Edward L. Kean

Enzymatic deglycosylation of bovine rhodopsin.

We have investigated the action of three endo N-acetylglucosaminidases on rhodopsin. The oligosaccharide chains of native and denatured opsin and rhodopsin, both solubilized and membrane-bound, were shown to be cleaved by endohexosaminidase H, endohexosaminidase F, and peptide-N-glycosidase F (PNGase F) as revealed by SDS-PAGE. These enzymes were shown to be free of protease activity. Under correct conditions, the endoglycosidases could release one or both carbohydrate chains. Rhodopsin and opsin at concentrations between 2 and 65 nmol ml-1 were cleaved, with more complete deglycosylation occurring at the higher concentrations.

Effect of tunicamycin on the glycosylation of rhodopsin.

Abstract The incorporation of [3H]glucosamine, [3H]mannose, and [35S]methionine into rhodopsin was investigated in retinas which had been incubated in the presence and absence of the antibiotic, tunicamycin. In its presence, the incorporation of glucosamine was inhibited 70% and mannose, 96% compared to controls. In the presence of tunicamycin the attachment of glucosamine to core-region sites was virtually eliminated. The formation of unglycosylated rhodopsin was also indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concanavalin A-Sepharose chromatography. These findings are consistent with the participation of the lipid-linked pathway in the glycosylation of this well-characterized intrinsic glycoprotein of the membranes of the disk of the rod outer segment. As indicated by the incorporation of [35S]methionine, the synthesis of rhodopsin apoprotein was inhibited by a much lesser amount. This suggests that the glycosylation of rhodopsin is not required for its insertion into the disk membrane.

Improved, rapid radioimmunoassay for rhodopsin

An improved radioimmunoassay for rhodopsin has been devised using [125I]rhodopsin, antibodyto purified bovine rhodopsin and protein A-bearing Staphylococcus aureus, serving as a solid phase immunoadsorbent in place of a second antibody. The method retains the high degree of sensitivity of the previous method (Lentrichia, Plantner and Kean, 1980), while being much more rapid and economical.

Regulation of the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol, feedback and product inhibition.

The assembly of the core oligosaccharide region of asparagine-linked glycoproteins proceeds by means of the dolichol pathway. The first step of this pathway, the reaction of dolichol phosphate with UDP-GlcNAc to formN-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-dolichol), is under investigation as a possible site of metabolic regulation. This report describes feedback inhibition of this reaction by the second intermediate of the pathway,N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-GlcNAc-P-P-dolichol), and product inhibition by GlcNAc-P-P-dolichol itself. These influences were revealed when the reactions were carried out in the presence of showdomycin, a nucleoside antibiotic, present at concentrations that block the de novo formation of GlcNAc-GlcNAc-P-P-dolichol but not that of GlcNAc-P-P-dolichol. The apparent K i values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol under basal conditions were 4.4 and 2.8 μm, respectively. Inhibition was also observed under conditions where mannosyl-P-dolichol (Man-P-dol) stimulated the biosynthesis of GlcNAc-P-P-dolichol; the apparent K i values for GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol were 2.2 and 11 μm, respectively. Kinetic analysis of the types of inhibition indicated competitive inhibition by GlcNAc-P-P-dolichol toward the substrate UDP-GlcNAc and non-competitive inhibition toward dolichol phosphate. Inhibition by GlcNAc-GlcNAc-P-P-dolichol was uncompetitive toward UDP-GlcNAc and competitive toward dolichol phosphate. A model is presented for the kinetic mechanism of the synthesis of GlcNAc-P-P-dolichol. GlcNAc-P-P-dolichol also exerts a stimulatory effect on the biosynthesis of Man-P-dol, i.e. a reciprocal relationship to that previously observed between these two intermediates of the dolichol pathway. This network of inhibitory and stimulatory influences may be aspects of metabolic control of the pathway and thus of glycoprotein biosynthesis in general.

The rhodopsin content of the human eye

The content of rhodopsin in the human retina was determined using a radioimmunoassay which measures both rhodopsin and opsin. A value of 0.5 nmole of rod visual pigment per mg of extractable protein was obtained, corresponding to 3.94 +/- 0.17 (mean +/- SEM, n = 42) nmole per eye. This value is slightly higher than those previously reported which were based on spectral determinations measuring only native rhodopsin. No significant difference was seen in the population studied with respect to age or sex. Human rhodopsin was partially purified by affinity chromatography on concanavalin A, and some of its properties were studied. A comparison of the immunological reactivity of human, bovine, rat and chicken rhodopsin indicated similarity among the three mammalian species, while the chicken was some 50 fold less reactive.

Radioimmunoassay for rhodopsin

Abstract A radioimmunoassay for rhodopsin was devised using [125I]rhodopsin and antibody to purified bovine rhodopsin in a double antibody procedure that can quantify the concentration of this visual pigment when present in picomolar amounts. The method demonstrated a high degree of sensitivity, reproducibility, and specificity, and is applicable to the determination of the rhodopsin concentration in crude extracts of tissue as well as in more purified preparations.

The enzymatic cleavage of rhodopsin by the retinal pigment epithelium. II. The carbohydrate composition of the glycopeptide cleavage product

The carbohydrate groups present in the glycopeptide cleaved from purified [3H]-GlcNAc-rhodopsin by an enzyme from the retinal pigment epithelium were analyzed in terms of the size of the oligosaccharide chains, the sequence of the sugars and their anomeric linkages. These analyses were performed also on intact rhodopsin and an enzyme control. The oligosaccharides were cleaved from the peptide by means of hydrazinolysis. After reduction with NaB3H4, the oligosaccharides, purified by paper electrophoretic and paper chromatographic means, were digested sequentially with exoglycosidases, and the resultant digestion products examined by high-resolution gel filtration on Bio-Gel P-4 (-400). The size, sequence and anomeric configuration of the constituents of the oligosaccharides present in the glycopeptide were essentially identical to those present in intact purified rhodopsin, indicating the retention by the glycopeptide of the sugar chains of the intact molecule.

On the existence of several isoelectric forms of bovine rhodopsin

Abstract Isoelectric focusing studies carried out on purified bovine rhodopsin revealed the presence of several forms which differed in their isoelectric points. Three major species of rhodopsin were detected having isoelectric points of 4·99, 5·33 and 5·91. Upon bleaching, only one form of opsin was generated which had an isoelectric point of 5·22.

The influence of carbohydrates on the binding of rod outer-segment (ROS) disc membranes and intact ROS by the cells of the retinal pigment epithelium of the embryonic chick*

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.

Mannosyl transferases of the retina: Mannolipid and complex glycan biosynthesis I. Kinetic properties; Product identification

Abstract The retina was shown to contain enzymes that catalyse the transfer of mannose from GDP-[U 14 C]mannose to endogenous acceptors resulting in the synthesis of a mannose-containing lipid. This product had the properties of the polyprenyl-phosphate monosaccharides. In addition, the transfer to a mannose-containing complex glycan was also observed. The optimal conditions for both of these enzymatic reactions were similar. The optimal pH for these reactions in 0·2 m -TES buffer was 7·0. Enzymatic activity did not require the addition of a metal ion, although both reactions were stimulated optimally by 3·3 m m -MnCl 2 . Both reactions were inhibited by EDTA. A high degree of specificity was observed for GDP-mannose as the substrate. The apparent K m for GDP-mannose in the formation of the mannolipid was 1·4 μ m . Mannose was the only radioactive sugar detected in both products. The complex glycan, present as an acid insoluble, chloroform/methanol (2:1)-extracted residue, was solubilized by pronase, from which a low molecular weight (about 7000), mannose containing product was obtained. The mannolipid had chromatographic and acid-base stability properties which were similar to those of dolichyl-phosphate mannose.