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BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of a human UDP-galactose:␤-N-acetylglucosamine ␤-1,3-galactosyltransferase, designated ␤3Gal-T1, revealed no ESTs with identical sequences but a large number with similarity. Three different sets of overlapping ESTs with sequence similarities to ␤3Gal-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:␤-N-acetylglucosamine ␤-1,3-galactosyltransferase (␤3Gal-T2) with similar kinetic properties as ␤3Gal-T1. Another gene represented a UDP-galactose:␤-N-acetyl-galactosamine ␤-1,3-galactosyltransferase (␤3Gal-T4) involved in G M1 /G D1 ganglioside synthesis, and this gene was highly similar to a recently reported rat G D1 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. ␤3Gal-T1 mRNA was expressed in brain, ␤3Gal-T2 was expressed in brain and heart, and ␤3Gal-T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons. ␤3Gal-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 ␤3-galactosyltransferase genes.

Differentiation and activation of lymphocytes are documented to result in changes in glycosylation associated with biologically important consequences. In this report, we have systematically examined global changes in N-linked glycosylation following activation of murine CD4 T cells, CD8 T cells, and B cells by MALDI-TOF mass spectrometry profiling, and investigated the molecular basis for those changes by assessing alterations in the expression of glycan transferase genes. Surprisingly, the major change observed in activated CD4 and CD8 T cells was a dramatic reduction of sialylated biantennary N-glycans carrying the terminal NeuGcα2-6Gal sequence, and a corresponding increase in glycans carrying the Galα1-3Gal sequence. This change was accounted for by a decrease in the expression of the sialyltransferase ST6Gal I, and an increase in the expression of the galactosyltransferase, α1-3GalT. Conversely, in B cells no change in terminal sialylation of N-linked glycans was evident, and the expression of the same two glycosyltransferases was increased and decreased, respectively. The results have implications for differential recognition of activated and unactivated T cells by dendritic cells and B cells expressing glycan-binding proteins that recognize terminal sequences of N-linked glycans.

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of the human UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferase, designated ␤4Gal-T1, revealed a large number of ESTs with identical as well as similar sequences. ESTs with sequences similar to that of ␤4Gal-T1 could be grouped into at least two non-identical sequence sets. Analysis of the predicted amino acid sequence of the novel ESTs with ␤4Gal-T1 revealed conservation of short sequence motifs as well as cysteine residues previously shown to be important for the function of ␤4Gal-T1. The likelihood that the identified ESTs represented novel galactosyltransferase genes was tested by cloning and sequencing of the full coding region of two distinct genes, followed by expression. Expression of soluble secreted constructs in the baculovirus system showed that these genes represented genuine UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferases, thus designated ␤4Gal-T2 and ␤4Gal-T3. Genomic cloning of the genes revealed that they have identical genomic organizations compared with ␤4Gal-T1. The two novel genes were located on 1p32-33 and 1q23. The results demonstrate the existence of a family of homologous galactosyltransferases with related functions. The existence of multiple ␤4-galactosyltransferases with the same or overlapping functions may be relevant for interpretation of biological functions previously assigned to ␤4Gal-T1.During the last decade, more than 40 mammalian glycosyltransferases have been cloned and characterized (1, 2). The initial strategy for cloning glycosyltransferases was cumbersome purification of labile enzyme proteins followed by screening of cDNA libraries with antibodies or DNA probes based on amino acid sequence information (3-11). The introduction of transfection cloning by Lowe and co-workers (12) resulted in a marked increase in the cloning of novel glycosyltransferase genes (13-17). A third successful approach has taken advantage of conserved sequences in glycosyltransferases that share donor and/or acceptor substrates. Thus, searches for novel members of homologous glycosyltransferase gene families utilizing conserved sequence motifs for RT-PCR 1 cloning with degenerate primers have resulted in the identification and cloning of a number of novel genes (18,19).One part of the human genome project is the establishment of a data base of expressed sequence tags (ESTs), which currently has over 700,000 unique sequences. ESTs represent short 5Ј-and 3Ј-sequences (200 -500 bp) of cDNA clones from a large variety of human and animal organs (20). The EST data base is now estimated to contain sequence information from more than half the human genes; it therefore provides a unique source for identifying novel members of homologous gene families (21). The EST data base has recently been successfully utilized in searches for novel glycosyltransferase genes of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, where several novel members of this homologous gene family have been iso...

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