Incorporation of l-[3H]fucose and d-[3H]glucosamine into cell-surface-associated glycoconjugates in epidermis of cultured pig skin slices

Abstract

EM autoradiography indicated that L-[3H]fucose and D-[3H]glucosamine were incorporated into cell-surface-associated glycoconjugates in the epidermis of cultured pig skin slices. Acid hydrolysis and paper chromatography of skin homogenates confirmed that there was little metabolic conversion of the labeled precursors to other sugars. Epidermis was separated from dermis using CaCl2 and was extracted with 8 M-urea/5% (wt/vol) sodium dodecyl sulfate and was analyzed by gel electrophoresis. The major component labeled with D-[3H]glucosamine had an apparent MW > 200,000. This material was not labeled with L-[3H]fucose. Lower MW components were labeled to a similar extent with L-[3H]fucose and D-[3H]glucosamine. The high MW material labeled with D-[3H]glucosamine was released into the medium when the epidermal cells were dispersed with trypsin, indicating that it was surface-associated or extracellular. It was labeled with D-[14H]glucuronic acid, 35SO42- and to a small extent with 14C-labeled amino acids indicating that it contained glycosaminoglycans derived from epidermal proteoglycans, since it was degraded by testicular hyaluronoglucosidase. It was not present in isolated membranes but was recovered in the soluble fraction from epidermal homogenates. It is very loosely bound at the cell surface or is present in the extracellular spaces. Membrane-bound [3H]glycoproteins were identified after differential centrifugation of epidermal homogenates. The radioactivity profiles of membrane glycoproteins were similar whether L-[3H]fucose or D-[3H]glucosamine were used and both consisted of a major heterogeneous peak in the apparent MW range 70,000-150,000. [3H]-Glycoproteins in this MW range were major components of a plasma membrane enriched fraction. These glycoproteins were probably bound to the membrane by hydrophobic interactions, since they were only solubilized by treatment with detergent or organic solvent. They contained terminal sialic acid residues since they were degraded by neuraminidase.