Lentrichia B B, Itoh Y, Plantner J J, Kean E L
Exp Eye Res. 1987 Jan;44(1):127-42. doi: 10.1016/s0014-4835(87)80031-3.
The role of carbohydrates in mediating the interaction of rhodopsin-containing membranes with retinal pigment epithelium (RPE) cells was investigated by studying the influence of various monosaccharides on their binding by RPE cells of the embryonic chick maintained in cell culture. Rod outer-segment (ROS) disc membranes were selected as a model rhodopsin-containing membrane system for these studies in view of their high concentration of rhodopsin and the relative purity with which they can be isolated. Disc membranes, frozen and thawed in order to expose the carbohydrate groups of rhodopsin which are oriented intraluminally in situ, were incubated with monolayers of RPE cells under various conditions, and the binding of the membranes by the cells was quantitated by radioimmunoassay for rhodopsin. Cell-membrane association was also verified by indirect immunofluorescence microscopy. The surface accessibility of the sugars in frozen-thawed discs was verified by succinyl concanavalin A-binding studies. From 15- to 20-fold increase in carbohydrate-reactive sites was obtained after freezing and thawing the discs. The RPE cell-membrane binding process was saturable, and time- and temperature-dependent. By means of competition studies carried out in the presence of high concentrations of various monosaccharides, and also by comparing the binding of disc membranes whose carbohydrate groups were either exposed (frozen-thawed) on the surface or inaccessible (native), it was concluded that the carbohydrates of rhodopsin, mannose and N-acetylglucosamine, were not involved in the interaction with the RPE. The possibility was also examined that enzymatically galactosylated rhodopsin might serve as a site for recognition by the RPE cell. The binding of ROS disc membranes modified in this manner was not enhanced, indicating that the presence of galactose groups on rhodopsin did not serve as a site for recognition by the RPE. The influence of monosaccharides on the binding of intact ROS by the RPE cells was also investigated. Similar to the results with the disc membranes, the process was not blocked by the presence in the incubation medium of high concentrations (up to 30,000-fold higher than that of rhodopsin) of mannose or GlcNAc, as with the disc membranes, or by glucose or galactose. Thus, from these studies it is concluded that a lectin-like carbohydrate-recognition process may not be involved in the interaction between rhodopsin-containing membranes and the RPE cells.
通过研究各种单糖对细胞培养中鸡胚视网膜色素上皮（RPE）细胞结合含视紫红质膜的影响，来探讨碳水化合物在介导含视紫红质膜与RPE细胞相互作用中的作用。鉴于视杆外段（ROS）盘膜中视紫红质浓度高且易于分离，相对纯净，因此选择其作为含视紫红质膜系统的模型用于这些研究。将盘膜冷冻和解冻，以暴露视紫红质在原位腔内定向排列的碳水化合物基团，然后在各种条件下与RPE细胞单层孵育，通过视紫红质放射免疫测定法定量细胞对膜的结合。细胞膜结合也通过间接免疫荧光显微镜进行验证。通过琥珀酰伴刀豆球蛋白A结合研究验证了冻融盘膜中糖的表面可及性。盘膜冷冻和解冻后，碳水化合物反应位点增加了15至20倍。RPE细胞膜结合过程是可饱和的，并且依赖时间和温度。通过在高浓度各种单糖存在下进行竞争研究，以及比较碳水化合物基团在表面暴露（冻融）或不可及（天然）的盘膜的结合情况，得出结论：视紫红质的碳水化合物、甘露糖和N-乙酰葡糖胺不参与与RPE的相互作用。还研究了酶促半乳糖基化视紫红质作为RPE细胞识别位点的可能性。以这种方式修饰的ROS盘膜的结合没有增强，表明视紫红质上半乳糖基团的存在不是RPE细胞识别的位点。还研究了单糖对RPE细胞结合完整ROS的影响。与盘膜的结果类似，该过程不会因孵育培养基中存在高浓度（比视紫红质高30,000倍）的甘露糖或N-乙酰葡糖胺、葡萄糖或半乳糖而受阻。因此，从这些研究得出结论，含视紫红质膜与RPE细胞之间的相互作用可能不涉及类凝集素碳水化合物识别过程。
