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The sialyl-Tn (sTn) antigen is a well known cancer-associated antigen, the expression of which is related to the prognosis of cancer patients. We aimed to isolate a human gene encoding an N -acetylgalactosamine alpha2,6-sialyltransferase which synthesizes sTn antigen, and to characterize the enzyme. Degenerate primers encoding sialyl motifs were used for the polymerase chain reaction to amplify complementary DNAs prepared from RNAs of human pyloric mucosae with intestinal metaplasia, which abundantly expressed sTn antigen, followed by screening of full-length cDNAs using the amplified DNA fragment as a probe. We isolated two human cDNA clones, long-form (2.46 kb) and short-form (2.23 kb) cDNAs. The former encodes an active enzyme with a predicted 600 amino acid sequence. The latter, a splice-variant of the long-form, encodes an inactive enzyme. HCT15 human colorectal cancer cells stably expressing the long-form cDNA expressed sTn epitopes on O -glycans. The long form cDNA was considered to encode a human homologue of chick ST6GalNAc I for the following reasons: (1) the putative amino acid sequence showed greater homology to that of chick ST6GalNAc I (55%) compared to other sialyltransferases, (2) it encodes the extraordinarily long stem region that is a typical feature of chick ST6GalNAc I, and (3) the substrate specificity was very similar to that of chick ST6GalNAc I. In situ hybridization demonstrated that the localization of transcripts correlated well with that of sTn antigen in gastric cancer cells and Goblet cells in intestinal metaplastic glands. Thus, we determined that the long-form cDNA of the human ST6GalNAc I gene encodes the probable candidate for the human sTn synthase(s).

Lewis b (Leb) antigens are gradiently expressed from the proximal to the distal colon, i.e., they are abundantly expressed in the proximal colon, but only faintly in the distal colon. In the distal colon, they begin to increase at the adenoma stage of cancer development and then increase with cancer progression. We aimed to clarify the molecular basis of Leb antigen expression in correlation with the expression of other type I Lewis antigens, such as Lewis a (Lea) and sialylated Lewis a (sLea), in colon cancer cells. Considering the Se genotype and the relative activities of the H and Se enzymes, the amounts of Leb antigens were proved to be determined by both the H and Se enzymes in noncancerous and cancerous colon tissues. But the Se enzyme made a much greater contribution to determining the Lebamounts than the H enzyme. In noncancerous colons, the Se enzyme were gradiently expressed in good correlation with the Leb expression, while the H enzyme was constantly expressed throughout the whole colon. In distal colon cancers, the H and Se enzymes were both significantly upregulated in comparison with in adjacent noncancerous tissues. In proximal colon cancers, expression of the H enzyme alone was highly augmented. The augmented expression of Leb antigens in distal colon cancers is caused mainly by upregulation of the Se enzyme and partly by the H enzymes, while it is caused by upregulation of the H enzyme alone in proximal colon cancers. The Se gene dosage profoundly influences the amounts of the Leb, Lea, and sLea antigens in whole colon tissues, regardless of whether they are noncancerous or cancerous tissues. It suggests that the Se enzyme competes with alpha2,3 sialyltransferase(s) and the Le enzyme for the type I acceptor substrates.

The expression of type-1 Lewis antigens on erythrocytes and in digestive organs is determined by a Lewis type alpha(1,3/1, 4)-fucosyltransferase (Lewis enzyme) encoded by the Fuc-TIII gene ( FUT3 gene; Lewis gene). We have classified the Lewis alleles in the Japanese population into four types, the wild-type allele ( Le ) and three mutated alleles, i.e., le1, which has missense mutations T59G and G508A, le2, which has T59G and T1067A, and le3, which has only T59G. Here we carried out an extensive study on the biological properties of the three mutant Lewis enzymes, the le1, le2, and le3 enzymes, using native tissues and obtained the following results. (1) In in vivo and in vitro experiments, the le1 and le2 enzymes were found to be susceptible to protease digestion probably because the one missense mutation in the catalytic domains, i.e., Gly170 to Ser in the le1 enzyme and Ile356 to Lys in the le2 enzyme, makes the three-dimensional structures of the enzymesunstable, while the le3 and wild-type Lewis enzymes wereresistant to protease digestion. (2) The le1 and le2 enzymes cannot synthesize type 1 Lewis antigens on either glycolipids or mucins. The le3 enzyme cannot synthesize Lewis-active glycolipids, which result in the Lewis antigen-negative phenotype of erythrocytes, while it can synthesize Lewis antigens on mucins in normal and cancerous colon tissues. The missense mutation, Leu20 to Arg, in the transmembrane domain reduces retention of the le3 enzyme in the Golgi membrane resulting in an apparent reduction of enzyme activity as revealed by the lack of Lewis antigen synthesis. (3) The Lewis gene dosage actually has effects in vivo on the amount of the Lewis enzyme, its activity, and finally the amounts of Lewis carbohydrate antigens. This is the first article that clearly demonstrates the gene dosage effects on the amount of the glycosyltransferase protein, its activity, and the amounts of carbohydrate products in vivo.