Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays
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Plant lectin recognition of glycans was evaluated by SPR imaging using a model array of N-biotinylated aminoethyl glycosides of β-d-glucose (negative control), α-d-mannose (conA-responsive), β-d-galactose (RCA120-responsive) and N-acetyl-β-d-glucosamine (WGA-responsive) printed onto neutravidin-coated gold chips. Selective recognition of the cognate ligand was observed when RCA120 was passed over the array surface. Limited or no binding was observed for the non-cognate ligands. SPR imaging of an array of 40 sialylated and unsialylated glycans established the binding preference of hSiglec7 for α2-8-linked disialic acid structures over α2-6-sialyl-LacNAcs, which in turn were recognized and bound with greater affinity than α2-3-sialyl-LacNAcs. Affinity binding data could be obtained with as little as 10–20 μg of lectin per experiment. The SPR imaging technique was also able to establish selective binding to the preferred glycan ligand when analyzing crude culture supernatant containing 10–20 μg of recombinant hSiglec7-Fc. Our results show that SPR imaging provides results that are in agreement with those obtained from fluorescence based carbohydrate arrays but with the added advantage of label-free analysis.
KeywordsCarbohydrate array Surface plasmon resonance (SPR) imaging Label-free detection Plant lectin Siglec 7
the 120 kDa component of Ricinus communis agglutinin
wheat germ agglutinin
This research was supported by Basic Technology Grant GR/S79268/02 from Research Councils UK (http://www.glycoarrays.org.uk/). Many of the biotinylated carbohydrate derivatives used in this study were prepared by the Carbohydrate Synthesis Core of the Consortium for Functional Glycomics (http://www.functionalglycomics.org/fg/) funded by the National Institute of General Medical Sciences grant GM62116. We thank colleagues from the John Innes Genome Laboratory for invaluable support.