以基質輔助雷射解吸電離質譜鑑定重組鱟血漿凝集素與脂多糖之作用關係

Abstract

從台灣鱟血漿中取得的重組鱟血漿凝集素(rHPL)已被成功地表現在大腸桿菌(E. coli)系統且能大量純化。rHPL已被證明是透過辨識鼠李醣(rhamnose)來識別病原體相關的分子模式(PAMPs)。此外rHPL還被證明有能力抑制如綠膿桿菌等病原細菌，這使得rHPL適合做為開發新型AMP的原型蛋白。不過rHPL和PAMP之間的結合機制尚未徹底理解。MALDI-TOF MS是具有高敏感度和準確性的鑑定技術，且被廣泛應用在生物分子的檢測。本篇論文中MALDI-TOF MS被應用到驗證rHPL和四個不同脂多醣(LPS)的結合：綠膿桿菌10血清型，綠膿桿菌PAO1血清型，綠膿桿菌PA14血清型和克雷伯氏肺炎菌。我們的結果證實該四種LPS的確會與rHPL相互作用，透過辨識Lipid A在MALDI-TOF MS上的圖譜進行鑑定。 rHPL對兩種傳染病菌確實作用的發現可能有助於發展新型病原微生物的診斷和治療策略。另外，本篇論文使用MALDI-TOF MS鑑定分子間作用的方法在未來也可被應用於其他脂多醣結合蛋白(LPS-binding protein)。此外，rHPL的實際蛋白分子模型尚未被解出。本篇論文利用電腦模擬的rHPL蛋白模型與受質進行對接，成功的印證了rHPL的活性接合部位氨基酸位置位於Y46及F103，並模擬出rHPL與受質結合時的分子模型。

Recombinant horseshoe crab plasma lectin (rHPL) derived from Taiwanese Tachypleus tridentatus has been successfully expressed and purified in E. coli system, and has been proven to recognize pathogen associated molecular patterns (PAMPs) through rhamnose. Also, rHPL is proven to inhibit bacterial growth such as P. aeruginosa, which makes rHPL an appropriate prototype in the development of a novel AMP. The binding mechanisms between rHPL and PAMPs are not yet thoroughly comprehended. MALDI-TOF MS with highly sensitive and precise features is applied to verify the inhibitory binding between rHPL and four different lipopolysaccharides (LPS): Pseudomonas aeruginosa 10, Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa PA14 and Klebsiella pneumoniae separately. Our results indicated that all four LPSs were confirmed to interact with rHPL and could be identified in the MALDI-TOF MS spectrum. The finding of rHPL interacting with two infectious strains could facilitate the applicability of novel diagnostic and therapeutic strategies for microbial pathogens in future development. Also our novel approach to verifying molecular interactions using MALDI-TOF MS could be applied to other LPS-binding proteins. Besides, 3-dimensional structure of rHPL has not been solved. We use molecular docking technique to predict ligand binding sites of rHPL and, successfully verify that Y46 and F103 are key amino acids in ligand binding.