Department of Nephrology, Kidney Research Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan 33333, Taiwan.
Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, National Tsing Hua University, Hsin Chu 30013, Taiwan.
Biochem J. 2020 Nov 13;477(21):4313-4326. doi: 10.1042/BCJ20200547.
Leptospirosis is the most common zoonotic disease caused by pathogenic Leptospira, which is classified into three groups according to virulence. Its pathogenic and intermediate species contain leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic strains. In this study, we presented the crystal structure of LSS_11580 (rLRR20) from pathogenic L. santarosai serovar Shermani. X-ray diffraction at a resolution of 1.99 Å revealed a horseshoe-shaped structure containing seven α-helices and five β-sheets. Affinity assays indicated that rLRR20 interacts with E-cadherin on the cell surface. Interestingly, its binds to the extracellular (EC) 1 domain in human epithelial (E)-cadherin, which is responsible for binding to another E-cadherin molecule in neighboring cells. Several charged residues on the concave face of LRR20 were predicted to interact with EC1 domain. In the affinity assays, these charged residues were replaced by alanine, and their affinities to E-cadherin were measured. Three vital residues and mutation variants of LRR20, namely D56A, E59A, and E123A, demonstrated significantly reduced affinity to E-cadherin compared with the control. Besides, we also demonstrated that rLRR20 induced the expression of neutrophil gelatinase-associated lipocalin (NGAL) in HK2 cells. The low ability of the three mutation variants to induce NGAL expression further demonstrates this induction. The present findings indicate that LRR20 from pathogenic Leptospira binds to E-cadherin and interacts with its EC1 domain. In addition, its induction of NGAL expression in HK2 cells is associated with acute kidney injury in human.
钩端螺旋体病是由致病性钩端螺旋体引起的最常见的人畜共患病，根据毒力可分为三组。其致病和中间种含有亮氨酸丰富重复（LRR）蛋白，在非致病株中很少表达。在这项研究中，我们展示了来自致病性 L. santarosai 血清型 Shermani 的 LSS_11580（rLRR20）的晶体结构。在 1.99 Å 的分辨率下进行的 X 射线衍射揭示了一个马蹄形结构，包含七个α-螺旋和五个β-折叠。亲和测定表明 rLRR20 与细胞表面的 E-钙粘蛋白相互作用。有趣的是，它与人类上皮（E）-钙粘蛋白的细胞外（EC）1 结构域结合，该结构域负责与相邻细胞中的另一个 E-钙粘蛋白分子结合。LRR20 凹面的几个带电残基预测与 EC1 结构域相互作用。在亲和测定中，这些带电残基被替换为丙氨酸，并测量它们与 E-钙粘蛋白的亲和力。与对照相比，LRR20 的三个重要残基和突变变体，即 D56A、E59A 和 E123A，对 E-钙粘蛋白的亲和力明显降低。此外，我们还证明 rLRR20 诱导 HK2 细胞中中性粒细胞明胶酶相关脂质运载蛋白（NGAL）的表达。这三个突变变体诱导 NGAL 表达的能力较低，进一步证明了这种诱导作用。本研究结果表明，致病性钩端螺旋体的 LRR20 与 E-钙粘蛋白结合并与它的 EC1 结构域相互作用。此外，它在 HK2 细胞中诱导 NGAL 表达与人的急性肾损伤有关。
