Rapid intramolecular turnover of N‐linked glycans in plasma membrane glycoproteins

Abstract

Plasma membrane glycoproteins of rat hepatocytes undergo a rapid terminal deglycosylation in that the terminal sugars of the oligosaccharide side chains are rapidly removed from the otherwise intact glycoproteins [Tauber, R., Park, C. S. Proc. Natl Acad. Sci. USA 80,. The present paper demonstrates that this rapid intramolecular turnover of plasma membrane glycoproteins is not restricted to peripheral sugars but, in contrast to liver, in hepatoma the core sugars of the oligosaccharide chains are also involved. lntramolecular turnover was measured in Morris hepatoma 7777 in five plasma membrane glycoproteins with M , of 85000 (hgp85), 105000 (hgpl05), 115000 (hgp115), 125000 (hgp125), 175000 (hgp175) (hgp = hepatoma glycoprotein) that were isolated and purified to homogeneity by concanavalin-A -Sepharose affinity chromatography and semipreparative SDS gel electrophoresis. Analysis of the carbohydrates of hgp85, hgpl05, hgpll5 and hgp125 revealed the presence of N-linked oligosaccharides containing L-fucose, D-galactose, Dmannose and N-acetyl-D-glucosamine, but only of trace amounts of N-acetyl-u-galactosamine; hgpl75 additionally contained significant amounts of N-acetyl-D-galactosamine, indicating the presence of both N-and 0-linked oligosaccharides. As shown by digestion with endoglucosaminidase H, the N-linked oligosaccharides of hgpl05, hgpll5, hgp125 and hgp175 were of the complex type, whereas hgp85 also contained oligosacharides of the highmannose type. Half-lives of the turnover of the oligosacharide chains and of the protein backbone of the five glycoproteins were measured in the plasma membrane in pulse-chase experiments in vivo, using ~-[~H]fucose as a marker of terminal sugars, ~-[~H]mannose as marker of a core sugar and ~-[~H]leucine for labelling the protein backbone. Protein backbones of the five glycoproteins were degraded with individual half-lives ranging over 41 -90 h with a mean of 66 h. Compared to the degradation of the polypeptide backbone, both the terminal sugar L-fucose and the core sugar D-mannose turned over with much shorter half-lives averaging about 20 h in the five glycoproteins. The data show that, conversely to liver, within plasma membrane glycoproteins of hepatoma not only peripheral sugars but also core sugars of the oligosaccharides are split off during the life-span of the protein backbone. It may therefore be assumed that this reprocessing of plasma membrane glycoproteins is sensitive to malignant transformation.Individual glycoproteins of the hepatocyte plasma membrane undergo continuous turnover, being degraded upon internalization and replaced by newly synthesized glycoproteins [1 -31. Superimposed upon this basic turnover is an additional rapid intramolecular turnover of the terminal sugar residues of the oligosaccharides side chains. As recently shown for several glycoproteins, including dipeptidylpeptidase IV, the terminal sugars L-fucose and N-acetylneuraminic acid are lost from the glycoproteins three to four times faster compared with the degradation of the protei...

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