Analysis of IgAl binding protein(BP)in IgA Nephropathy(IgA) patients using Mass Spectrometry(MS)

伊加肾病（IgAN）的发病机制主要是伊加的糖基化不足和WA免疫复合物（IC）的形成，但其发病机制尚不清楚。由于上呼吸道感染可导致IgAN，扁桃体切除术对本病有治疗作用，以往的研究已探讨了抗原在本病中的病理作用。然而，形成致肾炎性IgAIC的决定性抗原尚未鉴定。为了研究特异性外源性抗原是否参与IgAIC的形成，我们用质谱法（MS）分析了IgAN患者血清中的伊加结合蛋白（BP）。在通过jacalin分离IgA 1/IgA 1-BP后，我们进行一维（1D）电泳并分析用银染色的所有条带。6例IgAN患者与5例健康志愿者比较，两组均检测到载脂蛋白、免疫球蛋白（IG）重链（α. MS法检测IgG、Ig、Ig γ、Ig轻链、Ig IG J链、纤连蛋白（FN）、Clinhibitor（C1 INH）、C4结合蛋白（C4 bp）、α 1微球蛋白（MG），以IgA 1/IgA 1-BP表示。此外，治疗前后的样品用二维差异凝胶电泳（2D DIGE）系统进行分析，但我们不能鉴定新的蛋白质超过一维电泳。我们进一步对患者和健康志愿者进行了Western blot分析，以比较治疗前后的差异。IgAN患者血清IgA 1结合C4 bp、IgG、C3含量均高于正常对照组，治疗后下降。虽然在IgAl-BP中未检测到外源性抗原，但本研究结果提示，致肾IC中的IgG、C4 bp和C3可能在本病的发病中起关键作用。

Abnormalities of IgA including underglycosylatlon and polymeric WA immune complex (IC) formation are discussed in the pathogenesis of IgA nephropathy (IgAN), but the pathogenesis of this disease still remains unclear. Since upper respiratory infection exercerbates IgAN and tonsillectomy has therapeutic effects on this disease, the previous studies have examined the pathological role of antigens in this disease. However, definitive antigens that form nephritogenic IgAIC have not been identified. To examine whether specific exogenous antigens are involved in IgAIC formation, we analyzed IgA binding protein (BP) from IgAN patients serum by Mass Spectrometry (MS). After isolation of IgA1/IgA1-BP by jacalin, we performed one-dimensional (1D) electrophoresis and analyzed all bands stained with silver. When we compared 6 IgAN patients with 5 healthy volunteers, both groups identified apolipoprotein, immunoglobulin (Ig) heavy chain(α. Μ, γ) and light chain, Ig J chain, fibronectin (FN), Clinhibitor (C1INH), C4 binding protein(C4bp) and α1microglobulin (MG)as IgAl/IgAI-BP by MS. C4bp was frequently observed in the patients group. Moreover, samples before and after the therapies were analyzed by two dimensional difference gel electrophoresis (2D DIGE) system, but we could not identify new protein more than 1D electrophoresis. We further performed Western blot analysis to compare before and after the therapy in patients and healthy volunteers. Contents of IgAl binding C4bp, IgG, and C3 in IgAN patients were higher than those in the volunteers and decreased after the therapy. Although we could not detect exogenous antigen in IgAl-BP, the present findings suggested that IgG, C4bp and C3 in nephritogenic IC might play a key role in the pathogenesis of this disease.