Purification and characterization of a CMP-sialic:LcOse4Cer sialyltransferase from human colorectal carcinoma cell membranes

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Abstract
Purified lactotetraosylceramide (Gal.beta.1 .fwdarw. 3GlcNAc.beta.1 .fwdarw. 3Gal.beta.1 .fwdarw. 4Glc]-Cer) was tested for its ability to accept [14C]sialic acid from CMP-[14C]sialic into monosialoganglioside fractions in the presence of membrane fractions purified from human colorectal carcinoma cells (SW1116). Membrane fractions were isolated by three different methods: sucrose density centrifugation, CMP-agarose gel column chromatography, and LcOse4 gel chromatography. We optimized the incubation conditions for detergent dependency (taurocholate), pH (6.3), and acceptor concentration. The sialyltransferase activity was dependent on membrane protein and linear for time up to at least 4 h. The LcOse4 affinity chromatography of the crude microsomal membrane pellet from these cells yielded a membrane fraction that was 136-fold enriched in LcOse4 acceptor specific activity compared to cell homogenates. The apparent Km for the sialyltransferase activity with LcOse4Cer acceptor in the presence of affinity-purified membranes was 20 .mu.M and the Vmax was 7 pmol h-1 (100 .mu.g of protein)-1. Acceptor capabilities of other core structures were 5.sbd.10-fold lower: LcOse4Cer .mchgt. GgOse4Cer > nLcOse4Cer .mchgt. GbOse4Cer. The enzymatic activity was purified further (900-fold) by a combination of LcOse4 and CMP affinity gels. SDS-PAGE electrophoresis of this material showed a major set of closely migrating bands of M4 58,000-54,000 compared to authentic proteins, as well as a minor band at 27,000. We analyzed picomole quantities of the radioactive product by convenient controlled short-term hydrolyses with an endoglycoceramidase and sialidases (from four different sources) in comparison to sialylated tetrasaccharides of known structure. We conclude that the major product of the sialyltransferase activity, present in these affinity column purified membrane fractions, is IV3NeuNAcLcOse4Cer and that the enzyme specifically recognizes lactotetraosylceramide. This enzymatic activity may control the level of, at least, two ganglioside antigens of the lacto series reported previously to be "tumor associated", Ca50 and 19-9. Our results are the first to present extensive purification of this enzymatic activity from human carcinoma cells.

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