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Functional mapping of SPARC: peptides from two distinct Ca+(+)-binding sites modulate cell shape.
The Journal of Cell Biology
|December 1, 1990
Summary
Secreted Protein Acidic and Rich in Cysteine (SPARC) has two regions that inhibit cell spreading. One region also binds extracellular matrix components, suggesting SPARC uses multiple sites to regulate cell-matrix interactions.
Area of Science:
- Biochemistry
- Cell Biology
- Extracellular Matrix Research
Background:
- Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular glycoprotein involved in cell-matrix interactions.
- SPARC influences cell behavior, including adhesion, migration, and proliferation.
- The precise functional domains of SPARC mediating these effects are not fully elucidated.
Purpose of the Study:
- To identify specific regions of SPARC responsible for inhibiting cell spreading.
- To determine if these regions also mediate SPARC's binding to extracellular matrix (ECM) components.
- To investigate the mechanism by which SPARC regulates cell-matrix interactions.
Main Methods:
- Synthesis of specific peptide fragments representing distinct regions of SPARC.
- Treatment of endothelial cells and fibroblasts with synthesized peptides to assess cell spreading.
- Production and use of antipeptide antibodies to block SPARC activity.
- Solid-phase binding assays to evaluate peptide affinity for ECM molecules, including collagen.
- Ca++ dependency assays for peptide-ECM interactions.
Main Results:
- Two distinct regions of SPARC, represented by peptides 1.1 (N-terminus) and 4.2 (C-terminal EF-hand domain), significantly inhibited endothelial cell and fibroblast spreading.
- Peptide 4.2 demonstrated dose-dependent inhibition of cell spreading and competed with native SPARC for binding to ECM components.
- Peptide 4.2's binding to ECM molecules was calcium-dependent and showed specificities similar to native SPARC.
- Antipeptide antibodies against peptide 1.1 blocked SPARC-mediated anti-spreading activity.
Conclusions:
- Specific regions within the SPARC amino acid sequence are responsible for its anti-spreading activity and affinity for extracellular matrix components.
- SPARC likely employs multiple functional domains that act in concert to regulate cell-matrix interactions.
- These findings provide a deeper understanding of SPARC's role in modulating cell behavior through ECM interactions.