Scintillation proximity assay for E-, P-, and L-selectin utilizing polyacrylamide-based neoglycoconjugates as ligands

Analytical Biochemistry
S M GameN D Cook

Abstract

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 the...Continue Reading

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Citations

Aug 21, 2007·Biochimica Et Biophysica Acta·Sven EndersRudolf Tauber
Jun 25, 2002·Chemistry & Biology·Tatyana V PochechuevaNicolai V Bovin
Aug 8, 2006·Nature Medicine·Arne von BoninThomas M Zollner
Feb 14, 2007·Glycobiology·Albana CumashiUNKNOWN Consorzio Interuniversitario Nazionale per la Bio-Oncologia, Italy
Apr 23, 2008·Current Protocols in Protein Science·Neil CookKelvin Hughes
May 17, 2005·Biochemistry. Biokhimii︠a︡·N A UshakovaN V Bovin
Jan 15, 2021·Glycoconjugate Journal·Alexander TuzikovNicolai Bovin
May 6, 2003·Bioorganic & Medicinal Chemistry Letters·Tatyana V PochechuevaNicolai V Bovin

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