Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays.
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.
Karamanska
Rositsa
R
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, UK.
Clarke
Jonathan
J
Blixt
Ola
O
Macrae
James I
JI
Zhang
Jiquan Q
JQ
Crocker
Paul R
PR
Laurent
Nicolas
N
Wright
Adam
A
Flitsch
Sabine L
SL
Russell
David A
DA
Field
Robert A
RA
D019921:Biotinylation; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000032:analysis*; D054794:Glycomics / Q000379:methods*; D046228:Microarray Analysis / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors
D002241:Carbohydrates; D011506:Proteins