Lec15 cells transfer glucosylated oligosaccharides to protein.

B4-2-1 cells (Lec15 cells) are Chinese hamster ovary cells deficient in mannosylphosphoryldolichol synthase activity. They synthesize the truncated lipid intermediate Man5GlcNAc2-P-P-dolichol rather than the Glc3Man9GlcNAc2-P-P-dolichol synthesized by wild-type cells. In this report we present evidence that these cells did synthesize glucosylated Man5GlcNAc2-P-P-dolichol, but this species represented only a minor fraction of the labeled oligosaccharide-lipid. On the other hand, glucosylated oligosaccharides were a major species transferred to protein in these cells, showing that in vivo, glucosylated oligosaccharides are preferentially transferred to protein. The truncated oligosaccharides found in B4-2-1 cells were removed from the protein by N-glycanase treatment, since they were resistant to both endo-beta-N-acetylglucosaminidase H and F activity. B4-2-1 cells processed the glucosylated, truncated oligosaccharides transferred to G protein of vesicular stomatitis virus, leading to infectious virus.     

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.     

Monoglucosylated oligomannosides are released during the degradation process of newly synthesized glycoproteins

The Chinese hamster ovary mutant MI8-5 is known to synthesize Man(9)GlcNAc(2)-P-P-dolichol rather than the fully glucosylated lipid intermediate Glc(3)Man(9)GlcNAc(2)-P-P-dolichol. This nonglucosylated oligosaccharide lipid precursor is used as donor for N-glycosylation. In this paper we demonstrate that a significant part of the glycans bound to the newly synthesized glycoproteins in MI8-5 cells are monoglucosylated. The presence of monoglucosylated glycans on glycoproteins determines their binding to calnexin as part of the quality control machinery. Furthermore, we point out the presence of Glc(1)Man(5)GlcNAc(1) in the cytosol of MI8-5 cells. This indicates that part of the monoglucosylated glycoproteins can be directed toward a deglycosylation process that occurs in the cytosol. Besides studies on glycoprotein degradation based on the disappearance of protein moieties, MI8-5 cells can be used as a tool to elucidate the various step leading to glycoprotein degradation by studying the fate of the glycan moieties.     

Structure of Saccharomyces cerevisiae alg3, sec18 mutant oligosaccharides

Asparagine-linked oligosaccharides are synthesized by transfer of Glc3Man9GlcNAc2 from dolichol pyrophosphate to nascent polypeptides. Assembly of the precursor proceeds by highly ordered sequential addition of mannose and glucose to form Glc3Man9GlcNAc2-P-P-dolichol. Yeast mutants in asparagine-linked glycosylation (alg), generated by an 3H-Man suicide technique, were assigned to eight complementation groups which define steps in oligosaccharide-lipid synthesis (Huffaker, T.C., and Robbins, P.W. (1982) J. Biol. Chem. 257, 3203-3210). Alg3 invertase oligosaccharides are resistant to endo-beta-N-acetylglucosaminidase H, and the lipid-oligosaccharide pool yields Man5Glc-NAc2, suggesting its structure may be that from mammalian cells lacking Man-P-dolichol (Chapman, A., et al. (1980) J. Biol. Chem. 255, 4441-4446). To test this supposition, the endoplasmic reticulum form of invertase derepressed in alg3,sec18 yeast at 37 degrees C was isolated as a source of oligosaccharides whose processing beyond glucose and/or mannose trimming, if involved, would be prevented. Man8GlcNAc2 and Man5GlcNAc2 were released by peptide-N-glycosidase F from alg3,sec18 invertase in a 1:5 molar ratio. 1H NMR spectroscopy revealed Man8GlcNAc2 to be the alpha 1,2-mannosidase-trimming product described earlier (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666), while Man5GlcNAc2 was Man alpha 1, 2Man alpha 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc beta 1, 4GlcNAc. This provides a structural proof for the lipid-linked Man5GlcNAc2 originally proposed from enzymatic and chemical analyses of the radiolabeled mammalian precursor. Experimental evidence indicates that, unlike the mammalian cell mutants which are unable to synthesize Man-P-dolichol, alg3 yeast accumulate Man5GlcNAc2-P-P-dolichol due to a defective alpha 1,3-mannosyltransferase required for the next step in oligosaccharide-lipid elongation.     

Synthesis of dolichol derivatives in trypanosomatids. Characterization of enzymatic patterns

We have previously described that in certain parasitic protozoa, namely the trypanosomatids, the dolichol-P-P-linked oligosaccharides synthesized in vivo and transferred to protein are devoid of glucose residues and contain 6, 7, or 9 mannose units depending on the species. We have now conducted a cell-free characterization of the enzymatic patterns responsible for these phenotypes. Microsomes from Trypanosoma cruzi, Crithidia fasciculata, Leishmania enriettii, and Blastocrithidia culicis were found to synthesize dolichol-P-[14C]Man but not dolichol-P-[14C]Glc when incubated with rat liver dolichol-P and GDP-[14C]Man or UDP-[14C]Glc, thus providing for an explanation to the absence of glucosylated dolichol-P-P derivatives. Formation of dolichol-P-P-oligosaccharides was assayed in incubation mixtures containing rat liver dolichol-P, GDP-[14C]Man, microsomes, and unlabeled Man5-8GlcNAc2-P-P-dolichol from bovine liver. Membranes from species synthesizing dolichol-P-P-linked Man6GlcNAc2 or Man7GlcNAc2 in vivo were found to synthesize the same compounds but not the higher homologues in the cell-free assay. Species forming Man9GlcNAc2-P-P-dolichol in vivo were found to synthesize lipid-linked Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 in vitro. It is concluded that there are at least three and probably four different dolichol-P-Man-dependent enzymatic activities involved in the synthesis of dolichol-P-P-linked Man9GlcNAc2 and that microorganisms not forming that compound are devoid of all mannosyltransferases responsible for the addition of the missing residues and not only of the enzyme involved in the synthesis of the homologue higher than the oligosaccharide occurring in vivo by a single mannose unit.     

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.     

A block at Man5GlcNAc2-pyrophosphoryldolichol in intact but not disrupted castanospermine and swainsonine-resistant Chinese hamster ovary cells

A mutation in glycoprotein processing inhibitor-resistant (PIR) Chinese hamster ovary (CHO) cells was previously shown to result in a block at the Man5GlcNAc2 stage of the dolichol-oligosaccharide biosynthetic pathway (Lehrman, M.A., and Zeng, Y. (1989) J. Biol. Chem. 264, 1584-1593). These cells had normal mannose-P-dolichol synthase activity and were able to transfer the Man5GlcNAc2 oligosaccharides to protein. We have now characterized the mutation in greater detail. In PIR cells, biosynthesis of GDP-mannose and mannose-P-dolichol was normal, and pulse-chase analysis indicated that the rate of Man5GlcNAc2-P-P-dolichol formation in vivo was similar to that in parental CHO cells but without subsequent formation of larger intermediates. Cell fusion studies demonstrated that the PIR genotype was recessive and that PIR cells could complement the mutation in B4-2-1 cells, which fail to synthesize mannose-P-dolichol. In contrast to the results obtained with intact cells, incubation of membrane preparations of PIR cells with GDP-[3H]mannose resulted in the synthesis of intermediates containing up to 9 mannose residues, indicating that the cells contained active mannosyltransferases VI to IX. With a simplified assay for the formation of intermediates containing 6 to 9 mannoses, it was shown that physical disruption of PIR cells was able to eliminate the block at the pentamannosyl stage. Furthermore, although the temperature requirements of the reactions for the control CHO and PIR membranes were similar, Man5GlcNAc2-elongating activity in CHO membranes was inhibited by alkaline pH treatment, whereas this treatment irreversibly stimulated the activity in PIR membranes. Taken together, these results suggest that the PIR cells have a recessive defect, and that the missing gene product is required by mannosyltransferase VI in vivo for proper utilization of either mannose-P-dolichol or Man5GlcNAc2-P-P-dolichol. Since the defect was manifested in vivo but not in vitro, this requirement appears necessary for intact cells but not for disrupted cells or isolated membranes.     

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.     

Glycoprotein biosynthesis in yeast: purification and characterization of the endoplasmic reticulum Man9 processing alpha-mannosidase.

Saccharomyces cerevisiae Man9-alpha-mannosidase, responsible for trimming Man9GlcNAc2 in the endoplasmic reticulum to Man8GlcNAc2, the substrate for oligosaccharide elongation, has been purified to homogeneity from stabilized microsomal membranes without employing autolytic digestion. The activity was solubilized by the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulphonate (CHAPS), whose presence was necessary for maximal activity. Purification included Q-Sepharose ion-exchange chromatography, preparative isoelectric focusing and HPLC gel filtration on TSK 3000 matrix. Overall purification from post-nuclear supernatants was estimated to be 110,000-fold with a 50% recovery of activity. The purified enzyme hydrolysed Man9GlcNAc1,2 from thyroglobulin or oligosaccharide-lipid, but not invertase Man9GlcNAc, Man1 alpha 2Man1 alpha OCH3 or p-nitrophenyl-alpha-D-mannopyranoside. Conversion of thyroglobulin Man9GlcNAc to Man8GlcNAc was linear with time and enzyme concentration, with an apparent Km of 0.2 mM and a specific activity of 220 IU/mg. Glc3Man9GlcNAc2 from oligosaccharide-lipid was as good a substrate as Man9GlcNAc, but the lipid-linked Man7GlcNAc2 isomer was hydrolysed at only 10% of this rate. Hydrolysis of defined isomers of IgM and bovine thyroglobulin Man6,7,8GlcNAc indicated that, for maximal alpha 1,2-mannosidase activity, only the alpha 1,2-linked terminal mannoses on the alpha 3 branch of the Man9GlcNAc precursor were dispensable. Isomers lacking the terminal alpha 1,2-linked mannose on the alpha 6 branch were hydrolysed at only approximately 10% of the maximal rate. The enzyme exhibited a pI of 5.3 and a pH optimum at 6.5. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the absence of reducing agents gave a single sharp band at 66 kDa, while in the presence of beta-mercaptoethanol equimolar amounts of two peptides, one of 44 kDa and one of 23 kDa, were obtained. Sizing on Sephacryl SF300, Superose 12 and TSK 3000 provided a holoenzyme mol. wt of 60-68 kDa, indicating that the isolated active form of the Man9-alpha-mannosidase was composed of one each of the sulphydryl-bonded dissimilar peptides. The enzyme bound to concanavalin A (ConA)-Sepharose and was eluted with alpha-methylmannoside, indicating the presence of high-mannose oligosaccharides. The Man9-alpha-mannosidase required low levels of Ca2+, which could be removed by EGTA. Activity was restored by Ca2+ or Zn2+, but not by Mg2+ or Mn2+.     

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)     

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.     

Characterization of dolichol diphosphate oligosaccharide: protein oligosaccharyltransferase and glycoprotein-processing glucosidases occurring in trypanosomatid protozoa

We have previously reported that the oligosaccharides transferred in vivo from dolichol-P-P derivatives in protein N-glycosylation in trypanosomatids are devoid of glucose residues and contain 2 N-acetylglucosamine and 6, 7, or 9 mannose units depending on the species. In this respect trypanosomatids differ from wild type mammalian, plant, insect, and fungal cells in which Glc3Man9GlcNAc2 is transferred. We are now reporting that incubation of Glc1-3Man9GlcNAc2-P-P-dolichol and Man7-9GlcNAc2-P-P-dolichol with membranes of Trypanosoma cruzi, Leptomonas samueli, Crithidia fasciculata, and Blastocrithidia culicis and an acceptor hexapeptide leads to the transfer of the six above mentioned lipid-linked oligosaccharides at the same rate. Control experiments performed under similar conditions but with rat liver and Saccharomyces cerevisiae membranes showed that, as already known, Glc3Man9GlcNAc2 is preferentially transferred in the latter systems. We have also previously reported that, once transferred to protein, the oligosaccharides become transiently glucosylated in trypanosomatids. Depending on the species, protein-linked Glc1Man5-9GlcNAc2 have been transiently detected in cells incubated with [14C] glucose. We are now reporting that glucosidase activities degrading both Glc1Man9GlcNAc2 and Glc2Man9GlcNAc2 were detected in T. cruzi, L. samueli, and C. fasciculata. The enzymatic activities were associated with a membrane fraction; they had a neutral optimum pH value, and similarly to mammalian glucosidase II, the enzyme acting on the monoglucosylated substrate showed a decreased affinity when the latter contained fewer mannose residues. No glucosidase I-like enzyme acting on Glc3Man9GlcNAc2 was detected in any of the three above-mentioned protozoan species. This result is consistent with the fact that no oligosaccharides containing 3 glucose units occur in trypanosomatids.     

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.     

The accumulation of Man(6)GlcNAc(2)-PP-dolichol in the Saccharomyces cerevisiae Deltaalg9 mutant reveals a regulatory role for the Alg3p alpha1,3-Man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing

N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol. High-field (1)H NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Deltaalg9 yeast showed its structure to be Manalpha1,2Manalpha1,2Manalpha1, 3(Manalpha1,3Manalpha1,6)-Manbeta1,4GlcNAcbeta1, 4GlcNAcalpha/beta, confirming the addition of the alpha1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm alpha1,6-linked Man. This Man(6)GlcNAc(2) is the endoglycosidase H-sensitive product of the Alg3p step. The Deltaalg9 Hex(7-10)GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed. When compared with alg3 sec18 and wild-type core mannans, Deltaalg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha1,6-Man branch-initiating mannosyltransferase.     

Golgi endo-alpha-D-mannosidase from rat liver, a novel N-linked carbohydrate unit processing enzyme

An enzyme has been found in Triton-treated rat liver Golgi membranes which trims Glc1Man9GlcNAc to Man8GlcNAc with the release of Glc alpha 1-3Man. By removing a glucosylmannose disaccharide and yielding only one Man8GlcNAc isomer, this endo-alpha-D-mannosidase provides a processing route alternative to the sequential actions of alpha-glucosidase II and alpha-mannosidase I. The endomannosidase was fully active in the presence of 1-deoxynojirimycin and EDTA which inhibited exoglycosidase release of glucose and mannose, respectively, and these agents were, therefore, included in the standard assay. The specific activity of the endomannosidase was found to be 69-fold greater in Golgi than in rough endoplasmic reticulum (RER) membranes, and Golgi-RER mixing experiments excluded the possibility that the low activity in the RER was the result of some inhibitor present in this fraction. The neutral pH optimum (approximately 7.0) of the enzyme was consistent with a role in N-linked oligosaccharide processing. The existence of an endo-alpha-D-mannosidase pathway for glucose removal could provide an explanation for the incomplete block in oligosaccharide processing which is observed in cells with inhibited or deficient alpha-glucosidase.     

Induction of glycoprotein biosynthesis in activated B lymphocytes

Resting murine splenic B lymphocytes (B cells) can be stimulated to proliferate by exposure to a variety of polyclonal activators. To investigate changes in glycoprotein synthesis that occur during the activation process, N-glycosylation activity was assessed by following the incorporation of [2-3H]mannose into dolichol-linked oligosaccharide intermediates and glycoprotein after B cells were exposed to anti-immunoglobulin M (anti-mu). Stimulation of B cells by anti-mu resulted in a dramatic induction of N-glycosylation activity. The incorporation of radiolabeled mannose into oligosaccharide-lipid increased 9-fold while the rate of labeling of glycoprotein increased 27-fold between 18 and 38 h after exposure to anti-mu. Maximal stimulation of N-glycosylation activity was observed at an anti-mu concentration of 20-50 micrograms/ml. Similar results were obtained when B cells were activated by bacterial lipopolysaccharide (LPS), another polyclonal activating agent. The major dolichol-bound oligosaccharide labeled during the induction period was determined to be Glc3Man9GlcNAc2 by HPLC analysis. Nearly full induction of oligosaccharide-lipid synthesis and protein N-glycosylation was also seen when DNA synthesis was suppressed by activating B cells with anti-mu in a serum-free medium, or by activating with anti-mu or LPS in the presence of hydroxyurea. The results suggest that the N-glycosylation pathway is induced during the G0 to G1 transition or during the G1 period, and that entry into S phase is not required. These studies describe a striking developmental increase in N-glycosylation activity and extend the information on biochemical changes occurring during the activation of B cells.     

A new yeast mutation in the glucosylation steps of the asparagine-linked glycosylation pathway. Formation of a novel asparagine-linked oligosaccharide containing two glucose residues

We have isolated and characterized a new yeast mutation in the glucosylation steps of lipid-linked oligosaccharide biosynthesis, alg8-1. Cells carrying the alg8-1 mutation accumulate Glc1Man9GlcNAc2-lipid both in vivo and in vitro. We present evidence showing that the alg8-1 mutation blocks addition of the second alpha 1,3-linked glucose. alg8-1 cells transfer Glc1Man9GlcNAc2 to protein instead of the wild type oligosaccharide, Glc3Man9GlcNAc2. Pulse-chase studies indicate that the Glc1Man9GlcNAc2 transferred is processed more slowly than the wild type oligosaccharide. The yeast mutation gls1-1 lacks glucosidase I activity (Esmon, B., Esmon, P.C., and Schekman, R. (1984) J. Biol. Chem. 259, 10322-10327), the enzyme responsible for removing the alpha 1,2-linked glucose residues from protein-linked oligosaccharides. We demonstrate that gls1-1 cells contain glucosidase II activity (which removes alpha 1,3-linked glucose residues) and have constructed the alg8-1 gls1-1 haploid double mutant. The Glc1Man9GlcNAc2 oligosaccharide was trimmed normally in these cells, demonstrating that the alg8-1 oligosaccharide contained an alpha 1,3-linked glucose residue. A novel Glc2 compound was probably produced by the action of the biosynthetic enzyme that normally adds the alpha 1,2-linked glucose to lipid-linked Glc2Man9GlcNAc2. This enzyme may be able to slowly add alpha 1,2-linked glucose residue to protein-bound Glc1Man9GlcNAc2. The relevance of these findings to similar observations in other systems where glucose residues are added to asparagine-linked oligosaccharides and the possible significance of the reduced rate of oligosaccharide trimming in the alg mutants are discussed.     

Elicitors and suppressors of the defense response in tomato cells. Purification and characterization of glycopeptide elicitors and glycan suppressors generated by enzymatic cleavage of yeast invertase.

Cleavage of yeast invertase by alpha-chymotrypsin produced a number of small glycopeptides that were highly active as elicitors of ethylene biosynthesis and phenylalanine ammonia-lyase in suspension-cultured tomato cells. Five of these elicitors were purified and their amino acid sequence determined. They all had sequences corresponding to known sequences of yeast invertase, and all contained an asparagine known to carry a N-linked small high mannose glycan. The most active glycopeptide elicitor induced ethylene biosynthesis and phenylalanine ammonia-lyase half-maximally at a concentration of 5-10 nM. Structure-activity relationships of the peptide part were analyzed by further cleavage of a defined glycopeptide elicitor with various proteolytic enzymes. Removal of the C-terminal phenylalanine enhanced the elicitor activity, whereas removal of N-terminal arginine impaired it. A glycopeptide with the peptide part trimmed to the dipeptide arginine-asparagine was still fully active as elicitor. Glycopeptides with identical amino acid sequences were further separated into fractions differing in the oligosaccharide side chain. A given peptide had high elicitor activity when carrying a glycan with 10-12 mannosyl residues (Man10-12GlcNAc2), a 3-fold lower activity when carrying Man9GlcNAc2 and a 100-fold lower activity when carrying Man8GlcNAc2. The oligosaccharides, released by endo-beta-N-acetylglucosaminidase H from the pure glycopeptide elicitors, acted as suppressors of elicitor-induced ethylene biosynthesis and phenylalanine ammonia-lyase activity. A series of such oligosaccharides in the size range of Man8-13GlcNAc was purified. The structure and composition of the purified oligosaccharides corresponded to the known small high mannose glycans of yeast invertase as verified by 1H NMR spectroscopy at 600 MHz. The highest suppressor activities were obtained with the oligosaccharides containing 10-12 mannosyl residues (Man10-12GlcNAc). The oligosaccharide Man8 GlcNAc was ineffective as a suppressor. Thus, the structural requirements for the free oligosaccharides to act as efficient suppressors were the same as for the oligosaccharide side chains of the glycopeptides for high elicitor activity. We propose that the glycan suppressors bind to the same recognition site as the glycopeptide elicitors without inducing a response.     

Isolation of Chinese hamster ovary cell lines producing Man3GlcNAc2 asparagine-linked glycans.

Chinese hamster ovary lines with two mutations, one causing accumulation of Man5GlcNAc2-P-P-dolichol and a second resulting in defective N-acetylglucosaminyltransferase I activity, synthesize asparagine-linked glycans with the structure Man3GlcNAc2. As a result, the asparagine-linked glycans produced by these lines are smaller and less heterogeneous than those produced by other currently available animal cell lines.     

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.