Biosynthesis of N-acetylglucosamine-P-P-dolichol, the committed step of asparagine-linked oligosaccharide assembly.

Asparagine-linked glycosylation is initiated by the synthesis of N-acetylglucosaminylpyrophosphoryl dolichol (GlcNAc-P-P-dolichol), which is extended by a series of glycosyltransferases to yield Glc3Man9GlcNAc2-P-P-dolichol (where Glc is glucose and Man is mannose). The oligosaccharide unit is then transferred en bloc to asparagine residues of nascent polypeptides in the lumen of the rough endoplasmic reticulum. The question here is whether GlcNAc-P-P-dolichol biosynthesis is a fixed process unaffected by cellular events, or a regulated reaction responsive to cellular requirements for glycoprotein biosynthesis. Several lines of evidence indicate that the latter is the case and that GlcNAc-P-P-dolichol biosynthesis may be subject to multiple forms of regulation. Recent information about the N-acetylglucosamine-1-P transferase (GPT) responsible for this reaction and the cloning of cDNA candidates for this enzyme have provided further insight into these mechanisms. This review will examine current hypotheses dealing with GPT and its role in the committed step of asparagine-linked glycosylation.     

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.     

Characterization and partial purification of two enzymes transferring N-acetylglucosamine to dolichyl monophosphate and ribonuclease A

Two N-acetylglucosamine (GlcNAc) transferases which catalyze the incorporation of GlcNAc into GlcNAc-P-P-dolichol (dolichol enzyme) and into bovine pancreatic ribonuclease A (RNAseA enzyme) were solubilized from the rat liver microsomes in a non-ionic detergent, Triton X-100. Both enzyme activities were adsorbed on activated CH-Sepharose 4B, and could be eluted with a linear KCl gradient. Two enzyme activities were separated by this column with the dolichol enzyme eluting before the RNAseA enzyme. A 49-fold and 136-fold purification was achieved for the dolichol and the RNAseA enzyme, respectively. The addition of exogeneous dolichyl phosphate resulted in a 3-5-fold stimulation of the purified dolichol enzyme, but did not affect the purified RNAseA enzyme. The addition of RNAseA stimulated only the RNAseA enzyme. Whereas, tunicamycin could inhibit only the dolichol enzyme. The purified dolichol enzyme had a Km of 14 X 10(-6) M for UDP-GlcNAc and the reaction was saturated with about 0.25 M dolichyl phosphate. The purified RNAseA enzyme had a Km of 4.55 X 10(-6) M for UDP-GlcNAc and was saturated with about 0.36 mM RNAseA. The pH optima and the metal ion requirement for the two enzymes were different. These results suggest that because of the different properties of these two enzymes they may have distinct functions regarding the core glycosylation of N-linked glycoproteins. It is well established that the dolichol enzyme catalyzes the formation of the first dolichol-linked intermediate GlcNAc-P-P-dolichol, whereas according to the present finding, the RNAseA enzyme may catalyze the transfer of GlcNAc directly from UDP-GlcNAc into acceptor protein.     

Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway

Two complementing mutations in lipid-linked oligosaccharide biosynthesis have been isolated following a [3H]mannose suicide enrichment. Rather than making the wild type precursor oligosaccharide, Glc3man9Glc-NA2-P-P-dolichol, the mutants, alg5-1 and alg6-1, accumulate Man9GlcNAc2-P-P-dolichol as their largest lipid-linked oligosaccharide in vivo and in vitro. When UDP-[3H]Glc was added to microsomal membranes of each mutant, neither could elongate Man9GlcNAc2-P-P-dolichol and only alg6-1 could synthesize dolichol-phosphoglucose. When dolicholphospho[3H]glucose was added to microsomes from alg5-1, alg6-1, or the parental strain, only alg5-1 and the parental strain made glucosylated lipid-linked oligosaccharides. These results indicate that alg5-1 cells are unable to synthesize dolichol phosphoglucose while alg6-1 cells are unable to transfer glucose from dolichol phosphoglucose to the unglucosylated lipid-linked oligosaccharide. We also present evidence that both mutants transfer Man9GlcNAc2 to protein.     

Coupling of the dolichol-P-P-oligosaccharide pathway to translation by perturbation-sensitive regulation of the initiating enzyme,GlcNAc-1-P transferase

In mammalian cells, inhibition of translation interferes with synthesis of the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol as measured with radioactive sugar precursors. Conflicting hypotheses have been proposed, and the fundamental basis for this regulation has remained elusive. Here, fluorophore-assisted carbohydrate electrophoresis (FACE) was used to measure LLO concentrations directly in cells treated with translation blockers. Further, LLO biosynthetic enzymes were assayed in vitro with endogenous acceptor substrates using either cells gently permeabilized with streptolysin-O (SLO) or microsomes from homogenized cells. In Chinese hamster ovary (CHO)-K1 cells treated with translation blockers, FACE did not detect changes in concentrations of Glc3Man9GlcNAc2-P-P-dolichol or early LLO intermediates. These results do not support earlier proposals for feedback repression of LLO initiation by accumulated Glc3Man9GlcNAc2-P-P-dolichol, or inhibition of a GDP-mannose dependent transferase. With microsomes from cells treated with translation blockers, there was no interference with LLO initiation by GlcNAc-1-P transferase (GPT), mannose-P-dolichol synthase, glucose-P-dolichol synthase, or LLO synthesis in vitro, as reported previously. Surprisingly, inhibition of all of these was detected with the SLO in vitro system. Additional experiments with the SLO system showed that the three transferases shared a limited pool of dolichol-P that was trapped as Glc3Man9GlcNAc2-P-P-dolichol by translation arrest. Overexpression of GPT was unable to reverse the effects of translation arrest on LLO initiation, and experiments with FACE and the SLO system showed that overexpressed GPT was not functional in vivo, although it was highly active in microsomal assays. Thus, the combined use of the SLO in vitro system and FACE showed that LLO biosynthesis depends upon a limited primary pool of dolichol-P. Physical perturbation associated with microsome preparation appears to make available a secondary pool of dolichol-P, masking inhibition by translation arrest, as well as activating a nonfunctional fraction of GPT. The implications of these results for the organization of the LLO pathway are discussed.     

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. © 1998 Rapid Science Ltd     

Stimulation by dolichol phosphate-mannose and phospholipids of the biosynthesis of N-acetylglucosaminylpyrophosphoryl dolichol

Dolichol phosphate-mannose (dol-P-mannose) has been shown previously to stimulate the reaction: dolichol phosphate + UDP-[3H]GlcNAc----[3H]GlcNAc-P-P-polyprenols (Kean, E. L. (1982) J. Biol. Chem. 257, 7952-7954). Further studies on this phenomenon are described, using microsomes from the retina of the embryonic chick as the major source of enzyme. Neither dolichol-P-glucose nor retinyl-P-mannose showed this stimulatory activity. Phosphatidylglycerol also stimulated this same process and was most active among a variety of phospholipids which were tested, in accord with previous reports. The presence of GDP-2-deoxy-2-fluoro-D-mannose or GTP had no effect on the reaction. The apparent activation constant for dolichol-P-mannose was 2.2 microM, and for phosphatidylglycerol, 401 microM. The major product (90% or greater) obtained under basal and stimulatory conditions was GlcNAc-P-P-dolichol and the site of the stimulatory effect was the glucosaminyltransferase catalyzing the formation of this compound. The effects of stimulation on the kinetic properties were similar for both activators: increases in the Vmax of the reactions of 7-10-fold; increases in apparent Km for UDP-GlcNAc of 5-7-fold; a 3-fold decrease in apparent Km for dolichol-phosphate. When present together, a mutual inhibition of stimulation was observed compared to the additive effect from dol-P-mannose or phosphatidylglycerol alone. Although a substrate for the reaction, dolichol phosphate repressed the stimulation by dolichol-P-mannose but not that by phosphatidylglycerol. Dol-P-glucose, while not an activator of the reaction, acted as a negative modifier of the stimulation by dol-P-mannose by acting as a competitive inhibitor of the stimulation. The stimulatory phenomenon was observed in microsomes prepared from a variety of tissues from the embryonic chick and from postnatal tissue after partial delipidation. The addition of pyrophosphatase inhibitors did not bring about stimulation of GlcNAc-lipid synthesis, but did enhance the effect. These studies extend the previous observations of the participation of dolichol-P-mannose and phosphatidylglycerol as allosteric activators of GlcNAc-lipid synthesis and indicate additional aspects of metabolic regulation of the dolichol pathway.     

Influence of metal ions on the biosynthesis of N-acetylglucosaminyl polyprenols by the retina

The following enzymatic process was investigated, catalyzed by an enzyme preparation from the retina of the embryonic chick: UDP-GlcNAc + dolichol phosphate GDPmannose leads to metal ions GlcNAc-P-P-polyprenol + (GlcNAc)2-P-P-polyprenol + Man-(GlcNAc)2-P-P-polyprenol. These reactions were carried out in the presence of a dolichol phosphate mannose-synthesizing system, shown previously to be an activator of GlcNAc-lipid synthesis. The process was also strongly influenced by the choice of the divalent cation used during the reactions. In the presence of Mg2+, not only was the extent of incorporation of radioactivity from UDP-[3H]GlcNAc increased 4-fold into the GlcNAc lipids, as compared to Mn2+, but the relative distribution of the products was affected as well. In the presence of Mg2+ the reaction was driven mainly in the direction of the formation of the first intermediate of the dolichol pathway, GlcNAc-P-P-polyprenol. Many of the other characteristics of the GlcNAc-transferases, such as pH optimum, requirement for dolichol phosphate and specificity for stimulation by sugar nucleotides, were similar for either the Mn2+ or Mg2+ systems. Retinol phosphate could not replace the requirement for dolichol phosphate. The influence of metal ions, in addition to the stimulation by dolichol phosphate mannose, on GlcNAc-lipid synthesis may be aspects of metabolic regulation of the dolichol pathway.     

Evidence that transient glucosylation of protein-linked Man9GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2 occurs in rat liver and Phaseolus vulgaris cells

Formation of protein-linked Glc1Man9GlcNAc2 , Glc1Man8GlcNAc2 , and Glc1Man7GlcNAc2 was detected in rat liver slices and Phaseolus vulgaris seeds incubated with [U-14C]glucose. Similar compounds were not synthesized in Saccharomyces cerevisiae cells incubated under similar conditions. Rat liver microsomes were incubated with [glucose-U-14C] Glc3Man9GlcNAc2-P-P-dolichol or UDP-[U-14C]Glc as glycosyl donors. Only in the latter condition protein-linked Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 were formed. Addition of mannooligosaccharides that strongly inhibited alpha 1-2-mannosidases to incubation mixtures containing rat liver microsomes and UDP-[U-14C]Glc did not prevent formation of protein-bound Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 . Furthermore, the presence of amphomycin in reaction mixtures containing liver membranes and UDP-[U-14C]Glc completely abolished synthesis of glucosylated derivatives of dolichol without affecting formation of protein-linked Glc1Man9GlcNAc2 , Glc1Man8GlcNAc2 , and Glc1Man7GlcNAc2 . The results reported above indicated that under the experimental conditions employed protein-bound Glc1Man9GlcNAc2 , Glc1Man8GlcNAc2 , and Glc1Man7GlcNAc2 were formed by glucosylation of unglucosylated oligosaccharides. Results obtained in pulse-chase experiments performed in vitro also supported this conclusion. UDP-Glc appeared to be the donor of the glucosyl residues. The rough endoplasmic reticulum was found to be the main subcellular site of protein glucosylation. It is tentatively suggested that this process could prevent extensive degradation of oligosaccharides by mannosidases during transit of glycoproteins through the endoplasmic reticulum.     

Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide- lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol- P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.      

Recycling of dolichyl monophosphate to the cytoplasmic leaflet of the endoplasmic reticulum after the cleavage of dolichyl pyrophosphate on the lumenal monolayer

During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc(3)Man(9)GlcNAc(2)-P-P-Dol (DLO) biosynthesis.     

Glucuronosyl-N-acetylglucosaminyl pyrophosphoryldolichol. Formation in SV40-transformed human lung fibroblasts and biosynthesis in rat lung microsomal preparations.

Incubation of SV40-transformed human lung fibroblasts with [3H]glucosamine for 1 h. followed by chloroform:methanol extraction and thin layer chromatographic analysis, revealed the presence of a major radioactive lipid that was isolated and characterized as GIcUA-(1 leads to 4)-GlcNAc-P-P-dolichol. An identical lipid was formed in smaller quantities under similar incubation conditions in several fibroblastic lines, HeLa cells, and in mouse L cells. Rat lung microsomal preparations catalyze the synthesis of the disaccharide lipid in the following sequence of reactions: UDP-[3H]GlcNAc + dolichol-P leads to [3H]GlcNAc-P-P-dolichol (1) [3H]GlcNAc-P-P-dolichol + UDP-[14C]GlcUA leads to [14C]GlcUA-[3H]GlcNAc-P-P-dolichol (2) The double-labeled lipid was identical to the lipid isolated from SV40-transformed fibroblasts with regard to its behavior on thin layer and silicic acid chromatography. Further, the double-labeled disaccharide released from the lipid by mild acid hydrolysis was identical to GlcUA-(1 leads to 4)-GlcNAc in its chromatographic and electrophoretic behavior and in its composition. The occurrence of a polyprenol derivative of GlcUA-(1 leads to 4)-GlcNAc suggests a possible role for this lipid in the biosynthesis of the repeating disaccharide units of proteoglycans, such as heparin.     

Teaching dolichol-linked oligosaccharides more tricks with alternatives to metabolic radiolabeling

The dolichol cycle involves synthesis of the lipid-linked oligosaccharide (LLO) Glc(3)Man(9)GlcNAc(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol), transfer of G(3)M(9)Gn(2) to asparaginyl residues of nascent endoplasmic reticulum (ER) polypeptides by oligosaccharyltransferase (OT), and recycling of the resultant Dol-P-P to Dol-P for new rounds of LLO synthesis. The importance of the dolichol cycle in secretory and membrane protein biosynthesis, ER function, and human genetic disease is now widely accepted. Elucidation of the fundamental properties of the dolichol cycle in intact cells was achieved through the use of radioactive sugar precursors, typically [(3)H]-labeled or [(14)C]-labeled d-mannose, d-galactose, or d-glucosamine. However, difficulties were encountered with cells or tissues not amenable to metabolic labeling, or in experiments influenced by isotope dilution, variable rates of LLO turnover, or special culture conditions required for the use of radioactive sugars. This article will review recently developed alternatives for LLO analysis that do not rely upon metabolic labeling with radioactive precursors, and thereby circumvent these problems. New information revealed by these methods with regard to regulation, genetic disorders, and evolution of the dolichol cycle, as well as caveats of radiolabeling techniques, will be discussed.     

A biochemical chimera suggesting the existence of at least two dolichol-P-glucose:dolichol-P-P-oligosaccharide glucosyltransferases

As previously reported, incubation of liver dolichol-P, UDP-[14C]Gal, and a particulate preparation of Acetobacter xylinum leads to the synthesis of dolichol-P-[14C]Gal (P. Romero, R. Garcia, and M. Dankert (1977) Mol. Cell. Biochem. 16, 205-212). It is now reported that upon incubation of the latter with rat liver microsomes, [14C-galactose]-Gal1Man9GlcNAc2-P-P-dolichol and [14C-galactose]Gal1Glc1Man9GlcNAc2-P-P-dolichol are formed. The galactosyl residues appeared to be (1,3)-linked in the same positions as the glucose units in the respective physiological compounds. No lipid-linked Gal1Glc2Man9GlcNAc2 was formed, thus strongly suggesting the presence of at least two dolichol-P-Glc:dolichol-P-P-oligosaccharide glucosyltransferases, only one of which is able to use dolichol-P-Gal as substrate. Incubation of the galactosylated dolichol-P-P derivatives with rat liver microsomes led to the transfer of the oligosaccharides to microsomal proteins. No endogenous, membrane-bound glycosidases were able to remove the galactose residues but mannose units were excised by endogenous neutral mannosidases.     

Control of glycoprotein synthesis

Hen oviduct membranes have been shown to catalyze the transfer of GlcNAc from UDP-GlcNAc to GlcNAc-beta 1-2Man alpha 1-6(GlcNAc beta 1-2 Man alpha 1-3) Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X (GnGn) to form the triantennary structure GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. The enzyme has been named UDP-GlcNAc:GnGn (GlcNAc to Man alpha 1-3) beta 4-N-acetylglucosaminyltransferase IV (GlcNAc-transferase IV) to distinguish it from three other hen oviduct GlcNAc-transferases designated I, II, and III. Since GlcNAc-transferases III and IV both act on the same substrate, concanavalin A/Sepharose was used to separate the products of the two enzymes. At pH 7.0 and at a Triton X-100 concentration of 0.125% (v/v), GlcNAc-transferase IV activity in hen oviduct membranes is 7 nmol/mg of protein/h. The product was characterized by high resolution proton NMR spectroscopy at 360 MHz and by methylation analysis. In addition to triantennary oligosaccharide, hen oviduct membranes produced about 20% of bisected triantennary material, GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3] [GlcNAc beta 1-4]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. Maximal GlcNAc-transferase IV activity requires the presence of both terminal beta 1-2-linked GlcNAc residues in the substrate. Removal of the GlcNAc residue on the Man alpha 1-6 arm or of both GlcNAc residues reduces activity by at least 80%. A Gal beta 1-4GlcNAc disaccharide on the Man alpha 1-6 arm reduces activity by 68% while the presence of this disaccharide on the Man alpha 1-3 arm reduces activity to negligible levels. A similar substrate specificity was found for GlcNAc-transferase III, the enzyme which adds a bisecting GlcNAc in beta 1-4 linkage to the beta-linked Man residue. Since a bisecting GlcNAc was found to prevent GlcNAc-transferase IV action, the bisected triantennary material found in the incubation must have been formed by the sequential action of GlcNAc-transferase IV followed by GlcNAc-transferase III. Activities similar to GlcNAc-transferase IV were also detected in rat liver Golgi-rich membranes (0.4 nmol/mg/h) and pig thyroid microsomes (0.1 nmol/mg/h).     

Comparative studies on mannosylphosphoryl dolichol and glucosylphosphoryl dolichol synthases

Factors affecting the synthesis of mannosylphosphoryl dolichol and glucosylphosphoryl dolichol hen oviduct microsomes were compared in order to gain insight into the properties of their respective synthases. A stabilized form of mannosylphosphoryl dolichol synthase, but not glucosylphosphoryl dolichol synthase, was released from microsomes by freezing the membranes after exposure to the detergent CHAPSO. The activation energy for mannosylphosphoryl dolichol synthesis in membranes was 9.4 glucosylphosphoryl dolichol synthesis in membranes had a similar activation energy, 8.1 kcal/mol, but below 18 degrees C the value was 16.7 kcal/mol. Tryptic digestion of sealed microsomes preferentially inactivated mannosylphosphoryl dolichol synthase; however, both synthases were equally inactivated in detergent-permeabilized microsomes. Periodate-oxidized UDP-Glc was used to probe the topological orientation of glucosylphosphoryl dolichol synthase in rat liver microsomes. Sealed microsomes treated with oxidized UDP-Glc were inactive in synthesis of glucosylphosphoryl dolichol. However, when these treated microsomes were permeabilized, glucosylphosphoryl dolichol synthase activity was readily detected. From these studies we conclude that although mannosyl- and glucosylphosphoryl dolichol synthases catalyze chemically similar reactions in the endoplasmic reticulum, they differ in several respects. These differences were interpreted in terms of a topological model in which the active sites of the two enzymes reside on opposite faces of the endoplasmic reticulum, with that of the glucosyl lipid synthase facing the lumen and that of the mannosyl lipid synthase facing the cytosol.     

In vitro evidence for the dual function of Alg2 and Alg11: essential mannosyltransferases in N-linked glycoprotein biosynthesis

The biosynthesis of asparagine-linked glycoproteins utilizes a dolichylpyrophosphate-linked glycosyl donor (Dol-PP-GlcNAc(2)Man(9)Glc(3)), which is assembled by the series of membrane-bound glycosyltransferases that comprise the dolichol pathway. This biosynthetic pathway is highly conserved throughout eukaryotic evolution. While complementary genetic and bioinformatic approaches have enabled identification of most of the dolichol pathway enzymes in Saccharomyces cerevisiae, the roles of two of the mannosyltransferases in the pathway, Alg2 and Alg11, have remained ambiguous because these enzymes appear to catalyze only two of the remaining four unannotated transformations. To address this issue, a biochemical approach was taken using recombinant Alg2 and Alg11 from S. cerevisiae and defined dolichylpyrophosphate-linked substrates. A cell-membrane fraction isolated from Escherichia coli overexpressing thioredoxin-tagged Alg2 was used to demonstrate that this enzyme actually carries out an alpha1,3-mannosylation, followed by an alpha1,6-mannosylation, to form the first branched pentasaccharide intermediate of the pathway. Then, using thioredoxin-tagged Alg2 for the chemoenzymatic synthesis of the dolichylpyrophosphate pentasaccharide, it was thus possible to define the biochemical function of Alg11, which is to catalyze the next two sequential alpha1,2-mannosylations. The elucidation of the dual function of each of these enzymes thus completes the identification of the entire ensemble of glycosyltransferases that comprise the dolichol pathway.     

Control of glycoprotein synthesis. UDP-GlcNAc:glycopeptide beta 4-N-acetylglucosaminyltransferase III, an enzyme in hen oviduct which adds GlcNAc in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-glycosyl oligosaccharides

Hen oviduct membranes are shown to catalyze the following enzyme reaction: GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn + UDP-GlcNAc leads to GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)GlcNAc beta 1-4)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn + UDP. The enzyme catalyzing this reaction has been named UDP-GlcNAc:glycopeptide beta 4-N-acetylglucosaminyltransferase III (GlcNAc-transferase III) to distinguish it from two other GlcNAc-transferases (I and II) present in hen oviduct and previously described in several mammalian tissues. GlcNAc-transferases I and II, respectively, attach GlcNAc in beta 1-2 linkage to the Man alpha 1-3 and Man alpha 1-6 arms of Asn-linked oligosaccharide cores. A specific assay for GlcNAc-transferase III was devised by using concanavalin A/Sepharose columns to separate the product of transferase III from other interfering radioactive glycopeptides formed in the reaction. The specific activity of GlcNAc-transferase III in hen oviduct membranes is about 5 nmol/mg of protein/h. Substrate specificity studies have shown that GlcNAc-transferase III requires both terminal beta 1-2-linked GlcNAc residues in its substrate for maximal activity. Removal of the GlcNAc residue on the Man alpha 1-6 arm reduces activity by at least 85% and removal of both GlcNAc residues reduces activity by at least 93%. Two large scale preparations of product were subjected to high resolution proton NMR spectroscopy to establish the incorporation by the enzyme of a GlcNAc in beta 1-4 linkage to the beta-linked Man. This GlcNAc residue is called a "bisecting" GlcNAc and appears to play important control functions in the synthesis of complex N-glycosyl oligosaccharides. Several enzymes in the biosynthetic scheme are unable to act on glycopeptide substrates containing a bisecting GlcNAc residue.     

Glucosylation of glycoproteins by mammalian, plant, fungal, and trypanosomatid protozoa microsomal membranes

An assay for UDP-Glc:glycoprotein glucosyltransferase was developed. Incubation of rat liver microsomes with UDP-[14C]Glc led to the formation of hot trichloroacetic acid insoluble material identified as protein-linked Glc1Man7-9GlcNAc2. Addition of 8 M urea-denatured thyroglobulin to the incubation mixtures stimulated up to 10-12-fold the formation of the same compounds but only in the presence of detergents. Native thyroglobulin was ineffective. Several experiments indicated that the stimulation was due to the transfer of glucose residues from UDP-Glc to high-mannose oligosaccharides in urea-denatured thyroglobulin and that this transfer reaction did not involve dolichol mono- or diphosphate derivatives as intermediates. The glycoprotein glucosylating activity was mainly located in the endoplasmic reticulum and could glucosylate glycopeptides derived from the digestion of thyroglobulin with an unspecific protease. Glucosylation of oligosaccharides in those glycopeptides occurred, however, at a rate at least 2 orders of magnitude slower than that of the same compounds in urea-denatured thyroglobulin. Tryptic digestion of urea-denatured thyroglobulin did not affect its glucosylation rate. The structure of Glc1Man9GlcNAc2 linked to urea-denatured thyroglobulin was identical with that of Glc1Man9GlcNAc2-P-P-dolichol. The assay of UDP-Glc:glycoprotein glucosyltransferase allowed detection of the activity in microsomal membranes in which endogenous acceptors appeared to be absent or almost absent, such as those derived from mung bean, Mucor rouxii, Crithidia fasciculata, and Trypanosoma cruzi cells.(ABSTRACT TRUNCATED AT 250 WORDS)