Characterization of a soluble class I alpha-mannosidase in human serum.

Class I alpha-mannosidases are thought to exist exclusively as integral membrane proteins that play intracellulary an essential role in the N-glycan biosynthesis. Using [3H]Man9GlcNAc2 as a substrate, we were able to identify a soluble alpha-mannosidase in human serum that trims the substrate Man9GlcNAc2 to Man(5-8)GlcNAc2 with Man6GlcNAc2 being the major product. This serum mannosidase is Ca2+-dependent, sensitive to 1-deoxymannojirimycin but insensitive to the class II inhibitor swainsonine and, hence, belongs to class I mannosidases. The enzymatic properties of the serum class I mannosidase are similar to that of the membrane bound class I mannosidases Golgi-mannosidase IA and IB and Man9-mannosidase.     

The purification and characterization of mannosidase IA from rat liver Golgi membranes

Rat liver Golgi membranes contain two alpha 1,2-specific mannosidases (IA and IB) (Tulsiani, D. R. P., Hubbard, S. C., Robbins, P. W., and Touster, O. (1982) J. Biol. Chem. 257, 3660-3668). Mannosidase IA has now been purified to apparent homogeneity by detergent extraction and (NH4)2SO4 precipitation, followed by Sephacryl S-300, ion-exchange, and hydroxylapatite chromatography. The enzyme was homogeneous by nondenaturing polyacrylamide gel electrophoresis with different gel concentrations, and Ferguson plot analysis indicated an Mr of 230,000 for the native enzyme. Although electrophoresis under denaturing conditions generally gave a subunit Mr of 57,000, electrophoresis of less than 1 microgram of protein yielded a faint doublet of Mr 57,000 and 58,000. Thus, the enzyme appears to be a tetramer with four very similar subunits. The enzyme bound to concanavalin A-Sepharose 4B only when it was kept in contact with the lectin for 16 h. Endoglycosidase H treatment resulted in loss of its binding to the lectin, without leading to a detectable change in the size of the enzyme subunit. On electrophoretic gels, the enzyme gave a faint positive stain with periodic acid-Schiff's base. The enzyme contained about 0.9% hexose by direct analysis. It did not bind to affinity resins specific for neuraminic acid, galactose, or N-acetylglucosamine. All these studies suggest that the enzyme is a glycoprotein containing only one or two clusters of high mannose oligosaccharide. Mannosidase IA is active toward oligosaccharides containing alpha 1,2-linked mannosyl residues. [3H]Man9GlcNAc, [3H] Man8GlcNAc, [3H]Man7GlcNAc, and [3H]Man6GlcNAc are good substrates. Man9GlcNAc, the best substrate, yields Man8, Man7, and Man6 derivatives with structures suggesting that the sequence of release of mannose residues is rather specific. Immunoprecipitation studies using polyclonal antibody (IgG) prepared against homogeneous mannosidase IA cross-reacted with mannosidase IB, a result suggesting that these two enzymes share antigenic determinants. However, no cross-reactivity was observed with rat liver cytosolic and lysosomal alpha-D-mannosidases or with Golgi mannosidase II.     

Substrate specificities of recombinant murine Golgi alpha1, 2- mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing alpha1,2-mannosidases

The catalytic domains of murine Golgi alpha1,2-mannosidases IA and IB that are involved in N-glycan processing were expressed as secreted proteins in P.pastoris . Recombinant mannosidases IA and IB both required divalent cations for activity, were inhibited by deoxymannojirimycin and kifunensine, and exhibited similar catalytic constants using Manalpha1,2Manalpha-O-CH3as substrate. Mannosidase IA was purified as a 50 kDa catalytically active soluble fragment and shown to be an inverting glycosidase. Recombinant mannosidases IA and IB were used to cleave Man9GlcNAc and the isomers produced were identified by high performance liquid chromatography and proton-nuclear magnetic resonance spectroscopy. Man9GlcNAc was rapidly cleaved by both enzymes to Man6GlcNAc, followed by a much slower conversion to Man5GlcNAc. The same isomers of Man7GlcNAc and Man6GlcNAc were produced by both enzymes but different isomers of Man8GlcNAc were formed. When Man8GlcNAc (Man8B isomer) was used as substrate, rapid conversion to Man5GlcNAc was observed, and the same oligosaccharide isomer intermediates were formed by both enzymes. These results combined with proton-nuclear magnetic resonance spectroscopy data demonstrate that it is the terminal alpha1, 2-mannose residue missing in the Man8B isomer that is cleaved from Man9GlcNAc at a much slower rate. When rat liver endoplasmic reticulum membrane extracts were incubated with Man9GlcNAc2, Man8GlcNAc2was the major product and Man8B was the major isomer. In contrast, rat liver Golgi membranes rapidly cleaved Man9GlcNAc2to Man6GlcNAc2and more slowly to Man5GlcNAc2. In this case all three isomers of Man8GlcNAc2were formed as intermediates, but a distinctive isomer, Man8A, was predominant. Antiserum to recombinant mannosidase IA immunoprecipitated an enzyme from Golgi extracts with the same specificity as recombinant mannosidase IA. These immunodepleted membranes were enriched in a Man9GlcNAc2to Man8GlcNAc2- cleaving activity forming predominantly the Man8B isomer. These results suggest that mannosidases IA and IB in Golgi membranes prefer the Man8B isomer generated by a complementary mannosidase that removes a single mannose from Man9GlcNAc2.      

N-Glycan processing by a lepidopteran insect alpha1,2-mannosidase

Protein glycosylation pathways are relatively poorly characterized in insect cells. As part of an overall effort to address this problem, we previously isolated a cDNA from Sf9 cells that encodes an insect alpha1,2-mannosidase (SfManI) which requires calcium and is inhibited by 1-deoxymannojirimycin. In the present study, we have characterized the substrate specificity of SfManI. A recombinant baculovirus was used to express a GST-tagged secreted form of SfManI which was purified from the medium using an immobilized glutathione column. The purified SfManI was then incubated with oligosaccharide substrates and the resulting products were analyzed by HPLC. These analyses showed that SfManI rapidly converts Man(9)GlcNAc(2)to Man(6)Glc-NAc(2)isomer C, then more slowly converts Man(6)GlcNAc(2)isomer C to Man(5)GlcNAc(2). The slow step in the processing of Man(9)GlcNAc(2)to Man(5)GlcNAc(2)by SfManI is removal of the alpha1,2-linked mannose on the middle arm of Man(9)GlcNAc(2). In this respect, SfManI is similar to mammalian alpha1,2-mannosidases IA and IB. However, additional HPLC and(1)H-NMR analyses demonstrated that SfManI converts Man(9)GlcNAc(2)to Man(5)GlcNAc(2)primarily through Man(7)GlcNAc(2)isomer C, the archetypal Man(9)GlcNAc(2)missing the lower arm alpha1,2-linked mannose residues. In this respect, SfManI differs from mammalian alpha1,2-mannosidases IA and IB, and is the first alpha1,2-mannosidase directly shown to produce Man(7)GlcNAc(2)isomer C as a major processing intermediate.     

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.     

Mutation of Arg(273) to Leu alters the specificity of the yeast N-glycan processing class I alpha1,2-mannosidase

Class I alpha1,2-mannosidases (glycosyl hydrolase family 47) involved in the processing of N-glycans during glycoprotein maturation have different specificities. Enzymes in the endoplasmic reticulum of yeast and mammalian cells remove a single mannose from Man(9)GlcNAc(2) to form Man(8)GlcNAc(2) isomer B (lacking the alpha1, 2-mannose residue of the middle alpha1, 3-arm), whereas other alpha1,2-mannosidases, including Golgi alpha1,2-mannosidases IA and IB, can convert Man(9)GlcNAc(2) to Man(5)GlcNAc(2). In the present work, it is demonstrated that with a single mutation in its catalytic domain (Arg(273) --> Leu) the yeast endoplasmic reticulum alpha1,2-mannosidase acquires the ability to transform Man(9)GlcNAc to Man(5)GlcNAc. High resolution proton nuclear magnetic resonance analysis of the products shows that the order of removal of mannose from Man(9)GlcNAc is different from that of other alpha1, 2-mannosidases that remove four mannose from Man(9)GlcNAc. These results demonstrate that Arg(273) is in part responsible for the specificity of the endoplasmic reticulum alpha1,2-mannosidase and that small differences in non-conserved amino acids interacting with the oligosaccharide substrate in the active site of class I alpha1, 2-mannosidases are responsible for the different specificities of these enzymes.     

Characterization of a novel alpha-D-mannosidase from rat brain microsomes

A new alpha-D-mannosidase has been identified in rat brain microsomes. The enzyme was purified 70-100-fold over the microsomal fraction by solubilization with Triton X-100, followed by ion exchange, concanavalin A-Sepharose, and hydroxylapatite chromatography. The purified enzyme is very active towards mannose-containing oligosaccharides and has a pH optimum of 6.0. Unlike rat liver endoplasmic reticulum alpha-D-mannosidase and both Golgi mannosidases IA and IB, which have substantial activity only towards alpha 1,2-linked mannosyl residues, the brain enzyme readily cleaves alpha 1,2-, alpha 1,3-, and alpha 1,6-linked mannosyl residues present in high mannose oligosaccharides. The brain enzyme is also different from liver Golgi mannosidase II in that it hydrolyzes (Man)5GlcNAc and (Man)4GlcNAc without their prior N-acetylglucosaminylation. Moreover, the facts that the ability of the enzyme to cleave GlcNAc(Man)5GlcNAc, the biological substrate for Golgi mannosidase II, is not inhibited by swainsonine, and that p-nitrophenyl alpha-D-mannoside is a poor substrate provide further evidence for major differences between the brain enzyme and mannosidase II. Inactivation studies and the co-purification of activities towards various substrates suggest that a single enzyme is responsible for all the activities found. In view of these results, it seems possible that, in rat brain, a single mannosidase cleaves asparagine-linked high mannose oligosaccharide to form the core Man3GlcNAc2 moiety, which would then be modified by various glycosyl transferases to form complex type glycoproteins.     

Structure of Penicillium citrinum alpha 1,2-mannosidase reveals the basis for differences in specificity of the endoplasmic reticulum and Golgi class I enzymes.

Class I alpha1,2-mannosidases (glycosylhydrolase family 47) are key enzymes in the maturation of N-glycans. This protein family includes two distinct enzymatically active subgroups. Subgroup 1 includes the yeast and human endoplasmic reticulum (ER) alpha1,2-mannosidases that primarily trim Man(9)GlcNAc(2) to Man(8)GlcNAc(2) isomer B whereas subgroup 2 includes mammalian Golgi alpha1,2-mannosidases IA, IB, and IC that trim Man(9)GlcNAc(2) to Man(5)GlcNAc(2) via Man(8)GlcNAc(2) isomers A and C. The structure of the catalytic domain of the subgroup 2 alpha1,2-mannosidase from Penicillium citrinum has been determined by molecular replacement at 2.2-A resolution. The fungal alpha1,2-mannosidase is an (alphaalpha)(7)-helix barrel, very similar to the subgroup 1 yeast (Vallée, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A., and Howell, P. L. (2000) EMBO J. 19, 581-588) and human (Vallée, F., Karaveg, K., Herscovics, A., Moremen, K. W., and Howell, P. L. (2000) J. Biol. Chem. 275, 41287-41298) ER enzymes. The location of the conserved acidic residues of the catalytic site and the binding of the inhibitors, kifunensine and 1-deoxymannojirimycin, to the essential calcium ion are conserved in the fungal enzyme. However, there are major structural differences in the oligosaccharide binding site between the two alpha1,2-mannosidase subgroups. In the subgroup 1 enzymes, an arginine residue plays a critical role in stabilizing the oligosaccharide substrate. In the fungal alpha1,2-mannosidase this arginine is replaced by glycine. This replacement and other sequence variations result in a more spacious carbohydrate binding site. Modeling studies of interactions between the yeast, human and fungal enzymes with different Man(8)GlcNAc(2) isomers indicate that there is a greater degree of freedom to bind the oligosaccharide in the active site of the fungal enzyme than in the yeast and human ER alpha1,2-mannosidases.     

Respiratory distress and neonatal lethality in mice lacking Golgi alpha1,2-mannosidase IB involved in N-glycan maturation

There are three mammalian Golgi alpha1,2-mannosidases, encoded by different genes, that form Man5GlcNAc2 from Man(8-9)GlcNAc2 for the biosynthesis of hybrid and complex N-glycans. Northern blot analysis and in situ hybridization indicate that the three paralogs display distinct developmental and tissue-specific expression. The physiological role of Golgi alpha1,2-mannosidase IB was investigated by targeted gene ablation. The null mice have normal gross appearance at birth, but they display respiratory distress and die within a few hours. Histology of fetal lungs the day before birth indicate some delay in development, whereas neonatal lungs show extensive pulmonary hemorrhage in the alveolar region. No significant histopathological changes occur in other tissues. No remarkable ultrastructural differences are detected between wild type and null lungs. The membranes of a subset of bronchiolar epithelial cells are stained with lectins from Phaseolus vulgaris (leukoagglutinin and erythroagglutinin) and Datura stramonium in wild type lungs, but this staining disappears in lungs from null mice. Mass spectrometry of N-glycans from different tissues shows no significant changes in global N-glycans of null mice. Therefore, only a few glycoproteins required for normal lung function depend on alpha1,2-mannosidase IB for maturation. There are no apparent differences in the expression of several lung epithelial cell and endothelial cell markers between null and wild type mice. The alpha1,2-mannosidase IB null phenotype differs from phenotypes caused by ablation of other enzymes in N-glycan biosynthesis and from other mouse gene disruptions that affect pulmonary development and function.     

Purification and properties of the glycoprotein processing N-acetylglucosaminyltransferase II from plants

The presence of an N-acetylglucosaminyltransferase (GlcNAc-transferase) capable of adding a GlcNAc residue to GlcNAcMan3GlcNAc was demonstrated in mung bean seedlings. This enzyme was purified about 3400-fold by using (diethylaminoethyl)cellulose and phosphocellulose chromatographies and chromatography on Concanavalin A-Sepharose. The transferase was assayed by following the change in the migration of the [3H]mannose-labeled GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc on Bio-Gel P-4, or by incorporation of [3H]GlcNAc from UDP-[3H]GlcNAc into a neutral product, (GlcNAc)2Man3GlcNAc. Thus, the purified enzyme catalyzed the addition of a GlcNAc to that mannose linked in alpha 1,6 linkage to the beta-linked mannose. GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc was an excellent acceptor while Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, Man alpha 1,6(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, and Man alpha 1,6(Man apha 1,3)Man alpha 1,6[GlcNAcMan alpha 1,3]Man beta 1,4GlcNAc were not acceptors. Methylation analysis and enzymatic digestions showed that both terminal GlcNAc residues on (GlcNAc)2Man3GlcNAc were attached to the mannoses in beta 1,2 linkages. The GlcNAc transferase had an almost absolute requirement for divalent cation, with Mn2+ being best at 2-3 mM. Mn2+ could not be replaced by Mg2+ or Ca2+, but Cd2+ showed some activity. The enzyme was also markedly stimulated by the presence of detergent and showed optimum activity at 0.15% Triton X-100. The Km for UDP-GlcNAc was found to be 18 microM and that for GlcNAcMan3GlcNAc about 16 microM.     

Structural heterogeneity in the Man8-13GlcNAc oligosaccharides from log-phase Saccharomyces yeast: a one- and two-dimensional 1H NMR spectroscopic study.

Previously, Man8-14GlcNAc oligosaccharides were isolated from highly purified Saccharomyces cerevisiae invertase and shown by one-dimensional 1H NMR spectroscopy and alpha 1,2-linkage-specific mannosidase digestion to constitute a homologous series of nearly homogeneous compounds, which appeared to define the intermediates in oligosaccharide core synthesis in yeast (Trimble, R.B. and Atkinson, P.H. (1986) J. Biol. Chem., 261, 9815-9824). To evaluate whether invertase oligosaccharides reflected global core processing of yeast glycans, the soluble glycoprotein pool of disrupted log-phase cells was digested with endo-beta-N-acetyl-glucosaminidase H and Man8-13GlcNAc were isolated by Bio-Gel P-4 chromatography. Although analysis of each size class by one-dimensional 400 MHz and two-dimensional 500 MHz phase-sensitive COSY 1H NMR spectroscopy revealed considerable structural heterogeneity in all but Man8GlcNAc, the major positional isomer in Man9-13GlcNAc (approximately 50%) was identical to that previously elucidated on invertase. The heterogeneity resided in four families of oligosaccharides: (i) Glc3Man9GlcNAc----Man8 GlcNAc trimming intermediates; (ii) alpha-mannosidase degradation products of the principal isomers; (iii) mannan elongation intermediates; (iv) core structures with the alpha 1,2-linked mannose usually removed by the processing alpha-mannosidase. The potential for the vacuolar alpha-mannosidase (AMS1 gene product) to generate heterogeneity in vitro was confirmed by isolating oligosaccharides from AMS1 and ams1 yeast strains in the presence of a Man13GlcNAc[3H]-ol marker (where GlcNAc[3H]-ol is N-acetylglucosamin [1-3H]itol). Degradation of the Man13GlcNAc[3H]-ol to Man9-12GlcNAc[3H]-ol occurred in the former, but not in the latter. A role for the vacuolar alpha-mannosidase in generating at least some heterogeneity in vivo was inferred from the 1H NMR spectrum of the AMS1 Man11GlcNAc pool, which showed more structural isomerism than seen in the spectrum of a comparable ams1 Man11GlcNAc preparation. Thus, the principal biosynthetic pathway of inner core mannan in Saccharomyces is defined by the Man8-13GlcNAc oligosaccharides found on external invertase, while structural heterogeneity in these size classes results from precursor processing in the endoplasmic reticulum, core extension in the Golgi and metabolic degradation in the vacuole.     

N-glycan structure of a short-lived variant of ribophorin I expressed in the MadIA214 glycosylation-defective cell line reveals the role of a mannosidase that is not ER mannosidase I in the process of glycoprotein degradation.

A soluble form of ribophorin I (RI(332)) is rapidly degraded in Hela and Chinese hamster ovary (CHO) cells by a cytosolic proteasomal pathway, and the N-linked glycan present on the protein may play an important role in this process. Specifically, it has been suggested that endoplasmic reticulum (ER) mannosidase I could trigger the targeting of improperly folded glycoproteins to degradation. We used a CHO-derived glycosylation-defective cell line, MadIA214, for investigating the role of mannosidase(s) as a signal for glycoprotein degradation. Glycoproteins in MadIA214 cells carry truncated Glc(1)Man(5)GlcNAc(2) N-glycans. This oligomannoside structure interferes with protein maturation and folding, leading to an alteration of the ER morphology and the detection of high levels of soluble oligomannoside species caused by glycoprotein degradation. An HA-epitope-tagged soluble variant of ribophorin I (RI(332)-3HA) expressed in MadIA214 cells was rapidly degraded, comparable to control cells with the complete Glc(3)Man(9)GlcNAc(2) N-glycan. ER-associated degradation (ERAD) of RI(332)-3HA was also proteasome-mediated in MadIA214 cells, as demonstrated by inhibition of RI(332)-3HA degradation with agents specifically blocking proteasomal activities. Two inhibitors of alpha1,2-mannosidase activity also stabilized RI(332)-3HA in the glycosylation-defective cell line. This is striking, because the major mannosidase activity in the ER is the one of mannosidase I, specific for a mannose alpha1,2-linkage that is absent from the truncated Man(5) structure. Interestingly, though the Man(5) derivative was present in large amounts in the total protein pool, the two major species linked to RI(332)-3HA shortly after synthesis consisted of Glc(1)Man(5 )and Man(4), being replaced by Man(4 )and Man(3) when proteasomal degradation was inhibited. In contrast, the untrimmed intermediate of RI(332)-3HA was detected in mutant cells treated with mannosidase inhibitors. Our results unambiguously demonstrate that an alpha1,2-mannosidase that is not ER mannosidase I is involved in ERAD of RI(332-)3HA in the glycosylation-defective cell line, MadIA214.     

Structure and function of Class I alpha 1,2-mannosidases involved in glycoprotein synthesis and endoplasmic reticulum quality control

Class I alpha 1,2-mannosidases (glycosylhydrolase family 47) are conserved through eukaryotic evolution. This protein family comprises three subgroups distinguished by their enzymatic properties. The first subgroup includes yeast (Saccharomyces cerevisiae) and human alpha 1,2-mannosidases of the endoplasmic reticulum that primarily form Man(8)GlcNAc(2) isomer B from Man(9)GlcNAc(2). The second subgroup includes mammalian Golgi alpha 1,2-mannosidases, as well as enzymes from insect cells and from filamentous fungi, that trim Man(9)GlcNAc(2) to Man(8)GlcNAc(2) isomers A and/or C intermediates toward the formation of Man(5)GlcNAc(2). Yeast and mammalian proteins of the third subgroup have no enzyme activity with Man(9)GlcNAc(2) as substrate. The members of subgroups 1 and 3 participate in endoplasmic reticulum quality control and promote proteasomal degradation of misfolded glycoproteins. The yeast endoplasmic reticulum alpha 1,2-mannosidase has served as a model for structure-function studies of this family. Its structure was determined by X-ray crystallography as an enzyme-product complex. It consists of a novel (alpha alpha)(7) barrel containing the active site that includes essential acidic residues and calcium. The structures of the subgroup 1 human endoplasmic reticulum alpha 1,2-mannosidase and of a subgroup 2 fungal alpha 1,2-mannosidase were determined by molecular replacement. Comparison of the enzyme structures is providing some insight into the reasons for their different specificities.     

Characterization of MOPC 315 IgA oligosaccharide processing intermediates

The structures of alpha 1,2-mannose containing partially processed asparagine-linked oligosaccharides on the alpha-chain of MOPC 315 IgA were characterized using specific glycosidases and acetolysis. Man6GlcNAc2, a substrate for a Golgi alpha 1,2-mannosidase, was found to be a single isomeric structure. Likewise, Man7-9GlcNAc2 were single isomers indicating an ordered sequence of removal of alpha 1,2-linked mannose residues on this murine immunoglobulin heavy chain.     

The effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins

The influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. In order to determine what structural features of the oligosaccharide were required for fucosylation or where in the processing pathway fucosylation occurred, influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, [5,6-3H]fucose and [1-14C]mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the [14C]mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the [14C]mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the [3H]fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the [14C]mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure.     

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

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

Importance of glycosidases in mammalian glycoprotein biosynthesis

Processing glycosidases play an important role in N-glycan biosynthesis in mammalian cells by trimming Glc(3)Man(9)GlcNAc(2) and thus providing the substrates for the formation of complex and hybrid structures by Golgi glycosyltransferases. Processing glycosidases also play a role in the folding of newly formed glycoproteins and in endoplasmic reticulum quality control. The properties and molecular nature of mammalian processing glycosidases are described in this review. Membrane-bound alpha-glucosidase I and soluble alpha-glucosidase II of the endoplasmic reticulum remove the alpha1,2-glucose and alpha1,3-glucose residues, respectively, beginning immediately following transfer of Glc(3)Man(9)GlcNAc(2) to nascent polypeptides. The alpha-glucosidases participate in glycoprotein folding mediated by calnexin and calreticulin by forming the monoglucosylated high mannose oligosaccharides required for the interaction with the chaperones. In some mammalian cells, Golgi endo alpha-mannosidase provides an alternative pathway for removal of glucose residues. Removal of alpha1,2-linked mannose residues begins in the endoplasmic reticulum where trimming of mannose residues in the endoplasmic reticulum has been implicated in the targeting of malfolded glycoproteins for degradation. Removal of mannose residues continues in the Golgi with the action of alpha1, 2-mannosidases IA and IB that can form Man(5)GlcNAc(2) and of alpha-mannosidase II that removes the alpha1,3- and alpha1,6-linked mannose from GlcNAcMan(5)GlcNAc(2) to form GlcNAcMan(3)GlcNAc(2). These membrane-bound Golgi enzymes have been cloned and shown to have very distinct patterns of tissue-specific expression. There are also broad specificity alpha-mannosidases that can trim Man(4-9)GlcNAc(2) to Man(3)GlcNAc(2), and provide an alternative pathway toward complex oligosaccharide formation. Cloning of the remaining alpha-mannosidases will be required to evaluate their specific functions in glycoprotein maturation.