MOLECULAR-CLONING OF A DEVELOPMENTALLY-REGULATED N-ACETYLGALACTOSAMINE ALPHA-2,6-SIALYLTRANSFERASE SPECIFIC FOR SIALYLATED GLYCOCONJUGATES

A cDNA encoding a novel sialyltransferase has been isolated employing the polymerase chain reaction using degenerate primers to conserved re gions of the sialylmotif that is present in all eukaryotic members of the sialyltransferase gene family examined to date. The cDNA sequence revealed an open reading frame coding for 305 amino acids, making it t he shortest sialyltransferase cloned to date. This open reading frame predicts all the characteristic structural features of other sialyltra nsferases including a type II membrane protein topology and both sialy lmotifs, one centrally located and the second in the carboxyl-terminal portion of the cDNA. When compared with all other sialyltransferase c DNAs, the predicted amino acid sequence displays the lowest homology i n the sialyltransferase gene family. Northern analysis shows this sial yltransferase to be developmentally regulated in brain with expression persisting through adulthood in spleen, kidney, and lung. Stable tran sfection of the full-length cDNA in the human kidney carcinoma eel lin e 293 produced an active sialyltransferase with marked specificity for the sialoside, Neu5Ac alpha 2,3Gal beta 1,3GalNAc and glycoconjugates carrying the same sequence such as G(M1b) and fetuin. The disialylate d tetrasaccharide formed by reacting the sialyltransferase with the af orementioned sialoside was analyzed by one- and two-dimensional H-1 an d C-13 NMR spectroscopy and was shown to be the Neu5Ac alpha 2,3Gal be ta 1,3(Neu5Ac alpha 2,6)GalNAc sialoside. This indicates that the enzy me is a GalNAc alpha 2,6-sialyltransferase. Since two other ST6GalNAc sialyltransferase cDNAs have been isolated, this sialyltransferase has been designated ST6GalNAc III. Of these three, ST6GalNAc III displays the most restricted acceptor specificity and is the only sialyltransf erase cloned to date capable of forming the developmentally regulated ganglioside G(D1 alpha) from G(M1b).