The
Sialyl-Tn antigen (Neu5Acalpha2-6GalNAc-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 alpha2,6-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 GalNAcalpha-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 [Galbeta1-3 (Neu5Acalpha2-6)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.