Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India.
PLoS One. 2013 Apr 25;8(4):e62829. doi: 10.1371/journal.pone.0062829. Print 2013.
Tryptophan-rich antigens play important role in host-parasite interaction. One of the Plasmodium vivax tryptophan-rich antigens called PvTRAg33.5 had earlier been shown to be predominantly of alpha helical in nature with multidomain structure, induced immune responses in humans, binds to host erythrocytes, and its sequence is highly conserved in the parasite population. In the present study, we divided this protein into three different parts i.e. N-terminal (amino acid position 24-106), middle (amino acid position 107-192), and C-terminal region (amino acid position 185-275) and determined the erythrocyte binding activity of these fragments. This binding activity was retained by the middle and C-terminal fragments covering 107 to 275 amino acid region of the PvTRAg33.5 protein. Eight non-overlapping peptides covering this 107 to 275 amino acid region were then synthesized and tested for their erythrocyte binding activity to further define the binding domains. Only two peptides, peptide P4 (at 171-191 amino acid position) and peptide P8 (at 255-275 amino acid position), were found to contain the erythrocyte binding activity. Competition assay revealed that each peptide recognizes its own erythrocyte receptor. These two peptides were found to be located on two parallel helices at one end of the protein in the modelled structure and could be exposed on its surface to form a suitable site for protein-protein interaction. Natural antibodies present in the sera of the P. vivax exposed individuals or the polyclonal rabbit antibodies against this protein were able to inhibit the erythrocyte binding activity of PvTRAg33.5, its fragments, and these two synthetic peptides P4 and P8. Further studies on receptor-ligand interaction might lead to the development of the therapeutic reagent.
富含色氨酸的抗原在宿主-寄生虫相互作用中起着重要作用。恶性疟原虫（Plasmodium vivax）的一种富含色氨酸的抗原称为 PvTRAg33.5，先前已表明其本质上主要是具有多结构域的α螺旋结构，能诱导人体产生免疫反应，与宿主红细胞结合，并且其序列在寄生虫群体中高度保守。在本研究中，我们将该蛋白分为三个不同部分，即 N 端（氨基酸位置 24-106）、中间（氨基酸位置 107-192）和 C 端区域（氨基酸位置 185-275），并测定了这些片段的红细胞结合活性。中间和 C 端片段保留了这种结合活性，涵盖了 PvTRAg33.5 蛋白的 107-275 个氨基酸区域。然后合成了 8 个不重叠的肽段，覆盖了 107-275 个氨基酸区域，并测试了它们与红细胞的结合活性，以进一步确定结合结构域。只有两个肽段，肽段 P4（位于 171-191 个氨基酸位置）和肽段 P8（位于 255-275 个氨基酸位置），被发现具有红细胞结合活性。竞争实验表明，每个肽段都识别自己的红细胞受体。这两个肽段被发现位于蛋白质一端的两个平行螺旋上，可以暴露在其表面，形成适合蛋白质-蛋白质相互作用的位点。来自恶性疟原虫暴露个体血清中的天然抗体或针对该蛋白的多克隆兔抗体能够抑制 PvTRAg33.5、其片段以及这两个合成肽段 P4 和 P8 的红细胞结合活性。对受体-配体相互作用的进一步研究可能会导致治疗试剂的开发。
