Electrophoretic, biosensor, and bioactivity analyses of perlecans of different cellular origins

Abstract

Three cellular sources of perlecan were examined in this study, namely human umbilical arterial endothelial cells (HUAEC), a transformed human umbilical venous endothelial cell line (C 1 1 STH) and a human colon carcinoma cell line (WiDr). Perlecans were immunopurified from conditioned media of the above cells and the purity of the perlecan preparations was examined by composite agarose polyacrylamide gel electrophoresis (CAPAGE) and semi-dry immunoblotting with monoclonal antibodies directed to either the perlecan core protein (mAb A76) or heparan sulphate (HS) side-chain (mAb10E4). The ability of each perlecan species to bind fibroblast growth factor-l (FGF-1) was examined using a biosensor (BIAcore). The bioactivity of perlecan FGF-1 interactions was also analysed using BaF3 cells transfected with fibroblast growth factor receptors FGFR1b and 1c. CAPAGE demonstrated subtle differences between the perlecans, indicating they had differing charge to mass ratios with C 11 STH perlecan being slightly more mobile in CAPAGE than the HUAEC and WiDr sample. BIAcore biosensor analysis demonstrated distinct differences in the ability of perlecan preparations to bind FGF-1; HUAEC and C 11 STH perlecan showed similar high binding responses as compared to WiDr perlecan, which bound FGF-1 very poorly. Binding of FGF-1 to endothelial perlecans was shown to be HS-dependent. Interestingly, HUAEC perlecan stimulated the growth of FGFR1b and FGFR1c expressing cells in the presence of FGF-1 comparable to heparin, whereas C 11 STH perlecan showed only very limited stimulation of FGFR 1b cells and was incapable of stimulating FGFR1c cells. WiDr perlecan exhibited no stimulation of growth in either cell line. Collectively the data presented herein indicate that. different cell types express perlecans which vary in the growth factor binding capabilities, which may suggest differences in their HS chain substructure. This may represent a subtle mechanism whereby cells can modulate the responsiveness of perlecan to a range of biologically important ligands and thus in a broader context may have important implications for cell signalling.