RESUMEN

A novel member of the human ppGalNAc-T family, ppGalNAc-T20, was identified and characterized. Amino acid alignment revealed a high sequence identity between ppGalNAc-T20 and -T10. In the GalNAc transfer assay towards mucin-derived peptide substrates, the recombinant ppGalNAc-T20 demonstrated to be a typical glycopeptide GalNAc-transferase that exhibits activity towards mono-GalNAc-glycosylated peptide EA2 derived from rat submandibular gland mucin but no activity towards non-modified EA2. The in vitro catalytic property of ppGalNAc-T20 was compared with that of ppGalNAc-T10 to show different acceptor substrate specificities and kinetic constants. The ppGalNAc-T20 transcript was found exclusively in testis and brain. In situ hybridization further reveals that ppGalNAc-T20 was specifically localized in primary and secondary spermatocytes of the two meiotic periods, suggesting that it may involve in O-glycosylation during mouse spermatogenesis.

Asunto(s)

Encéfalo/enzimología , N-Acetilgalactosaminiltransferasas/metabolismo , Testículo/enzimología , Secuencia de Aminoácidos , Animales , Catálisis , Glicosilación , Humanos , Hibridación in Situ , Masculino , Ratones , Datos de Secuencia Molecular , N-Acetilgalactosaminiltransferasas/clasificación , N-Acetilgalactosaminiltransferasas/genética , Filogenia , Ratas , Transcripción Genética , Polipéptido N-Acetilgalactosaminiltransferasa

RESUMEN

A sequence highly homologous to beta1,4-N-acetylgalactosaminyltransferase III (beta4GalNAc-T3) was found in a database of human expressed sequence tags. The full-length open reading frame of the gene, beta4GalNAc-T4 (GenBank accession number AB089939), was cloned using the 5' rapid amplification of cDNA ends method. It encodes a typical type II transmembrane protein of 1039 amino acids having 42.6% identity with beta4GalNAc-T3. The recombinant enzyme transferred N-acetylgalactosamine to N-acetylglucosamine-beta-benzyl with a beta1,4-linkage to form N,N'-diacetyllactosediamine as did beta4GalNAc-T3. In specificity toward oligosaccharide acceptor substrates, it was quite similar to beta4GalNAc-T3 in vitro, however, the tissue distributions of the two enzymes were quite different. These results indicated that the two enzymes have similar roles in different tissues.

Asunto(s)

Disacáridos/biosíntesis , Lactosa/análogos & derivados , Lactosa/biosíntesis , N-Acetilgalactosaminiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia de Carbohidratos , Línea Celular , Clonación Molecular , Humanos , Datos de Secuencia Molecular , N-Acetilgalactosaminiltransferasas/genética , Alineación de Secuencia , Especificidad por Sustrato , Distribución Tisular

RESUMEN

We found, using a BLAST search, a novel human gene (GenBank trade mark accession number BC029564) that possesses beta3-glycosyltransferase motifs. The full-length open reading frame consists of 500 amino acids and encodes a typical type II membrane protein. This enzyme has a domain containing beta1,3-glycosyltransferase motifs, which are widely conserved in the beta1,3-galactosyltransferase and beta1,3-N-acetylglucosaminyltransferase families. The putative catalytic domain was expressed in human embryonic kidney 293T cells as a soluble protein. Its N-acetylgalactosaminyltransferase activity was observed when N-acetylglucosamine (GlcNAc) beta1-O-benzyl was used as an acceptor substrate. The enzyme product was determined to have a beta1,3-linkage by NMR spectroscopic analysis, and was therefore named beta1,3-N-acetylgalactosaminyltransferase-II (beta3GalNAc-T2). The acceptor substrate specificity of beta3GalNAc-T2 was examined using various oligosaccharide substrates. Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-para-nitrophenyl (core 2-pNP) was the best acceptor substrate for beta3GalNAc-T2, followed by GlcNAcbeta1-4GlcNAcbeta1-O-benzyl, and GlcNAcbeta1-6GalNAcalpha1-O-para-nitrophenyl (core 6-pNP), among the tested oligosaccharide substrates. Quantitative real time PCR analysis revealed that the beta3Gal-NAc-T2 transcripts was restricted in its distribution mainly to the testis, adipose tissue, skeletal muscle, and ovary. Its putative orthologous gene, mbeta3GalNAc-T2, was also found in a data base of mouse expressed sequence tags. In situ hybridization analysis with mouse testis showed that the transcripts are expressed in germ line cells. beta3GalNAc-T2 efficiently transferred GalNAc to N-glycans of fetal calf fetuin, which was treated with neuraminidase and beta-galactosidase. However, it showed no activity toward any glycolipid examined. Although the GalNAcbeta1-3GlcNAcbeta1-R structure has not been reported in humans or other mammals, we have discovered a novel human glycosyltransferase producing this structure on N- and O-glycans.

Asunto(s)

N-Acetilgalactosaminiltransferasas/genética , N-Acetilgalactosaminiltransferasas/metabolismo , Oligosacáridos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia de Carbohidratos , Biología Computacional , Humanos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Oligosacáridos/química , Filogenia , Protones , ARN Mensajero/análisis , Especificidad por Sustrato

RESUMEN

In order to investigate the relationship between glycosyltransferase families and the motif for them, we classified 47 glycosyltransferase families in the CAZy database into four superfamilies, GTS-A, -B, -C, and -D, using a profile Hidden Markov Model method. On the basis of the classification and the similarity between GTS-A and nucleotidylyltransferase family catalyzing the synthesis of nucleotide-sugar, we proposed that ancient oligosaccharide might have been synthesized by the origin of GTS-B whereas the origin of GTS-A might be the gene encoding for synthesis of nucleotide-sugar as the donor and have evolved to glycosyltransferases to catalyze the synthesis of divergent carbohydrates. We also suggested that the divergent evolution of each superfamily in the corresponding subcellular component has increased the complexities of eukaryotic carbohydrate structure.

Asunto(s)

Glicosiltransferasas/clasificación , Cadenas de Markov , Secuencia de Aminoácidos , Carbohidratos/análisis , Glicosilación , Glicosiltransferasas/análisis , Glicosiltransferasas/metabolismo , Datos de Secuencia Molecular , Alineación de Secuencia

RESUMEN

Recently, it has become evident that chondroitin sulfate (CS) glycosyltransferases, which transfer glucuronic acid and/or N-acetylgalactosamine residues from each UDP-sugar to the nonreducing terminus of the CS chain, form a gene family. We report here a novel human gene (GenBank trade mark accession number AB086062) that possesses a sequence homologous with the human chondroitin sulfate synthase-1 (CSS1) gene, formerly known as chondroitin synthase. The full-length open reading frame consists of 882 amino acids and encodes a typical type II membrane protein. This enzyme contains a beta 3-glycosyltransferase motif and a beta 4-glycosyltransferase motif similar to that found in CSS1. Both the enzymes were expressed in COS-7 cells as soluble proteins, and their enzymatic natures were characterized. Both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, CS polymer, and their corresponding oligosaccharides were used as the acceptor substrates, but no polymerization reaction was observed as in the case of CSS1. The new enzyme was thus designated chondroitin sulfate synthase-3 (CSS3). However, the specific activity of CSS3 was much lower than that of CSS1. The reaction products were shown to have a GlcUA beta 1-3GalNAc linkage and a GalNAc beta 1-4GlcUA linkage in the nonreducing terminus of chondroitin resulting from glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcript level of CSS3 was much lower than that of CSS1, although it was ubiquitously expressed in various human tissues. These results indicate that CSS3 is a glycosyltransferase having both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. It may make a contribution to CS biosynthesis that differs from that of CSS1.

Asunto(s)

Glicosiltransferasas/genética , Hexosiltransferasas/genética , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Sulfatos de Condroitina/biosíntesis , Clonación Molecular , ADN Complementario/genética , Femenino , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Hexosiltransferasas/química , Hexosiltransferasas/metabolismo , Humanos , Técnicas In Vitro , Cinética , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Filogenia , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Distribución Tisular

RESUMEN

Chondroitin sulfate is found in a variety of tissues as proteoglycans and consists of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues with sulfate residues at various places. We found a novel human gene (GenBank accession number AB086063) that possesses a sequence homologous with the human chondroitin sulfate glucuronyltransferase gene which we recently cloned and characterized. The full-length open reading frame encodes a typical type II membrane protein comprising 775 amino acids. The protein had a domain containing beta 3-glycosyltransferase motif but lacked a typical beta 4-glycosyltransferase motif, which is the same as chondroitin sulfate glucuronyltransferase, whereas chondroitin synthase had both domains. The putative catalytic domain was expressed in COS-7 cells as a soluble enzyme. Surprisingly, both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, chondroitin sulfate, and their oligosaccharides were used as the acceptor substrates. The reaction products were identified to have the linkage of GlcUA beta 1-3GalNAc and GalNAc beta 1-4GlcUA at the non-reducing terminus of chondroitin for glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcripts were ubiquitously expressed in various human tissues but highly expressed in the pancreas, ovary, placenta, small intestine, and stomach. These results indicate that this enzyme could synthesize chondroitin sulfate chains as a chondroitin sulfate synthase that has both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. Sequence analysis based on three-dimensional structure revealed the presence of not typical but significant beta 4-glycosyltransferase architecture.

Asunto(s)

Sulfatos de Condroitina/química , Hexosiltransferasas/química , Hexosiltransferasas/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoácidos , Animales , Células COS , Cationes , Bovinos , División Celular , Condroitín/química , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/metabolismo , Disacáridos/química , Relación Dosis-Respuesta a Droga , Epítopos , Vectores Genéticos , Ácido Glucurónico/química , Glucuronosiltransferasa/metabolismo , Glicosiltransferasas/metabolismo , Hexosiltransferasas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Monosacáridos , Oligosacáridos/química , Sistemas de Lectura Abierta , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Distribución Tisular , Uridina Difosfato/farmacología

RESUMEN

Heparan sulfate d-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, A., Shukla, D., and Liu, J. (2002) J. Biol. Chem. 277, 37912-37919). In the present study, we cloned putative catalytic domain of the human 3-OST-5 and expressed it in insect cells as a soluble enzyme. Recombinant 3-OST-5 only exhibited sulfotransferase activity toward heparan sulfate and heparin. When incubated heparan sulfate with [35S]PAPS, the highest incorporation of35S was observed, and digestion of the product with a mixture of heparin lyases yielded two major35S-labeled disaccharides, which were determined as DeltaHexA-GlcN(NS,3S,6S) and DeltaHexA(2S)-GlcN(NS,3S) by further digestion with 2-sulfatase and degradation with mercuric acetate. However, when used heparin as acceptor, we identified a highly sulfated disaccharide unit as a major product. This had a structure of DeltaHexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord, and at very low or undetectable levels in the other tissues. Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal heparan sulfate. To our knowledge, this is the first report to describe not only the natural occurrence of tetrasulfated disaccharide unit but also the enzymatic formation of this novel structure.

Asunto(s)

Disacáridos/biosíntesis , Sulfotransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Disacáridos/química , Femenino , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Sulfotransferasas/química , Sulfotransferasas/genética , Distribución Tisular

RESUMEN

The hinge region of human immunoglobulin A1 (*IgA1) possesses multiple O-glycans, of which synthesis is initiated by the addition of GalNAc to serine or threonine through the activity of UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). We found that six pp-GalNAc-Ts, pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9, were expressed in B cells, IgA-bearing B cells, and NCI-H929 IgA myeloma cells. pp-GalNAc-T activities of these six enzymes for a synthetic IgA hinge peptide, which has nine possible O-glycosylation sites, were examined using a reversed phase-high performance liquid chromatography, a matrix-assisted laser desorption ionization time of flight mass spectrometry, and peptide sequencing analysis. pp-GalNAc-T2 showed the strongest activity transferring GalNAc to a maximum of eight positions. Other pp-GalNAc-Ts exhibited different substrate specificities from pp-GalNAc-T2; however, their activities were extremely weak. It was reported that the IgA1 hinge region possesses a maximum of five O-glycans, and their amino acid positions have been determined. We found that pp-GalNAc-T2 selectively transferred GalNAc residues to the same five positions. These results strongly suggested that pp-GalNAc-T2 is an essential enzyme for initiation of O-linked glycosylation of the IgA1 hinge region.

Asunto(s)

Inmunoglobulina A/biosíntesis , Inmunoglobulina A/química , Linfocitos/inmunología , N-Acetilgalactosaminiltransferasas/metabolismo , Polisacáridos/biosíntesis , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Secuencia de Aminoácidos , Linfocitos B/enzimología , Linfocitos B/inmunología , Secuencia de Bases , Línea Celular , Humanos , Cinética , Linfocitos/enzimología , Datos de Secuencia Molecular , Mieloma Múltiple/inmunología , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad por Sustrato , Transcripción Genética , Células Tumorales Cultivadas , Polipéptido N-Acetilgalactosaminiltransferasa

RESUMEN

We found a novel glycosyltransferase gene having a hypothetical beta 1,4-galactosyltransferase motif (GenBank accession number ) by a BLAST search and cloned its full-length open reading frame using the 5'-rapid amplification of cDNA ends method. The truncated form was expressed in insect cells as a soluble enzyme. It transferred N-acetylgalactosamine, not galactose, to para-nitrophenyl-beta-glucuronic acid. The N-acetylgalactosamine-glucuronic acid linkage has been identified only in chondroitin sulfate; therefore, we examined its chondroitin elongation and initiation activities. N-Acetylgalactosaminyltransferase activity was observed toward chondroitin poly- and oligosaccharides, chondroitin sulfate oligosaccharides, and linkage tetrasaccharide (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), and the chondroitin polysaccharide and linkage tetrasaccharide were better acceptor substrates than the others. Northern blot analysis and quantitative real-time PCR analysis revealed that its 4-kb transcripts were highly expressed in thyroid and placenta, although they were ubiquitously expressed in various tissues and cells. These results suggest that this enzyme has N-acetylgalactosaminyltransferase activity in both the elongation and initiation of chondroitin sulfate synthesis. Furthermore, we performed enzymatic synthesis of chondroitin pentasaccharide in vitro. In one tube reaction with four enzymes, beta 1,4-galactosyltransferase-VII, beta 1,3-galactosyltransferase-VI, glucuronyltransferase-I, and this enzyme, and a synthetic xylose-peptide acceptor, the structure GalNAc-GlcA-Gal-Gal-Xyl-peptide was constructed. This is the first report of a chondroitin pentasaccharide constructed with recombinant glycosyltransferases in vitro.

Asunto(s)

Acetilgalactosamina/metabolismo , Condroitín/biosíntesis , Ácido Glucurónico/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Humanos , Datos de Secuencia Molecular , N-Acetilgalactosaminiltransferasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Espectrometría de Masa por Ionización de Electrospray , Distribución Tisular