Cloning and expression of SAT-3 involved in SA-Le(x) biosynthesis: inhibition studies with polyclonal antibody against GST-SAT-3 fusion protein.

ABSTRACT

The SAT-3 activity (CMP-NeuAcGal beta 1-4GlcNAc beta 1-3 Gal beta 1-4Glc-ceramide alpha 2-3 sialytransferase) involved in the biosynthesis of sialy Le(x) has been characterized in human colon carcinoma cells and embryonic chicken brains. Using RT-PCR-based strategy, we have isolated partial cDNA clones of SAT-3 from ECB and Colo-205 mRNAs. Suitable 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 (SAT-3) catalytic domain from ECB has been expressed as a glutathione S-transferase (GST) soluble fusion protein (62 kDa) in E. coli and purified over glutathione-agarose affinity matrix. Polyclonal antibody has been produced against affinity-purified catalytic domain of SAT-3 (GST-SAT-3 fusion protein). A concentration-dependent polydonal antibody binding to native SAT-3 has also been demonstrated by measuring the residual SAT-3 activity in the enzyme fractions from Colo-205. The marked inhibition (> 80%) of SAT-3 activity and relatively less inhibition (< 20%) of SAT-4 activity (CMP-NeuAcGgOse4Cer alpha 2-3sialyl transferase) suggests strongly the existence of two different gene products (SAT-3 and SAT-4) in human colon carcinoma Colo-205 cells and in embryonic chicken brains (ECB).