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

Abstract

S U M M A R Y 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 Ca 2 ϩ -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 deadhesive 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.

Matricellular Hevin Regulates Decorin Production and Collagen Assembly

“…Other chemicals were purchased from Fisher Scientific (Pittsburgh, PA). Recombinant murine hevin and rat IgG anti-hevin monoclonal antibody 12-51 were produced and purified according to established protocols (13). Recombinant human decorin core protein was from M. Höök (Center for Extracellular Matrix Biology and Department of Biochemistry and Biophysics, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX).…”

Novel Function of Alternatively Activated Macrophages: Stabilin-1-Mediated Clearance of SPARC

“…Recombinant human SPARC was coated onto microtiter wells (22,23), and phage-binding assays on purified proteins were performed as previously described (24). SPARC, hevin (25), bovine type I collagen (Vitrogen; Collaborative Biomedical Products), and BSA (Pierce Biotechnology) at 1 g in 50 l of PBS/0.1 mM Ca 2ϩ were coated onto microtiter wells overnight at 4°C. Wells were washed twice with PBS, blocked with PBS/3% BSA for 1 h at room temperature, and incubated with 2 ϫ 10 9 transducing units of phage (CIRFH-GTAC) or fd-tet phage (control) in 50 l of PBS/1.5% BSA, for 2 h at room temperature.…”

Genomic Profile of Matrix and Vasculature Remodeling in TGF-α–Induced Pulmonary Fibrosis

“…These transcripts included LOX1 and related enzyme protein family members, LOXL1, LOXL2, LOXL3, that can catalyze oxidative deamination of the epsilon-amino group in certain lysine and hydroxylysine residues of collagens and lysine residues of elastin, thereby leading to the cross-linking of fibrillar collagens and elastin (40). Also in this cluster was MGP, a mineral-, extracellular matrix-binding protein synthesized by vascular smooth muscle cells and chondrocytes involved in extracellular matrix mineralization, and SPARCL1, a collagen type I binding protein (41). Thus, genes in this cluster could be considered important in propagating and maturing the progressive fibrotic process.…”