Search

UDP-GlcNAc : α-3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) plays an essential role in the conversion of oligomannose to complex and hybrid N-glycans. Rabbit GnTI is 447 residues long and has a short four-residue N-terminal cytoplasmic tail, a 25-residue putative signal–anchor hydrophobic domain, a stem region of undetermined length and a large C-terminal catalytic domain, a structure typical of all glycosyltransferases cloned to date. Comparison of the amino acid sequences for human, rabbit, mouse, rat, chicken, frog and Caenorhabditis elegans GnT I was used to obtain a secondary structure prediction for the enzyme which suggested that the location of the junction between the stem and the catalytic domain was at about residue 106. To test this hypothesis, several hybrid constructs containing GnT I with N- and C-terminal truncations fused to a mellitin signal sequence were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcMNPV), Sf 9 insect cells were infected with the recombinant baculovirus and supernatants were assayed for GnT I activity. Removal of 29, 84 and 106 N-terminal amino acids had no effect on GnT I activity; however, removal of a further 14 amino acids resulted in complete loss of activity. Western blot analysis showed strong protein bands for all truncated enzymes except for the construct lacking 120 N-terminal residues indicating proteolysis or defective expression or secretion of this protein. The data indicate that the stem is at least 77 residues long. [ABSTRACT FROM AUTHOR]

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.

The dystrophin glycoprotein complex (DGC) is an assembly of proteins spanning the sarcolemma of skeletal muscle cells. Defects in the DGC appear to play critical roles in several muscular dystrophies due to disruption of basement membrane organization. O -mannosyl oligosaccharides on alpha-dystroglycan, a major extracellular component of the DGC, are essential for normal binding of alpha-dystroglycan to ligands (such as laminin) in the extracellular matrix and subsequent signal transmission to actin in the cytoskeleton of the muscle cell. Muscle-Eye-Brain disease (MEB) and Walker-Warburg Syndrome (WWS) have mutations in genes encoding glycosyltransferases needed for O -mannosyl oligosaccharide synthesis. Myodystrophic myd mice and humans with Fukuyama Congenital Muscular Dystrophy (FCMD), congenital muscular dystrophy due to defective fukutin-related protein (FKRP) and MDC1D have mutations in putative glycosyltransferases. These human congenital muscular dystrophies and the myd mouse are associated with defective glycosylation of alpha-dystroglycan. It is expected other congenital muscular dystrophies will prove to have mutations in genes involved in glycosylation.

UDP-GlcNAc:alpha6-D-mannoside beta1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) is a medial-Golgi resident enzyme that catalyses an essential step in the biosynthetic pathway leading from high mannose to complex N-linked oligosaccharides. Screening a cDNA library from Xenopus laevis ovary with a human GnT II DNA probe resulted in the isolation of two cDNA clones encoding two closely related GnT II isoenzymes, GnT II-A and GnT II-B. Analysis of the corresponding genomic DNAs revealed that the open reading frame of both X. laevis GnT II genes resides within a single exon. The GnT II-A gene was found to be transcriptionally active in all X. laevis tissues tested. In contrast, expression of the GnT II-B gene was detected only in a limited number of tissues. Both GnT II-A and GnT II-B exhibit a type II transmembrane protein topology with a putative N-terminal cytoplasmic tail of 9 amino acids followed by a transmembrane domain of 18 residues, and a C-terminal luminal domain of 405 residues. The two proteins differ at 28 amino acid positions within their luminal regions. Heterologous expression of soluble forms of the enzymes in insect cells showed that GnT II-A and GnT II-B are both catalytically active and exhibit similar specific activities. Both recombinant proteins are modified with N-linked oligosaccharides. N-terminal deletion studies demonstrated that the first 49 amino acid residues are not essential for proper folding and enzymatic activity of X. laevis GnT II.

We have isolated a murine cDNA coding for a beta1,3-N-acetylglucosaminyltransferase enzyme ( beta3GnT). This enzyme is similar in sequence to Drosophila melanogaster Brainiac and to the murine and human beta1,3-galactosyltransferase family of proteins. The mouse beta 3GnT protein is 397 amino acids in length and contains 7 cysteine residues that are conserved in the human orthologue. beta 3GnT is a type II membrane protein localized to the Golgi apparatus. Enzyme assays with recombinant mouse beta 3GnT reveal that it has a preference for acceptors with Gal(beta1-4)Glc(NAc) at the non-reducing termini. Proton NMR analysis of product showed incorporation of GlcNAc in beta1,3 linkage to the terminal Gal of Gal(beta1-4)Glc(beta1-O-benzyl). Northern blot analysis revealed the presence of a single 3.0[emsp4 ]kb transcript in all adult mouse and human organs tested, with highest levels in the kidney, liver, heart and placenta. The beta 3GnT gene is also expressed in a number of tumor cell lines. The human orthologue of beta 3GnT is located on chromosome 2pl5.

This review covers discoveries made over the past 30-35 years that were important to our understanding of the synthetic pathway required for initiation of the antennae or branches on complex N-glycans and O-glycans. The review deals primarily with the author's contributions but the relevant work of other laboratories is also discussed. The focus of the review is almost entirely on the glycosyltransferases involved in the process. The following topics are discussed. (1) The localization of the synthesis of complex N-glycan antennae to the Golgi apparatus. (2) The "evolutionary boundary" at the stage in N-glycan processing where there is a change from oligomannose to complex N-glycans; this switch correlates with the appearance of multicellular organisms. (3) The discovery of the three enzymes which play a key role in this switch, N-acetylglucosaminyltransferases I and II and mannosidase II. (4) The "yellow brick road" which leads from oligomannose to highly branched complex N-glycans with emphasis on the enzymes involved in the process and the factors which control the routes of synthesis. (5) A short discussion of the characteristics of the enzymes involved and of the genes that encode them. (6) The role of complex N-glycans in mammalian and Caenorhabditis elegans development. (7) The crystal structure of N-acetylglucosaminyltransferase I. (8) The discovery of the enzymes which synthesize O-glycan cores 1, 2, 3 and 4 and their elongation.

UDP-GlcNAc:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) is essential for the normal assembly of complex Asn-linked glycans. Northern analysis showed a major transcript at 2.0 kb and a minor band at approximately 2.9 kb in five different human cell lines. The gene (MGAT2) has three AATAAA polyadenylation sites at 68, 688 and 846 bp downstream of the translation stop codon. 3'-RACE (rapid amplification of cDNA ends) using RNA from the human cell line LS-180 indicated that all three sites were utilized for transcription termination. 5'-RACE and RNase protection analyses showed multiple transcription initiation sites at -440 to -489 bp relative to the ATG translation start codon (+1). The data show that the entire GnT II gene is on a single exon. The gene has a CCAAT box at -587 bp but lacks a TATA box and the 5'-untranslated region is GC-rich and contains consensus sequences suggestive of multiple binding sites for Sp1; these properties are typical for housekeeping genes. A series of chimeric constructs containing different lengths of the 5'-untranslated region fused to the chloramphenicol acetyltransferase (CAT) reporter gene were tested in transient transfection experiments using HeLa cells. The CAT activity of the construct containing the longest insert (-1076 bp relative to the ATG start codon) showed a approximately 38-fold increase as compared to that of the control. Removal of the region between -636 and -553 bp caused a dramatic decrease in CAT activity indicating this to be the main promoter region of the gene.

Connective tissue of the freshwater pulmonate Lymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in alpha 1 -2 linkage to terminal Gal of type 3 (Gal beta 1-3GalNAc) acceptors, and in alpha 1-3 linkage to GlcNAc ot type 2 (Gal beta 1-4GlcNAc) acceptors. The alpha 1-2 fucosyltransferase was active with Gal beta 1-3GalNAc beta 1-OCH2CH=CH2 (Km = 12mM, V(max) = 1.3 mUml-1) and Gal beta 1-3GalNAc (km =20 mM, V(max) = 2.1 mUml-1), whereas the alpha 1-3 fucosyltransferase was active with Gal beta 1-4GlcNAc (Km = 23 mM, V(max) = 1.1 mUml-1). The products formed from from Gal beta 1-3GalNAc beta 1-OCH2CH=CH2 and Gal beta 1-4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz 1H-NMR spectroscopy and methylation analysis to be Fucalpha1-2Gal beta 1-3GalNAc beta 1-OCH2CH=CH2 and Gal beta 1-4(Fucalpha1-3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.

UDP-GlcNAc:Man alpha 1-3R beta 1-2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) catalyses the conversion of [Man alpha 1-6(Man alpha 1-3)Man alpha 1-6][Man alpha 1-3]Man beta-O-R to [Man alpha 1-6(Man alpha 1-3)Man alpha 1-6] [GlcNAc beta 1-2Man alpha 1-3]Man beta-O-R (R = 1-4GlcNAc beta 1-4GlcNAc- Asn-X) and thereby controls the conversion of oligomannose to complex and hybrid asparagine-linked glycans (N-glycans). GlcNAc-T I also catalyses the conversion of Man alpha 1-6(Man alpha 1-3)Man beta-O-octyl to Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta-O-octyl. We have therefore tested a series of synthetic analogues of Man"alpha 1-6(Man'alpha 1-3)Man beta-O-octyl as substrates and inhibitors for rat liver GlcNAc-T I. The 2"-deoxy and the 3"-, 4"- and 6"-O-methyl derivatives are all good substrates confirming previous observations that the hydroxyl groups of the Man"alpha 1-6 residue do not play major roles in the binding of substrate to enzyme. In contrasts, all four hydroxyl groups on the Man'alpha 1-3 residue are essential since the corresponding deoxy derivatives either do not bind (2'- and 3'-deoxy) or bind very poorly (4'- and 6'-deoxy) to the enzyme. The 2'- and 3'-O-methyl derivatives also do not bind to the enzyme. However, the 4'-O-methyl derivative is a substrate (KM = 2.6 mM) and the 6'-O-methyl compound is a competitive inhibitor (Ki = 0.76 mM). We have therefore synthesized various 4'- and 6'-O-alkyl derivatives, some with reactive groups attached to an O-pentyl spacer, and tested these compounds as reversible and irreversible inhibitors of GlcNAc-T I. The 6'-O-(5-iodoacetamido-pentyl) compound is a specific time dependent inhibitor of the enzyme. Four other 6'-O-alkyl compounds showed competitive inhibition while the remaining compounds showed little or no binding indicating that the electronic properties of the attached O-pentyl groups influence binding.

UDP-GlcNAc:GlcNAc beta 1-2Man alpha 1-6R (GlcNAc to Man) beta 1,6- N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAc beta 1-6 branch to bi- and triantennary N-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAc beta 1-2Man alpha 1-6Glc/Man beta-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Man alpha 1-3 residue and the 4-hydroxyl of the Man beta- residue of the Man alpha 1-6(Man alpha 1-3)Man beta-R N-glycan core are not essential for catalysis but influence substrate binding. GlcNAc beta 1-2(4,6-di-O-methyl-)Man alpha 1-6Glc beta-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.

Filamentous fungi are capable of secreting relatively large amounts of heterologous recombinant proteins. Recombinant human glycoproteins expressed in this system, however, carry only carbohydrates of the oligomannose type limiting their potential use in humans. One approach to the problem is genetic engineering of the fungal host to permit production of complex and hybrid N-glycans. UDP-GlcNAc:alpha 3-D-mannoside beta- 1,2-N-acetylglucosaminyltransferase I (GnT I) is essential for the conversion of oligomannose to hybrid and complex N-glycans in higher eukaryotic cells. Since GnT I is not produced by fungi, we have introduced into the genome of Aspergillus nidulans the gene encoding full-length rabbit GnT I and demonstrated the expression of GnT I enzyme activity at levels appreciably higher than occurs in most mammalian tissues. All the GnT I activity in the Aspergillus transformants remains intracellular suggesting that the rabbit trans-membrane sequence may be capable of targeting GnT I to the fungal Golgi apparatus.

UDP-GlcNAc:Man alpha 1-6R beta(1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complex N-glycans. We have tested a series of synthetic analogues of the substrate Man"'alpha 1-6(GlcNAc"beta 1-2Man'alpha 1-3)Man beta-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attaches N-acetylglucosamine in beta 1-2 linkage to the 2"'-OH of the Man"'alpha 1-6 residue. The 2"'-deoxy analogue is a competitive inhibitor (Ki = 0.13 mM). The 2"'-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3"'-, 4"'- and 6"'-OH groups are not essential for binding or catalysis since the 3"'-, 4"'- and 6"'-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3"'-position to pentyl and substituted pentyl groups causes competitive inhibition (Ki = 1.0-2.5 mM). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3"'-O-(4,4-azo)pentyl group and a 3"'-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man"'alpha 1-6 residue are essential for binding although the 2"'- and 3"'-OH face the catalytic site of the enzyme. The 4-OH group of the Man beta-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind.(ABSTRACT TRUNCATED AT 250 WORDS)

UDP-GlcNAc: Man alpha 3R beta 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M(r) 42,000). The Vmax for the pure enzyme with [Man alpha 6(Man alpha 3)Man alpha 6](Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc beta-Asn as substrate was 4.6 mumol min-1 mg-1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds an N-acetylglucosamine (GlcNAc) residue in beta 1-2 linkage to the Man alpha 3Man beta-terminus of the substrate. Several derivatives of Man alpha 6(Man alpha 3)Man beta-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the beta-linked mannose of Man alpha 6(Man alpha 3)Man beta-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the alpha 3-linked mannose (Man) of the substrate increases the KM 20-fold. Modifications on the alpha 6-linked mannose or on the core structure affect mainly the KM and to a lesser degree the Vmax, e.g., substitutions of the Man alpha 6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower the KM, whereas various other substitutions at the 3-position increase the KM slightly. Man alpha 6(Man alpha 3)4-O-methyl-Man beta 4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.