Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain

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.

Fluorescent Multiplex Cell Rolling Assay: Simultaneous Capturing up to Seven Samples in Real-Time Using Spectral Confocal Microscopy

“…In contrast to previous studies that showed interexperimental variations using the standard PPFC assay, − we believe the FMCR assay and analysis pipeline has eliminated this variation between samples and the need for normalization via running multiple samples side by side. The improved statistical power and the elimination of interexperimental variation will help enhance the sensitivity of the assay to detect differences in rolling behavior, which could have significant consequences and lead to discoveries of biologically relevant phenomena.…”

“…The software recorded videos of the flow run with the tethering and rolling events of interest. For controls, to detect nonspecific binding events of E-selectin to its ligands, we infused the chamber with EDTA after the flow run to remove the specific Ca 2+ -dependent binding. , This setup allows for imaging under dynamic flow conditions down to 900 ms/frame (at 512 × 512 pixels) and using the new Airyscanning system (Zeiss) would allow for even lower frame sizes. Interestingly, a high-throughput multiwell microfluidics system has been described , to study multiple samples individually in a multiwell setting.…”

“…Our novel technique significantly increases the number of degrees of freedom from <10 to few hundreds, which brought about a significant increase in the statistical power of the assay by many orders of magnitude. For example, in the PPFC assay the number of events used to calculate the different rolling parameters are on the order tens or less, ,,− whereas in the FMCR assay described here, the numbers of events are in the range of hundreds and thousands.…”

Early Detection of Negative Smoking Impacts: Vascular Adaptation Deviation Based on Quantification of Circulated Endothelial Activation Markers

“…Still, there is no explanation of E-selectin’s biological activity and clearance. 39 In addition to showing endothelial activation, E-selectin’s increase may provide an overview of vascular inflammatory alteration. 17 …”

P-selectin glycoprotein ligand 1 deficiency prevents development of acute pancreatitis by attenuating leukocyte infiltration

“…We found that PSGL-1 deficiency significantly decreased acinar cell death, indicating the protective effect of PSGL-1 deficiency against acinar cell damage. PSGL-1, as an initiating factor of the leukocyte-endothelial cell adhesion cascade, has been well described to mediate the inflammatory response in immune cells[ 28 ]. In this study, we found that PSGL-1 deficiency not only attenuated local inflammation, but also attenuated the systemic inflammation.…”