Mapping of SPARC/BM-40/Osteonectin-binding Sites on Fibrillar Collagens

Abstract

The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix ␣A of the extracellular calciumbinding domain of SPARC and is partially masked by helix ␣C. Previously, we found that the removal of helix ␣C caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadowing, CNBr peptides, and synthetic peptides to map binding sites of SPARC onto collagens I, II, and III. Rotary shadowing and electron microscopy of SPARC-collagen complexes identified a major binding site ϳ180 nm from the C terminus of collagen. SPARC binding was also detected with lower frequency near the matrix metalloproteinase cleavage site. These data fit well with our analysis of SPARC binding to CNBr peptides, denaturation of which abolished binding, indicating triple-helical conformation of collagen to be essential. SPARC binding was substantially decreased in two of seven ␣2(I) mutant procollagen I samples and after N-acetylation of Lys/Hyl side chains in wild-type collagen. Synthetic peptides of collagen III were used to locate the binding sites, and we found SPARC binding activity in a synthetic triple-helical peptide containing the sequence GPOG-PSGPRGQOGVMGFOGPKGNDGAO (where O indicates 4-hydroxyproline), with affinity for SPARC comparable with that of procollagen III. This sequence is conserved among ␣ chains of collagens I, II, III, and V. In vitro collagen fibrillogenesis was delayed in the presence of SPARC, suggesting that SPARC might modulate collagen fibril assembly in vivo.Collagens are major components of the extracellular matrix, comprising ϳ30% of the protein mass of vertebrates. At least 28 collagens have been identified so far (1-3) encoded by 46 separate genes. The defining feature of collagens is a triple-helical domain assembled from three polypeptide strands, called ␣-chains, which each consist of repeating Gly-X-Y sequences, where X is often proline and Y is often hydroxyproline. This primary structure facilitates left-handed poly-proline II helical conformation in each ␣-chain, and three ␣-chains form a righthanded triple helix stabilized by interchain hydrogen bonds (4). To form this structure, Gly is required as every third residue. Its lack of side chains allows contact between ␣-chains, whereas the side chains of residues X and Y are exposed on the surface of the triple helix where they can interact with various molecules. Collagens are major structural proteins, endowing mechanical strength upon the extracellular matrix and also regulating cell behavior through the binding of cell surface receptors and other proteins, such as discoidin domain receptors, a subset of the integrins ␣1␤1, ␣2␤1, ␣10␤1, and ␣11␤1, NG2 proteoglycan, and many matrix molecules (5-7). Nearly 50 different ligands have been shown to interact with collagen, and the binding sites for half of these have been mapped onto collagen I (8). The bind...

Structural basis of sequence-specific collagen recognition by SPARC

“…SPARC FS-EC ⌬␣C binds with high affinity to residues 564-590 of the human collagen ␣1(III) chain (corresponding to 397-423 of the triplehelical domain) (13). A 33-residue peptide containing this collagen III region preceded by 2 GPO repeats forms a stable triple-helix and a 1:1 complex with SPARC FS-EC ⌬␣C (data not shown).…”

“…The only other plausible site from sequence analysis is a GATGFO sequence (GAAGFO in collagen III) halfway between the GVMGFO motif and the C terminus. Rotary shadowing electron micrographs indeed show a secondary SPARC-binding site in this region (13). The 6 distinct mammalian collagen IV chains assemble into 3 heterotrimers, of which the [␣1(IV)] 2 ␣2(IV) heterotrimer is the most abundant; invertebrates have only 1 type of collagen IV heterotrimer (1,32).…”

“…The detailed collagen sequence requirements for SPARC binding have not been probed biochemically (13). From our structure, we predict an absolute requirement for phenylalanine at position 23 of the trailing chain.…”

“…Collagens II and III are homotrimers. The major SPARC-binding site in the fibrillar collagens I-III, GVMGFO, is Ϸ600 residues from the C terminus of the triple helix (13). The only other plausible site from sequence analysis is a GATGFO sequence (GAAGFO in collagen III) halfway between the GVMGFO motif and the C terminus.…”

“…The atomic details of this important interaction were revealed by a crystal structure of the integrin ␣2 I-domain bound to a 21-residue triple helical peptide, providing the only example to date of a vertebrate protein-collagen complex (10). More recent studies have identified a GVMGFO motif, conserved in collagens I-III and situated Ϸ100 residues (Ϸ30 nm) upstream of the GFOGER motif, as a binding hotspot for 3 structurally and functionally distinct proteins: the plasma protein von Willebrand factor (VWF), whose interaction with collagen contributes to haemostasis (11); the receptor tyrosine kinase DDR2 (12); and the extracellular matrix protein SPARC (13).…”