Identification of carbohydrates binding to lectins by using surface plasmon resonance in combination with HPLC profiling.
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. Labeling 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 Man(5)GlcNAc(2) to Man(9)GlcNAc(2), as monitored by HPLC. In subsequent experiments and using well-defined labeled as well as nonlabeled 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 Manalpha2Manalpha6(Manalpha3)Man[D(3)B(A)4'], rather than the generally accepted trimannoside Manalpha6 (Manalpha3)Man [B(A)4' or 4(4')3]. In a similar experimental setup, 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 blood group 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.
Gutiérrez Gallego
Ricardo
R
Bijvoet Center, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.
Haseley
Simon R
SR
van Miegem
Vincent F L
VF
Vliegenthart
Johannes F G
JF
Kamerling
Johannis P
JP
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A / Q000737:chemistry; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D005643:Fucose / Q000737:chemistry; D006801:Humans; D008358:Mannose / Q000737:chemistry; D008895:Milk, Human / Q000737:chemistry; D009077:Mucins / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry*; D037121:Plant Lectins / Q000737:chemistry*; D020349:Surface Plasmon Resonance
D005456:Fluorescent Dyes; D009077:Mucins; D009844:Oligosaccharides; D037121:Plant Lectins; D003208:Concanavalin A; D005643:Fucose; D008358:Mannose