Institute of Parasitology, McGill University, Macdonald Campus, Sainte Anne de Bellevue, Quebec, Canada.
PLoS One. 2011;6(11):e27119. doi: 10.1371/journal.pone.0027119. Epub 2011 Nov 1.
A novel glutamate-binding protein was identified in Schistosoma mansoni. The protein (SmGBP) is related to metabotropic glutamate receptors from other species and has a predicted glutamate binding site located within a Venus Flytrap module but it lacks the heptahelical transmembrane segment that normally characterizes these receptors. The SmGBP cDNA was cloned, verified by 5' and 3' Rapid Amplification of cDNA Ends (RACE) and shown to be polyadenylated at the 3'end, suggesting the transcript is full-length. The cloned cDNA was subsequently expressed in bacteria and shown to encode a functional glutamate-binding protein. Other studies, using a specific peptide antibody, determined that SmGBP exists in two forms, a monomer of the expected size and a stable but non-covalent dimer. The monomer and dimer are both present in the membrane fraction of S. mansoni and are resistant to extraction with high-salt, alkaline pH and urea, suggesting SmGBP is either an integral membrane protein or a peripheral protein that is tightly associated with the membrane. Surface biotinylation experiments combined with western blot analyses and confocal immunolocalization revealed that SmGBP localized to the surface membranes of adult male schistosomes, especially the dorsal tubercles. In contrast, we detected little or no expression of SmGBP either in the females or larval stages. A comparative quantitative PCR analysis confirmed that the level of SmGBP expression is several-fold higher in male worms than cercariae, and it is barely detectable in adult females. Together, the results identify SmGBP as a new type of schistosome glutamate receptor that is both gender- and stage-specific. The high-level expression of this protein in the male tubercles suggests a possible role in host-parasite interaction.
在曼氏血吸虫中鉴定出一种新型的谷氨酸结合蛋白。该蛋白（SmGBP）与来自其他物种的代谢型谷氨酸受体相关，并且在 Venus Flytrap 模块内具有预测的谷氨酸结合位点，但它缺乏通常表征这些受体的七螺旋跨膜片段。SmGBP cDNA 已被克隆，并通过 5' 和 3' 快速扩增 cDNA 末端（RACE）得到验证，并且在 3' 末端被聚腺苷酸化，表明该转录物是全长的。随后在细菌中表达了克隆的 cDNA，并显示其编码一种功能性的谷氨酸结合蛋白。其他研究使用特异性肽抗体确定 SmGBP 存在两种形式，即预期大小的单体和稳定但非共价的二聚体。单体和二聚体都存在于曼氏血吸虫的膜部分中，并且对高盐、碱性 pH 和尿素的提取具有抗性，表明 SmGBP 要么是一种完整的膜蛋白，要么是一种与膜紧密相关的外周蛋白。表面生物素化实验结合 Western blot 分析和共聚焦免疫定位显示，SmGBP 定位于成年雄性血吸虫的表面膜，特别是背突。相比之下，我们在雌性或幼虫阶段检测到 SmGBP 的表达很少或没有。比较定量 PCR 分析证实，SmGBP 在雄性蠕虫中的表达水平比尾蚴高几个数量级，在成年雌性中几乎检测不到。总之，这些结果表明 SmGBP 是一种新型的血吸虫谷氨酸受体，具有性别和阶段特异性。该蛋白在雄性结节中的高水平表达表明其在宿主-寄生虫相互作用中可能具有重要作用。
