Fragaki Konstantina, Ferrua Bernard, Mograbi Baharia, Waldispühl Julie, Kubar Joanna
Groupe de Recherche en Immunopathologie de la Leishmaniose (EA 2675), Faculté de Médecine, Nice, France.
BMC Microbiol. 2003 Apr 30;3:7. doi: 10.1186/1471-2180-3-7.
We report cloning and characterization of a novel Leishmania infantum protein which we termed Lepp12, and we examine its possible implication in the interference with intramacrophage signaling pathways.
The protein Lepp12 contains 87 amino acid sequence and exhibits 5 potential phosphorylation sites by protein kinase C (PKC). Recombinant GST-Lepp12 is phosphorylated in vitro by exogenous PKC and by PKC-like activities present in promastigote and in the myelomonocytic THP-1 cell line, indicating that at least one phosphorylation site is functional on the recombinant Lepp12. The natural Lepp12 protein is present in L. infantum promastigotes, as evidenced using specific anti-Lepp12 antibodies produced by immunopurification from acute phase VL patient sera. Interestingly, human patient sera are strongly reactive with GST-Lepp12, demonstrating immunogenic properties of Lepp12 in man, but no immune response to Lepp12 is detectable in experimentally infected animals. When isolated from promastigotes, Lepp12 migrates as two species of apparent MW of 18.3 kDa (major) and 14 kDa (minor), localizes in the nuclear fraction and appears constitutively phosphorylated. Natural Lepp12 is phosphorylable in vitro by both exogenous PKC and PKC-like activity present in THP-1 extracts. The intracellular Lepp12 transfected into THP-1 cells activates these cells to produce IL-1beta and induces an enhancing effect on PMA stimulated IL-1beta synthesis, as demonstrated using GST-Lepp12 transfectants.
Together these results indicate that Lepp12 represents a substrate for PKC or other PKC-like activities present in the promastigote form and the host cell and therefore may interfere with signal transduction pathways involving PKC.
我们报道了一种新型婴儿利什曼原虫蛋白的克隆与特性分析，我们将其命名为Lepp12，并研究了其在干扰巨噬细胞内信号通路方面的可能作用。
Lepp12蛋白包含87个氨基酸序列，具有5个蛋白激酶C（PKC）潜在磷酸化位点。重组GST-Lepp12在体外可被外源性PKC以及前鞭毛体和骨髓单核细胞THP-1细胞系中存在的PKC样活性磷酸化，这表明重组Lepp12上至少有一个磷酸化位点具有功能。天然Lepp12蛋白存在于婴儿利什曼原虫前鞭毛体中，通过从急性期VL患者血清中免疫纯化产生的特异性抗Lepp12抗体得以证实。有趣的是，人类患者血清与GST-Lepp12强烈反应，表明Lepp12在人体内具有免疫原性，但在实验感染的动物中未检测到对Lepp12的免疫反应。当从前鞭毛体中分离时，Lepp12以两种表观分子量分别为18.3 kDa（主要）和14 kDa（次要）的形式迁移，定位于细胞核部分，且似乎是组成性磷酸化的。天然Lepp12在体外可被外源性PKC以及THP-1提取物中存在的PKC样活性磷酸化。转染到THP-1细胞中的细胞内Lepp12激活这些细胞产生IL-1β，并对PMA刺激的IL-1β合成产生增强作用，使用GST-Lepp12转染体得以证明。
这些结果共同表明，Lepp12是前鞭毛体形式和宿主细胞中存在的PKC或其他PKC样活性的底物，因此可能干扰涉及PKC的信号转导通路。
