致病性鉤端螺旋體外膜脂質蛋白LipL41的生物功能與分子結構之研究

Functional and Structural Studies of Lipoprotein LipL41 from Pathogenic Leptospira

林明興(Ming-Hsing Lin)

指導教授 ： 孫玉珠

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2014年)

摘要

鉤端螺旋體病是溫熱帶國家常見的人畜共通疾病之一，其致病的病原菌為鉤端螺旋體。此疾病可能導致多重器官衰竭，而腎臟是感染最好發之處。目前對於鉤端螺旋體如何引起宿主的先天免疫反應，以及感染腎臟的生理影響仍不清楚。鉤端螺旋體病相關致病因子多數為外膜蛋白，外膜蛋白位於病原體的表面，被免疫系統辨識而誘發免疫反應的機率較其他蛋白高，故常扮演毒素因子的角色，也經常被用來作為疫苗研發之標的；除此之外，外膜蛋白在病原體與宿主間交互作用中伴演著重要的角色。LipL41為鉤端螺旋體中高度保留且是主要的外膜脂質蛋白之一。先前的研究發現LipL41具有高鐵血紅素（hemin）的結合能力，且有可能參與病原體的鐵離子調控與儲存。然而，LipL41與高鐵血紅素結合的詳細機制尚未被明確地探討。在本研究中，我們透過高鐵血紅素結合分析法求得了LipL41和高鐵血紅素之間的解離常數（Dissociation constant, Kd）為0.59 ± 0.14 μM；同時，我們也鑑定出位於LipL41之上的可能參與高鐵血紅素結合的結合位（HRMs, heme regulatory motif）。藉由突變重組蛋白LipL41，我們也證實了LipL41上的兩個胺基酸Cys140以及Cys220同時參與在與高鐵血紅素的結合。我們也運用了穿透式電子顯微鏡發現了LipL41會形成一具有36個單體的超分子聚合體。此外，藉由圓二色光譜及核磁共振的分析，發現LipL41碳端包含兩個TPR motif的區域（LipL41-C100）具有熔球態蛋白（molten globule protein）的特性。

關鍵字

高鐵血紅素 ； 脂質蛋白 ； 穿透式電子顯微鏡 ； 致病因子

並列摘要

Leptospirosis is one of the most widespread zoonotic diseases in the world. It is caused by the pathogen Leptospira that results in multiple-organ failure, in particular of the kidney. Outer membrane lipoprotein is the suspected virulence factor of Leptospira. In Leptospira spp LipL41 is one major lipoprotein and is highly conserved. Previous study suggests that LipL41 bears hemin-binding ability and might play a possible role in iron regulation and storage. However, the characterization of hemin-binding ability of LipL41 is still unclear. Here the hemin-binding ability of LipL41 was examined, yielding a Kd = 0.59 ± 0.14 μM. Two possible heme regulatory motifs (HRMs), C[P/S], were found in LipL41 at 140Cys-Ser and 220Cys-Pro. The mutational study indicates that Cys140 and Cys220 might be cooperatively involved in hemin binding. A supramolecular assembly of LipL41 was determined by transmission electron microscopy. The LipL41 oligomer consists of 36 molecules and folds as a double-layered yo-yo particle. At the C-terminus of LipL41, there are two tetratricopeptide repeats (TPRs), which might be involved in the protein-protein interaction of the supramolecular assembly. Besides, the TPR-containing fragment, LipL41-C100, shows thermal stability and reversibility by circular dichroism and NMR. The flexible folding of LipL41-C100 can be stabilized by adding 50% TFE. The thermal dynamics and flexible folding consider that LipL41-C100 is a molten globule protein, which conserves a native-like secondary structure content but without the tightly packed protein interior.

並列關鍵字

hemin binding protein ； lipoprotein ； TEM ； virulence factor

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