Cloning and expression of a human gene encoding an N-acetylgalactosamine- 2,6-sialyltransferase (ST6GalNAc I): a candidate for synthesis of cancer-associated sialyl-Tn antigens

Abstract

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).

Focal amplifications are associated with high grade and recurrences in stage Ta bladder carcinoma

“…The cancer-associated sialyl-Tn (sTn) antigen is formed by ST6GALNAC1-catalyzed sialylation of GalNAc residues on mucins. 47 STn is overexpressed in several cancers and currently associated with more aggressive diseases and poor prognosis in breast cancer. 48 Although indications for no association between tumor progression to invasion and the expression of sTn-antigen have been reported, 49 the potential clinical value of overexpression of ST6GALNAC1 in bladder carcinoma has not been thoroughly investigated.…”

The ST6GalNAc-I Sialyltransferase Localizes throughout the Golgi and Is Responsible for the Synthesis of the Tumor-associated Sialyl-Tn O-Glycan in Human Breast Cancer

“…The amplification of cDNA was performed with the Expand Long template PCR system (Roche Applied Science) in a total volume of 50 l containing 0.3 M of a primer set, 5Ј-GCTAGCCGACCCACCACCATGAGG-3Ј (corresponding to bp (Ϫ12 to Ϫ6) of the ST6GalNAcI sequence) and 5Ј-CTCGAGTCAATTACCAAGACGATTCATTTCAATATCAGT-ATAAAGTTGACGATTGGGCGGGTTCTTGGCTTTGGCAGTT-C-3Ј (corresponding to bp 1761-1800 of the ST6GalNAc-I sequence). The 5Ј-end of the published cDNA sequence (accession number Y 11339 (25)) was found to disagree with the genomic ST6GalNAc-I (accession number AC005837). The sequence of the 5Ј-primer was taken from the genomic sequence, because use of the published 5Ј cDNA sequence did not result in a product.…”

“…Thus the STn O-glycan has been seen as a possible target for therapeutic intervention (21)(22)(23)(24). In human cells, the enzyme ST6GalNAc-I has been identified as a glycosyltransferase able to add sialic acid in ␣2,6 linkage to GalNAc linked to serine or threonine, thus creating the STn epitope (25). Other sialyltransferases active in the pathway of mucin type O-glycosylation, including hST6GalNAc-II, can add sialic acid in ␣2,6 linkage to GalNAc in vitro (26) but show a preference for GalNAc residues already modified by the addition of galactose or galactose followed by sialic acid in ␣2,3 linkage (26,27).…”

Golgi Localization of Carbohydrate Sulfotransferases Is a Determinant of L-selectin Ligand Biosynthesis

“…At saturating levels, highly expressed enzymes may distribute into Golgi subcompartments they would not otherwise occupy, thereby gaining access to novel substrates (52)(53)(54)(55). To exclude the possibility that dramatic differences in expression levels account for the distinct substrate preferences of GlcNAc6ST-1, -2, and -3 chimeras, we analyzed protein levels in the stable CHO cell lines by Western blot using anti-GFP serum.…”