瓜氨酸化修饰对纤维蛋白原多肽抗原性的影响

Impact of citrullination upon antigenicity of fibrinogen

目的研究瓜氨酸化修饰对人纤维蛋白原（Fg）多肽抗原性的影响，探讨针对瓜氨酸化瞻的自身免疫应答在类风湿关节炎（RA）发病中的分子机制。方法体外对人心进行瓜氨酸化修饰，用修饰后Fg刺激RA患者及对照组外周血单个核细胞，通过^3H掺入法观察细胞增殖情况；通过计算机软件对人Fgα链进行瓜氨酸化修饰前后的抗原性分析，从中筛选出1条多肽，并体外合成原型肽和瓜氨酸替换肽，通过在表达HLA—DRB1细胞系中的直接肽结合试验和抗体竞争肽结合试验，评价瓜氨酸化修饰对Fg多肽与HLA—DRB1亲合力的影响；另外，在体外采用抗原提呈及活化系统，通过检测细胞增殖情况观察瓜氨酸化修饰对Fg多肽激活T细胞作用的影响。结果瓜氨酸化修饰的瞻和野生型Fg对RA患者外周血单个核细胞的激活作用略高于对照组，但差异无统计学意义（P〉0．05）。多肽结合试验和细胞活化试验证实，瓜氨酸替换肽与HLA—DRB1的结合力明显高于原型肽，而且瓜氨酸替换肽对T细胞的活化作用强于原型肽，二者差异有统计学意义（SI为2．26±0．14比1．65±0．53，P〈0．01）。结论瓜氨酸化修饰能够增加Fg多肽与HLA—DRB1的结合力，诱导T细胞活化，从而增强Fg多肽的抗原性。

Objective To investigate the impact of citrullination upon the antigenicity of fibrinogen peptides and explore the molecular mechanism of autoimmunity against citrullinated fibrinogen in the pathogenesis of rheumatoid arthritis （RA）. Methods Human fibrinogen was citmllinated in vitro by peptidylarginine deiminase （PAD）. Stimulation of eitrullinated fibrinogen on PBMC （peripheral blood mononuclear cell） from RA patients and controls was studied by measuring cellular proliferation. Then the bindings of peptides derived from fibrinogen alpha chain and its citrullinated substitute to HLA-DR4 molecule were analyzed by the prediction software. One peptide was chosen and its arginine residues were altered by citrulline residues. Wild-type and altered peptides were synthesized and their bindings to HLA-DR4 molecules examined by peptide binding assay respectively. The effects of two peptides upon T cells were determined by T cell proliferation assay. Results Compared with controls, the cellular responses to citrullinated fibrinogen or wild protein were slightly higher in RA patients. However no statistical difference was found （P 〉 0. 05 ）. As predicted, the citrullinated substitute peptide from fibrinogen alpha chain was more prone to bind to HLA-DR4 molecule than wild-type peptide and could induce a stronger proliferation of T cells than wild-type peptide （ SI, 2. 26±0. 14 vs 1.65 ±0. 53, P 〈 0. 01 ） in the experiments of cell lines. Conclusions Citrullination can enhance the antigenieity of fibrinogen peptides by increasing the binding of fibrinogen peptides to HLA-DR4 molecules and inducing the specific T cell activation.