Gilson Paul R, Nebl Thomas, Vukcevic Damjan, Moritz Robert L, Sargeant Tobias, Speed Terence P, Schofield Louis, Crabb Brendan S
The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia.
Mol Cell Proteomics. 2006 Jul;5(7):1286-99. doi: 10.1074/mcp.M600035-MCP200. Epub 2006 Apr 7.
Most proteins that coat the surface of the extracellular forms of the human malaria parasite Plasmodium falciparum are attached to the plasma membrane via glycosylphosphatidylinositol (GPI) anchors. These proteins are exposed to neutralizing antibodies, and several are advanced vaccine candidates. To identify the GPI-anchored proteome of P. falciparum we used a combination of proteomic and computational approaches. Focusing on the clinically relevant blood stage of the life cycle, proteomic analysis of proteins labeled with radioactive glucosamine identified GPI anchoring on 11 proteins (merozoite surface protein (MSP)-1, -2, -4, -5, -10, rhoptry-associated membrane antigen, apical sushi protein, Pf92, Pf38, Pf12, and Pf34). These proteins represent approximately 94% of the GPI-anchored schizont/merozoite proteome and constitute by far the largest validated set of GPI-anchored proteins in this organism. Moreover MSP-1 and MSP-2 were present in similar copy number, and we estimated that together these proteins comprise approximately two-thirds of the total membrane-associated surface coat. This is the first time the stoichiometry of MSPs has been examined. We observed that available software performed poorly in predicting GPI anchoring on P. falciparum proteins where such modification had been validated by proteomics. Therefore, we developed a hidden Markov model (GPI-HMM) trained on P. falciparum sequences and used this to rank all proteins encoded in the completed P. falciparum genome according to their likelihood of being GPI-anchored. GPI-HMM predicted GPI modification on all validated proteins, on several known membrane proteins, and on a number of novel, presumably surface, proteins expressed in the blood, insect, and/or pre-erythrocytic stages of the life cycle. Together this work identified 11 and predicted a further 19 GPI-anchored proteins in P. falciparum.
覆盖人类疟原虫恶性疟原虫细胞外形式表面的大多数蛋白质通过糖基磷脂酰肌醇（GPI）锚定连接到质膜上。这些蛋白质会暴露于中和抗体中，其中几种是先进的疫苗候选物。为了鉴定恶性疟原虫的GPI锚定蛋白质组，我们结合了蛋白质组学和计算方法。聚焦于生命周期中与临床相关的血液阶段，对用放射性葡糖胺标记的蛋白质进行蛋白质组学分析，确定了11种蛋白质（裂殖子表面蛋白（MSP）-1、-2、-4、-5、-10、棒状体相关膜抗原、顶端寿司蛋白、Pf92、Pf38、Pf12和Pf34）上存在GPI锚定。这些蛋白质约占GPI锚定裂殖体/裂殖子蛋白质组的94%，是该生物体中迄今为止最大的一组经过验证的GPI锚定蛋白质。此外，MSP-1和MSP-2的拷贝数相似，我们估计这些蛋白质一起约占总膜相关表面涂层的三分之二。这是首次对MSP的化学计量进行研究。我们观察到，在预测恶性疟原虫蛋白质上的GPI锚定方面，现有软件表现不佳，而蛋白质组学已验证了这些蛋白质上的这种修饰。因此，我们开发了一种基于恶性疟原虫序列训练的隐马尔可夫模型（GPI-HMM），并用它根据所有蛋白质被GPI锚定的可能性对恶性疟原虫完整基因组中编码的所有蛋白质进行排名。GPI-HMM预测了所有经过验证的蛋白质、几种已知膜蛋白以及生命周期血液、昆虫和/或前红细胞阶段表达的许多新的、可能位于表面的蛋白质上的GPI修饰。这项工作共鉴定出恶性疟原虫中11种GPI锚定蛋白质，并预测了另外19种。
