Asparagine-linked Oligosaccharides Research Articles

Transglycosylation Activity of Mucor Hiemalis Endo-β-N-Acetylglucosaminidase Which Transfers Complex Oligosaccharides to the N-Acetylglucosamine Moieties of Peptides

A novel endo-β-N-acetylglucosaminidase in the culture fluid of Mucor hiemalis isolated from soil was found to have transglycosylation activity. This endo-β-N-acetylglucosaminidase, Endo-M, could liberate the complex type of asparagine-linked oligosaccharides by hydrolysis of diacetylchitobiose linkage from glycoproteins. The treatment of Endo-M with N-acetylglucosamine and asialotransferrin glycopeptide having the complex type of oligosaccharides resulted in the transfer of the released oligosaccharide from the glycopeptide to N-acetylglucosamine. The structure of the product after transfer was deduced to be (GlcNAc)2-Man-(Gal-GlcNAc-Man)2 by a combination method of pyridylamination and high perfomance liquid chromatography, and mass-spectrometry. The enzyme could transfer the complex type of oligosaccharide from asialotransferrin glycopeptide to peptidyl-N-acetylglucosamine prepared from bovine ribonuclease with the high-mannose type of oligosaccharide. This will lead to the construction of neoglycoproteins containing different types of oligosaccharides.

Microbial endoglycosidases for analyses of oligosaccharide chains in glycoproteins.

Microbial endoglycosidases are useful for elucidating the structure and function of the oligosaccharide chains of glycoconjugates. Most of the microbial endo-beta-N-acetylglucosaminidases including Endo-H can preferentially act on high-mannose type chains of asparagine-linked oligosaccharides of various glycoproteins. Among them, Flavobacterium sp. enzyme is produced in large amounts by the inducing cultivation. Using this enzyme, the role of oligosaccharide chains in various microbial glycoenzymes such as Rhizopus glucoamylase, and yeast invertase was examined. The findings suggested that the oligosaccharide chains of them are essential participants in the stabilization of the enzyme and in the protection from proteolytic inactivation. Novel endo-beta-N-acetylglucosaminidases were also found in the culture broths of microorganisms. Unlike most microbial endo-beta-N-acetylglucosaminidases, Endo-M of Mucor hiemalis could act on a complex type oligosaccharide chains, which is similar to Endo-F2 in multiple form of Endo-F from Flavobacterium meningosepticum. The complete amino acid sequences of Endo-F1, -F2, -F3, -H, and Flavobacterium sp. enzyme were determined. All of them had two highly conserved regions common to a number of chitinases. Endo-alpha-N-acetylgalactosaminidase which hydrolyzes the O-glycosidic linkages in glycoproteins was found in the culture broth of only a few microorganisms. The production of Alcaligenes sp. enzyme was highly induced by the addition of porcine gastric mucin in the culture medium. There is some evidence that endo-alpha-N-acetylgalactosaminidases may recognize not only the glycon but also the aglycon amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

A transmembrane model for an ionotropic glutamate receptor predicted on the basis of the location of asparagine-linked oligosaccharides.

Several different models have been proposed for the transmembrane structure of receptors for the neurotransmitter L-glutamate. In this study, the sites of N-linked oligosaccharides on GluR6, a member of the kainate class of ionotropic glutamate receptors, were examined. Site-directed mutagenesis was utilized to alter the consensus sequence at three potential sites for N-linked glycosylation in the carboxyl-terminal half of the molecule. The presence of a carbohydrate substitution was monitored by shifts in the relative molecular weight of the mutant receptors on immunoblots. Molecular weight shifts were observed for the mutants N515Q and N720Q and for two companion mutants, T517A and T722A, which also eliminate the consensus sequence for N-linked glycosylation. No shift in molecular weight was observed in the the mutant N574Q. These results indicate that asparagines 515 and 720 are glycosylated and thus are likely located extracellularly. In immunocytochemical analyses of GluR6 expressed in baculovirus-infected cells, permeabilization with detergents was required for immunostaining with a carboxyl-terminal antibody, indicating that the carboxyl terminus is located intracellularly. Electrophysiological recordings of the mutant receptors expressed in human embryonic kidney cells demonstrated that the amplitudes of the kainate-activated currents mediated by the N574Q, N720Q, and the T722A mutants were not significantly different from currents mediated by wild type GluR6 receptors, while the currents mediated by the N515Q and T517A mutants were significantly depressed. Based on these findings, we propose a model for the transmembrane topology of GluR6.

A Mucor pusillus mutant defective in asparagine-linked glycosylation.

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

Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development.

Complex asparagine (N)-linked oligosaccharides appear late in phylogeny and are highly regulated in vertebrates. Variations in these structures are found on the majority of cell-surface and secreted proteins. Complex N-linked oligosaccharide biosynthesis is initiated in the Golgi apparatus by the action of Mgat-1-encoded UDP-N-acetylglucosamine:alpha-3-D- mannoside beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI). To determine if these structures govern ontogenic processes in mammals, mouse embryos were generated that lacked a functional Mgat-1 gene. Inactivation of both Mgat-1 alleles produced deficiencies in GlcNAc-TI activity and complex N-linked oligosaccharides. Embryonic lethality occurred by day 10.5, thus establishing that complex N-linked oligosaccharides are required during post-implantation development. Remarkably, embryonic development proceeded into day 9 with the differentiation of multiple cell types. Complex N-linked oligosaccharides are important for morphogenic processes as neural tube formation, vascularization and the determination of left-right body plan asymmetry were impaired in the absence of a functional Mgat-1 gene.

Comparative study of the asparagine-linked oligosaccharide structures of normal and acute-phase rat plasma thiostatin

Rat plasma thiostatin is a 68 kDa glycoprotein with kinin donor and cysteine proteinase inhibitor properties. Thiostatin is an acute-phase plasma protein (APPP) with dramatically elevated plasma levels in response to inflammatory stimuli. APPPs have been shown to possess altered glycan structures in inflammation. This study compares the carbohydrate structure of normal thiostatin with that expressed during the acute-phase response. Thiostatin from both normal and acute-phase plasma was purified by carboxymethyl-papain Sepharose 4B affinity chromatography. Sugar composition analysis by gas chromatography and the Warren method yielded similar mean values for both proteins on a mole sugar per mole protein basis (normal/acute phase): fucose, 2.4/1.7; mannose, 7.5/8.0; galactose, 11.2/10.6; and sialic acid, 14.2/13.0. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot identified a homogeneous 68–70 kDa molecular species for normal and acute-phase thiostatin. Inter-sugar linkage analysis was carried out for permethylated oligosaccharides released by hydrazinolysis. Gas chromatography yielded the following partially methylated alditol acetates relative to 1.0 mole of 1,3,6-tri-O-linked mannose (mean normal/mean acute phase): galactose: 1,3-di-O-, 1.44/1.01; 1,6-di-O-, 1.02/0.68; mannose: 1,2-di-O-, 1.64/1.42; 1,2,4-tri-O-, 0.24/0.13; 1,3,6-tri-O-, 1.0/1.0; 2-deoxy-2-N-methylacetamidoglucose: 1,4-di-O-, 1.42/1.12. These analytical studies indicated that corresponding carbohydrate structures are present in normal and acute-phase thiostatin. Crossed affinoimmunoelectrophoresis (CAIE) further confirmed the structural similarity between the glycan moieties.

The Saccharomyces cerevisiae oligosaccharyltransferase is a protein complex composed of Wbp1p, Swp1p, and four additional polypeptides.

Asparagine-linked glycosylation of proteins in the lumen of the endoplasmic reticulum is catalyzed by the oligosaccharyltransferase. Previously, the mammalian oligosaccharyltransferase was shown to co-purify with a protein complex consisting of three integral membrane proteins: ribophorin I and ribophorin II and a nonglycosylated 48-kDa polypeptide designated OST48. Here, we describe the purification of the oligosaccharyltransferase from Saccharomyces cerevisiae. The yeast oligosaccharyltransferase complex is composed of six subunits (alpha, beta, gamma, delta, epsilon, and zeta). The alpha subunit of the yeast oligosaccharyltransferase complex is a heterogeneously glycosylated protein with three glycoforms of 64, 62, and 60 kDa that contain, respectively, four, three, and two asparagine-linked oligosaccharide chains. The beta and delta subunits were shown to correspond to the 45-kDa Wbp1 glycoprotein and the 30-kDa Swp1 protein, respectively. The Wbp1 and Swp1 proteins were previously shown to be essential for asparagine-linked glycosylation in vivo. The nonglycosylated gamma, epsilon, and zeta subunits have apparent molecular masses of 34, 16, and 9 kDa. Homology between the yeast and mammalian oligosaccharyltransferase complexes first became evident when the 48-kDa subunit of the mammalian enzyme was found to be 25% identical in sequence with the Wbp1 protein. Here we present an alignment between the Swp1 protein and the carboxyl-terminal half of human ribophorin II that reveals that these two proteins are related gene products.

Isolation and expression of murine and rabbit cDNAs encoding an alpha 1,2-mannosidase involved in the processing of asparagine-linked oligosaccharides

We have isolated a full-length cDNA clone encoding a murine alpha 1,2-mannosidase involved in the processing of mammalian Asn-linked oligosaccharides. Oligonucleotide primers were designed based on peptide sequences derived from the purified rabbit liver enzyme and were used to generate a 1011-base pair probe using the polymerase chain reaction. This probe was used to isolate clones from rabbit and mouse cDNA libraries. The full-length murine cDNA clone encodes a 655-amino acid type II transmembrane protein with a 43-amino acid cytoplasmic tail, a single transmembrane domain, and a large COOH-terminal catalytic domain containing two potential N-glycosylation sites. Stable transfection of the murine alpha 1,2-mannosidase cDNA into mouse L cells resulted in a approximately 22-fold overexpression of alpha 1,2-mannosidase activity. Three transcripts were detected in rabbit tissues, whereas two were found in rat and mouse tissues. The sequences of the rabbit and mouse cDNA clones indicate that the multiple transcripts differ in the length of their 3' sequences as a result of the use of multiple polyadenylation signals. Immunolocalization detected cross-reactive material in a juxtanuclear pattern consistent with the Golgi complex. The catalytic portion of the murine alpha 1,2-mannosidase was found to bear a strong similarity to the processing alpha 1,2-mannosidase from Saccharomyces cerevisiae.

Major Defect of Carbohydrate-Deficient-Glycoprotein Syndrome is Not Found in the Synthesis of Dolichyl Phosphate or N-Acetylglucosaminyl-Pyrophosphoryl-Dolichol

The contents of dolichyl phosphate and UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosamine 1-phosphate transferase (GlcNAc-1-P transferase) activity in fibroblasts from patients with carbohydrate-deficient-glycoprotein (CDG) syndrome were analyzed. The amount of dolichyl phosphate and GlcNAc-1-P transferase activity in CDG syndrome fibroblasts were similar to those in normal fibroblasts, suggesting that CDG syndrome may not be due to a deficiency of a biosynthetic enzyme for dolichol-oligosaccharide intermediates, but to a metabolic error in assembly of asparagine-linked oligosaccharide.

Role of N-glycosylation in the structure and function of the methotrexate membrane transporter from CCRF-CEM human lymphoblastic leukemia cells

The carrier protein for methotrexate and tetrahydrofolate cofactors (GP-MTX) in CCRF-CEM human lymphoblastic leukemia cells is a 117kDa glycoprotein containing both N- and O-linked oligosaccharides (Matherly et al., J Biol Chem 267: 23253–23260, 1992). Tunicamycin, an inhibitor of N-glycosylation, was used to investigate the roles of asparagine-linked oligosaccharides in the structure, intracellular routing, and transport function of GP-MTX. Tunicamycin was growth inhibitory toward CCRF-CEM cells ( ic 50 = 0.80 μ g/mL ) and caused a potent suppression of [ 3H]mannose incorporation into nascent glycoproteins. From 1–3 μg/mL, inhibition of [ 3H]mannose incorporation was 66–87%, exceeding that for [ 35S]methionine incorporation by 2 to 4-fold. Tunicamycin (1 and 2 μg/mL) exposures decreased the median molecular masses of GP-MTX on immunoblots (to 82 and 67 kDa, respectively) and were accompanied by reduced maximal rates of methotrexate uptake (31 and 37%, respectively, of control levels). Conversely, the K t values for methotrexate binding to the transporter were unaffected by tunicamycin treatments. The effects of tunicamycin on methotrexate influx closely correlated with lower levels of immunoreactive GP-MTX in plasma membranes and specific [ 3H]methotrexate binding to intact cells, suggesting that the transport effect was due to decreased numbers of carrier proteins at the membrane surface. The reduced molecular mass values for GP-MTX, which accompanied tunicamycin exposures, were further decreased (to 55 and 50 kDa at 1 and 2 μg/mL, respectively) by digestions with N-glycanase. Hence, despite the large loss of N-glycan from GP-MTX in tunicamycin-treated cells, residual core oligosccharides remained. The sizes of hypoglycosylated GP-MTX following both treatments were similar to that of the functionally homologous methotrexate membrane carrier previously identified in L1210 murine leukemia cells.

Association between calnexin and a secretion-incompetent variant of human alpha 1-antitrypsin.

The naturally occurring nullHong Kong variant of human alpha 1-antitrypsin is truncated at its carboxyl terminus, and is retained and degraded in a pre-Golgi compartment of stably transfected murine hepatoma cells (Le, A., Graham, K. S., and Sifers, R. N. (1990) J. Biol. Chem. 265, 14001-14007). Long-term metabolic radiolabeling with [35S]methionine or [32P]orthophosphate in combination with low stringency immunoprecipitation of the nullHong Kong variant has resulted in the co-precipitation of a radiolabeled 90-kDa protein designated p90. Several criteria, including mobility in SDS-polyacrylamide gel electrophoresis, absence of asparagine-linked oligosaccharides, and immunoreactivity with peptide-specific antiserum, have indicated that co-precipitating p90 is identical to calnexin, a calcium-binding phosphoprotein of the endoplasmic reticulum membrane (Wada, I. W., Rindress, P. H., Ou, W.-J., Doherty, J. J., Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). Finally, results from co-immunoprecipitation analyses and velocity sedimentation experiments have verified that approximately 30% of the retained nullHong Kong variant polypeptides are associated with calnexin in a 1:1 molar ratio and can be dissociated with either deoxycholate or chelation of calcium ions at 37 degrees C. Overall, these findings may extend our current understanding of the molecular pathogenesis of serum alpha 1-antitrypsin deficiency.

Characterization of the high mannose asparagine-linked oligosaccharides synthesized by microfilariae of Dirofilaria immitis.

This report describes the structures of high-mannose-type N-linked oligosaccharides in glycoproteins synthesized by the microfilariae of Dirofilaria immitis. Microfilariae of D. immitis were incubated in vitro in media containing 2-[3H] mannose to allow metabolic radiolabeling of the oligosaccharide moieties of newly synthesized glycoproteins. Glycopeptides were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionation by chromatography on concanavalin A-Sepharose. Thirty eight percent of 2-[3H] mannose incorporated into the microfilariae of D. immitis glycopeptides was recovered in high mannose-type asparagine-linked oligosaccharides which were bound to the immobilized lectin. Upon treatment of 2-[3H] mannose labeled glycopeptides with endo-beta-N-acetylglucosaminidase H, the high mannosetype chains were released and their structures were determined by high performance liquid chromatography and exoglycosidase digestion. The major species of high mannosetype chains synthesized by microfilariae of D. immitis have the composition Man5GlcNAc2, Man6ClcNAc2, Man7GlcNAc2, and Man8GlcNAc2. Structural analyses indicate that these oligosaccharides are similar to high mannose-type chains synthesized by vertebrates.

Characterization of the oligosaccharide structures on a recombinant tissue-type plasminogen activator analog synthesized in chinese hamster ovary cells

The asparagine-linked oligosaccharides on a recombinant tissue-type plasminogen activator analog, lacking the epidermal growth factor-like and kringle 1 domains (FK 2P), synthesized by Chinese hamster ovary cells, have been characterized. In vivo [ 3H]mannose labeled glycopeptides were extracted from SDS-PAGE gels of antibody precipitated FKZP and the major oligosaccharide structures were characterized by serial lectin affinity chromatography, size-exclusion chromatography in combination with exoglycosidase digestions, ion exchange chromatography, HPLC and methylation analysis. 44% of the oligosaccharides on the molecule are triantennary (35% 2,4-branched, 9% 2,6-branched), 34% are biantennary and 20% are tetraantennary complex type, all of which are core-fucosylated. Approximately 1% of the structures are high mannose type, in the form of Man 6GlcNAc 2, Man 7GlcNAc 2 and Man 8GlcNac 2. The remaining 1% of the structures appear to be hybrid type. The complex structures contain varying numbers of sialic acid residues, ranging from non- to fully sialylated. No N-acetylgalactosamine was detected in acid hydrolysates of FK 2P, consistent with a lack of serine/threonine-linked oligosaccharides. Sequence analysis of tryptic peptides prepared from purified FK 2P revealed that only one of the two consensus sites for N-linked glycosylation on the molecule is utilized.

Inhibitory Effects of Sialic Acid- or N-Acetylglucosamine-Specific Lectins on Histamine Release Induced by Compound 48/80, Bradykinin and a Polyethylenimine in Rat Peritoneal Mast Cells

The effects of seven lectins with various sugar-specificities on histamine release from rat peritoneal mast cells induced by non-immunologic stimuli were studied. The non-immunologic stimuli used were three basic secretagogues, compound 48/80, bradykinin and PEI6 (polyethylenimine with a molecular weight of 600). In this study, we observed inhibition of the histamine release by Macckia amurensis mitogen and Solanum tuberosum agglutinin (100 micrograms/ml at 37 degrees C for 10 min), which are specific for sialic acid-alpha 2,3-N-acetyl galactosamine (Sia alpha 2,3GalNAc) and N-acetyl glucosamine (GlcNAc) oligomers, respectively. The effects of Phytolacca americana mitogen and Sambucus sieboldiana agglutinin were different. Three lectins specific for mucin type oligosaccharides inhibited the histamine release induced by compound 48/80 but not that induced by bradykinin or PEI6. Since bradykinin and PEI6 additively enhanced the histamine release induced by compound 48/80, they partially shared the same signalling pathways. Glycoproteins with bisecting GlcNAc and Sia residues, as described previously (Jpn. J. Pharmacol. 57, 79-90, 1991), seemed to be one of the action sites for compound 48/80, bradykinin and PEI6. In addition to the direct activation of the pertussis toxin-sensitive G proteins, we propose another mechanism of non-immunologic stimuli via specific glycoproteins on rat peritoneal mast cells. The apparent sugar residues involved were asparagine-linked oligosaccharides with Sia (especially Sia alpha 2,3Gal), GlcNAc oligomers and/or bisecting GlcNAc.

Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells.

Recombinant human erythropoietin (rHuEPO) is used abundantly in the clinic to stimulate red blood cell growth in anaemic patients. The efficacy of the drug depends strongly on the extent of sialylation of its carbohydrate moiety. Prompted by conflicting literature reports on the issue, we reinvestigated the structures of the intact sialylated carbohydrate chains of rHuEPO expressed in Chinese hamster ovary (CHO) cells. The asparagine-linked oligosaccharides were released from rHuEPO with N-glycanase and fractionated by anion-exchange chromatography. The O-linked oligosaccharides were released under alkaline borohydride conditions. The primary structures of the major sialylated N- and O-type oligosaccharides were identified by 500-MHz 1H-NMR spectroscopy, supported by data from composition analysis, methylation analysis, low- and high-pH anion-exchange chromatography, and fast atom bombardment-mass spectrometry. The most abundant N-linked oligosaccharides in CHO cell-derived rHuEPO were found to be di-antennary, 2,4-branched tri-antennary, 2,6-branched tri-antennary and tetra-antennary chains (in the ratio of 7:6:5:82), with the latter containing between zero and three repeating N-acetyllactosamine units, in well-defined branches. The major (> 95%) di-, tri- and tetra-antennary structures are fully sialylated, i.e. they have two, three and four sialic acid residues, respectively, linked exclusively alpha(2-->3) to galactose residues. The majority (> 95%) of N-linked structures contain alpha(1-->6)-linked fucose at the proximal GlcNAc residue. The O-type mono- and disialyl oligosaccharides were characterized as a linear tri- and a branched tetra-saccharide, respectively.

Novel specificities of Mucor hiemalis endo-beta-N-acetylglucosaminidase acting complex asparagine-linked oligosaccharides.

Mucor hiemalis endo-beta-N-acetylglucosaminidase (Endo-M) was proved to act on complex type biantennary oligosaccharides of glycoproteins by using dansylated asparagine-linked and pyridylaminated oligosaccharides, as the substrate. The enzyme could act on both asialo- and sialo-biantennary oligosaccharides. This is the only endo-beta-N-acetylglucosaminidase known to act on sialo glycans, though their activity for them was weak. The enzyme could liberate complex type biantennary oligosaccharides from native human asialotransferrin, which was ascertained by a combination of the pyridylaminated method and HPLC. The enzyme had substrate specificity for high-mannose type oligosaccharides different from those of the endo-beta-N-acetylglucosaminidases of other microorganisms: ovalbumin glycopeptide-IV was a better substrate for Endo-M than glycopeptide-V. The enzyme could act on complex type triantennary oligosaccharides of dansylated glycopeptide prepared from calf fetuin. The enzyme had various novel specificities in regard to activities on complex type and high-mannose type oligosaccharides in glycoproteins.

14] Synthetic neoglycoconjugates in glycosyltransferase assay and purification

This chapter discusses the use of synthetic neoglycoconjugates for the assay and isolation of N-acetylglucosaminyltransferase V [GnT-V; EC 2.4.1.155, α(1,3(6)-mannosylglycoprotein β-1,6-N-acetylglucosaminyl transferase], an enzyme involved in the branching of asparagine-linked oligosaccharides. Synthetic neoglycoconjugates are useful tools for both the isolation and assay of glycosyltransferase enzymes. The main advantage in using a synthetic approach is that a variety of natural as well as unnatural oligosaccharide structures can be prepared. These will be free of cross reacting acceptors within the detection limits of chemical characterization, typically nuclear magnetic resonance (NMR) spectroscopy, high-performance liquid chromatography (HPLC), or mass spectrometry. In addition, incorporation levels can be controlled and a variety of proteins can be used for conjugation. In the laboratories, the most commonly used protein for conjugate preparation is the readily available bovine serum albumin (BSA).

Processing of asparagine-linked oligosaccharides in insect cells. N-acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells.

The levels of beta 1,2-N-acetylglucosaminyltransferase (GlcNAc-T) I and II activities in cultured cells from Bombyx mori (Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda (Sf-9 and Sf-21) were investigated. Apart from initial experiments with Man alpha-3(Man alpha 1-6)-Man beta 1-O(CH2)8COOH3 and 3H-labelled UDP-GlcNAc as substrates, GlcNAc-T I activity was measured with a non-radioactive HPLC method using pyridylaminated Man3-GlcNAc2 and Man5GlcNAc2 as acceptor oligosaccharides. It was shown by reversed-phase HPLC, exoglycosidase digestion and methylation analysis that the product obtained with Man3GlcNAc2 contained a terminal GlcNAc residue linked beta 1,2 to the alpha 1,3 arm of the acceptor. Compared to the enzyme from the human hepatoma cell line HepG2, insect cell GlcNAc-T I exhibited a much higher preference for the Man5 substrate. The GlcNAc-T I from Mb-0503 cells had apparent Km and Vmax values for pyridylaminated Man3- and Man5GlcNAc2 of 2.15 and 0.21 mM, and of 3.4 and 11.4 nmol/h/mg of cell protein, respectively. When Man5GlcNAc2 was used as the acceptor substrate, the levels of GlcNAc-T I activity in the four insect cell lines ranged between 7.5 and 14.7 nmol/h/mg of cell protein, and thus were comparable to that of HepG2 cells. Evidence is presented for the dependence of lepidopteran fucosyltransferase on the presence of terminal N-acetylglucosamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Intramolecular High-Mannose Chains in the Folding and Assembly of Soybean (Glycine max) Lectin Polypeptides: Studies by the Combined Use of Spectroscopy and Gel-Filtration Size Analysis

It was previously reported [Nagai, K. & Yamaguchi, H. (1993) J. Biochem. 113, 123-125] that intramolecular high-mannose chains are essential for reconstitution of soybean lectin from denatured subunits. To obtain more detailed information on the role of the intramolecular high-mannose chains in the folding and assembly of soybean lectin polypeptides, the effects of asparagine-linked oligosaccharides, Man9GlcNAc2Asn (M9-Asn) and Glc1-3Man9GlcNAc2Asn (GM9-Asn), on the reconstitution of soybean lectin from denatured subunits were examined by comparison with the denaturation features of the lectin with varying concentrations of guanidine hydrochloride. The combined use of spectroscopy and size-analysis by gel filtration revealed that both the folding and assembly of denatured subunit polypeptides were completely prevented in the presence of 300 microM M9-Asn, whereas the same concentration of GM9-Asn only interfered with the polypeptide assembly, exhibiting no significant effect on the polypeptide folding. These results, considered together with those in the previous report, indicate that the sugar branch Man alpha 1-2Man-alpha 1-2Man linked to the 3 position of the beta-mannosyl residue of the high-mannose chains functions in the folding of the subunit polypeptides, and that other branches participate in the subunit assembly.

A structural study of the asparagine-linked oligosaccharide moiety of duck ovomucoid

Asparagine-linked oligosaccharides of duck ovomucoid were released quantitatively from the protein by digestion with glycoamidase A (from almond), the reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated using two different types of high performance liquid chromatography (HPLC) on a reversed phase column and an amide adsorption column. More than sixteen different oligosaccharides were separated and the structures were characterized by a combination of the 2-dimensional sugar mapping technique using HPLC, exoglycosidase digestion, and proton nuclear magnetic resonance measurements (1- and 2-dimensional). Furthermore, the HPLC profile of duck ovomucoid oligosaccharides was compared with previously reported profiles obtained from quail and chicken ovomucoids.