Characterization of Two Glycolipid:α2-3Sialyltransferases, SAT-3 (CMP-NeuAc:nLcOse4Cer α2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer α2-3sialyltransferase), from Human Colon Carcinoma (Colo 205) Cell Line

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Abstract
Sialyltransferase activities, SAT-3 (CMP-NeuAc:nLcOse4Cer α2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer α2-3sialyltransferase), in Colo 205 cells catalyze the transfer of sialic acid to the terminal galactose of GlcNAc- 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 taurocholate 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 α1-acid glycoprotein, indicating that this enzyme is different from the sialyltransferase (ST3N) that synthesizes NeuAcα2-3Gal 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 α2-3sialyltransferase (SAT-3 or STZ) that 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 α2-3 linkage between sialic acid and terminal galactose of glycolipid substrates.