Role of the human ST6GalNAc-I and ST6GalNAc-II in the synthesis of the cancer-associated sialyl-Tn antigen.
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.
Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
Nuno T
NT
Marcos
Sandra
S
Pinho
Catarina
C
Grandela
Andrea
A
Cruz
Bénédicte
B
Samyn-Petit
Anne
A
Harduin-Lepers
Raquel
R
Almeida
Filipe
F
Silva
Vanessa
V
Morais
Julia
J
Costa
Jan
J
Kihlberg
Henrik
H
Clausen
Celso A
CA
Reis
D015295:Antigens, Tumor-Associated, Carbohydrate / Q000096:biosynthesis*; D002240:Carbohydrate Sequence; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D006020:Glycopeptides / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012799:Sialyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013274:Stomach Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D015295:Antigens, Tumor-Associated, Carbohydrate; D006020:Glycopeptides; D011994:Recombinant Proteins; C055491:sialosyl-Tn antigen; D012799:Sialyltransferases; C088391:galactosyl-1-3-N-acetylgalactosaminyl-specific 2,6-sialyltransferase; C038525:CMP-N-acetylneuraminate-alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase