Department of Pharmacy, Faculty of Medicine, University Hasan Prishtina, Pristina, Kosovo.
Department of Pharmaceutical Sciences, Università degli Studi di Milano, 20133 Milan, Italy.
J Neuroimmunol. 2018 Apr 15;317:77-83. doi: 10.1016/j.jneuroim.2018.01.006. Epub 2018 Jan 9.
Immunoproteomics is become a potent methodology used for identifying immunoreactive proteins. In this study, an immunoproteomic approach based on 2-dimensional gel electrophoresis (2D-PAGE) and immunoblotting combined with high resolution mass spectrometry (MS) was used to identify immunoreactive proteins that might be involved in mechanisms of Guillain-Barré syndrome (GBS) development, regardless of their potential reciprocal molecular mimicry. Proteins isolated from C. jejuni and human peripheral nerve tissue (HPN) were separated with 2D SDS-PAGE and subjected to western blotting using serum samples from GBS patients. The peptides generated after proteolysis of the immunoreactive proteins were submitted to nanoflow-high performance liquid chromatography-nano electrospray ionization coupled to high resolution mass spectrometry (nHPLC-nESI-MS and MS/MS) followed by SEQUESTdata analysis for proteins identification. In C. jejuni, immunoreactivity was found for GroEL and DnaK, structural proteins (MOMP), key enzymatic proteins necessary for the microbial proliferation (adenylate kinase, enolase, inorganic pyrophosphatase and aspartate ammonia-lyase), and antioxidant enzymes (alkyl hydroperoxide reductase-AhpC and DNA protection during starvation protein - DNA protection factor against Fe-mediated oxidative stress). HPN immunoreactive proteins identified were heat shock proteins (HSP), intermediate filaments (vimentin and desmin), and other proteins and enzymes such as troponin/tropomyosin complex and ATP synthase subunit beta and the keratan sulfate proteoglycan lumican. The targeting of vimentin and desmin, suggested that the neuronal autoimmune damage is specifically directed to intermediate neuronal (vimentin) and neuromuscular IF, probably localized nearby cell surface, affording increased accessibility to autoantibodies. These findings suggest that the post-infectious development of GBS may be also associated to additional concomitant immune factors that lead to nerve damage generated by auto-immune trigger(s) different from molecular mimicry.
免疫蛋白质组学已成为一种用于鉴定免疫反应性蛋白的有效方法。在这项研究中，采用了基于 2 维凝胶电泳（2D-PAGE）和免疫印迹与高分辨率质谱（MS）相结合的免疫蛋白质组学方法，以鉴定可能参与格林-巴利综合征（GBS）发病机制的免疫反应性蛋白，而不考虑它们潜在的相互分子模拟。从空肠弯曲菌和人周围神经组织（HPN）中分离的蛋白质用 2D SDS-PAGE 分离，并使用 GBS 患者的血清样本进行 Western 印迹。在免疫反应性蛋白经蛋白水解后生成的肽段，经纳流高效液相色谱-纳升电喷雾电离与高分辨率质谱（nHPLC-nESI-MS 和 MS/MS）联用，然后通过 SEQUEST 数据分析进行蛋白质鉴定。在空肠弯曲菌中，GroEL 和 DnaK、结构蛋白（MOMP）、微生物增殖所必需的关键酶蛋白（腺苷酸激酶、烯醇酶、无机焦磷酸酶和天冬氨酸氨-裂合酶）和抗氧化酶（烷基氢过氧化物还原酶-AhpC 和饥饿期间的 DNA 保护蛋白-DNA 保护因子，防止 Fe 介导的氧化应激）有免疫反应性。在 HPN 中鉴定出的免疫反应性蛋白为热休克蛋白（HSP）、中间丝（波形蛋白和结蛋白）以及其他蛋白和酶，如肌钙蛋白/原肌球蛋白复合物和 ATP 合酶亚基β以及硫酸角质素蛋白聚糖亮氨酸。波形蛋白和结蛋白的靶向性表明，神经元自身免疫损伤是特异性地针对中间神经元（波形蛋白）和神经肌肉 IF，可能位于细胞表面附近，使自身抗体更容易接近。这些发现表明，感染后 GBS 的发展可能也与其他伴随的免疫因素有关，这些因素导致的神经损伤是由不同于分子模拟的自身免疫触发因素引起的。
