Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

Dina Bashir Kamil Abu Samra, Alia Al Kilani, Samir Hamdan, Kosuke Sakashita, Samah Gadhoum, Jasmeen Merzaban

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow onand off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Original languageEnglish (US)
Pages (from-to)21213-21230
Number of pages18
JournalJournal of Biological Chemistry
Volume290
Issue number35
DOIs
StatePublished - Jun 29 2015

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: KAUST, King Abdullah University of Science and Technology

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