A remodeling system for the oligosaccharide chains on glycoproteins with microbial endo-beta-N-acetylglucosaminidases

Endo-M, endo-beta-N-acetylglucosaminidase from Mucor hiemalis, transferred the complex type oligosaccharide of sialoglycopeptide to partially deglycosylated proteins (N-acetylglucosamine-attached proteins), which were prepared by excluding high-mannose type oligosaccharides from glycoproteins with Endo-H, endo-beta-N-acetylglucosaminidase from Streptomyces plicatus. This finding indicated that the high-mannose type oligosaccharides on glycoproteins can be changed to complex type ones by the transglycosylation activity of Endo-M. This is the first report of the establishment of a remodeling system for the different types of oligosaccharides on glycoproteins with microbial endo-beta-N-acetylglucosaminidases having different substrate specificities. Endo-M is a powerful tool for the in vitro synthesis of glycoproteins containing complex type oligosaccharides from glycoproteins produced by yeast.     

The position of the oligosaccharide side-chains of phytohemagglutinin and their accessibility to glycosidases determines their subsequent processing in the Golgi

Phytohemagglutinin (PHA), the glycoprotein lectin of Phaseolus vulgaris has two types of asparagine-linked oligosaccharides per polypeptide: a high-mannose chain with the formula (Man)8-9(GlcNAc)2 on Asn12 and a modified chain with fewer mannose residues and additional fucose and xylose residues on Asn60. Glycosylation of PHA is a cotranslational process, which occurs in the endoplasmic reticulum, and newly synthesized PHA has two high-mannose chains. Transport of PHA to the protein bodies via the Golgi complex is accompanied by the modification of one of the two high-mannose chains. Why is only one chain modified, while the other remains in the high-mannose configuration? By determining the effect of digestion with various glycosidases (alpha-mannosidase, endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F) on native and denatured PHA we obtained evidence consistent with the interpretation that the accessibility of oligosaccharide chains to modifying enzymes is of major importance in determining whether a high-mannose chain becomes modified or not. The high-mannose chain of mature undenatured PHA is only partially accessible to glycosidases, while PHA obtained from the endoplasmic reticulum has one high-mannose chain, which is readily accessible to alpha-mannosidase and endoglycosidases H and F. We show that this readily accessible chain is in the same position on the polypeptide (Asn60) as the modified oligosaccharide on mature PHA. Thus, accessibility of the oligosaccharide side-chains to processing enzymes in the Golgi determines whether a particular oligosaccharide side-chain is processed or not.     

The major yeast exoglucanase: an extracellular glycoprotein lacking the carbohydrate outer chain

The major exoglucanase (1,3-beta-D-glucan glucanohydrolase, EC 3.2.1.39) secreted by Saccharomyces cerevisiae contains protein, mannose and phosphate in a molar ratio of 1:27:1. When digested with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96) it sequentially released two asparagine-linked oligosaccharide chains. Oligosaccharides were fractionated into a neutral and acidic component, each one accounting for 50% of the total carbohydrate. The neutral oligosaccharide consisted of a mixture of three homologues ranging from GlcNAc-(Man)12 to GlcNAc-(Man)14. The acidic carbohydrate was, in turn, split into two components. The major one (45% of the initial material) contained a phosphodiester bond and released only mannose when subjected to mild acid hydrolysis. From the filtration pattern, it was shown to be a mixture of oligosaccharides ranging from GlcNAc-(Man)11-P to GlcNAC-(Man)13-P. The minor phosphorylated component, which represented the residual carbohydrate (5%), contained a phosphomonoester bond. It was also heterogeneous in size, the several homologues having one mannose less than their counterparts from the phosphodiester oligosaccharide. These results clearly indicate that the addition of an outer chain of carbohydrate is not a requirement for the externalization of yeast glycoproteins.     

Oligosaccharides in lysosomal enzymes. Distribution of high-mannose and complex oligosaccharides in cathepsin D and beta-hexosaminidase

The distribution of the different types of oligosaccharides in cathepsin D and in beta-hexosaminidase synthesized in cultured human fibroblasts was studied by using endo-beta-N-acetylglucosaminidase H as a probe for high-mannose oligosaccharides. The enzymes were specifically labelled in the protein or the carbohydrate moiety. In both enzymes, resistant and cleavable oligosaccharides were found. The resistant oligosaccharides prevailed in the secreted enzymes. Precursor molecules of cathepsin D contained two oligosaccharide side chains. Multiple forms of the precursor are synthesized with both, one or none of two oligosaccharides sensitive to the action of the endo-beta-N-acetylglucosaminidase H. In fibroblasts unable to phosphorylate lysosomal enzymes (mucolipidosis II) the excessively secreted lysosomal enzymes contained predominantly oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H.     

Type analysis of the oligosaccharide chains on microheterogeneous components of bovine pancreatic DNAase by the lectin-nitrocellulose sheet method.

The oligosaccharide chains of microheterogeneous bovine pancreatic DNAases were characterized by the lectin-nitrocellulose sheet method. The active fractions of the DNAases from column chromatography showed four major and several minor spots on a two-dimensional polyacrylamide gel. They were transferred on to nitrocellulose sheets and treated with glycosidases (neuraminidase, endo-beta-N-acetyl glucosaminidase H or F, or peptide N-glycosidase F) and treated with peroxidase-coupled lectins (concanavalin A, Ricinus communis agglutinin or wheat-germ agglutinin). From the results, the most probable oligosaccharide types were proposed to be as follows: the four major spots contained components which had high-mannose type or hybrid-type oligosaccharides, such as those susceptible to endo-beta-N-acetylglucosaminidase H. In addition, spot 1 contained a complex-type biantennary oligosaccharide without sialic acid and spot 3 contained a tri- or tetra-antennary complex-type oligosaccharide with sialic acid. The component corresponding to spot 2 had a hybrid-type oligosaccharide chain with a 'bisecting' acetylglucosamine, linked 1-4 to the beta-mannose residue of the trimannosyl core, and the component corresponding to spot 4 had a high-mannose-type oligosaccharide chain.     

Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N-glycosidase F

Peptide N-glycosidase from Flavobacterium meningosepticum cleaves complex as well as neutral glycoproteins (Plummer, T.H., Jr., Elder, J.H., Alexander, S., Phelan, A.W., and Tarentino, A.L. (1984) J. Biol. Chem. 259, 10700-10704). Examples of neutral glycoprotein substrates include ribonuclease B (one high mannose oligosaccharide chain) and yeast external invertase (nine chains/invertase subunit). The rate of deglycosylation by the glycosidase was greatly enhanced if the glycoprotein substrate was denatured prior to enzyme treatment, from a low of 11-fold for external invertase to a high of 844-fold for ribonuclease B. Peptide N-glycosidase F was unable to cleave the asparaginyl-N-acetylglucosamine bond in endo-beta-N-acetylglucosaminidase H-modified external invertase or ribonuclease B, although that in similarly modified glycopeptide substrate was cleaved. Ribonuclease B was digested sequentially with various exoglycosidases to produce an oligosaccharide chain of varied length. Using the resulting forms of ribonuclease B as substrates for peptide N-glycosidase F, the minimum oligosaccharide chain for cleavage was the di-N-acetyl-chitobiosyl core unit.     

Primate cytomegalovirus glycoproteins: lectin-binding properties and sensitivities to glycosidases.

The lectin-binding properties and glycosidase sensitivities of the virion glycoproteins of primate cytomegaloviruses (CMVs) were examined. Three simian CMV (SCMV) strains, including Colburn, and four human CMV (HCMV) strains were compared. Their proteins were separated in denaturing polyacrylamide gels and electrotransferred onto nitrocellulose, and the glycosylated species were visualized with iodinated concanavalin A or wheat germ agglutinin (WGA). Virions of both HCMV and SCMV strains contained six principal and several minor lectin-reactive bands. Neuraminidase treatment abolished WGA binding and reduced the charge and charge heterogeneity of the SCMV (i.e., Colburn) virion glycoproteins and had a similar, although less dramatic, effect on those of HCMV. The specificities of concanavalin A and WGA in these assays were evaluated with endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F, and a combination of lectins and glycosidases was used to demonstrate that many of the primate CMV glycoproteins contain both high-mannose and complex, N-linked oligosaccharides. Results suggest that the HCMV virion glycoproteins are more extensively glycosylated or have more completely processed carbohydrate side chains, or both, than their SCMV counterparts.     

Structures of oligosaccharides on beta-galactosidase from Aspergillus oryzae

The carbohydrate portions of beta-galactosidase from Aspergillus oryzae were found to be composed of two types of sugar chains. They were released equally well with endo-beta-N-acetylglucosaminidase H, but were distinct in their chain length. The long sugar chains (fraction I), corresponding to 4% of the total carbohydrate chains, were composed of galactomannan-type oligosaccharides, which consisted of mannose, galactose, glucose, and glucosamine in the molar ratios of 30.0, 16.4, 1.4, and 2.1 per mol of aspartic acid, respectively. The short sugar chains (fraction II), corresponding to 96% of the total carbohydrate chains, consisted of mannose, galactose, glucose, and glucosamine in the molar ratios of 9.4, 0.6, 0.3, and 1.7 per mol of aspartic acid, respectively. Both types of sugar chains were fractionated into neutral and acidic subfractions. The neutral subfraction of fraction I (I-N), corresponding to 1% of the total carbohydrate chains, was very heterogeneous in length and was resistant to digestion with alpha-mannosidase and beta-galactosidase. The neutral subfraction of fraction II (II-N), corresponding to 91% of the total carbohydrate, was composed of a mixture of oligosaccharides with oligomanneoside chains (Mann GlcNAcol). The major components were similar to high mannose-type oligosaccharides of mammalian origin in their composition and size (n = 5-9). However, digestion of II-N with alpha 1,2-mannosidase produced considerable amounts of Man6GlcNAcol, an unusual product in the case of high mannose-type oligosaccharides of mammalian origin, in addition to the common one, Man5GlcNAcol.     

Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.     

Structural analysis of the N-glycans of the major cysteine proteinase of Trypanosoma cruzi. Identification of sulfated high-mannose type oligosaccharides

Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease, contains a major cysteine proteinase, cruzipain. This lysosomal enzyme bears an unusual C-terminal extension that contains a number of post-translational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of UV-MALDI-TOF mass spectrometry in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N-linked oligosaccharides present in this domain. The UV-MALDI-TOF MS analysis in the negative-ion mode, using nor-harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In accordance, after chemical or enzymatic desulfation, the corresponding signals disappeared. In addition, by UV-MALDI-TOF MS analysis (a) a main population of high-mannose type oligosaccharides was shown in the positive-ion mode, (b) lactosaminic glycans were also identified, among them, structures corresponding to monosialylated species were detected, and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number of oligosaccharides occurring in cruzipain was shown to be much higher than previous estimates. This constitutes the first report on the presence of sulfated glycoproteins in Trypanosomatids.     

Changes in N-linked oligosaccharides during seed development of Ginkgo biloba

Structural changes in N-linked oligosaccharides of glycoproteins during seed development of Ginkgo biloba have been explored to discover possible endogenous substrate(s) for the Ginko endo-beta-N-acetylglucosaminidase (endo-GB; Kimura, Y., et al. (1998) Biosci. Biotechnol. Biochem., 62, 253-261), which should be involved in the production of high-mannose type free N-glycans. The structural analysis of the pyridylaminated oligosaccharides with a 2D sugar chain map, by ESI-MS/MS spectroscopy, showed that all N-glycans expressed on glycoproteins through the developmental stage of the Ginkgo seeds have the xylose-containing type (GlcNAc2 approximately 0Man3Xyl1Fuc1 approximately 0GlcNAc2) but no high-mannose type structure. Man3Xyl1Fuc1GlcNAc2, a typical plant complex type structure especially found in vacuolar glycoproteins, was a dominant structure through the seed development, while the amount of expression of GlcNAc2Man3Xyl1Fuc1GlcNAc2 and GlcNAc1Man3Xyl1Fuc1GlcNAc2 decreased as the seeds developed. The dominantly occurrence of xylose-containing type structures and the absence of the high-mannose type structures on Ginkgo glycoproteins were also shown by lectin-blotting and immunoblotting of SDS-soluble glycoproteins extracted from the developing seeds at various developmental stages. Concerning the endogenous substrates for plant endo-beta-N-acetylglucosaminidase, these results suggested that the endogenous substrates might be the dolicol-oligosaccharide intermediates or some glycopeptides with the high-mannose type N-glycan(s) derived from misfolded glycoproteins in the quality control system for newly synthesized glycoproteins.     

Carbohydrate structures of HVJ (Sendai virus) glycoproteins

The carbohydrate structures of two membrane glycoproteins (HANA protein and F protein) of HVJ have been determined on materials purified from virions grown in the allantoic sac of embryonated chicken eggs. Both glycoproteins contain fucose, mannose, galactose, and glucosamine but not galactosamine, indicating that their sugar chains are exclusively of the asparagine-linked type. The radioactive oligosaccharide fractions obtained from the two glycoproteins by hydrazinolysis followed by NaB[3H]4 reduction gave quite distinct fractionation patterns after paper electrophoresis. More than 75% of the oligosaccharides from F protein were acidic and separated into at least four components by paper electrophoresis. Only 18% of the oligosaccharide from HANA protein was an acidic single component. These acidic oligosaccharides could not be converted to neutral oligosaccharides by sialidase digestion. Structural studies of the neutral oligosaccharide fractions from HANA and F proteins revealed that both of them are mixtures of a series of high mannose type oligosaccharides and of complex type oligosaccharides with Gal beta 1 leads to (Fuc alpha 1 leads to 3) GlcNAc group in their outer chain moieties.     

Phosphorylated high mannose-type and hybrid-type oligosaccharide chains of human thyroglobulin isolated from malignant thyroid tissue

Structures of anionic oligosaccharides released by endo-beta-N-acetylglucosaminidase H from human thyroglobulin (19 S) purified from normal and malignant (papillary carcinoma) thyroid tissues were studied by sequential exoglycosidase digestion and lectin affinity chromatography. It was revealed that sialylated hybrid-type oligosaccharides were present in thyroglobulin from normal thyroid tissue, whereas, in the case of thyroglobulin from malignant thyroid tissue, phosphorylated high-mannose type and hybrid-type oligosaccharides each containing one phosphate group in a diester linkage were present instead of sialylated oligosaccharides. The possible meaning of the phosphorylated oligosaccharides was discussed in relation to the low thyroglobulin content in malignant thyroid tissue.     

Echinococcus granulosus: antigen characterization by chemical treatment and enzymatic deglycosylation.

Parasite antigenic fractions obtained by biochemical purification of sheep hydatid fluid were subjected to enzymatic digestion. The relative mobilities of the 5 and B antigens, before and after treatment, were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Antigenic fractions transferred to nitrocellulose were also treated with sodium metaperiodate and concanavalin A. The results indicate that antigen 5 contains a substantial amount of carbohydrates covalently linked to a polypeptide backbone, which strongly bind to concanavalin A and is removed by N-glycosidase F (PNGase F). Antigen 5 possesses complex N-linked oligosaccharides (PNGase F sensitive), without terminal N-acetyl-D-glucosamine residues (N-acetyl-D-glucosaminidase nonsensitive) and has no high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H nonsensitive). In contrast, the antigen B of low molecular weight is not susceptible to either enzymatic digestions (PNGase F, Endo H, and N-acetyl-D-glucosaminidase) or sodium metaperiodate oxidation and it does not bind to concanavalin A. Polyclonal antibodies prepared against the two antigens reacted with the deglycosylated antigen 5 in Western blot. The dominant epitopes are, therefore, polypeptides, although the presence of carbohydrate epitopes in the native glycoproteins cannot be excluded.     

Castanospermine inhibits glucosidase I and glycoprotein secretion in human hepatoma cells.

We studied the effect of the plant alkaloid castanospermine on the biosynthesis and secretion of human hepatoma glycoproteins. The HepG-2 cells, grown in the presence or absence of the alkaloid, were labelled with [2-3H]mannose and then the labelled glycopeptides were prepared by Pronase digestion. This material was analysed by gel filtration on Bio-Gel P-4 before and after treatment with endo-beta-N-acetylglucosaminidase H. Castanospermine caused an accumulation of high-mannose oligosaccharides, by 70-75% over control. The major accumulated product, which could also be labelled with [3H]galactose and was only partially susceptible to alpha-mannosidase digestion, was identified by h.p.l.c. as a Glc3Man9GlcNAc. Thus the alkaloid inhibits glucosidase I in the human hepatoma cells. Analysis of total glycoproteins secreted by the cells into the medium revealed the presence of only complex oligosaccharides in both control and treated cultures, and the amount of the oligosaccharides labelled with radioactive mannose, galactose or N-acetylmannosamine, secreted by treated cells, was decreased by about 60%. The rate of secretion of total protein labelled with [35S]methionine and precipitated from the medium with trichloroacetic acid was inhibited by up to 40% in the presence of castanospermine. Pulse-chase studies utilizing [35S]methionine labelling were performed to study the effect of the alkaloid on secretion of individual plasma proteins. Immunoprecipitation at different chase times with monospecific antisera showed that castanospermine markedly decreased the secretion rates of alpha 1-antitrypsin, caeruloplasmin and, to a lesser extent, that of antithrombin-III. Secretions of apolipoprotein E, a glycoprotein containing only O-linked oligosaccharide(s), and albumin, a non-glycosylated protein, were not affected by the drug. It is suggested that castanospermine inhibits secretion of at least some glycoproteins containing N-linked oligosaccharides, owing to the inhibition of oligosaccharide processing.     

Dissecting glycoprotein biosynthesis by the use of specific inhibitors

It is possible to interfere with different steps in the dolichol pathway of protein glycosylation and in the processing of asparagine-linked oligosaccharides. Thus some clues about the role of protein-bound carbohydrate can be obtained by comparing the biochemical fates and functions of glycosylated proteins with their non-glycosylated counterparts, or with proteins exhibiting differences in the type of oligosaccharide side chains. Cells infected with enveloped viruses are good systems for studying both aspects of protein glycosylation, since they contain a limited number of different glycoproteins, often with well-defined functions. Tunicamycin, an antibiotic, as well as several sugar analogues have been found to act as inhibitors of protein glycosylation by virtue of their anti-viral properties. They interfere with various steps in the dolichol pathway resulting in a lack of functional lipid-linked oligosaccharide precursors. Compounds that interfere with oligosaccharide trimming represent a second generation of inhibitors of glycosylation. They are glycosidase inhibitors that interfere with the processing glucosidases and mannosidases and, as a result, the conversion of high-mannose into complex-type oligosaccharides is blocked. Depending upon the compound used, glycoproteins contain glucosylated-high-mannose, high-mannose or hybrid oligosaccharide structures instead of complex ones. The biological consequences of the alterations caused by the inhibitors are manifold: increased susceptibility to proteases, improper protein processing and misfolding of polypeptide chains, loss of biological activity and alteration of the site of virus-budding, to name but a few.     

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Fractionation of oligosaccharides liberated by hydrazinolysis and structural studies of the neutral oligosaccharides

The asparagine-linked sugar chains of the plasma membrane glycoproteins of rat erythrocytes were released as oligosaccharides by hydrazinolysis and labeled by NaB³H₄ reduction. The radioactive oligosaccharides were separated into a neutral and at least four acidic fractions by paper electrophoresis. The neutral oligosaccharide fraction was separated into at least 11 peaks upon Bio-Gel P-4 column chromatography. Structural studies of them by sequential exoglycosidase digestion in combination with methylation analysis revealed that they were a mixture of three high mannose-type oligosaccharides and at least 11 complex type oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc as their cores and Galβ1 → 4GlcNAc, Galβ1 → 3Galβ1 → 4GlcNAc, and various lengths of Galβ1 → 4GlcNAc repeating chains in their outer chain moieties. Most of the complex-type Oligosaccharides were biantennary, and the tri- and tetraantennary Oligosaccharides contain only the Galβ1 → 3Galβ1 → 4GlcNAc group in their outer chain moieties.     

Carbohydrates of human immunodeficiency virus. Structures of oligosaccharides linked to the envelope glycoprotein 120

Human T-cells (H9), persistently infected with the HTLV-III strain of human immunodeficiency virus, were metabolically labeled with D-[2-3H]mannose or D-[6-3H]glucosamine. The viral envelope glycoprotein, gp120, was isolated either from cell lysates or from cell-free culture supernatant. After proteolytic digestion, the radiolabeled oligosaccharides were sequentially liberated from glycopeptides by treatment with endo-beta-N-acetylhexosaminidase H and peptide:N-glycosidase F. Oligosaccharides released were separated from residual (glyco)peptides and fractionated according to size, charge, and fucose content. The individual oligosaccharide species obtained were characterized by digestion with exoglycosidases and by chromatographic comparison with standard oligosaccharides. Our results demonstrate that the intracellular gp120 carries predominantly oligomannosidic glycans comprising nine or eight mannose residues. The secreted glycoprotein is equally substituted by oligomannosidic species, containing seven to nine mannose residues, and by fucosylated, partially sialylated bi- and triantennary complex-type oligosaccharides.     

Identification of the ZPC oligosaccharide ligand involved in sperm binding and the glycan structures of Xenopus laevis vitelline envelope glycoproteins

The Xenopus laevis egg vitelline envelope is composed of five glycoproteins (ZPA, ZPB, ZPC, ZPD, and ZPX). As shown previously, ZPC is the primary ligand for sperm binding to the egg envelope, and this binding involves the oligosaccharide moieties of the glycoprotein (Biol. Reprod., 62:766-774, 2000). To understand the molecular mechanism of sperm-egg envelope binding, we characterized the N-linked glycans of the vitelline envelope (VE) glycoproteins. The N-linked glycans of the VE were composed predominantly of a heterogeneous mixture of high-mannose (5-9) and neutral, complex oligosaccharides primarily derived from ZPC (the dominant glycoprotein). However, the ZPA N-linked glycans were composed of acidic-complex and high-mannose oligosaccharides, ZPX had only high-mannose oligosaccharides, and ZPB lacked N-linked oligosaccharides. The consensus sequence for N-linked glycosylation at the evolutionarily conserved residue N113 of the ZPC protein sequence was glycosylated solely with high-mannose oligosaccharides. This conserved glycosylation site may be of importance to the three-dimensional structure of the ZPC glycoproteins. One of the complex oligosaccharides of ZPC possessed terminal beta-N-acetyl-glucosamine residues. The same ZPC oligosaccharide species isolated from the activated egg envelopes lacked terminal beta-N-acetyl-glucosamine residues. We previously showed that the cortical granules contain beta-N-acetyl-glucosaminidase (J. Exp. Zool., 235:335-340, 1985). We propose that an alteration in the oligosaccharide structure of ZPC by glucosaminidase released from the cortical granule reaction is responsible for the loss of sperm binding ligand activity at fertilization.     

Alterations in N-linked oligosaccharides of glycoproteins during rat liver regeneration

[3H]Mannose-labeled glycopeptides in the slices after partial hepatectomy were characterized by column chromatography using Sephadex G-50, DE-52 and Con A-Sepharose, and further by digestion with alpha-mannosidase and endo-beta-N-acetylglucosaminidase H. They contained both 'complex type' and 'high-mannose type' oligosaccharides. A higher proportion of 'complex type' oligosaccharides was contained in regenerating liver 24 h after partial hepatectomy than in control. This tendency was increased gradually with time and was most pronounced at 144 h. In our previous studies, the activities of microsomal N-acetylglucosaminyltransferase towards endogenous and exogenous acceptors at 144 h after partial hepatectomy were shown to exceed most prominently that in control. No differences in the oligosaccharides were observed at 240 h when the deficit of liver had been restored. The oligosaccharides of glycopeptides in the incubation media were mostly 'complex type' and the differences between regenerating liver and control were observed only at 144 h. These results suggest that oligosaccharide processing of glycoproteins is regulated at the transfer step of peripheral N-acetylglucosamine to core oligosaccharides 144 h after partial hepatectomy, and that these alterations in oligosaccharides of glycoproteins may be related to hypertrophy and hyperplasia of hepatic cells in liver regeneration.