The carbohydrate chains of rhodopsin are unique as compared to other asparagine-linked glycoproteins, suggesting the presence of special mechanisms which control their biosynthesis. We wish to investigate this regulatory process. (1) We shall examine the possibility that the assembly of its oligosaccharides is influenced by signals encoded in the polypeptide backbone of opsin. A key step in the formation of the complex type of oligosaccharide chain would be the addition of a GlcNAc residue to the alpha (1- 6)-mannose arm of the major oligosaccharide of rhodopsin. What directs the retina not to have this structure on rhodopsin? (a) In order to explore the influence of the polypeptide on this reaction, we will examine the activity of purified GlcNAc-transferase II from rat liver as well as that of Golgi preparations from the retina using the purified oligosaccharide of rhodopsin as acceptor of GlcNAc, as compared to using opsin, rhodopsin, or glycopeptides which contain this same oligosaccharide. (b) We shall also examine the galactosylation of rhodopsin in the same manner. (c) We will alter the primary structure of opsin by incorporating amino acid analogs into the polypeptide and examine the effect of this alteration on the glycosylation of bovine rhodopsin. (2) We shall explore the Golgi of the retina for the presence of beta-galactosidase and exohexosaminidase activities using pertinent analogs of opsin as substrates. (3) We shall characterize the oligosaccharides of the processing intermediates involved in the glycosylation of rhodopsin. Pre- opsins synthesized in the inner segment region of the rod cell will be isolated by immunological techniques. The oligosaccharides will be liberated enzymatically, and their structures investigated. The effect of inhibitors of processing will aslo be examined. The techniques of analysis by HPLC, plus enzymatic and chemical processing will also be examined. The techniques of analysis by HPLC, plus enzymatic and chemical degradation, will be used in all of these studies. We shall continue our studies dealing with metabolic regulation of the dolichol pathway. Previously, we have shown that dolichol-P- mannose acts as an allosteric activator of the enzyme that catalyzes the first reaction of the pathway. We shall examine the influence of other intermediates of the dolichol pathway on the activating phenomenon. These investigations may reveal additional aspects of control for this key aspect of glycoprotein biosynthesis. The activity of glucosidases of the retina toward Glc-oligosaccharide-lipids will be examined. All of these studies may provide information concerning the biosynthesis of complex carbohydrates by the retina.
