Mutants of mouse fibroblasts altered in the synthesis of cell surface glycoproteins. Preliminary evidence for a defect in the acetylation of glucosamine 6-phosphate.

作者：Jacques Pouysségur, Ira H Pastan · 发表于：Journal of Biological Chemistry · 年份：1977 · DOI：10.1016/s0021-9258(17)40597-7 · 被引用次数：64 · 研究领域：Glycosylation and Glycoproteins Research、Ubiquitin and proteasome pathways、Chemical Synthesis and Analysis

We hve reported the isolation and preliminary biochemical characterization of two mutants of mouse fibroblasts selected for a decrease incell-to-substrate adhesion (Pouysségur, J., and Pastan, I. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 544-548). We attributed the adhesive defect of these mutants (AD6 and AD8) to the absence of iodinatable cell surface proteins. This study demonstrates that a defect in glycoprotein synthesis is the biochemical basis for the reduction in proteins exposed at the outer surface of the mutant cells. When D-glucosamine or L-fucose was used as radioactive precursor, analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the labeling of all the glycoproteins of both clones. This decrease in glycoprotein labeling was not due to a defect in D-glucosamine uptake, since this precursor was taken up at a higher rate in the mutants than in the wild type. In spite of this high uptake, the rate of D-glucosamine incorporation into macromolecules was decreased by 60% and the carbohydrate content of membranes (mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid) from clone AD6 was diminished by 40 to 60%. When the various cell lines were labeled for 1 to 3 h with glucosamine and the acid-soluble pool analyzed, the wild type cells were found to accumulate UDP-N-acetylhexosamine as the major component. In contrast, clones AD6 and AD8 accumulated glucosamine 6-phosphate as the major component. This l...