Zemzoumi K, Sereno D, François C, Guilvard E, Lemesre J L, Ouaissi A
CJF INSERM 96-04, Montpellier, France.
Biol Cell. 1998 Jun;90(3):239-45. doi: 10.1016/s0248-4900(98)80020-8.
In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms. In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (e.g. logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes). The reactive antigen was also detected in Trypanosoma cruzi extracts. Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used. No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti-LmSIR2rp immune serum against parasite nuclear extracts. Furthermore, immunoprecipitation of [35S]methionine-labeled promastigote antigens after pulse chase experiments, using anti-LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted-secreted antigens (ESA). Moreover, immunofluorescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface. The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues.
在先前的研究中，我们已对几种硕大利什曼原虫多肽进行了表征，并表明该组中的一个成员（LmSIR2rp）与酿酒酵母的沉默信息调节因子2（SIR2）具有显著同源性，SIR2蛋白在这些生物体的端粒和交配型基因座抑制中均发挥作用。在本研究中，通过使用分子和免疫学方法，我们能够在不同的利什曼原虫物种和发育阶段（例如对数期（LP）和静止期前鞭毛体（SP）以及无鞭毛体）中鉴定出LmSIR2rp同源物。在克氏锥虫提取物中也检测到了反应性抗原。令人惊讶的是，免疫荧光分析显示，根据所使用寄生虫的生命阶段，LmSIR2rp主要与大小和数量不同的细胞质颗粒相关。在细胞核中未观察到反应性，这与蛋白质印迹结果一致，即抗LmSIR2rp免疫血清对寄生虫核提取物无免疫反应性。此外，在脉冲追踪实验后，使用抗LmSIR2rp融合蛋白抗体对[35S]甲硫氨酸标记的前鞭毛体抗原进行免疫沉淀，结果表明该蛋白属于寄生虫分泌-排泄抗原（ESA）。此外，用纯化的LmSIR2rp或活的前鞭毛体与小鼠巨噬细胞进行短时间孵育后进行的免疫荧光分析表明，LmSIR2rp可以与巨噬细胞表面结合。LmSIR2rp意外的细胞质定位及其在ESA中的存在可能暗示了沉默信息调节因子同源物的一种新作用模式。
