The Sialyl-Tn antigen (Neu5Ac␣2-6GalNAc-O-Ser/Thr) is highly expressed in several human carcinomas and is associated with carcinoma aggressiveness and poor prognosis. We characterized two human sialyltransferases, CMP-Neu5Ac:GalNAc-R ␣2,6-sialyltransferase (ST6GalNAc)-I and ST6GalNAc-II, that are candidate enzymes for Sialyl-Tn synthases. We expressed soluble recombinant hST6GalNAc-I and hST6GalNAc-II and characterized the substrate specificity of both enzymes toward a panel of glycopeptides, glycoproteins, and other synthetic glycoconjugates. The recombinant ST6GalNAc-I and ST6GalNAc-II showed similar substrate specificity toward glycoproteins and GalNAc␣-O-Ser/Thr glycopeptides, such as glycopeptides derived from the MUC2 mucin and the HIVgp120. We also observed that the amino acid sequence of the acceptor glycopeptide contributes to the in vitro substrate specificity of both enzymes. We additionally established a gastric cell line, MKN45, stably transfected with the full length of either ST6GalNAc-I or ST6GalNAc-II and evaluated the carbohydrate antigens expression profile induced by each enzyme. MKN45 transfected with ST6GalNAc-I showed high expression of Sialyl-Tn, whereas MKN45 transfected with ST6GalNAc-II showed the biosynthesis of the Sialyl-6T structure [Gal␤1-3 (Neu5Ac␣2-6)GalNAc-O-Ser/Thr].In conclusion, although both enzymes show similar in vitro activities when Tn antigen alone is available, whenever both Tn and T antigens are present, ST6GalNAc-I acts preferentially on Tn antigen, whereas the ST6GalNAc-II acts preferentially on T antigen. Our results show that ST6GalNAc-I is the major Sialyl-Tn synthase and strongly support the hypothesis that the expression of the Sialyl-Tn antigen in cancer cells is due to ST6GalNAc-I activity.

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Helicobacter pylori exploits host glycoconjugates to colonize the gastric niche. Infection can persist for decades promoting chronic inflammation, and in a subset of individuals lesions can silently progress to cancer. This study shows that H. pylori chronic infection and gastric tissue inflammation result in a remodeling of the gastric glycophenotype with increased expression of sialyl-Lewis a/x antigens due to transcriptional up-regulation of the B3GNT5, B3GALT5, and FUT3 genes. We observed that H. pylori infected individuals present a marked gastric local proinflammatory signature with significantly higher TNF-α levels and demonstrated that TNF-induced activation of the NF-kappaB pathway results in B3GNT5 transcriptional up-regulation. Furthermore, we show that this gastric glycosylation shift, characterized by increased sialylation patterns, favors SabA-mediated H. pylori attachment to human inflamed gastric mucosa. This study provides novel clinically relevant insights into the regulatory mechanisms underlying H. pylori modulation of host glycosylation machinery, and phenotypic alterations crucial for life-long infection. Moreover, the biosynthetic pathways here identified as responsible for gastric mucosa increased sialylation, in response to H. pylori infection, can be exploited as drug targets for hindering bacteria adhesion and counteract the infection chronicity.