Structural and functional dissection of the adhesive domains of Plasmodium falciparum thrombospondin-related anonymous protein (TRAP)
Structural and functional dissection of the adhesive domains of Plasmodium falciparum thrombospondin-related anonymous protein (TRAP)
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DOI：
10.1042/bj20031500
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发表时间：
2004-05-01
影响因子：
4.1
通讯作者：
Sharma, A
中科院分区：
文献类型：
--
作者：
Akhouri, RR;Bhattacharyya, A;Sharma, A
TRAP(血栓反应蛋白相关匿名蛋白)是一种在肝细胞侵袭中起核心作用的子孢子表面蛋白。我们已经开发了分别使用细菌和杆状病毒表达系统重组生产整个ECD(胞外区)和TRAP的A结构域的程序。ECD和A结构域被纯化为均一，并在凝胶过滤柱上以非聚集的单体蛋白的形式迁移。这些黏附模块以剂量依赖和二价阳离子非依赖的方式与HepG2细胞结合。ECD和A结构域与HepG2细胞的结合被过量的硫化物类似物抑制得很差，这表明肝细胞上存在尚未确定的A结构域受体。利用基于表面等离子体共振的传感器技术(Biacore)，我们证明了TRAP ECD对肝素的亲和力(K-D=40 nM)比A结构域(K-D=79 nM)更高。我们还根据同源的von Willebrand因子A1结构域的晶体结构，提出了A结构域的三维结构。陷阱A结构域显示两个空间上不同的配体结合表面。A结构域的一个表面含有MlDAS(金属离子依赖的黏附位点)基序，其中Thr(131)和Asp(162)的点突变与子孢子对细胞感染性的损害有关。另一个表面包含一个推测的肝素结合位点，并由一个碱性残基簇组成。我们的研究表明，在肝细胞侵袭过程中，TRAP与多个受体相互作用。我们的结果也为将这些高质量的重组TRAP结构域包括在基于亚单位的疟疾疫苗中铺平了道路。
TRAP (thrombospondin-related anonymous protein) is a sporozoite surface protein that plays a central role in hepatocyte invasion. We have developed procedures for recombinant production of the entire ECD (extracellular domain) and A domain of TRAP using bacterial- and baculovirus-expression systems respectively. The ECD and A domain were purified to homogeneity and migrated on gel-filtration columns as non-aggregated, monomeric proteins. These adhesive modules bound to HepG2 cells in a dose-dependent and bivalent cation-independent manner. The binding of ECD and the A domain to HepG2 cells was inhibited poorly by an excess of sulphatide analogues, suggesting the presence of as yet unidentified receptors for the A domain on hepatocytes. Using surface-plasmon-resonance-based sensor technology (Biacore), we demonstrate that TRAP ECD has higher affinity for heparin (K-D = 40 nM) compared with the A domain (K-D = 79 nM). We also present a three-dimensional struc- lure of the A domain based on the crystal structure of the homologous von Willebrand factor A1 domain. The TRAP A domain shows two spatially distinct ligand-binding surfaces. One surface on the A domain contains the MlDAS (metal-ion-dependent adhesion site) motif, where point mutations of Thr(131) and Asp(162) correlate with impairment of cell infectivity by sporozoites. The other surface contains a putative heparin-binding site and consists of a basic residue cluster. Our studies suggest that TRAP interacts with multiple receptors during the hepatocyte invasion process. Our results also pave the way for inclusion of these high-quality recombinant TRAP domains in subunit-based vaccines against malaria.