The invention provides polypeptides derived from the perlecan core protein as coatings for scaffolds, such as implantable tissue scaffolds. The inventive coatings allow strong and selective cell adhesion to the scaffold. For example, the inventive polypeptides can be coated on matrices implanted in damaged tissue such as bone or cartilage to increase the adhesion of desirable biological materials to the repair site.
The inventive polypeptide is derived from the perlecan core protein. Perlecan is a heparan sulfate proteoglycan (Noonan, D. M. (1991) J. Biol. Chem. (266) 34: 22939-22947). The proteoglycan family contains more than 30 members that perform an assortment of biological functions and are widely distributed in basement membranes. Proteoglycans act as tissue organizers and as biological filters, affect cell growth, and have growth factor activities (Iozzo, R. V. (1998) Annu. Rev. Biochem. 67: 609-652). Perlecan, also known as HSPG2, is a constituent of all basement membranes and plays a role in cell adhesion, angiogenesis, cell proliferation, and tumor development (Sharma, B. (1998) J. Clin. Invest. (102) 8: 1599-1608). Perlecan consists of three heparan sulfate side chains attached to a large core protein consisting of five modular domains.
Perlecan Domains
Domain I contains a signal peptide and a unique 172 amino acid sequence that contains the heparan sulfate binding sites. Domain I has no internal repeats and is devoid of cysteine residues. This domain is enriched with acidic amino acid residues that facilitate the heparan sulfate attachment (Iozzo, R., supra). Domain II contains four cysteines and acidic amino acid rich repeats similar to those found in the low-density lipoprotein receptor (LDLr). Domain III contains cysteine-rich globular regions similar to the short arm of the laminin A chain. Domain IV is the largest and most repetitive domain because it contains 14 and 21 immunoglobulin repeats in the murine and human species, respectively (Iozzo, R., supra). Domain V contains three laminin type repeats and four repeats similar to epidermal growth factor. This domain is homologous to the globular terminus of the laminin A chain.
Rotary shadowing revealed perlecan to be a series of globules separated by rods creating the appearance of beads on a string, hence the name perlecan. Perlecan is derived from the Middle English “perle” meaning pearl and “can” signifying the posttranslational glycosaminoglycan modification (Noonan, D. M., supra).
Work by Cohen et al. showed that perlecan has multiple sites of transcription initiation, which suggests that the various transcripts observed with perlecan are more likely due to alternative splicing of internal exons than due to differential usage of polyadenylation sites (Cohen, I. (1993) Proc. Nat. Acad. Sci. 90: 10404-10408).
Cell Adhesion
Work by SundarRaj et al. showed that purified perlecan promoted the attachment of immortalized rat chondrocytes in vitro (SundarRaj, N. (1995) J. Cell Sci. 108 (Pt 7): 2663-2672). Chakravarti et al. studied an Arginine-Glycine-Aspartic Acid-Serine (“RGDS”) amino acid sequence located in domain III that was associated with cell adhesion (Chakravarti, S. (1995) J. Biol. Chem. (270) 1: 404-409). Chakravarti et al. produced domain III as a recombinant protein and evaluated its cell adhesion activity. Recombinant domain III coated on tissue culture dishes supported adhesion of a mouse mammary tumor cell line in a dose dependent manner. Forty percent of the cells attached at the maximum dose. Furthermore, all of the attachment could be abolished with synthetic RGDS peptide. Chakravarti et al. concluded that the RGDS sequence is the only binding site in their recombinant domain III protein.
Hayashi et al. studied full-length perlecan and reported a higher percentage attachment of cells than Chakravarti et al. reported with just domain III (Hayashi, K. (1992) J. Cell Biol. 119 (4): 945-959).
Hopf et al. assessed the protein-protein interaction between domain IV of mouse perlecan and fibronectin, nidogen-1, nidogen-2, laminin-1-nidogen-1 complex, fibulin-2, and collagen IV (Hopf, M (1999) Eur. J. Biochem. 259 (3): 917-925). Hopf et al. created two separate recombinant molecules from domain IV. One consisted of the IG 2-9 modules of domain IV and had a molecular weight of 100 kD (IV-1). The second consisted of the IG modules 10-15 and had a molecular weight of 66 kDa (IV-2). Hopf et al. found that there was strong protein-protein interaction between IV-1 and fibronectin, nidogen-1, nidogen-2 and the laminin-1-nidogen-1 complex, while the IV-2 fragment had a much more restricted protein-protein interaction with weaker binding to fibronectin and fibulin-2.
Scaffolding for Tissue Repair
Numerous skeletal and connective-tissue related disorders have been treated with engineered implants. For example, implants made of substrates such as ceramics, metals, polymers, and biological composites have been used to repair bone and tissue. Improved understanding of cellular and molecular events that occur at the interface between tissues and implants is beginning to allow new approaches to implant design. Preferably, implants are designed to elicit specific, clinically-desirable responses from living cells and tissues in a patient's body. For example, it is desirable for osteoblasts to rapidly deposit mineralized matrix on the surface of (or in close apposition to) newly implanted prostheses. The swift deposition of bone stabilizes the prosthesis and minimizes motion-induced damage to surgically traumatized tissue at the implantation site.
Anchorage-dependent cells (such as osteoblasts) must first adhere to a surface in order to perform subsequent cellular functions (e.g., proliferation, deposition of bone tissue, etc.). Because cell adhesion is needed for subsequent events, methods for promoting cell adhesion are of considerable interest. The effects on cell adhesion of peptides immobilized on the surfaces of substrates have been reported. Substrates have included polymers (Massia et al. (1991) J. Biomed. Natl. 25: 223-242) and dental/orthopedic implant materials such as Cobalt-Chromium-Molybdenum alloy (Mikos et al. (1994) Biomatls. Cell and Drug Delivery 331: 269-274). Adhesion-related peptides that have been attached to substrates have included integrin-binding peptides, such as those that contain the Arginine-Glycine-Aspartic Acid (RGD) sequence. U.S. Pat. No. 6,262,017 “Peptides for Altering Osteoblast Adhesion” issued Jul. 17, 2001 discloses polypeptide coatings for controlling osteoblast adhesion to implants.
Although it is known that perlecan is involved in cell adhesion, the intact molecule is too large to exploit commercially as a cellular adhesive. Its size does not allow for efficient and cost effective commercial production. The present invention avoids this problem and meets the needs of the art by providing a small molecule with strong and selective cell adhesion properties.