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).     

N-acetylglucosaminyltransferase III, IV and V activities in Novikoff ascites tumour cells, mouse lymphoma cells and hen oviduct. Application of a sensitive and specific assay by use of high-performance liquid chromatography

A specific and fast method for the determination of N-acetylglucosaminyltransferase III, IV and V activity in one assay is described. The method is based on the separation by HPLC of the three transferase products formed from the common acceptor oligosaccharide substrate GlcNAc beta 1----2Man alpha 1----3(GlcNAc beta 1----2Man alpha 1---- 6)Man beta 1----4GlcNAc. Assays are not interfered with by substances that result from enzymatic or chemical breakdown of the donor substrate UDP-[14C]GlcNAc. Using this assay system N-acetylglucosaminyltransferase III, IV and V activities were estimated in Novikoff ascites tumour cells, mouse lymphoma BW 5147 cells and hen oviduct.     

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.     

Control of glycoprotein synthesis. Detection and characterization of a novel branching enzyme from hen oviduct, UDP-N-acetylglucosamine:GlcNAc beta 1-6 (GlcNAc beta 1-2)Man alpha-R (GlcNAc to Man) beta-4-N-acetylglucosaminyltransferase VI

Hen oviduct membranes were shown to contain high activity of a novel enzyme, UDP-GlcNac:GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-R (GlcNAc to Man) beta 4-GlcNAc-transferase VI. The enzyme was shown to transfer GlcNAc in beta 1-4 linkage to the D-mannose residue of GlcNAc beta 1-6 (GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or methyl. Radioactive enzyme products were purified by several chromatographic steps, including high performance liquid chromatography, and structures were determined by proton nmr, fast atom bombardment-mass spectrometry, and methylation analysis to be GlcNAc beta 1-6 ([14C]GlcNAc beta 1-4) (GlcNAc beta 1-2) Man alpha-R. The enzyme is stimulated by Triton X-100 and has optimum activity at a relatively high MnCl2 concentration of about 100 mM; Co2+, Mg2+, and Ca2+ could partially substitute for Mn2+. A tissue survey demonstrated high GlcNAc-transferase VI activity in hen oviduct and lower activity in chicken liver and colon, duck colon, and turkey intestine. No activity was found in mammalian tissues. Hen oviduct membranes cannot act on GlcNAc beta 1-6Man alpha-R but have a beta 4-GlcNAc-transferase activity that converts GlcNAc beta 1-2Man alpha-R to GlcNAc beta 1-4(GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or 1-6Man beta methyl. The latter activity is probably due to GlcNAc-transferase IV which preferentially adds GlcNAc in beta 1-4 linkage to the Man alpha 1-3 arm of the GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn core structure of asparagine-linked glycans. The minimum structural requirement for a substrate of beta 4-GlcNAc-transferase VI is therefore the trisaccharide GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-; this trisaccharide is found on the Man alpha 6 arm of many branched complex asparagine-linked oligosaccharides. The data suggest that GlcNAc-transferase VI acts after the synthesis of the GlcNAc beta 1-2Man alpha 1-3-, GlcNAc beta 1-2Man alpha 1-6-, and GlcNAc beta 1-6 Man alpha 1-6-branches by GlcNAc-transferases I, II, and V, respectively, and is responsible for the synthesis of branched oligosaccharides containing the GlcNAc beta 1-6(GlcNAc beta 1-4)(GlcNAc beta 1-2)Man alpha 1-6Man beta moiety.     

Chemical structure of neutral sugar chains isolated from human mature milk kappa-casein

The carbohydrate chains linked to human kappa-casein from mature milk were released by alkaline borohydride treatment as reduced oligosaccharides. The neutral oligosaccharides of lower molecular weight were fractionated and purified by gel filtration and preparative thin layer chromatographies. Seven neutral oligosaccharides (a di- (0.5%), two tetra- (30.5%), two penta- (5.4%) and two hexasaccharide alditols (10.9%] were obtained in homogeneity, and followed by methylation analysis with gas-liquid chromatography-mass spectrometry and by anomer analysis with 13C nuclear magnetic resonance. Their chemical structures were identified to be Gal beta 1----3GalNAc-ol (I), Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (II), Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (III), GlcNAc beta 1----3/6Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (IV), GlcNAc beta 1----3/6Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (V), Fuc alpha 1----4GlcNAc beta 1----3/6Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (VI) and Fuc alpha 1----4GlcNAc beta 1----3/6Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (VII). Five oligosaccharide alditols (III-VII) were the novel carbohydrate chains of kappa-casein from mammalian milk.     

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.     

Control of glycoprotein synthesis

Hen oviduct membranes have been shown to catalyze the transfer of GlcNAc from UDP-GlcNAc to GlcNAc-beta 1-2Man alpha 1-6(GlcNAc beta 1-2 Man alpha 1-3) Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X (GnGn) to form the triantennary structure GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. The enzyme has been named UDP-GlcNAc:GnGn (GlcNAc to Man alpha 1-3) beta 4-N-acetylglucosaminyltransferase IV (GlcNAc-transferase IV) to distinguish it from three other hen oviduct GlcNAc-transferases designated I, II, and III. Since GlcNAc-transferases III and IV both act on the same substrate, concanavalin A/Sepharose was used to separate the products of the two enzymes. At pH 7.0 and at a Triton X-100 concentration of 0.125% (v/v), GlcNAc-transferase IV activity in hen oviduct membranes is 7 nmol/mg of protein/h. The product was characterized by high resolution proton NMR spectroscopy at 360 MHz and by methylation analysis. In addition to triantennary oligosaccharide, hen oviduct membranes produced about 20% of bisected triantennary material, GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3] [GlcNAc beta 1-4]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. Maximal GlcNAc-transferase IV activity requires the presence of both terminal beta 1-2-linked GlcNAc residues in the substrate. Removal of the GlcNAc residue on the Man alpha 1-6 arm or of both GlcNAc residues reduces activity by at least 80%. A Gal beta 1-4GlcNAc disaccharide on the Man alpha 1-6 arm reduces activity by 68% while the presence of this disaccharide on the Man alpha 1-3 arm reduces activity to negligible levels. A similar substrate specificity was found for GlcNAc-transferase III, the enzyme which adds a bisecting GlcNAc in beta 1-4 linkage to the beta-linked Man residue. Since a bisecting GlcNAc was found to prevent GlcNAc-transferase IV action, the bisected triantennary material found in the incubation must have been formed by the sequential action of GlcNAc-transferase IV followed by GlcNAc-transferase III. Activities similar to GlcNAc-transferase IV were also detected in rat liver Golgi-rich membranes (0.4 nmol/mg/h) and pig thyroid microsomes (0.1 nmol/mg/h).     

Structures of sugar chains of human kidney gamma-glutamyltranspeptidase

gamma-Glutamyltranspeptidase purified from human kidneys contains 4-5 asparagine-linked sugar chains in each molecule. The sugar chains were released from the polypeptide portion of the enzyme by hydrazinolysis as oligosaccharides and separated by paper electrophoresis into one neutral and two acidic fractions. By sequential exoglycosidase digestion and methylation analysis, the neutral fraction, which comprised 69% of total oligosaccharides, was shown to be a mixture of bisected bi- and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups in their outer chain moieties. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of bisected triantennary complex-type oligosaccharides with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc group in their outer chain moieties. Some of the outer chains of the acidic oligosaccharides were considered to be sialylated X-antigenic structures.     

The structures of the carbohydrate moieties of mouse kidney gamma-glutamyltranspeptidase: occurrence of X-antigenic determinants and bisecting N-acetylglucosamine residues

Paper electrophoresis and Bio-Gel P-4 column chromatography of the oligosaccharides released from mouse kidney gamma-glutamyltranspeptidase by hydrazinolysis gave fractionation patterns quite distinct from those of the bovine and rat kidney enzymes. Structural studies of the fractionated oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis showed that mouse kidney gamma-glutamyltranspeptidase contains a series of bisected complex-type asparagine-linked sugar chains with the following oligosaccharides as their outer chain moieties: GlcNAc beta 1----, Sia alpha 2----Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, and sialylated N-acetyllactosamine repeating sugar chains. Some of these sugar chains were found for the first time in glycoproteins.     

Enzymatic basis for the structural changes of asparagine-linked sugar chains of membrane glycoproteins of baby hamster kidney cells induced by polyoma transformation

Previous studies indicated that enrichment of the GlcNAc beta 1----6Man alpha 1---- group with concomitant decrease of the GlcNAc beta 1----4Man alpha 1---- group occurs in the complex-type asparagine-linked sugar chains of the membrane glycoproteins of baby hamster kidney cells transformed by polyoma virus. The enzymatic basis of the chemical change is reported in this paper. By using oligosaccharides isolated from the urine of patients with a variety of exoglycosidase deficiencies, beta-N-acetylglucosaminyltransferases in the cell homogenate were successfully assayed separately. Both baby hamster kidney cells and their polyoma transformants contain beta-N-acetylglucosaminyltransferases I, II, IV, V, and VI, but not beta-N-acetylglucosaminyltransferase III. The beta-N-acetylglucosamine residue added by each beta-N-acetylglucosaminyltransferase (GnT) is shown below. (formula see text) Comparative studies of the specific activities of the five beta-N-acetylglucosaminyltransferases in the two cell lines revealed that the value of beta-N-acetylglucosaminyltransferase V in the polyoma transformant was twice of that in the normal cells, while those of the other four transferases in the two cell lines were not significantly different. Therefore the increase in beta-N-acetylglucosaminyltransferase V may be the direct cause of the changes found in the sugar chains of surface glycoproteins in baby hamster kidney cells transformed by polyoma virus.     

Biosynthesis of blood group i-active polylactosaminoglycans. Partial purification and properties of an UDP-GlcNAc:N-acetyllactosaminide beta 1----3-N-acetylglucosaminyltransferase from Novikoff tumor cell ascites fluid

An N-acetylglucosaminyltransferase has been partially purified from Novikoff tumor cell ascites fluid by affinity chromatography on concanavalin A-Sepharose. The enzyme was obtained in a highly concentrated form after lyophilization. The enzyme appeared to be highly specific for acceptor oligosaccharides and glycoproteins carrying a terminal Gal beta 1----4GlcNAc beta 1----R unit. Characterization of products formed by the enzyme in vitro by methylation analysis and 1H NMR spectroscopy revealed that the enzyme catalyzed the formation of a GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-R sequence. The enzyme therefore could be described as an UDP-GlcNAc:Gal beta 1----4GlcNAc beta-R beta 1----3-N-acetylglucosaminyltransferase. Acceptor specificity studies with oligosaccharides that form part of N-glycans revealed that the presence of a Gal beta 1----4GlcNAc beta 1----2(Gal beta 1----4GlcNAc beta 1----6)Man pentasaccharide in the acceptor structure is a requirement for optimal activity. Studies on the branch specificity of the enzyme showed that the branches of this pentasaccharide structure, when contained in tri- and tetraantennary oligosaccharides, are highly preferred over other branches for attachment of the 1st and 2nd mol of GlcNAc into the acceptor molecule. The enzyme also showed activity toward oligosaccharides related to blood group I- and i-active polylactosaminoglycans. In addition the enzyme together with calf thymus UDP-Gal:GlcNAc beta-R beta 1----4-galactosyltransferase was capable of catalyzing the synthesis of a series of oligomers of N-acetyllactosamine. Competition studies revealed that all acceptors were acted upon by a single enzyme species. It is concluded that the N-acetylglucosaminyltransferase functions in both the initiation and the elongation of polylactosaminoglycan chains of N-glycoproteins and possibly other glycoconjugates.     

Systematic fractionation of oligosaccharides of human immunoglobulin G by serial affinity chromatography on immobilized lectin columns

Human immunoglobulin G is known to contain 16 different biantennary complex-type asparagine-linked sugar chains, each of which occurs in a nonsialylated, monosialylated, or disialylated form. These oligosaccharides can be separated into 14 fractions by sequential affinity chromatography with Aleuria aurantia lectin (AAL)-Sepharose, RCA120-WG003, and E4-phytohemagglutinin-agarose columns. Twelve of them were found to contain a single oligosaccharide, while the fraction which passed through all three columns was shown to contain two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT. The fraction, which bound to the AAL-Sepharose column and passed through the remaining two lectin columns, also contained two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (Fuc alpha 1----6)GlcNAcOT. These results indicated that serial affinity chromatography with the three lectin columns can be used effectively to detect changes in the sugar chains of IgG resulting from diseases such as rheumatoid arthritis.     

Comparative study of the oligosaccharides released from baby hamster kidney cells and their polyoma transformant by hydrazinolysis

The asparagine-linked sugar chains of the membrane of baby hamster kidney cells and their polyoma transformant were quantitatively released as oligosaccharides by hydrazinolysis and labeled by NaB3H4 reduction. The radioactive oligosaccharides thus obtained were fractionated by paper electrophoresis. The neutral oligosaccharides of both cells were exclusively of high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6 (Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (+/- Fuc alpha 1----6) GlcNAc as their cores and Gal beta 1----4 GlcNAc and various lengths of Gal beta 1----4 GlcNAc repeating chains in their outer-chain moieties. Prominent features of these acidic oligosaccharides are that all sialic acid residues were N-acetylneuraminic acid and were linked exclusively at C-3 of the nonreducing terminal galactose residues of the outer chains. Comparative study of oligosaccharides of the two cells by Bio-Gel P-4 column chromatography revealed that transformation of baby hamster kidney cells leads to a reduction in high mannose-type oligosaccharides and an increase in tetraantennary oligosaccharides. Increase of the outer chains linked at C-6 of the Man alpha 1----6 residue of the core is the cause of increase in the relative amount of highly branched oligosaccharides in the polyoma transformant.     

Purification and characterization of UDP-GlcNAc: GlcNAcbeta 1-6(GlcNAcbeta 1-2)Manalpha 1-R [GlcNAc to Man]-beta 1, 4-N-acetylglucosaminyltransferase VI from hen oviduct

A new beta1,4-N-acetylglucosaminyltransferase (GnT) responsible for the formation of branched N-linked complex-type sugar chains has been purified 64,000-fold in 16% yield from a homogenate of hen oviduct by column chromatography procedures using Q-Sepharose FF, Ni(2+)-chelating Sepharose FF, and UDP-hexanolamine-agarose. This enzyme catalyzes the transfer of GlcNAc from UDP-GlcNAc to tetraantennary oligosaccharide and produces pentaantennary oligosaccharide with the beta1-4-linked GlcNAc residue on the Manalpha1-6 arm. It requires a divalent cation such as Mn(2+) and has an apparent molecular weight of 72,000 under nonreducing conditions. The enzyme does not act on biantennary oligosaccharide (GnT I and II product), and beta1,6-N-acetylglucosaminylation of the Manalpha1-6 arm (GnT V product) is essential for its activity. This clearly distinguishes it from GnT IV, which is known to generate a beta1-4-linked GlcNAc residue only on the Manalpha1-3 arm. Based on these findings, we conclude that this enzyme is UDP-GlcNAc:GlcNAcbeta1-6(GlcNAcbeta1-2)Manalpha1-R [GlcNAc to Man]-beta1,4-N-acetylglucosaminyltransferase VI. This is the only known enzyme that has not been previously purified among GnTs responsible for antenna formation on the cores of N-linked complex-type sugar chains.     

Detection of bisected biantennary form in the asparagine-linked oligosaccharides of fibronectin isolated from human term amniotic fluid

Fibronectin purified from human term amniotic fluid contains 10 asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction and fractionated by anion-exchange column chromatography and serial lectin affinity chromatography. The structures of these sugar chains were determined by sequential exoglycosidase digestion in combination with methylation analysis. The results indicated that they are a mixture of bisected and non-bisected bi- and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Gal beta 1----4GlcNAc beta 1----, GlcNAc beta 1----, Neu5Ac alpha 2----3Gal beta 1----4GlcNAc beta 1----, and Neu5Ac alpha 2----6Gal beta 1----4GlcNAc beta 1---- groups in their outer chain moieties.     

Complex N-glycans: the story of the “yellow brick road”

The synthesis of complex asparagine-linked glycans (N-glycans) involves a multi-step process that starts with a five mannose N-glycan structure: [Manα1-6(Manα1-3)Manα1-6][Manα1-3]-R where R?=?Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-protein. N-acetylglucosaminyltransferase I (GlcNAc-TI) first catalyzes addition of GlcNAc in β1-2 linkage to the Manα1-3-R terminus of the five-mannose structure. Mannosidase II then removes two Man residues exposing the Manα1-6 terminus that serves as a substrate for GlcNAc-T II and addition of a second GlcNAcβ1-2 residue. The resulting structure is the complex N-glycan: GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)-R. This structure is the precursor to a large assortment of branched complex N-glycans involving four more N-acetylglucosaminyltransferases. This short review describes the experiments (done in the early 1970s) that led to the discovery of GlcNAc-TI and II.     

Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma

The sugar chains of transferrin samples, purified from sera of patients with hepatocellular carcinoma and of healthy individuals, were released quantitatively as radioactive oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Comparative study of their structure by serial lectin column chromatography, by Bio-Gel P-4 column chromatography, and by sequential exoglycosidase digestion revealed that prominently altered glycosylation is commonly found in the hepatoma transferrins, although they all contain two complex-type asparagine-linked sugar chains in one molecule like in the case of normal transferrins. The alteration is quite various, including the increase of highly branched sugar chains, of those with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc beta 1----groups in their outer chain moieties and of those with a fucosylated trimannosyl core. Many but not all of the hepatoma transferrin samples contained a small amount of a bisected biantennary sugar chain, which was not detected in the normal transferrin samples.     

Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains

Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.     

Occurrence of poly-N-acetyllactosamine synthesis in Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase I, II, III, IV, V and VI cDNAs

Lectin blot analysis of membrane glycoprotein samples from Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase (beta-1,4-GalT) I, II, III, IV, V et VI cDNAs showed that the endogenous N-linked oligosaccharides are galactosylated (Guo et al., Glycobiology (2001), in press). Further analysis revealed that membrane glycoprotein samples from all the gene-transfected cells are also reactive to Lycopersicon esculentum agglutinin (LEA) et Datura stramonium agglutinin (DSA), both of which bind to oligosaccharides with poly-N-acetyllactosamine chains while no lectin reactive protein bands are detected when blots are pretreated with a mixture of diplococcal beta-1,4-galactosidase et jack bean beta-N-acetylhexosaminidase or N-glycanase. Analysis of endo-beta-galactosidase-digestion products revealed the presence of the Gal1-->GlcNAc1-->Gal and/or GlcNAc1-->Gal structures in the gene-transfected cells. When the homogenates of the gene-transfected cells were used as enzyme sources towards oligosaccharides with the GlcNAc beta 1-->(3Gal beta 1-->4GlcNAc)(1-3) structures, human recombinant beta-1,4-GalTs I et II galactosylated these oligosaccharides more effectively than other beta-1,4-GalTs. These results indicate that beta-1,4-GalTs I-VI can synthesize poly-N-acetyllactosamine chains with beta-1,3-N-acetylglucosaminyltransferase.