Functional mapping of SPARC: peptides from two distinct Ca+(+)-binding sites modulate cell shape.
Using synthetic peptides, we have identified two distinct regions of the glycoprotein SPARC (Secreted Protein Acidic and Rich in Cysteine) (osteonectin/BM-40) that inhibit cell spreading. One of these sites also contributes to the affinity of SPARC for extracellular matrix components. Peptides representing subregions of SPARC were synthesized and antipeptide antibodies were produced. Immunoglobulin fractions of sera recognizing an NH2-terminal peptide (designated 1.1) blocked SPARC-mediated anti-spreading activity. Furthermore, when peptides were added to newly plated endothelial cells or fibroblasts, peptide 1.1 and a peptide corresponding to the COOH terminal EF-hand domain (designated 4.2) inhibited cell spreading in a dose-dependent manner. These peptides exhibited anti-spreading activity at concentrations from 0.1 to 1 mM. The ability of peptides 1.1 and 4.2 to modulate cell shape was augmented by an inhibitor of protein synthesis and was blocked by specific antipeptide immunoglobulins. In addition to blocking cell spreading, peptide 4.2 competed for binding of [125I]SPARC and exhibited differential affinity for extracellular matrix molecules in solid-phase binding assays. The binding of peptide 4.2 to matrix components was Ca+(+)-dependent and displayed specificities similar to those of native SPARC. These studies demonstrate that both anti-spreading activity and affinity for collagens are functions of unique regions within the SPARC amino acid sequence. The finding that two separate regions of the SPARC protein contribute to its anti-spreading activity lead us to propose that multiple regions of the protein act in concert to regulate the interactions of cells with their extracellular matrix.
Lane
T F
TF
Department of Biological Structure, University of Washington, Seattle 98195.
Sage
E H
EH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D015676:Osteonectin / Q000737:chemistry / Q000502:physiology*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D013329:Structure-Activity Relationship
D000906:Antibodies; D015676:Osteonectin; D010446:Peptide Fragments; D003094:Collagen; D002118:Calcium