三种调节昆虫免疫应答的新型serpin的分离纯化及生物学功能研究
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摘要
与人类的凝血途径和补体系统同理,许多昆虫的免疫应答由丝氨酸蛋白酶(serine protease, SP)级联介导,如黑化反应和Toll信号通路等。过度产生免疫应答会对宿主造成自身伤害,所以对激活免疫应答的上游信号进行精确调节至关重要。Serpin(serine protease inhibitor)是蛋白酶抑制因子中最大也是最重要的超家族,serpin使用自杀底物原理与靶酶形成稳定的共价复合物,导致靶酶失活。大量研究证实serpin参与脊椎动物中多种SP级联的活性调控,但对于serpin调节无脊椎动物的先天免疫应答的具体分子机理,我们仍然所知甚少,其原因主要是难以确定serpin的天然特异靶酶。
最近,本实验室报导了黄粉虫中的由三种SP组成的级联反应可以特异的激活Toll信号通路和黑色素合成。利用以上三种SP,我们从黄粉虫血淋巴中成功的检测到并纯化了三种新型的serpin,根据在SDS-PAGE胶上显示的分子量将它们分别命名为SPN40、SPN55和SPN48。
下一步我们克隆了新型serpin的cDNA序列,三种serpin的核苷酸序列在Genbank的登陆号分别是AB514563、AB514564和AB514565。这些serpin与Toll信号通路中的三种上游SP形成特异的复合物,如SPN40-MSP (Modular Serine Protease), SPN55-SAE(Spatzle-processing enzyme-Activating Enzyme)和SPN48-SPE (Spatzle-Processing Enzyme)三种配对。我们测定了靶酶在serpin的RCL(Reactive Center Loop)上的特异水解位点,出乎意料的是SPN55和SPN48分别在Tyr和Glu被特异水解,不符合它们的靶酶SAE和SPE的胰蛋白酶活性特点,这种现象归因于以上serpin使用外识别位点的机制增强自身的靶酶特异性。
在向黄粉虫中注射入Toll受体的天然配基Spatzle后,血淋巴中的SPN40和SPN55的含量大幅增加,说明了在Toll被激活后产生的细胞内信号能够诱导相关serpin的表达,大量的SPN40和SPN55以负反馈的机理从细胞进入血淋巴并阻断上游信号的传递。
体外活性重建实验证实了三种serpin可以共同阻断SP级联的信号传递,注射三种serpin混合物能够明显的抑制由β-1,3葡聚糖诱导的黑化反应并增加黄粉虫在黑化反应下的存活率。
综上所述,本研究发现并证实了三种新型serpin特异的调节昆虫的两种主要免疫应答Toll信号通路和黑化反应。在国际上对Toll信号通路的研究中首次阐明了完整的serpin-SP的配对关系,证实了本领域研究中对昆虫SP级联的每步反应都受到抑制因子精确调控的假想。
Analogous to blood coagulation and complement system in human, many insect defense responses such as melanin biosynthesis and Toll pathway signaling are mediated by serine proteinase (SP) cascades, however, excessive activation of the immune response is harmful to host tissues and cells, thus, a precise negative regulation of the upstream signal is extremely critical for host surviving. Serpins (serine protease inhibitors) are the largest and most important superfamily of protease inhibitors, they act as suicide substrates by binding covalently to their target proteases, leading inactivation of activity. As we known, SP cascades are usually regulated by the serpins in vertebrates, however, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins.
We recently reported the molecular activation mechanisms of three serine protease mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. By using these SPs, we detected and purified three novel serpins from the hemolymph of T. molitor, according their molecule sizes on SDS-PAGE gel, we named them SPN40, SPN55 and SPN48, respectively.
We then cloned the serpin genes. The nucleotide sequence of serpins has been submitted to Genbank with accession numbers of AB514563, AB514564 and AB514565. These serpins made specific serpin-SP pairs with three Toll cascade-activating serine proteases:such as SPN40-MSP (Modular Serine Protease), SPN55-SAE (Spatzle-processing enzyme-Activating Enzyme), and SPN48-SPE (Spatzle-Processing Enzyme). we determined cleavage sites of each serpin on their reactive center loops (RCL). Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in RCL, respectively, despite being targeted by trypsin-like SAE and SPE。
The levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll-ligand, processed Spatzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors.
Results of in vitro reconstitution experiment suggest three serpins cooperatively blocked the Toll signaling cascade, furthermore, injection of three serpins into Tenebrio larvae inhibitedβ-1, 3-glucan mediated melanin biosynthesis in vivo.
Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins, This is the first determination and functional study of specific SP-serpin pairs that are directly involved in the regulation of the pattern recognition protein-dependent Toll signaling cascade.
最近,本实验室报导了黄粉虫中的由三种SP组成的级联反应可以特异的激活Toll信号通路和黑色素合成。利用以上三种SP,我们从黄粉虫血淋巴中成功的检测到并纯化了三种新型的serpin,根据在SDS-PAGE胶上显示的分子量将它们分别命名为SPN40、SPN55和SPN48。
下一步我们克隆了新型serpin的cDNA序列,三种serpin的核苷酸序列在Genbank的登陆号分别是AB514563、AB514564和AB514565。这些serpin与Toll信号通路中的三种上游SP形成特异的复合物,如SPN40-MSP (Modular Serine Protease), SPN55-SAE(Spatzle-processing enzyme-Activating Enzyme)和SPN48-SPE (Spatzle-Processing Enzyme)三种配对。我们测定了靶酶在serpin的RCL(Reactive Center Loop)上的特异水解位点,出乎意料的是SPN55和SPN48分别在Tyr和Glu被特异水解,不符合它们的靶酶SAE和SPE的胰蛋白酶活性特点,这种现象归因于以上serpin使用外识别位点的机制增强自身的靶酶特异性。
在向黄粉虫中注射入Toll受体的天然配基Spatzle后,血淋巴中的SPN40和SPN55的含量大幅增加,说明了在Toll被激活后产生的细胞内信号能够诱导相关serpin的表达,大量的SPN40和SPN55以负反馈的机理从细胞进入血淋巴并阻断上游信号的传递。
体外活性重建实验证实了三种serpin可以共同阻断SP级联的信号传递,注射三种serpin混合物能够明显的抑制由β-1,3葡聚糖诱导的黑化反应并增加黄粉虫在黑化反应下的存活率。
综上所述,本研究发现并证实了三种新型serpin特异的调节昆虫的两种主要免疫应答Toll信号通路和黑化反应。在国际上对Toll信号通路的研究中首次阐明了完整的serpin-SP的配对关系,证实了本领域研究中对昆虫SP级联的每步反应都受到抑制因子精确调控的假想。
Analogous to blood coagulation and complement system in human, many insect defense responses such as melanin biosynthesis and Toll pathway signaling are mediated by serine proteinase (SP) cascades, however, excessive activation of the immune response is harmful to host tissues and cells, thus, a precise negative regulation of the upstream signal is extremely critical for host surviving. Serpins (serine protease inhibitors) are the largest and most important superfamily of protease inhibitors, they act as suicide substrates by binding covalently to their target proteases, leading inactivation of activity. As we known, SP cascades are usually regulated by the serpins in vertebrates, however, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins.
We recently reported the molecular activation mechanisms of three serine protease mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. By using these SPs, we detected and purified three novel serpins from the hemolymph of T. molitor, according their molecule sizes on SDS-PAGE gel, we named them SPN40, SPN55 and SPN48, respectively.
We then cloned the serpin genes. The nucleotide sequence of serpins has been submitted to Genbank with accession numbers of AB514563, AB514564 and AB514565. These serpins made specific serpin-SP pairs with three Toll cascade-activating serine proteases:such as SPN40-MSP (Modular Serine Protease), SPN55-SAE (Spatzle-processing enzyme-Activating Enzyme), and SPN48-SPE (Spatzle-Processing Enzyme). we determined cleavage sites of each serpin on their reactive center loops (RCL). Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in RCL, respectively, despite being targeted by trypsin-like SAE and SPE。
The levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll-ligand, processed Spatzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors.
Results of in vitro reconstitution experiment suggest three serpins cooperatively blocked the Toll signaling cascade, furthermore, injection of three serpins into Tenebrio larvae inhibitedβ-1, 3-glucan mediated melanin biosynthesis in vivo.
Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins, This is the first determination and functional study of specific SP-serpin pairs that are directly involved in the regulation of the pattern recognition protein-dependent Toll signaling cascade.
引文
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[3]Gettins, P.G. Serpin structure, mechanism, and function. Chem. Rev.2002,102,4751-4804
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[6]Irving JA, Pike RN, Dai W, Bromme D, Worrall DM, Silverman GA, et al. Evidencethat serpin architecture intrinsically supports papain-like cysteine protease inhibition:engineering alpha(1)-antitrypsin to inhibit cathepsin proteases. Biochemistry,2002,41:4998-5004.
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