Acosta Daniel, Cancela Martín, Piacenza Lucia, Roche Leda, Carmona Carlos, Tort José F
Unidad de Biología Parasitaria, Instituto de Higiene, Facultad de Ciencias, Uruguay.
Mol Biochem Parasitol. 2008 Mar;158(1):52-64. doi: 10.1016/j.molbiopara.2007.11.011. Epub 2007 Nov 22.
Leucyl aminopeptidases (LAP) from different parasitic organisms are attracting attention as relevant players in parasite biology, and consequently being considered as candidates for drug and vaccine design. In fact, the highest protection level achieved in ruminant immunization by a native antigen was previously reported by us, using a purified LAP as immunogen in a sheep trial against fasciolosis. Here, we report the cloning of a full-length cDNA from adult F. hepatica encoding a member of the M17 family of LAP (FhLAP) and functional expression and characterization of the corresponding enzyme. FhLAP was closely related to Schistosoma LAPs, but interestingly distant from their mammalian host's homologues, and was expressed in all stages of the parasite life cycle. The recombinant enzyme, functionally expressed in Escherichia coli, showed a marked amidolytic preference against the synthetic aminopeptidase substrate l-leucine-7-amino-4-methylcoumarin (Leu-AMC) and was also active against Cys-AMC and Met-AMC. Both native and recombinant enzyme were stimulated by the addition of divalent cations predominantly Mn(2+), and strongly inhibited by bestatin and cysteine. Physico-chemical properties, localization by immunoelectron microscopy, MALDI-TOF analysis, and cross-reactivity of anti-rFhLAP immune serum demonstrated that the recombinant enzyme was identical to the previously purified gut-associated LAP from adult F. hepatica. Vaccination trials using rFhLAP for rabbit immunization showed a strong IgG response and a highly significant level of protection after experimental infection with F. hepatica metacercariae, confirming that FhLAP is a relevant candidate for vaccine development.
来自不同寄生生物的亮氨酰氨基肽酶（LAP）作为寄生虫生物学中的相关参与者正受到关注，因此被视为药物和疫苗设计的候选对象。事实上，我们之前报道过，在反刍动物免疫中，使用纯化的LAP作为免疫原进行绵羊抗肝片吸虫病试验时，一种天然抗原实现了最高的保护水平。在此，我们报道了从成年肝片吸虫中克隆出一个全长cDNA，其编码LAP的M17家族成员（FhLAP），并对相应酶进行了功能表达和特性鉴定。FhLAP与血吸虫LAP密切相关，但有趣的是与它们哺乳动物宿主的同源物距离较远，并且在寄生虫生命周期的所有阶段都有表达。在大肠杆菌中功能性表达的重组酶对合成的氨肽酶底物L-亮氨酸-7-氨基-4-甲基香豆素（Leu-AMC）表现出明显的酰胺水解偏好，并且对Cys-AMC和Met-AMC也有活性。天然酶和重组酶都受到二价阳离子（主要是Mn2+）的刺激，并被贝抑素和半胱氨酸强烈抑制。重组酶的物理化学性质、免疫电子显微镜定位、基质辅助激光解吸电离飞行时间（MALDI-TOF）分析以及抗rFhLAP免疫血清的交叉反应性表明，该重组酶与先前从成年肝片吸虫中纯化的肠道相关LAP相同。使用rFhLAP对兔子进行免疫的疫苗试验显示，在感染肝片吸虫囊蚴后，产生了强烈的IgG反应和高度显著的保护水平，证实FhLAP是疫苗开发的相关候选对象。
