Molecular cloning and functional expression of human ST6GalNAc II. Molecular expression in various human cultured cells

A cDNA clone encoding a human Gal beta 1-3GalNAc alpha 2,6-sialyltransferas e (designated hST6GalNAc II) was identified employing the PCR with degenera ted primers to the sialylmotifs, followed by BLAST analysis of databanks. T his sialyltransferase sequence is similar to that of previously cloned ST6G alNAc II (chicken and mouse) and shows the sialylmotifs that are present in all eukaryotic members of the sialyltransferase gene family. The predicted amino acid sequence encodes a putative type II transmembrane protein as fo und for other eukaryotic sialyltransferases and shows significant similarit y to chicken (56.8% identity) and mouse (74.6% identity) enzymes. Expressio n of a secreted form of hST6GalNAc II in COS-7 cells showed that the gene p roduct had Gal beta 1-3GalNAc (sialyl to GalNAc) alpha 2,6-sialyltransferas e activity. In vitro analysis of substrate specificity revealed that the en zyme required a peptide aglycone fraction to be active and used both Gal be ta 1-3GalNAc and Neu5Ac alpha 2-3Gal beta 1-3GalNAc as acceptor substrates. Northern analysis revealed a restricted expression pattern of two hST6GalN Ac II transcripts, a 2.0 kb mRNA found mainly in skeletal muscle, heart and kidney and a 1.8 kb mRNA found in placenta, lung and leukocytes. No transc riptional expression was detected in brain, thymus or spleen. Transcription al expression of the ST6GalNAc II gene was followed in various human cell l ines and found to be expressed in almost all cell types with notable except ions for several myeloid and lymphoid cell lines. (C) 2000 Elsevier Science B.V. All rights reserved.