Function of the Carbohydrate Moieties of Glycoproteins

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To determine the function of the carbohydrate moiety of glycoproteins, we have used tunicamycin, an analog of N‐acetylglucosamine, to inhibit the glycosylation of N‐glycosidically linked glycoproteins. First, we examined the effect of this drug on the intracellular processing, export and biological activity of fibronectin–the major cell surface glycoprotein of chick embryo fibroblasts. Chick fibroblasts treated with tunicamycin produced only nonglycosylated fibronectin and the export or secretion of the carbohydrate‐free protein species was not totally impaired. We did observe that there was a substantial decrease in the absolute amount of nonglycosylated fibronectin on the cell surface and in the culture medium. This decrease was shown to be due to increased proteolytic degradation of the nonglycosylated protein species. To examine the biological activity of nonglycosylated fibronectin, we compared the activities of the glycosylated and nonglycosylated forms of this protein utilizing in vitro assay procedures. We have shown that isolated, nonglycosylated fibronectin retained the biological properties characteristic of the glycosylated protein; they are: 1) promotion of cell‐cell and cell‐substratum adhesion, 2) restoration of normal behavior and phenotype to transformed cells, and 3) promotion of cell binding to collagen. The isolated, nonglycosylated protein was shown to be more sensitive to degradation by proteolytic enzymes, in agreement with the data obtained “in vivo.” The requirement of glycosylation for the export of acetylcholine receptor was also examined. We found that treatment of embryonic muscle cells in culture with tunicamycin did not inhibit the export of this protein to the cell surface. As with fibronectin, there was a substantial decrease in the amount of receptor present on the cell surface, due to enhanced proteolysis of the nonglycosylated protein. The simultaneous treatment of cells with the protease inhibitor leupeptin diminished the rate of degradation of the nonglycosylated receptor and restored the expression of receptor on the cell surface. Finally, the requirement for N‐glycosidically linked glycoproteins during differentiation of embryonic myoblasts into multinucleated, functional muscle fibers was also investigated. Tunicamycin blocked the expression of glycoproteins on the cell surface and strongly inhibited fusion when added to cultures of differentiating muscle cells prior to fusion. The inhibition of fusion was partially prevented when tunicamycin was administered in the presence of protease inhibitors such as leupeptin and pepstatin. Both glycosylation and fusion were completely restored to normal after removal of tunicamycin from the medium. These studies provide strong support for the idea that myoblast fusion is partially mediated by surface glycoproteins with asparagine‐linked oligosaccharides. However, the requirement for the carbohydrate portion of the glycoprotein appears to be indirect in that it acts to stabilize the protein moiety against proteolytic degradation. To elucidate the mechanism responsible for the enhancement of proteolysis of cell surface glycoproteins following treatment with tunicamycin, we investigated the effect of tunicamycin on the intracellular processing of proteolytic enzymes. Treatment of chick embryo fibroblasts with tunicamycin resulted in more than a 10‐fold increase in the amount of protease activity released into the culture medium. The enzyme activity has been tentatively identified as cathepsin B based on substrate specificity, pH optimum and inhibition with leupeptin. These results as well as extensive work by other investigators [see references [1–11] for recent reviews] suggest that the carbohydrate moiety of surface glycoproteins is not required for their synthesis, secretion or biological function, but instead helps to protect the protein against proteolytic degradation. In contrast, in agreement with the results of Neufeld et al [12–24] and Sly et al [15, 16], the carbohydrate moiety of lysosomal enzymes is required for their intracellular retention. Copyright © 1982 Alan R. Liss, Inc.

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