Stallmach Robert, Kavishwar Manoli, Withers-Martinez Chrislaine, Hackett Fiona, Collins Christine R, Howell Steven A, Yeoh Sharon, Knuepfer Ellen, Atid Avshalom J, Holder Anthony A, Blackman Michael J
Division of Parasitology, MRC National Institute for Medical Research, London, NW7 1AA, UK.
Mol Microbiol. 2015 Apr;96(2):368-87. doi: 10.1111/mmi.12941. Epub 2015 Feb 11.
The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle.
疟原虫恶性疟原虫在红细胞内的寄生泡（PV）中进行复制。恶性疟原虫PV中最丰富的蛋白质称为SERA5，它在血液阶段至关重要，并且具有木瓜蛋白酶样结构域，这引发了人们对其作为蛋白水解酶发挥作用的猜测。然而，不同寻常的是，SERA5在木瓜蛋白酶样蛋白酶的典型催化半胱氨酸位置具有一个丝氨酸残基（Ser596），并且SERA5的功能或其是否发挥酶促作用尚不清楚。在本研究中，我们未能检测到与含有Ser596的寄生虫来源或重组蛋白相关的蛋白水解活性。然而，将Ser596替换为半胱氨酸残基产生了一种具有半胱氨酸蛋白酶特征的活性重组酶，表明SERA5可以结合肽。利用恶性疟原虫中的靶向同源重组，我们将Ser596替换为丙氨酸，没有产生表型后果，证明SERA5在寄生虫中不发挥必需的酶促作用。我们还可以用亲和纯化标签替换SERA5的内部片段。相比之下，使用几乎相同的靶向构建体，我们无法截短SERA5基因或在其C末端进行标记，也无法将Ser596替换为庞大的精氨酸残基。我们的研究结果表明，SERA5在恶性疟原虫血液阶段的生命周期中起着不可或缺但非酶促的作用。
