Roberts G P, Jenner L
Biochem J. 1983 May 15;212(2):355-63. doi: 10.1042/bj2120355.
Glycoproteins and proteoglycans synthesized by human keratinocytes in medium containing D-[1-14C]glucosamine were extracted and analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Extraction of the labelled keratinocytes with 0.5% Triton X-100 removed most of the glycoconjugates and left the cytoskeleton and nuclear residue adherent to the substratum. In addition to the cytoskeletal proteins, there was a relatively simple profile of glycoproteins and glycosaminoglycans associated with this adherent cytoskeleton. These consisted of eight glycoproteins in the mol.wt. range 99000-232000, five proteins in the keratin region (mol.wt. 42000-61000), hyaluronic acid and a sulphated glycosaminoglycan. Surface labelling of the keratinocytes with galactose oxidase (with or without neuraminidase)/KB3H4 revealed that many of the glycoproteins were exposed on the cell surface. The importance of the glycoproteins and proteoglycans in attaching the keratinocytes to the substratum was examined by studying their expression after incubation in medium containing tunicamycin and their degradation after digestion with trypsin and hyaluronidase. These studies, together with an examination of the glycoconjugates released by sequential extraction with 0.5% Triton X-100 followed by 0.2% sodium dodecyl sulphate, revealed that the glycoprotein of mol.wt. 232000 has an important role in mediating the attachment of keratinocytes to the substratum.
在含有D-[1-¹⁴C]葡糖胺的培养基中，由人角质形成细胞合成的糖蛋白和蛋白聚糖被提取出来，并在十二烷基硫酸钠存在的情况下通过聚丙烯酰胺凝胶电泳进行分析。用0.5% Triton X-100提取标记的角质形成细胞，去除了大部分糖缀合物，使细胞骨架和核残余物附着在基质上。除细胞骨架蛋白外，与这种附着的细胞骨架相关的糖蛋白和糖胺聚糖的图谱相对简单。这些包括分子量在99000 - 232000范围内的八种糖蛋白、角质蛋白区域的五种蛋白（分子量42000 - 61000）、透明质酸和一种硫酸化糖胺聚糖。用半乳糖氧化酶（有无神经氨酸酶）/KB³H₄对角质形成细胞进行表面标记，发现许多糖蛋白暴露在细胞表面。通过研究在含有衣霉素的培养基中孵育后糖蛋白和蛋白聚糖的表达以及用胰蛋白酶和透明质酸酶消化后的降解情况，来检验它们在角质形成细胞附着于基质中的重要性。这些研究，连同对先用0.5% Triton X-100然后用0.2%十二烷基硫酸钠顺序提取释放的糖缀合物的检查，表明分子量为232000的糖蛋白在介导角质形成细胞附着于基质中起重要作用。
