鉤端螺旋體病（Leptospirosis）是全世界常見的人畜共通傳染病，其病因由鉤端螺旋體（Leptospira）感染所引起的疾病，先前的研究指出鉤端螺旋體外套膜中存在一個主要脂蛋白LipL32，這個脂蛋白具有一個非典型的polyD 區域(D161DDDDGDD168)，從已解構的LipL32 晶體結構發現這個非典型的polyD 區域是鈣離子結合的區域，另外也有一些胺基酸參與在與鈣離子的結合，這些胺基酸包括了Asp132，Thr133，Asp164，Asp165 以及Tyr178。此篇論文的目的在於決定鈣離子在LipL32 所扮演的角色以及鈣離子參與在LipL32與第二型類鐸受體結合之間的關係，進而探討引發腎臟細胞發炎的機制。我們利用定點突變(Site-directed mutagenesis)將這些參與鈣離子結合的胺基酸進行突變並觀察這些突變後所產生的現象，首先由光譜學方法觀察這些胺基酸突變後會造成LipL32 的構型改變且使LipL32 失去與鈣離子結合的能力。此外，利用酵素連結免疫吸附法(ELISA)與原子力顯微鏡(AFM)分析LipL32 與第二型類鐸受體結合能力也會因這些胺基酸突變而減少，更進一步研究LipL32 所引發腎臟的發炎反應因子如hCXCL8/IL-8，hCCL2/MCP-1，hMMP7以及hTNF-α 等發現這些突變株也會降低這些發炎反應的產生。根據以上的研究所得到的結論，我們發現這些參與鈣離子結合的胺基酸在LipL32 中扮演重要的角色，除了與鈣離子結合外也會使LipL32 產生構型變化進而影響LipL32 與第二型類鐸受體的結合與其下游所產生的發炎反應因子及其後所造成的致病效應。

Leptospirosis is the most widespread zoonosis caused by the pathogenic Leptospira worldwide. LipL32, a 32-kDa lipoprotein, is the most abundant protein on the outer membrane of Leptospira and has an atypical poly(Asp) motif (161DDDDDGDD168). The x-ray crystallographic structure of LipL32 revealed that the calcium-binding cluster of LipL32 includes several essential residues Asp132, Thr133, Asp164, Asp165, and Tyr178. The goals of this study were to determine possible roles of the Ca2+-binding cluster for the interaction of LipL32 with Toll-like receptor 2 (TLR2) in induced inflammatory responses of human kidney cells. Site-directed mutagenesis was employed to individually mutate Ca2+-binding residues of LipL32 to Ala, and their effects were subsequently observed. These mutations abolished primarily the structural integrity of the calcium-binding cluster in LipL32. The binding assay and atomic force microscopy analysis further demonstrated the decreased binding capability of LipL32 mutants to TLR2. Inflammatory responses induced by LipL32 variants, as determined by TLR2 pathway intermediates hCXCL8/IL-8, hCCL2/MCP-1, hMMP7, and hTNF-α, were also lessened. In conclusion, the calcium-binding cluster of LipL32 plays essential roles in presumably sustaining LipL32 conformation for its proper association with TLR2 to elicit inflammatory responses in human renal cells.

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