Phylogenetic expression of Galalpha1-4Gal on avian glycoproteins: glycan differentiation inscribed in the early history of modern birds

Abstract

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.

Fig. 1.

Representative N-glycan structures of ovalbumins from chicken (36-38) and pigeon (12, 13). Specificity of lectins (Con A, RCA-I, and GS-I) and anti-P₁ mAb for the detection of oligosaccharides were also indicated. Monosaccharides were denoted as follows: M, mannose; GN, N-acetylglucosamine; G, galactose; NA, N-acetylneuraminic acid.

Fig. 2.

Example of lectin/immunoblottings of avian egg white glycoproteins. Major egg white glycoproteins were visualized with Coomassie brilliant blue (CBB) staining. Galα1-4Gal was detected by GS-I and anti-P₁ mAb. Abbreviations for samples based on the common (English) name of birds are shown in Table 1.

Fig. 3.

Phylogeny of modern birds and expression of Galα1-4Gal on avian egg white glycoproteins. The phylogenetic tree is based on DNA-DNA hybridization by Sibley et al. (29). The position of the Turniciformes in their classification is uncertain (p. 255 in ref. 29), and is indicated with dashed line. Avian orders belonging to Ratitae (^*) and Galloanserae (^**) are indicated with asterisks. Non-Ratitae/Galloanserae modern birds are sometimes called Neoaves. The examples of birds and recorded number of species in each order (27) are indicated in parentheses. Ciconiiformes in Sibley's classification (27, 28) are traditionally classified as Charadriiformes (e.g., gulls), Falconiformes (e.g., eagles), Podicipediformes (grebes), Pelecaniformes (e.g., tropicbirds), Ciconiiformes (e.g., storks), Sphenesciformes (penguins), Gaviiformes (loons), and Procellariformes (e.g., albatrosses). The number of Galα1-4Gal-positive species and the total species examined in this study are indicated in the right column. N/A, not available. [Modified from ref. with permission from Yale Univ. Press (Copyright 1990, Yale Univ. Press).]

Fig. 4.

Schematic models for acquisition/loss of Galα1-4Gal expression capability in the modern birds during their diversification. (A) The common ancestors of modern birds already possess the capability to express Galα1-4Gal glycoproteins (open circle, followed by thick lines) before the differentiation into Ratitae (R), Galloanserae (G), and non-Ratitae/Galloanserae (N), but this capability might have been lost (cross, followed by thin lines) in Ratitae and Galloanserae after they are separated from others. (B) The common ancestors of modern birds did not possess the capability, but it was acquired by non-Ratitae/Galloanserae after they are separated from others. In either models, some species of birds belonging to non-Ratitae/Galloanserae might have lost (thin lines) the capability to express Galα1-4Gal glycoproteins in egg whites at the levels of families and/or genera. The root position of Galloanserae was undefined.