Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development
Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development
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DOI：
10.1182/blood-2002-01-0101
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发表时间：
2002-08-01
期刊：
影响因子：
20.3
通讯作者：
Mizuno, A
中科院分区：
文献类型：
--
作者：
Hanspal, M;Dua, M;Mizuno, A
恶性疟原虫衍生的半胱氨酸蛋白酶falcipain-2在酸性pH下切割宿主红细胞血红蛋白，在中性pH下切割膜骨架的特定组分。对falcipain-2的这2种蛋白水解活性的阶段特异性表达的分析表明，血红蛋白水解活性在早期滋养体中最大，在晚期迅速下降，而膜骨架蛋白水解活性在滋养体晚期和滋养体期显著增加。在红细胞膜骨架蛋白中，锚蛋白和蛋白4.1在它们的C-末端附近被天然和重组的falcipain-2切割。为了鉴定蛋白4.1的水解位点处的精确肽序列，我们使用蛋白4.1的重组构建体作为底物，随后对切割产物进行MALDI-MS分析。我们发现，falcipain-2介导的蛋白4.1裂解后立即发生赖氨酸437，这是在一个区域内的血影蛋白结合域的红细胞膜稳定性的关键。含有切割位点的16-mer肽完全抑制酶活性并阻断falcipain-2诱导的红细胞血影碎片化。基于这些结果，我们提出，falcipain-2切割血红蛋白在早期滋养体阶段的酸性食物泡中，而它切割特定成分的红细胞骨架在晚期滋养体和滋养体阶段。正是骨骼蛋白的蛋白水解导致膜不稳定，这反过来又促进了体内寄生虫的释放。(C)2002年，美国血液学会。
Plasmodium falciparum-derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2-mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrinactin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2-induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo. (C) 2002 by The American Society of Hematology.