Rojas Agustina, García-Lugo Pablo, Crisante Gladys, Añez-Rojas Néstor, Añez Néstor
Investigaciones Parasitológicas J.F. Torrealba, Facultad de Ciencias, Departamento de Biología, Universidad de Los Andes, Mérida 5101, Venezuela.
Acta Trop. 2008 Feb;105(2):139-44. doi: 10.1016/j.actatropica.2007.10.009. Epub 2007 Nov 4.
GPI-anchored proteins from the plasma membrane of Leishmania (Viannia) braziliensis promastigotes were isolated, characterized and their migration pattern compared with those from other Leishmania species. In all cases the SDS-PAGE migration patterns were obtained under reducing and non-reducing conditions, using DL-dithiothreitol (DTT) as a reducer agent. Our results reveal that under reducing conditions the SDS-PAGE migration pattern is modified as a consequence of the disruption of disulphur-bonds and protein transformation. This is demonstrated when in non-reducing conditions the L. (V.) braziliensis-GPI-anchored proteins pattern showed a group of bands over the 100kDa, and two more bands of 52kDa and 50kDa in four different isolates, whereas under reducing conditions the major GPI-anchored protein fractions were detected as bands of 63kDa, 50kDa and an increase of peptides between 34kDa and 22kDa. Similar modifications were detected in the SDS-PAGE migration patterns of GPI-anchored protein fractions from L. (Leishmania) donovani, L. (L.) mexicana and L. (L.) amazonensis run under the same reducing conditions. Antigenic evaluation carried out by Western blot revealed the presence of two very specific L. (V.) braziliensis-GPI-anchored protein bands of 50kDa and 28kDa. These bands were specifically recognized by anti-L. (V.) braziliensis-GPI-anchored protein serum from experimentally immunized animals. These two peptides were not detected when GPI-anchored protein fractions from L. (L.) donovani, L. (L.) mexicana and L. (L.) amazonensis, were challenged with the same anti-serum. The present results lead us to suggest the use of these two peptides as biochemical markers to identify and differentiate leishmaniasis caused by L. (V.) braziliensis. The lack of immunogenicity observed here with the peptide gp63, a very common protein detected in Leishmania species, is considered.
从巴西利什曼原虫（维氏亚属）前鞭毛体的质膜中分离出糖基磷脂酰肌醇（GPI）锚定蛋白，对其进行表征，并将其迁移模式与其他利什曼原虫物种的进行比较。在所有情况下，使用二硫苏糖醇（DTT）作为还原剂，在还原和非还原条件下获得十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）迁移模式。我们的结果表明，在还原条件下，由于二硫键的破坏和蛋白质转化，SDS-PAGE迁移模式发生改变。这在非还原条件下得到证明，即巴西利什曼原虫（维氏亚属）-GPI锚定蛋白模式在四个不同分离株中显示出一组超过100kDa的条带，以及另外两条52kDa和50kDa的条带，而在还原条件下，主要的GPI锚定蛋白组分被检测为63kDa、50kDa的条带以及34kDa至22kDa之间肽段的增加。在相同还原条件下运行的来自杜氏利什曼原虫（利什曼亚属）、墨西哥利什曼原虫（利什曼亚属）和亚马逊利什曼原虫（利什曼亚属）的GPI锚定蛋白组分的SDS-PAGE迁移模式中也检测到类似的变化。通过蛋白质印迹法进行的抗原评估显示存在两条非常特异的50kDa和28kDa的巴西利什曼原虫（维氏亚属）-GPI锚定蛋白条带。这些条带被来自实验免疫动物的抗巴西利什曼原虫（维氏亚属）-GPI锚定蛋白血清特异性识别。当用相同的抗血清检测来自杜氏利什曼原虫（利什曼亚属）、墨西哥利什曼原虫（利什曼亚属）和亚马逊利什曼原虫（利什曼亚属）的GPI锚定蛋白组分时，未检测到这两种肽段。目前的结果使我们建议使用这两种肽段作为生化标志物来鉴定和区分由巴西利什曼原虫（维氏亚属）引起的利什曼病。同时考虑到这里观察到的在利什曼原虫物种中非常常见的gp63肽段缺乏免疫原性的情况。
