KR20060005595A - Polypeptide and derivatives thereof containing novel heparin-binding site derived from human tenacin c - Google Patents

Abstract

The present invention relates to a polypeptide comprising a novel heparin-binding site derived from human tenacin-C (TN) and derivatives thereof, and specifically, heparan sulfate proteoglycan (heparan sulfate). proteoglycan (HSPG) binding site and increases HSPG-dependent cell adhesion activity to tenasin-C type III domain 5 (TNIII5), including a part of the amino acid sequence of tenasin-C type III domain 4 (TNIII4) The present invention relates to a polypeptide and a derivative thereof. The polypeptide of the present invention can be usefully used as a cell adhesion peptide because it binds with heparin with high affinity and enhances cell adhesion.

Description

Polypeptide and derivatives containing containing novel heparin-binding site derived from human tenacin C}             

Figure 1a is a schematic diagram showing the configuration of a human tenacin-C type III protein,

EGF: epidermal growth factor, FNIII: fibronectin type III

Fbg: Single Fibrinogen-Like Domain

1B shows a schematic diagram of recombinant proteins comprising only some domains of human tenacin-C type III and the results of electrophoresis on SDS-PAGE,

Lane 1: molecular weight marker, lane 2: TNIII5,

Lane 3: TNIII4-5, lane 4: TNIII3-5

2 is a result of analyzing the binding affinity of recombinant proteins containing only a part of domains of human tenacin-C type III to heparin immobilized on chip yarn using a BIAcore system.

A: TNIII3-5, B: TNIII4-5, C: TNIII5

FIG. 3A shows the structure-based sequence alignment results of domains 3 to 5 of human tenacin-C type III and domain 10 of fibronectin type III,

3B compares the amino acid sequences of tenascin-C type III domain 4 in humans ( Homo sapiences ), mice ( Mus musculus ), chickens ( Gallus gallus ) and pigs ( Sus scrofa ),

Figure 4 shows the results of measuring the cell adhesion activity of recombinant proteins containing only a partial domain of human tenacin-C type III and its substitution mutants.

The present invention relates to a polypeptide comprising a novel heparin-binding site derived from human tenacin-C (TN) and derivatives thereof, and more particularly to heparan sulfate proteoglycan ( part of the amino acid sequence of tenascin-C type III domain 4 (TNIII4), including the binding site of heparan sulfate proteoglycan (HSPG) and increasing HSPG-dependent cell adhesion to tenasin-C type III domain 5 (TNIII5) It relates to a polypeptide and a derivative thereof.

Tenasin-C (TN) is a large extracellular matrix (ECM) glycoprotein with an epidermal growth factor (EGF), fibronectin type III domain (FNIII), and domains homologous to fibrinogen. (Jones FS, et al., Proc. Natl. Acad. Sci. USA 87: 6497-6501, 1990; Jones FS, et al., Proc. Natl. Acad. Sci. USA 85: 2186-2190, 1988). . The human tenasin-C gene (GenBank Accession NO: NM_002160) has a nucleotide sequence of 7560 bp set forth in SEQ ID NO: 1 , from which encodes a protein having an amino acid sequence set forth in SEQ ID NO: 2 . Tenasin-C has an oligomeric structure composed of six identical subtypes in six radially arranged structures called hexabrachion (Erickson HP, et al., Nature 311: 267-). 269, 1984). Each subtype exists as a series of small spherical domains including FNIII domains 8-15 (Gulcher JR, et al., Proc. Natl. Acad. Sci. USA 88: 9438-9442, 1991; Erickson HP, et al., Curr. Opin. Cell Biol. 5: 869-876, 1993). FNIII is found in a very broad range of proteins, including cell surface receptors and cell adhesion molecules (Bork P, et al., Protein Sci. 2: 1185-1187, 1993), with the central structure of the FNIII repeat units on one side. Four β-strands, three β-strands on the other, are conserved as they form β-sandwiches (Leahy DJ, et al., Science 258: 987-991, 1992; Dickson CD, et al. , J. Mol. Biol. 236: 1079-1092, 1994). The third FNIII repeat unit (TNIII3) of tenasin-C comprising an Arg-Gly-Asp (RGD) triple peptide sequence interacts with the cell through various integrins, for example, integrins such as αvβ3, αvβ6, α9β1 and α2β1. Seems to work (Prieto AL, et al., J. Cell Biol. 119: 663-678; Prieto AL, et al., Proc. Natl. Acad. Sci. USA 90: 10154-10158, 1993; Joshi P, et al., J. Cell Sci. 106: 389-400, 1993; Yokosaki Y, et al., J. Biol. Chem. 269: 26691-26696, 1994; Dorries U, et al., J. Neurosci.Res . 43: 420-438, 1996). Tenasin-C also contains CTB-prodeoglycans (Hoffman S, et al., J. Cell Biol. 106: 519-532, 1988), neurocans, phosphacans (Grumet M, et al., J. Biol. Chem. 269: 12142-12146, 1994) and syndecane-1 (Salmivirta M, et al., J. Biol. Chem. 266: 7733-7739, 1991). . Syndecane-1 and Tenasin-C are transiently co-expressed in primordial organs present at the interface between mesenchymal and epithelium (Thesleff I, et al., Development 101: 289-296, 1987; Vainio S, et. al., J. Cell Biol. 108: 1945-1953, 1989), the binding of syndecan-1 to tenasin-C depends on the heparan sulfate side chains of proteoglycans (Salmivirta M, et al., J. Biol. Chem. 266: 7733-7739, 1991).

Heparin / Heparan Sulfate and Heparan Sulfate Proteoglycans (HSPGs) are macromolecules with long, unique carbohydrate chains attached to the protein core (Gallagher JT, et al., Biochem. J. 236: 313-325, 1986; Hardingham TE, et al., FASEB J. 6: 861-870, 1992). They exist as constituents or membrane-binding proteins of the extracellular matrix (ECM) and participate in a variety of biochemical and physiological processes through interactions with several proteins, including ECM proteins (Rouslahti E, Annu. Rev. Cell Biol. 4: 229-255, 1988; Jackson RL, et al., Physiol. Rev. 71: 481-539, 1991; Kjellen L, et al., Annu. Rev. Biochem . 60: 443-475 , 1991). Heparin prevents peripheral neurons from attaching to tenasin-C in culture (Chiquet M, et al., Perspect. Dev. Neurobiol. 2: 67-74, 1994; Wehrler-Haller B, et al., J Cell Sci 106: 597-610, 1993). Heparin-protein interactions are made by ionic contact between the positively charged residues of the protein (Arg, Lys) and the negatively charged functional groups of heparin (sulfuric groups of glucosamine or carboxyl groups of iduronic acid) (Faham S, et al. , Science 271: 1116-1120, 1996). Therefore, it is very important to identify specific domains responsible for binding to glycosaminoglycans in order to characterize the biological function of tenasin-C. As part of this study, two heparin-binding sites have been identified in tenasin-C (Aukhil I, et al., J. Biol. Chem. 268: 2542-2553, 1993; Weber P, et al., J. Biol. Chem. 270: 4619-4623, 1995), one containing the fifth FNIII repeat unit (TNIII5) and the other containing the carboxyl-terminal fibrinogen-like domain (Fbg).

Heparin / heparan sulfate binding is identified as a common feature of ECM molecules, including tenasin-C. Although the binding affinity of tenasin-C to heparin has not been clarified yet, tenasin-C has been reported to bind heparin at physiological salt concentrations (Marton LS, et al., J. Biol). Chem. 264: 13145-13149, 2989).

The characterization of FNIII domains in tenasin-C that interacts with heparin is largely focused on the TNIII5 domain, although it is still unclear whether the TNIII5 domain is the only motif required for interaction with heparin. It is not true.

Accordingly, the present inventors have made a thorough study to investigate the interaction between heparin and tenasin-C and to estimate a new heparin-binding site in tenasin-C that mediates the interaction. Polypeptides comprising domain 4 fragments of C type III contain secondary heparin-binding sites and thus optimal for tenascin-C type III to heparin by co-action with tenasin-C type III domain 5 (TNIII5). The present invention was completed by revealing the binding activity of and increasing the HSPGs-dependent cell adhesion to TNIII5.

It is an object of the present invention to provide a novel heparin-binding domain in tenasin-C type III which exhibits high heparin-binding affinity and cell adhesion activity.

In order to achieve the above object, the present invention provides a polypeptide comprising a KEDK sequence showing a cell adhesion activity as a novel heparin-binding site involved in the interaction between tenasin-C and heparin and derivatives thereof.

The present invention also provides a pharmaceutical composition for cell attachment comprising the polypeptide and its derivatives as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a polypeptide comprising a KEDK sequence exhibiting cell adhesion activity as a novel heparin-binding site and derivatives thereof.

The present invention identifies the role of the TNIII4 domain in the interaction of heparin and tenasin-C and provides a polypeptide sequence in the TNIII4 domain that mediates heparin-binding jointly with the TNIII5 domain, previously known as the heparin-binding site.

Preferably, the polypeptide sequence of the present invention is a polypeptide comprising a 969th to 972th amino acid sequence corresponding to domain 4 of tenasin-C type III in the amino acid sequence of SEQ ID NO: 2 as the heparin-binding site, more preferably. Comprises the KEDK amino acid sequence of SEQ ID NO: 3 .

Polypeptides of the present invention are tena by a cooperative mechanism with the heparin-binding site of tenacin-C type III domain 5 (TNIII5), including the binding site of heparan sulfate proteoglycan (HSPG). It is characterized by increasing heparin-binding activity of syn-C and enhancing HSPG-dependent cell adhesion to TNIII5.

Specifically, in the present invention, in order to select a polypeptide including a specific sequence in TNIII4 showing cell adhesion activity as a heparin-binding site, a recombinant protein of fibronectin type III domains of tenasin-C (TNIII) was prepared ( 1A and 1B ), their binding affinity to heparin was investigated. As a result, as previously reported, the TNIII5 domain containing the main heparin-binding site binds to heparin at physiological salt concentrations and pH conditions. However, the binding affinity of the TNIII5 domain alone for heparin was significantly lower than the fragments containing both TNIII5 and TNIII4 domains (see Figure 2 ). In addition, as a result of kinetic analysis, the recombinant fragments containing the TNIII3-5 and TNIII4-5 domains showed a fast binding rate constant for heparin, while showing a slow dissociation rate constant, indicating that they are very stable (see Table 1 ). The binding affinity for heparin in recombinant fragments containing only the TNIII5 domains was much lower than the binding affinity of presynthetic fragments containing the TNIII3-5 or TNIII4-5 domains, indicating that the TNIII4 domains are optimal between tenasin-C and heparin. It means that it contributes considerably to the combination of.

Therefore, the present inventors judge that the TNIII4 domain enhances the heparin-binding activity of the TNIII5 domain by a kind of cooperative mechanism, and assumes that one site in the TNIII4 and TNIII5 domain that can simultaneously bind to heparin exists. It was. This co-mechanism has also been observed in several domains, where the primary heparin-binding site in the thirteenth type III repeat unit of fibronectin (FNIII13) and the tentative site (~ 60 Hz) in the fourteenth type III repeat unit (FNIII14) of fibronectin interact with each other. Maximal induction of heparin-binding activity (Sharma A, et al., EMBO J. 18: 1468-1479, 1999) and the tenth and eleventh type III repeat units of tenasin-X (TN-X) It has been reported that two adjacent heparin-binding sites in the mice act similarly (Lethias C, et al., J. Biol. Chem. 276: 16432-16438, 2001).

The overall structure of the FNIII repeating molecule molecules in tenasin-C can vary in various shapes depending on the angle of inclination between the repeating units and the length of the region between these repeating units. According to previous reports, TNIII4 and 5 repeat units are densely packed to form long, straight strands (Leahy DJ, et al., Science 258: 987-991, 1992). Structure-based sequence alignment of TNIII domains 3 to 5 and FNIII domain 10 revealed that the TNIII3 to 5 domains shared the same overall structure, with functional segments occupying the surface loop (see FIG. 3A ). The loop region is located on the surface of the protein and mainly contains hydrophilic amino acids, of which heparin-binding potential is the KEDK sequence in TNIII4 (969-972 of the amino acid sequence of SEQ ID NO: 2) , which has a remarkably positive surface charge. It was confirmed that it can act as a site. Although the KEDK sequence of TNIII4 and the KGR sequence of TNIII5 are slightly connected to each other, it can be seen that the KEDK sequence in the loop region between the F and G β-sheets of TNIII4 is located on the surface of the same molecule as the KGR sequence in TNIII5. (See FIG. 3A ). Moreover, KEDK loops on the TNIII4 domain show high sequence homology between different species, including chicks, mice, pigs and humans, and have been conserved (see FIG. 3B ).

As described above, the inventors of the present invention confirmed that the KEDK sequence in the TNIII4 domain induces the optimal binding activity for heparin of tenasin-C together with the TNIII5 domain. Was prepared and the heparin-binding activity was investigated. As a result, substitutional mutants having double mutations in the KEDK sequence exhibited significantly reduced heparin-binding activity, indicating that the 969-972th lysine residues of SEQ ID NO: 2 in the KEDK sequence of the TNIII4 domain are essential for heparin-binding activity. It was confirmed (see Table 2 ).

Thus, combining the structure-based sequence alignment, mutation and sequence homology analysis results, it can be seen that the KEDK sequence in TNIII4 is essential for showing a high binding affinity for heparin. Since the KEDK sequence in TNIII4 is located on the surface of the same molecule as the KGR sequence in TNIII5, the heparin-binding sites of tenasin-C exist across the TNIII4-5 domains, whereby TNIII4 derived KEDK residues are primarily in the TNIII5 domain. Co-operation with the phosphorus heparin-binding site leads to optimal heparin-binding activity.

The involvement of KGRHKSKPARVK sequences in TNIII5 with heparin-binding activity has already been reported (Weber P, et al., J. Biol. Chem. 270: 4619-4623, 1995). The present invention also confirmed that mutations in the 1057th lysine and 1059th arginine residues in the amino acid sequence of SEQ ID NO: 2 significantly reduced heparin-binding activity. From the above results, tenasin-C type III contains one site in TNIII5 and TNIII4 capable of simultaneously binding heparin, and the two binding sites are optimal for heparin of tenasin-C by co-action. It can be seen that it induces binding activity.

As a result of investigating whether the heparin-binding activity of TNIII4 identified as the heparin-binding domain is associated with cell adhesion, TNIII4-5 showed significantly higher cell adhesion activity than TNIII5, while having a double mutation in the KEDK sequence. TNIII4-5 substitution mutants significantly reduced cell adhesion activity. In addition, cell adhesion to TNIII4-5 was completely inhibited in the presence of heparin, which inhibited the binding activity of TNIII4-5 to HSPGs on the cell surface when water-soluble heparin pre-bound to heparin-binding sites in TNIII4-5. It can be. This was demonstrated from the result that the cell adhesion activity of TNIII4-5 was significantly reduced by Heparinase I treatment. From the above results, it was confirmed that TNIII4 contains a binding site of HSPGs, and co-mechanism increases the adhesion activity of TNIII5 to HSPGs.

Accordingly, a polypeptide having a high binding affinity for heparin of the present invention and capable of acting as a heparin-binding site, and exhibiting cell adhesion activity, may be a polynucleotide comprising a KEDK (Lys-Glu-Asp-Lys) sequence as set forth in SEQ ID NO: 3 . It is preferably a peptide, and includes all modified forms of the amino acid sequence and may be used repeatedly. In addition, in the present invention, the KEDK sequence described in SEQ ID NO: 3 may be used in whole or in part, but preferably includes first and fourth rhisin residues.

The present invention also encompasses functional derivatives of these polypeptides that exhibit high binding affinity for heparin and can enhance cell adhesion. Functional derivatives in the present invention means a variable, analog or chemical derivative of the polypeptide capable of binding to heparin with high binding affinity and cell adhesion activity.

As used herein, a variable refers to a glycosylation pattern in an amino acid sequence or to a molecule modified in a noncovalent or covalent manner, including mutations. If the ability to bind heparin in a way that has high binding affinity and promotes cell adhesion activity is included in the final structure, some of the variants belonging to the invention include amino acid substitutions, deletions or insertions.

As used herein, chemical derivatives refer to polypeptides that can be produced in place of one another by naturally occurring or various chemical modifications, such as alkyl, acyl or amine groups.

The present invention also provides a pharmaceutical composition for cell attachment containing the polypeptide and its derivatives as an active ingredient.

The polypeptide and its derivatives can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical formulations. That is, the peptides and derivatives thereof of the present invention may be administered in various parenteral formulations, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. It is prepared. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the polypeptide and its derivatives may be used in admixture with various carriers that are pharmaceutically acceptable such as physiological saline or organic solvent, and in order to increase the stability or absorption of the peptide, such as glucose, sucrose or dextran. Antioxidants such as carbohydrates, ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.

In the pharmaceutical composition of the present invention, the total effective amount of the polypeptide 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 polypeptide is determined by taking into consideration various factors such as the age and health status of the patient as well as the route of administration and the number of treatments of the drug, the person having ordinary knowledge in this field If so, it will be possible to determine the appropriate effective dosage for the particular use of the peptide as a pharmaceutical composition.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Dynamic Analysis of TNIII Domains

<1-1> Preparation of TNIII domain expression vector

Recombinant fragments were prepared that included only some domains of human tenacin-C FNIII (TNIII) to identify domains essential for interaction with heparin among the tenasin-C type III domains.

First, cDNAs of human tenacin-C (TN) were amplified by conventional PCR methods using adult human cDNA libraries as templates. At this time, the forward TN3F of SEQ ID NO: 4 designed to recognize TNIII domains 3 to 5 by PCR primers; Inverse TN3R of SEQ ID NO: 5 ; Reverse TN4R of SEQ ID NO: 6 ; And reverse TN5R of SEQ ID NO: 7 was used.

PCR was performed with 50 μl containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl ₂ , 100 μg / ml gelatin, 0.2 mM dNTPs, 1.25 unit Taq polymerase (Perkin-Elmer) and 50 pmol primer. It was carried out in the reaction solution. PCR conditions were repeated 30 times of 1 minute at 55 ℃, 1 minute at 72 ℃ and 1 minute at 94 ℃. After cleavage of the amplified cDNA products therefrom by Kpn Ⅰ and Eco RⅠ restriction enzymes were isolated using the PCR purification kit (Qiagen, Chatsworth, CA). The PCR product isolated above was cloned into a pBAD / HisA expression vector (Invitrogen) having 6 His labels at the C-terminus. TNIII3-5, TNIII4-5 and TNIII5 fusion proteins containing poly-His labels were expressed and purified using a Ni ²⁺ affinity column (Invitrogen) according to the manufacturer's instructions under denaturing conditions. Cell lysates and purified fusion proteins were separated by electrophoresis on 12% (w / v) acrylamide gel under reducing conditions and observed by staining with Coomassie Blue. As a result, as shown in Figure 1 , it was confirmed that the recombinant TNIII domains were expressed and homogeneously separated and purified.

<1-2> Heparin-binding activity assay of recombinant TNIII domains

The BIAcore X system based on the surface plasmon resonance (SPR) principle was used to analyze the binding activity of each of the recombinant TNIII domain fragments isolated and purified in Example <1-1> to heparin. . The BIAcore system enables quantitative analysis of intermolecular interactions in real time, from which the binding and dissociation rate constants are calculated immediately.

The chip used was a CM5 sensor chip coated with carboxymethyl-dextran (CM-Dex) for protein fixation. First, the chip surface was stabilized with HBS buffer (containing 10 mM HEPES, pH 7.4, 150 mM NaCl and 0.005% surfactant P20), and then 0.1 M NHS ( N -hydroxysuccnimide) and 0.4 M EDC ( N -ethyl- N 200 μl of a mixture of 9- [3- (dimethylamino) propyl] carbodiimide was flowed to activate the chip surface. In this state, streptavidin was coupled to the CM5 sensor chip using an amine coupling kit (BIAcore Inc.) according to the manufacturer's instructions. Two flow cells per sensor chip for the Biacore X system are used, the streptavidin is fixed in the first flow cell with a 20k resonance unit (RU), and the bovine serum albumin (BSA) is the background. A negative control for fertilization was immobilized in the second flow cell with 950 resonance units. Biotinylated heparin (BiHep, Sigma) was fixed to the streptavidin-binding CM5 sensor chip prepared as described above according to the manufacturer's instructions. Thereafter, 20 μl of 1.0 M ethanolamine was poured to replace the chip surface remaining in the activated state without adhering to BiHep. The binding affinity measurement of the recombinant TNIII domain fragments to heparin was determined by immobilizing BiHep on the CM5 sensor chip, followed by binding buffer (0.01 M HEPES, 0.15 mM NaCl, 3 mM EDTA, 0.005% polysorbate 20, pH). 7.4) was continually flowed to equilibrate and determined by flowing binding buffer in which the recombinant fragments were dissolved at 20, 40 and 80 mM concentrations. At this time, the peptides bind to each other at different speeds and slopes according to the binding affinity between the recombinant fragment and heparin, and the binding affinity for heparin was measured from each result.

Kinetic rate constants (k _a and k _d ) and equilibrium separation constants (k _D ) were determined using BIAcore kinetic evaluation software (BIAcore, Uppsala, Sweden). To determine the binding kinetics of each of the recombinant TNIII domain fragments, a series of samples of different concentrations were injected at a rate of 40 μl / min during the 5 minute association phase. The linker was run for at least 10 minutes. Flow cells were regenerated by injecting 1.0 mM NaCl for 3 minutes, leaving heparin intact while removing recombinant TNIII domain fragments. Kinetic analysis of BIAcore sensor sensors indicating the degree of interaction between various recombinant TNIII domain fragments and immobilized heparin was determined according to Equation 1 below.

dR / dt = k _a CR _max- ( k _a C + k _d ) R _t

Where dR / dt is the rate of change of the SRP signal (RU) due to the interaction between the fixed heparin and the sample at time t seconds, k _a and k _d represent the binding and dissociation rate constants respectively, and C is the Concentration, R _max represents the maximum analyte binding capacity of the sample for heparin immobilized at RU. Both binding and dissociation rate constants were determined from size analysis of the sensograms using the BIA evaluation software (version 3.0). All binding curves were corrected by subtracting the value of the flow cytometry for background and bulk refractive index donations.

TNIII domain 5 in tenasin-C has already been identified as the major heparin-binding site (Weber P, et al., J. Biol. Chem . 270: 4619-4623, 1995), and the SPR-based BIAcore of the present invention. System analysis also confirmed that TNIII5 acts as a binding site for interaction with heparin. Under physiological conditions, k _d for recombinant TNIII3-5 and TNIII4-5 for heparin immobilized on a streptavidin-binding CM5 sensor chip was measured at 30 nM and 40 nM, respectively, indicating high affinity for heparin ( FIG. 2). ). As shown in Table 1 , both TNIII3-5 and TNIII4-5 have fast binding rates to heparin on the chip surface ( k _a TNIII3-5, 8.64x10 ³ xM ^-1 ; k _a TNIII4-5, 4.46x10 ³ xM ^-1 s ^-1 ), while the slow dissociation rate ( k _d TNIII3-5, 0.29x10 ³ xs ^-1 ; k _d TNIII4-5, 0.2x10 ³ xs ^-1 ) was found to be very stable.

sample k _d (s ^-1 x10 ^-3 ) k _a (M ^-1 s ^-1 x10 ³ ) K _D (μM) TNⅢ3-5 0.29 8.64 0.03 TNⅢ4-5 0.2 4.46 0.04 TNⅢ5 0.82 0.19 0.25 TNⅢ4 NB NB NB TNⅢ3 NB NB NB k _d : dissociation rate constant, k _a : coupling rate constant, K _D : equilibrium constant, NB: not bind

Interestingly, TNIII5 exhibited 160- and 106-fold reduced affinity ( K _d = 4.25 μM) compared to TNIII3-5 and TNIII4-5, respectively. However, both TNIII3 and TNIII4 could not quantify binding and dissociation kinetics because heparin binds very poorly to the immobilized chip surface ( Table 1 ). The binding affinity for heparin in recombinant fragments containing TNIII5 was always much lower than the binding affinity of presynthetic fragments containing TNIII3-5 or TNIII4-5, indicating that the TNIII4 domain was optimal for tenacin-C and heparin. It is a significant contribution to the bond.

Example 2 Sequence Alignment and Homology Analysis

NMR and crystal structure analysis reported that the RGD sequence of fibronectin type III domain 10 (FIII10) was present in a loop extending from the domain surface (Sharma A, et al., EMBO J. 18: 1468-1479 , 1999; Grant RP, et al., J. Biol. Chem. 272: 6159-6166, 1997). Thus, sequence alignment and homology analysis based on the structures of FIII10 and TNIII3, TNIII4 and TNIII5 were performed and the results are shown in FIG. 3A . The amino acid residues indicated in green in FIG. 3A represent residues that are conserved in all of FIII10, TNIII3, TNIII4 and TNIII5, and yellow residues represent residues that are conserved in at least three of these domains.

As shown in FIG. 3A , the TNIII3-5 domains shared the same overall structure, with functional segments occupying the surface loop. The loop region is located on the surface of the protein and mainly contains hydrophilic amino acids. The KGRHKSKPARVK sequence in TNIII5 (corresponding to 1057-1068 in the amino acid sequence of SEQ ID NO: 2 ) has already been reported to be involved in heparin-binding activity (Weber P, et al., J. Biol. Chem . 270: 4619- 4623, 1995). These six positively charged residues provided from the loop between the F and G strands, K or R, protrude into the solvent. Therefore, the present inventors focused on the loop region in TNIII4 to identify a new binding site for heparin in tenasin-C, and from there, the KEDK sequence in TNIII4 (the amino acid sequence of SEQ ID NO: 2) has a significant surface positive charge therefrom. 969-972) can act as a potential heparin-binding site. Moreover, KEDK sequences on the TNIII4 domain showed high sequence homology between different species, including chicks, mice, pigs and humans ( FIG. 3B ).

Example 3 Identification of Heparin-Binding Sites in TNIII4

<3-1> Preparation of Substitution Mutants by Position-Specific Mutation

In order to further investigate the binding activity of the KEDK sequence to heparin in TNIII4 confirmed by the sequence alignment and homology analysis of Example 2, the 969th and 972th lysines (Lys) in the amino acid sequence of SEQ ID NO. Substitution mutants of the recombinant TNIII3-5 protein comprising a double mutation substituted with) were prepared.

Specifically, site-directed mutagenesis using PCR was performed to prepare mutants in which Lys ⁹⁶⁹ and Lys ⁹⁷² were replaced with Gln in the KEDK sequence of the recombinant TNIII4-5 protein. Position-directed mutations were performed using the Quickchange kit (Quickchange kit, Stratagene, La Jolla, Calif.) According to the manufacturer's instructions. PCR was performed using recombinant TNIII3-5 prepared in Example 1 as a template and using SEQ ID NO: 8 and SEQ ID NO: 9 as primer pairs. The primer pair of SEQ ID NO: 8 and SEQ ID NO: 9 is designed to introduce a substitution with Gln at the Lys ⁹⁶⁹ and Lys ⁹⁷² position in recombinant TNIII3-5. PCR was performed using 2.5 units of Pfu turbo DNA polymerase (Stratagen, Ca. No. 200518) as a polymerase and preliminarily denatured at 95 ° C. for 5 minutes, followed by 30 seconds at 95 ° C., 60 seconds at 55 ° C., and 72 ° C. The reaction of 2 minutes was carried out 30 times.

In the same manner as described above, mutants were prepared in which the 1057th lysine was replaced with glutamine and the 1059th arginine (Arg) was substituted with glutamine in the KGR sequence of the TNIII5 protein of SEQ ID NO: 2 . A primer pair of SEQ ID NO: 10 and SEQ ID NO: 11 designed to introduce a substitution of Gln at the Lys ¹⁰⁵⁷ position in recombinant TNIII5 as a primer pair of PCR for the position-directed mutation; And primer pairs of SEQ ID NO: 12 and SEQ ID NO: 13 designed to introduce a substitution with Gln at the Arg ¹⁰⁵⁹ position.

Double mutants K969Q / K972Q (KEDK), wherein Lys ⁹⁶⁹ and Lys ⁹⁷² were each substituted with Gln in the KEDK sequence of the recombinant TNIII4-5 protein; And mutants K1057Q (KGR) with Lys ¹⁰⁵⁷ substituted with Gln and mutant R1059Q (KGR) with Arg ¹⁰⁵⁹ substituted with Gln in the RGD sequence of the recombinant TNIII5 protein.

The amplified PCR products were recovered, digested with restriction enzymes KpnI and EcoRI, and separated using a PCR purification kit (Qiagen, Chatsworth, CA). PCR products thus isolated were cloned into pBAD / HisA expression vectors (Invitrogen) with 6 His labels at the C-terminus, respectively. Expressing K969Q / K972Q (KEDK), K1057Q (KGR) and R1059Q (KGR) substitutional mutant fusion proteins containing poly-His label and purifying according to manufacturer's instructions under denaturation conditions using Ni ²⁺ affinity column (Invitrogen) It was.

The nucleotide sequence of the substitution mutant fusion proteins purified as described above was confirmed that the desired mutation was introduced into each substitution mutant.

<3-2> Heparin-Binding Activity Assay

Heparin-binding activity of the TNIII4-5 mutants prepared above was measured using the BIAcore system in the same manner as in Example <1-2>, and the results are shown in Table 2 below. At this time, heparin-binding activity of the TNIII4-5 mutants was expressed as a relative value compared to the TNIII4-5 wild-type protein.

sample k _d (s ^-1 x10 ^-3 ) k _a (M ^-1 s ^-1 x10 ³ ) K _D (μM) K969Q / K972Q (KEDK) 0.01 1.38 9.2 K1057Q (KGR) NB NB B R1059Q (KGR) NB NB B

As shown in Table 2 above, k _d for the recombinant TNIII4-5 K969QA / K972Q double mutant showed a binding affinity ( k _d = 29.2 μM) that was about 70-fold reduced for heparin compared to wild-type TNIII4-5. , TNIII4-5 K1057Q and TNIII4-5 R1059Q with mutations in the KGR sequence in TNIII5 completely inhibited binding to heparin. From the above results, it was confirmed that the TNIII5 domain is the primary heparin-binding site, and that Arg ¹⁰⁵⁹ and Lys ¹⁰⁵⁷ residues of the KGR sequence in the domain play a critical role in the interaction with heparin. In addition, the fact that mutations in the KEDK sequence in the TNIII4 domain significantly reduced binding affinity for heparin indicates that KEDK residues act as secondary heparin-binding sites for optimal binding of tenacin-C to heparin. These results suggest that KEDK residues in the loop region between the F and G strands induce optimal binding activity between tenasin-C and heparin, and heparin-binding sites in tenasin-C are found in both the TNIII4 and TNIII5 domains. It is implied to extend over.

Example 4 Adhesion Activity of TNIII4 Heparin-Binding Domain

In order to investigate the biological significance of the KEDK sequence of TNIII4 identified above as the heparin-binding domain, the present inventors investigated whether heparin-binding activity of tenasin-C is associated with cell adhesion.

Human gingival fibroblasts (HGF) were cultured in minimal essential medium (α-MEN) containing 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin (Invitrogen). HGFs were again cultured serum-free in the same medium for 24 hours and then harvested by 0.02% trypsin and 1 mM EDTA treatment. Harvested HGFs were prepared by resuspending in α-MEN followed by three washes with α-MEN containing 100 μg / ml soybean trypsin inhibitor and 1% BSA. In a 24-well plate, the recombinant proteins TNIII4-5 and TNIII5 prepared in Example <1-1> and the double mutant K969Q / K972Q (KEDK) prepared in Example <3-2> , respectively, were added at a concentration of 1 μM. After incubation overnight at 4 ℃ coated. PBS containing 1% (w / v) BSA was added to the well plate and blocked for 30 minutes at room temperature, followed by washing with BSA-free PBS. The HGFs prepared above were added to each well of the recombinant protein coated plate at a concentration of 5 × 10 ⁴ cells / well. At this time, the cells were pretreated with heparinase I or chondroitinase ABC at a concentration of 2 units / ml for 1 hour at 37 ° C., or heparin at 5 μg / ml before the cells were added to the well plate. Co-incubated at 37 ° C. for 1 h. Suspensions of control cells were maintained in BSA-coated plates for a defined time. After incubation at 37 ° C. for 60 minutes, unattached cells were removed by washing twice with PBS. The attached cells were fixed with 3% paraformaldehyde (Sigma) and stained with 2% (v / v) ethanol aqueous solution containing 0.25% crystal violet. The well plate was sufficiently washed with distilled water and dried, and then the absorbance was measured at 570 nm. At this time, non-specific cell adhesion was determined in wells coated with 1% BSA as a negative control.

As a result, as shown in Figure 4 , TNIII4-5 showed significantly higher cell adhesion activity than TNIII5, while TNIII4-5 K969Q / K972Q double mutant significantly reduced cell adhesion activity. In addition, the adhesion of HGF to TNIII4-5 was completely inhibited in the presence of 5 μg / ml of heparin, indicating that TNIII4-5 was bound to cell surface HSPGs when water-soluble heparin was previously bound to heparin-binding sites in TNIII4-5. It suggests that binding activity may be inhibited. To investigate this possibility, cell surface was first treated with Heparinase I, an enzyme that acts on highly sulfurized HSPGs (Feitsma K, et al., J. Biol. Chem . 275: 9396-9402, 2000). HSPGs were removed. As a result, heparinase I treatment of HGF cells reduced TNIII4-5's adhesion activity to HGF cells by 75%. In addition, the results of HGF chondroitinase ABC treatment showed that the cell adhesion activity of TNIII4-5 was mediated not by chondroitin sulfate but by heparan sulfate on the cell surface ( FIG. 4 ). These results demonstrate that TNIII4 contains a binding site of HSPGs and that co-mechanisms increase the adhesion activity of TNIII5 to HSPGs.

As described above, the polypeptide including the tenasin-C domain 4 derived KEDK sequence of the present invention has a high affinity for heparin and can act as a heparin-binding site and enhances cell adhesion because it enhances cell adhesion activity. It can be usefully used for peptides.

<110> Seoul National University Industry Foundation <120> HUMAN TENACIN-C DERIVED POLYPEPTIDE AND A DERIVATIVE THEREOF AS A          NOVEL HEPARIN-BINDING SITE <160> 13 <170> KopatentIn 1.71 <210> 1 <211> 7560 <212> DNA <213> Homo sapiens tenascin C <220> <221> CDS (222) (314) .. (6916) <220> <221> misc_feature (222) (740) .. (2095) <223> Fibrillins and related proteins containing Ca2 + -binding EGF-like          domains <220> <221> misc_feature (222) (785) .. (1438) <223> Integrin beta subunit (N-terminal portion of extracellular          region) <220> <221> misc_feature (222) (968) .. (2665) <223> Teneurin-1 and related extracellular matrix proteins, contain          EGF-like repeats <220> <221> misc_feature (222) (2186) .. (3088) <223> Neural cell adhesion molecule L1 <220> <221> misc_feature (222) (2189) .. (2398) <223> Fibronectin type III domain <220> <221> misc_feature (2456) .. (2692) <223> Fibronectin type III domain <220> <221> misc_feature (222) (2729). (2965) <223> Fibronectin type III domain <220> <221> misc_feature (222) (2873) .. (3211) <223> Teneurin-1 and related extracellular matrix proteins, contain          EGF-like repeats <220> <221> misc_feature (222) (2999) .. (3241) <223> Fibronectin type III domain <220> <221> misc_feature (222) (3275) .. (3505) <223> Fibronectin type III domain <220> <221> misc_feature (222) (3545) .. (3775) <223> Fibronectin type III domain <220> <221> misc_feature (222) (3818) .. (4036) <223> Fibronectin type III domain <220> <221> misc_feature (222) (4091) .. (4294) <223> Fibronectin type III domain <220> <221> misc_feature (222) (4097) .. (5740) <223> Receptor mediating netrin-dependent axon guidance <220> <221> misc_feature (222) (4364) .. (4594) <223> Fibronectin type III domain <220> <221> misc_feature <222> (4637) .. (4837) <223> Fibronectin type III domain <220> <221> misc_feature (222) (4910) .. (5143) <223> Fibronectin type III domain <220> <221> misc_feature (222) (5180) .. (5413) <223> Fibronectin type III domain <220> <221> misc_feature (222) (5450) .. (5683) <223> Fibronectin type III domain <220> <221> misc_feature (222) (5486) .. (6196) <223> Receptor mediating netrin-dependent axon guidance <220> <221> misc_feature <222> (5717) .. (5929) <223> Fibronectin type III domain <220> <221> misc_feature (222) (5909). (6892) Ficolin and related extracellular proteins <400> 1 accggccaca gcctgcctac tgtcacccgc ctctcccgcg cgcagataca cgcccccgcc 60 tccgtgggca caaaggcagc gctgctgggg aactcggggg aacgcgcacg tgggaaccgc 120 cgcagctcca cactccaggt acttcttcca aggacctagg tctctcgccc atcggaaaga 180 aaataattct ttcaagaaga tcagggacaa ctgatttgaa gtctactctg tgcttctaaa 240 tccccaattc tgctgaaagt gaatccctag agccctagag ccccagcagc acccagccaa 300 acccacctcc acc atg ggg gcc atg act cag ctg ttg gca ggt gtc 346                       Met Gly Ala Met Thr Gln Leu Leu Ala Gly Val                         1 5 10 ttt ctt gct ttc ctt gcc ctc gct acc gaa ggt ggg gtc ctc aag aaa 394 Phe Leu Ala Phe Leu Ala Leu Ala Thr Glu Gly Gly Val Leu Lys Lys              15 20 25 gtc atc cgg cac aag cga cag agt ggg gtg aac gcc acc ctg cca gaa 442 Val Ile Arg His Lys Arg Gln Ser Gly Val Asn Ala Thr Leu Pro Glu          30 35 40 gag aac cag cca gtg gtg ttt aac cac gtt tac aac atc aag ctg cca 490 Glu Asn Gln Pro Val Val Phe Asn His Val Tyr Asn Ile Lys Leu Pro      45 50 55 gtg gga tcc cag tgt tcg gtg gat ctg gag tca gcc agt ggg gag aaa 538 Val Gly Ser Gln Cys Ser Val Asp Leu Glu Ser Ala Ser Gly Glu Lys  60 65 70 75 gac ctg gca ccg cct tca gag ccc agc gaa agc ttt cag gag cac aca 586 Asp Leu Ala Pro Pro Ser Glu Pro Ser Glu Ser Phe Gln Glu His Thr                  80 85 90 gta gat ggg gaa aac cag att gtc ttc aca cat cgc atc aac atc ccc 634 Val Asp Gly Glu Asn Gln Ile Val Phe Thr His Arg Ile Asn Ile Pro              95 100 105 cgc cgg gcc tgt ggc tgt gcc gca gcc cct gat gtt aag gag ctg ctg 682 Arg Arg Ala Cys Gly Cys Ala Ala Ala Pro Asp Val Lys Glu Leu Leu         110 115 120 agc aga ctg gag gag ctg gag aac ctg gtg tct tcc ctg agg gag caa 730 Ser Arg Leu Glu Glu Leu Glu Asn Leu Val Ser Ser Leu Arg Glu Gln     125 130 135 tgt act gca gga gca ggc tgc tgt ctc cag cct gcc aca ggc cgc ttg 778 Cys Thr Ala Gly Ala Gly Cys Cys Leu Gln Pro Ala Thr Gly Arg Leu 140 145 150 155 gac acc agg ccc ttc tgt agc ggt cgg ggc aac ttc agc act gaa gga 826 Asp Thr Arg Pro Phe Cys Ser Gly Arg Gly Asn Phe Ser Thr Glu Gly                 160 165 170 tgt ggc tgt gtc tgc gaa cct ggc tgg aaa ggc ccc aac tgc tct gag 874 Cys Gly Cys Val Cys Glu Pro Gly Trp Lys Gly Pro Asn Cys Ser Glu             175 180 185 ccc gaa tgt cca ggc aac tgt cac ctt cga ggc cgg tgc att gat ggg 922 Pro Glu Cys Pro Gly Asn Cys His Leu Arg Gly Arg Cys Ile Asp Gly         190 195 200 cag tgc atc tgt gac gac ggc ttc acg ggc gag gac tgc agc cag ctg 970 Gln Cys Ile Cys Asp Asp Gly Phe Thr Gly Glu Asp Cys Ser Gln Leu     205 210 215 gct tgc ccc agc gac tgc aat gac cag ggc aag tgc gtg aat gga gtc 1018 Ala Cys Pro Ser Asp Cys Asn Asp Gln Gly Lys Cys Val Asn Gly Val 220 225 230 235 tgc atc tgt ttc gaa ggc tac gcc ggg gct gac tgc agc cgt gaa atc 1066 Cys Ile Cys Phe Glu Gly Tyr Ala Gly Ala Asp Cys Ser Arg Glu Ile                 240 245 250 tgc cca gtg ccc tgc agt gag gag cac ggc aca tgt gta gat ggc ttg 1114 Cys Pro Val Pro Cys Ser Glu Glu His Gly Thr Cys Val Asp Gly Leu             255 260 265 tgt gtg tgc cac gat ggc ttt gca ggc gat gac tgc aac aag cct ctg 1162 Cys Val Cys His Asp Gly Phe Ala Gly Asp Asp Cys Asn Lys Pro Leu         270 275 280 tgt ctc aac aat tgc tac aac cgt gga cga tgc gtg gag aat gag tgc 1210 Cys Leu Asn Asn Cys Tyr Asn Arg Gly Arg Cys Val Glu Asn Glu Cys     285 290 295 gtg tgt gat gag ggt ttc acg ggc gaa gac tgc agt gag ctc atc tgc 1258 Val Cys Asp Glu Gly Phe Thr Gly Glu Asp Cys Ser Glu Leu Ile Cys 300 305 310 315 ccc aat gac tgc ttc gac cgg ggc cgc tgc atc aat ggc acc tgc tac 1306 Pro Asn Asp Cys Phe Asp Arg Gly Arg Cys Ile Asn Gly Thr Cys Tyr                 320 325 330 tgc gaa gaa ggc ttc aca ggt gaa gac tgc ggg aaa ccc acc tgc cca 1354 Cys Glu Glu Gly Phe Thr Gly Glu Asp Cys Gly Lys Pro Thr Cys Pro             335 340 345 cat gcc tgc cac acc cag ggc cgg tgt gag gag ggg cag tgt gta tgt 1402 His Ala Cys His Thr Gln Gly Arg Cys Glu Glu Gly Gln Cys Val Cys         350 355 360 gat gag ggc ttt gcc ggt ttg gac tgc agc gag aag agg tgt cct gct 1450 Asp Glu Gly Phe Ala Gly Leu Asp Cys Ser Glu Lys Arg Cys Pro Ala     365 370 375 gac tgt cac aat cgt ggc cgc tgt gta gac ggg cgg tgt gag tgt gat 1498 Asp Cys His Asn Arg Gly Arg Cys Val Asp Gly Arg Cys Glu Cys Asp 380 385 390 395 gat ggt ttc act gga gct gac tgt ggg gag ctc aag tgt ccc aat ggc 1546 Asp Gly Phe Thr Gly Ala Asp Cys Gly Glu Leu Lys Cys Pro Asn Gly                 400 405 410 tgc agt ggc cat ggc cgc tgt gtc aat ggg cag tgt gtg tgt gat gag 1594 Cys Ser Gly His Gly Arg Cys Val Asn Gly Gln Cys Val Cys Asp Glu             415 420 425 ggc tat act ggg gag gac tgc agc cag cta cgg tgc ccc aat gac tgt 1642 Gly Tyr Thr Gly Glu Asp Cys Ser Gln Leu Arg Cys Pro Asn Asp Cys         430 435 440 cac agt cgg ggc cgc tgt gtc gag ggc aaa tgt gta tgt gag caa ggc 1690 His Ser Arg Gly Arg Cys Val Glu Gly Lys Cys Val Cys Glu Gln Gly     445 450 455 ttc aag ggc tat gac tgc agt gac atg agc tgc cct aat gac tgt cac 1738 Phe Lys Gly Tyr Asp Cys Ser Asp Met Ser Cys Pro Asn Asp Cys His 460 465 470 475 cag cac ggc cgc tgt gtg aat ggc atg tgt gtt tgt gat gac ggc tac 1786 Gln His Gly Arg Cys Val Asn Gly Met Cys Val Cys Asp Asp Gly Tyr                 480 485 490 aca ggg gaa gac tgc cgg gat cgc caa tgc ccc agg gac tgc agc aac 1834 Thr Gly Glu Asp Cys Arg Asp Arg Gln Cys Pro Arg Asp Cys Ser Asn             495 500 505 agg ggc ctc tgt gtg gac gga cag tgc gtc tgt gag gac ggc ttc acc 1882 Arg Gly Leu Cys Val Asp Gly Gln Cys Val Cys Glu Asp Gly Phe Thr         510 515 520 ggc cct gac tgt gca gaa ctc tcc tgt cca aat gac tgc cat ggc cag 1930 Gly Pro Asp Cys Ala Glu Leu Ser Cys Pro Asn Asp Cys His Gly Gln     525 530 535 ggt cgc tgt gtg aat ggg cag tgc gtg tgc cat gaa gga ttt atg ggc 1978 Gly Arg Cys Val Asn Gly Gln Cys Val Cys His Glu Gly Phe Met Gly 540 545 550 555 aaa gac tgc aag gag caa aga tgt ccc agt gac tgt cat ggc cag ggc 2026 Lys Asp Cys Lys Glu Gln Arg Cys Pro Ser Asp Cys His Gly Gln Gly                 560 565 570 cgc tgc gtg gac ggc cag tgc atc tgc cac gag ggc ttc aca ggc ctg 2074 Arg Cys Val Asp Gly Gln Cys Ile Cys His Glu Gly Phe Thr Gly Leu             575 580 585 gac tgt ggc cag cac tcc tgc ccc agt gac tgc aac aac tta gga caa 2122 Asp Cys Gly Gln His Ser Cys Pro Ser Asp Cys Asn Asn Leu Gly Gln         590 595 600 tgc gtc tcg ggc cgc tgc atc tgc aac gag ggc tac agc gga gaa gac 2170 Cys Val Ser Gly Arg Cys Ile Cys Asn Glu Gly Tyr Ser Gly Glu Asp     605 610 615 tgc tca gag gtg tct cct ccc aaa gac ctc gtt gtg aca gaa gtg acg 2218 Cys Ser Glu Val Ser Pro Pro Lys Asp Leu Val Val Thr Glu Val Thr 620 625 630 635 gaa gag acg gtc aac ctg gcc tgg gac aat gag atg cgg gtc aca gag 2266 Glu Glu Thr Val Asn Leu Ala Trp Asp Asn Glu Met Arg Val Thr Glu                 640 645 650 tac ctt gtc gtg tac acg ccc acc cac gag ggt ggt ctg gaa atg cag 2314 Tyr Leu Val Val Tyr Thr Pro Thr His Glu Gly Gly Leu Glu Met Gln             655 660 665 ttc cgt gtg cct ggg gac cag acg tcc acc atc atc cag gag ctg gag 2362 Phe Arg Val Pro Gly Asp Gln Thr Ser Thr Ile Ile Gln Glu Leu Glu         670 675 680 cct ggt gtg gag tac ttt atc cgt gta ttt gcc atc ctg gag aac aag 2410 Pro Gly Val Glu Tyr Phe Ile Arg Val Phe Ala Ile Leu Glu Asn Lys     685 690 695 aag agc att cct gtc agc gcc agg gtg gcc acg tac tta cct gca cct 2458 Lys Ser Ile Pro Val Ser Ala Arg Val Ala Thr Tyr Leu Pro Ala Pro 700 705 710 715 gaa ggc ctg aaa ttc aag tcc atc aag gag aca tct gtg gaa gtg gag 2506 Glu Gly Leu Lys Phe Lys Ser Ile Lys Glu Thr Ser Val Glu Val Glu                 720 725 730 tgg gat cct cta gac att gct ttt gaa acc tgg gag atc atc ttc cgg 2554 Trp Asp Pro Leu Asp Ile Ala Phe Glu Thr Trp Glu Ile Ile Phe Arg             735 740 745 aat atg aat aaa gaa gat gag gga gag atc acc aaa agc ctg agg agg 2602 Asn Met Asn Lys Glu Asp Glu Gly Glu Ile Thr Lys Ser Leu Arg Arg         750 755 760 cca gag acc tct tac cgg caa act ggt cta gct cct ggg caa gag tat 2650 Pro Glu Thr Ser Tyr Arg Gln Thr Gly Leu Ala Pro Gly Gln Glu Tyr     765 770 775 gag ata tct ctg cac ata gtg aaa aac aat acc cgg ggc cct ggc ctg 2698 Glu Ile Ser Leu His Ile Val Lys Asn Asn Thr Arg Gly Pro Gly Leu 780 785 790 795 aag agg gtg acc acc aca cgc ttg gat gcc ccc agc cag atc gag gtg 2746 Lys Arg Val Thr Thr Thr Thr Arg Leu Asp Ala Pro Ser Gln Ile Glu Val                 800 805 810 aaa gat gtc aca gac acc act gcc ttg atc acc tgg ttc aag ccc ctg 2794 Lys Asp Val Thr Asp Thr Thr Ala Leu Ile Thr Trp Phe Lys Pro Leu             815 820 825 gct gag atc gat ggc att gag ctg acc tac ggc atc aaa gac gtg cca 2842 Ala Glu Ile Asp Gly Ile Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro         830 835 840 gga gac cgt acc acc atc gat ctc aca gag gac gag aac cag tac tcc 2890 Gly Asp Arg Thr Thr Ile Asp Leu Thr Glu Asp Glu Asn Gln Tyr Ser     845 850 855 atc ggg aac ctg aag cct gac act gag tac gag gtg tcc ctc atc tcc 2938 Ile Gly Asn Leu Lys Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Ser 860 865 870 875 cgc aga ggt gac atg tca agc aac cca gcc aaa gag acc ttc aca aca 2986 Arg Arg Gly Asp Met Ser Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr                 880 885 890 ggc ctc gat gct ccc agg aat ctt cga cgt gtt tcc cag aca gat aac 3034 Gly Leu Asp Ala Pro Arg Asn Leu Arg Arg Val Ser Gln Thr Asp Asn             895 900 905 agc atc acc ctg gaa tgg agg aat ggc aag gca gct att gac agt tac 3082 Ser Ile Thr Leu Glu Trp Arg Asn Gly Lys Ala Ala Ile Asp Ser Tyr         910 915 920 aga att aag tat gcc ccc atc tct gga ggg gac cac gct gag gtt gat 3130 Arg Ile Lys Tyr Ala Pro Ile Ser Gly Gly Asp His Ala Glu Val Asp     925 930 935 gtt cca aag agc caa caa gcc aca acc aaa acc aca ctc aca ggt ctg 3178 Val Pro Lys Ser Gln Gln Ala Thr Thr Lys Thr Thr Leu Thr Gly Leu 940 945 950 955 agg ccg gga act gaa tat ggg att gga gtt tct gct gtg aag gaa gac 3226 Arg Pro Gly Thr Glu Tyr Gly Ile Gly Val Ser Ala Val Lys Glu Asp                 960 965 970 aag gag agc aat cca gcg acc atc aac gca gcc aca gag ttg gac acg 3274 Lys Glu Ser Asn Pro Ala Thr Ile Asn Ala Ala Thr Glu Leu Asp Thr             975 980 985 ccc aag gac ctt cag gtt tct gaa act gca gag acc agc ctg acc ctg 3322 Pro Lys Asp Leu Gln Val Ser Glu Thr Ala Glu Thr Ser Leu Thr Leu         990 995 1000 ctc tgg aag aca ccg ttg gcc aaa ttt gac cgc tac cgc ctc aat tac 3370 Leu Trp Lys Thr Pro Leu Ala Lys Phe Asp Arg Tyr Arg Leu Asn Tyr    1005 1010 1015 agt ctc ccc aca ggc cag tgg gtg gga gtg cag ctt cca aga aac acc 3418 Ser Leu Pro Thr Gly Gln Trp Val Gly Val Gln Leu Pro Arg Asn Thr 1020 1025 1030 1035 act tcc tat gtc ctg aga ggc ctg gaa cca gga cag gag tac aat gtc 3466 Thr Ser Tyr Val Leu Arg Gly Leu Glu Pro Gly Gln Glu Tyr Asn Val                1040 1045 1050 ctc ctg aca gcc gag aaa ggc aga cac aag agc aag ccc gca cgt gtg 3514 Leu Leu Thr Ala Glu Lys Gly Arg His Lys Ser Lys Pro Ala Arg Val            1055 1060 1065 aag gca tcc act gaa caa gcc cct gag ctg gaa aac ctc acc gtg act 3562 Lys Ala Ser Thr Glu Gln Ala Pro Glu Leu Glu Asn Leu Thr Val Thr        1070 1075 1080 gag gtt ggc tgg gat ggc ctc aga ctc aac tgg acc gcg gct gac cag 3610 Glu Val Gly Trp Asp Gly Leu Arg Leu Asn Trp Thr Ala Ala Asp Gln    1085 1090 1095 gcc tat gag cac ttt atc att cag gtg cag gag gcc aac aag gtg gag 3658 Ala Tyr Glu His Phe Ile Gle Val Gln Glu Ala Asn Lys Val Glu 1100 1105 1110 1115 gca gct cgg aac ctc acc gtg cct ggc agc ctt cgg gct gtg gac ata 3706 Ala Ala Arg Asn Leu Thr Val Pro Gly Ser Leu Arg Ala Val Asp Ile                1120 1125 1130 ccg ggc ctc aag gct gct acg cct tat aca gtc tcc atc tat ggg gtg 3754 Pro Gly Leu Lys Ala Ala Thr Pro Tyr Thr Val Ser Ile Tyr Gly Val            1135 1140 1145 atc cag ggc tat aga aca cca gtg ctc tct gct gag gcc tcc aca ggg 3802 Ile Gln Gly Tyr Arg Thr Pro Val Leu Ser Ala Glu Ala Ser Thr Gly        1150 1155 1160 gaa act ccc aat ttg gga gag gtc gtg gtg gcc gag gtg ggc tgg gat 3850 Glu Thr Pro Asn Leu Gly Glu Val Val Val Ala Glu Val Gly Trp Asp    1165 1170 1175 gcc ctc aaa ctc aac tgg act gct cca gaa ggg gcc tat gag tac ttt 3898 Ala Leu Lys Leu Asn Trp Thr Ala Pro Glu Gly Ala Tyr Glu Tyr Phe 1180 1185 1190 1195 ttc att cag gtg cag gag gct gac aca gta gag gca gcc cag aac ctc 3946 Phe Ile Gln Val Gln Glu Ala Asp Thr Val Glu Ala Ala Gln Asn Leu                1200 1205 1210 acc gtc cca gga gga ctg agg tcc aca gac ctg cct ggg ctc aaa gca 3994 Thr Val Pro Gly Gly Leu Arg Ser Thr Asp Leu Pro Gly Leu Lys Ala            1215 1220 1225 gcc act cat tat acc atc acc atc cgc ggg gtc act cag gac ttc agc 4042 Ala Thr His Tyr Thr Ile Thr Ile Arg Gly Val Thr Gln Asp Phe Ser        1230 1235 1240 aca acc cct ctc tct gtt gaa gtc ttg aca gag gag gtt cca gat atg 4090 Thr Thr Pro Leu Ser Val Glu Val Leu Thr Glu Glu Val Pro Asp Met    1245 1250 1255 gga aac ctc aca gtg acc gag gtt agc tgg gat gct ctc aga ctg aac 4 138 Gly Asn Leu Thr Val Thr Glu Val Ser Trp Asp Ala Leu Arg Leu Asn 1260 1265 1270 1275 tgg acc acg cca gat gga acc tat gac cag ttt act att cag gtc cag 4186 Trp Thr Thr Pro Asp Gly Thr Tyr Asp Gln Phe Thr Ile Gln Val Gln                1280 1285 1290 gag gct gac cag gtg gaa gag gct cac aat ctc acg gtt cct ggc agc 4234 Glu Ala Asp Gln Val Glu Glu Ala His Asn Leu Thr Val Pro Gly Ser            1295 1300 1305 ctg cgt tcc atg gaa atc cca ggc ctc agg gct ggc act cct tac aca 4282 Leu Arg Ser Met Glu Ile Pro Gly Leu Arg Ala Gly Thr Pro Tyr Thr        1310 1315 1320 gtc acc ctg cac ggc gag gtc agg ggc cac agc act cga ccc ctt gct 4330 Val Thr Leu His Gly Glu Val Arg Gly His Ser Thr Arg Pro Leu Ala    1325 1330 1335 gta gag gtc gtc aca gag gat ctc cca cag ctg gga gat tta gcc gtg 4378 Val Glu Val Val Thr Glu Asp Leu Pro Gln Leu Gly Asp Leu Ala Val 1340 1345 1350 1355 tct gag gtt ggc tgg gat ggc ctc aga ctc aac tgg acc gca gct gac 4426 Ser Glu Val Gly Trp Asp Gly Leu Arg Leu Asn Trp Thr Ala Ala Asp                1360 1365 1370 aat gcc tat gag cac ttt gtc att cag gtg cag gag gtc aac aaa gtg 4474 Asn Ala Tyr Glu His Phe Val Ile Gln Val Gln Glu Val Asn Lys Val            1375 1380 1385 gag gca gcc cag aac ctc acg ttg cct ggc agc ctc agg gct gtg gac 4522 Glu Ala Ala Gln Asn Leu Thr Leu Pro Gly Ser Leu Arg Ala Val Asp        1390 1395 1400 atc ccg ggc ctc gag gct gcc acg cct tat aga gtc tcc atc tat ggg 4570 Ile Pro Gly Leu Glu Ala Ala Thr Pro Tyr Arg Val Ser Ile Tyr Gly    1405 1410 1415 gtg atc cgg ggc tat aga aca cca gta ctc tct gct gag gcc tcc aca 4618 Val Ile Arg Gly Tyr Arg Thr Pro Val Leu Ser Ala Glu Ala Ser Thr 1420 1425 1430 1435 gcc aaa gaa cct gaa att gga aac tta aat gtt tct gac ata act ccc 4666 Ala Lys Glu Pro Glu Ile Gly Asn Leu Asn Val Ser Asp Ile Thr Pro                1440 1445 1450 gag agc ttc aat ctc tcc tgg atg gct acc gat ggg atc ttc gag acc 4714 Glu Ser Phe Asn Leu Ser Trp Met Ala Thr Asp Gly Ile Phe Glu Thr            1455 1460 1465 ttt acc att gaa att att gat tcc aat agg ttg ctg gag act gtg gaa 4762 Phe Thr Ile Glu Ile Ile Asp Ser Asn Arg Leu Leu Glu Thr Val Glu        1470 1475 1480 tat aat atc tct ggt gct gaa cga act gcc cat atc tca ggg cta ccc 4810 Tyr Asn Ile Ser Gly Ala Glu Arg Thr Ala His Ile Ser Gly Leu Pro    1485 1490 1495 cct agt act gat ttt att gtc tac ctc tct gga ctt gct ccc agc atc 4858 Pro Ser Thr Asp Phe Ile Val Tyr Leu Ser Gly Leu Ala Pro Ser Ile 1500 1505 1510 1515 cgg acc aaa acc atc agt gcc aca gcc acg aca gag gcc ctg ccc ctt 4906 Arg Thr Lys Thr Ile Ser Ala Thr Ala Thr Thr Glu Ala Leu Pro Leu                1520 1525 1530 ctg gaa aac cta acc att tcc gac att aat ccc tac ggg ttc aca gtt 4954 Leu Glu Asn Leu Thr Ile Ser Asp Ile Asn Pro Tyr Gly Phe Thr Val            1535 1540 1545 tcc tgg atg gca tcg gag aat gcc ttt gac agc ttt cta gta acg gtg 5002 Ser Trp Met Ala Ser Glu Asn Ala Phe Asp Ser Phe Leu Val Thr Val        1550 1555 1560 gtg gat tct ggg aag ctg ctg gac ccc cag gaa ttc aca ctt tca gga 5050 Val Asp Ser Gly Lys Leu Leu Asp Pro Gln Glu Phe Thr Leu Ser Gly    1565 1570 1575 acc cag agg aag ctg gag ctt aga ggc ctc ata act ggc att ggc tat 5098 Thr Gln Arg Lys Leu Glu Leu Arg Gly Leu Ile Thr Gly Ile Gly Tyr 1580 1585 1590 1595 gag gtt atg gtc tct ggc ttc acc caa ggg cat caa acc aag ccc ttg 5146 Glu Val Met Val Ser Gly Phe Thr Gln Gly His Gln Thr Lys Pro Leu                1600 1605 1610 agg gct gag att gtt aca gaa gcc gaa ccg gaa gtt gac aac ctt ctg 5194 Arg Ala Glu Ile Val Thr Glu Ala Glu Pro Glu Val Asp Asn Leu Leu            1615 1620 1625 gtt tca gat gcc acc cca gac ggt ttc cgt ctg tcc tgg aca gct gat 5242 Val Ser Asp Ala Thr Pro Asp Gly Phe Arg Leu Ser Trp Thr Ala Asp        1630 1635 1640 gaa ggg gtc ttc gac aat ttt gtt ctc aaa atc aga gat acc aaa aag 5290 Glu Gly Val Phe Asp Asn Phe Val Leu Lys Ile Arg Asp Thr Lys Lys    1645 1650 1655 cag tct gag cca ctg gaa ata acc cta ctt gcc ccc gaa cgt acc agg 5338 Gln Ser Glu Pro Leu Glu Ile Thr Leu Leu Ala Pro Glu Arg Thr Arg 1660 1665 1670 1675 gac tta aca ggt ctc aga gag gct act gaa tac gaa att gaa ctc tat 5386 Asp Leu Thr Gly Leu Arg Glu Ala Thr Glu Tyr Glu Ile Glu Leu Tyr                1680 1685 1690 gga ata agc aaa gga agg cga tcc cag aca gtc agt gct ata gca aca 5434 Gly Ile Ser Lys Gly Arg Arg Ser Gln Thr Val Ser Ala Ile Ala Thr            1695 1700 1705 aca gcc atg ggc tcc cca aag gaa gtc att ttc tca gac atc act gaa 5482 Thr Ala Met Gly Ser Pro Lys Glu Val Ile Phe Ser Asp Ile Thr Glu        1710 1715 1720 aat tcg gct act gtc agc tgg agg gca ccc acg gcc caa gtg gag agc 5530 Asn Ser Ala Thr Val Ser Trp Arg Ala Pro Thr Ala Gln Val Glu Ser    1725 1730 1735 ttc cgg att acc tat gtg ccc att aca gga ggt aca ccc tcc atg gta 5578 Phe Arg Ile Thr Tyr Val Pro Ile Thr Gly Gly Thr Pro Ser Met Val 1740 1745 1750 1755 act gtg gac gga acc aag act cag acc agg ctg gtg aaa ctc ata cct 5626 Thr Val Asp Gly Thr Lys Thr Gln Thr Arg Leu Val Lys Leu Ile Pro                1760 1765 1770 ggc gtg gag tac ctt gtc agc atc atc gcc atg aag ggc ttt gag gaa 5674 Gly Val Glu Tyr Leu Val Ser Ile Ila Ala Met Lys Gly Phe Glu Glu            1775 1780 1785 agt gaa cct gtc tca ggg tca ttc acc aca gct ctg gat ggc cca tct 5722 Ser Glu Pro Val Ser Gly Ser Phe Thr Thr Ala Leu Asp Gly Pro Ser        1790 1795 1800 ggc ctg gtg aca gcc aac atc act gac tca gaa gcc ttg gcc agg tgg 5770 Gly Leu Val Thr Ala Asn Ile Thr Asp Ser Glu Ala Leu Ala Arg Trp    1805 1810 1815 cag cca gcc att gcc act gtg gac agt tat gtc atc tcc tac aca ggc 5818 Gln Pro Ala Ile Ala Thr Val Asp Ser Tyr Val Ile Ser Tyr Thr Gly 1820 1825 1830 1835 gag aaa gtg cca gaa att aca cgc acg gtg tcc ggg aac aca gtg gag 5866 Glu Lys Val Pro Glu Ile Thr Arg Thr Val Ser Gly Asn Thr Val Glu                1840 1845 1850 tat gct ctg acc gac ctc gag cct gcc acg gaa tac aca ctg aga atc 5914 Tyr Ala Leu Thr Asp Leu Glu Pro Ala Thr Glu Tyr Thr Leu Arg Ile            1855 1860 1865 ttt gca gag aaa ggg ccc cag aag agc tca acc atc act gcc aag ttc 5962 Phe Ala Glu Lys Gly Pro Gln Lys Ser Ser Thr Ile Thr Ala Lys Phe        1870 1875 1880 aca aca gac ctc gat tct cca aga gac ttg act gct act gag gtt cag 6010 Thr Thr Asp Leu Asp Ser Pro Arg Asp Leu Thr Ala Thr Glu Val Gln    1885 1890 1895 tcg gaa act gcc ctc ctt acc tgg cga ccc ccc cgg gca tca gtc acc 6058 Ser Glu Thr Ala Leu Leu Thr Trp Arg Pro Pro Arg Ala Ser Val Thr 1900 1905 1910 1915 ggt tac ctg ctg gtc tat gaa tca gtg gat ggc aca gtc aag gaa gtc 6106 Gly Tyr Leu Leu Val Tyr Glu Ser Val Asp Gly Thr Val Lys Glu Val                1920 1925 1930 att gtg ggt cca gat acc acc tcc tac agc ctg gca gac ctg agc cca 6154 Ile Val Gly Pro Asp Thr Thr Ser Tyr Ser Leu Ala Asp Leu Ser Pro            1935 1940 1945 tcc acc cac tac aca gcc aag atc cag gca ctc aat ggg ccc ctg agg 6202 Ser Thr His Tyr Thr Ala Lys Ile Gln Ala Leu Asn Gly Pro Leu Arg        1950 1955 1960 agc aat atg atc cag acc atc ttc acc aca att gga ctc ctg tac ccc 6250 Ser Asn Met Ile Gln Thr Ile Phe Thr Thr Ile Gly Leu Leu Tyr Pro    1965 1970 1975 ttc ccc aag gac tgc tcc caa gca atg ctg aat gga gac acg acc tct 6298 Phe Pro Lys Asp Cys Ser Gln Ala Met Leu Asn Gly Asp Thr Thr Ser 1980 1985 1990 1995 ggc ctc tac acc att tat ctg aat ggt gat aag gct cag gcg ctg gaa 6346 Gly Leu Tyr Thr Ile Tyr Leu Asn Gly Asp Lys Ala Gln Ala Leu Glu                2000 2005 2010 gtc ttc tgt gac atg acc tct gat ggg ggt gga tgg att gtg ttc ctg 6394 Val Phe Cys Asp Met Thr Ser Asp Gly Gly Gly Trp Ile Val Phe Leu            2015 2020 2025 aga cgc aaa aac gga cgc gag aac ttc tac caa aac tgg aag gca tat 6442 Arg Arg Lys Asn Gly Arg Glu Asn Phe Tyr Gln Asn Trp Lys Ala Tyr        2030 2035 2040 gct gct gga ttt ggg gac cgc aga gaa gaa ttc tgg ctt ggg ctg gac 6490 Ala Ala Gly Phe Gly Asp Arg Arg Glu Glu Phe Trp Leu Gly Leu Asp    2045 2050 2055 aac ctg aac aaa atc aca gcc cag ggg cag tac gag ctc cgg gtg gac 6538 Asn Leu Asn Lys Ile Thr Ala Gln Gly Gln Tyr Glu Leu Arg Val Asp 2060 2065 2070 2075 ctg cgg gac cat ggg gag aca gcc ttt gct gtc tat gac aag ttc agc 6586 Leu Arg Asp His Gly Glu Thr Ala Phe Ala Val Tyr Asp Lys Phe Ser                2080 2085 2090 gtg gga gat gcc aag act cgc tac aag ctg aag gtg gag ggg tac agt 6634 Val Gly Asp Ala Lys Thr Arg Tyr Lys Leu Lys Val Glu Gly Tyr Ser            2095 2100 2105 ggg aca gca ggt gac tcc atg gcc tac cac aat ggc aga tcc ttc tcc 6682 Gly Thr Ala Gly Asp Ser Met Ala Tyr His Asn Gly Arg Ser Phe Ser        2110 2115 2120 acc ttt gac aag gac aca gat tca gcc atc acc aac tgt gct ctg tcc 6730 Thr Phe Asp Lys Asp Thr Asp Ser Ala Ile Thr Asn Cys Ala Leu Ser    2125 2130 2135 tac aaa ggg gct ttc tgg tac agg aac tgt cac cgt gtc aac ctg atg 6778 Tyr Lys Gly Ala Phe Trp Tyr Arg Asn Cys His Arg Val Asn Leu Met 2140 2145 2150 2155 ggg aga tat ggg gac aat aac cac agt cag ggc gtt aac tgg ttc cac 6826 Gly Arg Tyr Gly Asp Asn Asn His Ser Gln Gly Val Asn Trp Phe His                2160 2165 2170 tgg aag ggc cac gaa cac tca atc cag ttt gct gag atg aag ctg aga 6874 Trp Lys Gly His Glu His Ser Ile Gln Phe Ala Glu Met Lys Leu Arg            2175 2180 2185 cca agc aac ttc aga aat ctt gaa ggc agg cgc aaa cgg gca taaa 6920 Pro Ser Asn Phe Arg Asn Leu Glu Gly Arg Arg Lys Arg Ala        2190 2195 2200 ttggagggac cactgggtga gagaggaata aggcggccca gagcgaggaa aggattttac 6980 caaagcatca atacaaccag cccaaccatc ggtccacacc tgggcatttg gtgagaatca 7040 aagctgacca tggatccctg gggccaacgg caacagcatg ggcctcacct cctctgtgat 7100 ttctttcttt gcaccaaaga catcagtctc caacatgttt ctgttttgtt gtttgattca 7160 gcaaaaatct cccagtgaca acatcgcaat agttttttac ttctcttagg tggctctggg 7220 atgggagagg ggtaggatgt acaggggtag tttgttttag aaccagccgt attttacatg 7280 aagctgtata attaattgtc attatttttg ttagcaaaga ttaaatgtgt cattggaagc 7340 catccctttt tttacatttc atacaacaga aaccagaaaa gcaatactgt ttccatttta 7400 aggatatgat taatattatt aatataataa tgatgatgat gatgatgaaa actaaggatt 7460 tttcaagaga tctttctttc caaaacattt ctggacagta cctgattgta tttttttttt 7520 aaataaaagc acaagtactt ttgaaaaaaa accggaattc 7560 <210> 2 <211> 2201 <212> PRT <213> Homo sapiens tenascin C <400> 2 Met Gly Ala Met Thr Gln Leu Leu Ala Gly Val Phe Leu Ala Phe Leu   1 5 10 15 Ala Leu Ala Thr Glu Gly Gly Val Leu Lys Lys Val Ile Arg His Lys              20 25 30 Arg Gln Ser Gly Val Asn Ala Thr Leu Pro Glu Glu Asn Gln Pro Val          35 40 45 Val Phe Asn His Val Tyr Asn Ile Lys Leu Pro Val Gly Ser Gln Cys      50 55 60 Ser Val Asp Leu Glu Ser Ala Ser Gly Glu Lys Asp Leu Ala Pro Pro  65 70 75 80 Ser Glu Pro Ser Glu Ser Phe Gln Glu His Thr Val Asp Gly Glu Asn                  85 90 95 Gln Ile Val Phe Thr His Arg Ile Asn Ile Pro Arg Arg Ala Cys Gly             100 105 110 Cys Ala Ala Ala Pro Asp Val Lys Glu Leu Leu Ser Arg Leu Glu Glu         115 120 125 Leu Glu Asn Leu Val Ser Ser Leu Arg Glu Gln Cys Thr Ala Gly Ala     130 135 140 Gly Cys Cys Leu Gln Pro Ala Thr Gly Arg Leu Asp Thr Arg Pro Phe 145 150 155 160 Cys Ser Gly Arg Gly Asn Phe Ser Thr Glu Gly Cys Gly Cys Val Cys                 165 170 175 Glu Pro Gly Trp Lys Gly Pro Asn Cys Ser Glu Pro Glu Cys Pro Gly             180 185 190 Asn Cys His Leu Arg Gly Arg Cys Ile Asp Gly Gln Cys Ile Cys Asp         195 200 205 Asp Gly Phe Thr Gly Glu Asp Cys Ser Gln Leu Ala Cys Pro Ser Asp     210 215 220 Cys Asn Asp Gln Gly Lys Cys Val Asn Gly Val Cys Ile Cys Phe Glu 225 230 235 240 Gly Tyr Ala Gly Ala Asp Cys Ser Arg Glu Ile Cys Pro Val Pro Cys                 245 250 255 Ser Glu Glu His Gly Thr Cys Val Asp Gly Leu Cys Val Cys His Asp             260 265 270 Gly Phe Ala Gly Asp Asp Cys Asn Lys Pro Leu Cys Leu Asn Asn Cys         275 280 285 Tyr Asn Arg Gly Arg Cys Val Glu Asn Glu Cys Val Cys Asp Glu Gly     290 295 300 Phe Thr Gly Glu Asp Cys Ser Glu Leu Ile Cys Pro Asn Asp Cys Phe 305 310 315 320 Asp Arg Gly Arg Cys Ile Asn Gly Thr Cys Tyr Cys Glu Glu Gly Phe                 325 330 335 Thr Gly Glu Asp Cys Gly Lys Pro Thr Cys Pro His Ala Cys His Thr             340 345 350 Gln Gly Arg Cys Glu Glu Gly Gln Cys Val Cys Asp Glu Gly Phe Ala         355 360 365 Gly Leu Asp Cys Ser Glu Lys Arg Cys Pro Ala Asp Cys His Asn Arg     370 375 380 Gly Arg Cys Val Asp Gly Arg Cys Glu Cys Asp Asp Gly Phe Thr Gly 385 390 395 400 Ala Asp Cys Gly Glu Leu Lys Cys Pro Asn Gly Cys Ser Gly His Gly                 405 410 415 Arg Cys Val Asn Gly Gln Cys Val Cys Asp Glu Gly Tyr Thr Gly Glu             420 425 430 Asp Cys Ser Gln Leu Arg Cys Pro Asn Asp Cys His Ser Arg Gly Arg         435 440 445 Cys Val Glu Gly Lys Cys Val Cys Glu Gln Gly Phe Lys Gly Tyr Asp     450 455 460 Cys Ser Asp Met Ser Cys Pro Asn Asp Cys His Gln His Gly Arg Cys 465 470 475 480 Val Asn Gly Met Cys Val Cys Asp Asp Gly Tyr Thr Gly Glu Asp Cys                 485 490 495 Arg Asp Arg Gln Cys Pro Arg Asp Cys Ser Asn Arg Gly Leu Cys Val             500 505 510 Asp Gly Gln Cys Val Cys Glu Asp Gly Phe Thr Gly Pro Asp Cys Ala         515 520 525 Glu Leu Ser Cys Pro Asn Asp Cys His Gly Gln Gly Arg Cys Val Asn     530 535 540 Gly Gln Cys Val Cys His Glu Gly Phe Met Gly Lys Asp Cys Lys Glu 545 550 555 560 Gln Arg Cys Pro Ser Asp Cys His Gly Gln Gly Arg Cys Val Asp Gly                 565 570 575 Gln Cys Ile Cys His Glu Gly Phe Thr Gly Leu Asp Cys Gly Gln His             580 585 590 Ser Cys Pro Ser Asp Cys Asn Asn Leu Gly Gln Cys Val Ser Gly Arg         595 600 605 Cys Ile Cys Asn Glu Gly Tyr Ser Gly Glu Asp Cys Ser Glu Val Ser     610 615 620 Pro Pro Lys Asp Leu Val Val Thr Glu Val Thr Glu Glu Thr Val Asn 625 630 635 640 Leu Ala Trp Asp Asn Glu Met Arg Val Thr Glu Tyr Leu Val Val Tyr                 645 650 655 Thr Pro Thr His Glu Gly Gly Leu Glu Met Gln Phe Arg Val Pro Gly             660 665 670 Asp Gln Thr Ser Thr Ile Ile Gln Glu Leu Glu Pro Gly Val Glu Tyr         675 680 685 Phe Ile Arg Val Phe Ala Ile Leu Glu Asn Lys Lys Ser Ile Pro Val     690 695 700 Ser Ala Arg Val Ala Thr Tyr Leu Pro Ala Pro Glu Gly Leu Lys Phe 705 710 715 720 Lys Ser Ile Lys Glu Thr Ser Val Glu Val Glu Trp Asp Pro Leu Asp                 725 730 735 Ile Ala Phe Glu Thr Trp Glu Ile Ile Phe Arg Asn Met Asn Lys Glu             740 745 750 Asp Glu Gly Glu Ile Thr Lys Ser Leu Arg Arg Pro Glu Thr Ser Tyr         755 760 765 Arg Gln Thr Gly Leu Ala Pro Gly Gln Glu Tyr Glu Ile Ser Leu His     770 775 780 Ile Val Lys Asn Asn Thr Arg Gly Pro Gly Leu Lys Arg Val Thr Thr 785 790 795 800 Thr Arg Leu Asp Ala Pro Ser Gln Ile Glu Val Lys Asp Val Thr Asp                 805 810 815 Thr Thr Ala Leu Ile Thr Trp Phe Lys Pro Leu Ala Glu Ile Asp Gly             820 825 830 Ile Glu Leu Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr         835 840 845 Ile Asp Leu Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys     850 855 860 Pro Asp Thr Glu Tyr Glu Val Ser Leu Ile Ser Arg Arg Gly Asp Met 865 870 875 880 Ser Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Leu Asp Ala Pro                 885 890 895 Arg Asn Leu Arg Arg Val Ser Gln Thr Asp Asn Ser Ile Thr Leu Glu             900 905 910 Trp Arg Asn Gly Lys Ala Ala Ile Asp Ser Tyr Arg Ile Lys Tyr Ala         915 920 925 Pro Ile Ser Gly Gly Asp His Ala Glu Val Asp Val Pro Lys Ser Gln     930 935 940 Gln Ala Thr Thr Lys Thr Thr Leu Thr Gly Leu Arg Pro Gly Thr Glu 945 950 955 960 Tyr Gly Ile Gly Val Ser Ala Val Lys Glu Asp Lys Glu Ser Asn Pro                 965 970 975 Ala Thr Ile Asn Ala Ala Thr Glu Leu Asp Thr Pro Lys Asp Leu Gln             980 985 990 Val Ser Glu Thr Ala Glu Thr Ser Leu Thr Leu Leu Trp Lys Thr Pro         995 1000 1005 Leu Ala Lys Phe Asp Arg Tyr Arg Leu Asn Tyr Ser Leu Pro Thr Gly    1010 1015 1020 Gln Trp Val Gly Val Gln Leu Pro Arg Asn Thr Thr Ser Tyr Val Leu 1025 1030 1035 1040 Arg Gly Leu Glu Pro Gly Gln Glu Tyr Asn Val Leu Leu Thr Ala Glu                1045 1050 1055 Lys Gly Arg His Lys Ser Lys Pro Ala Arg Val Lys Ala Ser Thr Glu            1060 1065 1070 Gln Ala Pro Glu Leu Glu Asn Leu Thr Val Thr Glu Val Gly Trp Asp        1075 1080 1085 Gly Leu Arg Leu Asn Trp Thr Ala Ala Asp Gln Ala Tyr Glu His Phe    1090 1095 1100 Ile Ile Gln Val Gln Glu Ala Asn Lys Val Glu Ala Ala Arg Asn Leu 1105 1110 1115 1120 Thr Val Pro Gly Ser Leu Arg Ala Val Asp Ile Pro Gly Leu Lys Ala                1125 1130 1135 Ala Thr Pro Tyr Thr Val Ser Ile Tyr Gly Val Ile Gln Gly Tyr Arg            1140 1145 1150 Thr Pro Val Leu Ser Ala Glu Ala Ser Thr Gly Glu Thr Pro Asn Leu        1155 1160 1165 Gly Glu Val Val Val Ala Glu Val Gly Trp Asp Ala Leu Lys Leu Asn    1170 1175 1180 Trp Thr Ala Pro Glu Gly Ala Tyr Glu Tyr Phe Phe Ile Gln Val Gln 1185 1190 1195 1200 Glu Ala Asp Thr Val Glu Ala Ala Gln Asn Leu Thr Val Pro Gly Gly                1205 1210 1215 Leu Arg Ser Thr Asp Leu Pro Gly Leu Lys Ala Ala Thr His Tyr Thr            1220 1225 1230 Ile Thr Ile Arg Gly Val Thr Gln Asp Phe Ser Thr Thr Pro Leu Ser        1235 1240 1245 Val Glu Val Leu Thr Glu Glu Val Pro Asp Met Gly Asn Leu Thr Val    1250 1255 1260 Thr Glu Val Ser Trp Asp Ala Leu Arg Leu Asn Trp Thr Thr Pro Asp 1265 1270 1275 1280 Gly Thr Tyr Asp Gln Phe Thr Ile Gln Val Gln Glu Ala Asp Gln Val                1285 1290 1295 Glu Glu Ala His Asn Leu Thr Val Pro Gly Ser Leu Arg Ser Met Glu            1300 1305 1310 Ile Pro Gly Leu Arg Ala Gly Thr Pro Tyr Thr Val Thr Leu His Gly        1315 1320 1325 Glu Val Arg Gly His Ser Thr Arg Pro Leu Ala Val Glu Val Val Thr    1330 1335 1340 Glu Asp Leu Pro Gln Leu Gly Asp Leu Ala Val Ser Glu Val Gly Trp 1345 1350 1355 1360 Asp Gly Leu Arg Leu Asn Trp Thr Ala Ala Asp Asn Ala Tyr Glu His                1365 1370 1375 Phe Val Ile Gln Val Gln Glu Val Asn Lys Val Glu Ala Ala Gln Asn            1380 1385 1390 Leu Thr Leu Pro Gly Ser Leu Arg Ala Val Asp Ile Pro Gly Leu Glu        1395 1400 1405 Ala Ala Thr Pro Tyr Arg Val Ser Ile Tyr Gly Val Ile Arg Gly Tyr    1410 1415 1420 Arg Thr Pro Val Leu Ser Ala Glu Ala Ser Thr Ala Lys Glu Pro Glu 1425 1430 1435 1440 Ile Gly Asn Leu Asn Val Ser Asp Ile Thr Pro Glu Ser Phe Asn Leu                1445 1450 1455 Ser Trp Met Ala Thr Asp Gly Ile Phe Glu Thr Phe Thr Ile Glu Ile            1460 1465 1470 Ile Asp Ser Asn Arg Leu Leu Glu Thr Val Glu Tyr Asn Ile Ser Gly        1475 1480 1485 Ala Glu Arg Thr Ala His Ile Ser Gly Leu Pro Pro Ser Thr Asp Phe    1490 1495 1500 Ile Val Tyr Leu Ser Gly Leu Ala Pro Ser Ile Arg Thr Lys Thr Ile 1505 1510 1515 1520 Ser Ala Thr Ala Thr Thr Glu Ala Leu Pro Leu Leu Glu Asn Leu Thr                1525 1530 1535 Ile Ser Asp Ile Asn Pro Tyr Gly Phe Thr Val Ser Trp Met Ala Ser            1540 1545 1550 Glu Asn Ala Phe Asp Ser Phe Leu Val Thr Val Val Asp Ser Gly Lys        1555 1560 1565 Leu Leu Asp Pro Gln Glu Phe Thr Leu Ser Gly Thr Gln Arg Lys Leu    1570 1575 1580 Glu Leu Arg Gly Leu Ile Thr Gly Ile Gly Tyr Glu Val Met Val Ser 1585 1590 1595 1600 Gly Phe Thr Gln Gly His Gln Thr Lys Pro Leu Arg Ala Glu Ile Val                1605 1610 1615 Thr Glu Ala Glu Pro Glu Val Asp Asn Leu Leu Val Ser Asp Ala Thr            1620 1625 1630 Pro Asp Gly Phe Arg Leu Ser Trp Thr Ala Asp Glu Gly Val Phe Asp        1635 1640 1645 Asn Phe Val Leu Lys Ile Arg Asp Thr Lys Lys Gln Ser Glu Pro Leu    1650 1655 1660 Glu Ile Thr Leu Leu Ala Pro Glu Arg Thr Arg Asp Leu Thr Gly Leu 1665 1670 1675 1680 Arg Glu Ala Thr Glu Tyr Glu Ile Glu Leu Tyr Gly Ile Ser Lys Gly                1685 1690 1695 Arg Arg Ser Gln Thr Val Ser Ala Ile Ala Thr Thr Ala Met Gly Ser            1700 1705 1710 Pro Lys Glu Val Ile Phe Ser Asp Ile Thr Glu Asn Ser Ala Thr Val        1715 1720 1725 Ser Trp Arg Ala Pro Thr Ala Gln Val Glu Ser Phe Arg Ile Thr Tyr    1730 1735 1740 Val Pro Ile Thr Gly Gly Thr Pro Ser Met Val Thr Val Asp Gly Thr 1745 1750 1755 1760 Lys Thr Gln Thr Arg Leu Val Lys Leu Ile Pro Gly Val Glu Tyr Leu                1765 1770 1775 Val Ser Ile Ile Ala Met Lys Gly Phe Glu Glu Ser Glu Pro Val Ser            1780 1785 1790 Gly Ser Phe Thr Thr Ala Leu Asp Gly Pro Ser Gly Leu Val Thr Ala        1795 1800 1805 Asn Ile Thr Asp Ser Glu Ala Leu Ala Arg Trp Gln Pro Ala Ile Ala    1810 1815 1820 Thr Val Asp Ser Tyr Val Ile Ser Tyr Thr Gly Glu Lys Val Pro Glu 1825 1830 1835 1840 Ile Thr Arg Thr Val Ser Gly Asn Thr Val Glu Tyr Ala Leu Thr Asp                1845 1850 1855 Leu Glu Pro Ala Thr Glu Tyr Thr Leu Arg Ile Phe Ala Glu Lys Gly            1860 1865 1870 Pro Gln Lys Ser Ser Thr Ile Thr Ala Lys Phe Thr Thr Asp Leu Asp        1875 1880 1885 Ser Pro Arg Asp Leu Thr Ala Thr Glu Val Gln Ser Glu Thr Ala Leu    1890 1895 1900 Leu Thr Trp Arg Pro Pro Arg Ala Ser Val Thr Gly Tyr Leu Leu Val 1905 1910 1915 1920 Tyr Glu Ser Val Asp Gly Thr Val Lys Glu Val Ile Val Gly Pro Asp                1925 1930 1935 Thr Thr Ser Tyr Ser Leu Ala Asp Leu Ser Pro Ser Thr His Tyr Thr            1940 1945 1950 Ala Lys Ile Gln Ala Leu Asn Gly Pro Leu Arg Ser Asn Met Ile Gln        1955 1960 1965 Thr Ile Phe Thr Thr Ile Gly Leu Leu Tyr Pro Phe Pro Lys Asp Cys    1970 1975 1980 Ser Gln Ala Met Leu Asn Gly Asp Thr Thr Ser Gly Leu Tyr Thr Ile 1985 1990 1995 2000 Tyr Leu Asn Gly Asp Lys Ala Gln Ala Leu Glu Val Phe Cys Asp Met                2005 2010 2015 Thr Ser Asp Gly Gly Gly Trp Ile Val Phe Leu Arg Arg Lys Asn Gly            2020 2025 2030 Arg Glu Asn Phe Tyr Gln Asn Trp Lys Ala Tyr Ala Ala Gly Phe Gly        2035 2040 2045 Asp Arg Arg Glu Glu Phe Trp Leu Gly Leu Asp Asn Leu Asn Lys Ile    2050 2055 2060 Thr Ala Gln Gly Gln Tyr Glu Leu Arg Val Asp Leu Arg Asp His Gly 2065 2070 2075 2080 Glu Thr Ala Phe Ala Val Tyr Asp Lys Phe Ser Val Gly Asp Ala Lys                2085 2090 2095 Thr Arg Tyr Lys Leu Lys Val Glu Gly Tyr Ser Gly Thr Ala Gly Asp            2100 2105 2110 Ser Met Ala Tyr His Asn Gly Arg Ser Phe Ser Thr Phe Asp Lys Asp        2115 2120 2125 Thr Asp Ser Ala Ile Thr Asn Cys Ala Leu Ser Tyr Lys Gly Ala Phe    2130 2135 2140 Trp Tyr Arg Asn Cys His Arg Val Asn Leu Met Gly Arg Tyr Gly Asp 2145 2150 2155 2160 Asn Asn His Ser Gln Gly Val Asn Trp Phe His Trp Lys Gly His Glu                2165 2170 2175 His Ser Ile Gln Phe Ala Glu Met Lys Leu Arg Pro Ser Asn Phe Arg            2180 2185 2190 Asn Leu Glu Gly Arg Arg Lys Arg Ala        2195 2200 <210> 3 <211> 4 <212> PRT <213> minor heparin-binding site <400> 3 Lys Glu Asp Lys   One <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer TN3F <400> 4 agccatatgg ctgttcctcc tcccactgac ctg 33 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer TN3R <400> 5 gccgaattca tgttgtgaag gtctc 25 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer TN4R <400> 6 ggaattcctc acttgggcgt gtccaac 27 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer TN5R <400> 7 tcgaattcta tgttcggtaa ttaat 25 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gctgtgcagc aaaacaagga gagcaatc 28 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tgctctcctt gttttgctgc acagcaga 28 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gacagccgag caaggcagac ac 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gtctgccttg ctcggctgtc 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gacagccgag aaaggccgac ac 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gtcggccttt ctcggctgtc 20  

Claims (6)

Polypeptides and derivatives thereof comprising a heparin-binding site derived from human tenacin C. The polypeptide and derivative thereof according to claim 1, wherein the heparin binding site comprises a binding site of heparan sulfate proteoglycan (HSPG). The polypeptide and derivative thereof according to claim 1, wherein the heparin-binding site comprises the KEDK amino acid sequence of SEQ ID NO. The polypeptide of claim 1, wherein the polypeptide increases heparin-binding activity of tenasin-C by a cooperative mechanism with a heparin-binding site of tenasin-C type III domain 5 (TNIII5). And derivatives thereof. The polypeptide and derivative thereof according to claim 1, which increases HSPG-dependent cell adhesion activity against tenasin-C type III domain 5 (TNIII5). A pharmaceutical composition for promoting cell adhesion comprising the polypeptide of claim 1 or a derivative thereof as an active ingredient.

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