Processing of the Phalloidin Proprotein by Prolyl Oligopeptidase from the Mushroom Conocybe albipes
Processing of the Phalloidin Proprotein by Prolyl Oligopeptidase from the Mushroom Conocybe albipes
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DOI：
10.1074/jbc.m109.006460
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发表时间：
2009-07-03
影响因子：
4.8
通讯作者：
Walton, Jonathan D.
中科院分区：
文献类型：
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作者：
Luo, Hong;Hallen-Adams, Heather E.;Walton, Jonathan D.
有毒鹅膏菌的肽毒素是双环八肽（鹅膏毒素）或七肽（鬼笔毒素）。在双孢伞菌中，α-鹅膏菌素和鬼笔酸丁分别合成为 35 个和 34 个氨基酸的前蛋白，其中成熟毒素中发现的氨基酸序列两侧是保守的氨基酸序列。紧邻毒素区域上游的不变 Pro 残基的存在以及毒素区域本身中最后预测的氨基酸表明 Pro 特异性肽酶负责鹅膏菌毒素前蛋白的初始翻译后加工。我们从产生鬼笔环肽的蘑菇 Conocybe albipes 中纯化了一种酶，该酶可裂解合成的 22 聚体鬼笔环肽，释放成熟的毒素肽 (AWLATCP)。对纯化蛋白的质谱分析结合编码基因的分离和测序表明，负责的加工酶是蛋白酶的脯氨酰寡肽酶 (POP) 亚家族 (EC 3.4.21.26) 的成员。该加工酶能够使用显色 POP 底物苯甲氧基羰基-甘氨酸-脯氨酸-对硝基苯胺，并被特定 POP 抑制剂苯甲氧基羰基-脯氨酸-脯氨酸抑制。蛋白原中的两个 Pro 键均由相同的酶裂解，C 端 Pro 键首先裂解或比 N 端 Pro 键裂解得快得多。 N 末端中间体的瞬时积累表明裂解不是很强的进行性。代表鬼笔酸原蛋白的合成肽也被白色念珠菌的 POP 裂解，但鹅膏菌素的前体（不是由白色念珠菌产生）被低效裂解。
The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, alpha-amanitin and phallacidin are synthesized as 35-and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstream of the toxin regions and as the last predicted amino acid in the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide and was inhibited by the specific POP inhibitor benzyloxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently.