Oligosaccharide processing at individual glycosylation sites on MOPC 104E immunoglobulin M. Differences in alpha 1,2-linked mannose processing

Processing of the asparagine-linked oligosaccharides at the known glycosylation sites on the mu-chain of IgM secreted by MOPC 104E murine plasmacytoma cells was investigated. Oligosaccharides present on intracellular mu-chain precursors were of the high mannose type, remaining susceptible to endo-beta-N-acetylglucosaminidase H. However, only 26% of the radioactivity was released from [3H]mannose-labeled secreted IgM glycopeptides, consistent with the presence of high mannose-type and complex-type oligosaccharides on the mature mu-chain. [3H]Mannose-labeled cyanogen bromide glycopeptides derived from mu-chains of secreted IgM were isolated and analyzed to identify the glycopeptide containing the high mannose-type oligosaccharide from those containing complex-type structures. [3H]Mannose-labeled intracellular mu-chain cyanogen bromide glycopeptides corresponding to those from secreted IgM were isolated also, and the time courses of oligosaccharide processing at the individual glycosylation sites were determined. The major oligosaccharides on all intracellular mu-chain glycopeptides after 20 min of pulse labeling with [3H]mannose were identified as Man8GlcNAc2, Man9GlcNAc2, and Glc1Man9GlcNAc2. Processing of the oligosaccharide destined to become the high mannose-type structure on the mature protein was rapid. After 30 min of chase incubation the predominant structures of this oligosaccharide were Man5GlcNAc2 and Man6GlcNAc2 which were also identified on the high mannose-type oligosaccharide of the secreted mu-chain. In contrast, processing of oligosaccharides destined to become complex type was considerably slower. Even after 180 min of chase incubation, Man7GlcNAc2 and Man8GlcNAc2 were the predominant structures at some of these glycosylation sites. The isomeric structures of Man8GlcNAc2 obtained from all of the glycosylation sites were identical. Thus, the different rates of processing were not the result of a different sequence of alpha 1,2-mannose removal.     

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.     

Regulation of Myelination: Schwann Cell Transition from a Myelin-Maintaining State to a Quiescent State After Permanent Nerve Transection

Permanent nerve transection of the adult rat sciatic nerve forces Schwann cells in the distal nerve segment from a myelin-maintaining to a quiescent state. This transition was followed by serial morphometric evaluation of the percentage fascicular area having myelin (myelin percent of area) in transverse sections of the distal nerve segment and revealed a rapid decline from a normal value of 36.6% to 3.2% by 14 days for the sciatic nerve to less than 1.0% throughout the remaining time course (up to 105 days). No evidence of axonal reentry into the distal nerve segment or new myelin formation was observed at times under 70 days. In some of the distal nerve segments at 70, 90, and 105 days, new myelinated fibers were observed that usually consisted of only a few myelinated fibers at the periphery and in the worst case amounted to 1.6% (myelin percent of area). Radioactive precursor incorporation of [3H]mannose into endoneurial slices at 4 and 7 days after transection revealed two species of the major myelin glycoprotein, P0, with Mr of 28,500 and 27,700. By 14 days after nerve transection, only the 27,700 Mr species remained. Incorporation of [3H]mannose into the 27,700 Mr species increased progressively to 35 days after transection and then began to decline at 70 and 105 days. Alterations in the oligosaccharide structure of this down-regulated myelin glycoprotein accounted for the progressive increase in mannose incorporation. Lectin affinity chromatography of pronase-digested P0 glycopeptides on concanavalin A-Sepharose revealed that the 28,500 Mr species of P0 had the complex-type oligosaccharide as the predominant oligosaccharide structure (92%). In contrast, the high mannose-type oligosaccharide was the predominate structure for the 27,700 Mr form, which increased to 70% of the total radioactivity by 35 days after nerve transection. Since the biosynthesis of the complex-type oligosaccharide chains on glycoproteins involves high mannose-type intermediates, the mechanism of down-regulation in the biosynthesis of this major myelin glycoprotein, therefore, results in a biosynthetic switch from the complex-type oligosaccharide structure as an end product to the predominantly high mannose-type oligosaccharide structure as a biosynthetic intermediate. This biosynthetic switch occurs gradually between 7 and 14 days after nerve transection and likely reflects a decreased rate of processing through the Golgi apparatus. It remains to be determined if the high mannose-type oligosaccharide chain on P0 can undergo additional processing steps in this permanent nerve transection model.     

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.     

Processing of N-linked oligosaccharides in soybean cultured cells

Evidence, based on both in vivo and in vitro studies with suspension-cultured soybean cells, is presented to demonstrate the processing of the oligosaccharide chain of plant N-linked glycoproteins. Following a 1-h incubation of soybean cells with [2-3H]mannose, the predominant glycopeptide obtained by pronase digestion of the membrane fraction was a Man7- or Man8GlcNAc2-Asn (GlcNAc, N-acetylglucosamine). However, the major oligosaccharide isolated from the lipid-linked oligosaccharides of these cells was a Glc2- or Glc3Man9GlcNAc2. Soybean cells were incubated with [2-3H]mannose and the incorporation of mannose into Pronase-released glycopeptides was followed during a 2-h chase. During the first 10 min of labeling, the radioactivity was mostly in a large-sized glycopeptide that appeared to be a Glc1Man9GlcNAc2-peptide. During the next 60 to 90 min of chase, this radioactivity was shifted to smaller and smaller-sized glycopeptides indicating that removal of sugars (i.e., processing) had occurred. Both glucosidase and mannosidase activity was detected in membrane preparations of soybean cells. Nine different glycopeptides were isolated from Pronase digests of soybean cell membrane fractions. These glycopeptides were purified by repeated gel filtration on columns of Bio-Gel P-4. Partial characterization of these glycopeptides by endoglucosaminidase H and alpha-mannosidase digestion, and by analysis of the products, suggested the following glycopeptides: Glc1Man9GlcNAc2-Asn, Man8GlcNAc2-Asn, Man7GlcNAc2-Asn, Man6GlcNAc2-Asn, and Man5GlcNAc2-Asn.     

Structures of the asparagine-linked sugar chain of glucose transporter from human erythrocytes

The asparagine-linked sugar chain of glucose transporter from human erythrocytes was quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted to radioactive oligosaccharides by NaB3H4 reduction after N-acetylation and fractionated by anion-exchange column chromatography and Bio-Gel P-4 column chromatography after sialidase treatment. Structural study of each oligosaccharide by exo- and endoglycosidase digestion and methylation analysis indicated that the glycoprotein contains a high-mannose-type oligosaccharide, Man9.GlcNAc.GlcNAc, and biantennary complex-type oligosaccharides with Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3) Man beta beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their cores and the poly-N-acetyllactosamine composed of about 16 N-acetyllactosaminyl units as their outer chains. These structural features of the sugar moiety of glucose transporter are quite different from those of two major intrinsic glycoproteins of human erythrocytes, glycophorin A and band 3.     

Regulation of Myelination: Biosynthesis of the Major Myelin Glycoprotein by Schwann Cells in the Presence and Absence of Myelin Assembly

Schwann cell biosynthesis of the major myelin glycoprotein, P0, was investigated in the crush-injured adult rat sciatic nerve, where there is myelin assembly, and in the permanently transected nerve, where there is no myelin assembly. Endoneurial fractions from desheathed rat sciatic nerves distal to the crush were compared with similar fractions from the permanently transected nerves at 7, 14, 21, 28, and 35 days after injury. The Schwann cell expression of this asparagine-linked glycoprotein was evaluated after sodium dodecyl sulfate-pore gradient electrophoresis by Coomassie Blue and silver stain and by autoradiography after direct overlay of radioiodinated lectins [wheat germ agglutinin, gorse agglutinin, and concanavalin A (Con A)]. As evaluated by these parameters, the concentration of P0 after crush decreased and subsequently increased as a function of time after injury, corresponding to the events of demyelination and remyelination. After permanent transection, the P0 concentration decreased following the same time course found after crush. At subsequent time points, P0 could not be detected with Coomassie Blue stain, silver stain, or wheat germ agglutinin. Both gorse agglutinin and Con A, however, showed binding to P0. Radioactive precursor incorporation studies with [3H]fucose or [3H]-mannose into endoneurial slices at 35 days posttransection revealed active oligosaccharide processing of P0 glycoprotein by Schwann cells in this permanent transection model. Compared with other Schwann cell glycoproteins in the transected nerve, the highest level of incorporation of [3H]mannose was found in P0 which accounted for 42.7% of the incorporated label. In contrast, incorporation of [3H]mannose into endoneurial slices at 35 days after crush accounted for only 13.3% in P0. In addition, higher levels of Con A binding were observed in P0 in the transected nerve compared with the contralateral control or the crushed nerve. Both the [3H]fucose incorporation and gorse agglutinin binding to P0 in the transected nerve suggest posttranslational processing of this glycoprotein in the Golgi apparatus; however, the absence of wheat germ agglutinin binding, the high level of mannose incorporation, and the high level of binding by Con A imply that additional processing steps are required prior to its assembly into myelin.     

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

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

Glycoprotein biosynthesis in quiescent and stimulated thymocytes and a T-cell lymphoma.

Quiescent thymocytes, mitogen-stimulated thymocytes and acute-leukaemic lymphoblasts provide a model for the study of protein glycosylation in quiescent cells, mitotically active non-malignant and malignant cells respectively. The biosynthesis of both complex and high-mannose-type oligosaccharides was monitored by metabolic labelling with [6-3]fucose and [2-3H]mannose. Bio-Gel P6 elution profiles of [6-3H]fucose-labelled glycopeptides showed that quiescent thymocytes and stimulated thymocytes synthesized qualitatively and quantitatively similar glycopeptides; however, higher-molecular-weight glycopeptides were synthesized by the acute-leukaemic lymphoblasts. The amount of [2(-3)H]mannose incorporated into glycopeptide by quiescent thymocytes was less than 10% of that incorporated by stimulated thymocytes. The Bio-Gel P6 elution profile of [2(-3)H]mannose-labelled glycopeptides from acute leukaemic lymphoblasts was qualitatively similar to that of stimulated thymocytes, with about 40% of the radioactivity incorporated into one glycopeptide peak. This glycopeptide was characterized by Bio-Gel P6 and concanavalin A affinity chromatography, radioactive-sugar analysis, sensitivity to alpha-mannosidase and endoglycosidase H and resistance to beta-glucosaminidase as containing a high-mannose oligosaccharide, possible of Man7-8GlcNAc2 structure. Pulse/chase experiments indicated that this high-mannose oligosaccharide was an end product and not a biosynthetic intermediate. It is concluded that higher-molecular-weight fucose-labelled glycopeptides are characteristic of the malignant cell type, and the synthesis of high-mannose oligosaccharide, Man7-8GlcNAc2, in stimulated thymocytes and acute-leukaemic lymphoblasts is associated with mitotically active cells.     

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.     

The oligosaccharide moieties of the epidermal growth factor receptor in A-431 cells. Presence of complex-type N-linked chains that contain terminal N-acetylgalactosamine residues

The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides.     

Structural analysis of the asparagine-linked oligosaccharides of rat haptoglobin metabolically labeled in a hepatocyte culture system

We analyzed the asparagine-linked oligosaccharide chains of rat haptoglobin which were synthesized and secreted by hepatocytes in primary culture. When the cells were incubated with either [3H]mannose, [3H]galactose, or [3H]fucose, all the radioactive precursors were incorporated into the beta subunit of haptoglobin. [3H]Mannose-labeled haptoglobin was purified from the culture medium by immunoaffinity chromatography, and [3H]oligosaccharides were prepared by strong alkali-borohydride treatment. The oligosaccharides obtained were analyzed by anion-exchange high-performance liquid chromatography, concanavalin-A--Sepharose chromatography and Bio-Gel P-4 chromatography before and after sequential exoglycosidase digestions. The oligosaccharides labeled with [3H]fucose or [3H]galactose were also characterized by the above methods. The results indicate that rat haptoglobin contains two complex-type oligosaccharide chains in each beta subunit; one with a possible structure of ( +/- NeuAc----Gal beta----GlcNAc beta----)3(Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc and the other with ( +/- NeuAc----Gal beta----GlcNAc beta----Man alpha----)2 Man beta----GlcNAc----( +/- Fuc alpha----)GlcNAc.     

Schistosoma mansoni synthesizes novel biantennary Asn-linked oligosaccharides containing terminal beta-linked N-acetylgalactosamine.

This report describes the structure of novel complex-type Asn-linked oligosaccharides in glycoproteins synthesized by the human blood fluke, Schistosoma mansoni. Adult schistosome worm pairs (male and female) isolated from infected hamsters were metabolically radiolabelled with either [3H]glucosamine, [3H]mannose or [3H]galactose. The glycopeptides prepared by pronase digestion of the total glycoprotein fraction were isolated by affinity chromatography on columns of immobilized Concanavalin A (Con A) and Wisteria floribunda agglutinin (WFA). A subset of glycopeptides, designated IIb, that bound to both Con A and WFA was isolated. WFA has been shown to have affinity for oligosaccharides containing beta 1,4-linked N-acetylgalactosamine (GalNAc) at their non-reducing termini. Compositional analysis of IIb glycopeptides demonstrated that they contained N-acetylglucosamine (GlcNAc), GalNAc, mannose (Man) and fucose (Fuc), but no galactose (Gal) or N-acetylneuraminic acid (NeuAc). Methylation analyses and exoglycosidase digestions indicated that IIb glycopeptides were complex-type biantennary structures with branches containing the sequence GalNAc beta 1-4-[+/- Fuc alpha 1-3]GlcNAc beta 1-2Man alpha 1-R. The discovery of these unusual oligosaccharides synthesized by a human parasite, which appear to be similar to some newly discovered mammalian cell-derived oligosaccharides, may shed light on future studies related to the role oligosaccharides may play in host-parasite interactions.     

Developmental regulation of asparagine-linked oligosaccharide synthesis in Dictyostelium discoideum

In the preceding report we demonstrated that the expression of two developmentally regulated alpha-mannosidase activities is induced in Dictyostelium discoideum during its differentiation from single-cell amoebae to multicellular organism (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18477-18484). These activities, designated membrane alpha-mannosidase I (MI) and membrane alpha-mannosidase II (MII), were shown to have several properties in common with rat liver Golgi alpha-mannosidases I and II, respectively, suggesting that MI and MII may play a role in the processing of asparagine-linked oligosaccharides in developing D. discoideum. In this study we analyzed the structures of the asparagine-linked oligosaccharides synthesized by D. discoideum at various stages of development to determine the timing and extent of asparagine-linked oligosaccharide processing. Cells were labeled with [2-3H] mannose, and then total cellular glycoproteins were digested with Pronase to generate glycopeptides that were fractionated on concanavalin A-Sepharose. Glycopeptides from each fraction were digested with endoglycosidase H, both before and after desulfation by solvolysis, and the released, neutral oligosaccharides were sized by high pressure liquid chromatography. At early stages of development, D. discoideum contain predominantly large high mannose-type oligosaccharides (Man9GlcNAc and Man8GlcNAc). Some of these are modified by GlcNAc residues attached beta 1-4 to the mannose-linked alpha 1-6 to the beta-linked core mannose (the "intersecting" position), as well as by fucose, sulfate, and phosphate. In contrast, the oligosaccharides found at late stages of development (18-24 h) have an array of sizes from Man9GlcNAc to Man3GlcNAc. These are still modified by GlcNAc, fucose, sulfate, and phosphate, but the percent of larger high mannose oligosaccharides that are modified with GlcNAc in the intersecting position decreases after 6 h of development, in parallel with the decrease in the intersecting GlcNAc transferase activity. Similarly, the changes in the size of asparagine-linked oligosaccharides synthesized during development correlate well with the appearance of MI and MII activities and suggest that these developmentally regulated alpha-mannosidase activities function in the processing of these oligosaccharides. This is supported further by the observation that oligosaccharide processing was inhibited in late stage cells labeled in the presence of either deoxymannojirimycin, an inhibitor of MI, or swainsonine, an inhibitor of MII.     

1,4-Dideoxy-1,4-imino-D-mannitol inhibits glycoprotein processing and mannosidase

1,4-Dideoxy-1,4-imino-D-mannitol (DIM) was synthesized chemically from benzyl-alpha-D-mannopyranoside [Fleet et al (1984) J. Chem. Soc. Chem. Commun., 1240-1241], and was tested in vitro as an inhibitor of various alpha-mannosidases and in cell culture as an inhibitor of glycoprotein processing. DIM proved to be an effective inhibitor of jack bean alpha-mannosidase, with 50% inhibition requiring 25 to 50 ng/ml inhibitor. It also inhibited lysosomal alpha-mannosidase, but in this case 50% inhibition required about 1 to 2 micrograms/ml. In both cases, the inhibition was of the competitive type when p-nitrophenyl-alpha-D-mannopyranoside was used as the substrate. The inhibition was better at higher pH values, suggesting that DIM was more effective when the nitrogen in the ring was in the unprotonated form. In addition, rat liver processing mannosidase I was also inhibited by DIM as measured by the release of [3H]mannose from [3H]mannose-labeled Man9GlcNAc. Glycoprotein processing was examined in influenza virus-infected MDCK cells. Infected cells were incubated in various concentrations of DIM and labeled with [2-3H]mannose. Viral and cell pellets were digested with Pronase and glycopeptides were isolated by gel filtration on columns of Bio-Gel P-4. The glycopeptides were then treated with endoglucosaminidase H (Endo H) and rechromatographed on the Bio-Gel column in order to distinguish complex from high-mannose structures. As the DIM concentration in the medium was raised, more and more of the [3H]mannose was incorporated into high-mannose oligosaccharides, and less and less radioactivity was in the complex chains. Most of the Endo H-released oligosaccharides induced by DIM were of the Man9GlcNAc structure, as determined by gel filtration, HPLC, and digestion by alpha-mannosidase. Thus, DIM also appears to inhibit mannosidase I in cell culture. However, about 15% of the Endo H-released oligosaccharides appear to be hybrid types of oligosaccharides, suggesting that DIM may also inhibit mannosidase II.     

Age-Dependent Changes in the Oligosaccharide Structure of the Major Myelin Glycoprotein, P0

The single oligosaccharide moiety of the major myelin glycoprotein, P0, resides in an immunoglobulin-like domain that appears to participate in homophilic binding. The studies presented here indicate that the structure of the P0 oligosaccharide from rat nerve changes as a function of Schwann cell age. Examination of 5-day-old nerve revealed that P0 contained predominantly endo-beta-N-acetylglucosaminidase H (endo H)-resistant, complex-type oligosaccharide. In contrast, P0 from adult rats had mostly endo H-sensitive carbohydrate, indicating the presence of appreciable high-mannose and/or hybrid-type oligosaccharide on the glycoprotein. The endo H-sensitive and -resistant P0 of adult nerve could be readily phosphorylated by protein kinase C, as could the complex-type P0 from 5-day-old nerve. This suggests that the glycoprotein progresses to the plasma membrane and myelin regardless of the type of oligosaccharide chain. Analysis of 35SO4(2-)-labeled P0 showed that the sulfate group was found on both endo H-sensitive and -resistant oligosaccharide. The endo H-sensitive P0 carbohydrate from adult nerve appears to be primarily of the hybrid type, as evidenced by (a) the elution profile of [3H]mannose-labeled P0 glycopeptides from adult nerve during concanavalin A chromatography and (b) the inability of P0 from adult nerve to interact with Galanthus nivalis agglutinin. The observed age-dependent changes of P0 oligosaccharide may modify the binding properties of this myelin glycoprotein.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

Sertoli cell glycosylation patterns as affected by culture age and extracellular matrix

This study evaluated the responsiveness of Sertoli cell glycosylation in vitro to changes in culture age and to the presence of a reconstituted basement membrane (Matrigel) or collagen IV/laminin substrata. Primary Sertoli cell cultures were prepared from 20-day-old rats and incubated with [3H]mannose, a monosaccharide specific for asparagine-linked oligosaccharides. The cells were harvested on Days 4, 6, or 10 of culture life. A supernatant enriched in cell-surface glycopeptides (the trypsinate) and a cell pellet stripped of surface glycoconjugates were evaluated separately. Glycopeptides derived from a Pronase digest of the two samples were fractionated using concanavalin-A lectin affinity chromatography into three major classes: multiantennary complex-type, biantennary complex-type, and high-mannose-type oligosaccharide structures. The proportion of radiolabeled glycopeptides appearing in each of the three classes did not differ between Days 4 and 6 of culture. In contrast, a significant increase in the percentage of radiolabeled glycopeptides containing multiantennary complex-type oligosaccharides was observed in cells harvested from the 10-day-old cultures. In other experiments, Sertoli cells were grown on various substrata: plastic; collagen IV/laminin; or Matrigel, a reconstituted basement membrane (RBM) composed of laminin, collagen IV, proteoglycan sulfate, entactin, and nidogen. Growth on RBM significantly increased multiantennary complex-type oligosaccharide formation compared to plastic, whereas the high-mannose-type glycopeptides increased in cells grown on collagen IV/laminin. These studies suggest that environmental and physiological conditions such as culture age and the presence of extracellular matrix significantly affect glycosylation patterns in Sertoli cell cultures.     

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.     

The effect of mannosamine on the formation of lipid-linked oligosaccharides and glycoproteins in canine kidney cells

Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with [2-3H]mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Methylation analysis also indicated that this Man5GlcNAc2 contained 1----3 linked mannose residues. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps by inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with [3H]mannose or [3H]galactose. In contrast, control cells produced complex and high-mannose structures but no hybrid oligosaccharides were detected. The inhibition by mannosamine could be overcome by adding high concentrations of glucose to the medium.