Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays

Abstract

Plant lectin recognition of glycans was evaluated by SPR imaging using a model array of N-biotinylated aminoethyl glycosides of beta-D-glucose (negative control), alpha-D: -mannose (conA-responsive), beta-D-galactose (RCA(120)-responsive) and N-acetyl-beta-D-: glucosamine (WGA-responsive) printed onto neutravidin-coated gold chips. Selective recognition of the cognate ligand was observed when RCA(120) 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 alpha2-8-linked disialic acid structures over alpha2-6-sialyl-LacNAcs, which in turn were recognized and bound with greater affinity than alpha2-3-sialyl-LacNAcs. Affinity binding data could be obtained with as little as 10-20 microg 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 microg 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.

Optimization of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis

“…Lectins can be implemented for glycoanalysis in a number of different formats including lectin microarray techniques (21)(22)(23)(24)(25). However, the most simplistic format in which lectins can be employed for glycoanaylsis is the Enzyme-Linked Lectin Assay (ELLA).…”

Surface plasmon resonance imaging of glycoarrays identifies novel and unnatural carbohydrate-based ligands for potential ricin sensor development

“…13 To briefly summarise, the library assessed was composed of a range of glycans with non-reducing termini based on: Gal-b-1,4 (7 compounds), Gal-b-1,3 (3), GalNAc (1), GlcNAc (4), sulfo-Gal (3), NeuAc-2,3 (12), NeuGc-2,3/2,6 (2/1), NeuAc-2,6 (4), and NeuAc-2,8 (3) ( Table 1). The array was used to screen for RCA 120 binders in the anticipation that glycans possessing a non-reducing terminal b-linked galactose residue (black and orange), the previously determined simple ligand for this lectin, would stand out as hits.…”

“…The array was used to screen for RCA 120 binders in the anticipation that glycans possessing a non-reducing terminal b-linked galactose residue (black and orange), the previously determined simple ligand for this lectin, would stand out as hits. 13,18,19 Data for equilibrium binding of RCA 120 and the a-2,6-NeuAc-specific Sambucus nigra agglutinin (SNA) to the glycoarray are presented in Fig. 1.…”

“…Data errors were typically in the AE10-15% range. 13 maintained the highest levels of saturation (100 and 72%, respectively), suggesting that these positions were integral to ligand recognition. Agreement with this observation can be found in lack of binding to RCA 120 of a2,3Neu5Ac-terminating oligosaccharides (red bars, Fig.…”

Glycosylation Characterization of Human and Porcine Fibrinogen Proteins by Lectin-Binding Biophotonic Microarray Imaging

“…In combination with surface plasmon resonance (SPR) 22 , lectins can provide useful quantitative data on protein glycosylation without the need for the more conventional fluorescent labelling of probes 21,23 . Using the SPR label-free technology in an array format allows the study of a large numbers of carbohydrate-protein interactions including determination of binding kinetics, affinity and avidity constants and relative energies of interactions.…”