Brandley B K, Schnaar R L
J Biol Chem. 1985 Oct 15;260(23):12474-83.
Cell-cell adhesion is a multi-step process which may be initiated by binding of cell surface carbohydrates to complementary carbohydrate receptors on apposing cell surfaces. We have modeled such interactions using polyacrylamide gels covalently derivatized with glycosides, to which intact cells specifically adhere; chicken hepatocytes adhere to gels derivatized with N-acetylglucosamine (GlcNAc). Initially adhesion is blocked (or reversed) by soluble GlcNAc, but becomes sugar-resistant rapidly at 37 degrees C, perhaps due to cellular modification of the carbohydrate-derivatized surface (Guarnaccia, S. P., Kuhlenschmidt, M. S., Slife, C. W., and Schnaar, R. L. (1982) J. Biol. Chem. 257, 14293-14299). We report here that, subsequent to recognition and adhesion, intact chicken hepatocytes transfer phosphate covalently to GlcNAc-derivatized gels. Metabolically radiolabeled cells (32Pi) were incubated on polyacrylamide gels derivatized with various aminohexyl glycosides. Noncovalently bound material was then removed from the gels by extensive washing in detergents and salt solutions. Subsequent radiochemical analysis revealed that phosphate was transferred selectively to GlcNAc-derivatized gels (up to 20-fold more than to glucose-, galactose-, or mannose-derivatized gels). Soluble GlcNAc (but not other sugars) or low temperature inhibited phosphate transfer. The phosphorylation was mediated by intact cells; cell lysate was itself incapable of specific phosphate transfer and attenuated specific transfer when added to intact cells. When GlcNAc was immobilized using a cleavable (disulfide-containing) linker arm the transferred phosphate radiolabel could be solubilized by disulfide reduction and recovered for further analysis. The released phosphorylated product migrated as a single low molecular weight species upon gel permeation chromatography, paper electrophoresis, and cellulose thin layer chromatography. Acid hydrolysis of the phosphorylated product generated a compound with the mobility of GlcNAc-6-P in five different separation systems. Treatment with alkaline phosphatase converted the radiolabel to a compound with the properties of inorganic phosphate. These data indicate that; subsequent to carbohydrate recognition and adhesion, intact hepatocytes generate phosphomonoesters of recognized carbohydrates outside of their plasma membranes.
细胞间黏附是一个多步骤过程，它可能由细胞表面碳水化合物与相邻细胞表面互补碳水化合物受体的结合引发。我们利用共价衍生有糖苷的聚丙烯酰胺凝胶对这种相互作用进行了建模，完整细胞会特异性地黏附于该凝胶；鸡肝细胞会黏附于用N - 乙酰葡糖胺（GlcNAc）衍生的凝胶。最初，黏附会被可溶性GlcNAc阻断（或逆转），但在37℃时会迅速变得对糖不敏感，这可能是由于碳水化合物衍生表面的细胞修饰（瓜尔纳恰，S. P.，库伦施密特，M. S.，斯利夫，C. W.，以及施纳尔，R. L.（1982年）《生物化学杂志》257卷，14293 - 14299页）。我们在此报告，在识别和黏附之后，完整的鸡肝细胞会将磷酸共价转移至GlcNAc衍生的凝胶上。将经代谢放射性标记的细胞（³²Pi）在衍生有各种氨基己基糖苷的聚丙烯酰胺凝胶上孵育。然后通过在去污剂和盐溶液中大量洗涤从凝胶上去除非共价结合的物质。随后的放射化学分析表明，磷酸被选择性地转移至GlcNAc衍生的凝胶上（转移量比转移至葡萄糖、半乳糖或甘露糖衍生的凝胶上多20倍）。可溶性GlcNAc（而非其他糖类）或低温会抑制磷酸转移。磷酸化作用由完整细胞介导；细胞裂解物本身无法进行特异性磷酸转移，并且当添加到完整细胞中时会减弱特异性转移。当使用可裂解（含二硫键）连接臂固定GlcNAc时，转移的磷酸放射性标记可通过二硫键还原而溶解并回收用于进一步分析。在凝胶渗透色谱、纸电泳和纤维素薄层层析中，释放的磷酸化产物以单一低分子量物质的形式迁移。磷酸化产物的酸水解在五种不同的分离系统中产生了一种具有GlcNAc - 6 - P迁移率的化合物。用碱性磷酸酶处理将放射性标记转化为具有无机磷酸性质的化合物。这些数据表明，在碳水化合物识别和黏附之后，完整的肝细胞在其质膜外生成被识别碳水化合物的磷酸单酯。
