Uzcanga Graciela L, Perrone Trina, Noda José Alfredo, Pérez-Pazos Jacqueline, Medina Rafael, Hoebeke Johan, Bubis José
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela.
Biochemistry. 2004 Jan 27;43(3):595-606. doi: 10.1021/bi0301946.
Salivarian trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as a defense against the host immune system. Although about 1000 VSG and pseudo-VSG genes are scattered throughout the trypanosome genome, each trypanosome expresses only one VSG, while the rest of the genes are transcriptionally silent. A 64-kDa glycosylated cross-reacting antigen between Trypanosoma evansi and Trypanosoma vivax (p64), which was purified from the TEVA1 T. evansi Venezuelan isolate, was proven here to represent the soluble form of a VSG. Initially, a biochemical characterization of p64 was carried out. Gel filtration chromatography, sedimentation, and chemical cross-linking provided evidences of the dimeric nature of p64. The hydrodynamic parameters indicated that p64 is asymmetrical with a frictional ratio f/fo = 1.57. Isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis revealed that p64 contained two isoforms with isoelectric points of 6.8-6.9 and 7.1-7.2. When p64 and three p64 Staphylococcus aureus V8 proteolytic fragments were sequenced, the same N-termini sequence was obtained: Ala-Pro-Ile-Thr-Asp-Ala-Asp-Leu-Gly-Pro-Ala-Gln-Ile-Ala-Asp, which displayed a significant homology with a putative Trypanosoma brucei VSG gene located on chromosome 4. Additionally, immunofluorescence microscopy on T. evansi and T. vivax established that p64 and its T. vivax homologue were confined to the surface of both parasites. An immunological characterization of this antigen was also carried out using several Venezuelan T. evansi isolates expressing different VSGs, which were obtained from naturally infected animals. Although sera from animals infected with the various T. evansi isolates recognized p64, only one isolate, besides TEVA1, contained polypeptides that were recognized by anti-p64 antibodies. All these results together with prior evidences [Uzcanga, G. et al. (2002) Parasitology 124, 287-299] confirmed that p64 is the soluble form of a T. evansi VSG, containing common epitopes recognized by sera from animals infected with T. evansi or T. vivax. Despite the huge repertoire of VSG genes existing on bloodstream trypanosomes, our data also demonstrated the potential use of a VSG variant from the TEVA1 T. evansi isolate as a diagnostic reagent.
涎源性锥虫利用其可变特异性表面糖蛋白（VSG）外壳的抗原变异来抵御宿主免疫系统。尽管约1000个VSG和假VSG基因分散在锥虫基因组中，但每个锥虫仅表达一种VSG，而其余基因转录沉默。从委内瑞拉伊氏锥虫TEVA1分离株中纯化出的伊氏锥虫和活泼锥虫之间的一种64 kDa糖基化交叉反应抗原（p64），在此被证实代表一种VSG的可溶性形式。最初，对p64进行了生化特性分析。凝胶过滤色谱、沉降和化学交联提供了p64为二聚体性质的证据。流体动力学参数表明p64不对称，摩擦比f/fo = 1.57。等电聚焦和二维聚丙烯酰胺凝胶电泳显示p64包含两种等电点分别为6.8 - 6.9和7.1 - 7.2的同工型。对p64及其三个金黄色葡萄球菌V8蛋白酶水解片段进行测序时，获得了相同的N端序列：丙氨酸 - 脯氨酸 - 异亮氨酸 - 苏氨酸 - 天冬氨酸 - 丙氨酸 - 天冬氨酸 - 亮氨酸 - 甘氨酸 - 脯氨酸 - 丙氨酸 - 谷氨酰胺 - 异亮氨酸 - 丙氨酸 - 天冬氨酸，该序列与位于4号染色体上的一个推测的布氏锥虫VSG基因具有显著同源性。此外，对伊氏锥虫和活泼锥虫的免疫荧光显微镜检查证实p64及其活泼锥虫同源物局限于两种寄生虫的表面。还使用从自然感染动物获得的几种表达不同VSG的委内瑞拉伊氏锥虫分离株对该抗原进行了免疫学特性分析。尽管血液中锥虫存在大量的VSG基因库，但我们的数据也证明了来自伊氏锥虫TEVA1分离株的一种VSG变体作为诊断试剂的潜在用途。
