Distribution of the Galß1-4Gal epitope among birds: species-specific loss of the glycan structure in chicken and its relatives.

The Galß1-4Gal epitope is rarely found in mammals, and the natural antibody against Galß1-4Gal is rich in human. In contrast, we have previously demonstrated the presence of Galß1-4Gal in pigeon and ostrich, and the absence of this epitope in chicken. Here, to further investigate the expression of this glycan among birds, egg white glycoproteins and egg yolk IgG from nine species of birds, namely, chicken, duck, emu, guineafowl, ostrich, peafowl, pigeon, quail, and turkey, were analyzed by western blot using an anti-(Galß1-4Gal) antibody. The results indicated that some egg white glycoproteins from emu, ostrich, and quail, and heavy chains of IgG from all of the birds, except chicken and quail, were stained with the antibody. The presence of Galß1-4Gal on N-glycans of IgGs from guineafowl, peafowl, and turkey were confirmed by mass spectrometry (MS), MS/MS, and MS(n) analyses. In quail, the presence of Galß1-4Gal was confirmed by detecting the activities of UDP-galactose: ß-galactoside ß1,4-galactosyltransferase (ß4GalT(Gal)) in various tissues, and by detecting Galß1-4Gal by western blotting. In contrast, bamboo partridge, which is a close relative of chicken, did not show any detectable activities of ß4GalT(Gal) or Galß1-4Gal on glycoproteins. Because quail, peafowl, turkey, chicken, and bamboo partridge belong to the same family, i.e., Phasianidae, expression of Galß1-4Gal was most likely differentiated within this family. Considering that Galß1-4Gal is also expressed in ostrich, emu, and pigeon, which are phylogenetically distant relatives within modern birds, Galß1-4Gal expression appears to be widely distributed among birds, but might have been abolished in the ancestors of chicken and bamboo partridge.

Generation of monoclonal antibodies against the Galß1-4Gal epitope: a key tool in studies of species-specific glycans expressed in fish, amphibians and birds.

Whereas the Galß1-4Gal epitope is rarely found in mammalian glycans, it has been found in glycans of various species of non-mammalian vertebrates, such as fish, amphibians and birds. Although glycans containing Galß1-4Gal in these vertebrates were detected by precise structural analysis of the glycans using mass spectrometry and/or NMR spectrometry, there are no convenient methods to detect Galß1-4Gal from various samples. To analyze systematically the distribution of Galß1-4Gal in nature, we generated mouse monoclonal antibodies (mAbs) specific for Galß1-4Gal using extracts of medaka eggs as an immunogen. Four mAbs (two immunoglobulin (Ig)Ms and two IgG1s) were obtained by enzyme-linked immunosorbent assay-based screening. The specificities of these mAbs were evaluated by frontal affinity chromatography using 142 kinds of 2-aminopyridine (PA)-derivatized oligosaccharides. While all mAbs interacted with (Galß1-4Gal)-containing oligosaccharides at their non-reducing termini with dissociation constants (K(d)) ranging from 1.0 x 10⁻5 to 2.8 x 10⁻4 M, no apparent interaction was observed with any other glycans. The number of branches containing Galß1-4Gal on N-glycans did not significantly affect K(d) of mAbs of IgG1 subclasses, but those of IgM mAbs were decreased by ∼1 order of magnitude, in increments of the number of branches present. Using the mAbs, we established that Galß1-4Gal is also expressed on glycoproteins in various tissues from the African clawed frog. Immunohistochemical staining of medaka sections revealed that Galß1-4Gal epitopes were expressed in the endothelium, epithelium and epidermis, which directly contact the external environment or invading organisms. Thus, these mAbs are useful for systematically investigating the species-specific expression of glycans, which may act as a barrier against infection.

Distinct expression profiles of UDP-galactose: ß-D-galactoside α1,4-galactosyltransferase and UDP-galactose: ß-D-galactoside ß1,4-galactosyltransferase in pigeon, ostrich and chicken.

We previously identified two novel enzymes in pigeon, α1,4- and ß1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Galα1-4Gal and Galß1-4Gal sequences on glycoproteins, respectively. No such glycan structures and/or enzymes have been found in mammals, suggesting that the expression of these enzymes diverged during the course of vertebrate evolution. To compare their expression profiles among avian species, we first established a method for detecting the activities of these two GalTs based on the two-dimensional high pressure liquid chromatography mapping technique, using 2-aminopyridine-derivatized asialo-biantennary N-glycans as an acceptor substrate. When we analyzed the activities of GalTs in pigeon liver extracts in the presence of UDP-Gal, 13 different products containing Galα1-4Galß1-4GlcNAc, Galß1-4Galß1-4GlcNAc and/or Galα1-4Galß1-4Galß1-4GlcNAc branches were identified. The newly formed glycosidic linkages of the enzymatic products were determined by nuclear magnetic resonance and methylation analysis, as well as by galactosidase digestions. The activities of both α1,4- and ß1,4-GalTs were detected in various tissues in pigeon, although their relative activities were different in each tissue. In contrast, ostrich expressed ß1,4-GalT, but not α1,4-GalT, in all tissues analyzed, whereas neither α1,4- nor ß1,4-GalT activity was detected in chicken. These results indicate that α1,4- and ß1,4-GalTs are expressed in a species-specific manner and are distributed throughout the entire body of pigeon or ostrich when the enzymes are present.

Stable interaction of the cargo receptor VIP36 with molecular chaperone BiP.

VIP36 is an intracellular lectin that cycles between the endoplasmic reticulum (ER) and the Golgi apparatus, and is thought to act as a cargo receptor in the transport and sorting of glycoproteins. Here we sought to identify the proteins that interact with VIP36 during the quality control of secretory proteins. VIP36 was crosslinked and immunoprecipitated from HEK293 cells that expressed Myc-tagged VIP36. An approximately 80 kDa protein coprecipitated with VIP36 and LC/MS/MS analysis revealed it to be immunoglobulin-binding protein (BiP), a major protein of the Hsp70 chaperone family. A VIP36 mutant with defective lectin activity was also proficient for the coimmunoprecipitation of an equivalent amount of BiP, indicating that the interaction between VIP36 and BiP was carbohydrate-independent. Immunoelectron microscopy experiment demonstrated that the interaction between VIP36 and BiP occurred in the ER. However, the VIP36 coprecipitated with BiP was resistant to endo beta-N-acetylglucosaminidase H treatment. A pulse-chase experiment revealed that the amount of BiP interacting with VIP36 did not change over more than 2 h. These results suggest that the interaction of VIP36 and BiP is not due to chaperone-substrate complex. Surface plasmon resonance analysis using recombinant proteins confirmed these binding characteristics of VIP36 and BiP in vitro. The interaction between recombinant soluble VIP36 and BiP is dependent on divalent cations but not on ATP. This mode of interaction is also different from that observed between BiP and its chaperone substrates. These observations suggest a new role for VIP36 in the quality control of secretory proteins.

Detection of weak sugar binding activity of VIP36 using VIP36-streptavidin complex and membrane-based sugar chains.

High mannose-type glycan-lectin interactions play important roles especially in quality control of glycoproteins. VIP36 is a receptor with homology to plant leguminous lectins in its luminal region. The luminal region of VIP36 with a C-terminal biotinylation-tag (sVIP36) was expressed in Escherichia coli and oligomerized with R-phycoerythrin (PE)-labelled streptavidin. Flow cytometric analysis revealed that PE-labelled sVIP36-SA complex (sVIP36-SA) bound to deoxymannojirimycin (DMJ)- and kifunensine (KIF)-treated HeLaS3 cells. The binding of sVIP36-SA to HeLaS3 cells treated with DMJ or KIF was abolished by endo-beta-N-acetylglucosaminidase H treatment of the cells. Furthermore, the binding of sVIP36-SA to the cells was inhibited by high mannose-type glycans especially Man(7-9) GlcNAc(2), indicating that the binding of sVIP36-SA to cell surfaces was mediated by high mannose-type glycans. Although VIP36 has the lower affinity for ligands than typical homologous plant lectins, we were able to monitor the sugar-binding activity of VIP36 using less than 100 ng of the sVIP36-SA. This method is highly sensitive and suitable for detecting interactions between lectins and sugar chains of low affinity.