Phylogenetic expression of Galα1-4Gal on avian glycoproteins: Glycan differentiation inscribed in the early history of modern birds

Glycoproteins containing Galalpha1-4Gal (galabiose) had been rarely found in vertebrates, except in a few species of birds and amphibians. We had previously reported that pigeon (Columba livia) egg white and serum glycoproteins are rich in N-glycans with Galalpha1-4Gal at nonreducing termini. To investigate the origin of Galalpha1-4Gal expression in avian evolution, we examined the presence of Galalpha1-4Gal glycoproteins in egg whites from 20 orders, 88 families, 163 genera, and 181 species of birds, as probed by Western blot with Griffonia simplicifolia-I lectin (terminal alpha-Gal/GalNAc-specific) and anti-P(1) mAb (Galalpha1-4Galbeta1-4GlcNAcbeta1-specific). One of the significant observations is the total absence of Galalpha1-4Gal glycoproteins in Struthioniformes (four species), Tinamiformes (three species), Craciformes (two species), Galliformes (14 species), and Anseriformes (10 species), which are phylogenetically separated from other orders at earlier stage of modern bird diversification (100-65 million years ago). The presence or absence of Galalpha1-4Gal glycoproteins in other avian orders varied by the species (104 species positive, and 44 species negative), even though some of them belong to the same order or family. Our results revealed that the expression of Galalpha1-4Gal glycoproteins is not rare among avians, and is correlated with the phylogeny. The expression was most likely differentiated at earlier stage of diversification in modern birds, but some birds might have lost the facility for the expression relatively recently.