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Glycobiology Advance Access published online on January 21, 2004
Glycobiology, doi:10.1093/glycob/cwh052
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Submitted on February 24, 2003
Revised on November 19, 2003
Accepted on December 30, 2003

© 2004 Oxford University Press

ORIGINAL ARTICLES

Identification of carbohydrates binding to lectins by using surface plasmon resonance in combination with HPLC profiling

Ricardo Gutiérrez Gallego 1, Simon R. Haseley 1, Vincent F. L. van Miegem 1, Johannes F. G. Vliegenthart 1, and Johannis P. Kamerling 1*

1 Bijvoet Center, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands

* To whom correspondence should be addressed. E-mail: j.p.kamerling{at}chem.uu.nl.

Abstract

A new, powerful method is presented for screening the binding in real time and taking place under dynamic conditions of oligosaccharides to lectins. The approach combines an SPR biosensor and HPLC profiling with fluorescence detection, and is applicable to complex mixtures of oligosaccharides in terms of "ligand-fishing". Labelling the oligosaccharides with 2-aminobenzamide ensures a detection level in the fmol range. In an explorative study the binding of RNAse B-derived oligomannose-type N-glycans to biosensor-immobilized concanavalin A (Con A) was examined, and an affinity ranking could be established for Man5GlcNAc2 to Man9GlcNAc2, as monitored by HPLC. In subsequent experiments and using well-defined labelled as well as non-labelled oligosaccharides, it was found that the fluorescent tag does not interfere with the binding and that the optimum epitope for the interaction with Con A comprises the tetramannoside unit Man{alpha}2Man{alpha}6(Man{alpha}3)Man [D3B(A)4'], rather than the generally accepted trimannoside Man{alpha}6(Man{alpha}3)Man [B(A)4' or 4(4')3]. In a similar experimental set-up, the interaction of various fucosylated human milk oligosaccharides with the fucose-binding lectin from Lotus tetragonolobus purpureaus was studied, and it appeared that oligosaccharides containing bloodgroup H could selectively be retained and eluted from the lectin-coated surface. Finally, using the same lectin and a mixture of O-glycans derived from bovine submaxillary gland mucin, minor constituents but containing fucose, could selectively be picked from the analyte solution as demonstrated by HPLC profiling.


HPLC profiling, lectins, oligosaccharides, Surface Plasmon Resonance, interaction
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