Evaluation of the role of rat liver Golgi endo-alpha-D-mannosidase in processing N-linked oligosaccharides

Golgi membranes from rat liver have been shown to contain an endo-alpha-D-mannosidase which can convert Glc1Man9GlcNAc to Man8GlcNAc with the release of Glc alpha 1----3Man (Lubas, W. A., and Spiro, R. G. (1987) J. Biol. Chem. 262, 3775-3781). We now report that this enzyme has the capacity to cleave the alpha 1----2 linkage between the glucose-substituted mannose residue and the remainder of the polymannose branch in a wide range of oligosaccharides (Glc3Man9GlcNAc to Glc1Man4GlcNAc) as well as glycopeptides and oligosaccharide-lipids. Whereas the tri- and diglucosylated species (Glc3Man9GlcNAc and Glc2Man9GlcNAc), which yielded Glc3Man and Glc2Man, respectively, were processed more slowly than Glc1Man9GlcNAc, the monoglucosylated components with truncated mannose chains (Glc1Man8GlcNAc to Glc1Man4GlcNAc) were trimmed at an increased rate which was inversely related to the number of mannose residues present. The endomannosidase was not inhibited by a number of agents which are known to interfere with N-linked oligosaccharide processing by exoglycosidases, including 1-deoxynojirimycin, castanospermine, bromoconduritol, 1-deoxymannojirimycin, swainsonine, and EDTA. However, Tris and other buffers containing primary hydroxyl groups substantially decreased its activity. After Triton solubilization, the endomannosidase was observed to be bound to immobilized wheat germ agglutinin, indicating the presence of a type of carbohydrate unit consistent with Golgi localization of the enzyme. The Man8GlcNAc isomer produced by endomannosidase action was found to be processed by Golgi enzymes through a different sequence of intermediates than the rough endoplasmic reticulum-generated Man8GlcNAc variant, in which the terminal mannose of the middle branch is absent. Whereas the latter oligosaccharide is converted to Man5GlcNAc via Man7GlcNAc and Man6GlcNAc at an even rate, the processing of the endomannosidase-derived Man8GlcNAc stalls at the Man6GlcNAc stage due to the apparent resistance to Golgi mannosidase I of the alpha 1,2-linked mannose of the middle branch. The results of our study suggest that the Golgi endomannosidase takes part in a processing route for N-linked oligosaccharides which have retained glucose beyond the rough endoplasmic reticulum; the distinctive nature of this pathway may influence the ultimate structure of the resulting carbohydrate units.     

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.     

Impairment of dolichyl saccharide synthesis and dolichol-mediated glycoprotein assembly in the aortic smooth muscle cell in culture by inhibitors of cholesterol biosynthesis

25-Hydroxycholesterol treatment of the aortic smooth muscle cell inhibited the incorporation of acetate but not mevalonate into dolichol and cholesterol by 91 and 82%, respectively, and diminished the synthesis from glucose of cholesterol, dolichylpyrophosphoryl oligosaccharide, and dolichol-dependent glycoproteins. The dolichol-bound oligosaccharide unit contained approximately 10 Man/2 Glc/2 GlcNAc residues and appeared to be a precursor to protein-bound saccharide units which contained on the average 8 Man/1 Glc/2 GlcNAc residues. Mevalonate was found to protect the cells against the effect 25-hydroxycholesterol and to restore normal cellular synthesis of dolichyl saccharides and glycoproteins. It is suggested that hydroxymethylglutaryl coenzyme A reductase may function as a rate-controlling enzyme in the biosynthesis of not only sterols but also dolichols, and may as a result regulate the assembly of certain cellular glycoproteins.     

Biosynthesis of the core region of yeast mannoproteins. Formation of a glucosylated dolichol-bound oligosaccharide precursor, its transfer to protein and subsequent modification

A new membrane preparation from Saccharomyces cerevisiae was developed, which effectively catalyzes the synthesis of large oligosaccharide-lipids from GDP-Man and UDP-Glc allowing a detailed study of their formation and size. The oligosaccharide from an incubation with GDP-Man could be separated by gel filtration chromatography into several species consisting of two N-acetylglucosamine (GlcNAc) residues at the reducing end and differing by one mannos unit; the major compound formed has the composition (Man)9(GlcNAc)2. Upon incubation with UDP-Glc, three oligosaccharides corresponding to the size of (Glc)1-3(Man)9(GlcNAc)2 are formed. Thus, the oligosaccharides generated in vitro by the yeast membranes appear to be identical in size with the oligosaccharides found in animal systems. In addition the results indicate that dolichyl phosphate mannoe (DolP-Man) is the immediate donor in assembling the oligosaccharide moiety from (Man)5(GlcNAc)2 to (Man)9(GlcNAc)2. All three glucose residues are transferred from DolP-Glc. Experiments with isolated [Glc-14C]oligosaccharide-lipid as substrate demonstrated that the oligosaccharide chain is transferred to an endogenous membrane protein acceptor. Moreover, transfer is followed by an enzymic removal of glucose residues, due to a glucosidase activity associated with the membranes. Glucose release from the free [Glc-14C]oligosaccharide is less effective than from protein-bound oligosaccharide. Glycosylation was also observed using [Man-14C]oligosaccharide-lipid or DolPP-(GlcNAc)2 as donor. However, transfer in the presence of glucose seems to be more rapid. The mannose-containing oligosaccharide, released from the lipid, was shown to function as a substrate for further chain elongation reactions utilizing GDP-Man but not DolPP-Man as donor. It is suggested that the immediate precursor in the synthesis of the heterogeneous core region, (Man)12-17(GlcNAc)2, of yeast mannoproteins is a glucose-containing lipid-oligosaccharide with the composition (Glc)3(Man)9(GlcNAc)2, i.e. only part of what has been defined as inner core is built up on the lipid carrier. After transfer to protein the oligosaccharide is modified by excision of the glucose residues, followed subsequently by further elongation from GDP-Man to give the size of th oligosaccharide chains found in native mannoproteins.     

Potential regulation of N-glycosylation precursor through oligosaccharide-lipid hydrolase action and glucosyltransferase-glucosidase shuttle.

The potential role of degradative mechanisms in controlling the level of the dolichyl pyrophosphate-linked Glc3Man9GlcNAc2 required for protein N-glycosylation has been explored in thyroid slices and endoplasmic reticulum (ER) vesicles, focusing on cleavage of the oligosaccharide from its lipid attachment and on the enzymatic removal of peripheral monosaccharide residues. Vesicle incubations demonstrated a substantial release of free Glc3Man9GlcNAc2 (at 30 min approximately 35% of that transferred to protein) which was inhibited in the presence of exogenous peptide acceptor and was sensitive to disruption of membrane integrity by detergent. In thyroid slices glucosylated oligosaccharides terminating in the di-N-acetylchitobiose sequence were also noted and these continued to be formed even during inhibition by puromycin of both protein synthesis and the attendant N-glycosylation. These observations indicated that the oligosaccharide originated from the lipid donor and suggested, together with previously reported similarities in substrate specificity and cofactor requirements, that the oligosaccharyltransferase can carry out in vivo both the hydrolytic and transfer functions. In addition to the release of the intact Glc3Man9GlcNAc2, we also obtained evidence that the lipid-linked oligosaccharide can be modified by the in vivo action of ER glycosidases. Since radiolabeling of the oligosaccharide-lipid in thyroid slices indicated a preferential turnover of the glucose residues, the possible existence of a glucosyltransferase-glucosidase shuttle was explored with the use of castanospermine. In the presence of this glucosidase inhibitor, the formation of under-glucosylated and nonglucosylated oligosaccharides was not observed, even under conditions of energy deprivation in which they accumulate. Glucosidase inhibition in ER vesicle incubations likewise prevented the appearance of incompletely glucosylated oligosaccharide-lipids. Studies employing the mannosidase inhibitor 1-deoxymannojirimycin in thyroid slices furthermore indicated that in vivo removal of at least one mannose residue from the dolichyl pyrophosphate-linked oligosaccharide can occur.     

Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices

Incubations of thyroid microsomes with radiolabeled dolichyl pyrophosphoryl oligosaccharide (Glc3Man9-GlcNAc2) under conditions optimal for the N-glycosylation of protein resulted in the release, by apparently independent enzymatic reactions, of two types of neutral glucosylated polymannose oligosaccharides which differed from each other by terminating either in an N-acetylglucosamine residue (Glc3Man9GlcNAc1) or a di-N-acetylchitobiose moiety (Glc3Man9GlcNAc2). The first mentioned oligosaccharide, which was released in a steady and slow process unaffected by the addition of EDTA, appeared to be primarily the product of endo-beta-N-acetylglucosaminidase action on newly synthesized glycoprotein and such an enzyme with a neutral pH optimum capable of hydrolyzing exogenous glycopeptides and oligosaccharides (Km = 18 microM) was found in the thyroid microsomal fraction. The Glc3Man9GlcNAc2 oligosaccharide, in contrast, appeared to originate from the oligosaccharide-lipid by a rapid hydrolysis reaction which closely paralleled the N-glycosylation step, progressing as long as oligosaccharide transfer to protein occurred and terminating when carbohydrate attachment ceased either due to limitation of lipid-saccharide donor or addition of EDTA. There was a striking similarity between oligosaccharide release and transfer to protein with lipid-linked Glc3Man9GlcNAc2 serving as a 10-fold better substrate for both reactions than lipid-linked Man9-8GlcNAc2. The coincidence of transferase and hydrolase activities suggest the possibility of the existence of one enzyme with both functions. The physiological relevance of oligosaccharide release was indicated by the formation of such molecules in thyroid slices radiolabeled with [2-3H]mannose. Large oligosaccharides predominated (12 nmol/g) and consisted of two families of components; one group terminating in N-acetylglucosamine, ranged from Glc1Man9GlcNAc1 to Man5GlcNAc1 while the other contained the di-N-acetylchitobiose sequence and included Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, and Man9GlcNAc2.     

Regulation of glycosylation. Three enzymes compete for a common pool of dolichyl phosphate in vivo

We examined changes in the levels of the dolichol forms in Chinese hamster ovary cells containing alterations in the levels of activity of two enzymes in the oligosaccharyl-P-P-dolichol biosynthetic pathway, namely UDP-GlcNAc:dolichyl phosphate:GlcNAc-phosphotransferase (GlcNAc-1-phosphotransferase) and mannosylphosphoryldolichol (Man-P-Dol) synthase. Under normal conditions in wild type cells, Glc3Man9GlcNAc2-pyrophosphoryldolichol was the most abundant form. Of the other anionic forms of dolichols, dolichyl phosphate, Man-P-Dol, glucosylphosphoryldolichol, and Man5GlcNAc2-pyrophosphoryl dolichol were approximately equally abundant. When 3E11 cells (a tunicamycin-resistant Chinese hamster ovary line containing 15 times more GlcNAc-1-phosphotransferase activity than wild type), B4-2-1 cells (a mutant lacking Man-P-Dol synthase activity), and wild type cells incubated with or without tunicamycin were utilized, significant changes in the levels of most of the anionic dolichol derivatives, with the exception of dolichyl phosphate, were found. Since changes in dolichyl phosphate levels were not detected under a variety of conditions where the levels of enzyme activity utilizing this substrate were varied, all three enzymes appear to have access to the same pool of dolichyl phosphate, and further, to have similar Km values for dolichyl phosphate.     

Processing of N-linked oligosaccharides in soybean cultured cells

Evidence, based on both in vivo and in vitro studies with suspension-cultured soybean cells, is presented to demonstrate the processing of the oligosaccharide chain of plant N-linked glycoproteins. Following a 1-h incubation of soybean cells with [2-3H]mannose, the predominant glycopeptide obtained by pronase digestion of the membrane fraction was a Man7- or Man8GlcNAc2-Asn (GlcNAc, N-acetylglucosamine). However, the major oligosaccharide isolated from the lipid-linked oligosaccharides of these cells was a Glc2- or Glc3Man9GlcNAc2. Soybean cells were incubated with [2-3H]mannose and the incorporation of mannose into Pronase-released glycopeptides was followed during a 2-h chase. During the first 10 min of labeling, the radioactivity was mostly in a large-sized glycopeptide that appeared to be a Glc1Man9GlcNAc2-peptide. During the next 60 to 90 min of chase, this radioactivity was shifted to smaller and smaller-sized glycopeptides indicating that removal of sugars (i.e., processing) had occurred. Both glucosidase and mannosidase activity was detected in membrane preparations of soybean cells. Nine different glycopeptides were isolated from Pronase digests of soybean cell membrane fractions. These glycopeptides were purified by repeated gel filtration on columns of Bio-Gel P-4. Partial characterization of these glycopeptides by endoglucosaminidase H and alpha-mannosidase digestion, and by analysis of the products, suggested the following glycopeptides: Glc1Man9GlcNAc2-Asn, Man8GlcNAc2-Asn, Man7GlcNAc2-Asn, Man6GlcNAc2-Asn, and Man5GlcNAc2-Asn.     

The effect of mannosamine on the formation of lipid-linked oligosaccharides and glycoproteins in canine kidney cells

Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with [2-3H]mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Methylation analysis also indicated that this Man5GlcNAc2 contained 1----3 linked mannose residues. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps by inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with [3H]mannose or [3H]galactose. In contrast, control cells produced complex and high-mannose structures but no hybrid oligosaccharides were detected. The inhibition by mannosamine could be overcome by adding high concentrations of glucose to the medium.     

Evidence for an incomplete dolichyl-phosphate pathway of lipoglycan formation in Volvox carteri f. nagariensis

Crude membrane fractions from Volvox carteri in the presence of detergent and metal complexing agent catalyze the transfer of glucose from dolichyl phosphate glucose to branched dolichyl diphosphate chitobiosyl pentamannoside Dol-PP-(GlcNAc)2-(Man)5, a known intermediate of the lipid-mediated pathway of N-glycosylation of proteins, resulting in the formation of Dol-PP-(GlcNAc)2-(Man)5-(Glc)1. Under the various conditions tested, neither Dol-P-Man nor other known mannosyl donors of the nucleoside-activated or lipid-activated type can serve as donor molecules for the elongation of the lipid-linked heptasaccharide. On the other hand, calf liver microsomes in similar experiments mannosylated the heptasaccharide further with Dol-P-Man up to a nonamannoside, Dol-PP-(GlcNAc)2-(Man)9. A direct glucosylation of the acceptor, however, with Dol-P-Glc failed in this system. The (GlcNAc)2-(Man)5-(Glc)1, obtained after mild acid hydrolysis of the above glycolipid is not significantly split by an unspecific alpha-glucosidase from yeast. However, Volvox microsomes liberated most of the glucose indicating a specific glucosidase in the membranes of the alga. This enzyme does not act on (GlcNAc)2-(Man)9-(Glc)1, the usual protein-linked carbohydrate intermediate of trimming processes of N-glycosidic glycoproteins. The data on glycolipid formation let us postulate that in Volvox the normal N-glycosylation pathway differs from that found in higher plants and animals either by a lack of evolution or by mutation in the genes coding for the mannosyl transferases involved.     

Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2

Endoplasmic reticulum-associated degradation of misfolded or misprocessed glycoproteins in mammalian cells is prevented by inhibitors of class I alpha-mannosidases implicating mannose trimming from the precursor oligosaccharide Glc3Man9GlcNAc2 as an essential step in this pathway. However, the extent of mannose removal has not been determined. We show here that glycoproteins subject to endoplasmic reticulum-associated degradation undergo reglucosylation, deglucosylation, and mannose trimming to yield Man6GlcNAc2 and Man5GlcNAc2. These structures lack the mannose residue that is the acceptor of glucose transferred by UDP-Glc:glycoprotein glucosyltransferase. This could serve as a mechanism for removal of the glycoproteins from folding attempts catalyzed by cycles of reglucosylation and calnexin/calreticulin binding and result in targeting of these molecules for proteasomal degradation.     

Characterization of lipid-linked octa- through undecasaccharides implicated in the biosynthesis of Saccharomyces cerevisiae mannoproteins.

The lipid-linked oligosaccharide Glc3-Man9(GlcNAc)2 (Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine) serves as a precursor for the biosynthesis of the inner core portion of the asparagine-linked polysaccharide of Saccharomyces cerevisiae mannoproteins. It has been shown previously that incubation of a microsomal preparation from this organism with UDP-N-acetylglucosamine and GDP-[14C]mannose gives rise to a series of lipid-linked oligosaccharides of the general structure Mann(GlcNAc)2, with n from 1 to 9. A structural characterization of Man1- to Man5(GlcNAc)2 oligosaccharides indicated that the major structures among these were identical to the intermediates proposed for the biosynthesis of animal glycoproteins (C. Prakash and I. K. Vijay, Biochemistry 21:4810-4818, 1982). In the present study, the structural characterization of the Man6- through Man9(GlcNAc)2 species was conducted. The Man6- through Man8(GlcNAc)2 species have two isomers, whereas Man9(GlcNAc)2 is monoisomeric. One isomer each of Man6- through Man8(GlcNAc)2 and the monoisomeric Man9(GlcNAc)2 are identical to the intermediates for the biosynthesis of asparagine-linked glycoproteins in animal systems. It is proposed that the steps of the lipid-linked assembly of the carbohydrate precursor for S. cerevisiae mannoproteins are identical to those of the major pathway in animal systems. A lack of acceptor substrate specificity by the mannosyltransferases, as observed with in vitro studies with animal systems, also might be responsible for the biosynthesis of multiple isomers reported here.     

Mannosyl carrier functions of retinyl phosphate and dolichyl phosphate in rat liver endoplasmic reticulum

Of the subcellular fractions of rat liver the endoplasmic reticulum was the most active in GDP-mannose: retinyl phosphate mannosyl-transfer activity. The synthesis of retinyl phosphate mannose reached a maximum at 20-30 min of incubation and declined at later times. Retinyl phosphate mannose and dolichyl phosphate mannose from endogenous retinyl phosphate and dolichyl phosphate could also be assayed in the endoplasmic reticulum. About 1.8 ng (5 pmol) of endogenous retinyl phosphate was mannosylated per mg of endoplasmic reticulum protein (15 min at 37 degrees C, in the presence of 5 mM-MnCl2), and about 0.15 ng (0.41 pmol) of endogenous retinyl phosphate was mannosylated with Golgi-apparatus membranes. About 20 ng (13.4 pmol) of endogenous dolichyl phosphate was mannosylated in endoplasmic reticulum and 4.5 ng (3 pmol) in Golgi apparatus under these conditions. Endoplasmic reticulum, but not Golgi-apparatus membranes, catalysed significant transfer of [14C]mannose to endogenous acceptor proteins in the presence of exogenous retinyl phosphate. Mannosylation of endogenous acceptors in the presence of exogenous dolichyl phosphate required the presence of Triton X-100 and could not be detected when dolichyl phosphate was solubilized in liposomes. Dolichyl phosphate mainly stimulated the incorporation of mannose into the lipid-oligosaccharide-containing fraction, whereas retinyl phosphate transferred mannose directly to protein.     

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.     

Characterization of the endomannosidase pathway for the processing of N-linked oligosaccharides in glucosidase II-deficient and parent mouse lymphoma cells.

Studies on N-linked oligosaccharide processing in the mouse lymphoma glucosidase II-deficient mutant cell line (PHAR2.7) as well as the parent BW5147 cells indicated that the former maintain their capacity to synthesize complex carbohydrate units through the use of the deglucosylation mechanism provided by endomannosidase. The in vivo activity of this enzyme was evident in the mutant cells from their production of substantial amounts of glucosylated mannose saccharides, predominantly Glc2Man; moreover, in the presence of 1-deoxymannojirimycin or kifunensine to prevent processing by mannosidase I, N-linked Man8GlcNAc2 was observed entirely in the form of the characteristic isomer in which the terminal mannose of the alpha 1,3-linked branch is missing (isomer A). In contrast, parent lymphoma cells, as well as HepG2 cells in the presence of 1-deoxymannojirimycin accumulated Man9GlcNAc2 as the primary deglucosylated N-linked oligosaccharide and contained only about 16% of their Man8GlcNAc2 as isomer A. In the presence of the glucosidase inhibitor castanospermine the mutant released Glc3Man instead of Glc2Man, and the parent cells converted their deglucosylation machinery to the endomannosidase route. Despite the mutant's capacity to accommodate a large traffic through this pathway no increase in the in vitro determined endomannosidase activity was evident. The exclusive utilization of endomannosidase by the mutant for the deglucosylation of its predominant N-linked Glc2Man9GlcNAc2 permitted an exploration of the in vivo site of this enzyme's action. Pulse-chase studies utilizing sucrose-D2O density gradient centrifugation indicated that the Glc2Man9GlcNAc2 to Man8GlcNAc2 conversion is a relatively late event that is temporally separated from the endoplasmic reticulum-situated processing of Glc3Man9GlcNAc2 to Glc2Man9GlcNAc2 and in contrast to the latter takes place in the Golgi compartment.     

Glycosyltransfer by pea membranes from sugar nucleotides to added prenyl phosphates

Pea membranes supplied with GDP-[14C]mannose, UDP-N-[14C]acetylglucosamine or UDP-[14C]glucose catalyze the transfer of 14C-labeled sugars or sugar phosphates to endogenous lipid acceptors as well as to exogenously added dolichyl phosphates. Fully unsaturated polyprenyl phosphates were not used as effective acceptors by this system. Mannosyl-P-dolichol was formed most rapidly in the presence of long-chained dolichyl-P while mannosyl-PP-, glucosyl-PP- and GlcNAc-PP-dolichol were preferentially formed from relatively short-chained dolichyl phosphate acceptors. Glucosyl-PP- and mannosyl-PP-dolichol accumulated in the preparation without further metabolism, but GlcNAc-PP-dolichol was lengthened by addition of a second GlcNAc plus several [14C]mannose units to form an oligosaccharide fraction susceptible to the action of endoglycosidase H. This lipid-linked oligosaccharide could then be glycosylated in the presence of UDP-[14C]glucose to form a longer oligosaccharide. It is concluded that levels of endogenous dolichyl phosphates in pea membranes are rate-limiting for several of the key glycosyltransferases required for oligosaccharide assembly.     

Protein glycosylation in Trypanosoma cruzi. The mechanism of glycosylation and structure of protein-bound oligosaccharides

As reported previously (Parodi, A.J., and Cazzulo, J.J. (1982) J. Biol. Chem. 257, 7641-7645), label was incorporated first to the glucose residues of protein-bound Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 when Trypanosoma cruzi cells, the causative agent of Chagas disease, were incubated with [U-14C]glucose. It is now reported that the glucose residues are removed from the oligosaccharides after a chase period. The relative proportion of Man9GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, and Man6GlcNAc2 appeared to be the same after 120 and 180 min of chase, thus indicating that these compounds were the fully processed protein-bound oligosaccharides. No complex type protein-bound oligosaccharides were detected. Evidence is presented indicating that Glc1Man7GlcNAc2 was formed mainly by glucosylation of Man7GlcNAc2 and not by demannosylation of Glc1Man9GlcNAc2. Man9GlcNAc2 was the first oligosaccharide to be labeled when cells were incubated with [2-3H]mannose. Based on these and previous results, the overall mechanism of protein N-glycosylation appeared to be: (formula; see text) The structure of the oligosaccharides appeared to be similar to some of those present in human glycoproteins. T. cruzi cells isolated from distant locations in South America were found to share a common mechanism of protein glycosylation.     

Glycoprotein biosynthesis. Rat liver microsomal glucosidases which process oligosaccharides.

Asparagine-linked oligosaccharides of glycoproteins undergo extensive modification or "processing" following their attachment to protein. A key step in post-glycosylation processing is the sequential removal of glucose residues from the protein-linked oligosaccharide. We have studied rat liver preparations which catalyze removal of glucose from Glc3Man9GlcNAc, Glc2Man9GlcNAc, and Glc1Man9GlcNAc. Detergent solubilization studies, inhibitor studies, and temperature-activity profiles indicate that at least two distinct glucosidases are present in the membranes. One of these glucosidases removes the distal glucose from Glc3Man9GlcNAc, and the other glucosidase sequentially removes glucose from Glc2Man9GlcNAc and Glc1Man9GlcNAc. The latter glucosidase has been solubilized from the microsomal memrbranes and purified 12-fold. The glucosidases, which are integral membrane proteins, are localized in the rough and smooth microsomes and appear to be located on the cisternal surface of the microsomal vesicles. These glucosidases are suggested to be of biological importance in catalyzing the initial events in the post-glycosylation processing of cellular glycoprotein.     

Transfer of nonglucosylated oligosaccharide from lipid to protein in a mammalian cell

We have previously shown that the glucosidase inhibitor, N-methyl-1-deoxynojirimycin (MedJN), only partially inhibited N-linked complex oligosaccharide biosynthesis in F9 teratocarcinoma cells whereas the alpha-mannosidase I inhibitor, manno-1-deoxynojirimycin, completely prevented this synthesis (Romero, P. A. and Herscovics, A. (1986) Carbohydr. Res. 151, 21-28). In order to determine whether a pathway independent of processing glucosidases can occur, F9 cells were pulse-labeled for 2 min with D-[2-3H]mannose in the presence or absence of 2 mM MedJN. In control cells, Man7GlcNAc was identified in the protein-bound oligosaccharides released with endo-beta-N-acetylglucosaminidase H, in addition to the expected Glc1-3Man9GlcNAc and Man9GlcNAc arising from processing of Glc3Man9GlcNAc. MedJN completely prevented the removal of glucose residues from Glc3Man9GlcNAc, but did not greatly affect the appearance of Man7GlcNAc associated with protein. Labeled Man7GlcNAc was also found in the lipid-linked oligosaccharides of both control and treated cells. The 2-min pulse-labeled Man7GlcNAc obtained from both the lipid and protein fractions were shown to have identical structures by concanavalin A-Sepharose chromatography and by acetolysis and were clearly different from the Man7GlcNAc obtained from the usual processing pathway. These results demonstrate that transfer of a nonglucosylated oligosaccharide (Man7GlcNAc2) from dolichyl pyrophosphate to protein occurs in F9 cells.     

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.