Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- or L- selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin–ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin–ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.
KAUST Repository Item: Exported on 2020-10-01
Acknowledged KAUST grant number(s): OCRF-2014-CRG3-2276
Acknowledgements: This research was supported by a King Abdullah University of Science and Technology (KAUST) Faculty Baseline Research Funding Program to J.S.M. and by a Competitive Research Grant (OCRF-2014-CRG3-2276) awarded to J.S.M. We would like to express our gratitude to Mr. Vlad-Stefan Raducanu for discussion about E-S0 sample aggregates; Ms. Maryam Mih, Ms. Samar A. Rustum and Ms. Umme Habiba for their support in the management of the lab. We would also like to thank Veronica Tremblay from the KAUST Academic Writing Service for editing the manuscript.