Interference of nucleoside diphosphate derivatives of 2-deoxy-D-glucose with the glycosylation of virus-specific glycoproteins in vivo.

The predominant effect of 2-deoxy-D-glucose on chick embryo cells infected with Semliki Forest virus is an interference with glycosylation of virus-specific glycoproteins; this results in a block of synthesis of infectious virus. Incorporation of radioactive mannose is blocked severely in the presence of 2-deoxyglucose in the cultural medium although it is readily phosphorylated and subsequently activated by GTP to yield GDP-mannose, which accumulates under these conditions. The intracellular concentrations of GDP-mannose and UDP-N-acetyl-D-hexosamine are not reduced in the presence of the inhibitor. An equimolar concentration of mannose in the cultural medium competes with the inhibitory effect of the deoxysugar and drops the cellular pool of GDP-2-deoxy-D-glucose below the level of detection, at the same time restoring the synthesis of infectious virus. When the intracellular concentration of UDP-2-deoxyglucose is reduced by addition of glucose into the cultural medium the inhibition of virus synthesis by the deoxysugar and the concentration of GDP-2-deoxyglucose within the cells remain near to the values when the inhibitor is present alone. It is concluded that among the metabolites of 2-deoxyglucose which occur in vivo after addition of 2-deoxyglucose to the culture medium, GDP-2-deoxyglucose is the agent responsible for inhibition of glycosylation of viral glycoproteins.     

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein.     

Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18.

The glycosylation of human cytokeratin (CK) 8 and 18 was studied after metabolic labeling of HT29 colonic cells with [3H]glucosamine. Labeling of CK8/18 was not inhibited by tunicamycin, suggesting that glycosylation was not N-linked. Acid hydrolysis of CK8 and CK18, purified from [3H]glucosamine-labeled cells, generated free glucosamine. In the presence of UDP-[3H]galactose, galactosyltransferase catalyzed the labeling of cytokeratin 8 and 18. beta-Elimination of the [3H]galactose- labeled CK8/18 generated the disaccharide N-acetyllactosaminitol, indicating that cytokeratin 8 and 18 contain single O-linked N-acetylglucosamine residues. Using chemical analysis, the stoichiometry of glycosylation was found to be 1.5 and 2 molecules of N-acetylglucosamine/protein molecule of CK8 and CK18, respectively. Peptide maps of [3H]glucosamine-labeled CK8/18 showed that multiple peptides were labeled with the amino sugar. The biosynthetic and degradation rates of the carbohydrate moiety were faster than the protein core as determined by metabolic radiolabeling or pulse-chase experiments, respectively. Our results show that CK8 and 18 are glycosylated at multiple sites with a single O-linked N-acetylglucosamine. Furthermore, CK8/18 glycosylation is a dynamic process which is likely to have functional relevance.     

Control of amino sugar metabolism in Escherichia coli and isolation of mutants unable to degrade amino sugars

1. Growth of Escherichia coli on glucosamine results in an induction of glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] and a repression of glucosamine 6-phosphate synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16); glucose abolishes these control effects. 2. Growth of E. coli on N-acetylglucosamine results in an induction of N-acetylglucosamine 6-phosphate deacetylase and glucosamine 6-phosphate deaminase, and in a repression of glucosamine 6-phosphate synthetase; glucose diminishes these control effects. 3. The synthesis of amino sugar kinases (EC 2.7.1.8 and 2.7.1.9) is unaffected by growth on amino sugars. 4. Glucosamine 6-phosphate synthetase is inhibited by glucosamine 6-phosphate. 5. Mutants of E. coli that are unable to grow on N-acetylglucosamine have been isolated, and lack either N-acetylglucosamine 6-phosphate deacetylase (deacetylaseless) or glucosamine 6-phosphate deaminase (deaminaseless). Deacetylaseless mutants can grow on glucosamine but deaminaseless mutants cannot. 6. After growth on glucose, deacetylaseless mutants have a repressed glucosamine 6-phosphate synthetase and a super-induced glucosamine 6-phosphate deaminase; this may be related to an intracellular accumulation of acetylamino sugar that also occurs under these conditions. In one mutant the acetylamino sugar was shown to be partly as N-acetylglucosamine 6-phosphate. Deaminaseless mutants have no abnormal control effects after growth on glucose. 7. Addition of N-acetylglucosamine or glucosamine to cultures of a deaminaseless mutant caused inhibition of growth. Addition of N-acetylglucosamine to cultures of a deacetylaseless mutant caused lysis, and secondary mutants were isolated that did not lyse; most of these secondary mutants had lost glucosamine 6-phosphate deaminase and an uptake mechanism for N-acetylglucosamine. 8. Similar amounts of (14)C were incorporated from [1-(14)C]-glucosamine by cells of mutants and wild-type growing on broth. Cells of wild-type and a deaminaseless mutant incorporated (14)C from N-acetyl[1-(14)C]glucosamine more efficiently than from N[1-(14)C]-acetylglucosamine, incorporation from the latter being further decreased by acetate; cells of a deacetylaseless mutant showed a poor incorporation of both types of labelled N-acetylglucosamine.     

Glycosylation requirements for intracellular transport and function of the hemagglutinin of influenza virus.

The contribution of each of the seven asparagine-linked oligosaccharide side chains on the hemagglutinin of the A/Aichi/68 (X31) strain of influenza virus was assessed with respect to its effect on the folding, intracellular transport, and biological activities of the molecule. Twenty mutant influenza virus hemagglutinins were constructed and expressed, each of which had one or more of the seven glycosylation sites removed. Investigations of these mutant hemagglutinins indicated that (i) no individual oligosaccharide side chain is necessary or sufficient for the folding, intracellular transport, or function of the molecule, (ii) at least five oligosaccharide side chains are required for the X31 hemagglutinin molecule to move along the exocytic pathway to the plasma membrane, and (iii) mutant hemagglutinins having less than five oligosaccharide side chains form intracellular aggregates and are retained in the endoplasmic reticulum.     

Biosynthesis of sulfated glycoprotein in the endometrium of rabbit uterus.

The endometrial scrapings obtained from the uteri of estrogen-treated rabbits were incubated with N-acetyl-d[1-3H]glucosamine and [35S]sulfate, and then the incubation medium (M-Fr) was separated from the tissue. The tissue was subsequently homogenized exhaustively in 0.25m sucrose, and the insoluble residue (R-Fr) was separate. The supernatant at 8,500Xg for 10 min of the homogenate was subjected to subcelular fractionation by discontinuous sucrose gradient ultracentrifugation, and a thiamine pyrophosphatase-rich fraction (g-fr) was obtained. Complex carbohydrates were then separated from M-Fr, R-Fr, and G-Fr. The radioactivities incorporated into these complex carbohydrates suggested that sulfated glycoprotein synthesized in G-Fr was secreted into M-Fr. In order to confirm the above observation, labelled sulfated glycoprotein was isolated from the incubation medium. Subsequently, N-ACETYL-D[1-3H]glucosamine was incorporated into N-acetylglucosamine residues and [35S]sulfate into sulfates located most probably at the 6-position of N-acetylglucosamine residues of sulfated glycoprotein.     

Role of sulfation in the processing of gastric mucins.

The role of sulfation in the processing of mucus glycoprotein in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in MEM at various medium sulfate concentrations in the presence of [35S]Na2SO4, [3H]glucosamine and [3H]proline, with and without chlorate an inhibitor of PAPS formation. The results revealed that the mucin sulfation attained maximum at 300 microM medium sulfate concentration. Introduction of chlorate into the incubation medium, while having no effect on the protein synthesis as evidenced by [3H]proline incorporation, caused at its optimal concentration of 2 mM a 90% decrease in mucin sulfation and a 40% drop in mucin glycosylation. Evaluation of mucin molecular forms distribution indicated the predominance of the high molecular mucin form in the intracellular fraction and the low molecular mucin from in the extracellular fraction. Increase in medium sulfate caused an increase in the high molecular weight mucin form in both fractions, and this effect was inhibited by chlorate. Also, higher medium sulfate concentrations led to a higher degree of sulfation in the high molecular weight mucin form, the effect of which was inhibited by chlorate. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and is required for mucin polymer formation. Hence, the disturbances in mucin sulfation process could be detrimental to the maintenance of gastric mucus coat integrity.     

Proteins specified by herpes simplex virus. XIII. Glycosylation of viral polypeptides.

In the course of herpes simplex virus 1 (HSV-1) replication in human epidermoid carcinoma no. 2 cells, the synthesis and glycosylation of host cell proteins ceases and is replaced by the synthesis and glycosylation of virus-specified polypeptides. Analyses of the synthesis of viral glycoproteins show that the glycosylation of viral polypeptides occurs late in the virus growth cycle and that certain of the precursors to major vital glycoproteins are members of the gamma group of polypeptides, i.e., polypeptides synthesized at increasing rates until 12 to 15 h postinfection. Viral glycoproteins are formed by stepwise additions of heterosaccharide chains to completed precursor polypeptides. The precursor and the highly glycosylated product are separable by gel electrophoresis and are localized in different fractions of infected cells. Within 15 min of their synthesis, precursor polypeptides acquire heterosaccharide chains of about 2,000 molecular weight, which contain glucosamine but little or nor fucose or sialic acid. Both precursor and product of this first stage of glycosylation are absent or present in low concentrations in the surface membranes of the infected cell and in the virion. The partially glycosylated product is then conjugated further in a slow, discontinuous process to form the mature glycoprotein of the virion and plasma membrane. These mature products bear large heterosaccharide units with molecular weights greater than 4,000 to 5,000; these contain fucose and sialic acid as well as glucosamine. Heterosaccharide chains from infected and uninfected cells are distributed among discrete size classes and the smallest chains consist of multiple saccharide residues.     

Role of sulfation in post-translational processing of gastric mucins.

1. Gastric mucosal segments were incubated in MEM supplemented with various sulfate concentrations in the presence of [3H]glucosamine, [3H]proline and [35S]Na2SO4, with and without chlorate, an inhibitor of 3'-phosphoadenosine-5'-phosphosulfate formation. 2. Incorporation of glucosamine and sulfate depended upon the sulfate content of the medium and reached a maximum at 300 microM sulfate. Introduction of chlorate into the medium, while having no effect on protein synthesis as evidenced by proline incorporation, caused, at its optimal concentration of 2 mM, a 90% decrease in mucin sulfation and a 40% drop in glycosylation. 3. At low sulfate content in the medium and in the presence of chlorate, the incorporation of sulfate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulfate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulfation in its carbohydrate chain. 4. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and that sulfate availability is essential for the formation of the high molecular-weight mucin polymer.     

Metabolic control of the potassium permeability in pancreatic islet cells

The K(+) permeability of pancreatic islet cells was studied by monitoring the efflux of (86)Rb(+) (used as tracer for K(+)) from perifused rat islets and measuring the uptake of (42)K(+). Glucose markedly and reversibly decreased (86)Rb(+) efflux from islet cells and this effect was antagonized by inhibitors of the metabolic degradation of the sugar, i.e. mannoheptulose, iodoacetate, glucosamine and 2-deoxyglucose. Among glucose metabolites, glyceraldehyde reduced the K(+) permeability even more potently than did glucose itself; pyruvate and lactate alone exhibited only a small effect, but potentiated that of glucose. Other metabolized sugars, like mannose, glucosamine and N-acetylglucosamine, also decreased (86)Rb(+) efflux from islet cells. Fructose was effective only in the presence of glucose. Non-metabolized sugars like galactose, 2-deoxyglucose and 3-O-methylglucose had no effect. The changes in K(+) permeability by agents known to modify the concentrations of nicotinamide nucleotides, glutathione or ATP in islet cells were also studied. Increasing NAD(P)H concentrations in islet cells by pentobarbital rapidly and reversibly reduced (86)Rb(+) efflux; exogenous reduced glutathione produced a similar though weaker effect. By contrast, oxidizing nicotinamide nucleotides with phenazine methosulphate or Methylene Blue, or oxidizing glutathione by t-butyl hydroperoxide increased the K(+) permeability of islet cells. Uncoupling the oxidative phosphorylations with dicumarol also augmented (86)Rb(+) efflux markedly. In the absence of glucose, but not in its presence, methylxanthines reduced (86)Rb(+) efflux from the islets; such was not the case for cholera toxin or dibutyryl cyclic AMP. Glucose and glyceraldehyde had no effect on (42)K(+) uptake after a short incubation (10min), but augmented it after 60min; the effect of glucose was suppressed by mannoheptulose and not mimicked by 3-O-methylglucose. The results clearly establish the importance of the metabolic degradation of glucose and other substrates for the control of the K(+) permeability in pancreatic islet cells and support the concept that a decrease in the K(+) permeability represents a major step of the B-cell response to physiological stimulation.     

Induction of the nag regulon of Escherichia coli by N-acetylglucosamine and glucosamine: role of the cyclic AMP-catabolite activator protein complex in expression of the regulon.

The divergent nag regulon located at 15.5 min on the Escherichia coli map encodes genes necessary for growth on N-acetylglucosamine and glucosamine. Full induction of the regulon requires both the presence of N-acetylglucosamine and a functional cyclic AMP (cAMP)-catabolite activator protein (CAP) complex. Glucosamine produces a lower level of induction of the regulon. A nearly symmetric consensus CAP-binding site is located in the intergenic region between nagE (encoding EIINag) and nagB (encoding glucosamine-6-phosphate deaminase). Expression of both nagE and nagB genes is stimulated by cAMP-CAP, but the effect is more pronounced for nagE. In fact, very little expression of nagE is observed in the absence of cAMP-CAP, whereas 50% maximum expression of nagB is observed with N-acetylglucosamine in the absence of cAMP-CAP. Two mRNA 5' ends separated by about 100 nucleotides were located before nagB, and both seem to be similarly subject to N-acetylglucosamine induction and cAMP-CAP stimulation. To induce the regulon, N-acetylglucosamine or glucosamine must enter the cell, but the particular transport mechanism used is not important.     

Uncoating of influenza virus in endosomes

The intracellular uncoating site of influenza virus was studied by measuring the fluorescence intensity of probes conjugated to the virus or the isolated hemagglutinin and also by assaying virus replication under various incubation conditions. Acidification of the viral environment was monitored by the decrease in the fluorescence intensity of fluorescein isothiocyanate, and transport of the virus particles into secondary lysosomes was assayed by the increase in the fluorescence intensity of fluorescein isothiocyanate diphosphate. The intracellular pH was estimated by the ratio of fluorescence intensities excited at two different wavelengths. It was found that the viral environment became acidified to a pH value of 5.1 to 5.2 within 10 min at 37 degrees C or 1 h at 20 degrees C after endocytosis. Addition of ammonium chloride to the medium rapidly raised the pH to 6.7. Transport of the virus particles into the secondary lysosomes was slower and negligibly low during those incubation periods. Virus replication occurred when the cells were incubated for 10 min at 37 degrees C or for 1 h at 20 degrees C, followed by incubation in the presence of ammonium chloride for a total of 12 h. These results indicate the uncoating of influenza virus in endosomes before reaching the secondary lysosomes.     

Intracellular production of virus particles and viral components in NIH/3T3 cells chronically infected with Moloney murine leukemia virus: effect of interferon.

The effect of interferon on the biochemical properties and the maturation process of intracellular viral particles isolated from the cytoplasmic fraction of NIH/3T3 cells chronically infected with Moloney murine leukemia virus was investigated. By labeling these virions with either [35S]methionine or [3H]glucosamine, we demonstrated that they contain the same viral proteins and glycoproteins found in extracellular virions. Interferon treatment was found to reduce the rate of intracellular virus assembly. This effect was not a consequence of an interferon inhibition of viral RNA synthesis or its translation or a consequence of an interference with the posttranslational cleavage processing of viral precursor proteins, since all of these steps were not affected by interferon. However, the reduced rate of virus assembly could be attributed to the inhibition of viral protein glycosylation observed in interferon-treated cells. Nevertheless, despite this reduced rate, virus particles accumulated in interferon-treated cells. This accumulation was probably due to the strong inhibition of their final release from such cells.     

Assay and properties of N-acetylglucosamine-6-phosphate deacetylase from rat liver

A single-vial assay has been developed for N-acetylglucosamine-6-phosphate deacetylase, in which [3H]acetate released from 3H-acetyl-labeled substrate is measured in a biphasic liquid scintillation counting system after acidification of the reaction mixture. The deacetylase was partially purified from rat liver, and some of its properties were determined. Chromatography on a calibrated Sepharose CL-6B column indicated a molecular weight of 345,000. The Km for the substrate at pH 8.0 was 0.3 mM. Glucosamine 6-phosphate and glucose 6-phosphate inhibited the enzyme, whereas N-acetylgalactosamine, N-acetylglucosamine, N-acetylglucosamine 1-phosphate, and glucosamine 1-phosphate were without effect. The effects of several divalent cations were also examined. Under the conditions tested, Ca2+, Mg2+, and Ba2+ had essentially no effect, whereas Mn2+, Ni2+, and Cu2+ were inhibitory and Co2+ stimulated activity at low concentrations but inhibited above 5 mM. An increase in the ionic strength of the reaction mixture to 0.3 M decreased the activity by 40%.     

Vesicular stomatitis virus envelope glycoprotein alterations induced by host cell transformation

Vesicular stomatitis virus (VSV) contains a single structural glycoprotein in which the sugar sequences are largely host specified. We have used VSV as a probe to study the changes in cell glycoprotein metabolism induced by virus transformation. Analysis of purified VSV grown in baby hamster kidney (BHK) or polyoma transformed BHK cells showed that the virus glycoproteins have identical apparent molecular weights. The glycopeptides derived from the glycoproteins by extensive pronase digestion have an identical molecular weight distribution.On the basis of labeling experiments with fucose, mannose, and glucosamine, the oligosaccharide moieties of the VSV glycoprotein were different in virus from the two cell lines. The VSV glycopeptides from transformed cells showed an increased resistance to cleavage by an endoglycosidase, indicating structural changes in the core region of the oligosaccharides. They also showed an increased ratio of sialic acid to N-acetylglucosamine.VSV grows in a wide variety of cell types, and the carbohydrate structures of its single glycoprotein are amenable to analysis with specific glycosidases. The virus thus provides an excellent tool with which to study alterations induced by cell transformation in the glycosylation of membrane proteins.     

The effect of losing glycosylation sites near the receptor-binding region on the receptor phenotype of the human influenza virus H1N1

The receptor properties of influenza virus (IF) isolates/SSSR/90/77 are studied. The isolates are peculiar for losing glycosylation sites (GS) at the Asn131 receptor-binding region (GS131) after passaging in mice and at the Asn158 region (GS158) after cultivation in the presence of mouse serum. The loss of each carbohydrate residue increases the influenza virus affinity for carbohydrate chains with the terminal group Neu5Ac alpha 2-6Gal and reduces its affinity for Neu5Ac alpha 2-3Gal receptors. The effect is more pronounced in the GS158-depleted virus. Upon substitution of asparagine by aspartic acid, the electrostatic component of virus binding to the receptor is altered because of the increased negative charge on hemagglutinin. The virus receptor phenotype changes depending on the cultivation conditions. The isolate adapted to mice has higher affinity to mouse lung cell receptors, while the virus propagated in chick embryos in the presence of inhibitors has higher affinity to allantoic membrane cells.     

Carbohydrates of Influenza Virus. I. Glycopeptides Derived from Viral Glycoproteins After Labeling with Radioactive Sugars

The carbohydrate moiety of the influenza glycoproteins NA, HA₁, and HA₂ were analyzed by labeling with radioactive sugars. Analysis of glycopeptides obtained after digestion with Pronase indicated that there are at least two different types of carbohydrate side chains. The side chain of type I is composed of glucosamine, mannose, galactose, and fucose. It is found on NA, HA₁, and HA₂. The side chain of type II contains a high amount of mannose and is found only on NA and HA₂. The molecular weights of the corresponding glycopeptides obtained from virus grown in chicken embryo cells are 2,600 for type I and 2,000 for type II. The glycoproteins of virus grown in MDBK cells have a higher molecular weight than those of virus grown in chicken embryo cells, and there is a corresponding difference in the molecular weights of the glycopeptides. Under conditions of partial inhibition of glycosylation, virus particles were isolated that contained hemagglutinin with reduced carbohydrate content. Glycopeptide analysis indicated that this reduction is due to the lack of whole carbohydrate side chains and not to the incorporation of incomplete ones. This observation suggests that glycosylation of the viral glycoproteins involves en bloc transfer of the core sugars to the polypeptide chains.     

Magnesium uptake by pancreatic islet cells is modulated by stimulators and inhibitors of the B-cell function

28Mg2+ uptake by rat islets was measured during incubation with various stimulators or inhibitors of insulin release. D-Glucose induced a dose-dependent increase in 28Mg2+ uptake after 10 min or 120 min. The threshold concentration was around 6 mM and the maximum effect was observed with 15-20 mM glucose. After 120 min 28Mg2+ uptake was also stimulated by the metabolized sugars mannose, N-acetylglucosamine or glyceraldehyde, was unaffected by the non-metabolized or poorly metabolized L-glucose, galactose, 3-O-methylglucose, 2-deoxyglucose, fructose or mannoheptulose and was inhibited by glucosamine. The effect of glucose was markedly impaired by mannoheptulose, glucosamine, aminooxyacetate and NH4Cl, but was only partially decreased by D600 or diazoxide, which were ineffective in a glucose-free medium. Tolbutamide or KCl slightly increased 28Mg2+ uptake. Alanine, leucine alone or with glutamine, and ketoisocaproate also stimulated 28Mg2+ uptake, whereas arginine and lysine decreased it. These changes in 28Mg2+ uptake, brought about by various modifiers of the B-cell function, are thus similar but not identical to the changes in Ca2+ uptake, and are not the consequence of insulin release. The stimulatory effect of glucose requires glucose metabolism by islet cells, but is only partially due to depolarization of the B-cell membrane.     

Synthesis of five nona-β-(1→6)-d-glucosamines with various patterns of N-acetylation corresponding to the fragments of exopolysaccharide of Staphylococcus aureus

A series of five 3-acetamidopropyl β-glycosides of nona-β-(1→6)-glucosamines containing two N-acetylglucosamine residues separated by a different number of glucosamine units with free amino groups have been synthesized using a convergent blockwise approach. Oxazoline glycosylation was used to introduce N-acetylglucosamine residues. These nonasaccharides are structurally related to the poly-N-acetylglucosamine (PNAG) extracellular polysaccharide of Staphylococcus aureus and can be used as models for biochemical and immunological studies.     

Glycosylation of vesicular stomatitis virus glycoprotein in virus-infected HeLa cells.

Glycosylation of the envelope glycoprotein of vesicular stomatitis virus was examined using virus-infected HeLa cells that were pulse-labeled with radioactive sugar precursors. The intracellular sites of glycosylation and the stepwise elongation of the carbohydrate side chains of the G protein were monitored by membrane fractionation and gel filtration of Pronase-digested glycopeptides. The results with short pulses of sugar label (5 to 10 mtein linkage (glucosamine and mannose) are added to G which was associated with the rough endoplasmic reticulum-enriched membrane fraction, whereas the more distal sugars (galactose, sialic acid, fucose, and possibly more glucosamine) are added in the light-density internal membrane fraction. Accumulation of mature G was observed in the plasma membrane-enriched fraction. The gel filtration studies indicated that the initial glycosylation event may be the en bloc addition of a mannose and glucosamine oligomer, followed by the stepwise addition of the more distal sugars.