Eric Paul Bennett

Cloning of a Human UDP-N-Acetyl-α-d-Galactosamine:PolypeptideN-Acetylgalactosaminyltransferase That Complements Other GalNAc-Transferases in Complete O-Glycosylation of the MUC1 Tandem Repeat

A fourth human UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, and -T3, which contain multiple coding exons, in that the coding region is contained in a single exon. GalNAc-T4 was placed at human chromosome 12q21.3-q22 by in situ hybridization and linkage analysis. GalNAc-T4 expressed in Sf9 cells or in a stably transfected Chinese hamster ovary cell line exhibited a unique acceptor substrate specificity. GalNAc-T4 transferred GalNAc to two sites in the MUC1 tandem repeat sequence (Ser in GVTSA and Thr in PDTR) using a 24-mer glycopeptide with GalNAc residues attached at sites utilized by GalNAc-T1, -T2, and -T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). Furthermore, GalNAc-T4 showed the best kinetic properties with an O-glycosylation site in the P-selectin glycoprotein ligand-1 molecule. Northern analysis of human organs revealed a wide expression pattern. Immunohistology with a monoclonal antibody showed the expected Golgi-like localization in salivary glands. A single base polymorphism, G1516A (Val to Ile), was identified (allele frequency 34%). The function of GalNAc-T4 complements other GalNAc-transferases in O-glycosylation of MUC1 showing that glycosylation of MUC1 is a highly ordered process and changes in the repertoire or topology of GalNAc-transferases will result in altered pattern of O-glycan attachments.

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

The UDP-GalNAc:polypeptideN-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:polypeptideN-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.

A Family of Human β3-Galactosyltransferases CHARACTERIZATION OF FOUR MEMBERS OF A UDP-GALACTOSE:β-N-ACETYL-GLUCOSAMINE/β-NACETYL-GALACTOSAMINE β-1,3-GALACTOSYLTRANSFERASE FAMILY

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of a human UDP-galactose:β-N-acetyl-glucosamine β-1,3-galactosyltransferase, designated β3Gal-T1, revealed no ESTs with identical sequences but a large number with similarity. Three different sets of overlapping ESTs with sequence similarities to β3Gal-T1 were compiled, and complete coding regions of these genes were obtained. Expression of two of these genes in the Baculo virus system showed that one represented a UDP-galactose:β-N-acetyl-glucosamine β-1,3-galactosyltransferase (β3Gal-T2) with similar kinetic properties as β3Gal-T1. Another gene represented a UDP-galactose:β-N-acetyl-galactosamine β-1,3-galactosyltransferase (β3Gal-T4) involved in GM1/GD1 ganglioside synthesis, and this gene was highly similar to a recently reported rat GD1 synthase (Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794–24799). Northern analysis of mRNA from human organs with the four homologous cDNA revealed different expression patterns. β3Gal-T1 mRNA was expressed in brain, β3Gal-T2 was expressed in brain and heart, and β3Gal-T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons. β3Gal-T2, -T3, and -T4 were localized to 1q31, 3q25, and 6p21.3, respectively, by EST mapping. The results demonstrate the existence of a family of homologous β3-galactosyltransferase genes.

Control of O-Glycan Branch Formation MOLECULAR CLONING OF HUMAN cDNA ENCODING A NOVEL β1,6-N-ACETYLGLUCOSAMINYLTRANSFERASE FORMING CORE 2 AND CORE 4

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.