N-Glycan Structures of Pigeon IgG

Abstract

We had shown previously that all major glycoproteins of pigeon egg white contain Gal␣1-4Gal epitopes (Suzuki, N., Khoo, K. H., Chen, H. C., Johnson, J. R., and Lee, Y. C. (2001) J. Biol. Chem. 276, 23221-23229). We now report that Gal␣1-4Gal-bearing glycoproteins are also present in pigeon serum, lymphocytes, and liver, as probed by Western blot with Griffonia simplicifolia-I lectin (specific for terminal ␣-Gal) and anti-P 1 (specific for Gal␣1-4Gal␤1-4GlcNAc␤1-) monoclonal antibody. One of the major glycoproteins from pigeon plasma was identified as IgG (also known as IgY), which has Gal␣1-4Gal in its heavy chains. High pressure liquid chromatography, mass spectrometric (MS), and MS/MS analyses revealed that N-glycans of pigeon serum IgG included (i) high mannose-type (33.3%), (ii) disialylated biantennary complex-type (19.2%), and (iii) ␣-galactosylated complex-type N-glycans (47.5%). Bi-and tri-antennary oligosaccharides with bisecting GlcNAc and ␣1-6 Fuc on the Asn-linked GlcNAc were abundant among N-glycans possessing terminal Gal␣1-4Gal sequences. Moreover, MS/MS analysis identified Gal␣1-4Gal␤1-4Gal␤1-4GlcNAc branch terminals, which are not found in pigeon egg white glycoproteins. An additional interesting aspect is that about two-thirds of high mannose-type N-glycans from pigeon IgG were monoglucosylated. Comparison of the N-glycan structures with chicken and quail IgG indicated that the presence of high mannose-type oligosaccharides may be a characteristic of these avian IgG. Pigeon (Columba livia) egg white (PEW)1 is a rich source of glycoproteins containing Gal␣1-4Gal (galabiose). All four major PEW glycoproteins, ovotransferrin, two ovalbumins, and ovomucoid, contain Gal␣1-4Gal (1), and all of their constituent N-glycans were found to possess Gal␣1-4Gal at the non-reducing termini of tri-, tetra-, and penta-antennary structures (2). No sialylation is found on the branch that contains the Gal␣1-4Gal sequence. Because biosynthesis and glycosyl modification of major egg white glycoproteins of chicken are carried out in tubular gland cells of oviduct (3), the most likely site of fabricating Gal␣1-4Gal linkages on the PEW glycoproteins would be in the corresponding cells of pigeon oviduct.Gal␣1-4Gal in glycoproteins is, however, uncommon in nature. Mammals (e.g. human, pig, rat, mouse, and hamster) express Gal␣1-4Gal mostly on glycolipids (4 -8), as in P 1 (Gal␣1-4Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc␤1-1Cer) and P k (Gal␣1-4Gal␤1-4Glc␤1-1Cer) antigens, but rarely on glycoproteins. An exception in mammals is the case of glycoproteins with Gal␣1-4Gal in hydatid cyst fluid and membrane infected by tapeworm Echnococcus granulosus (9 -11). In animals other than birds and mammals, the only recorded presence of Gal␣1-4Gal was in O-glycosides such as those found in an insect cell line from Mamestra brassicae (12) or egg jelly mucins from amphibians, such as Xenopus laevis (13), Rana clamitans (14), and Ambystoma mexicanum (15).The presence of Gal␣1-4Gal or substances similar to P 1 antigen on glycoproteins is limited even...

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

“…Pigeon liver was utilized as a source of mRNA to construct the cDNA library for expression cloning, because we detected (Gal␣1-4Gal)-containing glycoproteins in this tissue (12). After the selection using anti-P 1 mAb (specific for Gal␣1-4Gal␤1-4GlcNAc) by cell sorting, the plasmid designated as pcDNA-pigeon␣4GalT(Gal) was isolated.…”

“…Pigeon IgG possesses another unique glycan sequence, Gal␣1-4Gal␤1-4Gal␤1-4GlcNAc, at the nonreducing termini of N-glycans (12). The same sequence is found in O-glycans from the salivary gland mucin of the Chinese swiftlet (13).…”

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

“…Additionally, coenzymes or specific chaperons are sometimes also critical for the enzyme activity (58). Whereas pigeon expresses Gal␣1-4Gal glycoproteins not only in egg whites but also in other tissues and f luids (14), no Gal␣1-4Gal has been detected so far in chicken. The apparent absence of Gal␣1-4Gal glycoproteins in entire body of chicken probably ref lects inactivation or absence of the ␣-1,4-GalT gene.…”

“…We recently showed that pigeon (Rock Dove, Columba livia) express Gal␣1-4Gal (galabiose) antigens attached on glycoproteins from egg white (12,13) and serum (14), and analyzed their N-glycan structures (Fig. 1).…”

MALDI In-Source Decay, from Sequencing to Imaging

“…As glycans can be easily isolated and purified, the ISD and pMS 3 -sequencing approaches were also employed to characterize thoroughly the structures of both native [43,[54][55][56][57][58][59][60] and permethylated glycans [61][62][63][64][65][66], allowing unambiguous discrimination of isobaric species. In ref.…”