Molecular cloning and expression of a human hST8Sia VI (alpha2,8-sialyltransferase) responsible for the synthesis of the diSia motif on O-glycosylproteins.
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.
Teintenier-Lelièvre
Mélanie
M
Unité de Glycobiologie Structurale et Fonctionnelle, CNRS UMR 8576, IFR 118, GDR CNRS 2590, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France.
Julien
Sylvain
S
Juliant
Sylvie
S
Guerardel
Yann
Y
Duonor-Cérutti
Martine
M
Delannoy
Philippe
P
Harduin-Lepers
Anne
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002874:Chromosome Mapping; D002879:Chromosomes, Human, Pair 10; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010835:Phytohemagglutinins / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D012799:Sialyltransferases / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
D006023:Glycoproteins; D010835:Phytohemagglutinins; D011134:Polysaccharides; C100156:leukoagglutinins, plants; D012799:Sialyltransferases; C464440:alpha2,8-sialyltransferase VI