Scintillation proximity assay for E-, P-, and L-selectin utilizing polyacrylamide-based neoglycoconjugates as ligands.
In this study, a novel scintillation proximity assay (SPA) that uses radiolabeled soluble neoglycoconjugates as synthetic alternatives to the natural E-, P-, and L-selectin counterligands was developed. The neoglycoconjugates contained sialyl LewisX or sialyl LewisA attached via a three-carbon spacer to a poly[N-(hydroxyethyl)acrylamide] backbone, thus presenting the carbohydrates in a multivalent form. Selectin-ZZ fusion proteins were immobilized on anti-rabbit IgG-coated SPA beads via a rabbit IgG bridge. The neoglycoconjugate ligands bound to all three bead-immobilized selectins, with the highest binding levels apparent with E-selectin. Saturation binding studies with E-selectin revealed a complex interaction indicative of two or more binding affinities. The response to carbohydrate inhibitors was comparable in E-selectin assays that used either the neoglycoconjugates or the tritium-labeled HL60 cells as selectin counterligands. The incorporation of tyrosine sulfate groups into the backbone of the neoglycoconjugate resulted in enhanced binding avidity to both P- and L-selectin, indicating that the sulfate-containing neoglycoconjugates are viable synthetic mimics of the natural P- and L-selectin counterligands. The use of these radiolabeled neoglycoconjugates in conjunction with SPA results in a format ideally suited for the high-throughput screening for selectin antagonists. Furthermore, this approach can potentially be used to measure other low-avidity lectin-carbohydrate interactions.
Game
S M
SM
Cardiff Laboratories, Nycomed Amersham Research and Development, Forest Farm, Whitchurch, CF4 7YT, United Kingdom.
Rajapurohit
P K
PK
Clifford
M
M
Bird
M I
MI
Priest
R
R
Bovin
N V
NV
Nifant'ev
N E
NE
O'Beirne
G
G
Cook
N D
ND
D000180:Acrylic Resins; D000818:Animals; D001665:Binding Sites; D018395:CA-19-9 Antigen; D002240:Carbohydrate Sequence; D019040:E-Selectin / Q000032:analysis; D005069:Evaluation Studies as Topic; D006001:Glycoconjugates / Q000737:chemistry; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D019041:L-Selectin / Q000032:analysis; D008024:Ligands; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D019007:P-Selectin / Q000032:analysis; D011817:Rabbits; D012588:Scintillation Counting / Q000379:methods*; D019087:Selectins / Q000032:analysis*; D000080506:Sialyl Lewis X Antigen; D013431:Sulfates / Q000737:chemistry; D014316:Tritium; D000099166:Polyacrylamides
D000180:Acrylic Resins; D018395:CA-19-9 Antigen; D019040:E-Selectin; D006001:Glycoconjugates; D019041:L-Selectin; D008024:Ligands; D009844:Oligosaccharides; D019007:P-Selectin; D019087:Selectins; D000080506:Sialyl Lewis X Antigen; D013431:Sulfates; D014316:Tritium; D000099166:Polyacrylamides