Based on BLAST analysis of the human and mouse genome databases using the human
CMP sialic acid; alpha2,8-sialyltransferase
cDNA (hST8Sia I; EC 2.4.99.8), a putative
sialyltransferase gene, was identified on human chromosome 10. The genomic organization was found to be similar to that of hST8Sia I and hST8Sia V. Transcriptional expression analysis showed that the newly identified gene was constitutively expressed at low levels in various human tissues and cell lines. We have isolated a full-length
cDNA clone from the
breast cancer cell line MCF-7 that encoded a type II
membrane protein of 398
amino acid residues with the conserved motifs of
sialyltransferases. We have established a mammary cell line (MDA-MB-231) stably transfected with the full-length hST8Sia VI and the analysis of sialylated
carbohydrate structures expressed at the cell surface clearly indicated the disappearance of Neu5Acalpha2-3-sialylated structures. The transient expression of a truncated soluble form of the
enzyme in either COS-7 cells or insect Sf-9 cells led to the production of an active
enzyme in which substrate specificity was determined. Detailed substrate specificity analysis of the hST8Sia VI recombinant
enzyme in vitro, revealed that this
enzyme required the
trisaccharide Neu5Acalpha2-3Galbeta1-3GalNAc (where Neu5Ac is
N-acetylneuraminic acid and GalNAc is N-
acetylgalactosamine) to generate diSia (disialic
acid) motifs specifically on O-
glycans.