Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria-causing parasites.
Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria-causing parasites.
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DOI：
10.1016/j.jbc.2021.101550
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发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Odom John AR
中科院分区：
文献类型：
--
作者：
Frasse PM;Miller JJ;Polino AJ;Soleimani E;Zhu JS;Jakeman DL;Jez JM;Goldberg DE;Odom John AR
引起疟疾的寄生虫恶性疟原虫每年造成2亿多人感染和40万人死亡。在其复杂的生命周期的多个阶段，恶性疟原虫表达几种通过糖基磷脂酰肌醇（GPI）锚栓在其表面的必需蛋白质，这些蛋白质对于寄生虫排出和宿主红细胞再入侵等生物过程至关重要。针对这一途径进行治疗有可能广泛影响寄生虫在多个生命阶段的发育。在这里，我们表征了寄生虫GPI锚生物合成的上游组分，推定的磷酸甘露变位酶（PMM）（EC 5.4.2.8），HAD 5（PF 3D7_1017400）。我们证实了PMM和磷酸葡萄糖变位酶活性的纯化重组HAD 5通过开发新的连接酶的生化测定。通过用TetR-DOZI诱导的敲低系统调节HAD 5在转基因寄生虫中的表达，我们证明了HAD 5是疟疾寄生虫外出和红细胞再侵袭所必需的，并且我们通过放射性标记的葡萄糖胺的放射自显影和薄层色谱法评估了HAD 5在GPI锚合成中的作用。最后，我们确定了HAD 5的三维X射线晶体结构，并确定了一种底物类似物，该底物类似物与orthopathic人PMM相比以时间依赖性方式特异性抑制HAD 5。这些发现表明GPI锚生物合成途径对寄生虫中的抑制异常敏感，并且HAD 5具有作为特异性多阶段抗疟靶标的潜力。
The malaria-causing parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of parasite GPI anchor biosynthesis, the putative phosphomannomutase (PMM) (EC 5.4.2.8), HAD5 (PF3D7_1017400). We confirmed the PMM and phosphoglucomutase activities of purified recombinant HAD5 by developing novel linked enzyme biochemical assays. By regulating the expression of HAD5 in transgenic parasites with a TetR-DOZI-inducible knockdown system, we demonstrated that HAD5 is required for malaria parasite egress and erythrocyte reinvasion, and we assessed the role of HAD5 in GPI anchor synthesis by autoradiography of radiolabeled glucosamine and thin layer chromatography. Finally, we determined the three-dimensional X-ray crystal structure of HAD5 and identified a substrate analog that specifically inhibits HAD5 compared to orthologous human PMMs in a time-dependent manner. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition in parasites and that HAD5 has potential as a specific, multistage antimalarial target.
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DOI：
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发表时间：
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影响因子：
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