Cysteine proteinases from distinct cellular compartments are recruited to phagocytic vesicles by Entamoeba histolytica
Cysteine proteinases from distinct cellular compartments are recruited to phagocytic vesicles by Entamoeba histolytica
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DOI：
10.1016/s0166-6851(01)00387-5
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发表时间：
2002-01-01
影响因子：
1.5
通讯作者：
Reed, SL
中科院分区：
文献类型：
--
作者：
Que, XC;Brinen, LS;Reed, SL
半胱氨酸蛋白酶，这是由至少七个基因编码，发挥了关键作用，在侵袭性阿米巴病的发病机制所造成的溶组织内阿米巴。这些酶的研究一直受到阻碍，无法获得大量的个别天然蛋白酶。我们现在已经在杆状病毒表达载体中表达了具有功能活性的重组ACP 1（EhCP 3）和ACP 2（EhCP 2）蛋白酶。纯化的重组ACP1和ACP2蛋白酶表现出类似的荧光肽底物的活动，特别是在他们的偏好在P2位置的精氨酸残基。虽然ACP 1和ACP 2在结构上是组织蛋白酶L，但同源性建模显示S2口袋中的天冬氨酸将导致对带正电荷的氨基酸（如组织蛋白酶B）的底物特异性。肽底物的水解强烈抑制小肽抑制剂专门设计的寄生虫半胱氨酸蛋白酶。共聚焦和免疫电镜定位的蛋白酶与单克隆抗体和单特异性抗体提出的重组酶和肽表明，ACP 2是膜相关的，而ACP 1是细胞质。吞噬红细胞后，ACP 1以及膜相关半胱氨酸蛋白酶ACP 2被掺入吞噬囊泡中。这些研究表明E.溶组织菌具有用于细胞内消化的半胱氨酸蛋白酶的冗余，并且它们可以从不同的细胞区室募集到被吞噬细胞的消化位点。杆状病毒中活性蛋白酶的产生和细菌中大规模重组酶的产生将进一步加深我们对不同半胱氨酸蛋白酶基因产物在毒力中作用的理解。(C)2002 Elsevier Science B.V.保留所有权利。
Cysteine proteinases, which are encoded by at least seven genes, play a critical role in the pathogenesis of invasive amebiasis caused by Entamoeba histolytica. The study of these enzymes has been hampered by the inability to obtain significant quantities of the individual native proteinases. We have now expressed functionally active recombinant ACP1 (EhCP3) and ACP2 (EhCP2) proteinases in baculoviral expression vectors. The purified recombinant ACP1 and ACP2 proteinases exhibited similar activities for fluorogenic peptide substrates, especially in their preference for an arginine residue at the P2 position. Although ACP1 and ACP2 are structurally cathepsin L, homology modeling revealed that the aspartic acid in the S2 pocket would result in a substrate specificity for positively charged amino acids, like cathepsin B. The hydrolysis of peptide substrates was strongly inhibited by small peptidyl inhibitors specifically designed for parasitic cysteine proteinases. Confocal and immunoelectron microscopy localization of the proteinases with monoclonal and monospecific antibodies raised to the recombinant enzymes and peptides demonstrated that ACP2 was membrane-associated while ACP1 was cytoplasmic. Following phagocytosis of erythrocytes, ACP1, as well as the membrane-associated cysteine proteinase, ACP2, were incorporated into phagocytic vesicles. These studies suggest that E. histolytica has a redundancy of cysteine proteinases for intracellular digestion and that they may be recruited from different cellular compartments to the site of digestion of phagocytosed cells. The production of active proteinases in baculovirus and large scale recombinant enzymes in bacteria should further our understanding of the role of different cysteine proteinase gene products in virulence. (C) 2002 Elsevier Science B.V. All rights reserved.