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...

Sperm motility and maturation are known to be affected by a host of factors encountered en route in both male and female genital tracts prior to fertilization. Using a concerted proteomics and glycomics approach with advanced mass spectrometry-based glycan sequencing capability, we show in this work that 24p3, an abundant mouse uterine luminal fluid (ULF) glycoprotein also called lipocalin 2 (Lcn2), is highly fucosylated in the context of carrying multiple Lewis X and Y epitopes on complex type N-glycans at its single glycosylation site. The predominance of Lewis X/Y along with Neu5Ac␣2-6 sialylation was found to be a salient feature of the ULF glycome, and several other protein carriers were additionally identified including the highly abundant lactotransferrin, which is N-glycosylated at two sites, both with a similar range of highly fucosylated N-glycans. A comparative glycomics analysis of the male genital tract fluids revealed that there is a gradient of glycomic complexity from the cauda to caput regions of the epididymis, varying from high mannose to sialylated complex type N-glycans but mostly devoid of fucosylation. The seminal vesicle fluid glycome, on the other hand, carries equally abundant multimeric Lewis X structures but is distinctively lacking in additional fucosylation of the terminal galactose to give the Lewis Y epitope typifying the glycome of female ULF. One-dimensional shotgun proteomics analysis identified over 40 proteins in the latter, many of which are reported for the first time, and a majority are notably involved in immune defense and antigen processing. Further sperm binding and motility assays suggest that the Lewis X/Y epitopes do contribute to the sperm motility-enhancing activity of 24p3, whereas lactotransferrin is largely inactive in this context despite being similarly glycosylated. These findings underline the importance of glycoproteomics in delin-