Chemoenzymatic synthesis of CD52 glycoproteins carrying native N-glycans

A facile synthesis of homogeneous CD52 glycoproteins carrying native N-glycans was achieved using an endolycosidase-catalyzed oligosaccharide transfer as the key step. The synthesis consists of two steps: the solid phase synthesis of GlcNAc-CD52 and the transfer of a high-mannose type or complex type N-glycan from Man(9)GlcNAc(2) Asn or a sialglycopeptide to the GlcNAc-CD52, under the catalysis of the endo-beta-N-acetylglucosaminidases from Arthrobacter (Endo-A) and Mucor hiemalis (Endo-M), respectively.     

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

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

Demonstration that Golgi endo-alpha-D-mannosidase provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins

Studies on N-linked oligosaccharide processing were undertaken in HepG2 cells and calf thyroid slices to explore the possibility that the recently described Golgi endo-alpha-D-mannosidase (Lubas, W.A., and Spiro, R.G. (1987) J. Biol. Chem. 262, 3775-3781) is responsible for the frequently noted failure of glucosidase inhibitors to achieve complete cessation of complex carbohydrate unit synthesis. We have found that in the presence of the glucosidase inhibitors, castanospermine (CST) or 1-deoxynojirimycin, there is a substantial production of the glucosylated mannose saccharides (Glc3Man, Glc2Man, and Glc1Man) which are the characteristic products of endomannosidase action. Furthermore, in HepG2 cells, a secretion of these components into the medium could be demonstrated. Characterization of the N-linked polymannose oligosaccharides produced by HepG2 cells in the presence of CST (as well as 1-deoxymannojirimycin to prevent processing by alpha-mannosidase I) indicated the occurrence, in addition to the expected glucosylated species, of substantial amounts of Man8GlcNAc and Man7GlcNAc. Since Man9GlcNAc was almost completely absent and the Man8GlcNAc isomer was shown to be identical with that formed by the in vitro action of endomannosidase on glucosylated polymannose oligosaccharides, we concluded that this enzyme was actively functioning in the intact cells and could provide a pathway for circumventing the glucosidase blockade. Indeed, quantitative studies in HepG2 cells supported this contention as the continued formation of complex carbohydrate units (50% of control) during CST inhibition could be accounted for by the deglucosylation effected by endomannosidase.     

Characterization of oligosaccharides from the urine of loco-intoxicated sheep

Two major oligosaccharides were isolated by preparative HPLC from the urine of a locoweed-fed sheep. Analysis by gas-liquid chromatography and mass-spectrometry indicated compositions of (Man)4(GlcNAc)2 and (Man)5(GlcNAc)2, respectively. Structures were determined by digestion with alpha-D-mannosidase and endo-beta-N-acetylglucosaminidases D and H, and comparison of the products by HPLC with synthetic standards, and oligosaccharides isolated from human mannosidosis urine. Incubation with an exo-beta-N-acetylglucosaminidase was without effect.     

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.     

Catabolic pathway of oligosaccharide-diphospho-dolichol. Subcellular sites of the degradation of the oligomannoside moiety

The degradation of oligosaccharide-diphospho-dolichol leads to the release of oligosaccharide material ranging from (Glc)3(Man)9(GlcNAc)2-P to (Man)3 species and further smaller species. The subcellular location of the glucosidases and mannosidases involved in this catabolic process has been investigated on the basis of their differential sensitivity towards specific inhibitors (castanospermine, deoxymannojirimycin and swainsonine). The results indicate that the first steps of degradation down to the (Man)6 species occurs in the rough endoplasmic reticulum. This result is supported by the fact that the (Man)6 species is the end product when lipid-intermediate-derived glucosylated oligosaccharides are incubated with purified rough endoplasmic reticulum membranes. Swainsonine and lysosomotropic agents (chloroquine and ammonium chloride) do not affect the degradation process, thus indicating that neither Golgi apparatus nor lysosomes are involved in this catabolism. The observation of the same degradation pattern of the released oligosaccharide material in mannosidosis fibroblasts, lacking lysosomal mannosidases, confirms these results. Finally, the subcellular distribution of the released oligosaccharide material indicates that the oligomannosides larger than (Man)6 species are sequestered in the particulate fraction whereas, in contrast, oligomannosides smaller than (Man)6 species are found predominantly in the cytosol. Taken altogether, the experiments demonstrate that the first steps of the degradation of oligosaccharide-diphospho-dolichol occurs in the rough endoplasmic reticulum producing oligomannosides of the (Man)6 species which are then translocated to the cytoplasm to be further degraded.     

Structural study of the asparagine-linked oligosaccharides of lipophorin in locusts

The complete structure of oligosaccharides from locust lipophorin was studied. The asparagine-linked oligosaccharides were first liberated from the protein moiety of lipophorin by digestion with almond glycopeptidase (N-oligosaccharide glycopeptidase, EC 3.5.1.52). Two major oligosaccharides (E and F), separated by subsequent thin-layer chromatography, were analyzed by methylation analysis and 1H-NMR. Based on the experimental data, the whole structure of oligosaccharide E was identified as Man alpha 1----2Man alpha 1----6(Man alpha 1----2Man alpha 1----3) Man alpha 1----6(Man alpha 1----2Man alpha 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc. The data also revealed that oligosaccharide F is identical with oligosaccharide E in the structure, except for one glucose residue that is linked to the nonreducing terminal Man alpha 1----2 residue.     

A comparative study of the N-linked oligosaccharide structures of human IgG subclass proteins.

Quantitative oligosaccharide profiles were determined for each of 18 human IgG paraproteins representing the four subclasses. Each paraprotein exhibits a unique profile that may be substantially different from that observed for polyclonal IgG. The IgG2 and some IgG3 proteins analysed exhibit a predominance of oligosaccharide moieties having galactose on the Man(alpha 1----3) arm rather than the Man(alpha 1----6) arm; it was previously held that galactosylation of the Man(alpha 1----6) arm is preferred, as observed for IgG1, IgG4 and polyclonal IgG. An IgG4 protein is reported that has galactosylated Man(alpha 1----3) and Man(alpha 1----6) arms on both Fc-localized carbohydrate moieties; previous findings suggested that such fully glycosylated structures could not be accommodated within the internal space of the C gamma 2 domains. Unusual monoantennary oligosaccharides present in IgG2 and IgG3 proteins were isolated and their structures determined.     

alpha-Glucosidase II-deficient cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.

The kinetics of N-linked oligosaccharide processing and the structures of the processing intermediates have been examined in normal parental BW5147 mouse lymphoma cells and the alpha-glucosidase II-deficient PHAR2.7 mutant cells. The mutant cells accumulated glucosylated intermediates but were able to deglucosylate and process about 40% of their oligosaccharides to complex-type. This processing was not due to residual alpha-glucosidase II activity since the alpha-glucosidase inhibitors 1-deoxynojirimycin (DNJ) and N-butyl-DNJ did not prevent it. Parent cells also showed alpha-glucosidase II-independent processing in the presence of DNJ and N-butyl-DNJ. Membrane preparations from both parent and mutant cells had endo alpha-mannosidase activity, that is, split Glc1,2Man9GlcNAc to Glc1,2Man plus Man8GlcNAc, indicating that this was a candidate for an alternate route to complex oligosaccharide formation in the mutant cells. A balance study in which the cellular glycoproteins, intracellular water soluble saccharides, and saccharides secreted into the medium were isolated and analyzed from [2-3H]mannose-labeled mutant cells showed that the cells formed the di- and trisaccharides Glc1Man and Glc2Man in amounts equivalent to the deglucosylated oligosaccharides found in the cellular glycoproteins. This result shows unequivocally that the alpha-glucosidase II-deficient mutant cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.     

Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity

The processing of asparagine-linked oligosaccharides on the alpha- chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N- acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2 was greatly inhibited in the presence of monensin.     

Purification to homogeneity and properties of glucosidase II from mung bean seedlings and suspension-cultured soybean cells

Glucosidase II was purified approximately 1700-fold to homogeneity from Triton X-100 extracts of mung bean microsomes. A single band with a molecular mass of 110 kDa was seen on sodium dodecyl sulfate gels. This band was susceptible to digestion by endoglucosaminidase H or peptide glycosidase F, and the change in mobility of the treated protein indicated the loss of one or two oligosaccharide chains. By gel filtration, the native enzyme was estimated to have a molecular mass of about 220 kDa, suggesting it was composed of two identical subunits. Glucosidase II showed a broad pH optima between 6.8 and 7.5 with reasonable activity even at 8.5, but there was almost no activity below pH 6.0. The purified enzyme could use p-nitrophenyl-alpha-D-glucopyranoside as a substrate but was also active with a number of glucose-containing high-mannose oligosaccharides. Glc2Man9GlcNAc was the best substrate while activity was significantly reduced when several mannose residues were removed, i.e. Glc2Man7-GlcNAc. The rate of activity was lowest with Glc1Man9GlcNAc, demonstrating that the innermost glucose is released the slowest. Evidence that the enzyme is specific for alpha 1,3-glucosidic linkages is shown by the fact that its activity on Glc2Man9GlcNAc was inhibited by nigerose, an alpha 1,3-linked glucose disaccharide, but not by alpha 1,2 (kojibiose)-, alpha 1,4(maltose)-, or alpha 1,6 (isomaltose)-linked glucose disaccharides. Glucosidase II was strongly inhibited by the glucosidase processing inhibitors deoxynojirimycin and 2,6-dideoxy-2,6-imino-7-O-(beta-D- glucopyranosyl)-D-glycero-L-guloheptitol, but less strongly by castanospermine and not at all by australine. Polyclonal antibodies prepared against the mung bean glucosidase II reacted with a 95-kDa protein from suspension-cultured soybean cells that also showed glucosidase II activity. Soybean cells were labeled with either [2-3H]mannose or [6-3H]galactose, and the glucosidase II was isolated by immunoprecipitation. Essentially all of the radioactive mannose was released from the protein by treatment with endoglucosaminidase H. The labeled oligosaccharide(s) released by endoglucosaminidase H was isolated and characterized by gel filtration and by treatment with various enzymes. The major oligosaccharide chain on the soybean glucosidase II appeared to be a Man9(GlcNAc)2 with small amounts of Glc1Man9(GlcNAc)2.     

Assembly of asparagine-linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases

We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544]. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean alpha-mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases.     

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.     

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.     

Cell type-dependent variations in the subcellular distribution of alpha- mannosidase I and II

alpha-mannosidases I and II (Man I and II) are resident enzymes of the Golgi complex involved in oligosaccharide processing during N-linked glycoprotein biosynthesis that are widely considered to be markers of the cis- and medial-Golgi compartments, respectively. We have investigated the distribution of these enzymes in several cell types by immunofluorescence and immunoelectron microscopy. Man II was most commonly found in medial- and/or trans- cisternae but showed cell type- dependent variations in intra-Golgi distribution. It was variously localized to either medial (NRK and CHO cells), both medial and trans (pancreatic acinar cells, enterocytes), or trans- (goblet cells) cisternae, or distributed across the entire Golgi stack (hepatocytes and some enterocytes). The distribution of Man I largely coincided with that of Man II in that it was detected primarily in medial- and trans- cisternae. It also showed cell type dependent variations in its intra- Golgi distribution. Man I and Man II were also detected within secretory granules and at the cell surface of some cell types (enterocytes, pancreatic acinar cells, goblet cells). In the case of Man II, cell surface staining was shown not to be due to antibody cross- reactivity with oligosaccharide epitopes. These results indicate that the distribution of Man I and Man II within the Golgi stack of a given cell type overlaps considerably, and their distribution from one cell type to another is more variable and less compartmentalized than previously assumed.     

The lipid-linked oligosaccharide donor specificities of Trypanosoma brucei oligosaccharyltransferases

We recently presented a model for site-specific protein N-glycosylation in Trypanosoma brucei whereby the TbSTT3A oligosaccharyltransferase (OST) first selectively transfers biantennary Man(5)GlcNAc(2) from the lipid-linked oligosaccharide (LLO) donor Man(5)GlcNAc(2)-PP-Dol to N-glycosylation sequons in acidic to neutral peptide sequences and TbSTT3B selectively transfers triantennary Man(9)GlcNAc(2) to any remaining sequons. In this paper, we investigate the specificities of the two OSTs for their preferred LLO donors by glycotyping the variant surface glycoprotein (VSG) synthesized by bloodstream-form T. brucei TbALG12 null mutants. The TbALG12 gene encodes the α1-6-mannosyltransferase that converts Man(7)GlcNAc(2)-PP-Dol to Man(8)GlcNAc(2)-PP-Dol. The VSG synthesized by the TbALG12 null mutant in the presence and the absence of α-mannosidase inhibitors was characterized by electrospray mass spectrometry both intact and as pronase glycopetides. The results show that TbSTT3A is able to transfer Man(7)GlcNAc(2) as well as Man(5)GlcNAc(2) to its preferred acidic glycosylation site at Asn263 and that, in the absence of Man(9)GlcNAc(2)-PP-Dol, TbSTT3B transfers both Man(7)GlcNAc(2) and Man(5)GlcNAc(2) to the remaining site at Asn428, albeit with low efficiency. These data suggest that the preferences of TbSTT3A and TbSTT3B for their LLO donors are based on the c-branch of the Man(9)GlcNAc(2) oligosaccharide, such that the presence of the c-branch prevents recognition and/or transfer by TbSTT3A, whereas the presence of the c-branch enhances recognition and/or transfer by TbSTT3B.     

A Mucor pusillus mutant defective in asparagine-linked glycosylation.

A Mucor pusillus mutant defective in asparagine-linked glycosylation was found in our stock cultures. This mutant, designated 1116, secreted aspartic proteinase (MPP) in a less-glycosylated form than that secreted by the wild-type strain. Analysis of enzyme susceptibility, lectin binding, and carbohydrate composition indicated that this mutant secreted three glycoforms of MPPs, one of which contained no carbohydrate; the other two had truncated asparagine-linked oligosaccharide chains such as Man0-1GlcNAc2. Further analysis using oligosaccharide processing inhibitors, such as castanospermine, 1-deoxynojirimycin and N-methyldeoxynojirimycin, suggested that MPPs in the mutant were glycosylated through a transfer of the truncated lipid-linked oligosaccharides, Man0-1GlcNAc2, to the MPP protein but not through an aberrant processing. In addition, genetic studies with forced primary heterokaryons indicated that the mutation in strain 1116 was recessive.     

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.     

Structural characterization of novel complex oligosaccharides accumulated in the caprine beta-mannosidosis kidney. Occurrence of tetra- and pentasaccharides containing a beta-linked mannose residue at the nonreducing terminus

Four oligosaccharide fractions were isolated and purified from the kidney of goats affected with beta-mannosidosis by repeating Bio-Gel P-2 column chromatography. The structural characterization of the purified oligosaccharide fractions (oligosaccharides A, B, C1,2, and D) included sugar composition analysis by gas chromatography, sugar sequence analysis by mass spectrometry of their permethylated alditols, and by methylation analysis as well as anomeric configuration studies by exoglycosidase digestions. Oligosaccharides A and B were the major oligosaccharides accumulating in the kidney and were elucidated as Man beta 1-4GlcNAc and Man beta 1-4GlcNAc beta 1-4GlcNAc, respectively (Matsuura, F., Laine, R. A., and Jones, M. Z. (1981) Arch. Biochem. Biophys. 211, 485-493). Oligosaccharide C1,2 was a mixture of two tetrasaccharides and oligosaccharide D was a pentasaccharide. The proposed structures are: oligosaccharide C1, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc; oligosaccharide C2, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc; oligosaccharide D, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc beta 1-4GlcNAc. Tetrasaccharide C1 and pentasaccharide D are heretofore undiscovered oligosaccharides. There is no precedent for these structures in glycoproteins or other glycoconjugates. One possibility which accounts for the presence of oligosaccharide C1 and D is that a bisecting N-acetylglucosamine (the beta-N-acetylglucosamine residue linked at the C-4 position of the beta-mannosyl residue of the trimannosyl core of the asparagine-linked sugar chains) is linked by a beta-mannosyl residue. Moreover, the detection of oligosaccharides containing two N-acetylglucosamine residues at the reducing terminus, together with those containing a single N-acetylglucosamine residue, is further corroboration of species-specific differences in glycoprotein catabolic pathways (Hancock, L. W., and Dawson, G. (1984) Fed. Proc. 43, 1552) or in glycoprotein structures.     

Studies on the effect of glycoprotein processing inhibitors on fusion of L6 myoblast cell lines

The effect of oligosaccharide processing inhibitors on the fusion of L6 myoblasts was studied. The glucosidase inhibitors, castanospermine, 1-deoxynojirimycin and N-methyl-deoxynojirimycin were potent inhibitors of myoblast fusion, as was the mannosidase II inhibitor, swainsonine. Inhibition of fusion was reversed when inhibitors were removed. However, the mannosidase I inhibitor, 1-deoxymannojirimycin did not inhibit fusion. Changes in cell membrane oligosaccharide structure were followed by monitoring the binding of concanavalin A (conA) and wheat germ agglutinin (WGA) to cell surface membranes in cells treated with processing inhibitors. All the processing inhibitors resulted in increased binding of conA and decreased binding of WGA; this is consistent with the known mechanisms of inhibition of the inhibitors used in the study. Inhibition of fusion by the processing inhibitors also resulted in reduced activities of creatine phosphokinase, an enzyme used as a marker for biochemical differentiation during fusion. Treatment of a non-differentiating conA-resistant cell line with processing inhibitors did not induce fusion, but the cells did show altered lectin-binding properties. The main conclusion drawn from these studies is that cell surface glycoproteins probably containing the mannose (Man)9 structure are important for the fusion reaction.