Purification and characterization of avian glycolipid: beta-galactosyltransferases (GalT-4 and GalT-3): cloning and expression of truncated betaGalT-4.
Acidic glycolipid of ganglio-(containing sialic acid) and sialyl-lactofucosyl-type, SA-Lex (containing sialic acid and fucose) are developmentally regulated and appear to be ubiquitous 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 beta1,4galactosyltransferase) from ECB and human Colo-204 were isolated. These cDNAs were expressed as a soluble glutathione-S-transferase fusion protein (48 kDa) in Escherichia coli. 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:GM2beta1,3galactosyltransferase) was purified from 19-day-old ECB, and a polyclonal antibody was produced against the peptide backbone for immunoscreening of a lambdaZAP 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.
Basu
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Department of Chemistry and Biochemistry, University of Notre Dame, IN, USA.
Dastgheib
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Ghosh
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Basu
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Kelly
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Basu
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D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005700:Galactosyltransferases / Q000235:genetics* / Q000302:isolation & purification*; D006017:Glycolipids / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D010957:Plasmids
D018076:DNA, Complementary; D006017:Glycolipids; D005700:Galactosyltransferases