Figure 1.
Structures of PA-oligosaccharides used as acceptor substrates for the GalTs assay.
PA-derivatized N-glycan A and N-glycan B were utilized as substrates of α/β4GalTs(Gal) and β4GalT(GlcNAc), respectively.
Figure 2.
Antibody/lectin-staining of avian egg white glycoproteins and isolated egg yolk IgG.
Egg white glycoproteins (A, 2.5 µg/lane) or egg yolk IgG (B, 1.5 µg/lane) from chicken, duck, emu, guineafowl, ostrich, peafowl, pigeon, quail, and turkey were blotted onto a membrane, and visualized with CBB-staining. Pigeon IgG (for CBB and anti-P1 mAb stainings) and α-galactosidase-treated pigeon IgG (for anti-(Galβ1-4Gal) mAb and ECA stainings) were used as controls [11].
Figure 3.
Digestion of quail ovomucoid and avian egg yolk IgG with exogalactosidases or glycoamidase F (GAF).
A, Quail egg white proteins and quail ovomucoid were untreated (−) or treated with β4-galactosidase (β). B, The isolated egg yolk IgGs from duck, emu, guineafowl, ostrich, peafowl, and turkey were untreated (−) or treated with β4-galactosidase (β) or GAF (N). In B, pigeon egg yolk IgG was treated with α-galactosidase (α), α-galactosidase and then β4-galactosidase (αβ), or GAF (N). Each sample was separated by SDS-PAGE, transferred onto PVDF membranes, and stained with CBB or anti-(Galβ1-4Gal) mAb.
Table 1.
List of birds whose eggs and/or tissues were used.
Table 2.
Identification of avian egg yolk IgGa.
Figure 4.
MALDI-MS profiles of permethylated total IgG N-glycans from turkey, guineafowl, and peafowl.
The avian IgG N-glycans before (A) and after (B) β4-galactosidase digestion were permethylated and analyzed with MALDI-MS. The most probable structures corresponding to the afforded major [M+Na]+ molecular ion signals are annotated along with their monoisotopic values. Assignments are based on a combination of inferred glycosyl compositions, susceptibility to β-galactosidase digestion (B) and MS/MS data obtained on the more abundant components (Figure 5). The MS/MS data positively identify N-glycans with a bisecting GlcNAc as the major complex type structures but do not rule out the presence of alternative non-bisected structures. Unassigned signals that did not give interpretable glycan-like MS/MS spectra are marked with an asterisk (*). Monosaccharide symbols used conform to the recommendations by the Consortium for Functional Glycomics.
Figure 5.
MALDI MS/MS and nanoESI-MSn analysis of the permethylated N-glycans of avian IgG.
High energy CID MS/MS analyses on MALDI-TOF/TOF readily identified the presence of bisecting GlcNAc and core fucosylation on most complex type structures, as shown by the representative spectra for the sodiated molecular ions at m/z 3054 (A) and 2850 (B) from permethylated turkey and peafowl IgG N-glycans, respectively. The presence of Gal-Gal-GlcNAc in the former but not the latter is confirmed by the non-reducing terminal B ion at m/z 690. The B ion at m/z 464 in (A), which corresponds to the non-reducing terminal oxonium ion of Hex-HexNAc, indicates that an alternative non-bisected, triantennary structural isomer was also present but at smaller amount since no other supporting ions could be detected. The linkage of the Gal-Gal was established by nanoESI-MSn analysis, by observing the characteristic 3,5A ion at m/z 329 at the level of MS4 (C). Sialylation at 6 and not 3 position of Gal was likewise established by detecting the characteristic 3,5A ion at m/z 486 at the level of MS4 (D). Assignments of all other major fragment ions are schematically illustrated on each of the Figures, adopting the ion nomenclature as described previously [18], [21].
Figure 6.
Analysis of the tissue distributions of α4GalT(Gal), β4GalT(Gal), and β4GalT(GlcNAc) from quail and bamboo partridge.
Microsomal fractions or tissue extracts were prepared from various tissues of quail (A) or bamboo partridge (B). The values represent the means ±S.D. of duplicate samples. Scales for β4GalT(Gal) (open bar) activities were indicated on the left y-axes, and those for β4GalT(GlcNAc) (hatched bar) activities on the right y-axes of each graph. No α4GalT(Gal) activities were detected in any tissues from quail or bamboo partridge. β4GalT(Gal) activities were detected only in tissues from quail.
Figure 7.
Antibody/lectin-staining of protein extracts from various tissues of quail (A) and bamboo partridge (B).
Proteins (20 µg/lane) blotted onto PVDF membranes were visualized with CBB-staining. Pigeon IgG (for CBB and anti-P1 mAb stainings) and α-galactosidase-treated pigeon IgG (for anti-(Galβ1-4Gal) mAb and ECA stainings) were used as controls.
Figure 8.
Phylogenetic relationship of birds used in this study.
The phylogenetic tree is based on documented literature [27], [28], [29], [30], [32], [34], but the relationships within Phasianidae (quail, bamboo partridge, chicken, peafowl, and turkey) were simplified due to the lack of consensus among proposed phylogeny. The presence (+) and absence (−) of Galα1-4Gal or Galβ1-4Gal on glycoproteins are based on detections in various tissues from species indicated with an asterisk (*) or on detections on egg while/yolk glycoproteins from all species except bamboo partridge. mya, million years ago.