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 form N-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 microM, 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 microM, 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.

Stimulation as well as inhibition by antibiotics of the formation of GlcNAc-lipids of the dolichol pathway.

The antiobiotics, diumycin, amphomycin, bacitracin, and showdomycin have been shown previously to block the synthesis of GlcNAc-P-P-dolichol and GlcNAc-GlcNAc-P-P-dolichol. In view of inconsistencies in the literature concerning the sites of inhibition, we have reinvestigated the influence of these drugs on the formation of these early intermediates of the dolichol pathway. Unexpectedly, when the individual products of the reactions were examined, instead of inhibition, showdomycin and bacitracin were found to stimulate the formation of GlcNAc-P-P-dolichol, and diumycin stimulated at low concentrations. Three derivatives of showdomycin were examined with similar results, showing stimulations of GlcNAc-P-P-dolichol formation of up to two-fold over controls. Amphomycin specifically inhibited GlcNAc-P-P-dolichol formation, an effect that was reversed by a high concentration of dolichyl phosphate. In contrast, with the exception of amphomycin, each compound directly inhibited the formation of GlcNAc-GlcNAc-P-P-dolichol. Using chemically synthesized GlcNAc-P-P-dolichol as substrate, the kinetics of inhibition of GlcNAc-GlcNAc-P-P-dolichol formation by showdomycin, bacitracin and diumycin was examined. The apparent Ki values calculated from these studies indicated that showdomycin was the most active inhibitor. These findings provide a new understanding of the action of these compounds on the GlcNAc-transferases of the dolichol pathway.

Kinetics of formation of GlcNAc-GlcNAc-P-P-dolichol by microsomes from the retina of the embryonic chick.

Little quantitative information is available concerning individual reactions of the dolichol pathway. We have investigated the kinetics of the GlcNAc-transferase that catalyzes the biosynthesis of GlcNAc-GlcNAc-P-P-dolichol using chemically synthesized GlcNAc-P-P-dolichol as the substrate. Using microsomal preparations from the retina of the embryonic chick as enzyme source, optimal incubation conditions of pH, metal ion and detergent concentrations were established, after which apparent kinetic constants (Km and Vmax) were determined under initial rate conditions for GlcNAc-P-P-dolichol and UDP-GlcNAc. These studies provide the first quantitative description of the kinetics of this reaction.

Analysis of the oligosaccharide chains of rhodopsin from normal rats and those with hereditary retinal dystrophy.

The nature of the oligosaccharides of rhodopsin from normal rats and from the Royal College of Surgeons (RCS) rats was examined by chemical, enzymatic and chromatographic procedures. This report is the first description of the structures of oligosaccharides of rat rhodopsin. The major oligosaccharide isomer of rat rhodopsin was shown to have the same structure as that from cow, human and frog. No neutral galactosylated species were detected. Although the site of the dystrophy in the RCS rat has been shown to be located at the retinal pigment epithelium, the possibility was examined that alterations in the glycosylation of rhodopsin might also be present. No differences were observed in either the amounts or structures of the rhodopsin oligosaccharide chains from young or adult control rats and RCS rats, although some differences were observed in the relative distribution of some oligosaccharide isomers between the RCS and controls. In addition, no differences in the amino acid content or SDS-PAGE patterns were detected.

Site of stimulation by mannosyl-P-dolichol of GlcNAc-lipid formation by microsomes of embryonic chick retina.

Mannosyl-P-dolichol (man-P-dol) has been shown to stimulate the early reactions of the dolichol pathway, specifically, the biosynthesis of GlcNAc-P-P-dol and GlcNAc-GlcNAc-P-P-dol, and thus may play a regulatory role in glycoprotein biosynthesis. The site of action of man-P-dol has previously been suggested to be the GlcNAc-transferase concerned with the formation of the monoglucosaminyl derivative. Since the concentration of the chitobiosyl compound also increases as a result of the presence of man-P-dol, the immediate site of the activation was reexamined. The effect of man-P-dol on the formation of GlcNAc-GlcNAc-P-P-dol using GlcNAc-P-P-dol synthesized in situ or added exogenously as the substrate was investigated. In addition, the distribution of radioactivity in the glucosaminyl constituents of the products under the stimulatory conditions was determined. The results of these studies supported the conclusion that the stimulation of GlcNAc-lipid synthesis by man-P-dol is due to the enhanced synthesis of GlcNAc-P-P-dol. It is not a result of the activation of the GlcNAc-transferase catalyzing the attachment of the second GlcNAc residue for the biosynthesis of the chitobiosyl derivative.

Galactosylation of rhodopsin by the human retina.

The major oligosaccharide chains of bovine, frog and human rhodopsins are abridged, hybrid, asparagine-linked structures that contain only mannose and N-acetylglucosamine as the constituent sugars. Isomers that also contain galactose have been detected in varying amounts in rhodopsins from different species. As part of studies to examine the mechanisms used by the human retina to control the glycosylation of rhodopsin, the kinetics of the retinal galactosyltransferase was examined using Golgi-enriched fractions of human retina as the enzyme source and rhodopsin, opsin, and the oligosaccharide isolated from rhodopsin as acceptors. These reactions were compared to those using bovine retina and rat liver Golgi. A comparison of the Vmax/Km ratios revealed that the efficiency of the human retinal galactosyltransferase was some 20-fold lower than that of the rat liver. In addition, consistent with the higher state of galactosylation of human as compared to bovine rhodopsin, greater efficiency was observed with the human preparation. While the conformation of the visual pigment exerted an effect, its low galactosylation was not due to a major directing influence on glycosylation by the polypeptide matrix as indicated by the even lower activity toward the isolated oligosaccharide. The galactosylated oligosaccharides obtained by these procedures were identified by chromatographic methods. The relatively low activity of the retinal galactosyltransferases observed in this study may help explain the limited glycosylation that is typical of rhodopsin.

Retinal GlcNAc-transferases and the glycosylation of rhodopsin.

The major oligosaccharide chains of bovine, frog and human rhodopsins are identical in structure. These molecules are unique in terms of their abridged size as compared to other asparagine-linked glycoproteins, a property which must be reflected in the activities and properties of the glycosyltransferases of the retina that are concerned with their biosynthesis. One of the unusual characteristics of the major oligosaccharide chain is its lack of branching. A key enzymatic step required for branching to occur would be the transfer of a residue of N-acetylglucosamine to the terminal unsubstituted mannose residue of the major rhodopsin oligosaccharide. We have investigated the kinetic properties of enzymes that would catalyse this process. The N-acetylglucosaminyl-transferases (GlcNAc-transferases) of Golgi-enriched fractions of bovine retina, human retina, rat liver Golgi and a partially purified GlcNAc-transferase II from rat liver were examined using as acceptors rhodopsin, opsin and the oligosaccharide isolated from rhodopsin. The identification of the substrates and products of the reactions was carried out by chromatographic means. From an evaluation of the Vmax/K(m) ratios it was observed that bovine and human retinas have very limited abilities compared to the rat liver enzymes to carry out the transfer of GlcNAc to these acceptors, showing from 100- to about 800-fold lower efficiency in this regard. A comparison of the activities obtained with intact rhodopsin or opsin and the rhodopsin oligosaccharide indicated that the activity of GlcNAc-transferase II was not appreciably influenced by the polypeptide portion of the molecule. It was also observed that prior galactosylation of rhodopsin blocked the addition of GlcNAc to rhodopsin. It is suggested that these properties contribute to the assembly of the abridged structures that have been observed in the oligosaccharides of rhodopsins of all species thus far examined.

Structural studies of the N-linked sugar chains of human rhodopsin.

Human rhodopsin is a glycoprotein containing two N-linked sugar chains. After the isolation and purification of rhodopsins from human retinas, structural studies of their N-linked sugar chains were performed. The sugar moieties, quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis, were converted to radioactive oligosaccharides by reduction with NaB3H4 after N-acetylation. As indicated by high-voltage paper electrophoresis, > 96% of the sugar chains were free of sialic acid and the remaining were sialylated derivatives. Structural studies of each oligosaccharide by lectin affinity column chromatography, and sequential exoglycosidase digestion in combination with methylation analysis, revealed that almost all of the oligosaccharides were hybrid-type sugar chains. While the major oligosaccharide species of bovine and human rhodopsin are identical, in contrast to the sugar chains of bovine rhodopsin, human rhodopsin also contains sialylated isomers and a high concentration of a galactosylated isomer. These results suggest that species-specific processing of the sugar chains of rhodopsin occurs.

Activation of GlcNAc-P-P-dolichol synthesis by mannosylphosphoryldolichol is stereospecific and requires a saturated alpha-isoprene unit.

Exogenous mannosylphosphoryldolichol (Man-P-Dol) has previously been shown to stimulate UDP-GlcNAc:dolichyl phosphate N-acetylglucosamine 1-phosphate transferase (GPT1), the enzyme catalyzing the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-P-P-Dol). To define the structural specificity of the mannolipid-mediated activation of GPT1, the ability of a variety of mannosylphosphorylisoprenols to stimulate GlcNAc-lipid biosynthesis in microsomal preparations from retinas of the embryonic chick has been tested. For these comparisons several Man-P-isoprenols were synthesized enzymatically and chemically. The catalytic efficiency of activation expressed as the Vmax/Ka ratio was substantially higher for Man-P-Dol95 than for mannosylphosphorylpolyprenol95 (Man-P-Poly95), demonstrating that the saturated alpha-isoprene unit of the dolichyl moiety influences the mannolipid-enzyme interaction. The degree of activation increased with chain length and hydrophobicity of the dolichyl moiety when Man-P-dolichols containing 2, 11, and 19 isoprene units were evaluated. A strict stereospecificity was exhibited as beta-Man-P-Dol95 provided a 100-fold greater stimulation than the corresponding alpha-stereoisomer. The recognition of the saturated alpha-isoprene unit, the influence of chain length, and the strict stereospecificity of the interaction between beta-Man-P-Dol and GPT1 extend the description of the mannolipid-enzyme interaction and provide strong new evidence that Man-P-Dol levels can influence the rate of GlcNAc-P-P-Dol synthesis.

Topography of basal and stimulated biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol: variation in effect of proteolysis.

Evidence has recently appeared indicating the cytoplasmic orientation in microsomal vesicles of the GlcNAc-transferases that participate in the biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol. The topography of the stimulation of these activities brought about by dolichol-P-mannose and phosphatidylglycerol, however, is not known. The present report continues studies of the topography of these early reactions of the dolichol pathway, examined under basal and stimulatory conditions after proteolysis of intact microsomes isolated from livers of the embryonic chick. Under all conditions which were examined, the GlcNAc-transferase concerned with the biosynthesis of the chitobiosyl derivative was completely inhibited, consistent with its cytoplasmic orientation. The effect of proteolysis on the formation of GlcNAc-P-P-dolichol, however, varied with different proteolytic enzymes, ranging from no inhibition to over 90% inhibition. This variation in response suggests that the GlcNAc-transferase may be partially buried within the microsomal bilayer which only some preparations of proteolytic enzymes can penetrate. Evidence was obtained indicating the cytoplasmic orientation of the stimulation of GlcNAc-P-P-dolichol biosynthesis. These studies support the conclusion that both the catalytic and allosteric activating sites of the GlcNAc-transferase have a similar topography.

In vitro galactosylation of rhodopsin and opsin: kinetics, properties and characterization.

At best, only trace amounts of galactose have been detected as constituents of rhodopsin as analysed by several laboratories. Nevertheless, the enzymatic galactosylation of rhodopsin proceeds readily in vitro, a process which can be catalysed by galactosyltransferases from several sources. Little information is available, however, concerning the properties of the in vitro reaction. We have examined characteristics of the latter process with the hope of shedding light on the capacity of the retina to carry out this reaction. Kinetic properties of the galactosyltransferases of bovine and embryonic chick retinas, bovine milk and rat liver-Golgi were examined using rhodopsin, opsin, N-acetylglucosamine and ovalbumin as exogenous acceptors. All of these studies demonstrated the very limited activity of the galactosyltransferases of the retina as compared to the milk and rat liver systems. The subcellular distribution of the galactosyltransferases of bovine retina was examined. The influence of compounds that might modulate the reaction was also examined. alpha-Lactalbumin, a modifier of the galactosyltransferase in milk, acted as a competitive inhibitor of the galactosylation of opsin. Analogs of vitamin A, shown to inhibit galactosyltransferases in other systems, did not have this effect on the galactosylation of opsin. Similarly, mixing experiments could not demonstrate the presence of endogenous material that inhibited the reaction in the retina. The conformation of the visual pigment was shown to influence the reaction. After bleaching by visible light, opsin was preferred over rhodopsin as an acceptor of galactose by the galactosyltransferases of bovine and embryonic chick retinas and by rat liver. This distinction was only minimally demonstrated by the milk enzyme. The galactosylation of ovalbumin was unaffected by conditions of light or dark by any of the enzymes. While the mode ratio of galactose to rhodopsin after catalysis by the milk enzyme was about 1.6, this ratio was only about 0.01 after reaction with the enzyme from bovine retina. The linkage of galactose in enzymatically galactosylated rhodopsin and opsin was beta(1-4).

Studies on the activation by dolichol-P-mannose of the biosynthesis of GlcNAc-P-P-dolichol and the topography of the GlcNAc-transferases concerned with the synthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol: a review.

A review is presented of research carried out in this laboratory on two aspects of the dolichol pathway that may have regulatory influences on these events. (i) The validity of the phenomenon of the activation of the biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol by dolichol-P-mannose is supported by experiments carried out on the Thy-1-negative mouse lymphoma cell. While this cell cannot synthesize the activating compound, this capacity was retained and revealed upon the addition of exogenous dolichol-P-mannose. (ii) The topographical orientation of the GlcNAc-transferases that catalyze the biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol was investigated in microsomes from the liver of the embryonic chick using dolichol phosphate liposomes as an exogenous substrate. The formation of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol was inhibited by trypsinization under conditions where the native orientation of the microsome was maintained, as indicated by the latency of mannose-6-phosphatase. Both GlcNAc-lipids were detected on free liposomes after incubation with intact microsomes, and in the same proportions as found on the microsome. From these and other studies, evidence was obtained indicating the cytoplasmic orientation of the GlcNAc-transferases that catalyze the synthesis of the first two intermediates of the dolichol pathway.

Effect of enzymatic deglycosylation on the regenerability of bovine rhodopsin.

The influence of the carbohydrate groups of rhodopsin on its ability to regenerate upon incubation with 11-cis retinaldehyde after photobleaching was examined. Rhodopsin was deglycosylated enzymatically with peptide-N-glycosidase F (PNGase F). Verification of deglycosylation was established by: (a) SDS-PAGE; (b) carbohydrate compositional analysis using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD); (c) isolation and carbohydrate analysis by HPAEC-PAD and fast atom bombardment-mass spectrometry of the oligosaccharides liberated from rhodopsin; and (d) absence of reactivity with lectins. Deglycosylated rhodopsin, when present either in rod outer segments or after purification, exhibited the same absorption spectrum as the native molecule. After photobleaching, deglycosylated rhodopsin reacted with 11-cis retinaldehyde in a manner similar to the native material, restoring the spectral properties lost after light-exposure. The carbohydrate portion, therefore, was not required for expressing the spectral properties of rhodopsin nor for regeneration of the photobleached visual pigment.

Reductively methylated, tritiated rhodopsin of high specific activity; a convenient sensitive tracer for use in the radioimmunoassay of rhodopsin.

Bovine rhodopsin was subjected to reductive methylation in the dark using formaldehyde and high specific activity sodium borotritide. After purification by gel filtration and affinity chromatography on Concanavalin A-Sepharose, the product retained its immunoreactive properties. [3H]-Reductively methylated rhodopsin (specific activity, 32 Ci/mmole) was suitable for use in radioimmunoassays for rhodopsin, having many advantages over radioiodinated rhodopsin for this purpose. The site of the reductive methylation was shown to be the non-active site lysines with the production of tritiated N-epsilon-dimethyllysine and tritiated N-epsilon-methyllysine in a molar ratio of about 1.3:1, respectively. In terms of stability, ease of preparation, and specificity, tritiated, reductively methylated rhodopsin presents itself as a preferable ligand to radioiodinated rhodopsin in many applications, such as the radioimmunoassay.

Sialic acid activation.

Cytidine 5'-monophosphosialic acid (CMP-sialic acid) is the activated form of sialic acid which is required for the biosynthesis of sialic acid-containing complex carbohydrates. Its discovery over 30 years ago by the laboratory of Dr Saul Roseman was a landmark in research dealing with the biosynthesis of these compounds. A review is presented of the salient features concerning this molecule: its discovery, chemistry, biosynthesis, subcellular location, enzymatic cleavage, transport and molecular biology. This report does not deal with its utilization by the sialyltransferases.

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.

Topographical orientation in microsomal vesicles of the N-acetylglucosaminyltransferases which catalyze the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol and N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol.

The orientation of the N-acetylglucosaminyltransferases which catalyze the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol and N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol was examined using microsomes isolated from livers of the embryonic chick. Dolichol phosphate liposomes were used as the exogenous substrate, thus avoiding the use of detergents. The formation of both of these intermediates of the dolichol pathway was almost completely inhibited after trypsinization of the microsomes under conditions in which microsomal integrity was maintained as evaluated by determining the latency of mannose-6-phosphatase. Using dolichol [32P]phosphate liposomes, it was shown that trypsinization did not interfere with the binding of liposomes to the microsome, suggesting that the effect of trypsinization was on the GlcNAc-transferases. Although dolichol phosphate liposomes associate with the microsomes during incubation, it was possible by sucrose density centrifugation to separate a population of liposomes which were not bound in this manner. GlcNAc lipids isolated from the free liposomes were shown to contain both the mono-GlcNAc and (GlcNAc)2 derivatives in proportions similar to those obtained from the microsomes. The possibility of transfer of the GlcNAc lipids from endogenous sources on the microsome to the liposome was examined and shown to play little role in these processes. This study strongly suggests a nonluminal, cytoplasmic orientation of the N-acetylglucosaminyltransferases concerned with the biosynthesis of the first two intermediates of the dolichol pathway.

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.