Topography of proteoglycan and glycosaminoglycan free chain expression in 3T3 fibroblasts and human keratinocytes.
Synthesis of heparan sulfate-free chains by human keratinocytes is upregulated during terminal differentiation. The cellular location of this product class and the significance of the differentiation effect are unknown. Differential plasma membrane shearing with cationized colloidal silica was used to evaluate the compartmentalization of the heparan and chondroitin sulfate free chains and their respective proteoglycans in 3T3 fibroblasts and human keratinocytes. The method exploits the topologic segregation of plasma membranes of adherent cells into ventral, dorsal, and intracellular domains and the selective binding of the silica to the dorsal membranes, which by shearing can be separated from ventral membranes adherent to the substratum. Analysis of membrane preparations from sheared cells that had been prelabeled with [35S]-sulfate revealed the proteoglycans to be predominantly ventral, at which location a matrix binding function could be accommodated. Proteoglycans were also recovered from dorsal and intracellular membranes, suggesting active trafficking between intra- and extra-cellular sites. In contrast, the major fraction of heparan and chondroitin sulfate free chains was either cytosolic or associated with intracellular membranes, with the remaining approximately 20% segregated to dorsal and ventral membranes. These results suggest different cellular functions for the proteoglycans and glycosaminoglycan free chains. The partial localization of the free chains to peripheral membranes is compatible with our prior hypothesis that they arise by processing of precursor proteoglycans on cell surfaces. Following this origin, the free glycosaminoglycan polymers could be available to bind ligands such as cytokines prior to transport to intracellular sites of action.
Piepkorn
M
M
Department of Medicine (Dermatology), University of Washington, Seattle 98195.
Hovingh
P
P
Linker
A
A
D016475:3T3 Cells / Q000737:chemistry*; D000818:Animals; D001345:Autoradiography; D000078404:Epidermal Cells; D006025:Glycosaminoglycans / Q000032:analysis*; D019763:Heparin Lyase; D015603:Keratinocytes / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D011133:Polysaccharide-Lyases / Q000494:pharmacology; D011509:Proteoglycans / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D053667:Syndecans
D006025:Glycosaminoglycans; D008562:Membrane Glycoproteins; D011509:Proteoglycans; D053667:Syndecans; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase