Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases.

The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum.     

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.     

The effect of deoxymannojirimycin on the processing of the influenza viral glycoproteins

Deoxymannojirimycin (dMM) was tested as an inhibitor of the processing of the oligosaccharide portion of viral and cellular N-linked glycoproteins. The NWS strain of influenza virus was grown in MDCK cells in the presence of various amounts of dMM, and the glycoproteins were labeled by the addition of 2-[3H]mannose to the medium. At levels of 10 micrograms/ml dMM or higher, most of the viral glycopeptides became susceptible to digestion by endoglucosaminidase H, and the liberated oligosaccharide migrated mostly like a Hexose9GlcNAc on a calibrated column of Bio-Gel P-4. This oligosaccharide was characterized as a typical Man9GlcNAc by a variety of chemical and enzymatic procedures. Deoxymannojirimycin gave rise to similar oligosaccharide structures in the cellular glycoproteins. In both the viral and the cellular glycoproteins, this inhibitor caused a significant increase in the amount of [3H]mannose present in the glycoproteins. Deoxymannojirimycin did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it affect the yield or infectivity of NWS virus particles. However, its effect on mannose incorporation into lipid-linked saccharides depended on the incubation time, the virus strain, and the cell line. Thus, high concentrations of dMM showed some inhibition of mannose incorporation into lipid-linked oligosaccharides with the NWS strain in a 3-h incubation, but no inhibition was observed after 48 h of incubation. On the other hand, the PR8 strain was much more sensitive to dMM inhibition, and mannose incorporation into lipid-linked oligosaccharides was strongly inhibited when the virus was raised in chick embryo cells, but less inhibition was observed when this virus was grown in MDCK cells. Nevertheless, in these cases also, the major oligosaccharide structure in the glycoproteins was the Man9GlcNAc2 species.     

Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris

N-glycans are synthesized in both yeast and mammals through the ordered assembly of a lipid-linked core Glc(3)Man(9)GlcNAc(2) structure that is subsequently transferred to a nascent protein in the endoplasmic reticulum. Once folded, glycoproteins are then shuttled to the Golgi, where additional but divergent processing occurs in mammals and fungi. We cloned the Pichia pastoris homolog of the ALG3 gene, which encodes the enzyme that converts Man(5)GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP. Deletion of this gene in an och1 mutant background resulted in the secretion of glycoproteins with a predicted Man(5)GlcNAc(2) structure that could be trimmed to Man(3)GlcNAc(2) by in vitro alpha-1,2-mannosidase treatment. However, several larger glycans ranging from Hex(6)GlcNAc(2) to Hex(12)GlcNAc(2) were also observed that were recalcitrant to an array of mannosidase digests. These results contrast the far simpler glycan profile found in Saccharomyces cerevisiae alg3-1 och1, indicating diverging Golgi processing in these two closely related yeasts. Finally, analysis of the P. pastoris alg3 deletion mutant in the presence and absence of the outer chain initiating Och1p alpha-1,6-mannosyltransferase activity suggests that the PpOch1p has a broader substrate specificity compared to its S. cerevisiae counterpart.     

Engineering of the yeast Yarrowia lipolytica for the production of glycoproteins lacking the outer-chain mannose residues of N-glycans

In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative alpha-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man8GlcNAc2, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man8GlcNAc2 to Man12GlcNAc2. Digestion with alpha-1,2- and alpha-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first alpha-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.     

The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells

The mannose analogue, 1-deoxymannojirimycin, which inhibits Golgi alpha-mannosidase I but not endoplasmic reticulum (ER) alpha-mannosidase has been used to determine the role of the ER alpha-mannosidase in the processing of the asparagine-linked oligosaccharides on glycoproteins in intact cells. In the absence of the inhibitor, the predominant oligosaccharide structures found on the ER glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase in UT-1 cells are single isomers of Man6GlcNAc and Man8GlcNAc. In the presence of 150 microM 1-deoxymannojirimycin, the Man8GlcNAc2 isomer accumulates indicating that the 1-deoxymannojirimycin-resistant ER alpha-mannosidase is responsible for the conversion of Man9GlcNAc2 to Man8GlcNAc2 on reductase. The processing of Man8GlcNAc2 to Man6GlcNAc2, however, must be attributed to a 1-deoxymannojirimycin-sensitive alpha-mannosidase. When cells were radiolabeled with [2-(3)H]mannose for 15 h in the presence of 1-deoxymannojirimycin and then further incubated for 3 h in nonradioactive medium without inhibitor, the Man8GlcNAc2 oligosaccharides which accumulated during the labeling period were partially trimmed to Man6GlcNAc. This finding suggests that a second alpha-mannosidase, sensitive to 1-deoxymannojirimycin, resides in the crystalloid ER and is responsible for trimming the reductase oligosaccharide chain from Man8GlcNAc2 to Man6GlcNAc2. To determine if ER alpha-mannosidase is responsible for trimming the oligosaccharides of all glycoproteins from Man9GlcNAc to Man8GlcNAc, the total asparagine-linked oligosaccharides of rat hepatocytes labeled with [2-(3)H]mannose in the presence or absence of 1.0 mM 1-deoxymannojirimycin were examined. the inhibitor prevented the formation of complex oligosaccharides and caused a 30-fold increase in the amount of Man9GlcNAc2 and a 13-fold increase in the amount of Man8GlcNAc2 present on secreted glycoproteins. This result suggests that only one-third of the secreted glycoproteins is initially processed by ER alpha-mannosidase, and two-thirds are processed by Golgi alpha-mannosidase I or another 1-deoxymannojirimycin-sensitive alpha-mannosidase. The inhibitor caused only a 2.6-fold increase in the amount of Man9GlcNAc2 on cellular glycoproteins suggesting that a higher proportion of these glycoproteins are initially processed by the ER alpha-mannosidase. We conclude that some, but not all, hepatocyte glycoproteins are substrates for ER alpha-mannosidase which catalyzes the removal of a specific mannose residue from Man9GlcNAc2 to form a single isomer of Man8GlcNAc2.     

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.     

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.     

The effect of glycoprotein-processing inhibitors on the secretion of glycoproteins by Madin-Darby canine kidney cells

The effects of various glycoprotein-processing inhibitors on the biosynthesis and secretion of N-linked glycoproteins was examined in cultured Madin-Darby canine kidney (MDCK) cells. Since incorporation of [2-3H]mannose into lipid-linked saccharides and into glycoproteins was much greater in phosphate-buffered saline (PBS) than in serum-supplemented basal medium (BME), most experiments were done in PBS. Castanospermine, an inhibitor of glucosidase I, caused the formation of glycoproteins having mostly Glc3Man7-9(GlcNAc)2 structures; deoxymannojirimycin, an inhibitor of mannosidase I, gave mostly glycoproteins with Man9(GlcNAc)2 structures; swainsonine, an inhibitor of mannosidase II, caused the accumulation of hybrid types of oligosaccharides. Castanospermine and swainsonine, either in PBS or in BME medium, had no effect on the incorporation of [2-3H]mannose or [5,6-3H]leucine into the secreted glycoproteins and, in fact, there was some increase in mannose incorporation in their presence. These inhibitors also did not affect mannose incorporation into cellular glycoproteins nor did they affect the biosynthesis as measured by mannose incorporation into lipid-linked saccharides. On the other hand in PBS medium, deoxymannojirimycin, at 25 micrograms/mL, caused a 75% inhibition in mannose incorporation into secreted glycoproteins, but had no effect on the incorporation of [3H]leucine into the secreted glycoproteins. Since deoxymannojirimycin also strongly inhibited mannose incorporation into lipid-linked oligosaccharides in PBS, the decreased amount of radioactivity in the secreted and cellular glycoproteins may reflect the formation of glycoproteins with fewer than normal numbers of oligosaccharide chains, owing to the low levels of oligosaccharide donor. However, in BME medium, there was only slight inhibition of mannose incorporation into lipid-linked saccharides and into cellular and secreted glycoproteins.     

ALG9 mannosyltransferase is involved in two different steps of lipid-linked oligosaccharide biosynthesis

N-linked protein glycosylation follows a conserved pathway in eukaryotic cells. The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for the oligosaccharyltransferase (OST), is catalyzed by different glycosyltransferases located at the membrane of the endoplasmic reticulum (ER). The substrate specificity of the different glycosyltransferase guarantees the ordered assembly of the branched oligosaccharide and ensures that only completely assembled oligosaccharide is transferred to protein. The glycosyltransferases involved in this pathway are highly specific, catalyzing the addition of one single hexose unit to the lipid-linked oligosaccharide (LLO). Here, we show that the dolichylphosphomannose-dependent ALG9 mannosyltransferase is the exception from this rule and is required for the addition of two different alpha-1,2-linked mannose residues to the LLO. This report completes the list of lumen-oriented glycosyltransferases required for the assembly of the LLO.     

Inhibitors of the biosynthesis and processing of N-linked oligosaccharides

A number of glycoproteins have oligosaccharides linked to protein in a GlcNAc----asparagine bond. These oligosaccharides may be either of the complex, the high-mannose or the hybrid structure. Each type of oligosaccharides is initially biosynthesized via lipid-linked oligosaccharides to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol and transfer of this oligosaccharide to protein. The oligosaccharide portion is then processed, first of all by removal of all three glucose residues to give a Man9GlcNAc2-protein. This structure may be the immediate precursor to the high-mannose structure or it may be further processed by the removal of a number of mannose residues. Initially four alpha 1,2-linked mannoses are removed to give a Man5 - GlcNAc2 -protein which is then lengthened by the addition of a GlcNAc residue. This new structure, the GlcNAc- Man5 - GlcNAc2 -protein, is the substrate for mannosidase II which removes the alpha 1,3- and alpha 1,6-linked mannoses . Then the other sugars, GlcNAc, galactose, and sialic acid, are added sequentially to give the complex types of glycoproteins. A number of inhibitors have been identified that interfere with glycoprotein biosynthesis, processing, or transport. Some of these inhibitors have been valuable tools to study the reaction pathways while others have been extremely useful for examining the role of carbohydrate in glycoprotein function. For example, tunicamycin and its analogs prevent protein glycosylation by inhibiting the first step in the lipid-linked pathway, i.e., the formation of Glc NAc-pyrophosphoryl-dolichol. These antibiotics have been widely used in a number of functional studies. Another antibiotic that inhibits the lipid-linked saccharide pathway is amphomycin, which blocks the formation of dolichyl-phosphoryl-mannose. In vitro, this antibiotic gives rise to a Man5GlcNAc2 -pyrophosphoryl-dolichol from GDP-[14C]mannose, indicating that the first five mannose residues come directly from GDP-mannose rather than from dolichyl-phosphoryl-mannose. Other antibodies that have been shown to act at the lipid-level are diumycin , tsushimycin , tridecaptin, and flavomycin. In addition to these types of compounds, a number of sugar analogs such as 2-deoxyglucose, fluoroglucose , glucosamine, etc. have been utilized in some interesting experiments. Several compounds have been shown to inhibit glycoprotein processing. One of these, the alkaloid swainsonine , inhibits mannosidase II that removes alpha-1,3 and alpha-1,6 mannose residues from the GlcNAc- Man5GlcNAc2 -peptide. Thus, in cultured cells or in enveloped viruses, swainsonine causes the formation of a hybrid structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transitory effects of glucose starvation on the synthesis of dolichol-linked oligosaccharides in mammalian cells

The distribution of lipid-linked oligosaccharide intermediates in cultured mammalian cells has been studied under conditions of glucose deprivation. It was found that at low to moderate cell densities within 20 min of glucose starvation, the major species of lipid-linked oligosaccharide shifted from mainly a single species containing three glucose, nine mannose, and two N-acetylglucosamine residues to a pattern dominated by two species containing either five mannose and two N-acetylglucosamine residues or two mannose and two N-acetylglucosamine residues. At high cell densities, this effect was not evident. Continued glucose starvation at low density resulted in a second shift in distribution in which the proportions of these two species decreased and that of the original major species (Glc3Man9GlcNAc2) increased. Addition of glucose or mannose, but not pyruvate, glutamine, galactose, inositol, or glycine, prevented the shift to the Man5GlcNAc2 and Man2GlcNAc2 species. The intermediates that accumulate during glucose starvation were identified by their elution position on gel filtration columns, sensitivity to digestion with alpha-mannosidase, resistance to digestion with endo-beta-N-acetylglucosaminidase H, and by the products of Smith degradation. These data suggest that a regulatory point in the lipid-linked oligosaccharide synthetic pathway exists at the reaction in which Man5GlcNAc2-P-P-dolichol is converted to Man6GlcNAc2-P-P-dolichol.     

Characterization of an alg2 mutant of the zygomycete fungus Rhizomucor pusillus

The zygomycete fungus Rhizomucor pusillus secretes an aspartic proteinase (MPP) that contains asparagine ( N )-linked oligosaccharides at two sites. Mutant strain 1116 defective in N -glycosylation secretes MPP with truncated oligo-saccharide chains. Lipid-linked oligosaccharides in mutant 1116 were labeled with [6-(3)H]glucosamine and [2-(3)H]mannose, prepared by cycles of solvent extraction, and analyzed by gel filtration chromatography on a Bio-Gel P-4 column after mild acid-hydrolysis. Mutant 1116 accumulated an intermediate, Man(1)GlcNAc(2)-dolichol pyrophosphate (PP-Dol), whereas wild-type strain F27 synthesized the fully assembled oligosaccharide precursor Glc(3)Man(9)GlcNAc(2)-PP-Dol. Consistent with this, alg2 encoding a mannosyltransferase in the lipid-linked oligosaccharide biosynthetic pathway in mutant 1116 had a 5 bp insertion that generated a stop codon in the middle of the coding sequence. Transformation of mutant 1116 with the intact alg2 gene on a pUC19-derived plasmid generated transformants that contained multicopies of alg2 at the alg2 locus. Glycosylation of the total proteins in the transformants was recovered to the same level as in strain F27, as determined with peroxidase-concanavalin A. These transformants produced MPP mainly with the same N -linked oligosaccharides as that produced by strain F27, but still with truncated oligosaccharides in small amounts. All of these data show that Alg2 is an alpha-1,3 or alpha-1,6 mannosyltransferase that elongates Man(1)GlcNAc(2)-PP-Dol to Man(2)GlcNAc(2)-PP-Dol. The slower growth of mutant 1116 was significantly recovered on introduction of alg2. The viability of the alg2 mutants of the zygomycete R.pusillus makes a contrast with the lethal effect of ALG2 mutations in the yeast Saccharomyces cerevisiae.     

The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man5GlcNAc2-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum

The initial steps in N-linked glycosylation involve the synthesis of a lipid-linked core oligosaccharide followed by the transfer of the core glycan to nascent polypeptides in the endoplasmic reticulum (ER). Here, we describe alg11, a new yeast glycosylation mutant that is defective in the last step of the synthesis of the Man(5)GlcNAc(2)-PP-dolichol core oligosaccharide on the cytosolic face of the ER. A deletion of the ALG11 gene leads to poor growth and temperature-sensitive lethality. In an alg11 lesion, both Man(3)GlcNAc(2)-PP-dolichol and Man(4)GlcNAc(2)-PP-dolichol are translocated into the ER lumen as substrates for the Man-P-dolichol-dependent sugar transferases in this compartment. This leads to a unique family of oligosaccharide structures lacking one or both of the lower arm alpha1,2-linked Man residues. The former are elongated to mannan, whereas the latter are poor substrates for outerchain initiation by Ochlp (Nakayama, K.-I., Nakanishi-Shindo, Y., Tanaka, A., Haga-Toda, Y., and Jigami, Y. (1997) FEBS Lett. 412, 547-550) and accumulate largely as truncated biosynthetic end products. The ALG11 gene is predicted to encode a 63.1-kDa membrane protein that by indirect immunofluorescence resides in the ER. The Alg11 protein is highly conserved, with homologs in fission yeast, worms, flies, and plants. In addition to these Alg11-related proteins, Alg11p is also similar to Alg2p, a protein that regulates the addition of the third mannose to the core oligosaccharide. All of these Alg11-related proteins share a 23-amino acid sequence that is found in over 60 proteins from bacteria to man whose function is in sugar metabolism, implicating this sequence as a potential sugar nucleotide binding motif.     

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.     

Processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus SA11

The synthesis and oligosaccharide processing of the glycoproteins of SA11 rotavirus in infected Ma104 cells was examined. Rotavirus assembles in the rough endoplasmic reticulum (RER) and encodes two glycoproteins: VP7, a component of the outer viral capsid, and NCVP5, a nonstructural protein. A variety of evidence suggests the molecules are limited to the ER, a location consistent with the high mannose N-linked oligosaccharides modifying these proteins. VP7 and NCVP5 were shown to be integral membrane proteins. In an in vitro translation system supplemented with dog pancreas microsomes, they remained membrane associated after high salt treatment and sodium carbonate-mediated release of microsomal contents. In infected cells, the oligosaccharide processing of these molecules proceeded in a time-dependent manner. For VP7, Man8GlcNAc2 and Man6GlcNAc2 were the predominant intracellular species after a 5-min pulse with [3H]mannose and a 90 min chase, while in contrast, trimming of NCVP5 halted at Man8GlcNAc2. VP7 on mature virus was processed to Man5GlcNAc2. It is suggested that the alpha-mannosidase activities responsible for the formation of these structures reside in the ER. In the presence of the energy inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), processing of VP7 and the vesicular stomatitis virus G protein was blocked at Man8GlcNAc2. After a 20-min chase of [3H]mannose-labeled molecules followed by addition of CCCP, trimming of VP7 could continue while processing of G protein remained blocked. Thus, an energy-sensitive translocation step within the ER may mark the divergence of the processing pathways of these glycoproteins.     

Posttranslational Protein Modification: Biosynthetic Control Mechanisms in the Glycosylation of the Major Myelin Glycoprotein by Schwann Cells

The posttranslational processing of the asparagine-linked oligosaccharide chain of the major myelin glycoprotein (P0) by Schwann cells was evaluated in the permanently transected, adult rat sciatic nerve, where there is no myelin assembly, and in the crush injured nerve, where there is myelin assembly. Pronase digestion of acrylamide gel slices containing the in vitro labeled [3H]mannose and [3H]fucose P0 after electrophoresis permitted analysis of the glycopeptides by lectin affinity and gel filtration chromatography. The concanavalin A-Separose profile of the [3H]mannose P0 glycopeptides from the transected nerve revealed the high-mannose-type oligosaccharide as the predominant species (72.9%), whereas the normally expressed P0 glycoprotein that is assembled into the myelin membrane in the crushed nerve contains 82.9-91.9% of the [3H]mannose radioactivity as the complex-type oligosaccharide chain. Electrophoretic analysis of immune precipitates verified the [3H]mannose as being incorporated into P0 for both the transected and crushed nerve. The high-mannose-type glycopeptides of the transected nerve isolated from the concanavalin A-Sepharose column were hydrolyzed by endo-beta-N-acetylglucosaminidase H, and the oligosaccharides were separated on Biogel P4. Man8GlcNAc and Man7GlcNAc were the predominant species with radioactivity ratios of 12.5/7.2/1.4/1.0 for the Man8, Man7, Man6, and Man5 oligosaccharides, respectively. Jack bean alpha-D-mannosidase gave the expected yields of free Man and ManGlcNAc from these high-mannose-type oligosaccharides. The data support the notion that at least two alpha-1,2-mannosidases are responsible for converting Man9GlcNAc2 to Man5GlcNAc2. The present experiments suggest distinct roles for each mannosidase and that the second mannosidase (I-B) may be an important rate-limiting step in the processing of this glycoprotein with the resulting accumulation of Man8GlcNAc2 and Man7GlcNAc2 intermediates. Pulse chase experiments, however, demonstrated further processing of this high-mannose-type oligosaccharide in the transected nerve. The [3H]mannose P0 glycoprotein with Mr of 27,700 having the predominant high-mannose-type oligosaccharide shifted its Mr to 28,500 with subsequent chase. This band at 28,500 was shown to have the complex-type oligosaccharide chain and to contain fucose attached to the core asparagine-linked GlcNAc residue. The extent of oligosaccharide processing of this down-regulated glycoprotein remains to be determined.     

Engineering Yarrowia lipolytica to produce glycoproteins homogeneously modified with the universal Man3GlcNAc2 N-glycan core

Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as "generally recognized as safe." Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man(3)GlcNAc(2) structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man(5)GlcNAc(2) and GlcMan(5)GlcNAc(2) glycans, and to a lesser extent with Glc(2)Man(5)GlcNAc(2) glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man(3)GlcNAc(2) structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.     

Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha

Presently almost no information is available on the oligosaccharide structure of the glycoproteins secreted from the methylotrophic yeast Hansenula polymorpha, a promising host for the production of recombinant proteins. In this study, we analyze the size distribution and structure of N-linked oligosaccharides attached to the recombinant glycoprotein glucose oxidase (GOD) and the cell wall mannoproteins obtained from H. polymorpha. Oligosaccharide profiling showed that the major oligosaccharide species derived from the H. polymorpha-secreted recombinant GOD (rGOD) had core-type structures (Man(8-12)GlcNAc(2)). Analyses using anti-alpha 1,3-mannose antibody and exoglycosidases specific for alpha 1,2- or alpha 1,6-mannose linkages revealed that the mannose outer chains of N-glycans on the rGOD have very short alpha 1,6 extensions and are mainly elongated in alpha 1,2-linkages without a terminal alpha 1,3-linked mannose addition. The N-glycans released from the H. polymorpha mannoproteins were shown to contain mostly mannose in their outer chains, which displayed almost identical size distribution and structure to those of H. polymorpha-derived rGOD. These results strongly indicate that the outer chain processing of N-glycans by H. polymorpha significantly differs from that by Saccharomyces cerevisiae, thus generating much shorter mannose outer chains devoid of terminal alpha 1,3-linked mannoses.     

The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation

The biosynthesis of the lipid-linked oligosaccharide substrate for N- linked protein glycosylation follows a highly conserved pathway at the membrane of the endoplasmic reticulum. Based on the synthetic growth defect in combination with a reduced oligosaccharyltransferase activity (wbp1), we have identified alg10 mutant strains which accumulate lipid- linked Glc2Man9GlcNAc2. We cloned the corresponding wild-type gene and show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose- dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide. Hypoglycosylation of secreted proteins in alg10 deletion strains demonstrates that the terminal alpha-1,2-linked glucose residue is a key element in substrate recognition by the oligosaccharyltransferase. This ensures that primarily completely assembled oligosaccharide is transferred to protein.