KR101055319B1 - L1 domain and active peptide of human laminin α2 chain to promote cell adhesion - Google Patents

Abstract

The present invention relates to a domain that promotes cell adhension and an active peptide thereof, and more particularly, to an LG1 domain of a human laminin-2 α2 chain that promotes cell adhesion and an active peptide in the LG1 domain. The domain that promotes cell adhesion of the present invention and its active peptides exhibit high cell adhesion activity, bind syndecan-1 with heparan sulfate and dermatane sulfate to the cell receptor, and through the binding Interacts with decan-1 to regulate the intracellular locations of protein kinase C-α and PKC-δ and to induce tyrosine phosphorylation of PKC-δ As a result, it can be very useful for research on adhesion activity of cells centered on neurons mediated by various extracellular matrix proteins including laminin, artificial neural conduit, burn treatment, wound treatment and tissue regeneration. have.

Adhesion, Peptides, LG1 Domain, Human Activity Array of Human Laminin-2 α2 Chain

Description

Human Laminin α2 Chain LG1 Domain and an Active Peptide Promoting Cell Adhesion

The present invention relates to a domain that promotes cell adhesion and an active peptide thereof, and more particularly, to an LG1 domain of a human laminin-2 α2 chain that promotes cell adhesion and an active peptide in the LG1 domain.

Laminin is a type of heterotrimeric glycoprotein predominantly found in the basement membrane and a type of extracellular matrix protein that adheres, migrates, grows, differentiates, wounds heals and tumors penetrate through interactions with specific integrins or other receptors on the cell surface. Regulate various biological functions such as (Timpl, R., Curr . Opin . Cell Biol . 8, 618-624, 1996; Howe, A., et al, Curr . Opin . Cell Biol . 10, 220-231, 1998; Colognato, H., and Yurchenco, PD, Dev . Dyn . 218, 213-2341, 2000). Laminin is composed of three different subunits of α, β and γ chains, which form a cross-shaped structure that is connected to each other by disulfide bonds (Ekblom, M., et al., ann. NY Acad. Sci. 857 , 194-211, 1998). So far, 5α, 3β and 3γ chains have been identified, and it has been reported that at least 15 isoforms (laminins 1-15) are formed by various combinations of these subunits (Colognato, H., and Yurchenco, PD, Dev . Dyn. 218, 213-2341, 2000; Burgeson, RE, et al., Matrix Biol . 14, 209-211, 1994; Miner, JH, et al., J. Cell Biol . 137, 685-7015, 1997). The 15 isoforms are composed of chains of one each of 5α chains (α1-α5), 3β chains (β1-β3) and 3γ chains (γ1-γ3) (Suzuki, N.et. al., Connect Tissue Res . 46, 142-152, 2005).

Laminin α2 chain constitutes laminin-2 (α2β1γ1), laminin-4 (α2β2γ1) and laminin-12 (α2β1γ3) and is expressed in skeletal, cardiac muscle, peripheral nerves, brain and placenta (Leivo, I. et.al) , Proc . Natl . Acad . Sci . USA 85, 1544-1548, 1998). Mutations in the laminin α2 chain gene cause congenital muscular atrophy that is deficient in merosin in both humans and mice (Sewry, CA et.al., Neuropediatrics 28, 217-222, 1997; Edwards, JP et.al) ., Brain Res . 788, 262-268, 1998). Laminin-2 is not expressed in both peripheral nerves, skeletal and muscle of rats with muscular dystrophy. Phenotypes of rats with muscular dystrophy are manifested by incomplete basement membrane and peripheral nerve demyelinization in muscular dystrophy, muscle and nerves (Feltri, ML et.al., J. Peripher . Nerv . Syst. 10, 128-143, 2005; Masaki, T et.al., Acta Neuropathol . (Berl) 99, 289-295, 2000 ; Matsumura, K. et.al., Neuromuscul . Disord . 7, 7-12, 1997). The studies indicate that laminin-2 is crucial for basal membrane formation and myelin formation of peripheral nerves.

The α2 chain contains an LG domain at the carboxyl terminus, which consists of a series of repeats of five similar LG domains (LG1-LG5), each domain containing an autonomous folding unit of 200 amino acid residues (Vuolteenaho, R et.al., J. Cell Biol . 124, 381-394, 1994). The LG domains of laminin α chains bind to integrin, α-dystroglycan and heparin / heparan sulfate proteoglycans and act as active sites for various biological functions (Wizemann, H. et.al., J. Mol . Biol . 332, 635-642, 2003; Tisi, D. et.al., Embo J. 19, 1432-1440, 2000).

Laminin α2 chain LG1-3 domains promote several integrin-related cell binding activities such as α3β1, α6β1 and α7β1. And the domain requires acetylcholine receptor clustering (Smirnov, SP et.al., J. Biol . Chem . 277, 18928-18937, 2002). Several synthetic peptides derived from the murine laminin α2 chain LG domain promote cell adhesion, heparin binding, neurite products and alveolar formation. For example, peptide MG-73 (KNRLTIELEVRT) derived from the rat laminin α2 chain LG4 domain (residues 2780-2791) promotes cell adhesion and neurite products and binds to cell surface heparan sulfate proteoglycan, Sindecan-1. (Nomizu, M. et.al., FEBS Lett . 396, 37-42, 1996; Richard, BL et al . , Exp . Cell Res . 228, 98-105, 1996; Hoffman, MP et . Al . , J. Biol . Chem . 273, 28633-28641, 1998). Similarly, peptide EF-2 (DFGTVQLRNGFPFFSYDLG) located at the linked loop site of the rat laminin α2 chain LG4 domain (residue 2808-2826) indicates binding to cell adhesion and syndecane-2 (Suzuki, N. et . Al . , J. Biol . Chem . 278, 45697-45705, 2003). These results indicate that the murine laminin α2 chain LG4 domain contains two heparin binding sites and has various biological functions. However, little is known about the biological function of human α2 chain LG domains, their cellular receptors and downstream signaling pathways.

Thus, the present inventors have identified recombinant human laminin-2 α2 chain LG1 domains expressed in the form of a water-soluble fusion protein of monomers in order to identify the function of human laminin-2 α2 chain LG domains and to determine key sequences in biological activity. Of these, LG1 domain-derived synthetic peptides were prepared and their cell adhesion activities were investigated. As a result, the present inventors showed high cell adhesion activity in the Ln2-P3 sequence (DLTIDDSYWYRI, residues 2221-2232) in the human laminin-2 α2 chain LG1 domain, and the cells were synthesized with hedecane sulfate and dermatan sulfate sulfate. It binds to the receptor, interacts with syndecane-1 through the binding, regulates the intracellular location of PKC-α and PKC-δ, and induces tyrosine phosphorylation of PKC-δ. The present invention has been completed by confirming that it can be usefully used for treatment, wound healing and tissue regeneration.

It is an object of the present invention to identify an active domain in a human laminin-2 α2 chain that promotes cell adhesion and an active peptide arrangement in the domain, and to provide a cell adhesion promoter comprising the domain or peptide.

In order to achieve the above object, the present invention provides a cell adhesion promoter comprising a polypeptide or a derivative thereof composed of the LG1 domain sequence of the human laminin-2 α2 chain.

In addition, the present invention provides a cell adhesion promoter comprising the peptide described in SEQ ID NO: 2 or a derivative thereof.

In another aspect, the present invention provides a device for promoting cell adhesion comprising the polypeptide, peptide or derivatives thereof.

In addition, the present invention provides a method for promoting cell adhesion, comprising attaching a cell after fixing the polypeptide, peptide or derivatives thereof to a solid support.

The present invention also provides a burn or wound treatment comprising the cell adhesion promoter.

The present invention also provides a coating for burn treatment or wound treatment comprising the cell adhesion promoter.

The present invention also provides an artificial neural conduit or scaffolds thereof comprising the cell adhesion promoter.

In addition, the present invention provides a support for tissue engineering comprising the cell adhesion promoter.

The domain promoting cell adhesion of the present invention and its active peptides have high cell adhesion activity, bind syndecan-1 with heparan sulfate and dermatane sulfate to the cell receptor, and through the binding Cells mediated by various extracellular matrix proteins, including laminin, by interacting with decan-1 to regulate the intracellular locations of PKC-α and PKC-δ and induce tyrosine phosphorylation of PKC-δ It can be very useful for adhesion activity research, burn treatment, wound treatment, and tissue regeneration using artificial neural conduit or its support.

The present invention provides a cell adhesion promoter comprising a polypeptide or derivative thereof composed of the LG1 domain sequence of the human laminin-2 α2 chain as an active ingredient.

The domain is set forth in SEQ ID NO: 1, and the derivative is preferably a polypeptide having at least 80% homology with the sequence described in SEQ ID NO: 1 and having cell adhesion activity, but is not limited thereto.

The cell is preferably a neuron, more preferably a PC12 cell, but is not limited thereto.

In the present invention, in order to identify the active domain in the human laminin-2 α2 chain that promotes cell adhesion, after preparing three independently expressed recombinant LG proteins (rLG1, rLG2 and rLG3) of human laminin (FIG. 1A or FIG. 1B), laminin, and cell adhesion activity of the respective LG domains were investigated by the reported method (Okazaki, I., et al., J. Biol . Chem . 277, 37070-37078, 2002). Adhesion activity of PC12 cells was concentration-dependent on laminin, rLG1, rLG2 and rLG3 domains, laminin showed maximum cell adhesion activity, and rLG1 and rLG3 domains showed moderate cell adhesion activity. rLG2 showed pharmaceutical cell adhesion activity. The domains showed the maximum adhesion activity at a concentration of 25 μg / ml, and the adhesion activity showed a concentration-dependent increase until the concentration of the domains was 25 μg / ml (see FIG. 1C).

Therefore, it can be seen that the cell adhesion site exists in the rLG1 and rLG3 domains.

In addition, the present invention provides a cell adhesion promoter containing the peptide or the derivative thereof represented by SEQ ID NO: 2 as an active ingredient.

The peptide is a peptide having cell adhesion activity present in the LG1 domain of the human laminin-2 α2 chain, and is described by SEQ ID NO: 2, and the derivative thereof has at least 80% homology with the sequence described by SEQ ID NO. It is preferably a polypeptide having, but is not limited thereto.

The cell is preferably a neuron, more preferably a PC12 cell, but is not limited thereto.

In the present invention, in order to find the minimum amino acid sequence of the cell attachment site in the human laminin α2 LG1 domain, peptides having 12 amino acids overlapping six amino acids within the amino acid residues 2209-2256 site derived from the LG1 domain are selected. After synthesis (see FIG. 2A), the synthesized peptides were synthesized with Ln2-P1 (DVGSGVGRVEYP, residues 2209-2220), Ln2-p2 (GRVEYPDLTIDD, residues 2215-2226), Ln2-P3 (DLTIDDSYWYRI, residues 2221-2232), Ln2-P4 (SYWYRIVASRTG, residues 2227-2238), Ln2-P5 (VASRTGRNGTIS, residues 2233-2244) and Ln2-P7 (VRALDGPKASIV, residues 2245-2256) were named respectively and cell adhesion activities were studied. As a result, it was confirmed that the synthetic Ln2-P3 peptide promoted cell adhesion (see FIG. 2B). Adhesion activity of peptide Ln2-P3 to neurons showed maximum adhesion activity at 100 μg / ml concentration of the peptide, and the adhesion activity increased concentration-dependently until the concentration of the peptide was 100 μg / ml. It is shown in the aspect. On the other hand, the other peptides did not show cell adhesion activity despite the high concentration.

Therefore, it can be seen that the essential peptide for promoting cell adhesion activity in the human laminin α2 LG1 domain is Ln2-P3.

In the present invention, to investigate the effect of peptide Ln2-P3 on the cell adhesion activity of human laminin α2 LG1 domain, it was investigated whether peptide Ln2-P3 competitively affects the cell adhesion activity of rLG1 domain on neurons. As a result, peptide Ln2-P3 markedly reduced the cell adhesion activity of the rLG1 domain on neurons, while other peptides did not reduce the adhesion activity.

Thus, it can be seen that peptide Ln2-P3 acts as a cell binding site in the original human laminin-2 α2 chain rLG1 domain.

The rLG1 domain, peptide Ln2-P3 or derivatives thereof of the present invention are preferably cell adhesion by a cell receptor having heparan sulfate or dermatane sulfate, but the characteristics of the receptor are not limited thereto.

In the present invention, in order to identify cell receptors that adhere to the rLG domain or peptide Ln2-P3, neurons were pretreated with various proteins, and then the degree of inhibition of cell adhesion to rLG1 protein or peptide Ln2-P3 was confirmed.

First, to determine whether cell adhesion is caused by integrin, the divalent cation, which is important for integrin activation, is removed, followed by pretreatment of neurons with an integrin β1 antibody that inhibits cell adhesion, followed by laminin, rLG protein and peptide. As a result of cell adhesion analysis for each of Ln2-P3, cell adhesion was inhibited for laminin, rLG2 and rLG3, while cell adhesion was not inhibited by integrin β1 for rLG1 and Ln2-P3 (FIG. 3A).

In addition, in order to determine whether the cellular receptor to which the rLG domain or the peptide Ln2-P3 attaches has a heparin type molecule, pretreatment of neurons with heparin, followed by cell adhesion analysis as described above, results in laminin Cell adhesion was inhibited in a concentration-dependent manner except rLG1, rLG2, rLG3 and Ln2-P3 (see FIG. 3B). In addition, solid phase heparin binding assays were used to investigate whether peptide Ln2-P3 could bind biotinylated heparin. As a result, the rLG1 domain and peptide Ln2-P3 showed heparin binding activity that was increased in a concentration-dependent manner.

Thus, it can be seen that the cellular receptors that attach to the rLG domain or peptide Ln2-P3 have heparin type molecules, and the cell adhesion is mediated by heparin type molecules (see FIG. 3C or FIG. 3D).

In addition, after pretreatment of neurons with various glycosaminoglycans, the cell adhesion assay was performed. As a result, heparan sulfate and dermatan sulfate were either rLG1 or Ln2-. Cell adhesion to P3 was inhibited (see FIG. 3E).

In addition, heparan sulfate and chondroitin sulfate ABC present in neuronal receptors were cut with heparintinase I or chondroitinase ABC enzyme, respectively, and then cell adhesion assays were performed as described above. As a result, cell adhesion to rLG1 or Ln2-P3 was inhibited as the enzyme activity was increased (see FIG. 3F). In particular, the cell adhesion inhibition was higher in cells treated with heparinase I than in cells treated with chondroitinase ABC.

Therefore, it can be seen that the cell adhesion occurs by the cell receptor having the rLG1 domain or the peptide Ln2-P3 having heparan sulfate or dermatane sulfate.

The LG1 domain, peptide Ln2-P3 or derivatives thereof of the present invention preferably bind to syndecan-1 to cause cell adhesion, but the binding is not limited thereto.

Several studies have reported that synthetic peptides derived from LG domains and laminin α chains bind to decane in many cells and cell types (Okamoto, O. et.al., J. Biol . Chem . 278, 44168-44177, 2003; Hozumi, K. et.al., J. Biol . Chem. 281, 32929-32940, 2003; Utani, A. et.al., J. Biol . Chem . 276, 28779-28788, 2001; Yamashita, H. et.al., Biochem . J. 382, 933-943, 2004). In addition, it has been reported that glypican-1, including glycosaminoglycan (GAG), is expressed in neurons (Malave, C. et.al., Brain). Res . 983, 74-83, 2003).

In the present invention, in order to find a receptor on the cell surface that is attached to the rLG1 domain or peptide Ln2-P3, a syndecan family and glycican having heparan sulfate proteoglycans expressed in neurons Expression of -1 was analyzed using FACS, immunofluorescence and immunodot blot analysis. As a result, it was confirmed that only syndecane-1 was strongly expressed on the surface of neurons, and that syndecane-4 and glypican-1 were expressed very weakly (see FIGS. 4A to 4C).

In the present invention, in order to determine that the cell receptor of the rLG1 domain or peptide Ln2-P3 is syndecan-1, it was confirmed by double staining and immunodot blot analysis, rLG1 domain or Ln2-P3 binds to the syndecane-1 It can be seen that (see Fig. 4D to 4H). In addition, neuronal cells were treated with syndecane-1 siRNA to reduce the expression of syndecane-1, and cell adhesion assays for rLG1 or Ln2-P3 showed about 50% inhibition of adhesion, whereas syndecane. As a result of cell adhesion analysis of rLG1 or Ln2-P3 of neurons with reduced expression of -4 and glypican-1 as described above, adhesion inhibition was not observed (see FIGS. 5A to 5E).

Therefore, it can be seen that syndecan-1 is a very important receptor for cell adhesion to rLG1 domain or peptide Ln2-P3 in neurons.

The LG1 domain, peptide Ln2-P3, or derivatives thereof of the present invention preferably bind to syndecane-1 to regulate the intracellular location of PKC-α or PKC-δ, but the action is not limited thereto.

In the present invention, rLG1 domain or peptide Ln2-P3 is bound to syndecan-1, and then in order to investigate the intracellular signaling induced, first when treated with calphostin C (calphostin C), a PKC inhibitor to neurons, rLG1 or Ln2 Cell adhesion to -P3 was confirmed to be inhibited (see Figure 6A). In addition, observing the protein levels of various PKC subtypes in the cytoplasmic and membrane sections of rLG1 or Ln2-P3 resulted in a decrease in the protein levels of PKC-α and PKC-δ in the cytoplasmic sections of cells attached to rLG1. , Increased in the membrane portion (see FIG. 6B) and decreased protein levels of PKC-α and PKC-δ in the cytoplasmic portion of cells attached to Ln2-P3, but did not change in the membrane portion (see FIG. 6C). In contrast, the remaining PKC subtypes did not change protein levels in the cytosolic and membrane portions of cells attached to rLG1 or Ln2-P3.

Thus, it can be seen that the rLG1 domain or peptide Ln2-P3 can regulate the intracellular location of PKC-α and PKC-δ by interacting with syndecane-1.

In the present invention, in order to determine whether syndecan-1 forms a complex with PKC-α or PKC-δ in neurons, immunoprecipitation method using a cell attached to rLG1 or Ln2-P3 and a cell not attached Confirmed by. As a result, syndecane-1 and PKC-δ specifically formed a complex (see FIGS. 6D to 6F). In addition, when neuronal cells were treated with inhibitors of PKC-α and PKC-δ, cell adhesion to rLG1 or Ln2-P3 was inhibited only when the PKC-δ inhibitor was treated (see FIG. 6H or 6G). . In addition, as a result of immunofluorescence analysis in neurons attached to rLG1 or Ln2-P3, it can be seen that syndecane-1 and PKC-δ are in the same position (see FIGS. 6I to 6J).

Therefore, it can be seen that when neurons adhere to the rLG1 domain or peptide Ln2-P3, PKC-δ migrates from the cytoplasmic part to the membrane part and binds to synthecan-1.

The LG1 domain, peptide Ln2-P3, or derivatives thereof of the present invention are preferably combined with syndecane-1 to induce tyrosine phosphorylation of PKC-δ, but the action is not limited thereto.

In the present invention, to determine whether rLG1 or Ln2-P3 activates PKC-δ by binding to syndecane-1, immunoprecipitation using PKC-δ antibody to cells attached to rLG1 or Ln2-P3 and those not attached As a result of the analysis, it was confirmed that tyrosine phosphorylation of PKC-δ is induced in cells attached to rLG1 or Ln2-P3 (see FIGS. 6K to 6L). In addition, when PKC-δ tyrosine phosphorylation was inhibited using a PLC inhibitor to the cells, cell adhesion to rLG1 or Ln2-P3 was inhibited (Fig. 6M).

Therefore, it can be seen that the neuronal cell adhesion with rLG1 domain or peptide Ln2-P3 induces tyrosine phosphorylation of PKC-δ.

The present invention also provides a device for promoting cell adhesion comprising an rLG1 domain, peptide Ln2-P3 or derivatives thereof.

The cell is preferably a neuron, more preferably a PC12 cell, but is not limited thereto.

The device is any one selected from the group consisting of cell culture dishes, petri dishes, tissue culture flasks, plates, containers, slides, filter chambers, slide chambers, roller bottles, harvesters and tubes. But is not limited thereto.

In addition,

1) fixing the rLG1 domain, peptide Ln2-P3 or derivatives thereof to a solid support; And

2) provides a cell adhesion promoting method comprising the step of attaching cells to the solid support.

The cell is preferably a neuron, more preferably a PC12 cell, but is not limited thereto.

In addition, the present invention provides a burn treatment, wound treatment, artificial neural conduit or tissue engineering support containing the cell adhesion promoter as an active ingredient.

The polypeptide of SEQ ID NO: 1, the peptide of SEQ ID NO: 2 and derivatives thereof of the present invention can be used for various medical purposes by promoting cell adhesion.

The polypeptide, peptide or derivatives thereof may be useful for treating burns or wounds because they promote adhesion of nerve cells.

If the polypeptide, peptide or derivatives thereof are used by coating, attaching or chemically binding the artificial neural conduit, it promotes the adhesion of nerve cells and thus may cause nerve regeneration when the artificial neural conduit is inserted in vivo.

The support for tissue engineering according to the present invention includes all supports that can be used in the field of tissue engineering aimed at maintaining, improving or restoring the function of the body by making and transplanting a substitute for living tissue. The support for tissue engineering is a synthetic biodegradable polymer, polyamino acid, polyanhydride, poly ε-caprolactone, polyorthoester, polyglycolic acid (PGA), polylactic acid (PLA) and copolymers thereof polylactic- And a porous support made of glycolic acid (PLGA) and the like and natural biodegradable polymers such as alginic acid, chitosan, hyaluronic acid and collagen. The tissue engineering support may include a shielding film, and the shielding film may be a porous polylactic acid shielding film, a regenerated film made of chitin or chitosan nanofibers, or a film in the form of a film of chitin or chitosan, but is not limited thereto.

The active ingredient of the pharmaceutical composition of the present invention can be administered parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation. In other words, the cell adhesion promoter of the present invention may be administered in various parenteral formulations, and when formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used. do. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, and lyophilized preparations. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, ethyl oleate and the like can be used.

Adhesion promoters can be used in admixture with a carrier that is acceptable as a medicament, such as physiological saline or organic solvents, and glucose, sucrose or dextran to increase the stability or absorption of the adhesion promoters. Carbohydrates such as (dextran), antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments. have.

In the pharmaceutical composition of the present invention, the total effective amount of the cell adhesion promoter may be administered to the patient in a single dose by bolus form or by infusion for a relatively short period of time. The dose may be administered by a fractionated treatment protocol that is administered for a long time. Since the concentration of the cell adhesion promoter is determined in consideration of various factors such as the route of administration and the number of treatments of the drug, as well as the age and health of the patient, the effective dose of the cell adhesion promoter may be determined by those skilled in the art. Growing up, one may determine the appropriate effective dosage for the particular use of the cell adhesion promoter as a pharmaceutical composition.

Since the pharmaceutical composition containing the cell adhesion promoter of the present invention prepared as an active ingredient is a parenteral preparation, no toxicity experiment was performed.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples specifically illustrate the present invention, and the content of the present invention is not limited by the Examples and Experimental Examples.

< Example > Human Laminin  α2 chain LG  Preparation of the Domain

<1-1> cell culture

5% at 37 ° C in RPMI 1640 culture (BioWhittaker Cambrex, Walkersville, MD) supplemented with antibiotics and 10% fetal serum (FBS) with PC12 cell line (CRT-1721 ^TM , ATCC) derived from transplanted mouse pheochromocytoma It was incubated under the condition of humid air of CO ₂ .

<1-2> human Laminin  α2 chain LG  Vector Preparation Expressing Domains

Human laminin α2 chain cDNA was cloned using RT-PCR with Superscript II reverse transcriptase (Invitrogen, Carlsbad, Calif.) Under conditions recommended by the manufacturer using mRNAs isolated from human keratinocytes (epithelial cells) and fibroblasts. . Three C-terminal LG cDNA fragments (LG1-LG3) of the laminin a2 chain were amplified by PCR using the laminin a2 chain cDNA as a template and linked to the pGEM-T Easy vector (Promega, Madison, WI). PCR reaction primers used are shown in Table 1 below:

Gene name PCR primer sequence LG1 Sense (SEQ ID NO: 4) 5'-GCCACTCGAGCAGGAGGTGACTG-3 ' Antisense (SEQ ID NO: 5) 5'-GCCACCATGGTCAACTGACAGTGCATCC-3 ' LG2 Sense (SEQ ID NO: 6) 5'-GCCACTCGAGCAGTCCTCAGGT-3 ' Antisense (SEQ ID NO: 7) 5'-GCCACCATGGTCACTCCACAAAACCAGGCTTA-3 ' LG3 Sense (SEQ ID NO: 8) 5'-GCCACTCGAGTGTGGAGCTCTCCCCTGT-3 ' Antisense (SEQ ID NO: 9) 5'-GCCACCATGGTCAAACTGGGGTGGGCGTAGGA-3 '

LG1 was amplified by nested PCR using two primer combinations. Nucleotide sequences of all plasmid preparations were confirmed by sequencing. PGEM-T Easy vectors containing LG cDNA fragments were assimilated with Xho I and Nco I. The cDNA fragments subsequently replicated the corresponding sites of the mammalian expression plasmid vector pRSET (Invitrogen). The correct direction of insertion was confirmed by sequence analysis.

<1-3> human Laminin  α2 chain LG  Expression and Purification of the Domain

Expression and purification of rLG domains have been previously reported (Kim, JM et.al., Exp. Cell Res . 304, 317-327, 2005). Specifically, rLG proteins induced expression in Escherichia coli BL21 growing at the MIDlog stage in Luria-Bertani culture using 1 mM IPTG (isopropyl- β- D-thiogalactopyranoside, Promega). After inducing protein expression at 37 ° C. for 5 hours, cells were harvested by centrifugation at 6,000 rpm for 10 minutes. Cell precipitates were stored at -80 ° C until use.

For protein purification, the cell precipitate was lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (Sigma) (lysis buffer, 100 mM NaH ₂ PO ₄ , 10 mM Tris-HCl, 8 M urea, pH 8.0) and then suspended. rLG proteins Ni ^{2 +} - was purified using nitrilotriacetic acid agarose chromatography ^{(Ni 2 + -nitrilotriacetic acid agarose,} Qiagen, Valencia, CA). LG proteins tagged with purified recombinant histidine 6 (His ₆ ) were 100 mM NaH ₂ PO ₄ And dialysis with urea from high to low concentrations (3 M, 2 M, 1 M or 0.5 M) in 10 mM Tris-HCl, pH 3.0 buffer containing 1 mM phenylmethylsulfonyl fluoride Afterwards, regeneration was performed by dialysis with PBS (pH 3.0) containing 1 mM phenylmethylsulfonyl fluoride. The dialyzed rLG proteins were concentrated to a concentration of 0.5 μg / μl using a Centricon instrument (Centricon YM-10, Millipore, Bedford, Mass.) And stored at −80 ° C. until use. Protein concentration was measured using a protein analysis kit (Bio-Rad protein assay kit, BioRad, Hercules, CA).

As a result, human laminin α2 chain C-terminal LG domains (LG1, LG2 and LG3) were respectively expressed as monomers in E. coli . The amino acid positions of the recombinant proteins LG1, LG2 and LG3 in the whole laminin α2 chain molecule are as shown in FIG. 1A. It was confirmed that the molecular weight of the purified rLG1, rLG2 and rLG3 proteins are 27, 32 and 33 kDa, respectively (see Figure 1B). rLG3 represents several bands, with the largest band corresponding to the expected molecular size, whereas the small bands correspond to partially degraded products. We expressed the rLG3 domain in E. coli to eliminate protease activity and obtained the original rLG3 domain with high efficiency (average 8 mg / 0.5 L of bacterial culture).

< Experimental Example  1> human Laminin  α2 chain LG  Cell adhesion analysis of domain

Cell adhesion assays were performed according to the method reported by Okazaki et al. (Okazaki, I., et al., J. Biol . Chem . 277, 37070-37078, 2002). Specifically, purely isolated rLG1, rLG2 and rLG3 proteins and human placental laminin were placed in 24-well culture plates (Nunc, Roskilde, Denmark) at 4 ° C. at 25 μg / ml and 5 μg / ml, respectively. Coating for 12 hours. Synthetic peptides coated on the plates were dried at room temperature for 12 hours. PBS containing 1% heat-inactivated bovine serum albumin (BSA, Sigma) was added to the substrate-coated plate, stored at 37 ° C. for 1 hour, blocked, and washed twice with PBS. It was. PC12 cells were removed from the culture dish with a solution containing 0.05% trypsin and 0.53 mM EDTA in PBS, resuspended in medium, and then added to each plate at a concentration of 2 × 10 ⁵ cells / 500 μl. Incubated for 1 hour at ℃. After incubation, the plates were washed twice with PBS to remove unattached cells. Cells attached to the plate were fixed with PBS containing 10% formalin for 15 minutes, then washed twice with PBS and stained with 0.5% crystal violet for 1 hour. The plate was washed three times lightly with DDW and then hemolyzed with 2% SDS for 5 minutes. Absorbance was measured at 570 nm using a Bio-Rad Model 550 microplate reader (Bio-Rad).

As a result, the cell adhesion activity of each of the rLG proteins of PC12 cells showed the maximum adhesion of laminin, and then the rLG1 and rLG3 showed moderate cell adhesion effects. In contrast, cell adhesion was weak in rLG2 (FIG. 1C). In addition, after coating the lLG1, rLG2 and rLG3 protein in a 24-well plate by concentration, and confirmed the cell adhesion activity as described above, rLG1 and rLG3 protein concentration-dependently increased cell adhesion, at 25 ㎍ / ㎖ Maximum adhesion effect was shown (FIG. 1D). Thus, it can be seen that the cell attachment sites exist in each of the rLG1 and rLG3 domains.

< Experimental Example  2> human Laminin -2 α2 chain rLG1  Determination of site of cell adhesion in domain

The present inventors synthesized peptides having 12 amino acids overlapping 6 by 6 within the amino acid residues 2209-2256 site in the LG1 domain, in order to find the minimum amino acid sequence having cell adhesion activity in the LG1 domain. Figure 2A) after each cell adhesion activity was examined. Synthesis of the peptides was carried out using a Pioneer peptide synthesizer (Cioner peptide synthesizer, Applied Biosystems, Forster City, CA) using C-terminal amide at Korea Basic Science Institute, Seoul, Korea. Was performed by a Fmoc-based solid-phase method (9-fluorenylmethoxycarbonyl-based solid-phase method).

As a result of the cell adhesion experiment in the same manner as in <Experimental Example 1>, Ln2-P3 peptide (DLTIDDSYWYRI, residues 2221-2232) among the synthesized peptides increased cell adhesion in a concentration-dependent manner, and the concentration of 100 ㎍ / ml Maximum cell adhesion activity was shown in (Fig. 2B).

In addition, after pretreatment of peptide Ln2-P3 to PC12 cells preincubated at 100 µg / ml for 15 minutes at room temperature, the rLG1 domain was treated to measure the cell adhesion activity of the rLG1 domain. As a result, peptide Ln2-P3 inhibited cell adhesion of the rLG1 domain by about 86%, while other peptides hardly inhibited the cell adhesion of the rLG1 domain (FIG. 2C).

Therefore, it can be seen that the Ln2-P3 sequence plays a role as a cell adhesion site in the human laminin-2 α2 chain rLG1 domain.

< Experimental Example  3> rLG1  Domains and Peptides Ln2 - P3 Characterization of cellular receptors attached to

<3-1> About cell adhesion Integrin  effect

Pretreatment of PC12 cells with an integrin β1 antibody that binds to the metal-chelating agents EDTA and integrin β1 to inhibit cell adhesion to remove bivalent cations (Ca ^{2 +} , Mg ^{2 +} , Mn ^{2 +} ), which are important for integrin activation, The degree of cell adhesion was examined in 24-well plates coated with, laminin, rLG proteins and peptide Ln2-P3.

As a result, laminin, rLG2 and rLG3 inhibited cell adhesion by integrin β1, whereas rLG1 and Ln2-P3 did not inhibit cell adhesion on integrin β1 (FIG. 3A).

<3-2> Effect of Heparin Molecule on Cell Attachment

PC12 cells were pretreated with heparin followed by 24-wells coated with various concentrations (5, 10, 25, 50 and 100 μg / ml) of laminin, rLG proteins (rLG1, rLG2 and rLG3), and peptide Ln2-P3, respectively. The degree of cell adhesion to the plate was examined.

As a result, cell adhesion was inhibited in a concentration-dependent manner in rLG1, rLG2, rLG3 and Ln2-P3 except laminin (FIG. 3B).

In addition, a solid-phase heparin binding assay was performed to examine in more detail whether cell adhesion is caused by a receptor having a heparin type substance. Specifically, 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated with various concentrations of rLG proteins at 4 ° C. for 12 hours. Synthetic peptides coated on the plates were dried at room temperature for 12 hours. Substrate-coated plates were washed twice with PBS containing 0.05% Tween 20 and then 3% heat-inactivated bovine serum albumin in PBS containing 0.05% Tween 20 for 2 hours at 37 ° C. , BSA, Sigma). The wells were washed twice with PBS containing 0.05% Tween 20 and 10 ng biotin attached heparin (Calbiochem, San Diego, Calif.). After incubation for 1 hour at 37 ° C., the supernatant was removed and the wells washed three times with PBS containing 0.05% Tween 20. To investigate the binding of biotin-bound heparin, 10 ng horseradish-peroxidase-coupled streptavidin (streptavidin-conjugated horseradish peroxidase, Calbiochem) was added and further incubated for 1 hour at 37 ° C. After washing three times with PBS containing 0.05% Tween 20, 0.4 mg / ㎖ O-phenylenediamine ( O -phenylenediamine, Sigma-Aldrich) was added and incubated for 10 minutes at room temperature. After adding 3 MH ₂ SO ₄ , absorbance was measured at 450 nm using a Bio-Rad Model microplate reader (Bio-Rad).

As a result, rLG1 and Ln2-P3 showed heparin adhesion in a concentration-dependent manner (FIGS. 3C and 3D).

<3-3> heparan sulfate on cell adhesion and Of dermatane sulfate  effect

To identify cellular receptors that attach to rLG1 proteins and peptide Ln2-P3, various glycosaminoglycans (GAG), heparinase I or chondroitinase ABC (chondroitinase ABC) As a competitor, a cell adhesion inhibition assay of rLG1 protein and peptide Ln2-P3 was performed. The cell adhesion inhibition assay specifically, PC12 cells (2 × 10 ⁵ cells / 500 ul) to 5 mM EDTA, 10 ug / ml of integrin β1 (BD Biosciences, San Jose, Calif.), Go 6976 ( Calbiochem, San Diego, CA), Rottlerin (Calbiochem), Calphostin C, U73122, or 100 ug / ml synthetic peptide, heparin, heparan sulfate, dermatane sulfate, hyaluronic acid ( hyaluronic acid), chondroitin sulfate A (chondroitin sulfate A), chondroitin sulfate C or de- N -sulfate heparin (Sigma-Aldrich) for 15 minutes pre-incubation. The precultured cells were transferred to plates precoated with either rLG protein (25 μg / ml) or peptide Ln2-P3 (100 μg / ml) and further incubated for 1 hour at 37 ° C. Attached cells were quantified by cell adhesion assay.

As a result, heparan sulfate and dermatane sulfate inhibited cell adhesion to rLG1 and Ln2-P3 (FIG. 3E).

In addition, in order to confirm in more detail the relationship between heparan sulfate and dermatan sulfate on cell adhesion, after enzymatic immersion of the cells with heparinase I or chondrinase ABC for 90 minutes at 37 ° C., the rLG1 protein and The degree of cell adhesion of peptide Ln2-P3 was examined. Enzymatic treatment of the cells was previously incubated for 2 hours with cells (6 × 10 ⁵ cells / 200 μl) with 2 μg / ml cycloheximide, followed by 37 ° C. (heparitinase I, chondroitinase ABC) or 30 ° C. Heparintinase I (0.1, 0.5 or 1 unit in PBS containing 2 mg / ml cycloheximide, 2 mM CaCl ₂ , and 0.1% BSA for 90 minutes at (PI-PLC) / Ml), chondroitinase ABC (0.1, 0.5 or 1 unit / ml) or phosphatidylinositol-phospholipase C (PI-PLC; 1 unit / ml, Sigma-Aldrich) Reacted. The cells were suspended in serum depleted RPMI 1640 culture and used for cell adhesion assays.

As a result, cell adhesion to rLG1 and Ln2-P3 was inhibited as the enzyme activity was increased (FIG. 3F). In particular, this inhibition of adhesion was higher in cells treated with heparinase I than in cells treated with chondrinase ABC. Therefore, it can be seen that rLG1 domain and peptide Ln2-P3 are adhered to the cell receptors with heparan sulfate and dermatane sulfate, and heparan sulfate has greater adhesion to rLG1 and Ln2-P3 than dermatan sulfate. .

< Experimental Example  4> rLG1  Domains and Peptides Ln2 - P3 Of cell receptors that adhere to

<4-1> Cindecan  Series and Glypican -1 expression investigation

In order to find receptors on the cell surface that adhere to rLG1 and Ln2-P3, the syndecan family and glypican-1 with heparan sulfate proteoglycans expressed in PC12 cells Expression patterns were measured using flow cytometyr analysis. Specifically, the flow cytometry, PC12 cells were isolated by treatment with trypsin / EDTA, washed with PBS, and then incubated with the primary antibody for 45 minutes at 4 ℃. The cells were washed with PBS and then incubated with FITC bound secondary antibody at 4 ° C. for 45 minutes. After two additional washes with PBS, flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, Calif.).

As a result, only syndecane-1 was expressed on the PC12 cell surface, and no remaining syndecane-2, syndecane-3, syndecane-4 and glypican-1 were found (FIG. 4A).

In addition, in order to examine the expression of the syndecane family and glypican-1 in more detail, it was observed using immuno-dot blotting and immunofluorescene.

Specifically, the immunodot blot analysis is as follows: First, the cells are washed with PBS and then buffered with a protease inhibitor cocktail (50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 1). % Triton X-100). The cell lysates were preincubated with 50 μl of fixed Protein A / G beads (Pierce, Rockford, IL) at 4 ° C. for 30 minutes. After centrifugation of the lysate, 1 ml of the supernatant was incubated with 30 μg of His ₆ or His ₆ -rLG1 protein at 4 ° C. for 2 hours. The bound materials were immunoprecipitated with 100 μl of immobilized Protein A / G beads carrying 1 μg anti-His ₆ antibody (Roche, Mannheim, Germany) for 12 hours at 4 ° C. After centrifugation, the beads carrying the immunocomplex were washed four times with buffer (25 mM Tris-HCl, pH 5.0, and 150 mM NaCl). To elute the immune complexes derived from the beads, 100 μl of elution buffer (Pierce) was added and then centrifuged at 2,500 g for 1 minute. After the supernatant was collected, 10 μl of neutralization buffer (1 M Tris-HCl) was added. The supernatant was dot-blotted onto the nitrocellulose membrane and then probed with a primary antibody. All blots were detected using horseradish-peroxidase bound secondary antibody and developed with ECL reagent (iNtRON Biotechnology, Korea).

The immunofluorescence method is specifically as follows: First, cells were cultured in a Lab-Tek chamber slide (Nalge Nunc International, Rochester, NY). For syndecan family and glypican-1 staining, the cells were treated with anti-decancan-1, -4, and anti-glycancan-1 antibodies (1:25) in PBS containing 1% BSA for 1 hour at 4 ° C. , Santa Cruz), washed with PBS, and fixed with 3.7% formalin for 20 minutes at room temperature. After washing with PBS, cells were FITC-bound anti rabbit IgG antibody (1:25, Jackson ImmunoResearch Laboratories, West Grove, PA) and FITC-bound anti goat IgG antibody (1:25, Santa) for 1 hour at room temperature. Cruz).

As a result, it was confirmed that syndecane-4 and glypican-1 are very weakly expressed on the cell surface (FIGS. 4B and 4C).

<4-2> rLG1  And Ln2 - P3 of Cindecan Binding to -1

In order to determine that the cellular receptors of rLG1 and Ln2-P3 are syndecane-1, the above-described immunodotblotting analysis and double immunostaining method were used.

The dual immunostaining is specifically as follows: To visualize the colocalization of rLG1 and syndecane-1, cells were fixed with 3.7% formalin for 20 minutes at room temperature, followed by 1% BSA for 1 hour. Block with PBS containing. Cells were precultured with heparin, scrambled peptide (Asp Val Gly Ser Gly Val Gly Arg Val Glu Tyr Pro: SEQ ID NO: 3), or peptide LN2-P3 for 2 hours at room temperature. rLG1 protein (25 μg / ml) and anti-His ₆ antibody (1:50, Roche) were added and then incubated at 4 ° C. for 12 hours. After washing with PBS, cells were incubated with anti-Synthecan-1 antibody (1:25, Santa Cruz) at 4 ° C. for 4 hours and then FITC-bound anti rabbit IgG antibody (1: 1) at room temperature for 1 hour. 25, Jackson ImmunoResearch Laboratories) and TexasRed-bound anti mouse IgG antibodies (1: 100, Jackson ImmunoResearch Laboratories).

As a result, it was found that rLG1 and Ln2-P3 are combined with syndecane-1 (FIGS. 4D to 4H).

Thus, it can be seen that syndecane-1 acts as a cell receptor for the rLG1 domain and peptide Ln2-P3.

<4-3> rLG1  And Ln2 - P3 of Cindecan Cell adhesion assay through -1

To confirm that syndecane-1 is a PC12 cell receptor of rLG1 domain and peptide Ln2-P3, treatment with syndecane-1 siRNA reduces the expression of syndecane-1, followed by 24-coated rLG1 and Ln2-P3. Cell adhesion assays were performed on well plates. The method for synthesizing the siRNA is specifically as follows:

RT - PCR

Total RNA was isolated from cells using TRI-reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol. After denaturing the RNA by incubating at 70 ° C. for 10 minutes, cDNA was prepared using reverse transcriptase (Supertranscript II, Invitrogen, Carlsbad, Calif.) And random hexamer, followed by Taq polymerase (Novagen). PCR amplified by. PCR primers used were as follows: Syndecan-1: 5'-CCCCACAACCCCTGCCACTCACTA-3 '(SEQ ID NO: 10) and 5'-AAGGTCTGCTGGGGCTCTAAAACA-3' (antisense) (SEQ ID NO: 11); Syndecan-4 5'-CATGGGGAGAGGAGTTGAGGATTG-3 '(SEQ ID NO: 12) and 5'-TGCCCCGAGATACACCACAGAGAC-3' (antisense) (SEQ ID NO: 13); Glypican-1 5'-GTGGCGCCTACGGTGGAAATGATG-3 '(sense) (SEQ ID NO: 14); 5'-TGCCCCTGAGGTCCCTGAAAAACA-3 '(antisense) (SEQ ID NO: 15). Primers were prepared to normalize expression levels: GAPDH; 5'-TGTCAGCAATGCATCCTGTACCACC-3 '(SEQ ID NO: 16) and 5'-AAAGTCCGTGGAGACCACCTGGTCC-3' (antisense) (SEQ ID NO: 17). The PCR product was dissolved in 1% agarose gel, stained with EtBr (Ethidium bromide), and then visualized using a UV illuminator.

siRNA  Synthetic and transfection ( transfect )

Small interfering RNA (siRNA) and nonspecific controls specific for syndecan-1, -4, or glypican-1 were purchased from Invitrogen. The RNA sequence is as follows: syndecan-1, 5'-AUUUGCGGUGACAAUUUGCGGGAGG-3 '(SEQ ID NO: 18); Syndecane-4, 5'-UGUCAUCUAGAUCUCCGGAACCCGA-3 '(SEQ ID NO: 19); Glypican-1, 5'-UUGGAAAUCCCAUUCCAGCAGCGGU-3 '(SEQ ID NO: 20). Specifically, PC12 cells (5 × 10 ⁵ cells) were inoculated into 60-mm dishes and then transfected with 100 nM siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's recommendations. Five hours after the transfection, the medium was replaced with a medium containing 10% FBS. 48 hours after the transfection, it was used for cell adhesion analysis.

As a result, decreased expression of syndecane-1 inhibited the cell adhesion of PC12 to rLG1 and Ln2-P3 by about 50% (FIGS. 5A-5C).

<4-4> rLG1  And Ln2 - P3 of Cindecan -4 and Glypican Cell adhesion assay through -1

Cadecan-4 and glypican-1, which are weakly expressed on the cell surface, were treated with cindcan-4 siRNA and glypican-1 siRNA, respectively, to reduce expression, and then in 24-well plates coated with LG1 and Ln2-P3. Cell adhesion assays were performed. Synthesis method of the siRNA is the same as in Experimental Example <4-3>.

As a result, decreased expression of syndecane-4 and glypican-1 did not inhibit cell adhesion of PC12 to rLG1 and Ln2-P3 (FIGS. 5D and 5E).

Therefore, it can be seen that syndecane-1 is an important receptor for cell adhesion of the rLG1 domain and peptide Ln2-P3 in PC12 cells.

< Experimental Example  5> rLG1  Domains and Peptides Ln2 - P3 of Cindecan Intracellular Signaling Through A-1

<5-1> rLG1  And Ln2 - P3 end PKC  Effect on protein location

To determine the intracellular signaling after rLG1 and Ln2-P3 binds to cindecan-1, 24-well coated with rLG1 and Ln2-P3 treated with concentrations of PKC inhibitor calphostin C Cell adhesion assays were performed on the plates.

As a result, it was found that inhibition of PKC inhibited cell adhesion of the rLG1 domain and peptide Ln2-P3 (FIG. 6A).

In addition, the protein levels of PKC subtypes (α, δ, ε, λ / τ) in the cytoplasmic and membrane portions of PC12 cells attached to rLG1 and Ln2-P3 were observed by western blotting. Classification of membrane and cytoplasmic proteins was performed using the plasma membrane protein extraction kit (BioVision, Mountain View, CA) according to the manufacturer's instructions. Specifically, after depleting nutrients for 12 hours before the experiment, the cells were separated and resuspended in serum-free medium containing 0.1% BSA. Cells were inoculated in dishes coated with rLG1 protein or coated with peptide Ln2-P3 (4 × 10 ⁶ / 100-mm dish) and then attached at 37 ° C. for 15 or 30 minutes. The cells were lysed with 200 ml Homogenize buffer (BioVision). Soluble cytosolic fractions were harvested after centrifugation at 10,000 g for 30 minutes at 4 ° C. The soluble membrane fraction was recovered after centrifugation at 14,000 g at 4 ° C. for 15 minutes. Protein concentrations were determined using Bradford reagent (BioRad), followed by Western blot analysis.

As a result, protein levels of PKC-α and PKC-δ decreased in the cytoplasmic portion of the cells attached to rLG1, and increased in the membrane portion (Fig. 6B). In addition, protein levels of PKC-α and PKC-δ decreased in the cytoplasmic portion of cells attached to Ln2-P3, but did not change in the membrane portion (Figure 6C). In contrast, the protein levels of PKC-ε and PKC-λ / τ did not change in the cytoplasmic and membrane portions of cells attached to rLG1 and Ln2-P3.

Thus, it can be seen that the rLG1 domain and Ln2-P3 can regulate the intracellular positions of PKC-α and PKC-δ by interacting with syndecane-1.

<5-2> Cindecan -1 and PKC -α or PKC complex formation of -δ

To determine whether syndecane-1 complexes with PKC-α or PKC-δ in PC12 cells, it was analyzed using immunoprecipitation method. The immunoprecipitation was specifically as follows: First, starve was depleted for 12 hours by replacing the cells with RPMI 1640 medium containing 0.1% FBS. Cells were trypsinized, washed and resuspended in serum free medium containing 0.1% BSA. Cells were plated (4 × 10 ⁶ ) in 100-mm dishes coated with rLG1 domain or peptide Ln2-P3 and then attached for 30 minutes. After washing the cells with ice-cold PBS, 500 ml of RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1%) containing the protease inhibitor cocktail SDS, 1 mM PMSF, 2 mM Na ₃ VO ₄ , and 1 mM glycerol phosphate). After lysing the lysate at 10,000 g for 15 minutes, the supernatant was used for immunoprecipitation. Cell lysates were pre-cleared with 20 ml of protein G agarose beads (Upstate Biotechnology Inc., Lake Placid, NY) to remove nonspecific binding. After centrifugation, the supernatant was washed for 2 hours with 1 μg of anti-His ₆ antibody (Roche), 10 μg of anti-syndecan antibody (Santa Cruz), or 5 μg of anti-PKC-α antibody or anti-PKC-δ antibody (Santa). Cruz). The immunocomplex was precipitated at 4 ° C. for 12 hours using 80 ml of Protein G agarose beads and then washed four times in RIPA buffer. The immunoprecipitates were eluted in 60 ml of SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue, and 100 mM β-mercaptoethanol) and then boiled for 5 minutes. , SDS / PAGE on 10% gels. The isolated protein was electroblotted on an Immobilon-P membrane (Millipore) and then probed with a primary antibody. All blots were detected using secondary antibodies conjugated with horseradish-peroxidase. The signal was detected by iNtRON Biotechnology (ECL).

As a result, it was found that syndecane-1 and PKC-δ specifically formed a complex (FIGS. 6D to 6F).

In addition, cell adhesion inhibition assays were performed on 24-well plates coated with rLG1 or Ln2-P3 with Go6976 and Rotlin, PKC-α and PKC-δ inhibitors, respectively.

As a result, adhesion was inhibited in cells treated with rotlerin (FIG. 6H), while adhesion was not inhibited in Go6976 treated cells (FIG. 6G).

In addition, immunofluorescence assays were performed on PC12 cells attached to rLG1 and Ln2-P3 for 30 minutes to determine whether syndecane-1 and PKC-δ are in the same position. The immunofluorescence assay was specifically as follows: PC12 cells were plated for 30 min on Lab-Tek chamber slides coated with rLG1 protein or peptide Ln2-P3. The cells were fixed with 3.7% formalin for 20 minutes at room temperature. Cells were incubated with anti-Synthecan-1 antibody (1/25 dilution; Santa Cruz) for 12 hours at 4 ° C., then washed with PBS and then FITC-bound anti rabbit IgG antibody (1 / 200 dilutions, Jackson ImmunoResearch Laboratories). The cells were washed with PBS and then permeabilized with 0.5% Triton X-100 in PBS for 5 minutes. Cells were incubated with anti PKC-δ antibody (1/500 dilution; BD biosciences) for 2 hours, followed by incubation with rhodamine-binding anti mouse IgG antibody (1/200 dilution; Santa Cruz). After washing three times with PBS, the stained cells were fixed on the slide and observed using a confocal laser scanning microscope (FV300; Olympus, Japan).

As a result, it was found that syndecane-1 and PKC-δ are at the same position (FIGS. 6I to 6J).

<5-3> rLG1  And Ln2 - P3 of Cindecan Through -1 PKC Effect on -δ

To determine whether rLG1 and Ln2-P3 are activated by PKC-δ by binding to syndecane-1, lysates obtained from cells attached to rLG1 and Ln2-P3 and non-attached cells were immunized with PKC-δ antibodies. It was settled and western blot was performed with the phosphotyrosine antibody.

As a result, it was found that tyrosine phosphorylation of PKC-δ was induced in cells attached to rLG1 and Ln2-P3 (FIGS. 6K to 6L).

In addition, PLC activity induces tyrosine phosphorylation of PKC-δ. Cellular adhesion to rLG1 and Ln2-P3 was inhibited by inhibiting tyrosine phosphorylation of PKC-δ by treating PLC inhibitor U73122 by concentration (Fig. 6M).

Therefore, it can be seen that when PC12 cells adhere to the rLG1 domain or Ln2-P3, PKC-δ migrates from the cytoplasm to the membrane and binds to synthecan-1, as well as induces tyrosine phosphorylation of PKC-δ. .

Figure 1 relates to the expression and cell adhesion activity of recombinant LG proteins (rLG1-rLG3) of the human laminin-2 α2 chain, Figure 1 A is a schematic diagram of the rLG1-rLG3 domain protein, Figure 1 B Is a graph showing the molecular weight of rLG1-rLG3 domain protein, Figure 1 C is a graph showing the PC12 cell adhesion activity of human laminin and rLG1-rLG3 domain, Figure 1 D is PC12 cell adhesion by concentration of rLG1-rLG3 domain Graph showing activity.

FIG. 2 relates to a novel motif of peptide Ln2-P3 (DLTIDDSYWYRI) in the LG1 domain that promotes cell adhesion, where A in FIG. 2 is a schematic of peptides with some amino acids in the rLG1 domain, and FIG. 2B is a synthetic peptide. Figure 2 is a graph showing the cell adhesion activity by concentration, Figure 2 C is a graph showing the inhibition of cell adhesion activity to the rLG1 domain by peptide Ln2-P3.

FIG. 3 is a graph showing inhibition of cell adhesion with rLG domain and peptide Ln2-P3 after pretreatment with GAG, heparinase I or chondrinase ABC. FIG. 3A is a graph showing cell adhesion inhibition by integrin β1. 3B is a graph showing inhibition of cell adhesion by heparin, C and D of FIG. 3 are graphs showing binding activity of heparin bound to biotin, and FIG. 3E is a graph of cell adhesion by GAGs. It is a graph which shows inhibitory activity, and F of FIG. 3 is a graph which shows the inhibitory activity of cell adhesion by heparinase I or chondroitin ABC.

Of Figure 4 as referring to the rLG domain and binds to a peptide Ln2-P3 Shin decane-1 in PC12 cells, a figure to indicate that A is expressed in renal decane -1 PC12 cell surface in Fig. 4, Fig. 4 B And C is a diagram showing that Shindecane-4 and Glypican-1 are very weakly expressed on the surface of the PC12 cell, and D to H of FIG. It is a graph.

Figure 5 is treated as the new decane -1 siRNA in PC12 cells indicating rLG domain and cell adhesion inhibiting peptides Ln2-P3 due to decreased expression of the new decane -1, A to C in Figure 5 is new decane siRNA -1 Figure 2 shows the inhibition of cell adhesion of the rLG domain and peptide Ln2-P3 due to the decrease in the expression of syndecane-1, and Figures D and E are synthecan-4 by syndecan-4 siRNA and glypican-1 siRNA. Figure 4 shows the inhibition of cell adhesion of rLG domain and peptide Ln2-P3 due to decreased expression of 4 and Glypican-1.

6 is in PC12 cells rLG domain and peptide Ln2-P3 renal relates to position control, and the tyrosine phosphorylation of the PKC-δ decane through -1, of Figure 6 A is a PKC inhibitory peptide of the rLG domain and Ln2-P3 Fig. 6B and C are graphs showing the level of PKC subtype protein in the cytoplasm and membrane portion of the cells attached to the rLG domain and peptide Ln2-P3, and D to D in Fig. 6. F is a diagram showing that Cindecan 1 specifically complexes with PKC-δ, and FIG. 6H shows that inhibition of PKC-α or PKC-δ inhibits cell adhesion of the rLG domain and peptide Ln2-P3. FIG. 6 is a graph showing synthecan-1 and PKC-δ at the same position, and K to L of FIG. 6 are PKC-δ in cells to which an rLG domain and peptide Ln2-P3 are attached. and the figure indicate that tyrosine phosphorylation is induced, M in FIG. 6 K It is a graph showing that inhibition of tyrosine phosphorylation of C-δ inhibits cell adhesion of the rLG domain and peptide Ln2-P3.

<110> SNU R & DB FOUNDATION <120> Human Laminin alpa 2 Chain LG1 Domain and an Active Peptide          within the LG1 Domain Promote Cell Adhesion <130> 8p-08-22 <150> KR10-2007-0089037 <151> 2007-09-03 <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 182 <212> PRT <213> Homo sapiens <400> 1 Ser Gly Gly Asp Cys Ile Arg Thr Tyr Lys Pro Glu Ile Lys Lys Gly   1 5 10 15 Ser Tyr Asn Asn Ile Val Val Asn Val Lys Thr Ala Val Ala Asp Asn              20 25 30 Leu Leu Phe Tyr Leu Gly Ser Ala Lys Phe Ile Asp Phe Leu Ala Ile          35 40 45 Glu Met Arg Lys Gly Lys Val Ser Phe Leu Trp Asp Val Gly Ser Gly      50 55 60 Val Gly Arg Val Glu Tyr Pro Asp Leu Thr Ile Asp Asp Ser Tyr Trp  65 70 75 80 Tyr Arg Ile Val Ala Ser Arg Thr Gly Arg Asn Gly Thr Ile Ser Val                  85 90 95 Arg Ala Leu Asp Gly Pro Lys Ala Ser Ile Val Pro Ser Thr His His             100 105 110 Ser Thr Ser Pro Pro Gly Tyr Thr Ile Leu Asp Val Asp Ala Asn Ala         115 120 125 Met Leu Phe Val Gly Gly Leu Thr Gly Lys Leu Lys Lys Ala Asp Ala     130 135 140 Val Arg Val Ile Thr Phe Thr Gly Cys Met Gly Glu Thr Tyr Phe Asp 145 150 155 160 Asn Lys Pro Ile Gly Leu Trp Asn Phe Arg Glu Lys Glu Gly Asp Cys                 165 170 175 Lys Gly Cys Thr Val Ser             180 <210> 2 <211> 12 <212> PRT <213> Homo sapiens <400> 2 Asp Leu Thr Ile Asp Asp Ser Tyr Trp Tyr Arg Ile   1 5 10 <210> 3 <211> 12 <212> PRT <213> Homo sapiens <400> 3 Asp Val Gly Ser Gly Val Gly Arg Val Glu Tyr Pro   1 5 10 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> LG1 sense primer <400> 4 gccactcgag caggaggtga ctg 23 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> LG1 antisense primer <400> 5 gccaccatgg tcaactgaca gtgcatcc 28 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LG2 sense primer <400> 6 gccactcgag cagtcctcag gt 22 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> LG2 antisense primer <400> 7 gccaccatgg tcactccaca aaaccaggct ta 32 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> LG3 sense primer <400> 8 gccactcgag tgtggagctc tcccctgt 28 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> LG3 antisense primer <400> 9 gccaccatgg tcaaactggg gtgggcgtag ga 32 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Syndecan-1 sense primer <400> 10 ccccacaacc cctgccactc acta 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Syndecan-1 antisense primer <400> 11 aaggtctgct ggggctctaa aaca 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Syndecan-4 sense primer <400> 12 catggggaga ggagttgagg attg 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Syndecan-4 antisense primer <400> 13 tgccccgaga tacaccacag agac 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Glypican-1 sense primer <400> 14 gtggcgccta cggtggaaat gatg 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> Glypican-1 antisense primer <400> 15 tgcccctgag gtccctgaaa aaca 24 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GAPDH sense primer <400> 16 tgtcagcaat gcatcctgta ccacc 25 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GAPDH antisense primer <400> 17 aaagtccgtg gagaccacct ggtcc 25 <210> 18 <211> 25 <212> RNA <213> Homo sapiens <400> 18 auuugcggug acaauuugcg ggagg 25 <210> 19 <211> 25 <212> RNA <213> Homo sapiens <400> 19 ugucaucuag aucuccggaa cccga 25 <210> 20 <211> 25 <212> RNA <213> Homo sapiens <400> 20 uuggaaaucc cauuccagca gcggu 25  

Claims (14)

A pharmaceutical composition for neuroregeneration comprising a polypeptide of SEQ ID NO: 1 as the LG1 domain sequence of the human laminin-2 α2 chain. delete A pharmaceutical composition for nerve regeneration comprising a peptide set forth in SEQ ID NO: 2. delete The pharmaceutical composition according to claim 1 or 3, wherein the polypeptide of SEQ ID NO: 1 or the peptide of SEQ ID NO: 2 binds to syndecan-1. According to claim 1 or claim 3, wherein the polypeptide described in SEQ ID NO: 1 or the peptide described in SEQ ID NO: 2 is protein kinase C-α (Protein kinase C-α, PKC-α through binding to syndecane-1) And the intracellular location of PKC-δ. The pharmaceutical composition of claim 1 or 3, wherein the polypeptide of SEQ ID NO: 1 or the peptide of SEQ ID NO: 2 induces tyrosine phosphorylation of PKC-δ. A container for promoting neuronal adhesion comprising the polypeptide of claim 1 or the peptide of claim 3. delete 1) fixing the polypeptide of claim 1 or the peptide of claim 3 to a solid support; 2) nerve cell adhesion promoting method comprising the step of attaching nerve cells to the solid support. delete A therapeutic agent for burns or wounds comprising the pharmaceutical composition of claim 1. An artificial neural conduit or scaffolds thereof comprising the pharmaceutical composition of claim 1. A support for tissue engineering, comprising the pharmaceutical composition of claim 1.

Images