芋頭半胱胺酸蛋白酶抑制劑多聚體形成及其抑制活性之探討

Study on Functional Domain of Tarocystatin for Oligomerization and Inhibition Activity

范凱雯(Kai-Wen Fan)

指導教授 ： 鄭貽生

國立臺灣大學/生命科學院/植物科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02823

摘要

植物半胱胺酸蛋白酶抑制劑參與植物體內蛋白質的代謝，以及作為植物對抗病原菌和植食性昆蟲的防禦機制。芋頭半胱胺酸蛋白酶抑制劑 (tarocystatin) 屬於第二群植物半胱胺酸蛋白酶抑制劑的成員，結構由高度保守性的胺基端 (NtD) 半胱胺酸蛋白酶抑制區域，以及羧基端 (CtE) 類似半胱胺酸蛋白酶抑制區域的延伸片段所組成。先前研究指出NtD與CtE的抑制蛋白酶活性的能力不同，在此研究進一步探討芋頭半胱胺酸蛋白酶抑制劑之蛋白質特性，及和目標蛋白木瓜酶之交互作用。利用示差掃描微量熱法 (differential scanning calorimetry, DSC) 測定tarocystatin全長 (FL), NtD和CtE的熔點都高於75ºC，顯示它們具有高度的熱穩定性；細胞內表現位置實驗結果表明，tarocystatin可同時出現在細胞質和細胞核中。以圓二色光譜 (circular dichroism spectroscopy) 分析其二級結構分布，證實其FL和CtE與已知結構之NtD類似，蛋白質折疊方式主要由β-strands組成。經膠體過濾分析 (gel filtration chromatography) 後，發現在不同pH溶液中FL、NtD和CtE皆能以單體和多聚體的形式存在，結合等溫滴定微量熱法 (isothermal titration calorimetry, ITC) 確認FL形成雙體並非NtD和CtE交互作用，而主要是NtD彼此交互作用所造成。進一步由ITC分析tarocystatin和木瓜酶的結合特性，結果顯示單體的FL和NtD對木瓜酶具有高度的結合力，雙體的FL和NtD以及單體的CtE則無法結合木瓜酶。抑制活性分析顯示，FL和NtD能有效抑制木瓜酶，且NtD具有強烈的抑制能力，其次是FL，然而CtE不但無法抑制木瓜酶，反而會強化蛋白質水解； FL和NtD經高於70ºC處理後，才會造成蛋白質不穩定，使得其抑制活性明顯下降。綜合以上的結果推論，tarocystatin可能以不同形態存在於植物體中，主要由NtD調控FL轉換成單體或多聚體，不同形式的tarocystatin與木瓜酶之間的結合特性亦不同；而羧基端多了胺基酸序列的延伸反而些微促進木瓜酶的活性，推測演化上，CtE的出現可能使tarocystatin更具抑制多樣性。

關鍵字

植物半胱胺酸蛋白酶抑制劑 ； 芋頭半胱胺酸蛋白酶抑制劑 ； 多聚體

並列摘要

Tarocystatin, a member of group-2 phytocystatin, contains a conserved N-terminal cystatin domain (NtD) and a C-terminal cystatin-like extension (CtE). It has been reported to act as defense proteins against pathogens. Melting temperature (Tm) of tarocystatin full-length (FL), NtD and CtE were measured using differential scanning calorimetry. Their Tm was higher than 75ºC and suggested they should have high thermal stability. Subcellular localization suggested the existence of tarocystatin in both the cytoplasm and the nucleus. Secondary structure analysis from circular dichroism spectroscopy revealed that FL and CtE have similar conformation to that of NtD with mainly composed of β-strands. Gel filtration chromatography combining with isothermal titration calorimetry (ITC) method indicated that tarocystatin might exist monomer and oligomer at ambient pH, and homodimeric FL could form by domain-domain interaction, especially by NtD and NtD, but not by NtD and CtE. In addition, ITC results also showed that monomeric FL and NtD can bind to papain with high binding affinity, in contrast with the dimeric FL, NtD and monomeric CtE did not show any binding affinity to papain. Inhibition activity assay revealed that NtD possess higher anti-papain activity than FL, but CtE could slightly increase papain activity, and FL and NtD both were unstable at > 70ºC. Thus, we propose that the presence of oligomerization of tarocystatin be relevant for inhibitory regulation in vivo and CtE provide inhibitory variety of tarocystatin in cystatin protein evolution.

並列關鍵字

phytocystatin ； tarocystatin ； oligomerization

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