Molecular cloning and characterization of a cathepsin B from Angiostrongylus cantonensis
Molecular cloning and characterization of a cathepsin B from Angiostrongylus cantonensis
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广州管圆线虫组织蛋白酶 B 的分子克隆和表征
DOI：
10.1007/s00436-011-2264-0
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发表时间：
2011-08-01
影响因子：
2
通讯作者：
Lv, Zhi-yue
中科院分区：
文献类型：
--
作者：
Han, Yan-ping;Li, Zheng-yu;Lv, Zhi-yue
半胱氨酸蛋白酶是水解酶的一个超家族，在寄生虫中具有多种功能。在此，我们报道了从广州管圆线虫第四期幼虫cDNA文库中编码组织蛋白酶B（AcCPB）的cDNA的克隆及特性。推导的氨基酸序列分析表明，AcCPB与其他组织蛋白酶B家族成员相关，具有整体保守的结构。AcCPB在进化上与其他寄生线虫的组织蛋白酶B比与宿主的更为接近，与其他生物的同源物具有43 - 53%的相似性。实时定量PCR分析显示，AcCPB在第四期幼虫（L4）和第五期幼虫（L5）中的表达显著高于第三期幼虫（L3）和成虫（Aw）。出乎意料的是，来自小鼠的L4和L5中AcCPB的表达水平高于来自大鼠的相同阶段的幼虫。在大肠杆菌中表达的重组AcCPB（rAcCPB）的蛋白酶活性显示出高热稳定性和最适酸性pH。广州管圆线虫的半胱氨酸蛋白酶抑制剂能明显抑制rAcCPB在卵清蛋白消化中的作用和酶活性。此外，我们发现rAcCPB增加了脂多糖刺激的树突状细胞（DCs）上CD40、MHC II和CD80的表达水平。在本研究中，我们为广州管圆线虫中组织蛋白酶B的表达提供了首个实验证据。除了在幼虫导致宿主血脑屏障功能障碍的L4和L5阶段高度特异性表达外，AcCPB在非适宜宿主和适宜宿主来源的幼虫阶段显示出不同的表达谱，并参与树突状细胞的成熟，这表明其在中枢神经系统入侵以及寄生虫 - 宿主相互作用过程中的免疫调节中具有潜在作用。
Cysteine proteases, a superfamily of hydrolytic enzymes, have numerous functions in parasites. Here, we reported the cloning and characterization of a cDNA encoding a cathepsin B (AcCPB) from Angiostrongylus cantonensis fourth-stage larvae cDNA library. The deduced amino acid sequence analysis indicated AcCPB is related to other cathepsin B family members with an overall conserved architecture. AcCPB is evolutionarily more close to other parasitic nematode cathepsin B than the ones from hosts, sharing 43-53% similarities to the homologues from other organisms. Real-time quantitative PCR analysis revealed that AcCPB was expressed significantly higher in the fourth-stage larvae (L4) and the fifth-stage larvae (L5) than that in the third-stage larvae (L3) and adult worms (Aw). Unexpectedly, AcCPB was expressed at a higher level in L4 and L5 derived from mice than the larvae at the same stages derived from rats. The protease activity of recombinant AcCPB (rAcCPB) expressed in Escherichia coli showed high thermostability and acidic pH optima. The role in ovalbumin digestion and enzyme activity of rAcCPB could be evidently inhibited by cystatin from A.cantonensis. Furthermore, we found rAcCPB increased the expression levels of CD40, MHC II, and CD80 on LPS-stimulated dendritic cells (DCs). In this study, we provided the first experimental evidence for the expression of cathepsin B in A.cantonensis. Besides its highly specific expression in the stages of L4 and L5 when the worms cause dysfunction of the blood-brain barrier of hosts, AcCPB displayed different expression profiles in non-permissive host- and permissive host-derived larval stages and was involved in the maturation of DCs, suggesting a potential role in the central nervous system invasion and the immunoregulation during parasite-host interactions.