Fasciola hepatica:: Parasite-secreted proteinases degrade all human IgG subclasses:: Determination of the specific cleavage sites and identification of the immunoglobulin fragments produced
Fasciola hepatica:: Parasite-secreted proteinases degrade all human IgG subclasses:: Determination of the specific cleavage sites and identification of the immunoglobulin fragments produced
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DOI：
10.1006/expr.1999.4479
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发表时间：
2000-02-01
影响因子：
2.1
通讯作者：
Goñi, F
中科院分区：
文献类型：
--
作者：
Berasain, P;Carmona, C;Goñi, F
本研究的重点是肝片形吸虫分泌的蛋白酶与人IgG亚类的关系。在不同的pH值和时间下，将IgG与成虫排泄产物或纯化的半胱氨酸蛋白酶组织蛋白酶L1和组织蛋白酶L2孵育。分离得到的Ig片段，用Western blot分析鉴定，用氨基酸序列分析确定具体的裂解位点。寄生虫排泄-分泌产物和两种组织蛋白酶L产生相似的降解模式，并在枢纽区域切割所有人IgG亚类，在pH 7.3和37℃下，IgG1和IgG3或Fab产生Fab和Fc片段，IgG2和IgG4产生Fc片段。IgG1和IgG3在有还原剂或不存在还原剂的情况下都容易被Ei S产物降解，而IgG2和IgG3对蛋白水解具有抗性，只有在0.1 M二硫苏糖醇的存在下才能被消化。组织蛋白酶L需要二硫苏糖醇的存在来消化IgG1， IgG2和IgG4，而IgG3在还原和非还原条件下都是相同的。E/S产物、CLI或CL2产生的主要切割位点分别位于肽键的位置：His237-Thr238、Glu237-Cys239、Gly233-Asp234和Ser241-Cys242，分别针对γ 1、γ 2、γ 3和γ 4。这些酶在IgG3的中间铰链上提供了额外的分裂位点，以产生更短的Fe片段，并对IgG4产生Fd降解。CL1和CL2之间没有发现切割特异性差异，但它们在IgG3降解动力学上存在差异。通过降低pH值，只有EIS产物在保留Fab部分的同时产生Fe的破坏。在所有测试条件下，酶产生一个14-15 kDa的Fc'样片段，与免疫球蛋白的整个CH3结构域相对应。与组织蛋白酶对消化蛋白的广泛降解相反，它对IgG亚类的作用是特异性和限制性的；因此，产生的所有片段可能潜在地参与了寄生虫用来逃避宿主免疫反应的机制，(C) 2000学术出版社。
The study was focused on the relationship of Fasciola hepatica-secreted proteinases and human IgG subclasses. Each IgG was incubated at different pH values and lengths of time with either the adult parasite excretion-secretion products or the purified cysteinyl proteinases cathepsin L1 and cathepsin L2. The Ig fragments produced were isolated and characterized by Western blot analysis, and the specific cleavage sites were determined by amino acid sequence analysis. Parasite excretion-secretion products and both cathepsins L produced similar degradation patterns and cleaved all human IgG subclasses at the hinge region, yielding at pH 7.3 and 37 degrees C Fab and Fc fragments in the case of IgG1 and IgG3 or Fab, and Fc in IgG2 and IgG4. While IgG1 and IgG3 were readily degraded by Ei S products either in the presence or in the absence of reducing agents, IgG2 and IgG3 were resistant to proteolysis and were only digested in the presence of 0.1 M dithiothreitol. The cathepsins L needed the presence of dithiothreitol to digest IgG1, IgG2, and IgG4 whereas IgG3 was identically cleaved under both reducing and nonreducing conditions. The main cleavage sites produced by E/S products, CLI, or CL2 were located at the positions peptide bonds: His237-Thr238, Glu237-Cys239, Gly233-Asp234, and Ser241-Cys242 for gamma 1, gamma 2, gamma 3, or gamma 4, respectively. The enzymes gave additional splitting sites on the middle hinge of IgG3 to produce shorter Fe fragments and also produce Fd degradation of the IgG4. No cleavage specificity differences were found between CL1 and CL2, but they differed in the kinetics of IgG3 degradation. By lowering the pH, only the EIS products produced concomitant destruction of the Fe while preserving the Fab portion. Under all the conditions assayed the enzymes produced an Fc'-like fragment of 14-15 kDa corresponding to the whole CH3 domain of the immunoglobulin. Contrary to the extensive degradation produced by cathepsins on digested proteins, its actions on IgG subclasses were specific and restricted; thus, all the fragments produced could be potentially involved in the mechanisms used by the parasite to evade the host immune response, (C) 2000 Academic Press.