Role of the Human ST6GalNAc-I and ST6GalNAc-II in the Synthesis of the Cancer-Associated Sialyl-Tn Antigen

Abstract

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.

Identification and expression of a sialyltransferase responsible for the synthesis of disialylgalactosylgloboside in normal and malignant kidney cells: downregulation of ST6GalNAc VI in renal cancers

“…Among the six members of the ST6GalNAc family cloned to date, ST6GalNAc I and II mainly utilize asialo-structures in Oglycans [36], whereas ST6GalNAc III-VI are considered to be involved in the synthesis of disialyl-structures in α-series gangliosides and O-glycans. [19,20,37,38].…”

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

“…Although both the mouse and human ST6GalNAc-II have been shown to synthesize STn in vitro (26,39), a secreted form of human ST6GalNAc-II apparently did not synthesize this disaccharide (27). To analyze the expression of the human ST6GalNAc-II enzyme in breast cancer 8 cases were selected from the 29 analyzed for expression of STn, representing tumors with high, low, or no expression of this O-glycan.…”

“…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).…”

CDX2 homeoprotein is involved in the regulation of ST6GalNAc-I gene in intestinal metaplasia

“…The biosynthesis of STn has been mostly attributed to ST6GalNAc-I activity, both in vitro and in vivo. [1][2][3][4] It is rarely observed in normal tissues but frequently accompanies cancer progression, being one of the most common features of premalignant lesions of the gastrointestinal tract, namely intestinal metaplasia (IM), 5,6 and of carcinomas, namely gastric carcinomas. [5][6][7] However, to date, little is known about ST6GalNAc-I regulation that might explain aberrant expression of STn in preneoplastic and cancer.…”