Sasak W, Herscovics A, Quaroni A
Biochem J. 1982 Feb 1;201(2):359-66. doi: 10.1042/bj2010359.
We studied mannose-containing glycopeptides and glycoproteins of subconfluent and confluent intestinal epithelial cells in culture. Cells were labelled with d-[2-(3)H]mannose for 24h and treated with Pronase or trypsin to release cell-surface components. The cell-surface and cell-residue fractions were then exhaustively digested with Pronase and the resulting glycopeptides were fractionated on Bio-Gel P-6, before and after treatment with endo-beta-N-acetylglucosaminidase H to distinguish between high-mannose and complex oligosaccharides. The cell-surface glycopeptides were enriched in complex oligosaccharides as compared with residue glycopeptides, which contained predominantly high-mannose oligosaccharides. Cell-surface glycopeptides of confluent cells contained a much higher proportion of complex oligosaccharides than did glycopeptides from subconfluent cells. The ability of the cells to bind [(3)H]concanavalin A decreased linearly with increasing cell density up to 5 days in culture and then remained constant. When growth of the cells was completely inhibited by either retinoic acid or cortisol, no significant difference was observed in the ratio of complex to high-mannose oligosaccharides in the cell-surface glycopeptides of subconfluent cells. Only minor differences were found in total mannose-labelled glycoproteins between subconfluent and confluent cells by two-dimensional gel analysis. The adhesion of the cells to the substratum was measured at different stages of growth and cell density. Subconfluent cells displayed a relatively weak adhesion, which markedly increased with increased cell density up to 6 days in culture. It is suggested that alterations in the structure of the carbohydrates of the cell-surface glycoproteins are dependent on cell density rather than on cell growth. These changes in the glycopeptides are correlated with the changes in adhesion of the cells to the substratum.
我们研究了培养中的亚汇合和汇合肠上皮细胞含甘露糖的糖肽和糖蛋白。用d-[2-(3)H]甘露糖标记细胞24小时，并用链霉蛋白酶或胰蛋白酶处理以释放细胞表面成分。然后用链霉蛋白酶彻底消化细胞表面和细胞残渣部分，所得糖肽在用内切β-N-乙酰葡糖胺酶H处理前后在Bio-Gel P-6上进行分级分离，以区分高甘露糖型和复合型寡糖。与主要含有高甘露糖型寡糖的残渣糖肽相比，细胞表面糖肽富含复合型寡糖。汇合细胞的细胞表面糖肽所含复合型寡糖的比例比亚汇合细胞的糖肽高得多。在培养长达5天的时间里，细胞结合[(3)H]伴刀豆球蛋白A的能力随细胞密度增加呈线性下降，然后保持恒定。当细胞生长被视黄酸或皮质醇完全抑制时，亚汇合细胞的细胞表面糖肽中复合型与高甘露糖型寡糖的比例没有显著差异。通过二维凝胶分析发现，亚汇合细胞和汇合细胞之间在总甘露糖标记的糖蛋白方面只有微小差异。在生长和细胞密度的不同阶段测量细胞对基质的黏附。亚汇合细胞表现出相对较弱的黏附，在培养长达6天的时间里随细胞密度增加而显著增加。提示细胞表面糖蛋白碳水化合物结构的改变取决于细胞密度而非细胞生长。这些糖肽的变化与细胞对基质黏附的变化相关。
