Preparation of a polyclonal antibody that recognizes a unique galactoseβ1-4fucose disaccharide epitope

Galactoseβ1-4fucose (Galβ1-4Fuc) is a unique disaccharide unit that has been found only in the N-glycans of protostomia. We demonstrated that this unit has a role as an endogenous ligand for Caenorhabditis elegans galectins. This unit is also recognized by fungal and mammalian galectins possibly as...

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Published in:Carbohydrate research 2015-08, Vol.412, p.50-55
Main Authors: Takeuchi, Tomoharu, Nishiyama, Kazusa, Saito, Saori, Tamura, Mayumi, Fuwa, Takashi J., Nishihara, Shoko, Takahashi, Hideyo, Natsugari, Hideaki, Arata, Yoichiro, Kasai, Ken-ichi
Format: Article
Language:English
Subjects:
Animals
anti-Glycan antibody
Antibodies - isolation & purification
Antibodies - metabolism
Ascaris suum - immunology
Caenorhabditis elegans
Chromatography, Affinity
Disaccharides - immunology
Disaccharides - metabolism
Drosophila melanogaster
Epitopes - immunology
Epitopes - metabolism
Frontal affinity chromatography
Galactoseβ1-4fucose
Mice, Inbred C57BL
Mites - immunology
Neoglycoconjugate
Protostomia
Rabbits
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Summary:Galactoseβ1-4fucose (Galβ1-4Fuc) is a unique disaccharide unit that has been found only in the N-glycans of protostomia. We demonstrated that this unit has a role as an endogenous ligand for Caenorhabditis elegans galectins. This unit is also recognized by fungal and mammalian galectins possibly as a non-self glycomarker. In order to clarify its biological function, we made a polyclonal antibody using (Galβ1-4Fuc)n-BSA as the antigen, which was prepared by crosslinking Galβ1-4Fuc-O–(CH2)2–SH and BSA. The binding specificity of the antibody was analyzed by frontal affinity chromatography, and it was confirmed that it recognizes naturally occurring N-glycans containing the Galβ1-4Fuc unit linked to the reducing-end GlcNAc via α1-6 linkage. By western blotting analysis, the antibody was also found to bind to (Galβ1-4Fuc)n-BSA but not to BSA or asialofetuin, which has N-glycan chains containing Galβ1-4GlcNAc. Western blotting experiments also revealed presence of stained proteins in crude extracts of C. elegans, the parasitic nematode Ascaris suum, and the allergenic mite Dermatophagoides pteronyssinus, while those from Drosophila melanogaster, Mus musculus, and the allergenic mites Dermatophagoides farinae and Tyrophagus putrescentiae were negative. This antibody should be a very useful tool for research on the distribution of the Galβ1-4Fuc disaccharide unit in glycans in a wide range of organisms. [Display omitted] •Anti-Galβ1-4Fuc polyclonal antibody was prepared.•Galβ1-4Fuc is expressed throughout development of Caenorhabditis elegans.•Galβ1-4Fuc bearing glycoproteins were not detected in fly and mouse.•Galβ1-4Fuc bearing glycoproteins were found in mite and ascaris.
ISSN:0008-6215
1873-426X
DOI:10.1016/j.carres.2015.04.015