| dc.description.abstract | The recruitment of human peripheral blood leukocytes to sites of infection and inflammation requires the surface expression of Sialyl Lewis x glycoconjugates (SLex) on white blood cells and their interaction with E- and P-selectins on activated endothelial cells. Human ST3Gal III, ST3Gal IV and ST3Gal VI genes code for a2,3?sialyltransferases potentially involved in the biosynthesis of the SLex epitope. In order to characterize these a2,3-sialyltransferases we have cloned and sequenced RNA transcripts of these genes. Our clones have revealed an unexpected heterogeneity in transcript isoforms. Among our ST3Gal IV clones we isolated nine alternatively spliced transcripts covering the coding region of the human ST3Gal IV gene. Five of these isotranscripts have not been described before. Within our ST3Gal III clones we isolated 26 different transcripts from peripheral blood leukocytes and fetal brain, which showed a wide variety of deletions from 45 to 896 nucleotides, and insertions of 26 to 173 nucleotides. Among the insertions we identified two new exons (E3, E6). Eight of the ST3Gal III isotranscripts coded for potentially active enzymes. ST3GalVI en mening??In order to investigate if the alternatively spliced isotranscripts were specific for human peripheral blood leukocytes, we analyzed by RT-PCR and laser-induced fluorescent capillary electrophoresis (LIF-CE) the expression in twenty-one human tissues. We found a highly tissue specific expression of both ST3Gal III and ST3Gal IV isotranscripts. For the ST3Gal III gene, neural and muscular tissues showed the most complex patterns and were distinctly different from all other tissues examined.We stably transfected HEK 293 cells with six of the potentially enzymatically active isoforms of ST3Gal III. Structural differences in the isoenzymes were limited to exon losses in the stem regions of these enzymes. Crude cell extracts from all transfected clones showed a2,3-sialyltransferase activities preferentially towards the type 1-chain disaccharide precursor (Galb1,3GlcNAc) but with different efficiencies and with varying specificities for the type 2-chain (Galb1,4GlcNAc) and Galb1,3GalNAc precursors. Other splice variants of ST3Gal III, unlikely to express enzymatic activities, but clearly exhibiting a very tissue specific expression, may have other biological functions but this remains to be clarified.The RT-PCR-LIF-CE technique, as we have used it, has an unique capacity to identify alternatively spliced transcripts with minor sequence differences as well as sufficient reproducibility and sensitivity for qualitative and semi-quantitative analysis of gene transcripts not provided by other methods. We have shown that redundancy of critical sialyltransferase activities not only appears between different genes but also between different isoenzymes obtained through alternative splicing of the coding sequence of such genes. | en |
