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 1-3 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-NeuAc:GgOse4Cer 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).
Basu
S S
SS
Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556, USA.
Basu
M
M
Dastgheib
S
S
Ghosh
S
S
Basu
S
S
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002642:Chick Embryo; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D000080506:Sialyl Lewis X Antigen; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*
D000906:Antibodies; D017931:DNA Primers; D018076:DNA, Complementary; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D000080506:Sialyl Lewis X Antigen; D012799:Sialyltransferases; C099134:neolactotetraosylceramide alpha-2,3-sialyltransferase; C019916:CMP-N-acetylneuraminic acid-monosialoganglioside sialyltransferase