Paul Gourab, Deshmukh Arunaditya, Kaur Inderjeet, Rathore Sumit, Dabral Surbhi, Panda Ashutosh, Singh Susheel Kumar, Mohmmed Asif, Theisen Michael, Malhotra Pawan
Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India.
Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India.
Malar J. 2017 Feb 16;16(1):79. doi: 10.1186/s12936-017-1716-0.
The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface.
Recombinant 6-cys proteins i.e. Pfs38, Pfs12, Pfs41 as well as PfMSP-1 were expressed and purified using Escherichia coli expression system and antibodies were raised against each of these proteins. These antibodies were used to immunoprecipitate the native proteins and their associated partners from parasite lysate. ELISA, Far western, surface plasmon resonance and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte binding assay with 6-cys proteins were undertaken to find out their possible role in host-parasite infection and seropositivity was assessed using Indian and Liberian sera.
Immunoprecipitation of parasite-derived polypeptides, followed by LC-MS/MS analysis, identified a large Pfs38 complex comprising of 6-cys proteins: Pfs41, Pfs38, Pfs12 and other merozoite surface proteins: GLURP, SERA5 and MSP-1. The existence of such a complex was further corroborated by several protein-protein interaction tools, co-localization and co-sedimentation analysis. Pfs38 protein of Pfs38 complex binds to host red blood cells (RBCs) directly via glycophorin A as a receptor. Seroprevalence analysis showed that of the six antigens, prevalence varied from 40 to 99%, being generally highest for MSP-1 and GLURP proteins.
Together the data show the presence of a large Pfs38 protein-associated complex on the parasite surface which is involved in RBC binding. These results highlight the complex molecular interactions among the P. falciparum merozoite surface proteins and advocate the development of a multi-sub-unit malaria vaccine based on some of these protein complexes on merozoite surface.
疟原虫基因组编码多种6 - Cys蛋白，这些蛋白含有由六个半胱氨酸残基组成的模块，形成三个分子内二硫键。这些蛋白在寄生虫生命周期的传播阶段和肝期已得到充分表征。在本研究中，在恶性疟原虫裂殖子表面鉴定出一个由6 - Cys蛋白Pfs41、Pfs38和Pfs12以及其他三种裂殖子表面蛋白：富含谷氨酸蛋白（GLURP）、SERA5和MSP - 1组成的大型复合物。
使用大肠杆菌表达系统表达并纯化重组6 - cys蛋白，即Pfs38、Pfs12、Pfs41以及PfMSP - 1，并针对每种蛋白制备抗体。这些抗体用于从寄生虫裂解物中免疫沉淀天然蛋白及其相关伴侣。进行酶联免疫吸附测定（ELISA）、Far western印迹法、表面等离子体共振和甘油密度梯度分级分离以确认各自的相互作用。此外，用6 - cys蛋白进行红细胞结合试验以确定它们在宿主 - 寄生虫感染中的可能作用，并使用印度和利比里亚血清评估血清阳性率。
对寄生虫来源的多肽进行免疫沉淀，随后进行液相色谱 - 串联质谱（LC - MS/MS）分析，鉴定出一个大型的Pfs38复合物，其由6 - Cys蛋白Pfs41、Pfs38、Pfs12以及其他裂殖子表面蛋白GLURP、SERA5和MSP - 1组成。几种蛋白质 - 蛋白质相互作用工具、共定位和共沉降分析进一步证实了这种复合物的存在。Pfs38复合物中的Pfs38蛋白通过血型糖蛋白A作为受体直接与宿主红细胞（RBC）结合。血清阳性率分析表明，在这六种抗原中，阳性率从40%到99%不等，一般以MSP - 1和GLURP蛋白最高。
这些数据共同表明在寄生虫表面存在一个与Pfs38蛋白相关的大型复合物，其参与红细胞结合。这些结果突出了恶性疟原虫裂殖子表面蛋白之间复杂的分子相互作用，并提倡基于裂殖子表面的一些这些蛋白复合物开发一种多亚基疟疾疫苗。
