Molecular Cloning of Pigeon UDP-galactose:β-D-Galactoside α1,4-Galactosyltransferase and UDP-galactose:β-D-Galactoside β1,4-Galactosyltransferase, Two Novel Enzymes Catalyzing the Formation of Galα1-4Galβ1-4Galβ1-4GlcNAc Sequence

We previously found that pigeon IgG possesses unique N-glycan structures that contain the Gal alpha 1-4Gal beta 1-4Gal beta 1-4GlcNAc sequence at their nonreducing termini. This sequence is most likely produced by putative alpha 1,4- and beta 1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Gal alpha 1-4Gal and Gal beta 1-4Gal sequences on the N-glycans, respectively. Because no such glycan structures have been found in mammalian glycoproteins, the biosynthetic enzymes that produce these glycans are likely to have distinct substrate specificities from the known mammalian GalTs. To study these enzymes, we cloned the pigeon liver cDNAs encoding alpha 4GalT and beta 4GalT by expression cloning and characterized these enzymes using the recombinant proteins. The deduced amino acid sequence of pigeon alpha 4GalT has 58.2% identity to human beta 4GalT and 68.0 and 66.6% identity to putative alpha 4GalTs from chicken and zebra finch, respectively. Unlike human and putative chicken alpha 4GalTs, which possess globotriosylceramide synthase activity, pigeon alpha 4GalT preferred to catalyze formation of the Gal alpha 1-4Gal sequence on glycoproteins. In contrast, the sequence of pigeon beta 4GalT revealed a type II transmembrane protein consisting of 438 amino acid residues, with no significant homology to the glycosyltransferases so far identified from mammals and chicken. However, hypothetical proteins from zebra finch (78.8% identity), frogs (58.9-60.4%), zebrafish (37.1-43.0%), and spotted green pufferfish (43.3%) were similar to pigeon beta 4GalT, suggesting that the pigeon beta 4GalT gene was inherited from the common ancestors of these vertebrates. The sequence analysis revealed that pigeon beta 4GalT and its homologs form a new family of glycosyltransferases.