Abstract

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- and/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.

Highlights

  • Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation

  • Analyses of the association and dissociation rates indicated that the short consensus repeats (SCRs) domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin–ligand binding, whereas just the lectin and epidermal growth factor (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

Read more

Summary

Edited by Ursula Jakob

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. By employing a comprehensive set of assays under static and physiological flow conditions, we showed that SCR domains, the conformational extension of the lectin and EGF domains, and protein dimerization contribute to the efficient binding of E-selectin to its glycosylated ligands. These three structural elements collectively contribute to the association rate, whereas the dissociation rate was mainly controlled by the lectin and EGF domains. These findings correlate, for the first time, a very important physiological role for the association rate in the functional binding of the selectin to its ligands

Results
Discussion
Experimental procedures
Cell lines
Flow cytometry
Western blotting and native polyacrylamide agarose gel electrophoresis
Glycan array analysis
Surface plasmon resonance
Cell rolling assay
Statistical analysis
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call