Hall M O, Burgess B L, Arakawa H, Fliesler S J
Jules Stein Eye Institute, UCLA Medical Center 90024-7008.
Glycobiology. 1990 Sep;1(1):51-61. doi: 10.1093/glycob/1.1.51.
Retinal pigment epithelial cells selectively phagocytize rod outer segments by a process that may be mediated by specific cell surface receptors. Since many receptors are glycoproteins, we have studied the effect of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, and of castanospermine and swainsonine, which are inhibitors of oligosaccharide processing, on the ability of cultured retinal pigment epithelial cells to phagocytize rod outer segment. Tunicamycin inhibits the glycosylation of newly synthesized glycoproteins by 85-90%; concomitantly, the phagocytosis of rod outer segments is inhibited by 70-80%. The effect of tunicamycin is to initially reduce rod outer segments binding, and therefore the subsequent ingestion of rod outer segments. SDS-PAGE analysis and autoradiography of [35S]methionine labelled extracts of tunicamycin-treated cells, demonstrates the disappearance of a number of glycoprotein bands, and the appearance of a number of protein bands of lower Mr. Kinetic analysis of the disappearance and reappearance of specific glycoproteins suggests that the lower Mr bands are the non-glycosylated forms of the higher Mr bands. By contrast, castanospermine and swainsonine have no effect on the ability of retinal pigment epithelial cells to phagocytize rod outer segments, or on the SDS-PAGE pattern of treated cells, although they were shown to inhibit oligosaccharide processing as expected. These results support the hypothesis that rod outer segment phagocytosis by retinal pigment epithelial cells is mediated by specific glycoprotein receptors. N-Glycosylation of these receptors is required for their function, or for their insertion into the plasma membrane, whereas processing of the N-linked oligosaccharide chains of these receptors is not crucial for rod outer segment phagocytosis by retinal pigment epithelial cells.
视网膜色素上皮细胞通过一个可能由特定细胞表面受体介导的过程选择性地吞噬视杆细胞外段。由于许多受体是糖蛋白，我们研究了衣霉素（一种N-连接寡糖合成抑制剂）、蓖麻毒蛋白和苦马豆素（寡糖加工抑制剂）对培养的视网膜色素上皮细胞吞噬视杆细胞外段能力的影响。衣霉素可抑制新合成糖蛋白的糖基化85%-90%；与此同时，视杆细胞外段的吞噬作用被抑制70%-80%。衣霉素的作用是最初减少视杆细胞外段的结合，从而减少随后对视杆细胞外段的摄取。对经衣霉素处理的细胞的[35S]甲硫氨酸标记提取物进行SDS-PAGE分析和放射自显影，显示许多糖蛋白条带消失，出现一些分子量较低的蛋白条带。对特定糖蛋白消失和重新出现的动力学分析表明，分子量较低的条带是分子量较高条带的非糖基化形式。相比之下，蓖麻毒蛋白和苦马豆素对视网膜色素上皮细胞吞噬视杆细胞外段的能力或对处理后细胞的SDS-PAGE图谱没有影响，尽管它们如预期那样抑制了寡糖加工。这些结果支持了以下假设：视网膜色素上皮细胞对视杆细胞外段的吞噬作用是由特定的糖蛋白受体介导的。这些受体的N-糖基化对于其功能或插入质膜是必需的，而这些受体的N-连接寡糖链的加工对于视网膜色素上皮细胞对视杆细胞外段的吞噬作用并不关键。
