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.     

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.     

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.     

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)     

Transient glucosylation of protein-bound Man9GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2 in calf thyroid cells. A possible recognition signal in the processing of glycoproteins

Calf thyroid slices incubated with [U-14C]glucose synthesized protein-bound Glc3Man9GlcNAc2, Glc2-Man9GlcNAc2, Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2. Although label in the glucose residues of the last three compounds could be detected within 5 min of incubation, appearance of radioactivity in the mannose residues of the alpha-mannosidase-resistant cores of Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 took more than 30 and 60 min, respectively, to appear after label was detected in the same mannose residues of Glc1Man9GlcNAc2. The glucose residues were removed upon chasing the slices with unlabeled glucose. The last compound to disappear was Glc1Man9GlcNAc2. Calf thyroid microsomes incubated with UDP-[U-14C]Glc synthesized the five protein-bound oligosaccharides mentioned above. Although addition to GDP-Man to the incubation mixtures greatly diminished the formation of Glc3Man9GlcNAc2 bound either to dolichol-P-P or to protein, labeling of Glc1Man9GlcNAc2, Glc1Man8GlcNAc2, and Glc1Man7GlcNAc2 was not affected. Addition of kojibiose prevented deglucosylation of protein-bound Glc3Man9GlcNAc2 without affecting the formation of Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 and only partially diminishing that of Glc1Man9GlcNAc2. These results indicate that Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 were formed by glucosylation of the unglucosylated species and not be demannosylation of Glc1Man9GlcNAc2 and that probably part of the latter compound was formed in the same way.     

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.     

Biosynthesis of the cancer-associated sialyl-Lea antigen

A cancer-associated glycolipid antigen defined by monoclonal antibody 19-9 has the structure NeuAc alpha 2-3Gal Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer. We have (formula; see text) studied its biosynthesis by testing the capacity of a crude microsomal fraction of SW 1116 cells to catalyze the addition of fucosyl or sialyl residues from GDP-fucose or CMP-sialic acid to glycolipid or oligosaccharide precursors. When the tetrasaccharide NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LSTa) is incubated with GDP-[14C]fucose and SW 1116 microsomes, a 14C-labeled oligosaccharide is formed that can be separated from the incubation mixture on an affinity column containing antibody 19-9 bound to protein A-Sepharose. The product migrates slower than LSTa when analyzed by paper or thin-layer chromatography. After treatment with neuraminidase, it co-migrates with the pentasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (formula; see text) (LNF II) in both chromatographic systems. Similar experiments demonstrate that SW 1116 microsomes catalyze the addition of a sialyl residue to the tetrasaccharide Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc to form LSTa. However, when LNF II is incubated with CMP-[14C]sialic acid and SW 1116 microsomes, no 19-9-active product is detected by affinity chromatography or by paper or thin-layer chromatography. Results using glycolipid precursors are consistent with these findings and also demonstrate the presence of the Lewis fucosyltransferase in SW 1116 cells. Thus, the biosynthesis of the sialyl-Lea antigen proceeds by addition of sialic acid to a type 1 precursor chain by a sialyltransferase, followed by addition of fucose by the Lewis fucosyltransferase.     

Structural characterization of several galactofuranose-containing, high-mannose-type oligosaccharides present in glycoproteins of the trypanosomatid Leptomonas samueli

It was reported before that cells of the trypanosomatid Leptomonas samueli incubated with [14C]glucose synthesized dolichol-P-P-linked Man9GlcNAc2 as the main and largest derivative. It is now reported that this protozoan is deficient in dolichol-P-Glc synthesis as judged from results obtained in a cell-free assay. We have structurally characterized several endo-beta-N-acetylglucosaminidase H sensitive oligosaccharides present in mature glycoproteins of this parasite. The compounds appeared to have the compositions Gal3Man9GlcNAc2, Gal2Man9GlcNAc2, Gal1Man9GlcNAc2, Man9GlcNAc2, Gal1Man8GlcNAc2, Man8GlcNAc2, Gal1Man7GlcNAc2, and Man7GlcNAc2. The galactose residues were in all cases in the furanose form and linked to mannoses in nonreducing ends. In the cases of Gal1Man8GlcNAc2 and Gal1Man7GlcNAc2, the galactose-substituted mannose units were the nonreducing residues originally present in the oligosaccharide transferred from dolichol-P-P (Man9GlcNAc2) and not the nonreducing termini generated by demannosylation of the latter oligosaccharide. Except for Gal3Man9GlcNAc2, the other galactosylated compounds appeared to be mixtures of several isomers.     

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.     

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.     

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.     

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.     

Plant glucosidase II catalyzes a transglucosylation reaction in addition to the hydrolytic reaction

When the purified plant glucosidase II was incubated with [3H]Glc2Man9GlcNAc in the presence of glycerol and the products were analyzed by gel filtration, a large peak of radioactivity emerged just before the glucose standard. The formation of this peak was dependent on both the presence of Glc2Man9GlcNAc and the presence of glycerol, and the amount of this product increased with time of incubation and amount of glucosidase II in the incubation. When the incubation was performed with [3H]Glc2Man9GlcNAc plus [14C]glycerol, the product contained both 14C and 3H. Strong acid hydrolysis of the purified product gave rise to [14C]glycerol and [3H]glucose. Various other chemical treatments and chromatographic techniques showed that the product was glucosyl----glycerol. Since the glucose was released by alpha-glucosidase, the product must be glucosyl-alpha-glycerol. This study demonstrates that the processing glucosidase II catalyzes a trans-glycosylation reaction in the presence of acceptors like glycerol. Since this transglycosylation reaction may give rise to unexpected products, investigators should be aware of its possible occurrence.     

Trypanosoma cruzi cells undergo an alteration in protein N-glycosylation upon differentiation

Trypanosoma cruzi epimastigotes (insect gut stage) incubated with [U-14C]glucose synthesized Man9GlcNAc2-P-P-dolichol as practically the sole dolichol-P-P derivative. On the other hand, amastigotes (intracellular stage) of the same parasite synthesized four to five times more Man7GlcNAc2-P-P-dolichol than Man9GlcNAc2-P-P-dolichol. Evidence is presented indicating that, whereas in epimastigotes only Man9GlcNAc2 was transferred to proteins, in amastigotes both Man7GlcNAc2 and Man9GlcNAc2 were transferred in direct proportion to their respective amounts bound to dolichol-P-P. The change in the mechanism of protein N-glycosylation could be observed upon in vitro differentiation of amastigotes to epimastigotes. The dissimilar size of the main oligosaccharides transferred to proteins in epimastigotes and amastigotes was responsible for differences in two structural features of high mannose-type oligosaccharides present in mature glycoproteins of both forms of the parasite, namely the average size of the compounds and the structure of the main species of some isomer oligosaccharides.     

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.      

N-linked high mannose-type oligosaccharides in the protozoa Crithidia fasciculata and Crithidia harmosa contain galactofuranose residues

Incubation of Crithidia fasciculata cells with [U-14C] glucose led to the synthesis of Man-P-dolichol but not of Glc-P-dolichol. The main and largest dolichol-P-P-linked oligosaccharide formed was Man7GlcNAc2 whether labeling was performed in 5 mM sodium pyruvate or 5.5 mM glucose. The protein-linked, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides isolated from mature glycoproteins were Man7GlcNAc and Gal1Man6GlcNAc, the latter being a mixture of two isomers. All the galactose residues were present in the furanose configuration, as judged by their extreme lability to acid hydrolysis, by the products obtained upon mild periodate oxidation, and by their sensitivity to beta-galactofuranosidase. Labeling cells for short times or at low temperature yielded a protein-bound, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide whose composition was Glc1Man7GlcNAc, of transient existence, and that was mainly labeled in the glucose residue. The latter oligosaccharide was detected on paper chromatography only as a smearing of Man7GlcNAc and Gal1Man6GlcNAc when cells were labeled with [2-3H] mannose, thus indicating that it was only present in minute amounts. Protein-bound endo beta-N-acetylglucosaminidase H-resistant oligosaccharides liberated, upon a mild acid treatment, galactose residues and an unidentified substituent. The treatment rendered the oligosaccharides sensitive to endo beta-N-acetylglucosaminidase H, which liberated Man7GlcNAc and two isomers of Man6GlcNAc. An almost similar mechanism of protein N-glycosylation, including the existence of galactofuranose residues in N-linked oligosaccharides, was found to occur in Crithidia harmosa.     

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.     

Biosynthesis of lipid-linked oligosaccharides. I. Preparation of lipid-linked oligosaccharide substrates.

In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using porcine pancreatic microsomes, while Glc3Man9(GlcNAc)2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6(Man alpha 1,3)Man alpha 1,6]Man beta 1,4GlcNAc beta 1,4GlcNAc-PP-dolichol; Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6]Man beta 1,4GlcNAc beta 1, 4GlcNac-PP-dolichol; and Man alpha 1,6(Man alpha 1,3)Man alpha 1, 6[Man alpha 1,3]Man beta 1,4GlcNAc-beta 1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described.     

Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing

Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1,6-Man backbone for elongation with additional alpha 1,6-linked mannose residues, according to the following scheme: (formula, see text).     

Bovine serum conglutinin is a lectin which binds non-reducing terminal N-acetylglucosamine, mannose and fucose residues.

Carbohydrate recognition by bovine serum conglutinin has been investigated by inhibition and direct binding assays using glycoproteins and polysaccharides from Saccharomyces cerevisiae (baker's yeast), and neoglycolipids derived from N-acetylglucosamine oligomers, mannobiose and human milk oligosaccharides. The results clearly show that conglutinin is a lectin which binds terminal N-acetylglucosamine, mannose and fucose residues as found in chitobiose (GlcNAc beta 1-4GlcNAc), mannobiose (Man alpha 1-3Man) and lacto-N-fucopentaose II [Fuc alpha 1-4(Gal beta 1-3)GlcNAc beta 1-3Gal beta 1-4Glc] respectively.