The MUC1 mucin represents a prime target antigen for cancer immunotherapy because it is abundantly expressed and aberrantly glycosylated in carcinomas. Attempts to generate strong humoral immunity to MUC1 by immunization with peptides have generally failed partly because of tolerance. In this study, we have developed chemoenzymatic synthesis of extended MUC1 TR glycopeptides with cancer-associated O-glycosylation using a panel of recombinant human glycosyltransferases. MUC1 glycopeptides with different densities of Tn and STn glycoforms conjugated to KLH were used as immunogens to evaluate an optimal vaccine design. Glycopeptides with complete O-glycan occupancy (five sites per repeat) elicited the strongest antibody response reacting with MUC1 expressed in breast cancer cell lines in both Balb/c and MUC1.Tg mice. The elicited humoral immune response showed remarkable specificity for cancer cells suggesting that the glycopeptide design holds promise as a cancer vaccine. The elicited immune responses were directed to combined glycopeptide epitopes, and both peptide sequence and carbohydrate structures were important for the antigen. A MAb (5E5) with similar specificity as the elicited immune response was generated and shown to have the same remarkable cancer specificity. This antibody may hold promise in diagnostic and immunopreventive measures.

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The completed fruit fly genome was found to contain up to 15 putative UDP-N-acetyl-␣-D-galactosamine:polypeptide N-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 GalNActransferases 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

In breast cancer, the O-glycans added to the MUC1 mucin are core 1-rather than core 2-based. We have analyzed whether competition by the glycosyltransferase, ST3Gal-I, which transfers sialic acid to galactose in the core 1 substrate, is key to this switch in MUC1 glycosylation that results in the expression of the cancer-associated SM3 epitope. Of the three enzymes known to convert core 1 to core 2, by the addition of GlcNAc to GalNAc in core1 C2GnT1 is the dominant enzyme expressed in normal breast tissue. Expression of C2GnT1 is low or absent in around 50% of breast cancers, whereas expression of ST3Gal-I is consistently increased. Mapping of ST3Gal-I and C2GnT1 within the Golgi pathway showed some overlap. To examine functional competition, the enzymes were overexpressed in T47D cells, which normally make core 1-based structures, have no detectable C2GnT1 activity and express the SM3 epitope. Overexpression of C2GnT1 resulted in loss of binding of SM3 to MUC1, accompanied by a decrease in the GalNAc/GlcNAc ratio, indicative of a switch to core 2 structures. Transfection of a C2GnT1 expressing line with ST3Gal

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.

A seventh member of the human ␤4-galactosyltransferase family, ␤4Gal-T7, was identified by BLAST analysis of expressed sequence tags. The coding region of ␤4Gal-T7 depicts a type II transmembrane protein with sequence similarity to ␤4-galactosyltransferases, but the sequence was distinct in known motifs and did not contain the cysteine residues conserved in the other six members of the ␤4Gal-T family. The genomic organization of ␤4Gal-T7 was different from previous ␤4Gal-Ts. Expression of ␤4Gal-T7 in insect cells showed that the gene product had ␤1,4-galactosyltransferase activity with ␤-xylosides, and the linkage formed was Gal␤1-4Xyl. Thus, ␤4Gal-T7 represents galactosyltransferase I enzyme (xylosylprotein ␤1,4-galactosyltransferase; EC 2.4. Six members of a family of human UDP-galactose:␤-Nacetylglucosamine/␤-glucosylceramide ␤1,4-galactosyltransferases (␤4Gal-Ts) 1 have previously been characterized (1-7).These six ␤4Gal-Ts catalyze biosynthesis of Gal␤1-4GlcNAc and/or Gal␤1-4Glc linkages in different glycoconjugates and free saccharides (for a review see Ref. 8). The six ␤4Gal-Ts have highly conserved sequence motifs in the putative catalytic domain including four conserved cysteine residues. The genomic organization of the first four genes is similar and includes conservation of spacing for five intron/exon boundaries in the coding regions (4, 7, 9, 10). This suggests that these genes arose late in evolutionary terms as a result of gene duplication and subsequent sequence divergence. Detailed analysis of the kinetic properties of these enzymes clearly show that each has a distinct function in biosynthesis of different glycoconjugates and saccharide structures, but in accordance with their close evolutionary relationships the linkages formed are similar. In the present study, a seventh homologue of the ␤4Gal-T gene family was characterized. The gene was identified by sequence analysis of the EST data base. The coding region of the novel gene, designated ␤4Gal-T7, exhibited distinct substitutions in the sequence motifs highly conserved among ␤4Gal-T1 to ␤4Gal-T6. Notably, none of the four cysteines conserved among other ␤4Gal-Ts were found in the ␤4Gal-T7 sequence. It was predicted that the enzymatic properties of ␤4Gal-T7 were different from other ␤4Gal-Ts. Analysis of the substrate specificity of recombinant ␤4Gal-T7 revealed that this enzyme formed the Gal␤1-4Xyl␤1-R linkage found in the linkage region of proteoglycans (GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser). ␤4Gal-T7 was proposed to encode a galactosyltransferase I (xylosylprotein ␤1,4-galactosyltransferase; EC 2.4.1.133) gene (11)(12)(13). 15) showed that partial inactivation of galactosyltransferase I represented the primary defect in one patient with progeroidal appearance and symptoms of the Ehlers-Danlos syndrome. As a consequence of the enzyme deficiency, only about half of the core proteins of the small proteoglycans decorin and biglycan were linked with glycosaminoglycan chains (16), 2 whereas no abnormality in the biosynthesis of large dermatan sulf...

A novel putative member of the human UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferase family, designated ␤4Gal-T4, was identified by BLAST analysis of expressed sequence tags. The sequence of ␤4Gal-T4 encoded a type II membrane protein with significant sequence similarity to other ␤1,4-galactosyltransferases. Expression of the full coding sequence and a secreted form of ␤4Gal-T4 in insect cells showed that the gene product had ␤1,4-galactosyltransferase activity. Analysis of the substrate specificity of the secreted form revealed that the enzyme catalyzed glycosylation of glycolipids with terminal ␤-GlcNAc; however, in contrast to ␤4Gal-T1, -T2, and -T3, this enzyme did not transfer galactose to asialo-agalacto-fetuin, asialo-agalactotransferrin, or ovalbumin. The catalytic activity of ␤4Gal-T4 with monosaccharide acceptor substrates, Nacetylglucosamine as well as glucose, was markedly activated in the presence of ␣-lactalbumin. The genomic organization of the coding region of ␤4Gal-T4 was contained in six exons. All intron/exon boundaries were similarly positioned in ␤4Gal-T1, -T2, and -T3. ␤4Gal-T4 represents a new member of the ␤4-galactosyltransferase family. Its kinetic parameters suggest unique functions in the synthesis of neolactoseries glycosphingolipids.A family of human UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferases (␤4Gal-Ts) 1 was recently identified (1-3). Four genes within this family encode ␤4-galactosyltransferases, which form the Gal␤1-4GlcNAc linkage (2, 4 -6). The kinetic parameters and expression patterns of these enzymes differ and they are predicted to show some degree of overlap in biological function (2, 3, 6). Two ␤4-galactosyltransferases, ␤4Gal-T1 and -T2, can function as lactose synthases in the presence of ␣-lactalbumin (2, 3, 7), whereas ␤4Gal-T3 and ␤4Gal-T5 2 are largely insensitive to ␣-lactalbumin modulation (2, 6, 8). ␤4Gal-T1, -T2, and -T3 catalyze transfer of galactose to lactoseries glycosphingolipids, but ␤4Gal-T3 only efficiently catalyzes synthesis of the first N-acetyllactosamine unit in lactoseries glycolipids (2). In contrast, ␤4Gal-T5 was reported to be inactive with a glycolipid substrate (Lc 3 Cer) 2 as well as with the glycoprotein acceptor, asialo-agalacto-transferrin (6). A rat lactosylceramide synthase was recently purified and cloned by Nomura et al. (9), and it appears to represent the ortholog of the human member of the gene ␤4-galactosyltransferase family designated ␤4Gal-T6 (10). Thus, the formation of Gal␤1-4Glc(NAc) linkages in different glycoconjugates and their varying oligosaccharide structures may be catalyzed by different ␤4-galactosyltransferases.Analysis of ESTs suggested the existence of additional members of the human ␤4Gal-T gene family (1-3), and recently, Lo et al. (10) compared the full coding sequences of six homologous human genes and suggested a nomenclature based on sequence similarity: ␤4Gal-T1 (5, 11, 12), ␤4Gal-T2 (2), ␤4Gal-T3 (2), ␤4Gal-T4, ␤4Gal-T5 (6), and ␤4Gal-T6 (9). So far, all genes except one, ...

Poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine repeats and provides the backbone structure for additional modifications such as sialyl Le x . Poly-N-acetyllactosamines in mucintype O-glycans can be formed in core 2 branched oligosaccharides, which are synthesized by core 2 ␤-1,6-Nacetylglucosaminyltransferase.Using a ␤-1,4-galactosyltransferase (␤4Gal-TI) present in milk and the recently cloned ␤-1,3-N-acetylglucosaminyltransferase, the formation of poly-N-acetyllactosamine was found to be extremely inefficient starting from a core 2 branched oligosaccharide, GlcNAc␤136-(Gal␤133)GalNAc␣3 R. Since the majority of synthesized oligosaccharides contained N-acetylglucosamine at the nonreducing ends, galactosylation was judged to be inefficient, prompting us to test novel members of the ␤4Gal-T gene family for this synthesis. Using various synthetic acceptors and recombinant ␤4Gal-Ts, ␤4Gal-TIV was found to be most efficient in the addition ofasinglegalactoseresiduetoGlcNAc␤136(Gal␤133)GalNAc␣3 R. Moreover, ␤4Gal-TIV, together with ␤-1,3-Nacetylglucosaminyltransferase, was capable of synthesizing poly-N-acetyllactosamine in core 2 branched oligosaccharides. On the other hand, ␤4Gal-TI was found to be most efficient for poly-N-acetyllactosamine synthesis in N-glycans. In contrast to ␤4Gal-TI, the efficiency of ␤4Gal-TIV decreased dramatically as the acceptors contained more N-acetyllactosamine repeats, consistent with the fact that core 2 branched O-glycans contain fewer and shorter poly-N-acetyllactosamines than N-glycans in many cells. These results, as a whole, indicate that ␤4Gal-TIV is responsible for poly-Nacetyllactosamine synthesis in core 2 branched O-glycans.Mucin-type O-glycans are present in a wide variety of cells and play various roles in different cells. Mucin-type glycoproteins are also present in the plasma membrane, and they are often involved in cell-cell interaction (1). For example, O-glycans present in eggs were shown to be a receptor for both mouse and sea urchin (2, 3). In granulocytes, monocytes, and certain T lymphocytes, mucin-type O-glycans can carry sialyl Le x , NeuNAc␣233Gal␤134(Fuc␣133)GlcNAc3 R, at their termini (4 -6). Sialyl Le x and its sulfated form are ligands for E-, P-, and L-selectin (7-11). Importantly, these selectins, in particular P-and L-selectin, preferentially bind to sialyl Le x in a limited number of mucin-type glycoproteins such as PSGL-1 (for P-selectin) and GlyCAM-1 and CD34 (for L-selectin) (12-14). As shown previously, sialyl Le x and its derivatives of O-glycans in blood cells can be only formed on core 2 branches, Gal␤134GlcNAc␤136(Gal␤133)GalNAc␣3 R (4, 5). Recent studies demonstrate that sialyl Le x and sialyl Le a in core 2 branches are highly correlated to tumor invasion and vessel invasion of colon carcinomas (15), probably because tumor cells utilize selectin-carbohydrate interaction for their adhesion.In patients with immunodeficiency such as Wiskott-Aldrich syndrome, AIDS, and leukemia, leukocytes in the peripheral blood...