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S Narasimhan

Publications and source records attributed to S Narasimhan.

At least 55 records · Page 3Linked to original sources
PubMed1988

Expression of N-acetylglucosaminyltransferase III in hepatic nodules during rat liver carcinogenesis promoted by orotic acid.

The activity of N-acetylglucosaminyltransferase III, which adds a "bisecting" GlcNAc in beta 1,4 linkage to the beta-linked Man of the core of Asn-linked oligosaccharides (Narasimhan, S. (1982) J. Biol. Chem. 257, 10235-10242), was determined in hepatic nodules of rats initiated by administration of a single dose of carcinogen 1,2-dimethylhydrazine.2HCl (100 mg/kg, intraperitoneal) 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. N-Acetylglucosaminyltransferase III was assayed using glycopeptide GlcNAc beta 1,2Man alpha 1,6(GlcNAc beta 1,2Man alpha 1,3)Man beta 1, 4GlcNAc beta 1,4GlcNAc-Asn as substrate and, as enzyme sources, microsomal membranes of the hepatic nodules, surrounding liver, regenerating liver, and age- and sex-matched control liver. The nodules had significant N-acetylglucosaminyltransferase III activity (0.78-2.18 nmol GlcNAc transferred/h/mg of protein), while the surrounding liver, the regenerating liver (24 h after partial hepatectomy), and the control liver had negligible activity (0.02-0.03 nmol/h/mg of protein). Product isolated from a large scale N-acetylglucosaminyltransferase III incubation with hepatic nodules as enzyme source showed the presence of the bisecting GlcNAc residue by 500 MHz proton NMR spectroscopy. Concomitant with the appearance of N-acetylglucosaminyltransferase III activity in the preneoplastic nodules, the activities of N-acetylglucosaminyltransferase I and II were decreased in these membranes when compared to those from surrounding liver, regenerating liver, and control liver. These results suggest that N-acetylglucosaminyltransferase III is induced at the preneoplastic stage in liver carcinogenesis promoted by orotic acid and are consistent with the reported presence of bisecting GlcNAc residues in the Asn-linked oligosaccharides of rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (Kobata, A., and Yamashita, K. (1984) Pure Appl. Chem. 56, 821-832).

1,2-Dimethylhydrazine↗
PubMed1988

The biosynthesis of highly branched N-glycans: studies on the sequential pathway and functional role of N-acetylglucosaminyltransferases I, II, III, IV, V and VI.

At least 6 N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V and VI) are involved in initiating the synthesis of the various branches found in complex asparagine-linked oligosaccharides (N-glycans), as indicated below: GlcNAc beta 1-6 GlcNAc-T V GlcNAc beta 1-4 GlcNAc-T VI GlcNAc beta 1-2Man alpha 1-6 GlcNAc-T II GlcNAc beta 1-4Man beta 1-4-R GlcNAc T III GlcNAc beta 1-4Man alpha 1-3 GlcNAc-T IV GlcNAc beta 1-2 GlcNAc-T I where R is GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAcAsn-X. HPLC was used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct (I. Brockhausen, J.P. Carver and H. Schachter (1988) Biochem. Cell Biol. 66, 1134-1151). The following sequential rules have been established: GlcNAc-T I must act before GlcNAc-T II, III and IV; GlcNAc-T II, IV and V cannot act after GlcNAc-T III, i.e., on bisected substrates; GlcNAc-T VI can act on both bisected and non-bisected substrates; both Glc-NAc-T I and II must act before GlcNAc-T V and VI; GlcNAc-T V cannot act after GlcNAc-T VI. GlcNAc-T V is the only enzyme among the 6 transferases cited above which can be essayed in the absence of Mn2+. In studies on the possible functional role of N-glycan branching, we have measured GlcNAc-T III in pre-neoplastic rat liver nodules (S. Narasimhan, H. Schachter and S. Rajalakshmi (1988) J. Biol. Chem. 263, 1273-1281). The nodules were initiated by administration of a single dose of carcinogen 1,2-dimethyl-hydrazine.2 HCl 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. The nodules had significant GlcNAc-T III activity (1.2-2.2 nmol/h/mg), whereas the surrounding liver, regenerating liver 24 h after partial hepatectomy and control liver from normal rats had negligible activity (0.02-0.03 nmol/h/mg). These results suggest that GlcNAc-T III is induced at the pre-neoplastic stage in liver carcinogenesis and are consistent with the reported presence of bisecting GlcNAc residues in N-glycans from rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (A. Kobata and K. Yamashita (1984) Pure Appl. Chem. 56, 821-832).

Animals↗
PubMed1988

Displacement of nutritional receptors on cell membrane as an initiation factor in carcinogenesis.

Carcinogenesis is a multi-step process, and initiation is the first step. The cell membrane may hold the key to the entry of carcinogens. According to our hypothesis, the cell membrane may have receptors for nutrients including the essential vitamins, in addition to hormones, immune modulators, prostaglandins and neurotransmitters. In addition, there may be cytoplasmic counter receptors to the above. Carcinogens after prolonged contact with cell membrane may displace nutritional receptors to gain an abnormal foothold on cell membrane before entering the cytoplasm and cell nucleus and activating the oncogenes. If this is proven to be correct, there is a possibility of cancer prevention at the cellular level. A possible experimental approach is also described.

Cell Membrane↗
PubMed1988

Beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase III activity in human B and T lymphocyte lines and in tonsillar B and T lymphocytes.

beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase III (GlcNAc-T III) catalyzes the incorporation of a "bisecting" N-acetylglucosamine (GlcNAc) residue in beta 1-4 linkage to the beta-linked mannose of the core of asparagine linked-protein bound oligosaccharides (N-glycans). The activity of GlcNAc-T III was determined in Triton X-100 extracts of four human Epstein-Barr virus (EBV)-infected B-cell lines, in four T-cell lines originally established from lymphocytes of patients with acute lymphatic leukemia, and in human tonsillar B and T lymphocytes. The four EBV-transformed B-cell lines showed appreciable GlcNAc-T III activities (ranging from 3.4 to 19.0 nmol.h-1.mg protein-1), while the tonsillar resting B lymphocytes had much less activity (0.68 nmol.h-1.mg protein-1). The four T-cell lines and the tonsillar T lymphocytes had negligible GlcNAc-T III activities (ranging from 0.02 to 0.25 nmol.h-1.mg protein-1). Enzyme product was identified by high resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. This is the first demonstration of GlcNAc-T III activity in human lymphocytes. The presence of GlcNAc-T III in B-cell lines correlates with the reported occurrence of bisecting GlcNAc residues in the oligosaccharides of human immunoglobulins G, A1, M, and D, tonsillar class II antigens, and membrane glycoproteins from B lymphocytes. The negligible GlcNAc-T III activity of the four human T-cell lines and of tonsillar T lymphocytes agrees with the reported absence of bisected structures in N-glycans from human T lymphocyte membrane glycoproteins.

Acetylglucosamine↗
PubMed1987

Identification of terminal N-acetylglucosamine residues of highly branched asparagine-linked oligosaccharides as immunoreactive domains of a chicken heterophile antigenic determinant.

Chicken heterophile antigenic determinant (CHAD-1) has been previously found in medullary lymphocytes of the bursa and thymus as well as in some non-lymphoid cells by the immunoperoxidase method, using rabbit antiserum to a complete Freund's adjuvant (CFA) as the first antibody. In this work we demonstrated that absorption of anti-CFA serum with highly purified preparations of hen egg white glycoproteins (ovomucoid, ovoinhibitor, ovalbumin) or chicken orosomucoid completely blocked immunoperoxidase staining for CHAD-1. Treatment of these glycoproteins with beta-N-acetylglucosaminidase suppressed their capacity to inhibit this staining. Absorption of anti-CFA serum with asparagine-linked glycopeptides which have the mannose alpha 1,3 arm disubstituted by GlcNAc residues and which have another GlcNAc residue linked beta 1,4 to the beta-linked mannose of the core also inhibited staining for CHAD-1. These data indicated that highly branched asparagine-linked oligosaccharides with terminal GlcNAc residues beta-linked to mannose represent immunoreactive domains of CHAD-1.

Absorption↗
PubMed1987

Analysis of asparagine-linked oligosaccharide structures of chronic lymphocytic leukemia cells.

The asparagine-linked oligosaccharide structures of chronic lymphocytic leukemia cells (CLL) were studied by sequential lectin affinity chromatography. Glycopeptides were isolated from a Concanavalin A (ConA)-binding glycoprotein fraction prepared from a soluble CLL extract. This fraction, which contained 1.8% of the proteins present in the soluble extract, greater than 30 polypeptides revealed by SDS-PAGE analysis, and known cell surface antigens such as HLA-DR and p85 glycoprotein, must include a major proportion of the glycoproteins of the CLL cells. Glycopeptides were prepared by digestion with pronase, were separated into four pools (A-D) by Bio-Gel P-6 filtration and were radiolabeled by N-14C-acetylation. Glycopeptide pools C and D (0.45 less than Kd less than 0.77) were 95-100% bound to ConA-Sepharose and 7-12% bound to Lens culinaris (Lens)-Sepharose, but did not interact with any of the other lectins tested, suggesting major amounts of high mannose structures and minor amounts of fucosylated biantennary complex structures with terminal GlcNAc on the Man alpha 1-6 arm. Structures with greater than 3 branches were suggested for pools A and B (0 less than Kd less than 0.44) which were 34-65% unbound to ConA-Sepharose and 37-40% bound to Lens-Sepharose. Analysis of the ConA-unbound glycopeptides on RCA-Agarose before and after acid hydrolysis indicated variable amounts of terminal galactose and sialic acid residues. Major components of pool A were structures with terminal GlcNAc on all branches (22%) and fully sialylated structures (20%). In pool B, 20% of the radioactivity interacted with L-Phaseolus vulgaris agglutinin (PHA)-Agarose and with Ricinus communis (RCA)-Agarose, indicative of a fully galactosylated triantennary structure with branches at C-2 and C-6 of the Man alpha 1-6 arm. Half of the L-PHA-interacting material also bound to Lens-Sepharose, indicative of a core fucose residue. A fully galactosylated biantennary complex structure in pool A (13%) was identified by weak binding to ConA-Sepharose and strong interaction with RCA-Agarose. The presence of the polylactosamine sequence (Gal beta 1-4GlcNAc beta 1-3)n on this structure was suggested by a sialic acid independent interaction with wheat germ agglutinin (WGA)-Agarose. A sialic acid dependent WGA-interaction was observed in the ConA-unbound glycopeptides of pool A (5%). Some of the structures identified in this study may be associated with the malignant CLL phenotype and/or with a distinct stage of B cell differentiation.

Asparagine↗
PubMed1986

Comparison of the N-linked glycopeptides of DQw1 and DR1 molecules.

HLA class II molecules have been isolated from a [3H]mannose-labeled GM3104 B lymphoblastoid cell line with the phenotype DQw1, DR1. The DQw1 molecules were purified by affinity to 77-34 IgG specifically reactive with the DQw1 specificity. The DR1 molecules were separated into two subsets, DR1a (70 to 80%) and DR1b (20 to 30%), by sequential affinity to 21r5-IgG and 21w4-IgG Sepharose. The alpha- and beta-chains of [3H]mannose-labeled DQw1, DR1a, and DR1b molecules were separated by SDS-PAGE and were recovered by electrophoretic elution. The isolated chains were digested with pronase and the glycopeptides were fractionated by sequential lectin chromatography on immobilized concanavalin A (Con A), Lens culinaris (Lens), and Ricinus communis agglutinin type I (RCA). The N-linked glycopeptides derived from the alpha-chains of DQw1, DR1a, or DR1b showed similar profiles on Con A Sepharose: 45% unbound (ConA I), 25% weakly bound (ConA II), and 30% tightly bound (ConA III). The glycopeptides derived from the beta-chains of DQw1 or DR1 molecules were found almost exclusively (80%) in the fraction unbound to Con A Sepharose, with only 11% and 9% in ConA II and ConA III fractions, respectively. The observation that most of the binding to Con A is associated with the alpha-chain glycopeptides suggests that binding of membrane-associated class II molecules to that lectin must be mediated by the alpha-chains. Binding to Lens Sepharose was higher for beta-(50%) than for alpha-(15%) chain glycopeptides, suggesting that within the intact glycoproteins, the beta-chains are responsible for the interaction with Lens. The ConA I fractions derived from the alpha-chain glycopeptides of either DQw1 or DR1 molecules were separated on RCA-agarose as follows: 60% unbound, 17% retarded, and 20% bound and eluted with 0.1 M galactose. The ConA I fractions derived from the beta-chain glycopeptides of either subset of class II molecules also had a similar profile on RCA-agarose: 70% unbound, 16% retarded, and 10% bound and eluted specifically. After removal of sialic acid residues, all of the ConA I fractions of alpha- and beta-chains bound to RCA-agarose. A high degree of similarity was observed between the corresponding glycopeptides of the three subsets of class II molecules and between the complex N-linked structures of alpha- and beta-chains. Minor variations were observed between DR1a and DR1b glycopeptides which appear greater than those observed between DR1 and DQw1 glycopeptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1986

Structural analysis of the oligosaccharides of DR1 and DQw1 molecules.

The major glycopeptide fractions of the alpha- and beta-chains of HLA-DR1 and DQw1 molecules were isolated on columns of immobilized concanavalin A (Con A), Lens culinaris (Lens), Ricinus communis agglutinin Type I (RCA), and leuko-phytohemagglutinin. Oligosaccharides were prepared from these fractions by enzymatic digestion with Endoglycosidases H or F and were analyzed on Bio-Gel P-6. The glycopeptides tightly bound to Con A (ConA III) were mostly associated with alpha-chains and were resolved as a single oligosaccharide peak (Kd = 0.72) on Bio-Gel P-6 after Endo H digestion. Man-5 is the minimal polymannosyl structure which can be deduced for the ConA III fractions of either DQw1 or DR1 oligosaccharides. The major component of the glycopeptides of the alpha-chains of either DR1 or DQw1 molecules which were weakly bound to Con A (ConA II fraction) did not interact with RCA before or after mild acid hydrolysis or neuraminidase treatment. This component represents a biantennary complex with neither terminal galactose nor sialic acid residues with a minimal structure terminating in N-acetyl glucosamine on the Mannose alpha 1----6 arm, referred to as GnM. The ConA II fractions, which constitute 10% of the total glycopeptides of beta-chains, are associated primarily with fucosylated, sialylated biantennary oligosaccharides not seen on the alpha-chains. The ConA I unbound fractions of either alpha- or beta-chains were mostly bound to RCA after mild acid hydrolysis, suggesting that the minimal structure was a sialylated triantennary structure. The major component associated with the beta-chains was bound to Lens such that a more definite structural assignment can be made, i.e., a triantennary structure with the Mannose on the alpha 1----6 arm substituted at C-2 and C-6. The oligosaccharides of alpha- and beta-chains were resolved as broad peaks on Bio-Gel P-6, suggesting that a mixture of tri- and tetraantennary structures with variable degrees of sialylation and galactosylation were present. The structural differences reported here between oligosaccharides of alpha- and beta-chains of DQw1 and of the two subsets of DR1 molecules could be responsible in part for the differential recognition properties expected of human class II molecules encoded by distinct loci.

Acrylic Resins↗
PubMed1986

The effect of a "bisecting" N-acetylglucosaminyl group on the binding of biantennary, complex oligosaccharides to concanavalin A, Phaseolus vulgaris erythroagglutinin (E-PHA), and Ricinus communis agglutinin (RCA-120) immobilized on agarose.

The effect of a "bisecting" 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, linked (1----4) to the beta-D-mannopyranosyl group of asparagine-linked complex and hybrid oligosaccharides, on the binding of [14C]acetylated glycopeptides to columns of immobilized concanavalin A (Con A), Phaseolus vulgaris erythroagglutinin (E-PHA), and Ricinus communis agglutinin-120 (RCA-120) was investigated. The presence of this "bisecting" GlcNAc group caused significant inhibition of the binding to ConA-agarose of biantennary complex glycopeptides in which the two branches are terminated at their nonreducing ends by two GlcNAc groups, or by a Gal and a GlcNAc group, or by two Gal groups, or by a Man and a GlcNAc group. Binding of biantennary, complex glycopeptides to E-PHA-agarose required a "bisecting" GlcNAc group, a Gal group at the nonreducing terminus of the alpha-D-Man-p-(1----6) branch, and a terminal or internal GlcNAc residue linked beta-(1----2) to the alpha-D-Manp-(1----3) branch. Binding to RCA-120-agarose occurred only when at least one nonreducing terminal Gal group was present, and increased as the proportion of terminal Gal groups increased; the presence of a "bisecting" GlcNAc group caused either enhancement or inhibition of these binding patterns. It is concluded that a "bisecting" GlcNAc group affects the binding of glycopeptides to all three lectin columns.

Acetic Anhydrides↗
PubMed1985

Control of glycoprotein synthesis. Bovine milk UDPgalactose:N-acetylglucosamine beta-4-galactosyltransferase catalyzes the preferential transfer of galactose to the GlcNAc beta 1,2Man alpha 1,3- branch of both bisected and nonbisected complex biantennary asparagine-linked oligosaccharides.

Bovine milk UDPgalactose:N-acetylglucosamine beta-4-galactosyltransferase has been used to investigate the effect of a bisecting GlcNAc residue (linked beta 1,4 to the beta-linked mannose of the trimannosyl core of asparagine-linked complex oligosaccharides) on galactosylation of biantennary complex oligosaccharides. Columns of immobilized lectins (concanavalin A, erythroagglutinating phytohemagglutinin, and Ricinus communis agglutinin 120) were used to separate the various products of the reactions. Preferential galactosylation of the GlcNAc beta 1,2Man alpha 1,3 arm occurred both in the absence and in the presence of a bisecting GlcNAc residue; the ratio of the rates of galactosylation of the Man alpha 1,3 arm to the Man alpha 1,6 arm was 6.5 in the absence of a bisecting GlcNAc and 2.8 in its presence. The bisecting GlcNAc residue reduced galactosylation of the Man alpha 1,3 arm by about 78% probably due to steric hindrance of the GlcNAc beta 1,2Man alpha 1,3 beta 1,4 region of the substrate by the bisecting GlcNAc. This steric hindrance prevents the action of four other enzymes involved in assembly of complex asparagine-linked oligosaccharides and indicates the importance of the bisecting GlcNAc residue in the control of glycoprotein biosynthesis. The Man alpha 1,3 arm of biantennary oligosaccharides is believed to be freely accessible to enzyme action whereas the Man alpha 1,6 arm is believed to be folded back toward the core. This may explain the preferential action of Gal-transferase on the Man alpha 1,3 arm of both bisected and nonbisected oligosaccharides.

Acetylglucosamine↗
PubMed1984

Branch specificity of purified rat liver Golgi UDP-galactose: N-acetylglucosamine beta-1,4-galactosyltransferase. Preferential transfer of of galactose on the GlcNAc beta 1,2-Man alpha 1,3-branch of a complex biantennary Asn-linked oligosaccharide.

In the final stages of the terminal glycosylation of N-linked complex oligosaccharides, UDP-galactose: N-acetylglucosamine beta-1,4-galactosyltransferase (galactosyltransferase) transfers galactose (Gal) onto the N-acetylglucosamine (GlcNAc) residue of each branch of a biantennary oligosaccharide. Purified rat liver Golgi galactosyltransferase was used with GlcNAc beta 1,2-Man alpha 1,6-(GlcNAc beta 1,2-Man alpha 1,3-)-Man beta 1,4-GlcNAc beta 1,4-(Fuc alpha 1,6-)-GlcNAc-Asn in order to determine the sequence of addition of Gal residues to the biantennary oligosaccharide. The different galactosylated products were separated by concanavalin A affinity chromatography and high voltage paper electrophoresis in borate. It was found that Gal was transferred at a much faster rate to the GlcNAc beta 1,2-Man alpha 1,3-branch than to the GlcNAc beta 1,2-Man alpha 1,6-branch, i.e. k1 was at least 5 times larger than k2. Also, k3 was larger than k4, indicating that most of the digalactosylated product "GG" was formed by the sequential addition of Gal to the Man alpha 1,3-branch followed by addition to the Man alpha 1,6-branch. The preferential galactosylation of the GlcNAc beta 1,2-Man alpha 1,3-branch may explain the formation of the asymmetrical oligosaccharides found in bovine and human IgG.

Animals↗
PubMed1983

Control of branching during the biosynthesis of asparagine-linked oligosaccharides.

Many mammalian and avian complex carbohydrates (glycoproteins and glycolipids) have highly branched oligosaccharides. Although the function of complex carbohydrates is not known, there is evidence to suggest that oligosaccharide branching may be an important factor in the process by which cells recognize one another and their environment. Asparagine-linked (N-glycosyl) oligosaccharides can be subdivided into at least 12 classes according to their branching patterns. It is presently believed that these classes all stem from a common precursor oligosaccharide containing three D-glucose, nine D-mannose, and two N-acetyl-D-glucosamine residues. This precursor is incorporated into the protein backbone in the rough endoplasmic reticulum and is then processed within the endoplasmic reticulum and Golgi apparatus by a series of highly specific glycosidases and glycosyltransferases to yield the various classes of N-glycosyl oligosaccharides. The branches that occur in N-glycosyl oligosaccharides are usually initiated by the incorporation of a N-acetylglucosamine (GlcNAc) residue. Our laboratory has studied four of the N-acetylglucosaminyltransferases (GlcNAc-transferases) involved in this initiation process. We have defined various factors which determine the synthetic pathway. There are at least three types of control that are commonly found. (i) Tissues differ in the relative activities of the different glycosyltransferases and glycosidases and, therefore, competition between two or more enzymes for a common intermediate often determines the synthetic route. (ii) The incorporation of a key glycosyl residue into an oligosaccharide may convert a nonsubstrate to a substrate for either a glycosyltransferase or a glycosidase. (iii) Conversely, the incorporation of a key residue may convert a substrate into a nonsubstrate. Other controls are undoubtedly operative during glycoprotein synthesis: e.g., the effect of the polypeptide sequence on transferase specificity, the distribution of transferases along the endomembrane system, and compartmentation and the availability of substrates and cofactors. These factors have not been studied in our laboratory. However, the oligosaccharides made by the hen oviduct correlate quite well with the control factors elucidated by our approach; other tissues are presently under investigation. Recent studies on the three-dimensional structures of N-glycosyl oligosaccharides have enabled us to explain certain features of glycosyltransferase substrate specificity on the basis of steric factors.

Animals↗
PubMed1982

Oligosaccharide branching of glycoproteins: biosynthetic mechanisms and possible biological functions.

One of the most striking features of N- and O-glycosyl oligosaccharides and of lipid-linked oligosaccharides is the high degree of branching of these complex structures. Both proteins and nucleic acids are essentially linear structures and are synthesized by template mechanisms. The branched nature of complex carbohydrates dictates a totally different mechanism of biosynthetic control. Although there are undoubtedly many factors controlling this assembly (e.g. subcellular compartmentation, availability of substrates, cations), our laboratory has studied primarily the enzymatic factors that control the assembly of branched N-glycosyl (Asn-GlcNAc type) and O-glycosyl (Ser[Thr]-GalNAc type) oligosaccharides. There are three basic types of control points that appear to direct biosynthesis. (a) There may be two or more enzymes capable of acting on a single common substrate. Control at this juncture is exerted by the relative activities of these enzymes in a particular tissue. (b) Addition of a specific sugar to the growing oligosaccharide may shut off one or more subsequent enzyme steps, thereby 'freezing' the structure at a certain stage in its synthesis. (c) Progression of the pathway may be impossible until a certain key sugar residue is inserted into the growing oligosaccharide chain. Examples of all three types of control occur in the assembly of both N- and O-glycosyl oligosaccharides. This paper discusses our work on the N-acetylglucosaminyltransferases, which initiate branches in N-glycosyl oligosaccharides, as well as some studies on glycosyltransferases that control the assembly of the four basic Ser(Thr)-GalNAc cores. Important features at all stages of control are the three-dimensional shape of the oligosaccharide, the effect of certain key sugar residues on this three-dimensional shape and the stereochemistry of the interaction of oligosaccharides with proteins. From a functional point of view, protein-oligosaccharide interaction is of vital importance not only to enzyme control mechanisms but to a variety of biological problems such as malignancy and cell-cell interactions, differentiation and development, and susceptibility of cells to hormones, drugs and toxins.

Animals↗
PubMed1982

Control of glycoprotein synthesis. UDP-GlcNAc:glycopeptide beta 4-N-acetylglucosaminyltransferase III, an enzyme in hen oviduct which adds GlcNAc in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-glycosyl oligosaccharides.

Hen oviduct membranes are shown to catalyze the following enzyme reaction: GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn + UDP-GlcNAc leads to GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)GlcNAc beta 1-4)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn + UDP. The enzyme catalyzing this reaction has been named UDP-GlcNAc:glycopeptide beta 4-N-acetylglucosaminyltransferase III (GlcNAc-transferase III) to distinguish it from two other GlcNAc-transferases (I and II) present in hen oviduct and previously described in several mammalian tissues. GlcNAc-transferases I and II, respectively, attach GlcNAc in beta 1-2 linkage to the Man alpha 1-3 and Man alpha 1-6 arms of Asn-linked oligosaccharide cores. A specific assay for GlcNAc-transferase III was devised by using concanavalin A/Sepharose columns to separate the product of transferase III from other interfering radioactive glycopeptides formed in the reaction. The specific activity of GlcNAc-transferase III in hen oviduct membranes is about 5 nmol/mg of protein/h. Substrate specificity studies have shown that GlcNAc-transferase III requires both terminal beta 1-2-linked GlcNAc residues in its substrate for maximal activity. Removal of the GlcNAc residue on the Man alpha 1-6 arm reduces activity by at least 85% and removal of both GlcNAc residues reduces activity by at least 93%. Two large scale preparations of product were subjected to high resolution proton NMR spectroscopy to establish the incorporation by the enzyme of a GlcNAc in beta 1-4 linkage to the beta-linked Man. This GlcNAc residue is called a "bisecting" GlcNAc and appears to play important control functions in the synthesis of complex N-glycosyl oligosaccharides. Several enzymes in the biosynthetic scheme are unable to act on glycopeptide substrates containing a bisecting GlcNAc residue.

Animals↗