Isolation and Characterization of Major Glycoproteins of Pigeon Egg White

Abstract

Ovotransferrin (POT), two ovalbumins (POA(hi) and POA(lo)), and ovomucoid (POM) were isolated from pigeon egg white (PEW). Unlike their chicken egg white counterparts, PEW glycoproteins contain terminal Gal␣1-4Gal, as evidenced by GS-I lectin (specific for terminal ␣-Gal), anti-P 1 (Gal␣1-4Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc␤1-1Cer) monoclonal antibody, and P fimbriae on uropathogenic Escherichia coli (specific for Gal␣1-4Gal). Gal␣1-4Gal on PEW glycoproteins were found in N-glycans releasable by treatment with glycoamidase F. The respective contents of N-glycans in each glycoprotein were 3.5%, POT; 17%, POA(hi); and 31-37%, POM. POA(hi) has four N-glycosylation sites, in contrast to chicken ovalbumin, which has only one. High performance liquid chromatography analysis showed that N-glycans on POA(hi) were highly heterogeneous. Mass spectrometric analysis revealed that the major N-glycans were monosialylated tri-, tetra-, and penta-antennary oligosaccharides containing terminal Gal␣1-4Gal with or without bisecting N-acetylglucosamine. Oligosaccharide chains terminating in Gal␣1-4Gal are rare among N-glycans from the mammals and avians that have been studied, and our finding is the first predominant presence of (Gal␣1-4Gal)-terminated N-glycans.In mammals, e.g. human, pig, rat, mouse, and hamster, Gal␣1-4Gal is normally found in the terminal and internal positions of glycolipids (1-5), as in P 1 (Gal␣1-4Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc␤1-1Cer), P k (Gal␣1-4Gal␤1-4Glc␤1-1Cer) antigens, and disialosyl galactosyl globoside (NeuAc␣2-3Gal␤1-3(NeuAc␣2-3)GalNAc␤1-3Gal␣1-4Gal␤1-4Glc␤1-1Cer) (6 -8).The glycolipids containing Gal␣1-4Gal are known as targets on host cells for infections of some pathogenic microbes, e.g. uropathogenic Escherichia coli (9, 10), Pseudomonas aeruginosa (PA-I lectin) (11), and Streptococcus suis (12)(13)(14). Bacterial enterotoxins, e.g. from E. coli (verotoxin) (15), Shigella dysenteriae Type 1 (Shiga toxin) (16), and Staphylococcus aureus (enterotoxin B) also specifically bind to these glycolipids (17). In contrast, glycoproteins with Gal␣1-4Gal were found only when the mammals were infected by tapeworm Echnococcus granulosus, in hydatid cyst fluid and membrane caused by the infection (18 -20).However, ovomucoids produced in pigeon (Columba livia) and turtle doves (Streptopelia resoria) possess high level of P 1 antigenic activity (21)(22)(23)(24). Pigeon ovomucoid (POM), 1 one of the major glycoproteins in pigeon egg white (PEW), was also found to bind some pathogenic microbes, e.g. uropathogenic E. coli (24 -26) and S. suis (12)(13)(14). Moreover, POM had been recently utilized for isolation of Shiga-like toxin type 1 (27). Although the presence of Gal␣1-4Gal on ovomucoids of turtle dove and pigeon has been evidenced and these glycoproteins can be utilized for the study of microbiology, complete carbohydrate structures of these ovomucoids are not yet known. Only a tentative structure had been proposed for the main oligosaccharide structure of turtle dove ovomucoid, which includes Gal␣1-4Gal sequence at ...

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 also confirmed that the putative chicken ␣4GalT(Gal), which was identified from chicken genome/protein data bases in NCBI, acts as a Gb3 synthase, like human ␣4GalT(Gal). This observation is consistent with the absence of Gal␣1-4Gal on glycoproteins in chicken (4,25). The high similarity between pigeon ␣4GalT(Gal) and chicken Gb3 synthase implies that they are most likely derived from a common ancestral gene relatively recently.…”

“…Mammals express Gal␣1-4Gal on glycolipids, such as globotriosylceramide (Gb3, 2 Gal␣1-4Gal␤1-4Glc-Cer) and P 1 antigen (Gal␣1-4Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc-Cer), but not on glycoproteins, except for those found in hydatid fluids caused by tapeworm (5,6). In contrast, we found that pigeon egg white is a rich source of glycoproteins that possess the Gal␣1-4Gal␤1-4GlcNAc sequence at the nonreducing termini of N-glycans (4). It was demonstrated that pigeon egg white glycoproteins are useful in preventing infection with uropathogenic E. coli (7).…”

“…Gal␣1-4Gal and Gal␤1-4Gal sequences are most likely biosynthesized in pigeon by the actions of putative UDP-Gal:␤-galactoside ␣1,4-galactosyltransferase (␣4GalT(Gal) 4 ) and UDP-Gal:␤-galactoside ␤1,4-galactosyltransferase (␤4GalT-(Gal)), respectively, which have not been reported so far. Because the Gal␣1-4Gal sequence is rarely attached to glycoproteins in mammals, the putative ␣4GalT(Gal) in pigeon is likely to have distinct acceptor substrate specificities from those of mammals.…”

“…Shiga toxins (see Ref. 4 and references therein). Mammals express Gal␣1-4Gal on glycolipids, such as globotriosylceramide (Gb3, 2 Gal␣1-4Gal␤1-4Glc-Cer) and P 1 antigen (Gal␣1-4Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc-Cer), but not on glycoproteins, except for those found in hydatid fluids caused by tapeworm (5,6).…”

Catch Bond-mediated Adhesion without a Shear Threshold

“…When P-fimbriated E. coli were allowed to accumulate on pigeon ovalbumin, a Gal-1,4-Gal-presenting glycoprotein (27,28), for 5 min at shears ranging from 0.01 to 4.3 pN/m 2 ( Fig. 7A), no shear threshold was seen, and accumulation decreased with increased shear.…”