Institute of Phytopathology and Applied Zoology, Justus Liebig University of Giessen, D-35392 Giessen, Germany.
Biol Chem. 2009 Dec;390(12):1303-11. doi: 10.1515/BC.2009.128.
The immune system can be stimulated by microbial molecules as well as by endogenously derived danger/alarm signals of host origin. Using the lepidopteran model insect Galleria mellonella, we recently discovered that fragments of collagen IV, resulting from hydrolysis by microbial metalloproteinases, represent danger/alarm signals in insects. Here, we characterized immune-stimulatory peptides generated by thermolysin-mediated degradation of collagen IV using nanospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) after separation by nanoscale liquid chromatography (nanoLC). The combination of FTICR MS analysis and de novo peptide sequencing resulted in the identification of 38 specific collagen IV fragments of which several peptides included the integrin-binding motif RGD/E known from numerous mammalian immune-related proteins. Custom-synthesized peptides corresponding either to the presently identified collagen peptide GIRGEHyp or to a well-known integrin-binding RGD peptide (GRGDS) were injected into G. mellonella to determine their immune-stimulatory activities in vivo. Both peptides stimulated immune cells and systemically the expression of lysozyme and a specific inhibitor of microbial metalloproteinases. Further examination using specific MAP kinase inhibitors indicated that MEK/ERK and p38 are involved in RGD/E-mediated immune-signaling pathways, whereas JNK seems to play only a minor role.
免疫系统可以被微生物分子以及宿主来源的内源性危险/警报信号所刺激。使用鳞翅目昆虫模式昆虫家蚕，我们最近发现，微生物金属蛋白酶水解产生的胶原 IV 片段是昆虫中的危险/警报信号。在这里，我们使用纳升喷雾电离傅里叶变换离子回旋共振质谱（FTICR MS）在纳米液相色谱（nanoLC）分离后，对热稳定素介导的胶原 IV 降解产生的免疫刺激肽进行了表征。FTICR MS 分析和从头测序肽的组合导致鉴定了 38 个特定的胶原 IV 片段，其中一些肽包含了已知来自许多哺乳动物免疫相关蛋白的整合素结合基序 RGD/E。合成的与当前鉴定的胶原肽 GIRGEHyp 或已知的整合素结合 RGD 肽（GRGDS）相对应的定制肽被注入家蚕中，以确定它们在体内的免疫刺激活性。这两种肽都刺激了免疫细胞，并系统地上调了溶菌酶和一种特定的微生物金属蛋白酶抑制剂的表达。使用特定的 MAP 激酶抑制剂进一步研究表明，RGD/E 介导的免疫信号通路涉及 MEK/ERK 和 p38，而 JNK 似乎只起次要作用。
