Inhibition of oligosaccharide processing and membrane morphogenesis in retinal rod photoreceptor cells.
Castanospermine (Cas), an inhibitor of alpha-glucosidase I, blocks "trimming" of the N-linked oligosaccharide Glc3Man9GlcNAc2, thus preventing normal glycoprotein maturation. With use of a dual-label protocol, Xenopus retinas incubated in the presence of Cas exhibited at least a 2.3-fold increase in the incorporation of [3H]mannose into total retina Cl3CCOOH-precipitable material, whereas incorporation of [14C]leucine was not significantly affected, relative to controls. Analysis of NaDodSO4/PAGE fluorograms of solubilized retinas and rod outer segment (ROS) membranes indicated a relatively selective effect of Cas on opsin (the rod visual pigment apoglycoprotein). The apparent molecular mass of opsin was increased by approximately 2500 in the presence of Cas; the incorporation of [3H]mannose into opsin was enhanced about 2.3-fold without a significant effect on [14C]leucine incorporation, relative to controls. Electron microscopic autoradiography of retinas incubated for 4 hr with [3H]mannose showed that the number of newly formed ROS discs in Cas-treated retinas was not significantly different from controls, but the silver grain density over those discs was about 2.6-fold greater than in controls. The morphology of the newly formed discs was comparable under both conditions. Thus, opsin bearing abnormally large oligosaccharides can be accommodated in the process of disc morphogenesis. These results suggest that the structural requirements for opsin's oligosaccharides, with regard to their potential role as determinants of disc morphogenesis, are not stringent. Furthermore, post-translational processing of N-linked oligosaccharides is not essential for the normal intracellular routing and cell surface expression of membrane glycoproteins.
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D005136:Eye Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D007212:Indolizines; D007425:Intracellular Membranes / Q000187:drug effects / Q000648:ultrastructure; D010786:Photoreceptor Cells / Q000378:metabolism* / Q000648:ultrastructure; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012160:Retina / Q000378:metabolism* / Q000648:ultrastructure; D017299:Rod Opsins; D014982:Xenopus laevis; D054836:Indolizidines
D000470:Alkaloids; D005136:Eye Proteins; D006023:Glycoproteins; D007212:Indolizines; D017299:Rod Opsins; C037806:castanospermine; D054836:Indolizidines