu Glu                1580 1585 1590 Asp Ile Arg Lys Thr Leu Pro Ser Gly Cys Phe Asn Thr Pro Ser                1595 1600 1605 Ile Glu Lys Pro            1609 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer for PCR amplification <400> 7 CAGGATCCAG TGGTGTCGTT AGA 23 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer for PCR amplification <400> 8 GGTCTAGATC ATCCATGATT GGCCTG 26 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer for PCR amplification <400> 9 CAGGATCCGT TCTGAGCTTG TAC 23 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer for PCR amplification <400> 10 CCTCTAGATC AGGAGAATGA GGCAGA 26

[Brief description of drawings]

FIG. 1 shows HT1 by two-dimensional SDS-PAGE.
Laminin α3 chain and γ secreted from 080 / WT cells
Analysis of two subunits is shown. Serum-free conditioned medium of HT1080 / WT cells was concentrated and subjected to 2D SDS-PAGE.
After analysis by (non-reducing and reducing SDS-PAGE), a monoclonal antibody against laminin α3 chain (L
Immunoblotting was performed using Sα3c4; panel A) or a monoclonal antibody against the γ2 chain (D4B5; panel B). Arrowheads indicate the estimated molecular size (k) of the protein spot in one dimension (horizontal axis) and two dimensions (vertical axis).
Da) is shown. Molecular size of marker protein (kD
a) is shown on the left.

FIG. 2 shows SDS-PAGE and immunoblotting analysis of purified mat-LN5, LN6 and LN6ΔG4-5. Panel A: mat on 5% gel
-LN5 (lane 1), LN6 (lane 2) and mouse LN1 (lane 3) non-reducing SDS-PAGE and silver staining. Panel B: reduced SDS-PAGE and silver staining of mat-LN5 (lane 1), LN6 (lane 2) and LN6ΔG4-5 (lane 3) on a 6% gel. Panel C: m
at-LN5 (lane 1), LN6 (lane 2) and L
Immunoblotting with monoclonal antibodies specific for laminin α3, β1, γ1, β3 and γ2 chains (from left to right) after reducing SDS-PAGE of N6ΔG4-5 (lane 3). The vertical axis represents the molecular size (kDa).

FIG. 3 is LN6, LN6ΔG4-5, LN6ΔG3 for HT1080 / WT cells and BRL cells.
5 shows the results of cell adhesion activity of -5, mat-LN5 and other three proteins. A 96-well plate is coated with each substrate at a predetermined concentration, and HT1080 / WT cells (panel A) and BRL cells (panel B) are serum-free at 37 ° C.
And incubated for 1 hour. After incubation,
The relative number of cells attached to the substrate was determined by measuring the fluorescence intensity. Each point is mean ± SD for triplicate measurements
Indicates. Other experimental conditions are described in "Materials and Methods". LN6 (white circle), LN6ΔG4-5 (black circle), LN
6ΔG3-5 (white diamond), mat-LN5 (black triangle),
Mouse LN1 (white triangle), LN10 / 11 (black square),
Fibronectin (white square).

FIG. 4 shows mat-LN5, LN6, LN6ΔG.
4 shows the morphology of HT1080 cells on 4-5 and LN10 / 11. Plastic plate with 0.5 μg / ml m
at-LN5 (a and e), 1 μg / ml LN6 (b
And f), 0.5 μg / ml LN6ΔG4-5 (c and g), or 10 μg / ml LN10 / 11 (d and h). HT1080 cells were plated on each substrate and incubated for 20 minutes (ad) or 60 minutes (fh).

FIG. 5 shows soluble and insoluble mat-LN5, L
The result of the dispersion | distribution of BRL cell by N6 and LN6 (DELTA) G4-5 is shown. 2 BRL cells (7 × 10 ³ cells / well)
No addition (a), 50 ng / m on 4-well plate
l mat-LN5 (b), LN6 (c), or LN6
Incubation was performed in the culture medium in the presence of 1% FBS supplemented with ΔG4-5 (d). Two days after incubation, the photographs were taken under a phase contrast microscope.

FIG. 6 shows soluble and insoluble mat-LN5, L
The result of the dispersion or migration of BRL cells by N6 and LN6ΔG4-5 is shown. Panel A: Cell dispersion activity when various laminins were added. mat-LN5 (white square), LN
6 (open circle) and LN6ΔG4-5 (black triangle) were added to the culture medium at a predetermined final concentration, and after 2 days of incubation,
The percentage of dispersed cells was measured as described in Materials and Methods. Panel B: Cell dispersal activity when various laminins were fixed. Each well of the 24-well plate has a mat-
LN5 (white square), LN6 (white circle), or LN6ΔG4
-5 (black triangle) was coated at a predetermined density. After incubating BRL cells for 2 days, the percentage of dispersed cells was measured. Panel C: Cell migration when various laminins are fixed. Each well of the plastic plate is
at-LN5 (dotting), LN6 (mesh), or LN6Δ
Coated with G4-5 (black). BRL cells were incubated with uncoated wells (clear) and laminin coated wells in medium containing 1% FBS. The migration of BRL cells was monitored by a video microscope for 10 hours to quantify the migration distance. Each bar represents the average migration rate ± SD for any 10 cells.

FIG. 7 shows mat-LN5 (A), LN6 with function-blocking antibodies against various integrin subunits.
HT108 on (B) and LN6ΔG4-5 (C) substrates
The result of having examined the effect in 0 cell adhesion is shown. HT1080 cells were treated with anti-α2 antibody (P1E6), anti-α
3 antibody (P1B5), anti-α5 antibody (P1D6), anti-α6
37 in suspension with antibody (G0H3), anti-β1 antibody (P4C10), or a mixture of anti-α3 and anti-α6 antibodies
Pre-incubate for 30 minutes at 0 ° C, 0.5 μg / ml
Mat-LN5, 1 μg / ml LN6 or 1 μg / ml
The cells were seeded on a 96-well plate precoated with ml of LN6ΔG4-5 and incubated in the presence of the antibody for 30 minutes. 100 cell adhesion in the presence of control mouse IgG
%. Each point represents the mean ± SD for triplicate measurements.
The other experimental conditions are the same as those described in FIG.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 37/06 A61P 43/00 105 43/00 105 A61K 37/02 ZNA C12N 5/06 C12N 5/00 E // C12N 15/09 15/00 AF term (reference) 4B024 AA01 BA80 CA04 DA03 EA04 GA11 4B065 AA93X AA93Y AB01 AC14 BA02 BB19 BC01 CA24 CA44 4C084 AA02 BA01 BA02 BA08 BA22 BA23 CA62 ZA01 ZA33 ZA89 B21 Za94 ZB02

Claims (16)

[Claims] 1. Cell adhesion activity and / or laminin-6-containing activity
Alternatively, a composition for regulating cell motility activity. 2. Laminin-6 is a protein comprising each subunit of α3 chain having the amino acid sequence of SEQ ID NO: 2, β1 chain having the amino acid sequence of SEQ ID NO: 4, and γ1 chain having the amino acid sequence of SEQ ID NO: 6. Or alternatively has an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in these sequences, and has weak cell adhesion activity, and / or substantially The composition according to claim 1, which is a protein having no cell motility activity. 3. Laminin-6 comprises an α3 chain having the amino acid sequence of SEQ ID NO: 2, a β1 chain having the amino acid sequence of SEQ ID NO: 4, and a γ1 chain having the amino acid sequence of SEQ ID NO: 6, and 50% in each amino acid sequence. A protein comprising each subunit having the above homology,
The composition according to. 4. Laminin-6 comprises an α3 chain having the amino acid sequence of SEQ ID NO: 2, a β1 chain having the amino acid sequence of SEQ ID NO: 4, and a γ1 chain having the amino acid sequence of SEQ ID NO: 6. The composition according to. 5. Laminin-6 comprises an α3 chain in which all or part of amino acid residues 1325-1713 are deleted in the amino acid sequence of SEQ ID NO: 2, a β1 chain having the amino acid sequence of SEQ ID NO: 4 and a sequence of SEQ ID NO: 6. It is a protein consisting of a γ1 chain having an amino acid sequence, or has an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in these sequences, and has cell adhesion activity. The composition according to claim 1, which is a protein having and / or having a weak cell motility activity. 6. Laminin-6 comprises an α3 chain in which all or part of amino acid residues 1325-1713 are deleted in the amino acid sequence of SEQ ID NO: 2, a β1 chain having the amino acid sequence of SEQ ID NO: 4 and a sequence of SEQ ID NO: 6. The composition according to claim 5, which is a protein consisting of a γ1 chain having an amino acid sequence and each subunit having 50% or more homology in each amino acid sequence. 7. Laminin-6 comprises an α3 chain in which all or part of amino acid residues 1149-1713 in the amino acid sequence of SEQ ID NO: 2 is deleted, a β1 chain having the amino acid sequence of SEQ ID NO: 4 and a SEQ ID NO: 6. A protein consisting of a γ1 chain having an amino acid sequence, or having an amino acid sequence in which one or more amino acid residues are deleted, added or substituted in these sequences, and is substantially a cell The composition according to claim 1, which is a protein having no adhesive activity. 8. The method according to claim 5, which has a cell-dispersing activity.
The composition according to any one of 1. 9. The composition of any one of claims 1-8, further comprising laminin-5. 10. The method according to any one of claims 1 to 9 for maintaining or treating the functions of epithelial tissue, nerve tissue and muscle.
The composition according to the item. 11. The composition according to claim 10, which is used for treatment or prevention of skin transplantation, retinal detachment, and epidermolytic varicella. 12. The method according to claim 1, which is used as an additive for culturing cells for transplantation or as an adhesive device.
The composition according to the item. 13. A method for regulating cell adhesion activity and / or cell motility activity using the composition according to any one of claims 1 to 12. 14. The method according to claim 13, wherein the composition according to any one of claims 5-6 and 8-9 is used to enhance cell adhesion activity. 15. The composition is fixed to a culture container, or
Alternatively, the method according to claim 14, which comprises adding to the culture medium. 16. A kit for regulating cell adhesion activity and / or cell motility activity, which comprises any one of claims 1 to 12.
A kit comprising the composition of claim 1.