Keith Brandon A, Ching John C H, Loewen Matthew E
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Biochem Biophys Rep. 2019 Mar 28;18:100630. doi: 10.1016/j.bbrep.2019.100630. eCollection 2019 Jul.
The human hCLCA1 gene is a member of the gene family that has a well-documented role in inflammatory airway diseases. Previously, we demonstrated that secreted hCLCA1 plays a role in regulating the innate immune response by activating airway macrophages. However, the mechanism of this regulation remains unclear. In this present study, recombinant proteins containing different hCLCA1 domains are expressed to determine the specific hCLCA1 domain(s) responsible for macrophage activation. Specifically, hCLCA1 constructs containing the hydrolase domain (HYD), the von Willebrand Factor Type A (VWA) domain, and the fibronectin type III (FN3) domain were heterologously expressed and affinity purified through fast protein liquid chromatography. Circular dichroism spectroscopy revealed that the purified hCLCA1 constructs exhibited secondary structure consistent with folded proteins. The VWA domain clearly demonstrated an ability to activate macrophages, inducing an increase in both IL-1β mRNA and protein expression. This activation was associated with the activation of MAPKs and NF-κB pathways, identifying potential mechanistic pathways by which hCLCA1's VWA domain exerts its signaling effect. Altogether, this work identifies a domain with signaling function within hCLCA1, providing a specific target to one of the most highly induced gene products of airway inflammatory disease.
人类hCLCA1基因是一个基因家族的成员，该家族在炎症性气道疾病中具有充分记载的作用。此前，我们证明分泌的hCLCA1通过激活气道巨噬细胞在调节先天免疫反应中发挥作用。然而，这种调节机制仍不清楚。在本研究中，表达了含有不同hCLCA1结构域的重组蛋白，以确定负责巨噬细胞激活的特定hCLCA1结构域。具体而言，含有水解酶结构域（HYD）、血管性血友病因子A（VWA）结构域和纤连蛋白III型（FN3）结构域的hCLCA1构建体被异源表达，并通过快速蛋白质液相色谱进行亲和纯化。圆二色光谱显示，纯化的hCLCA1构建体呈现出与折叠蛋白一致的二级结构。VWA结构域清楚地显示出激活巨噬细胞的能力，导致IL-1β mRNA和蛋白表达均增加。这种激活与MAPKs和NF-κB信号通路的激活相关，确定了hCLCA1的VWA结构域发挥其信号作用的潜在机制途径。总之，这项工作确定了hCLCA1内具有信号功能的一个结构域，为气道炎症疾病中诱导程度最高的基因产物之一提供了一个特定靶点。
