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.

中文翻译：

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E-选择蛋白与其配体的功能性结合通过其凝集素结构域以外的结构特征得到增强。

选择素是介导细胞粘附和迁移，免疫反应，转移和移植等基础过程相互作用的关键。选择素由凝集素结构域，表皮生长因子（EGF）样结构域，多个短共有重复序列（SCR），跨膜结构域和细胞质尾部组成。众所周知，凝集素和EGF结构域需要介导与配体的相互作用。但是，其他领域在调解这些交互作用方面的贡献仍然模糊。使用在新开发的基于蚕的表达系统中产生的各种E-选择素构建体，以及在静态和生理流动条件下进行的几种测定，包括流式细胞仪，聚糖阵列分析，表面等离振子共振和细胞滚动测定，我们在这里显示，SCR结构域数量的减少与功能性E-选择素与造血细胞E-或L-选择素配体（HCELL）和P-选择素糖蛋白配体-1（PSGL-1）结合的下降有关。而且，通过E-选择蛋白二聚化和通过模拟凝集素和EGF结构域的扩展构象的取代（A28H），显着改善了结合。对缔合和解离速率的分析表明，SCR结构域，构象延伸和二聚化共同促成E-选择素-配体结合的缔合速率，而仅凝集素和EGF域促成解离速率。这些发现为结合率在功能性E-选择素-配体相互作用中的关键作用提供了第一个证据，