Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense
Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense
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DOI：
10.1016/s0166-6851(01)00368-1
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发表时间：
2001-11-01
影响因子：
1.5
通讯作者：
McKerrow, JH
中科院分区：
文献类型：
--
作者：
Caffrey, CR;Hansell, E;McKerrow, JH
非洲锥虫寄生虫的半胱氨酸蛋白酶活性是使用合成蛋白酶抑制剂进行新化疗的靶标。为了支持这一努力并进一步表征该酶，我们从毕赤酵母中表达并纯化了罗得西亚蛋白酶，这是布鲁氏罗得西亚锥虫（MVAT4菌株）的靶蛋白酶。罗得西亚蛋白酶是一种活性的成熟蛋白酶。先前未发现的隐糖基化基序的定点突变允许生产适合结晶的罗得西亚蛋白酶。在rhodesain前肽序列中发现了一个不变的ER(A/V)FNAA基序，该基序是锥虫属特有的。罗得西亚锥虫的抗体将蛋白酶定位在溶酶体中，并在细长的罗得西亚锥虫和布氏锥虫的所有生命周期阶段中鉴定出一个40 kda的蛋白，在布氏锥虫中，蛋白酶在短小锥虫中的表达量是其他阶段的5倍。放射性标记的活性位点定向抑制剂鉴定了布鲁氏杆菌中主要的半胱氨酸蛋白酶是布鲁氏杆菌。拟肽类乙烯砜和环氧化物抑制剂被设计用于与活性位点裂缝的S-2、S-1和S'亚位相互作用，揭示了罗得西亚蛋白酶与相关的锥虫蛋白酶cruzain之间的差异。采用荧光二肽基底物，罗得西亚蛋白酶和cruzain具有最佳的酸性pH值。但与一些哺乳动物不同的是，组织蛋白酶在pH 8.0时仍保持显著的活性和稳定性，这与可能的细胞外功能一致。罗得西亚蛋白酶和cruzain与荧光肽基底物的S-2亚位图谱表明，在S-2上存在丙氨酸而不是谷氨酸阻止罗得西亚蛋白酶切割P-1为精氨酸的底物。(C) 2001 Elsevier Science B.V.版权所有
Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucci rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei, With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S-2, S-1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima. but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S-2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S-2 prevents rhodesain from cleaving substrates in which P-1 is arginine. (C) 2001 Elsevier Science B.V. All rights reserved.