Removal of glycosaminoglycans from cultures of human skin fibroblasts

Abstract

Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential medium containing [35S]sulphate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacter heparinum was added to the cell monolayers. This treatment removed most of the 35S-labelled glycosaminoglycans, with no appreciable removal of the 3H-labelled proteins or 3H-labelled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labelled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulphate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labelled glycosaminoglycans produced. Thus F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.

Evidence that cell surface heparan sulfate is involved in the high affinity thrombin binding to cultured porcine aortic endothelial cells.

“…The cell suspensions were centrifuged to obtain a supernate ("trypsinate" fraction) and a cell pellet ("cell" fraction). The trypsinate fraction includes glycosaminoglycans or proteins derived from the cell surface and from the solubilized matrix (28). The cultures were then treated with 0.5 ml of 0.1 N NaOH for removal of radioactivity remaining on the plates ("plate" fraction).…”

Cell Density-dependent Regulation of Proteoglycan Synthesis by Transforming Growth Factor-β1 in Cultured Bovine Aortic Endothelial Cells

“…The wash was combined with the corresponding cell suspension and centrifuged at 1500 ϫ g for 5 min. The supernatant includes GAGs derived from the cell surface and the solubilized extracellular matrix (37). The supernatant and the conditioned medium were used for the determination of radioactive GAGs by the CPC precipitation method (38) as follows: each fraction was incubated with 3 mg/ml Pronase at 50°C for 3 h. The Pronase digests were mixed with 4 mg/ml carrier chondroitin sulfate A and 0.5% CPC.…”

Involvement of cell surface heparin sulfate in the binding of lipoprotein lipase to cultured bovine endothelial cells.

“…Under (14). Furthermore, the cells treated with F. heparinum enzyme do not show any degradation of cellular protein and are able to continde normal glycosaminoglycan synthesis (14). Hence, it is very unlikely that the interference with binding by this treatment is due to proteinase activity in F. heparinum enzyme preparations.…”

“…Among various glycosaminoglycans, only heparin and heparan sulfate, but none ofthe other polysaccharides, were able to displace the endogenous polysaccharide (30). Under (14). Furthermore, the cells treated with F. heparinum enzyme do not show any degradation of cellular protein and are able to continde normal glycosaminoglycan synthesis (14).…”