Youakim A, Romero P A, Yee K, Carlsson S R, Fukuda M, Herscovics A
McGill Cancer Center, McGill University, Montreal, Quebec, Canada.
Cancer Res. 1989 Dec 15;49(24 Pt 1):6889-95.
The proportion of labeled polylactosaminoglycans found in glycoproteins decreases during spontaneous differentiation of CaCo-2 human colonic adenocarcinoma cells to enterocytes in culture (A. Youakim and A. Herscovics, Biochem. J., 247: 299-306, 1987). To identify polylactosaminoglycan-containing glycoproteins, CaCo-2 cells were incubated with [3H]glucosamine or [3H]fucose, for 24 h, and membrane glycoproteins solubilized with 0.5% Nonidet P-40 were fractionated by affinity chromatography on Datura stramonium (DSA)-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that a restricted set of glycoproteins with a molecular weight of about 100,000 bound to DSA-agarose. These labeled glycoproteins were shown to contain polylactosaminoglycans by DSA-agarose chromatography and endo-beta-galactosidase digestion of Pronase-derived glycopeptides. Immunoprecipitation of the [3H]glucosamine-labeled Nonidet P-40 extract with polyclonal antibodies to the lysosomal membrane proteins h-lamp-1 and h-lamp-2 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography also revealed a band with a molecular weight of about 100,000. The immunoprecipitates were digested with Pronase, and the resulting glycopeptides were first fractionated on Bio-Gel P-6 into excluded (Fraction I) and included (Fraction II) glycopeptides, and then by DSA-agarose affinity chromatography. A much greater proportion of labeled glycopeptides in undifferentiated cells (3 to 5 days in culture) than in differentiated cells (19 to 27 days in culture) was recovered in Fraction I; these glycopeptides were bound to DSA-agarose and were sensitive to endo-beta-galactosidase. This decrease in polylactosaminoglycans was associated primarily with h-lamp-1. These results indicate that h-lamp-1 of CaCo-2 cells contains polylactosaminoglycans and that it undergoes a change in glycosylation with differentiation.
在培养过程中，人结肠腺癌CaCo-2细胞自发分化为肠上皮细胞时，糖蛋白中标记的聚乳糖胺聚糖比例会降低（A. 尤亚金和A. 赫斯科维茨，《生物化学杂志》，247: 299 - 306, 1987）。为了鉴定含聚乳糖胺聚糖的糖蛋白，将CaCo-2细胞与[³H]葡糖胺或[³H]岩藻糖孵育24小时，用0.5% 非离子去垢剂P - 40溶解的膜糖蛋白通过在曼陀罗（DSA）-琼脂糖上进行亲和层析进行分离。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和荧光显影显示，一组分子量约为100,000的受限糖蛋白与DSA - 琼脂糖结合。通过DSA - 琼脂糖层析和对蛋白酶消化产生的糖肽进行内切β - 半乳糖苷酶消化，证明这些标记的糖蛋白含有聚乳糖胺聚糖。用针对溶酶体膜蛋白h - lamp - 1和h - lamp - 2的多克隆抗体对[³H]葡糖胺标记的非离子去垢剂P - 40提取物进行免疫沉淀，随后进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和荧光显影，也显示出一条分子量约为100,000的条带。用蛋白酶消化免疫沉淀物，将产生的糖肽首先在Bio - Gel P - 6上分离为排阻（组分I）和包含（组分II）糖肽，然后通过DSA - 琼脂糖亲和层析进行分离。与分化细胞（培养19至27天）相比，未分化细胞（培养3至5天）中回收的标记糖肽在组分I中的比例要高得多；这些糖肽与DSA - 琼脂糖结合且对内切β - 半乳糖苷酶敏感。聚乳糖胺聚糖的这种减少主要与h - lamp - 有关。这些结果表明，CaCo-2细胞的h - lamp - 1含有聚乳糖胺聚糖，并且其糖基化会随着分化而发生变化。
