Identification of a novel human UDP-GalNAc transferase with unique catalytic activity and expression profile.

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.

Immunolocalisation of members of the polypeptide N-acetylgalactosaminyl transferase (ppGalNAc-T) family is consistent with biologically relevant altered cell surface glycosylation in breast cancer.

An extensive family of UDP-N-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferases (polypeptide N-acetylgalactosaminyltransferases, ppGalNAc-T's) catalyse the attachment of the first N-acetylgalactosamine (GalNAc) monosaccharide to the polypeptide at the initiation of O-linked glycosylation of proteins. Some members of the family are broadly expressed while others are more restricted in their distribution, their expression and activity being confined to certain cells or tissues, being associated with physiological states or differentiation. Their careful regulation, which is not well understood, may mediate the synthesis of varied glycoforms of cellular proteins with different biological activities. Disruptions in glycosylation are a common feature of cancer and may have functional significance. Immunocytochemistry with confocal scanning laser microscopy was employed to detect members of the ppGalNAc-T family, ppGalNAc-T1, -T2, -T3, -T4 and -T6 in a range of breast cell lines. The cells were chosen to represent a range of phenotypes from 'normal'/benign (HMT 3,522), primary, non-metastatic breast cancer (BT 474), to aggressive, metastatic breast cancer (ZR75-1, T47D, MCF-7, DU 4,475). They stably synthesise varying levels, consistent with origin and phenotype, of aberrantly glycosylated glycoproteins featuring exposed, terminal GalNAc residues, including the cancer-associated Tn antigen, which, in numerous studies, have been associated with metastatic competence and poor cancer prognosis. GalNAc-T1 and -T2 were detectable at low levels in all cell lines studied. ppGalNAc-T4, which has never been described in breast, was very weakly detectable in BT 474, MCF7 and T47D. ppGalNAc-T3 and -T6 were weakly detectable or undetectable, respectively, in the cell line HMT 3,522 derived from 'normal'/benign breast epithelium, but were readily detectable in all malignant cell lines. Thus, a broader range of ppGalNAc-T's were detectable in the malignant cell lines in comparison to the 'normal'/benign cells, where only the 'housekeeping' ppGalNAc-T1 and -T2 were present. Expression of normally tightly restricted ppGalNAc-T's may result in initiation of O-linked glycosylation at normally unoccupied potential glycosylation sites leading to altered glycoforms of proteins with changed biological activity which may contribute to the pathogenesis of cancer.

Expression of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family is spatially and temporally regulated during Drosophila development.

The UDP-GalNAc : polypeptide N-acetylgalactosaminyltransferase (ppGaNTase or ppGalNAcT or pgant) enzyme family is responsible for the first committed step in the synthesis of mucin-type O-glycans on protein substrates. Previous work from our group has demonstrated both sequence and functional conservation between members of this family in mammals and the fruit fly, Drosophila melanogaster. One member of this family in Drosophila has been shown to be essential for viability and development. In an effort to understand the developmental stages and processes in which O-glycosylation is involved, we have determined the expression pattern of each functional family member as well as putative isoforms during Drosophila development. Our studies indicate that isoforms are expressed in discrete spatial and temporal fashions during development, with some isoforms being found uniquely in restricted areas of the developing embryo (brain, trachea, pharynx, esophagus, proventriculus, and amnioserosa), whereas others are found in multiple regions and overlap with the expression of other isoforms (salivary glands, posterior midgut, anterior midgut, and the fore-/hindgut) during embryogenesis. Additionally, we examined expression patterns in imaginal discs from third instar larvae, which will become the adult structures. Most isoforms are also expressed in the imaginal discs, with some showing unique transcript localization and spatial regulatory control. Thus, this report provides insight into the specific regions during Drosophila development that may require O-linked glycosylation in vivo as well as which isoforms may act cooperatively in certain tissues and which may be uniquely responsible for glycosylation in others.