PURIFICATION AND CHARACTERIZATION OF AVIAN GLYCOLIPID - BETA-GALACTOSYLTRANSFERASES (GALT-4 AND GALT-3) - CLONING AND EXPRESSION OF TRUNCATED BETA-GALT-4

Acidic glycolipid of ganglio-(containing sialic acid) and sialyl-lacto fucosyl-type, SA-Le(x) (containing sialic acid and fucose) are develop mentally regulated and appear to be ubiqitous on neuronal and cancer c ell 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 wer e believed to be due to expression of two gene products. A cDNA fragme nts (about 600 bp) encoding the catalytic domain of the GalT-4 (UDP-Ga l:LcOse3Cer beta 1,4galactosyl-transferase) from ECB and human Colo-20 4 were isolated. These cDNAs were expressed as a soluble glutathione-S -transferase fusion protein (48 kDa) in Eschericchia cold. Interaction s between GlcNAc-, UDP-hexanolamine-, and alpha-lactalbumin were studi ed with the purified fusion protein (recombinant and truncated). Funct ionally it was similar to that of native GalT-4 purified (40000-fold) from 11-day-old ECB. GalT-3 (UDP-Gal:G(M2)beta 1,3galactosyltransferas e was purified from 19-day-old ECB, and a polyclonal antibody was prod uced against the peptide backbone for immunoscreening of a lambda ZAP ECB cDNA expression library. Each of the GalT-3 peptides (62 and 65 kD a was analyzed by protein fingerprinting analysis indicating a similar peptide mapping pattern.