CLONING AND EXPRESSION OF SAT-3 INVOLVED IN SA-LE(X) BIOSYNTHESIS - INHIBITION STUDIES WITH POLYCLONAL ANTIBODY AGAINST GST-SAT-3 FUSION PROTEIN

The SAT-3 activity (CMP-NeuAc:Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4G lc-ceramide alpha 2-3 sialyltransferase) involved in the biosynthesis of sialyl Le(x) has been characterized in human colon carcinoma cells and embryonic chicken brains. Using RT-FCR-based strategy, we have iso lated partial cDNA clones of SAT-3 from ECB and Colo-205 mRNAs. Suitab le primers from sialylmotif and N-terminal sequence of human placenta SAT-3 (HP-SAT-3) were used. The 800 bp cDNA fragment encoding a region (90%) of alpha 2-3 sialyltransferase (SAT3) catalytic domain from ECB has been expressed as a glutathione S-transferase (GST) soluble fusio n protein (62 kDa) in E. coli and purified over glutathione-agarose af finity matrix. Polyclonal antibody has been produced against affinity- purified catalytic domain of SAT-3 (GST-SAT-3 fusion protein). A conce ntration-dependent polyclonal antibody binding to native SAT-3 has als o been demonstrated by measuring the residual SAT-3 activity in the en zyme fractions from Colo-205. The marked inhibition (> 80%) of SAT-3 a ctivity and relatively less inhibition (< 20%) of SAT-4 activity (CMP- NeuAc:GgOse4Cer alpha 2-3sialyl transferase) suggests strongly the exi stence of two different gene products (SAT-3 and SAT-4) in human colon carcinoma Colo-205 cells and in embryonic chicken brains (ECB).