Braschi Simon, Curwen Rachel S, Ashton Peter D, Verjovski-Almeida Sergio, Wilson Alan
Department of Biology, University of York, York, UK.
Proteomics. 2006 Mar;6(5):1471-82. doi: 10.1002/pmic.200500368.
The blood fluke Schistosoma mansoni can live for years in the hepatic portal system of its human host and so must possess very effective mechanisms of immune evasion. The key to understanding how these operate lies in defining the molecular organisation of the exposed parasite surface. The adult worm is covered by a syncytial tegument, bounded externally by a plasma membrane and overlain by a laminate secretion, the membranocalyx. In order to determine the protein composition of this surface, the membranes were detached using a freeze/thaw technique and enriched by sucrose density gradient centrifugation. The resulting preparation was sequentially extracted with three reagents of increasing solubilising power. The extracts were separated by 2-DE and their protein constituents were identified by MS/MS, yielding predominantly cytosolic, cytoskeletal and membrane-associated proteins, respectively. After extraction, the final pellet containing membrane-spanning proteins was processed by liquid chromatographic techniques before MS. Transporters for sugars, amino acids, ions and other solutes were found together with membrane enzymes and proteins concerned with membrane structure. The proteins identified were categorised by their function and putative location on the basis of their homology with annotated proteins in other organisms.
曼氏血吸虫这种血液吸虫能在人类宿主的肝门静脉系统中存活数年，因此必定拥有非常有效的免疫逃避机制。理解这些机制如何运作的关键在于确定暴露的寄生虫表面的分子结构。成虫被一层合胞体皮层覆盖，其外部由质膜界定，并被一层片状分泌物即膜被覆盖。为了确定该表面的蛋白质组成，使用冻融技术分离膜，并通过蔗糖密度梯度离心进行富集。所得制剂依次用三种溶解能力递增的试剂进行提取。提取物通过二维电泳分离，其蛋白质成分通过串联质谱鉴定，分别主要产生胞质、细胞骨架和膜相关蛋白。提取后，含有跨膜蛋白的最终沉淀在进行质谱分析之前通过液相色谱技术进行处理。发现了糖、氨基酸、离子和其他溶质的转运蛋白，以及与膜结构有关的膜酶和蛋白质。根据与其他生物体中注释蛋白的同源性，将鉴定出的蛋白质按其功能和假定位置进行分类。
