肝癌细胞67kD层粘连蛋白受体N-糖链结构与功能的变化

The Role of N-glycans in 67kD Laminin Receptor on Surface of Hepatocellular Carcinoma Cell in Laminin Binding

探讨肝癌细胞与正常肝细胞 6 7kD层粘连蛋白受体 (6 7LR)N 糖链结构与功能的差异 .采用流式细胞术检测SMMC 772 1肝癌细胞和L 0 2正常肝细胞膜表面 6 7LR的表达 ,并分别从这 2株细胞分离纯化到高亲和力的 6 7LR ,利用凝集素结合分析其糖链结构 ,并用肽 N 糖苷酶水解N 糖链 ,观察糖链在与层粘连蛋白结合过程中的作用 .结果发现 ,L 0 2细胞膜表面 6 7LR表达的阳性率为5 5 3% ,而SMMC 772 1细胞为 34.7% ,这两株细胞 6 7LR与伴刀豆素 (ConA)的结合能力无显著差异 ,但SMMC 772 1细胞的 6 7LR与麦胚凝集素的结合能力明显高于L 0 2细胞的 6 7LR ,说明 2株细胞 6 7LR的糖链结构存在显著差异 .当N 糖链被切除后 ,SMMC 772 1细胞的 6 7LR与层粘连蛋白的结合能力明显下降 ,而L 0 2细胞则没有变化 .这些资料表明 ,SMMC 772 1肝癌细胞和L 0 2正常肝细胞与层粘连蛋白结合能力的差别 ,以及两株细胞的 6 7LR与层粘连蛋白结合能力的不同 ,很可能是由于这两株细胞的层粘连蛋白受体的N

To investigate the difference of structure of Asn linked oligosaccharides in purified laminin receptor from SMMC 7721 hepatocellular carcinoma cell and L 02 hepatic cell and its role in laminin binding the expression of 67 kD laminin receptor (67 LR) on the surface of SMMC 7721 hepatocellular carcinoma cell and L 02 hepatic cell was detected by flow cytometric analysis. The structure of Asn linked oligosaccharides in 67 LR purified from these two cell lines was investigated by ELISA and Western blotting with lectins. After de N glycosylation by peptide N glycosidase F, competitive binding assay was carried out to explore the role of Asn linked oligosaccharides in laminin receptor binding to laminin. The expression of 67 LR in L 02 hepatic cell surface notably increased compared with SMMC 7721 HCC cell by flow cytometric analysis. The binding activity of 67 LR from SMMC 7721 cell with wheat germ agglutination (WGA) is significantly higher than that from L 02 cell by ELISA and Western blotting, while with concanavalin A (ConA) there is no significant difference. The result showed that the structure of oligosaccharides between 67 LR from SMMC 7721 and that from L 02 cell was different. De N glycosylation of 67LR from SMMC 7721 cell induced partial loss of laminin binding activity, but had not effect on that from L 02 cell, indicating that the difference of binding activity between SMMC 7721 HCC cell and L 02 hepatic cell with laminin, and binding affinity between 67 LR from the two cell lines with laminin, was likely due to the difference of the structure of Asn linked oligosaccharides in laminin receptors. It has been shown that the Asn linked oligosaccharides play an important role in laminin receptor function.