摘要：

Top of pageMaterials and methods Materials Preparation of asialo-glycoproteins Construction of expression vectors of hST6GalII and transfections Western blot analysis of a soluble ST6GalII Confocal microscopy Construction of recombinant baculoviruses and production of soluble ST6GalI and ST3GalIII in Sf9 Sialyltransferase assays Linkage analysis by sialidase digestion Multiple tissue expression array and northern analysis Results Identification and isolation of human ST6GalII cDNA ST6Gal-II gene expression Expression of a recombinant hST6GalII Linkage analysis ST6GalII induces the expression of NeuAcα2–6Gal structures at the cell surface of transfected cells Discussion The ST6Gal subfamily Sequence analysis Gene expression pattern Expression and specific activity of hST6GalII Acknowledgements References blast analysis of the human and mouse genome sequence databases using the sequence of the human CMP-sialic acid:β-galactoside α-2,6-sialyltransferase cDNA (hST6GalI, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresponding cDNA was also found as an expressed sequence tag of human brain. This gene contained a 1590bp open reading frame divided in five exons and the deduced amino-acid sequence didn't correspond to any sialyltransferase already known in other species. Multiple sequence alignment and subsequent phylogenic analysis showed that this new enzyme belonged to the ST6Gal subfamily and shared 48% identity with hST6Gal-I. Consequently, we named this new sialyltransferase ST6GalII. A construction in pFlag vector transfected in COS-7 cells gave raise to a soluble active form of ST6GalII. Enzymatic assays indicate that the best acceptor substrate of ST6GalII was the free disaccharide Galβ1–4GlcNAc structure whereas ST6GalI preferred Galβ1–4GlcNAc-R disaccharide sequence linked to a protein. The α2,6-linkage was confirmed by the increase of Sambucus nigra agglutinin-lectin binding to the cell surface of CHO transfected with the cDNA encoding ST6GalII and by specific sialidases treatment. In addition, the ST6GalII gene showed a very tissue specific pattern of expression because it was found essentially in brain whereas ST6GalI gene is ubiquitously expressed.

展开