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Eric P Bennett - One of the best experts on this subject based on the ideXlab platform.
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functional conservation of subfamilies of putative udp n acetylgalactosamine polypeptide n acetylgalactosaminylTransferases in drosophila caenorhabditis elegans and mammals one subfamily composed of l 2 35aa is essential in drosophila
Journal of Biological Chemistry, 2002Co-Authors: Tilo Schwientek, Eric P Bennett, Carlos Flores, John Thacker, Martin Hollmann, Celso A ReisAbstract:Abstract The completed fruit fly genome was found to contain up to 15 putative UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase (GalNAc-transferase) genes. Phylogenetic analysis of the putative catalytic domains of the large GalNAc-transferase enzyme families of Drosophila melanogaster (13 available), Caenorhabditis elegans(9 genes), and mammals (12 genes) indicated that distinct subfamilies of orthologous genes are conserved in each species. In support of this hypothesis, we provide evidence that distinctive functional properties of Drosophila and human GalNAc-transferase isoforms were exhibited by evolutionarily conserved members of two subfamilies (dGalNAc-T1 (l(2)35Aa) and GalNAc-T11;dGalNAc-T2 (CG6394) and GalNAc-T7).dGalNAc-T1 and novel human GalNAc-T11 were shown to encode functional GalNAc-Transferases with the same polypeptide acceptor substrate specificity, and dGalNAc-T2 was shown to encode a GalNAc-transferase with similar GalNAc glycopeptide substrate specificity as GalNAc-T7. Previous data suggested that the putative GalNAc-transferase encoded by l(2)35Aa had a lethal phenotype (Flores, C., and Engels, W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2964–2969), and this was substantiated by sequencing of three lethal alleles l(2)35Aa HG8,l(2)35Aa SF12, and l(2)35Aa SF32. The finding that subfamilies of GalNAc-Transferases with distinct catalytic functions are evolutionarily conserved stresses that GalNAc-transferase isoforms may serve unique biological functions rather than providing functional redundancy, and this is further supported by the lethal phenotype of l(2)35Aa.
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cloning and characterization of a close homologue of human udp n acetyl α d galactosamine polypeptide n acetylgalactosaminyltransferase t3 designated galnac t6 evidence for genetic but not functional redundancy
Journal of Biological Chemistry, 1999Co-Authors: Eric P Bennett, Helle Hassan, Michael A Hollingsworth, G F M Merkx, Ulla Mandel, Naoaki Akisawa, Yoshito Ikematsu, Ad Geurts Van Kessel, Sigvard Olofsson, Henrik ClausenAbstract:The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T3, exhibits unique functions. Specific acceptor substrates are used by GalNAc-T3 and not by other GalNAc-Transferases. The expression pattern of GalNAc-T3 is restricted, and loss of expression is a characteristic feature of poorly differentiated pancreatic tumors. In the present study, a sixth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T6, with high similarity to GalNAc-T3, was characterized. GalNAc-T6 exhibited high sequence similarity to GalNAc-T3 throughout the coding region, in contrast to the limited similarity that exists between homologous glycosyltransferase genes, which is usually restricted to the putative catalytic domain. The genomic organizations of GALNT3 and GALNT6 are identical with the coding regions placed in 10 exons, but the genes are localized differently at 2q31 and 12q13, respectively. Acceptor substrate specificities of GalNAc-T3 and -T6 were similar and different from other GalNAc-Transferases. Northern analysis revealed distinct expression patterns, which were confirmed by immunocytology using monoclonal antibodies. In contrast to GalNAc-T3, GalNAc-T6 was expressed in WI38 fibroblast cells, indicating that GalNAc-T6 represents a candidate for synthesis of oncofetal fibronectin. The results demonstrate the existence of genetic redundancy of a polypeptide GalNAc-transferase that does not provide full functional redundancy.
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localization of three human polypeptide galnac Transferases in hela cells suggests initiation of o linked glycosylation throughout the golgi apparatus
Journal of Cell Science, 1998Co-Authors: Sabine Rottger, Henrik Clausen, Eric P Bennett, Hans H Wandall, Jamie White, Jeanchristophe Olivo, Annika Stark, Caroline A Whitehouse, Eric G Berger, Tommy NilssonAbstract:O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminylTransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-Transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19). However, upon relocation of chimeric GalNAc-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficiencies indicating that all components required for initiation of O-glycosylation are present in the ER except for polypeptide GalNAc-Transferases.
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substrate specificities of three members of the human udp n acetyl α d galactosamine polypeptide n acetylgalactosaminyltransferase family galnac t1 t2 and t3
Journal of Biological Chemistry, 1997Co-Authors: Hans H Wandall, Eric P Bennett, Helle Hassan, Ekaterina Mirgorodskaya, Anne K Kristensen, Peter Roepstorff, Peter A Nielsen, Michael A Hollingsworth, Joy Burchell, Joyce TaylorpapadimitriouAbstract:Abstract Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptideN-acetylgalactosaminylTransferases (GalNAc-Transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-Transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-Transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-Transferases.
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biosynthetic basis of incompatible histo blood group a antigen expression anti a transferase antibodies reactive with gastric cancer tissue of type o individuals
Cancer Research, 1993Co-Authors: Leonor David, Thayer White, Eric P Bennett, Dina Leitao, Manuel Sobrinhosimoes, Ulla Mandel, Erik Dabelsteen, Henrik ClausenAbstract:The expression of incompatible A carbohydrate antigens in some adenocarcinomas may provide an explanation for the generally observed lower incidence of adenocarcinoma among types O and B versus type A individuals. The chemistry and genetic basis of incompatible A expression is largely unknown. Here, we have screened 31 cases of gastric tumors of phenotype O for the expression of blood group A gene-defined glycosyltransferase by immunohistology on frozen sections using newly developed monoclonal antibodies to the Transferases. Three cases were positive, and transferase expression was confirmed by enzyme analysis of extracts from the specimens. Blood group A carbohydrate antigens were also identified immunohistologically in these three cases as well as in five other cases. Thin-layer chromatography immunostaining analysis of glycolipid extracts from the three cases did not confirm the chemical presence of A antigen. The ABO genotype of all patients was found to be OO , showing that all carried O alleles with a structural defect at nucleotide position 261 leading to a shift in the reading frame. The data suggest that incompatible A antigen expression is a result of transferase expression derived from the ABO genes.
Henrik Clausen - One of the best experts on this subject based on the ideXlab platform.
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mucin type o glycosylation and its potential use in drug and vaccine development
Biochimica et Biophysica Acta, 2008Co-Authors: Mads Agervig Tarp, Henrik ClausenAbstract:Mucin-type O-glycans are found on mucins as well as many other glycoproteins. The initiation step in synthesis is catalyzed by a large family of polypeptide GalNAc-Transferases attaching the first carbohydrate residue, GalNAc, to selected serine and threonine residues in proteins. During the last decade an increasing number of GalNAc-transferase isoforms have been cloned and their substrate-specificities partly characterized. These differences in substrate specificities have been exploited for in vitro site-directed O-glycosylation. In GlycoPEGylation, polyehylene glycol (PEG) is transferred to recombinant therapeutics to specific acceptor sites directed by GalNAc-Transferases. GalNAc-Transferases have also been used to control density of glycosylation in the development of glycopeptide-based cancer vaccines. The membrane-associated mucin-1 (MUC1) has long been considered a target for immunotherapeutic and immunodiagnostic measures, since it is highly overexpressed and aberrantly O-glycosylated in most adenocarcinomas, including breast, ovarian, and pancreatic cancers. By using vaccines mimicking the glycosylation pattern of cancer-cells, it is possible to overcome tolerance in transgenic animals expressing the human MUC1 protein as a self-antigen providing important clues for an improved MUC1 vaccine design. The present review will highlight some of the potential applications of site-directed O-glycosylation.
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cloning and characterization of a close homologue of human udp n acetyl α d galactosamine polypeptide n acetylgalactosaminyltransferase t3 designated galnac t6 evidence for genetic but not functional redundancy
Journal of Biological Chemistry, 1999Co-Authors: Eric P Bennett, Helle Hassan, Michael A Hollingsworth, G F M Merkx, Ulla Mandel, Naoaki Akisawa, Yoshito Ikematsu, Ad Geurts Van Kessel, Sigvard Olofsson, Henrik ClausenAbstract:The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T3, exhibits unique functions. Specific acceptor substrates are used by GalNAc-T3 and not by other GalNAc-Transferases. The expression pattern of GalNAc-T3 is restricted, and loss of expression is a characteristic feature of poorly differentiated pancreatic tumors. In the present study, a sixth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T6, with high similarity to GalNAc-T3, was characterized. GalNAc-T6 exhibited high sequence similarity to GalNAc-T3 throughout the coding region, in contrast to the limited similarity that exists between homologous glycosyltransferase genes, which is usually restricted to the putative catalytic domain. The genomic organizations of GALNT3 and GALNT6 are identical with the coding regions placed in 10 exons, but the genes are localized differently at 2q31 and 12q13, respectively. Acceptor substrate specificities of GalNAc-T3 and -T6 were similar and different from other GalNAc-Transferases. Northern analysis revealed distinct expression patterns, which were confirmed by immunocytology using monoclonal antibodies. In contrast to GalNAc-T3, GalNAc-T6 was expressed in WI38 fibroblast cells, indicating that GalNAc-T6 represents a candidate for synthesis of oncofetal fibronectin. The results demonstrate the existence of genetic redundancy of a polypeptide GalNAc-transferase that does not provide full functional redundancy.
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control of o glycan branch formation molecular cloning of human cdna encoding a novel β1 6 n acetylglucosaminyltransferase forming core 2 and core 4
Journal of Biological Chemistry, 1999Co-Authors: Tilo Schwientek, Michael A Hollingsworth, Mitsuharu Nomoto, Steven B Levery, G F M Merkx, Ad Geurts Van Kessel, Eric Paul Bennett, Henrik ClausenAbstract:Abstract A novel human UDP-GlcNAc:Gal/GlcNAcβ1–3GalNAcα β1,6GlcNAc-transferase, designated C2/4GnT, was identified by BLAST analysis of expressed sequence tags. The sequence of C2/4GnT encoded a putative type II transmembrane protein with significant sequence similarity to human C2GnT and IGnT. Expression of the secreted form of C2/4GnT in insect cells showed that the gene product had UDP-N-acetyl-α-d-glucosamine:acceptor β1,6-N-acetylglucosaminyltransferase (β1,6GlcNAc-transferase) activity. Analysis of substrate specificity revealed that the enzyme catalyzed O-glycan branch formation of the core 2 and core 4 type. NMR analyses of the product formed with core 3-para-nitrophenyl confirmed the product core 4-para-nitrophenyl. The coding region of C2/4GnT was contained in a single exon and located to chromosome 15q21.3. Northern analysis revealed a restricted expression pattern of C2/4GnT mainly in colon, kidney, pancreas, and small intestine. No expression of C2/4GnT was detected in brain, heart, liver, ovary, placenta, spleen, thymus, and peripheral blood leukocytes. The expression of core 2O-glycans has been correlated with cell differentiation processes and cancer. The results confirm the predicted existence of a β1,6GlcNAc-transferase that functions in both core 2 and core 4O-glycan branch formation. The redundancy in β1,6GlcNAc-Transferases capable of forming core 2O-glycans is important for understanding the mechanisms leading to specific changes in core 2 branching during cell development and malignant transformation.
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localization of three human polypeptide galnac Transferases in hela cells suggests initiation of o linked glycosylation throughout the golgi apparatus
Journal of Cell Science, 1998Co-Authors: Sabine Rottger, Henrik Clausen, Eric P Bennett, Hans H Wandall, Jamie White, Jeanchristophe Olivo, Annika Stark, Caroline A Whitehouse, Eric G Berger, Tommy NilssonAbstract:O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminylTransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-Transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19). However, upon relocation of chimeric GalNAc-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficiencies indicating that all components required for initiation of O-glycosylation are present in the ER except for polypeptide GalNAc-Transferases.
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biosynthetic basis of incompatible histo blood group a antigen expression anti a transferase antibodies reactive with gastric cancer tissue of type o individuals
Cancer Research, 1993Co-Authors: Leonor David, Thayer White, Eric P Bennett, Dina Leitao, Manuel Sobrinhosimoes, Ulla Mandel, Erik Dabelsteen, Henrik ClausenAbstract:The expression of incompatible A carbohydrate antigens in some adenocarcinomas may provide an explanation for the generally observed lower incidence of adenocarcinoma among types O and B versus type A individuals. The chemistry and genetic basis of incompatible A expression is largely unknown. Here, we have screened 31 cases of gastric tumors of phenotype O for the expression of blood group A gene-defined glycosyltransferase by immunohistology on frozen sections using newly developed monoclonal antibodies to the Transferases. Three cases were positive, and transferase expression was confirmed by enzyme analysis of extracts from the specimens. Blood group A carbohydrate antigens were also identified immunohistologically in these three cases as well as in five other cases. Thin-layer chromatography immunostaining analysis of glycolipid extracts from the three cases did not confirm the chemical presence of A antigen. The ABO genotype of all patients was found to be OO , showing that all carried O alleles with a structural defect at nucleotide position 261 leading to a shift in the reading frame. The data suggest that incompatible A antigen expression is a result of transferase expression derived from the ABO genes.
Celso A Reis - One of the best experts on this subject based on the ideXlab platform.
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functional conservation of subfamilies of putative udp n acetylgalactosamine polypeptide n acetylgalactosaminylTransferases in drosophila caenorhabditis elegans and mammals one subfamily composed of l 2 35aa is essential in drosophila
Journal of Biological Chemistry, 2002Co-Authors: Tilo Schwientek, Eric P Bennett, Carlos Flores, John Thacker, Martin Hollmann, Celso A ReisAbstract:Abstract The completed fruit fly genome was found to contain up to 15 putative UDP-N-acetyl-α-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase (GalNAc-transferase) genes. Phylogenetic analysis of the putative catalytic domains of the large GalNAc-transferase enzyme families of Drosophila melanogaster (13 available), Caenorhabditis elegans(9 genes), and mammals (12 genes) indicated that distinct subfamilies of orthologous genes are conserved in each species. In support of this hypothesis, we provide evidence that distinctive functional properties of Drosophila and human GalNAc-transferase isoforms were exhibited by evolutionarily conserved members of two subfamilies (dGalNAc-T1 (l(2)35Aa) and GalNAc-T11;dGalNAc-T2 (CG6394) and GalNAc-T7).dGalNAc-T1 and novel human GalNAc-T11 were shown to encode functional GalNAc-Transferases with the same polypeptide acceptor substrate specificity, and dGalNAc-T2 was shown to encode a GalNAc-transferase with similar GalNAc glycopeptide substrate specificity as GalNAc-T7. Previous data suggested that the putative GalNAc-transferase encoded by l(2)35Aa had a lethal phenotype (Flores, C., and Engels, W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2964–2969), and this was substantiated by sequencing of three lethal alleles l(2)35Aa HG8,l(2)35Aa SF12, and l(2)35Aa SF32. The finding that subfamilies of GalNAc-Transferases with distinct catalytic functions are evolutionarily conserved stresses that GalNAc-transferase isoforms may serve unique biological functions rather than providing functional redundancy, and this is further supported by the lethal phenotype of l(2)35Aa.
Katrin Henze - One of the best experts on this subject based on the ideXlab platform.
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Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization.
Journal of Biological Chemistry, 2007Co-Authors: Koen W. A. Van Grinsven, Mark van der Giezen, William Martin, Aloysius G M Tielens, Silke Rosnowsky, Susanne W. H. Van Weelden, Simone Pütz, Jaap J. Van Hellemond, Katrin HenzeAbstract:Acetate:succinate CoA-Transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-Transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.
Juan M. Tomás - One of the best experts on this subject based on the ideXlab platform.
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a udp hexnac polyprenol p galnac 1 p transferase wecp representing a new subgroup of the enzyme family
Journal of Bacteriology, 2011Co-Authors: Susana Merino, Raquel Molero, Lamiaa Bouamama, Miguel Regue, Natalia Jimenez, Juan M. TomásAbstract:The Aeromonas hydrophila AH-3 WecP represents a new class of UDP-HexNAc:polyprenol-P HexNAc-1-P Transferases. These enzymes use a membrane-associated polyprenol phosphate acceptor (undecaprenyl phosphate [Und-P]) and a cytoplasmic UDP-d-N-acetylhexosamine sugar nucleotide as the donor substrate. Until now, all the WecA enzymes tested were able to transfer UDP-GlcNAc to the Und-P. In this study, we present in vitro and in vivo proofs that A. hydrophila AH-3 WecP transfers GalNAc to Und-P and is unable to transfer GlcNAc to the same enzyme substrate. The molecular topology of WecP is more similar to that of WbaP (UDP-Gal polyprenol-P transferase) than to that of WecA (UDP-GlcNAc polyprenol-P transferase). WecP is the first UDP-HexNAc:polyprenol-P GalNAc-1-P transferase described.
