Anthony W. Siadak

Functional Analysis of the Matricellular Protein SPARC with Novel Monoclonal Antibodies

SPARC (osteonectin, BM-40) is a matricellular glycoprotein that is expressed in many embryogenic and adult tissues undergoing remodeling or repair. SPARC modulates cellular interaction with the extracellular matrix (ECM), inhibits cell adhesion and proliferation, and regulates growth factor activity. To explore further the function and activity of this protein in tissue homeostasis, we have developed several monoclonal antibodies (MAbs) that recognize distinct epitopes on SPARC. The MAbs bind to SPARC with high affinity and identify SPARC by ELISA, Western blotting, immunoprecipitation, immunocytochemistry, and/or immunohistochemistry. The MAbs were also characterized in functional assays for potential alteration of SPARC activity. SPARC binds to collagen I and laminin-1 through an epitope defined by MAb 293; this epitope is not involved in the binding of SPARC to collagen III. The other MAbs did not interfere with the binding of SPARC to collagen I or III or laminin-1. Inhibition of the anti-adhesive effect of SPARC on endothelial cells by MAb 236 was also observed. Functional analysis of SPARC in the presence of these novel MAbs now confirms that the activities ascribed to this matricellular protein can be assigned to discrete subdomains.

Expression and Characterization of Murine Hevin (SC1), a Member of the SPARC Family of Matricellular Proteins

Hevin, also known as SC1, MAST 9, SPARC-like 1, RAGS1 and ECM2, is a member of the SPARC-related family of matricellular proteins. Mouse hevin is 53% identical to mouse SPARC, and both proteins share a follistatin-like module and an extracellular Ca2+-binding (E-C) domain. SPARC functions as a modulator of cell-matrix interactions, a regulator of growth factor activity, a de-adhesive protein, and a cell cycle inhibitor. Although the functions of mouse hevin are unknown, its human orthologue has been shown to be de-adhesive for endothelial cells. We now report the production of recombinant mouse hevin in insect cells through the use of a baculoviral expression system and its purification by anion-exchange, size-exclusion chromatography, and isoelectric focusing. Furthermore, we have produced rat anti-hevin monoclonal antibodies (MAbs) that have been characterized by indirect and capture ELISAs, immunoblotting, immunoprecipitation, and immunohistochemistry (IHC). Recombinant hevin, present as a soluble factor or bound to tissue-culture plastic, inhibited the spreading of bovine aortic endothelial cells in vitro. IHC analysis of hevin in normal human and mouse tissues revealed a limited expression pattern in many tissues, with particularly dominant staining in dermis, ducts, vasculature, muscle, and brain. In lung and pancreatic tumor xenografts, we found distinct reactivity with MAbs that were selective for stromal cells, tumor cells, and/or endothelial cells. Although similar to SPARC in its anti-adhesive activities, hevin nevertheless exhibits a distinctive histological distribution that, in certain invasive tumors, is associated with desmoplasia.