Casanova Carlo L, Xue Gongda, Taracha Evans L, Dobbelaere Dirk A
Molecular Pathology, Vetsuisse Faculty, University of Bern, CH-3012 Bern, Switzerland.
Mol Biochem Parasitol. 2006 Oct;149(2):144-54. doi: 10.1016/j.molbiopara.2006.05.005. Epub 2006 May 30.
The presence of the schizont stage of the obligate intracellular parasites Theileria parva or T. annulata in the cytoplasm of an infected leukocyte results in host cell transformation via a mechanism that has not yet been elucidated. Proteins, secreted by the schizont, or expressed on its surface, are of interest as they can interact with host cell molecules that regulate host cell proliferation and/or survival. The major schizont surface protein is the polymorphic immunodominant molecule, PIM, which contains a large glutamine- and proline-rich domain (QP-rd) that protrudes into the host cell cytoplasm. Analyzing QP-rd generated by in vitro transcription/translation, we found that the signal peptide was efficiently cleaved post-translationally upon addition of T cell lysate or canine pancreatic microsomes, whereas signal peptide cleavage of a control protein only occurred cotranslationally and in the presence of microsomal membranes. The QP-rd of PIM migrated anomalously in SDS-PAGE and removal of the 19 amino acids corresponding to the predicted signal peptide caused a decrease in apparent molecular mass of 24kDa. The molecule was analyzed using monoclonal antibodies that recognize a set of previously defined PIM epitopes. Depending on the presence or the absence of the signal peptide, two conformational states could be demonstrated that are differentially recognized, with N-terminal epitopes becoming readily accessible upon signal peptide removal, and C-terminal epitopes becoming masked. Similar observations were made when the QP-rd of PIM was expressed in bacteria. Our observations could also be of relevance to other schizont proteins. A recent analysis of the proteomes of T. parva and T. annulata revealed the presence of a large family of potentially secreted proteins, characterized by the presence of large stretches of amino acids that are also particularly rich in QP-residues.
专性细胞内寄生虫小泰勒虫或环形泰勒虫的裂殖体阶段存在于被感染白细胞的细胞质中，通过一种尚未阐明的机制导致宿主细胞转化。裂殖体分泌或在其表面表达的蛋白质备受关注，因为它们可与调节宿主细胞增殖和/或存活的宿主细胞分子相互作用。主要的裂殖体表面蛋白是多态性免疫显性分子PIM，它含有一个伸入宿主细胞质的富含谷氨酰胺和脯氨酸的大结构域（QP-rd）。通过体外转录/翻译产生QP-rd进行分析，我们发现加入T细胞裂解物或犬胰腺微粒体后，信号肽在翻译后被有效切割，而对照蛋白的信号肽切割仅在翻译过程中且在微粒体膜存在的情况下发生。PIM的QP-rd在SDS-PAGE中迁移异常，去除对应于预测信号肽的19个氨基酸导致表观分子量降低24kDa。使用识别一组先前定义的PIM表位的单克隆抗体对该分子进行分析。根据信号肽的存在与否，可以证明存在两种不同构象状态，它们被不同地识别，信号肽去除后N端表位变得易于接近，而C端表位被掩盖。当PIM的QP-rd在细菌中表达时也有类似的观察结果。我们的观察结果可能也与其他裂殖体蛋白相关。最近对小泰勒虫和环形泰勒虫蛋白质组的分析揭示了一个潜在分泌蛋白的大家族的存在，其特征是存在大量特别富含QP残基的氨基酸序列。
