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 Ca(2+)-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.
Brekken
Rolf A
RA
Department of Vascular Biology, The Hope Heart Institute, Seattle, Washington 98104-2046, USA.
Sullivan
Millicent M
MM
Workman
Gail
G
Bradshaw
Amy D
AD
Carbon
Juliet
J
Siadak
Anthony
A
Murri
Carrie
C
Framson
Paul E
PE
Sage
E Helene
EH
D000818:Animals; D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D006023:Glycoproteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D015151:Immunoblotting; D007150:Immunohistochemistry; D007313:Insecta / Q000166:cytology; D008297:Male; D051379:Mice; D009369:Neoplasms / Q000378:metabolism; D009928:Organ Specificity; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins; C503126:SPARCL1 protein, human; C503127:Sparcl1 protein, mouse; C503128:Sparcl1 protein, rat