Mutational analysis of a plant defensin from radish (Raphanus sativus L) reveals two adjacent sites important for antifungal activity
Mutational analysis of a plant defensin from radish (Raphanus sativus L) reveals two adjacent sites important for antifungal activity
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DOI：
10.1074/jbc.272.2.1171
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发表时间：
1997-01-10
影响因子：
4.8
通讯作者：
Broekaert, WF
中科院分区：
文献类型：
--
作者：
DeSamblanx, GW;Goderis, IJ;Broekaert, WF
使用基于聚合酶链反应的定点诱变和酵母作为异源表达系统，对Rs-AFP 2（一种萝卜抗真菌肽，属于被称为植物防御素的肽家族）进行突变分析。随后选择候选氨基酸残基进行取代的策略是基于Rs-AFP 2与表现出差异抗真菌特性的其他植物防御素的序列比较。鉴定了几种突变，其产生对黄色镰刀菌具有降低的抗真菌活性的肽变体。同时，尝试通过用精氨酸取代单个氨基酸来构建具有增强的抗真菌活性的变体。发现两个精氨酸取代变体在高离子强度的培养基中比野生型Rs-AFP 2更活跃。我们的数据表明，Rs-AFP 2具有两个相邻的位点，这两个位点似乎对抗真菌活性很重要，即一方面是连接β链2和3的VI型β转角周围的区域，另一方面是连接β链1和α螺旋的环上的残基以及α螺旋和β链3上的相邻残基形成的区域。在高离子强度的培养基中，Rs-AFP 2可使culmorum的Ca ~（2+）吸收增加20倍。增强抗真菌活性的精氨酸取代变体引起Ca 2+摄取增加高达50倍，而几乎没有抗真菌活性的变体不刺激Ca 2+摄取。
Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI beta-turn connecting beta-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting beta-strand 1 and the alpha-helix and contiguous residues on the alpha-helix and beta-strand 3, on the other hand, When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca2+ uptake.