Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Serbia.
Phytochemistry. 2013 Oct;94:53-9. doi: 10.1016/j.phytochem.2013.06.006. Epub 2013 Jul 3.
Plant proteinase inhibitors are considered important defense molecules against insect and pathogen attack. The cysteine proteinase inhibitor (CPI) from green kiwifruit (Actinidia deliciosa) belongs to the cystatin family and shows potent antifungal activity (in vitro and in vivo). However, the low abundance of this molecule in fruit (6μg/g of fresh fruit) seems to limit further investigations on the interaction between phytocystatin and photopathogenic fungi. In this paper the cDNA of the kiwi CPI was expressed in Escherichia coli. Fifteen N-terminal amino acids were identified by Edman degradation, and 77% of the rCPI primary structure was confirmed by mass fingerprint. The structural homology of recombinant CPI (rCPI) to its natural counterpart has been clearly demonstrated in immunological assays (immunoblot and ELISA inhibition). Biological activity of rCPI was demonstrated in inhibition assay with cysteine proteinase papain (EC50 2.78nM). In addition, rCPI reveals antifungal properties toward pathogenic fungi (Alternaria radicina and Botrytis cinerea), which designates it as an interesting model protein for the exploration of plant phytocystatins - pathogen interactions. Understanding the molecular mechanisms of natural plant resistance could lead to the development of ecologically safe fungicides for controlling post-harvest diseases and maintaining food quality.
植物蛋白酶抑制剂被认为是抵御昆虫和病原体攻击的重要防御分子。绿奇异果（Actinidia deliciosa）中的半胱氨酸蛋白酶抑制剂（CPI）属于半胱氨酸蛋白酶抑制剂家族，具有很强的抗真菌活性（体外和体内）。然而，这种分子在果实中的含量很低（6μg/g 新鲜果实），这似乎限制了对植物半胱氨酸蛋白酶与植物病原菌之间相互作用的进一步研究。本文在大肠杆菌中表达了奇异果 CPI 的 cDNA。通过 Edman 降解鉴定了 15 个 N 端氨基酸，通过质谱指纹图谱确定了 77%的 rCPI 一级结构。免疫印迹和 ELISA 抑制试验清楚地证明了重组 CPI（rCPI）与其天然对应物在结构上的同源性。rCPI 对木瓜蛋白酶（EC50 2.78nM）的抑制活性在抑制试验中得到了证实。此外，rCPI 对病原菌真菌（Alternaria radicina 和 Botrytis cinerea）具有抗真菌特性，这使其成为探索植物半胱氨酸蛋白酶-病原菌相互作用的有趣模型蛋白。了解天然植物抗性的分子机制可能会导致开发出生态安全的杀菌剂来控制采后病害并保持食品质量。
