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.     

Loss-of-function of an N-acetylglucosaminyltransferase,POMGnT1, in muscle-eye-brain disease

Muscle-eye-brain disease (MEB), an autosomal recessive disorder, is characterized by congenital muscular dystrophy, brain malformation, and ocular abnormalities. Previously, we found that MEB is caused by mutations in the gene encoding the protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), which is responsible for the formation of the GlcNAcbeta1-2Man linkage of O-mannosyl glycan. Although 13 mutations have been identified in patients with MEB, only the protein with the most frequently observed splicing site mutation has been studied. This protein was found to have no activity. Here, we expressed the remaining mutant POMGnT1s and found that none of them had any activity. These results clearly demonstrate that MEB is inherited as a loss-of-function of POMGnT1.     

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.     

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.     

A new beta-1,2-N-acetylglucosaminyltransferase that may play a role in the biosynthesis of mammalian O-mannosyl glycans

Recent studies have shown that O-mannosyl glycans are present in several mammalian glycoproteins. Although knowledge on the functional roles of these glycans is accumulating, their biosynthetic pathways are poorly understood. Here we report the identification and initial characterization of a novel enzyme capable of forming GlcNAc beta 1-2Man linkage, namely UDP-N-acetylglucosamine: O-linked mannose beta-1,2-N-acetylglucosaminyltransferase in the microsome fraction of newborn rat brains. The enzyme transfers GlcNAc to beta-linked mannose residues, and the formed linkage was confirmed to be beta 1-2 on the basis of diplococcal beta-N-acetylhexosaminidase susceptibility and by high-pH anion-exchange chromatography. Its activity is linearly dependent on time, protein concentration, and substrate concentration and is enhanced in the presence of manganese ion. Its activity is not due to UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT-I) or UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,2-D-acetylglucosaminyltransferase II (GnT-II), which acts on the early steps of N-glycan biosynthesis, because GnT-I or GnT-II expressed in yeast cells did not show any GlcNAc transfer activity against a synthetic mannosyl peptide. Taken together, the results suggest that the GlcNAc transferase activity described here is relevant to the O-mannosyl glycan pathway in mammals.     

Structural studies of two ovalbumin glycopeptides in relation to the endo-beta-N-acetylglucosaminidase specificity.

Heterogeneities of the two ovalbumin glycopeptides, (Man)5(GlcNAc)2Asn and (Man)6(GlcNAc)2Asn, were revealed by borate paper electrophoresis of oligosaccharide alcohols obtained from the glycopeptides by endo-beta-N-acetylglucosaminidase H digestion and NaB3H4 reduction. The structures of the major components of the oligosaccharides were determined by the combination of methylation analysis, acetolysis, and alpha-mannosidase digestion. Based on the results, the whole structures of the major components of (Man)5(GlcNAc)2Asn and (Man)6(GlcNAc)2Asn were elucidated as Manalpha1 leads to 6[Manalpha1 leads to 3]-Manalpha1 leads to 6[Manalpha1 leads to 3[Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc leads to Asn and Manalpha1 leads to 6[Manalpha1 leads to 3]Manalpha1 leads to 6[Manalpha1 leads to 2Manalpha1 leads to 3]Manbeta1 leads to 4GlcNAcbeta1 leads to GlcNAc leads to Asn, respectively. Since endo-beta-N-acetylglucosamini dase D hydrolyzes (Man)5(GlcNAc)2Asn but not (Man)6(GlcNAc)2Asn, the presence of the unsubstituted alpha-mannosyl residue linked at the C-3 position of the terminal mannose of Manbeta1 leads to 4GlcNAcbeta1 leads to 4 GlcNAcAsn core must be essential for the action of the enzyme.     

Deficiency of alpha-dystroglycan in muscle-eye-brain disease

Alpha-dystroglycan is a component of the dystrophin-glycoprotein-complex, which is the major mechanism of attachment between the cytoskeleton and the extracellular matrix. Muscle-eye-brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities and lissencephaly. We recently found that MEB is caused by mutations in the protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase (POMGnT1) gene. POMGnT1 is a glycosylation enzyme that participates in the synthesis of O-mannosyl glycan, a modification that is rare in mammals but is known to be a laminin-binding ligand of alpha-dystroglycan. Here we report a selective deficiency of alpha-dystroglycan in MEB patients. This finding suggests that alpha-dystroglycan is a potential target of POMGnT1 and that altered glycosylation of alpha-dystroglycan may play a critical role in the pathomechanism of MEB and some forms of muscular dystrophy.     

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.     

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.     

First evidence for occurrence of Galbeta1-3GlcNAcbeta1-4Man unit in N-glycans of insect glycoprotein: beta1-3Gal and beta1-4GlcNAc transferases are involved in N-glycan processing of royal jelly glycoproteins

On a way of structural analysis of total N-glycans linked to glycoproteins in royal jelly (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120 (2000), Kimura, M. et al., Biosci. Biotechnol. Biochem., 66, 1985-1989 (2002)), we found that some complex type N-glycans containing a beta1-3galactose residue occur on the insect glycoproteins. Up to date, it has been considered that naturally occurring insect glycoproteins do not bear the galactose-containing N-glycans, therefore, in this report we describe the structural analysis of the complex type N-glycans of royal jelly glycoproteins.By a combination of endo- and exo-glycosidase digestions, IS-MS analysis, and 1H-NMR spectroscopy, the structures of the beta1-3 galactose-containing N-glycan were identified as the following; GlcNAcbeta1-2Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, Manalpha1-3Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-3)Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc. To our knowledge, this is the first report showing that the Galbeta1-3GlcNAcbeta1-4Man unit occurs in N-glycans of insect glycoproteins, indicating a beta1-3 galactosyl transferase and beta1-4GlcNAc transferase (GNT-IV) are expressed in the honeybee cells.     

Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1.

Muscle-eye-brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities, and lissencephaly. Mammalian O-mannosyl glycosylation is a rare type of protein modification that is observed in a limited number of glycoproteins of brain, nerve, and skeletal muscle. Here we isolated a human cDNA for protein O-mannose beta-1,2-N-acetylglucosaminyltransferase (POMGnT1), which participates in O-mannosyl glycan synthesis. We also identified six independent mutations of the POMGnT1 gene in six patients with MEB. Expression of most frequent mutation revealed a great loss of the enzymatic activity. These findings suggest that interference in O-mannosyl glycosylation is a new pathomechanism for muscular dystrophy as well as neuronal migration disorder.     

Structure-function analysis of human protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1, POMGnT1

Protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) catalyzes the transfer of GlcNAc to O-mannose of glycoproteins. Mutations in the POMGnT1 gene cause a type of congenital muscular dystrophy called muscle-eye-brain disease (MEB). We evaluated several truncated mutants of POMGnT1 to determine the minimal catalytic domain. Deletions of 298 amino acids in the N-terminus and 9 amino acids in the C-terminus did not affect POMGnT1 activity, while larger deletions on either end abolished activity. These data indicate that the minimal catalytic domain is at least 353 amino acids. Single amino acid substitutions in the stem domain of POMGnT1 from MEB patients abolished the activity of the membrane-bound form but not the soluble form. This suggests that the stem domain of the soluble form of POMGnT1 is unnecessary for activity, but that some amino acids play a crucial role in the membrane-bound form.     

Isolation of null alleles of the Caenorhabditis elegans gly-12, gly-13 and gly-14 genes,all of which encode UDP-GlcNAc: alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I activity

UDP-GlcNAc: alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (GnT I) is a Golgi-resident enzyme which transfers a GlcNAc residue in beta1,2 linkage to the Manalpha1,3Manbeta-terminus of (Manalpha1,6(Manalpha1,3)Manalpha1,6)(Manalpha1,3)Manbeta1,4GlcNAcbeta1,4GlcNAc-Asn-protein, thereby initiating the synthesis of hybrid N-glycans. Three Caenorhabditis elegans genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14) have been cloned. All three cDNAs encode proteins with GnT I enzyme activity. We report in this paper the preparation by ultra-violet (UV) light irradiation in the presence of trimethylpsoralen, of mutants lacking either gly-12, gly-13 or gly-14. A double null mutation in the gly-12 and gly-14 genes (gly-14; gly-12) has also been prepared. These mutations are intragene deletions, removing large portions of the GnT I catalytic domain, and are therefore, all molecular nulls. The gly-12 and gly-14 mutants as well as the gly-14; gly-12 double mutant all displayed wild-type phenotypes, indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. In contrast, about 60% of the mutants lacking the gly-13 gene arrested as L1 larvae at 20 degrees C and the remaining 40% homozygous worms grew to adulthood but displayed severe morphological and behavioral defects despite the presence of the other two GnT I genes, gly-12 and gly-14. Attempts to rescue the gly-13 null phenotype with the wild type transgene were not successful. However, lethality co-segregated with the gly-13 deletion within 0.02 map units (mu) in genetic mapping experiments, suggesting that the gly-13 mutation is responsible for the phenotype.     

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.     

Deletion of the glucosidase II gene in Trypanosoma brucei reveals novel N-glycosylation mechanisms in the biosynthesis of variant surface glycoprotein

The trypanosomatids are generally aberrant in their protein N-glycosylation pathways. However, protein N-glycosylation in the African trypanosome Trypanosoma brucei, etiological agent of human African sleeping sickness, is not well understood. Here, we describe the creation of a bloodstream-form T. brucei mutant that is deficient in the endoplasmic reticulum enzyme glucosidase II. Characterization of the variant surface glycoprotein, the main glycoprotein synthesized by the parasite with two N-glycosylation sites, revealed unexpected changes in the N-glycosylation of this molecule. Structural characterization by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical and enzymatic treatments revealed that one of the two glycosylation sites was occupied by conventional oligomannose structures, whereas the other accumulated unusual structures in the form of Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, and Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc. The possibility that these structures might arise from Glc1Man9GlcNAc2 by unusually rapid alpha-mannosidase processing was ruled out using a mixture of alpha-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man9GlcNAc2 and Man5GlcNAc2 to the variant surface glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc3Man9GlcNAc2, the T. brucei UDP-Glc: glycoprotein glucosyltransferase and glucosidase II enzymes can use Man5GlcNAc2 and Glc1Man5GlcNAc2, respectively, as their substrates. The ability to transfer Man5GlcNAc2 structures to N-glycosylation sites destined to become Man(4-3)GlcNAc2 or complex structures may have evolved as a mechanism to conserve dolichol-phosphate-mannose donors for glycosylphosphatidylinositol anchor biosynthesis and points to fundamental differences in the specificities of host and parasite glycosyltransferases that initiate the synthesis of complex N-glycans.     

Tumor antigen occurs in N-glycan of royal jelly glycoproteins: honeybee cells synthesize T-antigen unit in N-glycan moiety

In our previous paper (Kimura, Y., et al., Biosci. Biotechnol. Biochem., 67, 1852-1856, 2003), we found that a complex type N-glycans containing beta1-3 galactose residue occurs on royal jelly glycoproteins. During structural analysis of minor components of royal jelly N-glycans, we found complex type N-glycans bearing both galactose and N-acetylgalactosamine residues. Detailed structural analysis of pyridylaminated oligosaccharide revealed that the newly found N-glycan had a complex type structure harboring a tumor marker (T-antigen) unit: Galbeta1-3GalNAcbeta1-4GlcNAcbeta1-2Manalpha1-6 (Galbeta1-3GalNAcbeta1-4GlcNAcbeta1-2Manalpha1-3) Manbeta1-4GlcNAcbeta1-4GlcNAc. To our knowledge, this may be the first report of the presence of the T-antigen unit in the N-glycan moiety of eucaryotic glycoproteins.     

Poly-N-acetyllactosamine synthesis in branched N-glycans is controlled by complemental branch specificity of I-extension enzyme and beta1,4-galactosyltransferase I.

Poly-N-acetyllactosamine is a unique carbohydrate that can carry various functional oligosaccharides, such as sialyl Lewis X. It has been shown that the amount of poly-N-acetyllactosamine is increased in N-glycans, when they contain Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->4GlcNAcbeta1 -->2)Manalpha1-->6 branched structure. To determine how this increased synthesis of poly-N-acetyllactosamines takes place, the branched acceptor was incubated with a mixture of i-extension enzyme (iGnT) and beta1, 4galactosyltransferase I (beta4Gal-TI). First, N-acetyllactosamine repeats were more readily added to the branched acceptor than the summation of poly-N-acetyllactosamines formed individually on each unbranched acceptor. Surprisingly, poly-N-acetyllactosamine was more efficiently formed on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain than in Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R, due to preferential action of iGnT on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain. On the other hand, galactosylation was much more efficient on beta1,6-linked GlcNAc than beta1,2-linked GlcNAc, preferentially forming Galbeta1-->4GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalph a1-->6Manbeta -->R. Starting with this preformed acceptor, N-acetyllactosamine repeats were added almost equally to Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R and Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chains. Taken together, these results indicate that the complemental branch specificity of iGnT and beta4Gal-TI leads to efficient and equal addition of N-acetyllactosamine repeats on both side chains of GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalpha1-->6Manbet a-->R structure, which is consistent with the structures found in nature. The results also suggest that the addition of Galbeta1-->4GlcNAcbeta1-->6 side chain on Galbeta1-->4GlcNAcbeta1-->2Man-->R side chain converts the acceptor to one that is much more favorable for iGnT and beta4Gal-TI.     

Conversion of brain-specific complex type sugar chains by N-acetyl-beta-D-hexosaminidase B

The N-linked sugar chains, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(Manalpha1++ +-3)Manbeta1-4GlcNAcb eta1-4(Fucalpha1-6)GlcNAc (BA-1) and GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(GlcNAcbeta1 -2Manalpha1-3)Manb eta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-2), were recently found to be linked to membrane proteins of mouse brain in a development-dependent manner [S. Nakakita, S. Natsuka, K. Ikenaka, and S. Hase, J. Biochem. 123, 1164-1168 (1998)]. The GlcNAc residue linked to the Manalpha1-3 branch of BA-2 is lacking in BA-1 and the removal of this GlcNAc residue is not part of the usual biosynthetic pathway for N-linked sugar chains, suggesting the existence of an N-acetyl-beta-D-hexosaminidase. Using pyridylaminated BA-2 (BA-2-PA) as a substrate the activity of this enzyme was found in all four subcellular fractions obtained. The activity was much greater in the cerebrum than in the cerebellum. To further identify the N-acetyl-beta-D-hexosaminidase, BA-1 and BA-2 in brain tissues of Hex gene-disrupted mutant mice were detected and quantified. PA-sugar chains were liberated from the cerebrum and cerebellum of the mutant mice by hydrazinolysis-N-acetylation followed by pyridylamination. PA-sugar chains were separated by anion-exchange HPLC, size-fractionation, and reversed-phase HPLC. Each peak was quantified by measuring the peaks at the elution positions of authentic BA-1-PA and BA-2-PA. BA-2-PA was detected in all the PA-sugar chain fractions prepared from Hexa, Hexb, and both Hexa and Hexb (double knockout) gene-disrupted mice, but BA-1 was not found in the fractions from Hexb gene-disrupted and double knockout mice. These results indicate that N-acetyl-beta-D-hexosaminidase B encoded by the Hexb gene hydrolyzed BA-2 to BA-1.