UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II in Caenorhabditis elegans

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I) and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II) are key enzymes in the synthesis of Asn-linked hybrid and complex glycans. We have cloned cDNAs from Caenorhabditis elegans for three genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14) and one gene homologous to mammalian GnT II. All four cDNAs encode proteins which have the domain structure typical of previously cloned Golgi-type glycosyltransferases and show enzymatic activity (GnT I and GnT II, respectively) on expression in transgenic worms. We have isolated worm mutants lacking the three GnT I genes by the method of ultraviolet irradiation in the presence of trimethylpsoralen (TMP); null mutants for GnT II have not yet been obtained. The gly-12 and gly-14 mutants as well as the gly-14;gly-12 double mutant displayed wild-type phenotypes indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. This finding and other data indicate that the GLY-13 protein is the major functional GnT I in C. elegans. The mutation lacking the gly-13 gene is partially lethal and the few survivors display severe morphological and behavioral defects. We have shown that the observed phenotype co-segregates with the gly-13 deletion in genetic mapping experiments although a second mutation near the gly-13 gene cannot as yet be ruled out. Our data indicate that complex and hybrid N-glycans may play critical roles in the morphogenesis of C. elegans, as they have been shown to do in mice and men.     

Molecular cloning and characterization of human GnT-IX,a novel beta1,6-N-acetylglucosaminyltransferase that is specifically expressed in the brain

A novel beta1,6-N-acetylglucosaminyltransferase (beta1, 6GnT) cDNA was identified by a BLAST search using the amino acid sequence of human GnT-V as a query. The full-length sequence was determined by a combination of 5'-rapid amplification of cDNA end analysis and a further data base search. The open reading frame encodes a 792 amino acid protein with a type II membrane protein structure typical of glycosyltransferases. The entire sequence identity to human GnT-V is 42%. When pyridylaminated (PA) agalacto biantennary N-linked oligosaccharide was used as an acceptor substrate, the recombinant enzyme generated a novel product other than the expected GnT-V product, (GlcNAcbeta1,2-Manalpha1,3-)[GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,6-]Manbeta1,4-GlcNAcbeta1,4-GlcNAc-PA. This new product was identified as [GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,3-][Glc-NAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,6-]Manbeta1,4-GlcNAcbeta1,4-GlcNAc-PA by mass spectrometry and 1H NMR. Namely, the new GnT (designated as GnT-IX) has beta1,6GnT activity not only to the alpha1,6-linked mannose arm but also to the alpha1,3-linked mannose arm of N-glycan, forming a unique structure that has not been reported to date. Northern blot analysis showed that the GnT-IX gene is exclusively expressed in the brain, whereas the GnT-V gene is expressed ubiquitously. These results suggest that GnT-IX is responsible for the synthesis of a unique oligosaccharide structure in the brain.     

The Saccharomyces cerevisiae alg12delta mutant reveals a role for the middle-arm alpha1,2Man- and upper-arm alpha1,2Manalpha1,6Man- residues of Glc3Man9GlcNAc2-PP-Dol in regulating glycoprotein glycan processing in the endoplasmic reticulum and Golgi apparatus

N-glycosylation in nearly all eukaryotes proceeds in the endoplasmic reticulum (ER) by transfer of the precursor Glc(3)Man(9)GlcNAc(2) from dolichyl pyrophosphate (PP-Dol) to consensus Asn residues in nascent proteins. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide lipid properly, and the alg12 mutant accumulates a Man(7)GlcNAc(2)-PP-Dol intermediate. We show that the Man(7)GlcNAc(2) released from alg12Delta-secreted invertase is Manalpha1,2Manalpha1,2Manalpha1,3(Manalpha1,2Manalpha1,3Manalpha1,6)-Manbeta1,4-GlcNAcbeta1-4GlcNAcalpha/beta, confirming that the Man(7)GlcNAc(2) is the product of the middle-arm terminal alpha1,2-mannoslytransferase encoded by the ALG9 gene. Although the ER glucose addition and trimming events are similar in alg12Delta and wild-type cells, the central-arm alpha1,2-linked Man residue normally removed in the ER by Mns1p persists in the alg12Delta background. This confirms in vivo earlier in vitro experiments showing that the upper-arm Manalpha1,2Manalpha1,6-disaccharide moiety, missing in alg12Delta Man(7)GlcNAc(2), is recognized and required by Mns1p for optimum mannosidase activity. The presence of this Man influences downstream glycan processing by reducing the efficiency of Ochlp, the cis-Golgi alpha1,6-mannosyltransferase responsible for initiating outer-chain mannan synthesis, leading to hypoglycosylation of external invertase and vacuolar protease A.     

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.     

The accumulation of Man(6)GlcNAc(2)-PP-dolichol in the Saccharomyces cerevisiae Deltaalg9 mutant reveals a regulatory role for the Alg3p alpha1,3-Man middle-arm addition in downstream oligosaccharide-lipid and glycoprotein glycan processing

N-Glycans in nearly all eukaryotes are derived by transfer of a precursor Glc(3)Man(9)GlcNAc(2) from dolichol (Dol) to consensus Asn residues in nascent proteins in the endoplasmic reticulum. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide-lipid properly, and the alg9 mutant, accumulates Man(6)GlcNAc(2)-PP-Dol. High-field (1)H NMR and methylation analyses of Man(6)GlcNAc(2) released with peptide-N-glycosidase F from invertase secreted by Deltaalg9 yeast showed its structure to be Manalpha1,2Manalpha1,2Manalpha1, 3(Manalpha1,3Manalpha1,6)-Manbeta1,4GlcNAcbeta1, 4GlcNAcalpha/beta, confirming the addition of the alpha1,3-linked Man to Man(5)GlcNAc(2)-PP-Dol prior to the addition of the final upper-arm alpha1,6-linked Man. This Man(6)GlcNAc(2) is the endoglycosidase H-sensitive product of the Alg3p step. The Deltaalg9 Hex(7-10)GlcNAc(2) elongation intermediates were released from invertase and similarly analyzed. When compared with alg3 sec18 and wild-type core mannans, Deltaalg9 N-glycans reveal a regulatory role for the Alg3p-dependent alpha1,3-linked Man in subsequent oligosaccharide-lipid and glycoprotein glycan maturation. The presence of this Man appears to provide structural information potentiating the downstream action of the endoplasmic reticulum glucosyltransferases Alg6p, Alg8p and Alg10p, glucosidases Gls1p and Gls2p, and the Golgi Och1p outerchain alpha1,6-Man branch-initiating mannosyltransferase.     

N-Acetylglucosaminyltransferase IX acts on the GlcNAc beta 1,2-Man alpha 1-Ser/Thr moiety, forming a 2,6-branched structure in brain O-mannosyl glycan

Mammals contain O-linked mannose residues with 2-mono- and 2,6-di-substitutions by GlcNAc in brain glycoproteins. It has been demonstrated that the transfer of GlcNAc to the 2-OH position of the mannose residue is catalyzed by the enzyme, protein O-mannose beta1,2-N-acetylglucosaminyltransferase (POMGnT1), but the enzymatic basis of the transfer to the 6-OH position is unknown. We recently reported on a brain-specific beta1,6-N-acetylglucosaminyltransferase, GnT-IX, that catalyzes the transfer of GlcNAc to the 6-OH position of the mannose residue of GlcNAcbeta1,2-Manalpha on both the alpha1,3- and alpha1,6-linked mannose arms in the core structure of N-glycan (Inamori, K., Endo, T., Ide, Y., Fujii, S., Gu, J., Honke, K., and Taniguchi, N. (2003) J. Biol. Chem. 278, 43102-43109). Here we examined the issue of whether GnT-IX is able to act on the same sequence of the GlcNAcbeta1,2-Manalpha in O-mannosyl glycan. Using three synthetic Ser-linked mannose-containing saccharides, Manalpha1-Ser, GlcNAcbeta1,2-Manalpha1-Ser, and Galbeta1,4-GlcNAcbeta1,2-Manalpha1-Ser as acceptor substrates, the findings show that (14)C-labeled GlcNAc was incorporated only into GlcNAcbeta1,2-Manalpha1-Ser after separation by thin layer chromatography. To simplify the assay, high performance liquid chromatography was employed, using a fluorescence-labeled acceptor substrate GlcNAcbeta1,2-Manalpha1-Ser-pyridylaminoethylsuccinamyl (PAES). Consistent with the above data, GnT-IX generated a new product which was identified as GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1-Ser-PAES by mass spectrometry and (1)H NMR. Furthermore, incorporation of an additional GlcNAc residue into a synthetic mannosyl peptide Ac-Ala-Ala-Pro-Thr(Man)-Pro-Val-Ala-Ala-Pro-NH(2) by GnT-IX was only observed in the presence of POMGnT1. Collectively, these results strongly suggest that GnT-IX may be a novel beta1,6-N-acetylglucosaminyltransferase that is responsible for the formation of the 2,6-branched structure in the brain O-mannosyl glycan.     

Schizosaccharomyces pombe produces novel Gal0-2Man1-3 O-linked oligosaccharides

Schizosaccharomyces pombe whole-cell glycoproteins, previously depleted of N-linked glycans by sequential treatment with endo-ss-N-acetylglucosaminidase H and peptide-N4-asparagine amidohydrolase F, were ss-eliminated with 0.1 M NaOH/1 M NaBH4 to release the O-linked oligosaccharides. The saccharide-alditols were separated by gel-exclusion chromatography into pools from Hexitol to Hex4Hexitol in size. Analysis of the Hexitol pool indicated Man to be the only sugar linked to Ser or Thr residues. The Hex1Hexitol pool contained two components, Galalpha1,2Man-ol (2A) and Manalpha1, 2Man-ol (2B). The Hex2Hexitol pool contained two components, Galalpha1,2Manalpha1,2Man-ol (3A) and Manalpha1,2Manalpha1,2Man-ol (3B). The two Hex3Hexitol components were Galalpha1,2(Galalpha1, 3)Manalpha1,2Man-ol (4A) and Manalpha1,2(Galalpha1,3)Manalpha1, 2Man-ol (4B). The Hex4Hexitol component was found to be a single isomer with the composition of Galalpha1,2(Galalpha1,3)Manalpha1, 2Manalpha1,2Man-ol (5AB). Surprisingly, galactobiose was not detected in any of these oligosaccharides. The gma12 (T. G. Chappell and G. Warren (1989) J. Cell Biol., 109, 2693-2707) and gth1 (T. G. Chappell personal communication) alpha1, 2-galactosyltransferase-deficient mutants and the gma12/gth1 double mutant S.pombe strains were similarly examined. The results indicated that gma12p is solely responsible for the addition of terminal alpha1,2-linked Gal in compound 2A, while one or both of gma12p and gth1p are required for the alpha1,2-linked Gal in 4A. Both transferases are largely responsible for terminal Gal in isomer 5AB. Neither gma12 nor gth1 had any discernible effect on the structure of the large N-linked galactomannans as determined by 1H NMR spectroscopy. Thus, while gth1p and gma12p appear responsible for adding alpha1,2-linked Gal to terminal Man, neither adds galactose side chains to the N-linked poly alpha1,6-Man outerchain, nor the O-linked branch-forming alpha1,3-linked Gal. Furthermore, the presence of Hexalpha1,2(Galalpha1,3)Manalpha1,2- structures in the O-linked glycans implies the presence of a novel branch-forming alpha1,3-galactosyltransferase in S.pombe.     

Oligosaccharide preferences of beta1,4-galactosyltransferase-I: crystal structures of Met340His mutant of human beta1,4-galactosyltransferase-I with a pentasaccharide and trisaccharides of the N-glycan moiety

beta-1,4-Galactosyltransferase-I (beta4Gal-T1) transfers galactose from UDP-galactose to N-acetylglucosamine (GlcNAc) residues of the branched N-linked oligosaccharide chains of glycoproteins. In an N-linked biantennary oligosaccharide chain, one antenna is attached to the 3-hydroxyl-(1,3-arm), and the other to the 6-hydroxyl-(1,6-arm) group of mannose, which is beta-1,4-linked to an N-linked chitobiose, attached to the aspargine residue of a protein. For a better understanding of the branch specificity of beta4Gal-T1 towards the GlcNAc residues of N-glycans, we have carried out kinetic and crystallographic studies with the wild-type human beta4Gal-T1 (h-beta4Gal-T1) and the mutant Met340His-beta4Gal-T1 (h-M340H-beta4Gal-T1) in complex with a GlcNAc-containing pentasaccharide and several GlcNAc-containing trisaccharides present in N-glycans. The oligosaccharides used were: pentasaccharide GlcNAcbeta1,2-Manalpha1,6 (GlcNAcbeta1,2-Manalpha1,3)Man; the 1,6-arm trisaccharide, GlcNAcbeta1,2-Manalpha1,6-Manbeta-OR (1,2-1,6-arm); the 1,3-arm trisaccharides, GlcNAcbeta1,2-Manalpha1,3-Manbeta-OR (1,2-1,3-arm) and GlcNAcbeta1,4-Manalpha1,3-Manbeta-OR (1,4-1,3-arm); and the trisaccharide GlcNAcbeta1,4-GlcNAcbeta1,4-GlcNAc (chitotriose). With the wild-type h-beta4Gal-T1, the K(m) of 1,2-1,6-arm is approximately tenfold lower than for 1,2-1,3-arm and 1,4-1,3-arm, and 22-fold lower than for chitotriose. Crystal structures of h-M340H-beta4Gal-T1 in complex with the pentasaccharide and various trisaccharides at 1.9-2.0A resolution showed that beta4Gal-T1 is in a closed conformation with the oligosaccharide bound to the enzyme, and the 1,2-1,6-arm trisaccharide makes the maximum number of interactions with the enzyme, which is in concurrence with the lowest K(m) for the trisaccharide. Present studies suggest that beta4Gal-T1 interacts preferentially with the 1,2-1,6-arm trisaccharide rather than with the 1,2-1,3-arm or 1,4-1,3-arm of a bi- or tri-antennary oligosaccharide chain of N-glycan.     

Biosynthesis of lipid-linked oligosaccharides. I. Preparation of lipid-linked oligosaccharide substrates.

In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using porcine pancreatic microsomes, while Glc3Man9(GlcNAc)2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6(Man alpha 1,3)Man alpha 1,6]Man beta 1,4GlcNAc beta 1,4GlcNAc-PP-dolichol; Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6]Man beta 1,4GlcNAc beta 1, 4GlcNac-PP-dolichol; and Man alpha 1,6(Man alpha 1,3)Man alpha 1, 6[Man alpha 1,3]Man beta 1,4GlcNAc-beta 1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described.     

Characterization of the oligosaccharide component of microsomal beta-glucuronidase from rat liver

The oligosaccharides of microsomal beta-glucuronidase were analysed by gel permeation and weak anion exchange chromatography following hydrazine release. N-linked glycans, constituted 80% of the total glycan pool and were mainly of the tri- and biantennary complex type with or without core and arm fucose. The major oligosaccharide, that comprised 30.6% of all the species analysed, was structurally identified by reagent array analysis method and found to be a triantennary complex structure, Galbeta1,4GlcNAcbeta1,2Manalpha1,6(3)(Galbeta1,4GlcNAcbeta1,4(Galbeta1,4GlcNAcbeta1,2) Manalpha1,3(6))Manbeta1,4GlcNAcbeta1,4 GlcNAc. O-Linked glycans comprised 20% of the total glycan pool, the major species being Galbeta1,3GalNAc. All of the N- and O-linked glycans were charged. Most of the negative charge was due to sialic acid (85.0%) with the remainder being phosphate present as phosphomonoesters (7.3%) and phosphodiesters (5%). This is the first report of O-linked carbohydrate chains in microsomal beta-glucuronidase. The presence of O-linked glycans and branched N-linked glycans in a microsomal enzyme, in relation to the current view of glycosyltransferase compartmentalization in the Golgi is discussed.     

Purification and properties of the glycoprotein processing N-acetylglucosaminyltransferase II from plants

The presence of an N-acetylglucosaminyltransferase (GlcNAc-transferase) capable of adding a GlcNAc residue to GlcNAcMan3GlcNAc was demonstrated in mung bean seedlings. This enzyme was purified about 3400-fold by using (diethylaminoethyl)cellulose and phosphocellulose chromatographies and chromatography on Concanavalin A-Sepharose. The transferase was assayed by following the change in the migration of the [3H]mannose-labeled GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc on Bio-Gel P-4, or by incorporation of [3H]GlcNAc from UDP-[3H]GlcNAc into a neutral product, (GlcNAc)2Man3GlcNAc. Thus, the purified enzyme catalyzed the addition of a GlcNAc to that mannose linked in alpha 1,6 linkage to the beta-linked mannose. GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc was an excellent acceptor while Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, Man alpha 1,6(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, and Man alpha 1,6(Man apha 1,3)Man alpha 1,6[GlcNAcMan alpha 1,3]Man beta 1,4GlcNAc were not acceptors. Methylation analysis and enzymatic digestions showed that both terminal GlcNAc residues on (GlcNAc)2Man3GlcNAc were attached to the mannoses in beta 1,2 linkages. The GlcNAc transferase had an almost absolute requirement for divalent cation, with Mn2+ being best at 2-3 mM. Mn2+ could not be replaced by Mg2+ or Ca2+, but Cd2+ showed some activity. The enzyme was also markedly stimulated by the presence of detergent and showed optimum activity at 0.15% Triton X-100. The Km for UDP-GlcNAc was found to be 18 microM and that for GlcNAcMan3GlcNAc about 16 microM.     

The Caenorhabditis elegans gene, gly-2, can rescue the N-acetylglucosaminyltransferase V mutation of Lec4 cells

UDP-N-acetylglucosamine:alpha-6-d-mannoside beta-1,6-N-acetylglucosaminyltransferase V (GlcNAc-TV) is a regulator of polylactosamine-containing N-glycans and is causally involved in T cell regulation and tumor metastasis. The Caenorhabditis elegans genome contains a single orthologous gene, gly-2, that is transcribed and encodes a 669-residue type II membrane protein that is 36.7% identical to mammalian GlcNAc-TV (Mgat-5). Recombinant GLY-2 possessed GlcNAc-TV activity when assayed in vitro, and protein truncations demonstrated that the N-terminal boundary of the catalytic domain is Ile-138. gly-2 complemented the Phaseolus vulgaris leucoagglutinin binding defect of Chinese hamster ovary Lec4 cells, whereas GLY-2(L116R), an equivalent mutation to that which causes the Lec4A phenotype, could not. We conclude that the worm gene is functionally interchangeable with the mammalian form. GlcNAc-TV activity was detected in wild-type animals but not those homozygous for a deletion allele of gly-2. Activity was restored in mutant animals by an extrachromosomal array that encompassed the gly-2 gene. Green fluorescent protein reporter transgenes driven by the gly-2 promoter were expressed by developing embryos from the late comma stage onward, present in a complex subset of neurons in larvae and, in addition, the spermathecal and pharyngeal-intestinal valves and certain vulval cells of adults. However, no overt phenotypes were observed in animals homozygous for deletion alleles of gly-2.     

Concanavalin A binding and endoglycosidase D resistance of beta1,2-xylosylated and alpha 1,3-fucosylated plant and insect oligosaccharides

The binding to concanavalin A (Con A) by pyridylaminated oligosaccharides derived from bromelain (Man alpha 1,6(Xyl beta 1, 2) Man beta 1, 4GlcNAc beta 1, 4(Fuc alpha 1, 3)GlcNAc), horseradish peroxidase (Man alpha 1,6(Man alpha 1, 3) (Xyl beta 1, 2)Man beta 1, 4GlcNAc beta 1,4(Fuc alpha 1, 3) GlcNAc), bee venom phospholipase A2 (Man alpha 1,6Man beta 1,4GlcNAc beta 1,4GlcNAc and Man alpha 1,6(Man alpha 1, 3)Man beta 1,4GlcNAc beta 1, 4 (Fuc alpha 1, 3)GlcNAc) and zucchini ascorbate oxidase (Man alpha 1,6(Man alpha 1, 3) (Xyl beta 1, 2)Man beta 1, 4 GlcNAc beta 1, 4GlcNAc) was compared to the binding by Man3GlcNAc2, Man5GlcNAc2 and the asialo-triantennary complex oligosaccharide from bovine fetuin. While the fetuin oligosaccharide did not bind, bromelain, zucchini, Man2GlcNAc2 and horseradish peroxidase were retarded (in that order). The alpha 1, 3-fucosylated phospholipase, Man3GlcNAc2 and Man5GlcNAc2 structures were eluted with 15 M alpha -methylmannoside. It is concluded that core alpha 1,3-fucosylation has little or no effect on ConA binding while xylosylation decreases affinity for ConA. In a parallel study comparing the endoglycosidase D (Endo D) sensitivities of Man3GlcNAc2, IgG-derived 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, the phospholipase Man alpha 1,6(Man alpha 1, 3)Man beta 1, 4GlcNAc beta 1,4(Fuc alpha 1,3)GlcNAc, and horseradish and zucchini pyridylaminated N-linked oligosaccharides, it was found that only the Man3GlcNAc2 structure was cleaved. The IgG structure was sensitive only when beta -hexosaminidase was also present. Thus, in contrast to core alpha 1,6-fucosylated structures, such as those present in mammals, the presence of core alpha 1,3-fucose, as found in structures from plants and insects, and/or beta 1,2-xylose, as found in plants, causes resistance to Endo D.     

X-ray crystal structures of rabbit N-acetylglucosaminyltransferase I (GnT I) in complex with donor substrate analogues

The Golgi-resident glycosyltransferase, UDP-N-acetyl-d-glucosamine:alpha-3-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I), initiates the conversion of high-mannose oligosaccharides to complex and hybrid structures in the biosynthesis of N-linked glycans. Reported here are the X-ray crystal structures of GnT I in complex with UDP-CH2-GlcNAc (a non-hydrolyzable C-glycosidic phosphonate), UDP-2-deoxy-2-fluoro-glucose, UDP-glucose and UDP. Collectively, these structures provide evidence for the importance of the GlcNAc moiety and its N-acetyl group in donor substrate binding, as well as insight into the role played by the flexible 318-330 loop in substrate binding and product release. In addition, the UDP-CH2-GlcNAc complex reveals a well-defined glycerol molecule poised for nucleophilic attack on the C1 atom of the donor substrate analogue. The position and orientation of this glycerol molecule have allowed us to model the binding of the Manalpha1,3Manbeta1 moiety of the acceptor substrate and, based on the model, to suggest a rationalization for the main determinants of GnT I acceptor specificity.     

Comparison of N-acetylglucosaminyltransferase V activities in Rous sarcoma-transformed baby hamster kidney (RS-BHK) and BHK cells

Recent studies have demonstrated that Rous sarcoma virus-transformed baby hamster kidney (RS-BHK) cells express twofold higher levels of those N-linked oligosaccharides that contain the sequence [GlcNAc-beta(1,6)Man (1,6)] compared to nontransformed parental BHK cells (Pierce and Arango, J. Biol.Chem. 261, 10772 [1986]). We have investigated in RS-BHK and BHK cells the activity of UDP-GlcNAc:alpha-D-mannoside beta(1,6)N-acetylglucosaminyltransferase V, the enzyme that begins the synthesis of the sequence that is increased in the RS-BHK cells. We have measured GnT V activity using UDP-[3H]-GlcNAc and a synthetic oligosaccharide acceptor, GlcNAc beta(1,2)Man alpha(1,6)Man beta-O-(Ch2)8COOCH3, separating the radioactive product by a newly devised reverse-phase chromatographic technique. Assayed under optimal conditions, the specific activity of GnT V is about fourfold higher in RS-BHK sonicates than in BHK sonicates, suggesting that this increase in activity may be the primary mechanism that causes the increase in [GlcNAc beta(1,6)Man] sequences in the RS-BHK cells. The apparent Km values of the enzymes in RS-BHK and BHK cell sonicates for UDP-GlcNAc and the synthetic acceptor are similar, as are the pH optima. These results suggest that the increase in GnT V-specific activity in RS-BHK cells is not caused by the presence in these cells of a GnT V with markedly different kinetic properties.     

Engineering of coordinated up- and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells

Production of O-linked oligosaccharides that interact with selectins to mediate cell-cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required.We report here the partial cloning of the CMP-sialic, acid:Galbeta1,3GalNAcalpha2,3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP-GlcNAc:Galbeta1,3GalNAc-R beta1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed "one-step" transient manipulation of multiple enzyme activities in the O-glycosylation pathway of a previously established CHO cell line already engineered to express alpha1,3-fucosyltransferase VI (alpha1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O-glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field.     

Stereochemistry of a methyl-group rearrangement during the biosynthesis of lanosterol.

1. (3RS,6R)-[6-2H1,6-3H1,6-14C], (3RS,6S)-[6-2H1,6-3H1,6-14C] and (3RS)-[6-3H1,6-14C]mevalonolactones were synthesised from R-[2H1,3H1,2-14C], S-[2H1,3H1,2-14C] and [3h1,2-14C]acetic acids respectively. 2. Each mevalonate was converted into cholesterol by a rat liver preparation. 3. Each cholesterol specimen was converted into androsta-1,4-diene-3,17-dione by incubation with Mycobacterium phlei in the presence of 2,2'.dipyridyl. Each specimen of androsta-1,4-diene-3,17-dione was converted into androsta-1,4-dien-3-one-17-ethylene ketail. 4. The samples of androsta-1,4-dien-3-one-17-ethylene ketal were each converted chemically into oestrones in which the methyl group at C-18 is the only carbon atom that originated from C-6 in mevalonolactone. 5. The oestrone from (3RS)-[6-3H1,6-14C]mevalonolactone was oxidised chemically to acetic acid which was converted into p-bromophenacyl acetate and the 3H/14C ratio was measured. 6. There was no overall loss of tritium from the methyl group of acetic acid, as measured by determining the 3H/14C ratios of the p-bromophenacyl esters, when the synthetic and degradative procedures 1 -- 5 were tested with [3H1,2-14C]acetic acid. 7. The oestrones derived from the 6R and 6S-mevalonolactones were oxidised. The chiralities of the resulting acetates were determined by an established procedure whereby the acetates were converted into 2S-malates which were examined for loss of tritium on equilibration with fumarate hydratase. 8. The oestrone from (3RS,6R)-[6-2H1,6-3H1,6-14C]mevalonate gave acetic acid which was converted into 2S-malate that retained 68.6% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was R. 9. The oestrone from (3RS,6S)-E16-2H1,6-3H1,6-14C]mevalonate was oxidised to acetic acid which was converted into 2S-malate that retained 31.9% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was S. 10. There was no overall change in the configuration of a chiral methyl group between C-6 of mevalonate and C-18 of oestrone. It is cncluded that the intramolecular migration of a chiral methyl group from C-15 in 2,3-oxidosqualene to C-13 in lanosterol is stereospecific and occurs with overall retention of configuration.     

The cytosolic and transmembrane domains of the beta 1,6 N-acetylglucosaminyltransferase (C2GnT) function as a cis to medial/Golgi-targeting determinant

The beta 1,6 N-acetylglucosaminyltransferase (C2GnT) has been recently mapped to the cis/medial-Golgi compartment. To analyze the Golgi-targeting determinants of C2GnT, we constructed various deletion mutants of the enzyme fused to the enhanced green fluorescent protein (EGFP) and localized these proteins by fluorescence microscopy in living cells. We found that the N-terminal peptide encompassing amino acids 1 to 32 represents the minimal Golgi-targeting signal sufficient to localize EGFP to the same compartment as the full-length C2GnT. This peptide makes up the cytoplasmic and the transmembrane domains of the enzyme and was referred to as CTd (cytoplasmic and transmembrane domains). We compared the Golgi-targeting efficiency of the C2GnT-derived CTd with its homologous domains from other glycosyltransferases, including the H-type alpha(1,2)-fucosyltransferase (FucTI), the polypeptide N-acetylgalactosaminyltransferase-I (GalNAcT-I), the alpha(1,3)-fucosyltransferase VII (FucTVII), and the alpha(2,6)-sialyltransferase (ST6Gal-I) and found that the Golgi-targeting determinants of these glycosyltransferases were also composed of their cytosolic and transmembrane domains. To investigate whether the CTd of C2GnT could serve as a cis to medial Golgi-specific signal, we tested its ability to mislocalize two late-Golgi acting glycosyltransferases FucTI and FucTVII. We show that fusing the C2GnT-derived CTd with the catalytic domain of FucTVII resulted in a complete mislocalization of the enzyme to the C2GnT compartment, with a parallel alteration of sialyl-Lewis x synthesis and P-selectin binding. The intracellular distribution and activity of FucTI, however, were not affected. Thus, CTds of either early or late-Golgi acting glycosyltransferases represent the Golgi-targeting domains of these enzymes. In addition, we show that C2GnT-derived CTd can function as a cis/medial Golgi-targeting determinant.     

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.     

Null mutations in Drosophila N-acetylglucosaminyltransferase I produce defects in locomotion and a reduced life span

UDP-GlcNAc:alpha3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (encoded by Mgat1) controls the synthesis of hybrid, complex, and paucimannose N-glycans. Mice make hybrid and complex N-glycans but little or no paucimannose N-glycans. In contrast, Drosophila melanogaster and Caenorhabditis elegans make paucimannose N-glycans but little or no hybrid or complex N-glycans. To determine the functional requirement for beta1,2-N-acetylglucosaminyltransferase I in Drosophila, we generated null mutations by imprecise excision of a nearby transposable element. Extracts from Mgat1(1)/Mgat1(1) null mutants showed no beta1,2-N-acetylglucosaminyltransferase I enzyme activity. Moreover, mass spectrometric analysis of these extracts showed dramatic changes in N-glycans compatible with lack of beta1,2-N-acetylglucosaminyltransferase I activity. Interestingly, Mgat1(1)/Mgat1(1) null mutants are viable but exhibit pronounced defects in adult locomotory activity when compared with Mgat1(1)/CyO-GFP heterozygotes or wild type flies. In addition, in null mutants males are sterile and have a severely reduced mean and maximum life span. Microscopic examination of mutant adult fly brains showed the presence of fused beta lobes. The removal of both maternal and zygotic Mgat1 also gave rise to embryos that no longer express the horseradish peroxidase antigen within the central nervous system. Taken together, the data indicate that beta1,2-N-acetylglucosaminyltransferase I-dependent N-glycans are required for locomotory activity, life span, and brain development in Drosophila.