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Glycobiology Advance Access originally published online on June 19, 2009
Glycobiology 2009 19(12):1417-1426; doi:10.1093/glycob/cwp091
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© The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

A simple micro-method for determining precise oligosaccharidic specificity of mannose-binding lectins

Henri Debray1,2, Bernadette Coddeville2, Liezelotte R Bomfim3 and Márcio V Ramos4

2 Unité de Glycobiologie Structurale et Fonctionnelle UMR CNRS/USTL n° 8576 Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France
3 Centro de Ciências, Faculdade de Educação de Crateús, Universidade Estadual do Ceará, Crateús Ceará 63700-000, Brasil
4 Departamento de Bioquímica e Biologia Molecular Universidade Federal do Ceará Campus do Pici, Caixa Postal 6033 Fortaleza, Ceará 60451-970, Brasil

1 To whom correspondence should be addressed: Tel: +33-(0)3-20-43-48-83; Fax: +33-(0)3-20-43-65-55; e-mail: henri.debray{at}univ-lille1.fr

Received on May 10, 2008; revised on June 15, 2009; accepted on June 17, 2009

A simple and inexpensive method was developed to rapidly define the specificity of mannose-specific lectins toward oligomannoside-type structures. The method involved the interaction of a mixture of N-[14C]-acetylated glycoasparagines, prepared by exhaustive pronase digestion of bovine pancreatic ribonuclease B and N-[14C]-acetylation with [14C]-acetic anhydride and containing all the possible oligomannoside-type N-glycans, with the lectin immobilized on Sepharose-4B. After exhaustive desalting, the obtained fractions were separated by high-performance thin-layer chromatography on silica gel plates and visualized by autoradiography with intensifying screen. As an example of the usefulness of this method, the fine specificity of artocarpin, the mannose-specificity lectin isolated from seeds of jackfruit (Artocarpus integrifolia) toward oligomannoside-type structures is presented. On the basis of such a determination, the best oligomannosidic ligand recognized by a mannose-specific lectin can be selected for studies of crystal structures of the lectin in complex with the defined ligand. Furthermore, some of these immobilized lectins, after definition of their precise specificities with the method, could represent valuable tools for the fractionation and characterization of oligomannose-type structures, present in complex mixtures.

Key words: artocarpin / autoradiography with intensifying screen / HPTLC / immobilized mannose-specific lectins / ribonuclease B glycoasparagines


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