Characterization of two glycolipid: alpha 2-3sialyltransferases, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), from human colon carcinoma (Colo 205) cell line.
Sialyltransferase activities, SAT-3 (CMP-NeuAc:nLcOse4Cer alpha 2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer alpha 2-3sialyltransferase), in Colo 205 cells catalyze the transfer of sialic acid to the terminal galactose of GlcNc-- and GalNAc-containing glycolipid substrates, respectively. Competition kinetic studies with nLcOse4Cer and GM1 as substrates in a sialyltransferase assay show that these two activities are catalyzed by two different catalytic entities. The two enzymes were co-solubilized with taurochlorate and resolved by DEAE--Cibacron Blue--Sepharose column chromatography into two elution peaks. The column eluent with SAT-3 activity failed to transfer sialic acid to asialo alpha(1)-acid glycoprotein, indicating that this enzyme is different from the sialyltransferase (ST3N) that synthesizes NeuAc alpha 2-3Gal linkage in asparagine-linked oligosaccharides of glycoprotein. However, SAT-3 activity can be immunoprecipitated with a polyclonal antibody produced against a protein expressed in Escherichia coli as GST-fusion protein from an ECB cDNA homolog of an alpha 2-3 sialyltransferase SAT-3 or STZ) the has been cloned from human melanoma cell and human placenta. Thus a concentration-dependent decrease in the residual SAT-3 activity relative to SAT-4 activity was observed in the supernatant after precipitation of the immune complex. Expression of SAT-3 (STZ) cDNA was also detected in Colo 205 cell by RT-PCR, followed by sequence analysis of the RT-PCR product. Characterization of the catalytic reaction products of SAT-3 and SAT-4 with thin-layer chromatography, sialidase treatment, and binding to specific antibodies indicates that both SAT-3 and SAT-4 catalyze the formation of alpha 2-3 linkage between sialic acid and terminal galactose of glycolipid substrates.
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Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
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D001483:Base Sequence; D002277:Carcinoma / Q000201:enzymology*; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism; D003110:Colonic Neoplasms / Q000201:enzymology*; D003429:Cross Reactions; D018076:DNA, Complementary / Q000032:analysis; D004791:Enzyme Inhibitors; D005677:G(M1) Ganglioside / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism; D006801:Humans; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D012794:Sialic Acids / Q000378:metabolism*; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity
D002518:Ceramides; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D006017:Glycolipids; D012794:Sialic Acids; D005677:G(M1) Ganglioside; D012799:Sialyltransferases; C099134:neolactotetraosylceramide alpha-2,3-sialyltransferase; C019916:CMP-N-acetylneuraminic acid-monosialoganglioside sialyltransferase; D019158:N-Acetylneuraminic Acid