JP5142246B2 - Laminin 6B (Laminin 3B11) protein - Google Patents

Description

  The present invention relates to laminin 6B (laminin 3B11) protein.

  In order for a multicellular organism to maintain its life, it is essential that cells adhere to an extracellular matrix (ECM). If the adherent cells lose their adhesion to the ECM, they stop growing and undergo apoptosis. That is, the ECM regulates an important function related to cell survival by interacting with the cell (Non-patent Document 1). ECM generally contains large and complex proteins such as proteins secreted from cells and complex polysaccharides. ECM molecules are roughly divided into two types, the basement membrane type that exists mainly in the basement membrane and the stromal type that exists in connective tissue. Another characteristic of ECM molecules is that they have the ability to self-associate. Thereby, a strong fiber or sheet-like structure can be made with a single molecular species. Furthermore, since it has many functional domain structures in the molecule and a domain that binds to other molecules, a strong network structure can be formed between ECM molecules.

  In the basement membrane existing between the epithelial tissue and the connective tissue, heparan sulfate proteoglycans such as type IV collagen and perlecan, and glycoproteins such as laminin and nidogen exist. These basement membrane molecules are complexly bonded to each other to form a vast network-like network.

  Laminin is a group of protein families consisting of three subunits, α chain, β chain, and γ chain. All laminins are bonded at the C-terminal of each constituent chain to form a coiled-coil structure and form a heterotrimer stabilized by disulfide bonds (Non-patent Documents 2 and 3). To date, 5 types of α chain, 3 types of β chain, and 3 types of γ chain have been confirmed, and 15 types of isoforms have been confirmed as laminin (Non-patent Document 4).

  The α chain of laminin has five globular domains (G1-5) in the C-terminal region (G1-5), and is mainly bound to cell membrane surface receptors such as integrins, syndecans, and α-dystroglycan. It is a part (Non-Patent Document 5). On the other hand, it has been revealed that β and γ chains have binding domains with other ECM molecules such as collagen, heparan sulfate, and nidogen.

  The laminin α3A chain binds to β3 and γ2 chains to form laminin 5A (or laminin 3A32), and binds to β1 and γ1 chains to form laminin 6A (or laminin 3A11) (FIG. 1) (Non-patent Document 4). . Laminin 5A and laminin 6A are secreted from various epithelial cells in tissues and in culture systems, and are mainly present in the basement membrane of the skin in vivo (Non-patent Documents 3 and 6). The α3A chain of laminin 5A is cleaved (processed) by protease from 190 kDa to 160 kDa or 145 kDa after secretion outside the cell (Non-patent Document 7). As a result, it has been reported that the motility activity and adhesion activity of laminin 5A to cells change. It has been reported that the cleavage site exists between the G3 domain and the G4 domain. On the other hand, it has been clarified that the α3 chain possessed by laminin 6A is mainly 190 kDa type (Non-patent Document 8).

It is known that laminin 5A present in the living body is present in a complex with laminin 6A (Non-patent Document 9). Furthermore, this laminin 5A-6A complex can interact with other laminins through the VI domains of the laminin 6A β1 and γ1 chains. In addition, it is considered that it is incorporated into a type IV collagen network through a bond between a γ1 chain and a nidogen (Non-patent Document 10). In addition, the G domain of the laminin 5A α3A chain in the laminin 5A-6A complex binds to the integrin α ₃ β ₁ or α ₆ β ₄ , but the α3A chain of laminin 6A does not bind (non-patent document). 11). From this, laminin 6A is considered to have a function of binding to other ECM molecules in the basement membrane of epithelial tissue and cross-linking network formation between ECM molecules. Therefore, by combining laminin 6A with laminin 5A and other ECM proteins, there is a possibility that the epidermis and other epithelial cells can be cultured under conditions close to those in the living body.

  Laminin α3A chain was known to have a laminin α3B chain with a long N-terminal region due to differences in mRNA splicing. Since this laminin α3B chain has a coiled coil domain (domain I / II) in common with the laminin α3A chain in its C-terminal region, it binds to the β3 and γ2 chains to form laminin 5B (or laminin 3B32), and β1, γ1 chains Although it is expected to bind to form laminin 6B (or laminin 3B11), the expression of these proteins in vivo is not known (FIG. 1). Recently, a full-length cDNA of human laminin α3B chain has been cloned, and recombinant laminin 5B has been prepared in human fetal kidney-derived cell line HEK293 (Non-patent Document 12). Laminin 5B exhibits higher cell adhesion activity and cell motility activity than laminin 5A.

Hood JD, Cheresh DA. (2002): Role of integrins in cell invasion and migration.Nat Rev Cancer.; 2 (2): 91-100. Colognato H, Yurchenco PD. (2000): Form and function: the laminin family of heterotrimers. Dev Dyn .; 218 (2): 213-34. Review. Aumailley M, Rousselle P. (1999): Laminins of the dermo-epidermal junction. Matrix Biol .; 18 (1): 19-28. Review. Aumailley, M., Bruckner-Tuderman, L., Carter, WG, Deutzmann, R., Edgar, D., Ekblom, P., Engel, J., Engvall, E., Hohenester, E., Jones, JC, Kleinman, HK, Marinkovich, MP, Martin, GR, Mayer, U., Meneguzzi, G., Miner, JH, Miyazaki, K., Patarroyo, M., Paulsson, M., Quaranta, V., Sanes, JR, Sasaki, T., Sekiguchi, K., Sorokin, LM, Talts, JF, Tryggvason, K., Uitto, J., Virtanen, I., von der Mark, K., Wewer, UM, Yamada, Y. and Yurchenco , PD (2005): A simplified laminin nomenclature. Matrix Biol. 24: 326-332. Review. Hirosaki T, Mizushima H, Tsubota Y, Moriyama K, Miyazaki K. (2000): Structural requirement of carboxyl-terminal globular domains of laminin alpha 3 chain for promotion of rapid cell adhesion and migration by laminin-5.J Biol Chem .; 275 (29): 22495-502. Marinkovich MP, Lunstrum GP, Keene DR, Burgeson RE. (1992) The dermal- epidermal junction of human skin contains a novel laminin variant. J Cell Biol .; 119 (3): 695-703. Miyazaki K. (2006) Laminin-5 (laminin-332): unique biological activity and role in tumor growth and invasion.Cancer Sci; 97 (2): 91-98. Hirosaki T, Tsubota Y, Kariya Y, Moriyama K, Mizushima H, Miyazaki K. (2002) Laminin-6 is activated by proteolytic processing and regulates cellular adhesion and migration differently from laminin-5.J Biol Chem .; 277 (51) : 49287-95. Champliaud MF, Lunstrum GP, Rousselle P, Nishiyama T, Keene DR, Burgeson RE. (1996): Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment .J Cell Biol .; 132 (6): 1189-98. Mayer U, Poschl E, Gerecke DR, Wagman DW, Burgeson RE, Timpl R. (1995): Low nidogen affinity of laminin-5 can be attributed to two serine residues in EGF-like motif gamma 2III4.FEBS Lett.; 365 ( 2-3): 129-32. Rousselle P, Keene DR, Ruggiero F, Champliaud MF, Rest M, Burgeson RE. (1997) Laminin 5 binds the NC-1 domain of type VII collagen.J Cell Biol .; 138 (3): 719-28. Kariya Y, Yasuda C, Nakashima Y, Ishida K, Tsubota Y, Miyazaki K. (2004) Characterization of laminin-5B (α3Bβ3γ2) and NH2-terminal proteolytic fragment of its α3B chain: Promotion of cellular adhesion, migration and proliferation. Biol Chem, 279 (23): 24774-24784.

  An object of the present invention is to provide a new species of laminin molecule that supports cell adhesion, migration, survival, and the like and plays an important role in tissue construction.

  The present inventors established a cell line LN6B-HEK that stably and forcibly expresses laminin 6B for the first time by introducing cDNAs of laminin α3B, β1, and γ1 chains into HEK293 cells. The laminin 6B protein produced by this cell was purified, and it was revealed that this laminin exhibits cell adhesion activity and cell motility activity. In addition, it was revealed that laminin 6B is localized in the vascular basement membrane and promotes the proliferation of vascular endothelial cells. In the present invention, the α3A chain cDNA was introduced into HEK293 cells instead of laminin α3B chain, thereby succeeding in expressing about 10 times as much laminin 6A as in the conventional method.

  In order for animal cells to survive and proliferate, a protein serving as a substrate (scaffold) is required, and this substrate protein varies depending on the cell type. In recent years, in the field of regenerative medicine, it has been considered that various cells are cultured in vitro and transplanted to the human body. However, it is particularly difficult to stably culture normal epithelial cells on a petri dish, and one of the factors is that an appropriate substrate protein cannot be obtained.

So far, fibronectin, collagen, laminin 1 and the like that can be easily prepared have been used as cell adhesion substrate proteins, but laminin 6B is not known for its existence and has never been used. Laminin 6B exhibits higher cell adhesion activity than laminin 1 (consisting of α1, β1, and γ1 chains). Due to differences in structure, activity, and tissue distribution, it is expected that laminin 6B will exhibit different properties from known cell adhesion molecules in cell culture.
As shown in Examples described later, laminin 6B is localized in the vascular basement membrane and promotes the proliferation of vascular endothelial cells. Therefore, there is a possibility that laminin 6B can be used as an adhesion substrate for stably culturing vascular endothelial cells and epidermal cells.
The gist of the present invention is as follows.

(1) The following protein (a) or (b):
(a) It is composed of each subunit of α3B chain having the amino acid sequence shown by SEQ ID NO: 2, β1 chain having the amino acid sequence shown by SEQ ID NO: 4, and γ1 chain having the amino acid sequence shown by SEQ ID NO: 6 Laminin 6B protein.
(b) In the protein of (a), at least one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 4, and the amino acid sequence represented by SEQ ID NO: 6 A protein having an amino acid deleted, substituted, or added and having the biological activity of laminin 6B protein.
However, the α3B chain may not include the LG4-5 domain (the 8833th to 10002nd amino acid sequence of SEQ ID NO: 2) present on the C-terminal side.

(2) A host cell transformed with the following DNAs (i), (ii) and (iii):
(i) DNA encoding an α3B chain having the amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids may be deleted, substituted or added
(ii) DNA encoding the β1 chain having the amino acid sequence represented by SEQ ID NO: 4 in which one or several amino acids may be deleted, substituted or added
(iii) DNA encoding the γ1 chain having the amino acid sequence represented by SEQ ID NO: 6 in which one or several amino acids may be deleted, substituted or added
However, the α3B chain may not include the LG4-5 domain (the 8833th to 10002nd amino acid sequence of SEQ ID NO: 2) present on the C-terminal side.

(3) The cell according to (2), wherein the host cell is a HEK cell.
(4) The method for producing a protein according to (1), comprising culturing the cell according to (2) or (3).
(5) A composition comprising the protein according to (1).
(6) The composition according to (5), which is used as a cell culture substrate, a cell growth promoter, an angiogenesis agent, a wound healing agent, a nerve regeneration agent, or a transplant base material.

    According to the present invention, a novel laminin 6B protein has been provided. Since laminin 6B promotes cell adhesion and migration, it may be used in the fields of regenerative medicine and cell engineering in the future.

Hereinafter, embodiments of the present invention will be described in more detail.
FIG. 1 shows the structures of laminin 5A, laminin 6B and laminin 6A of the present invention. Laminin 6B protein consists of an α3B chain, a β1 chain, and a γ1 chain. Laminin 5A is composed of α3A chain, β3 chain and γ2 chain, and laminin 6A is composed of α3A chain, β1 chain and γ1 chain. The α3B chain is about twice as large as the α3A chain, and the structure of about half the C-terminal side of the α3B chain is common to the α3A chain. There is a globular (G) domain in the C-terminal part of the α3B chain and α3A chain, which is divided into five subdomains (or modules) (G1-G5).

  The α3B chain has a structure in which three globular domains (domains LN, L4a and L4b from the N-terminal side) and two EGF-like domains (domains LEa and LEb from the N-terminal side) are added to the N-terminal side of the αA chain. These domains are thought to contribute to integrin binding and association with self or other laminins. The overall structure of the α3B chain is similar to α1, α2, α5 chains, which are other long chain α chains.

  The laminin 6B protein of the present invention can be produced by expressing each subunit using recombinant DNA technology. The cDNA encoding each subunit of laminin 6B of the present invention is designed by polymerase chain reaction (PCR) by designing a primer based on the nucleotide sequence of SEQ ID NO: 1, 3 or 5 and using an appropriate cDNA library as a template. Can be produced by amplifying the sequence. Such PCR techniques are well known in the art, see, for example, “PCR Protocols, A Guide to Methods and Applications”, Academic Press, Michael, et al. , Eds. , 1990.

  A DNA encoding each laminin 6B chain gene is incorporated into an appropriate expression vector and introduced into a host cell (eg, a eukaryotic or prokaryotic cell) to express each chain as desired. Of protein. Examples of host cells that can be used to express the protein of the present invention include, but are not limited to, prokaryotic hosts such as E. coli and Bacillus subtilis, and eukaryotic organisms such as yeast, fungi, insect cells and mammalian cells. A host. Mammalian cells useful as hosts include gastric cancer cell lines such as STKM-1 and MKN-45, fibrosarcoma cell lines such as HT1080, fetal kidney-derived cell lines such as HEK293, floating HEK293F cells, HeLa cells, fibroblasts Cell-derived cells, such as VERO or CHO-K1, or lymphocyte-derived cells, and derivatives thereof are included. Preferred mammalian host cells include human kidney-derived cell lines such as HEK293 and suspension HEK293F cells, fibroblast-derived cells such as VERO or CHO-K1. In addition, plant cells and insect cells such as Drosophila cells can also be used as hosts.

  A vector refers to a single-stranded or double-stranded nucleic acid molecule that can be transfected into a cell and replicate in the cell genome or independently thereof. The expression vector includes a promoter region that drives DNA expression, and may further include transcriptional and translational control sequences such as a TATA box, a capping sequence, a CAAT sequence, a 3 'non-coding region, an enhancer, and the like. Examples of promoters include bla promoter, cat promoter, lacZ promoter when used in prokaryotic hosts, mouse metallothionein I gene sequence promoter, herpesvirus TK promoter when used in eukaryotic hosts, Examples include SV40 early promoter and yeast glycolytic enzyme gene sequence promoter. Examples of vectors include, but are not limited to, pBR322, pUC118, pUC119, λgt10, λgt11, pMAM-neo, pKRC, BPV, vaccinia, SV40, 2-micron and the like.

The expression vector preferably has one or more markers so that a host cell containing it can be selected. As the marker, those that give nutrition to an auxotrophic host, antibiotic resistance (for example, ampicillin, tetracycline, neomycin, hygromycin, zeocin, etc.), or heavy metal resistance (for example, copper) can be used.
Furthermore, the vector can be constructed so that the protein of the present invention is secreted and expressed using a signal sequence, or the protein of the present invention is expressed in the form of a fusion protein with another protein. By using a fusion protein, the stability of the protein can be improved or purification can be facilitated. The construction of such expression vectors is well known in the art.

  The DNA encoding each strand of laminin 6B may be incorporated into a single expression vector for expression, or may be incorporated into separate expression vectors and transfected into the same cells for expression. . Since the subunits of α3B chain, β1 chain and γ1 chain are all very large polypeptides, the latter method is preferably used.

  A vector constructed to express laminin 6B of the present invention is introduced into an appropriate host cell by transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, etc. can do. The laminin 6B protein of the present invention can be obtained by growing cells containing the vector in an appropriate medium to produce the protein of the present invention, and recovering and purifying the desired recombinant protein from the cell or medium. Purification can be performed using ion exchange chromatography, heparin affinity chromatography, immunoaffinity chromatography, and the like.

Each chain of laminin 6B of the present invention has one or several amino acid sequences (1 or more, preferably 2 or more, preferably 30 or less, more preferably 2 or more and 10 or less) in the corresponding amino acid sequence. ) Amino acids may be deleted, added or substituted. However, regarding the α3B chain, a protein that does not contain the LG4-5 domain (the 8833th to 10002nd amino acid sequence of SEQ ID NO: 2) present on the C-terminal side is also included in this protein. Such a protein homologous to a natural protein is also within the scope of the present invention.
It is well known in the art that conservative changes in amino acids can be made to obtain proteins or polypeptides that retain their original function. Such substitutions include replacing an amino acid with a residue having similar physicochemical properties, eg, replacing one aliphatic residue (Ile, Val, Leu or Ala) with another, or basic residue. Substitution is included between the groups Lys and Arg, acidic residues Glu and Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe and Tyr.

  And at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the amino acid sequence set forth in SEQ ID NO: 2, 4 or 6. A protein having the biological activity of laminin 6B protein is also within the scope of the present invention. Identity is calculated by dividing the number of residues that are identical by the total number of residues in a known sequence or domain of a known sequence and multiplying by 100. Several computer programs for determining sequence identity using standard parameters, such as Gapped BLAST or PSI-BLAST (Altschul, et al. (1997) Nucleic Acids Res. 25: 3389-3402), BLAST (Altschul, et al. (1990) J. Mol. Biol. 215: 403-410), and Smith-Waterman (Smith, et al. (1981) J. Mol. Biol. 147: 195. -197) is available. Preferably, the default settings of these programs are used, but these settings may be changed as desired.

Examples of the biological activity of laminin 6B protein include cell adhesion activity, cell motility activity, and cell proliferation activity. In particular, it is a culture substrate suitable for the proliferation of vascular endothelial cells.
A protein in which a part of each chain constituting laminin 6B of the present invention is deleted is also within the scope of the present invention. For example, in the α3B chain of laminin 6B of the present invention, G4 and G5 are cleaved and removed by protease. However, as long as it has activity as laminin 6B, it is within the scope of the present invention regardless of whether it is cleaved or uncut.

  Furthermore, a protein having an activity similar to that of human laminin 6B derived from an organism other than human is also within the scope of the present invention. A gene encoding such a protein is easily isolated using a polynucleotide having the sequence shown in SEQ ID NO: 1, 3 or 5 or a fragment thereof as a probe or primer and using a technique such as hybridization or PCR. can do. The homologous gene thus obtained is at least 50% or more, preferably 60% or more, more preferably 70% or more, further preferably 80% or more with respect to the base sequence described in SEQ ID NO: 1, 3 or 5. The homology is particularly preferably 90% or more, most preferably 95% or more. Alternatively, the homologous gene can hybridize with a gene having the base sequence described in SEQ ID NO: 1, 3 or 5 under stringent hybridization conditions.

  The term hybridize means that DNA or its corresponding RNA binds to another DNA or RNA molecule by hydrogen bonding interaction in solution or on a solid support. The strength of such interaction can be evaluated by changing the stringency of hybridization conditions. Depending on the desired specificity and selectivity, various stringency hybridization conditions can be used, and the stringency can be adjusted by changing the concentration of salt or denaturant. Such methods for regulating stringency are well known in the art, for example, “Molecular Cloning: A Laboratory Manual”, 2nd edition. Cold Spring Harbor Laboratory, Sambrook, Fritsch, & Maniatis, eds. 1989).

Stringent hybridization conditions refer to conditions of 42 ° C. or equivalent in 700 mM NaCl in the presence of 50% formamide. An example of stringent hybridization conditions is 50% formamide, 5XSSC, 50 mM NaH ₂ PO ₄ , pH 6.8, 0.5% SDS, 0.1 mg / mL sonicated salmon sperm DNA, and 42 in 5X Denhardt's solution. Overnight hybridization at 0 ° C; 2XSSC, 0.1% SDS at 45 ° C wash; and 0.2XSSC, 0.1% SDS at 45 ° C wash.

  The laminin 6B protein of the present invention has cell adhesion activity, cell motility activity, cell proliferation activity and the like. The cell adhesion activity of laminin 6B can be assayed by coating the plate with laminin 6B of the present invention, seeding the plate with appropriate cells, incubating for a predetermined time, and then measuring the number of cells adhered. The cell motility promoting activity can be assayed by coating the plate with laminin 6B of the present invention, seeding appropriate cells on the plate, and measuring the degree of cell dispersion after a predetermined time. Alternatively, the state of cell movement may be taken by video and the speed of cell movement may be obtained by image analysis. Cell proliferation activity can be measured by seeding appropriate cells such as vascular endothelial cells on a plate coated with laminin 6B, culturing for several days, and counting the number of proliferated cells.

  When the laminin 6B protein of the present invention is used as a cell culture substrate or cell growth promoter, laminin 6B is added to the cell culture solution, or applied or immobilized on a culture plate or culture sheet. Can be used. Or you may apply | coat on the feeder cell grown on the culture plate or the culture sheet. Most animal cells must adhere to the surface of the incubator as a living condition, and further divide under stimulation by growth factors. Since laminin 6B has cell adhesion activity, it is effective as a proliferation promoter for various cells. For example, normal cells and cancer cells in skin, liver, pancreas, cartilage, nerves, blood vessels, etc., stem cells of various tissues, embryonic stem (ES) cells and the like can be used. On the other hand, laminin 6B of the present invention may be formulated as an external preparation together with a pharmaceutically acceptable carrier and applied to the surface of skin, mucous membrane or the like. The laminin 6B protein of the present invention can be used as a wound treatment preparation in the form of an ointment, a skin patch, a cultured cell sheet for transplantation, an artificial matrix or the like. Moreover, it can be used as a coating agent for various medical implants to be embedded in a living tissue using the high cell adhesion effect of laminin 6B. Furthermore, it can also be used as an angiogenic agent, a nerve regeneration agent and the like.

Without being limited thereto, the composition of the present invention can be used preferably at a concentration of 0.01 μg / ml to 20 μg / ml when used in vitro or ex vivo. When the composition of the present invention is used as a pharmaceutical composition, it contains a therapeutically effective amount of laminin 6B in a mixture with a pharmaceutically acceptable carrier. The compositions of the present invention may be administered systemically or locally, preferably intravenously, subcutaneously, intramuscularly or parenterally. Preparation of a parenterally administrable laminin solution can be performed within the technical scope of those skilled in the art in consideration of pH, isotonicity, safety and the like.
The dosage regimen of the composition of the present invention takes into account various factors that influence the action of the drug, eg, the nature and / or severity of the patient's symptoms, weight, sex, diet, time of administration, and other clinical effects. And can be determined by the examining physician. One skilled in the art can determine the dose of the composition of the invention based on these factors.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

[Example 1] Production of expression system of laminin 6B and properties of purified laminin 6B
Cell culture human fetal kidney cell line HEK293 was purchased from American Type Culture Collection (ATCC, CRL-1573) (Rockville, MD). Culture is Dalbecco's modified essential medium (DMEM) / Ham's F12 (1: 1) (Invitrogen, Carlsbad, CA) with 10% fetal calf serum (FCS; JRH Biosciences, Lenexa, KS), 100 U / ml penicillin G (Meiji Seika) , Tokyo) and a medium supplemented with 0.1 mg / ml streptomycin (Meiji Seika) (DMEM / F12 with 10% FCS) at 37 ° C. in the presence of 5% CO ₂ .

Antibodies and other materials Mouse monoclonal antibody (F7) against laminin α3B chain and mouse monoclonal antibody (BG5) against α3A chain (which reacts weakly with α3B chain) were prepared in our laboratory. Mouse monoclonal antibody against laminin γ2 chain (D4B5) (Mizushima, H. et al., Horm. Res. 50 Suppl. 2: 7-14, 1998) and mouse monoclonal antibody against laminin β3 chain (LE8A, LE12C) (Nakashima, Y. et al., J. Biochem. 138: 539-552, 2005) was used in this laboratory. LE8A was used for Western blotting, and LE12C was used for immunostaining. Rat monoclonal antibody (LT3) against laminin β1 chain and mouse monoclonal antibody (2E8) against laminin γ1 chain were purchased from Chemicon International (Temecula, CA, USA).

Preparation of cDNA expression vector A human laminin α3B chain expression vector (LNα3BpcDNA3.1 Hygro (+)) was prepared as described in Example 1 of WO2004 / 108927 (Non-patent Document 12). This α3B chain expression vector contains 8,832 bp cDNA (1st to 8832th in the base sequence of SEQ ID NO: 1) and encodes 2,944 amino acid sequences from the N-terminal side. However, this sequence does not include the LG4-5 domain (FIG. 1) (positions 2945 to 3333 of the amino acid sequence of SEQ ID NO: 2). β1 chain (full length 5,361 bp) and γ1 chain (full length 4,830 bp) cDNA were prepared as follows. Primers were designed to divide the β1 and γ1 chains into four fragments (β1-1 to 4 and γ1-1 to 4), respectively, and each of them was polymerized using a polymerase chain reaction (PCR) method from a human lung cDNA library. The fragment was amplified. Table 1 shows the combinations of the primers used and the prepared plasmids. At that time, for incorporation into an expression vector, a Kpn I site at the 5 ′ end of β1-1, an EcoR V site at the 3 ′ end of β1-4, a BamH I site at the 5 ′ end of γ1-1, and a γ1-4 An Xba I site was added to the 3 ′ end. The amplified fragment was incorporated into pGEM (registered trademark) T-Easy vector (Promega, Madison, Wis.), And the nucleotide sequence was determined using a DNA sequencer. The determined sequence was compared with the known β1 chain (GenBank ^TM accession no. M61916) and γ1 chain (GenBank ^TM accession no. J03202) base sequences to confirm the presence or absence of mutations. Bases with mutations involving amino acid substitutions were corrected to the correct base sequences by point mutation introduction methods or restriction enzyme linkage.

表１のプライマー１〜１３の塩基配列をそれぞれ配列番号７〜１９に示す。

表２のプライマー１〜１３の塩基配列をそれぞれ配列番号２０〜３２に示す。

表３のプライマー１〜９の塩基配列をそれぞれ配列番号３３〜４１に示す。 The base sequence was corrected by the point mutation introduction method by a method using PCR (Imai Yoshinori (1999): one-day mutagenesis). At that time, the primers (Tables 1, 2 and 3) containing the corrected bases were designed to contact each other back to back.

The base sequences of primers 1 to 13 in Table 1 are shown in SEQ ID NOs: 7 to 19, respectively.

The base sequences of primers 1 to 13 in Table 2 are shown in SEQ ID NOs: 20 to 32, respectively.

The base sequences of primers 1 to 9 in Table 3 are shown in SEQ ID NOs: 33 to 41, respectively.

As shown in FIGS. 2 and 3, the fragments confirmed to have the correct nucleotide sequence were bound using a restriction enzyme site, and the full length of human laminin β1 chain cDNA (β1full / pBS) and full length of human laminin γ1 chain cDNA (γ1full) / T-Easy). β1 and γ1 chain cDNAs excised from β1full / pBS with Kpn I and EcoR V and from γ1full / T-Easy with BamH I and Xba I, respectively, pcDNA3.1 / Zeo (+) (Invitrogen) or pcDNA3 (Invitrogen) Incorporated into the corresponding enzyme site of the expression vector.
The cDNA expression vector to be introduced into the cells was purified with QIAGEN (registered trademark) Plasmid Midi Kit (QIAGEN, Valencia, CA).

Construction of human recombinant LN6B stable expression cell line using adherent HEK293 cells (Fig. 4)
A γ1 chain cDNA expression vector was introduced into the human fetal kidney-derived cell line HEK293 using the lipofection method. After introduction, selection was performed with G418 which is a resistant drug, and the cells were further cloned. The secretion amount of γ1 chain in the culture supernatant collected from these clones was analyzed by Western blotting, the clone with the highest expression level was selected, and this clone was designated as γ1-HEK. Next, a β1-chain cDNA expression vector was introduced into the γ1-HEK cells, selection was performed with the resistant drug zeocin, and the cells were further cloned. The secretion amount of β1 chain in the culture supernatant collected from these clones was analyzed, and the clone with the highest expression level was designated as β1 · γ1-HEK. Furthermore, an α3B chain cDNA expression vector (LNα3BpcDNA3.1 Hygro (+) described in Example 1 of WO2004 / 108927) (Non-patent Document 12) is introduced into β1 · γ1-HEK, selection is performed with hygromycin, and cells are further selected. Was cloned. The expression level of LN6B in the culture supernatant collected from these clones was analyzed by Western blotting using antibodies of each constituent chain, and as a result, clones that stably expressed LN6B could be obtained (FIG. 5). . This clone was designated as cell line LN6B-HEK and used in the following experiments.

  In the present invention, an expression system for laminin 6A could also be established by incorporating α3A chain cDNA in the expression vector instead of α3B chain cDNA and performing the same operation as described above. When laminin 6A was expressed in HEK293 cells, the expression level was 10 times or more compared to the expression system (non-patent document 8) of laminin 6A in the HT1080 system established previously.

Purification of human recombinant LN6B
To purify laminin 6B from LN6B-HEK, the cells were cultured in serum-free medium, and 1.5 l of the collected culture supernatant was added to a Q Sepharose CL-4B anion exchange column (10 ml) (Amersham, Piscataway, NJ, USA). The protein adsorbed on the column was eluted with 0.5M NaCl. Thereafter, the fraction containing laminin 6B was applied to an anti-α3B antibody (F7) -coupled Sepharose column (2 ml), and the adsorbed laminin 6B was eluted with 0.05% trifluoroacetic acid (TFA). Immediately after elution, 20 μl of 20 mM Tris-HCl (pH 8.0) per 1 ml of the eluate was mixed and neutralized. Each fraction was separated by SDS electrophoresis under reducing conditions, and CBB dye staining and Western glotting were performed to analyze the expression of each constituent chain. As a result, bands having molecular weights corresponding to α3B, β1, and γ1 chains, which are constituent chains of laminin 6B, were detected (FIG. 6). As a result of protein quantification, about 350 μg of laminin 6B could be purified per 1.5 l of the culture supernatant.

Measurement of Cell Adhesion Activity of Human Recombinant Laminin 6B Using purified laminin 6B (LN6B), cell adhesion activity was examined by a previously reported method (Non-patent Document 13). The activity was measured using BRL cells (provided by Professor Gordon H. Sato (currently retired), University of California, San Diego), which is a rat liver-derived epithelial cell line that is highly responsive to laminin. For comparison, human laminin 5B (LN5B) with the same α3B chain and different β and γ chains (produced by the method of Non-Patent Document 12), laminin 1 (LN1) with β1, γ1 chain and different α chains (mouse EHS laminin) ; Chemicon International). Each laminin was coated at a specified concentration on a 96-well plate for ELISA (Coster, Cambridge, MA, USA), and further blocked with 1% bovine serum albumin. BRL cells (2 × 10 ⁴ cells / well) dispersed in serum-free DME / F12 medium were seeded on this plate and incubated at 37 ° C. for 1 hour. After removing non-adherent cells, the adherent cells were stained with a fluorescent dye (Hoechst 33342), and the fluorescence intensity was measured (FIG. 7). As a result, laminin 5B showed the highest cell adhesion activity, and laminin 6B showed almost intermediate activity between laminin 5B and laminin 1. From this result, it was revealed that purified laminin 6B has about twice the adhesive activity as compared to laminin 1 and about half that of laminin 5B.

Measurement of cell dispersion activity of human recombinant laminin 6B The cell motility activity of purified human laminin 6B (LN6B) was measured as cell dispersion activity. Cell dispersal activity was performed by a previously reported method using BRL cells (Non-patent Document 12). Laminin 6B, human laminin 5B (LN5B), and mouse laminin 1 (LN1) were coated at a specified concentration on a 24-well plate for cell culture by the same method as in the example of FIG. BRL cells (7,000 cells / well) dispersed in DME / F12 medium containing 1% FCS were seeded on this plate and incubated at 37 ° C. for 40 hours. In three different fields under the microscope, the proportion of cells that did not adhere to each other but were dispersed was determined. As a result, only laminin 5B showed high cell dispersion activity. Laminin 6B showed slightly higher cell dispersion activity than laminin 1 (FIG. 8).

[Example 2] Expression and function of laminin 6B in blood vessels
Tissue distribution of human laminin 6B mRNA for laminin α3B chain is known to be expressed in a wide range of tissues such as skin and lung, but no protein has been identified. Therefore, a specific mouse monoclonal antibody (F7) against human laminin α3B chain was prepared and immunohistochemical staining was performed on human skin. Formalin-fixed paraffin sections were purchased from BioChain (Wako Pure Chemicals, Osaka), and immunohistochemical staining was performed according to a previously reported method (Kagesato, Y. et al., Jpn. J. Cancer Res. 92: 184-192, 2001). As a result, laminin α3B protein signal was strongly detected in the basement membrane (arrow) of connective tissue microvessels in addition to the epidermal basement membrane (FIG. 9).

  In order to clarify whether the above distribution of laminin α3B chain is due to laminin 6B or laminin 5B, anti-α3A chain antibody BG5 (which reacts weakly to α3B chain), α3B chain specific antibody F7, β3 chain specific antibody LE12C, Immunohistochemical staining of human skin tissue sections was performed using γ2 chain specific antibody D4B5 (FIG. 10). As a result, the anti-α3A chain antibody BG5 specifically stained the epidermal basement membrane (arrow), while the α3B chain-specific antibody added to the basement membrane (arrow) of the connective tissue microvessel in addition to the epidermal basement membrane (arrow). Stained strongly. On the other hand, the β3 chain specific antibody LE12C and the γ2 chain specific antibody D4B5 strongly stained the epidermal basement membrane (arrow), but did not stain the vascular basement membrane (asterisk position). From the above results, it was found that laminin α3A chain, α3B chain, β3 chain, and γ2 chain exist in the epidermal basement membrane, but only α3B chain exists in the vascular basement membrane. Similar distributions were seen in the esophagus, mammary gland, and lungs. It is known that laminin 8 (α4β1γ1; laminin 411) and laminin 10 (α5β1γ1; laminin 511) having β1 chain and γ1 chain exist as main components in the basement membrane of blood vessels (Iivanainen, A. et. al., J. Biol. Biochem. 272: 27862-27878, 1997). Therefore, it is considered that the laminin α3B chain present in the vascular basement membrane exists as laminin 6B (α3Bβ1γ1), not laminin 5B (α3Bβ3γ2). On the other hand, in the epidermal basement membrane, it is considered that it exists mainly as laminin 5B because β3 chain and γ2 chain exist. The wide tissue distribution of laminin α3B chain is thought to be due to the expression of laminin 6B by blood vessels.

Measurement of the growth-promoting activity of human recombinant laminin 6B on microvascular endothelial cells The presence of laminin 6B in the basement membrane of the skin microvessels revealed the effect of laminin 6B on the proliferation of microvascular endothelial cells I investigated. Human skin microvascular endothelial cells (HMVEC) and growth medium (HuMedia-MvG) were purchased from Kurabo Industries (Osaka). Purified laminin 6B (LN6B), human laminin 5B (LN5B), and mouse laminin 1 (LN1) were coated on a 24-well plate for cell culture at a concentration of 2 μg / ml. Blocking with bovine serum albumin was not performed. The plate was seeded with HMVEC cells (5,000 cells / well) dispersed in 5% FCS-containing HuMedia-MvG medium, cultured at 37 ° C. for 4 days, and then the number of cells was counted. As shown in FIG. 11, laminin 5B and laminin 1 did not show a growth promoting effect as compared with the non-treated plate (None), but laminin 6B coated plate showed a high growth promoting effect. From this result, it became clear that laminin 6B exhibits a high proliferation promoting activity on microvascular endothelial cells.

Discussion In this Example 1, a cell line LN6B-HEK that stably and highly expresses human recombinant laminin 6B is obtained by introducing cDNA of each subunit of laminin 6B into HEK293 cells and selecting with a drug. I was able to establish it. Furthermore, it was possible to establish a system for culturing a large amount of these cells and stably purifying laminin 6B from the culture supernatant. To date, there are no reports of laminin 6B expression in vivo and in vitro.

  In the future, human recombinant laminin 6B can be easily obtained using the expression cells and purification procedures established this time. Laminin 6B purified this time has approximately twice the adhesion activity of rat liver-derived BRL cells as laminin 1 and about half that of laminin 5B, and is very low compared to laminin 5B. It became clear that it showed dispersion activity. From this, it is considered that laminin 6B and laminin 5B have different properties from each other although they have the same laminin α3B chain. The effect on cell proliferation and differentiation does not directly correlate with cell adhesion activity or cell motility activity, but varies depending on the cell type. Therefore, it is considered that laminin 6B and laminin 5B can be used complementarily as culture substrates for various cells.

  As shown in Example 2, laminin 5B was found to be present in epithelial basement membranes such as skin, esophagus, lung, and mammary gland, while laminin 6B was widely present in vascular basement membranes. In fact, it was revealed that laminin 6B has a higher cell proliferation promoting activity against microvascular endothelial cells than laminin 1 and laminin 5B. Such properties mean that laminin 6B serves as a culture substrate for efficiently culturing vascular endothelial cells. From these results, it is considered that laminin 6B may be useful as a pro-angiogenic agent in myocardial infarction, cerebral infarction, diabetes, wound healing and the like.

  Since laminin 6B promotes cell adhesion and migration, it may be used in the fields of regenerative medicine and cell engineering in the future. As shown in Example 2, it was revealed that laminin 6B is present in the basement membrane of blood vessels and promotes the proliferation of vascular endothelial cells. Therefore, laminin 6B can be used as an adhesion substrate for stably culturing vascular endothelial cells and other cells, and may be used as an angiogenesis promoter in cardiovascular failure.

The structure of laminin 6B of the present invention is shown along with the structures of laminin 5A and 6A. Preparation of β1 chain expression vector. Preparation of β1 chain expression vector. Construction of large-scale expression system of genetically modified laminin 6B. All the α3B chain, β1 chain, and γ1 chain, which are laminin 6B constituent chains, are introduced into HEK293 cells as foreign genes and expressed. Expression analysis of recombinant LN6B by Western blot. The culture supernatant of LN6B-HEK was separated by SDS-PAGE under 3 to 7.5% gel and non-reducing conditions, and Western blotting was performed using α3, β1, and γ1 antibodies. Purification of human recombinant LN6B. Purified LN6B was separated by SDS-PAGE under 5% gel and reducing conditions, and CBB staining and Western blotting using LNα3B, β1, and γ1 antibodies were performed. CBB: Coomasie brilliant blue Cell adhesion activity of human recombinant LN6B. Cell dispersion activity of human recombinant LN6B. Immunostaining of human skin tissue with anti-laminin α3B chain antibody F7. Immunostaining with α3B antibody. The basement membrane of blood vessels (arrows) and epidermis (arrowheads) is stained. Immunostaining of human skin tissue with anti-laminin α3A chain BG5, anti-laminin α3B chain F7, anti-laminin β3 chain LE12C, and anti-laminin γ2 chain antibody D4B5. The α3A chain antibody specifically stained the epidermal basement membrane (arrow), while the α3B chain antibody strongly stained the microvascular basement membrane (arrow) in addition to the epidermal basement membrane (arrow). On the other hand, the β3 chain antibody and the γ2 chain antibody strongly stained the epidermal basement membrane (arrow), but did not stain the vascular basement membrane (position of asterisk). Growth-promoting activity of laminin 6B on human skin microvascular endothelial cells HMVEC. Laminin 6B (LN6B) strongly promoted the proliferation of HMVEC cells compared to human laminin 5B (LN5B) and mouse laminin 1 (LN1). None, growth on untreated plates.

<SEQ ID NO: 1>
SEQ ID NO: 1 shows the full-length base sequence of human laminin α3B chain. In the base sequence of SEQ ID NO: 1, the first methionine codon is from the first to the third and the stop codon is from the 10,000th to the 10002nd.
<SEQ ID NO: 2>
SEQ ID NO: 2 shows the full-length amino acid sequence of human laminin α3B chain.
<SEQ ID NO: 3>
SEQ ID NO: 3 shows the full-length base sequence of human laminin β1 chain. In the base sequence of SEQ ID NO: 3, the first methionine codon is at positions 336 to 338, and the stop codon is at positions 5694 to 5696.
<SEQ ID NO: 4>
SEQ ID NO: 4 shows the full-length amino acid sequence of human laminin β1 chain.
<SEQ ID NO: 5>
SEQ ID NO: 5 shows the full-length base sequence of human laminin γ1 chain. In the base sequence of SEQ ID NO: 5, the first methionine codon is 300th to 302nd, and the stop codon is 5127th to 5129th.
<SEQ ID NO: 6>
SEQ ID NO: 6 shows the full-length amino acid sequence of human laminin γ1 chain.
<SEQ ID NOS: 7 to 19>
SEQ ID NOs: 7 to 19 show the base sequences of primers used for preparing a human laminin β1 chain cDNA expression vector.
<SEQ ID NOs: 20 to 32>
SEQ ID NOs: 20 to 32 show the base sequences of primers used for preparing a human laminin γ1 chain cDNA expression vector.
<SEQ ID NOs: 33-41>
Sequence number 33-41 shows the base sequence of the primer used for human laminin gamma 1 chain Oligo preparation.

Claims (1)

A composition comprising the following protein (a) or (b), wherein the composition is used as a cell culture substrate, a cell growth promoter, an angiogenesis agent, a wound healing agent, or a transplantation substrate .
(a) It is composed of each subunit of α3B chain having the amino acid sequence shown by SEQ ID NO: 2, β1 chain having the amino acid sequence shown by SEQ ID NO: 4, and γ1 chain having the amino acid sequence shown by SEQ ID NO: 6 Laminin 6B protein
(b) In the protein of (a), one or several of the amino acid sequence represented by SEQ ID NO: 2, the amino acid sequence represented by SEQ ID NO: 4, and the amino acid sequence represented by SEQ ID NO: 6 A protein having an amino acid deleted, substituted or added and having the biological activity of the laminin 6B protein. However, with respect to the α3B chain, the LG4-5 domain existing on the C-terminal side (from 8833 to 10002 in SEQ ID NO: 2) (The second amino acid sequence) may not be included.

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