Purification and characterization of avian glycolipid:: β-galactosyltransferases (GalT-4 and GalT-3):: cloning and expression of truncated βGalT-4

Acidic glycolipid of ganglio-(containing sialic acid) and sialyl-lactofucosyl-type, SA-Le(x) (containing sialic acid and fucose) are developmentally regulated and appear to be ubiqitous on neuronal and cancer cell surfaces of animals. Two glycolipid: beta-galactosyltransferases, GalT-3 and GalT-4, were characterized in embryonic chicken brain (ECB). Based on substrate competition experiments, these two activities were believed to be due to expression of two gene products. A cDNA fragments (about 600 bp) encoding the catalytic domain of the GalT-4 (UDP-Gal:LcOse3Cer beta 1,4galactosyl-transferase) from ECB and human Colo-204 were isolated. These cDNAs were expressed as a soluble glutathione-S-transferase fusion protein (48 kDa) in Eschericchia cold. Interactions between GlcNAc-, UDP-hexanolamine-, and alpha-lactalbumin were studied with the purified fusion protein (recombinant and truncated). Functionally it was similar to that of native GalT-4 purified (40000-fold) from 11-day-old ECB. GalT-3 (UDP-Gal:G(M2)beta 1,3galactosyltransferase was purified from 19-day-old ECB, and a polyclonal antibody was produced against the peptide backbone for immunoscreening of a lambda ZAP ECB cDNA expression library. Each of the GalT-3 peptides (62 and 65 kDa was analyzed by protein fingerprinting analysis indicating a similar peptide mapping pattern.