日本腦炎病毒封套蛋白第三區塊與葡萄胺聚醣和單株抗體鍵結的結構研究

當病毒感染宿主細胞時，會先與細胞表面上的蛋白質接受器鍵結，再進行胞吞作用進入細胞。根據之前的研究報告，我們可以得知日本腦炎病毒是利用其外套膜蛋白與細胞表面的多醣體結合，進一步完成感染細胞的動作。所以藉由研究細胞表面上的多醣體與日本腦炎病毒外套膜蛋白之間的結合機制，可以進而設計抑制病毒進入細胞的藥物或疫苗。葡萄胺聚醣(Glycosaminoglycan)是一纇由胺基醣衍生的重複雙醣單元構成的多醣體，包含透明質酸、硫酸軟骨素、肝素等等。在之前的研究指出日本腦炎病毒外套膜蛋白主要與肝素鍵結的位置，為第三區塊N端的前面279-297序列，而因為細胞表面的肝素是帶有大量負電荷的化學分子，所以我們推測，日本腦炎病毒外套膜蛋白主要與肝素鍵結的胺基酸為第三區塊N端前面279-297序列中，四個帶有正電荷的胺基酸。因此，本實驗進行蛋白質定點突變，將這四個帶正電荷的胺基酸，分別為K279、 K286、 R288、 and K290，將其置換成不帶電的胺基酸，然後利用核磁共振和螢光實驗分析，去研究這些胺基酸位置對於肝素的鍵結的重要性。實驗結果顯示這四個帶正電荷的胺基酸突變成不帶電的胺基酸後，對於肝素的鍵結能力變得較弱。而我們也合成了一段只包含日本腦炎病毒外套膜蛋白279-297的短鏈胜肽，進行與肝素鍵結的實驗。結果顯示其鍵結能力與日本腦炎病毒外套膜蛋白序列261-402相差不大，所以，可以證實日本腦炎病毒外套膜蛋白主要和肝素鍵結的位置，為序列279-297上帶正電荷的胺基酸。最後，為了比較日本腦炎病毒外套膜蛋白261-402對單株抗體E3.3及肝素鍵結能力的競爭情況，所以利用ELISA進行實驗分析，以期單株抗體E3.3能有效的抑制肝素鍵結。

關鍵字

日本腦炎；封套蛋白；葡萄胺聚醣；結構

並列摘要

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, responsible for yellow fever was believed to target host cell by the interaction of an envelope protein with a highly sulfated heparin sulfate. Thus, the heparin binding activity of JEV may play an important role in its function on cellular surfaces. Glycosaminoglycans (GAGs) are linear sulfated polysaccharides consisting of several repeating disaccharides units of uronic acid and hexosamines, such as heparin, which maintain a large of negative charges. It is known that the GAG-binding region of JEV envelop protein is located in E279-297. Hence, we propose that the four positive-charged amino acids, K279, K286, R288, and K290 may contribute to the direct interaction with GAGs. In order to demonstrate this hypothesis, we created four JEV E261-402 mutants (K279A, K286A, R288A, K290A) by site- directed mutagenesis, and then we employed NMR and fluorescence experiments to analyze the GAG-binding affinity of wild type JEV E261-402 and four mutants. In addition, we also synthesize a peptide fragment (JEV E279-297) to mimic the GAG-binding ability of wild type JEV E261-402. Furthermore, we employed ELISA analysis to identify the monoclonal antibody E3.3 competes with heparin to bind JEV E261-402. The results showed that the GAG-binding affinity of synthetic peptide is comparable to that of wild type, but which of four mutants are all declined. The ELISA experiments show that mAb E3.3 has significant competition with heparin. Taken together, we conclude that the four positive-charge amino acids are all important for direct interaction with heparin, and the completion of this study will provide a structural basis for design of vaccines.

並列關鍵字

Japanese encephalitis virus ； envelope protein ； GAGs ； structural

參考文獻

1. Bruke, D. S., and Monath, T. P. (2001) Field Virology 4th Ed, 1043-1125, Lippincott Williams & Wilkins, Philadelphia, PA

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日本腦炎病毒封套蛋白第三區塊與葡萄胺聚醣和單株抗體鍵結的結構研究

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