Cloning, expression and gene organization of a human Neu4Ac alpha 2-3Gal beta 1-3GalNAc alpha 2,6-sialyltransferase: hST6GalNAc IV

On the basis of the detection of expressed sequence tag ('EST') similar to the rat N-acetylgalactosamine alpha2,6-sialyltransferase (ST6GalNAc) III cD NA, we have identified a novel member of the human ST6GalNAc family. We hav e isolated a cDNA clone containing an open reading frame that codes for a t ype II membrane protein of 302 amino acids with a seven-amino-acid cytoplas mic domain, an 18-amino-acid transmembrane domain and the smallest describe d catalytic domain of 277 amino acids. This predicted sialyltransferase seq uence is similar to the rat ST6GalNAc III (46.6 %), but was found to be eve n more similar to the recently reported mouse ST6GalNAc IV (88.1 %) on the basis of amino acid sequence identity. Northern-blot analysis showed that t he newly identified gene is expressed constitutively in various adult human tissues as a 2.2 kb transcript, but was also found to be expressed at lowe r levels in brain, heart and skeletal muscle as a 2.5 kb transcript. Expres sion of the hST6GalNAc IV gene was investigated by reverse transcription PC R in Various human cancer cells, and was found to be present in the majorit y of cell types with the exception of the carcinoma cell line T47D and pro- monocyte THP cells. The transient expression in COS-7 cells of the full-len gth cDNA led to the production of an active enzyme sharing the acceptor spe cificity of the ST6GalNAc family towards Neu5Ac alpha2-3Gal beta1-3GalNAc a lpha -O-R (where 'R' denotes H, benzyl, or a peptidic chain). Detailed anal ysis in vitro of substrate specificity revealed that the enzyme required th e trisaccharide Neu5Ac alpha2-3Gal beta1-3GalNAc found on O-glycans and ary lglycosides. In addition, we have clarified the genomic organization of ST6 GalNAc IV gene.