IgG型甲状腺自身抗体的糖基化修饰差异对其Fc段介导的生物学效应的影响

Thyroglobulin antibody (TgAb) and thyroid peroxidase antibody are diagnositic markers of Hahsimoto's thyroiditis (HT), which are predominantly of IgG class.In our previous study, supported by National Natural Science Foundation for the Youth, it was shown that TgAb and TPOAb may play important roles in the pathogenesis of HT. IgG is a glycoprotein with a sugar moiety attached to each of the asparagin 297 residues in the CH2-domains of the two Fc-fragments. Variable attachment of outer arm sugars (sialic acid, galactose and fucose) to a heptasaccharide GlcNac2Man3GlcNac2 core structure can differ in their ef?cacy of effector function activation such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), as it influences binding of IgG molecules to Fcγ receptors (FcγRs) and C1q. The aim of our study is to compare the sugar moiety on TgAb and TPOAb IgG in sera from HT patients and health controls by lectin microarray and mass spectrometry. Further, sera TgAb and TPOAb IgG from HT patients will be isolated by affinity chromatography. And purified TgAb and TPOAb IgG with different levels of sugar moiety (core fucose, galactose and sialic acid) will be obtained by glycosyl transferase and glycosyl exonuclease. Then, peripheral blood monouclear cells (PBMC) from healthy donors will be incubated with TgAb or TPOAb IgG with different levels of sugar moiety, respectively. The expression of Fcγ receptors on PBMC will be detected and effectors functions such as ADCC and CDC mediated by TgAb and TPOAb IgG with different sugar moiety will be further studied. We hope that our reserach will provide more evidence on the glycosylation of thyroid IgG antibodies on IgG-Fc-mediated effector functions and it might be helpful to investigate the roles of thyroid antibodies in the pathogenesis of HT in a new view on glycobiology.

甲状腺球蛋白抗体（TgAb）和甲状腺过氧化物酶抗体（TPOAb）是桥本甲状腺炎（HT）的诊断标志，主要为IgG型。申请者在青年基金的资助下证实TgAb和TPOAb在HT发病机制中发挥重要作用。文献报道IgG Fc段Asn-297上糖基化修饰可影响其与Fc段受体（FcγRs）的结合，进而影响抗体依赖细胞介导的细胞毒作用（ADCC）和补体依赖的细胞毒作用（CDC）。本研究拟通过亲和层析纯化HT患者血清中的IgG型TgAb和TPOAb，进一步通过凝集素芯片及质谱分析的方法明确其糖基化修饰与健康献血者的差异；进而应用糖基外切酶及糖基转移酶通过去除和添加糖基，获得不同糖基化（岩藻糖、半乳糖及唾液酸）修饰水平的甲状腺抗体；观察甲状腺抗体的不同糖基化修饰水平对PBMC上FcγR表达的调节及对IgGFc段介导的生物学效应（ADCC及CDC）的影响，以期从糖生物学角度揭示甲状腺自身抗体在HT发病机制中的作用

桥本甲状腺炎（HT）是常见的器官特性自身免疫性疾病，也是甲状腺功能减退（简称甲减）最主要的病因。甲状腺自身抗体主要是IgG型，其是诊断HT的标志性抗体，但其在HT发病机制中的作用并不完全明确。IgG是糖蛋白，文献报道IgG Fc段CH2区Asn-297位点上存在双天线结构的糖链，且糖链上的糖基化修饰水平可影响IgG与Fc段受体（FcγRs）的结合，进而影响抗体依赖细胞介导的细胞毒作用（ADCC）等生物学效应。本研究通过收集HT及健康对照者的血清，利用亲和层析的方法纯化了IgG型TgAb，通过MALDI-QIT-TOF-MS质谱法和凝集素芯片的方法证实健康对照者和HT患者血清中IgG型TgAb的糖型一致，但是糖基化修饰水平存在差异；进一步研究组建立了检测ADCC作用的方法，并应用商品化的特异性糖苷酶，分别获得了无唾液酸、无半乳糖、无糖链的IgG型TgAb，观察不同糖基化修饰水平的TgAb对IgG Fc段介导的ADCC作用的影响。在FcγR的工作中，课题组发现HT患者外周血单个核细胞（PBMC）上FcγR的表达较健康对照者存在差异，B细胞上的FcγRⅡB（CD32b）的表达水平在HT患者中较健康对照者显著下降。进一步研究发现HT患者中双阴性记忆B细胞和浆细胞百分比较健康对照组升高，提示HT患者存在B细胞亚群分布异常，且HT组双阴性记忆B细胞表面CD32b的MFI水平显著低于健康对照组。体外实验证实细胞亚群CD32b的表达受损与B细胞活化信号和细胞因子的调控可能有关。此外课题组在观察HT患者PBMC的免疫状态时，发现HT患者的Tc17细胞和NK细胞可能参与了HT的疾病进程。而CD26在HT患者CD8+ T细胞及亚群上表达受损，且甲减组CD26的水平较甲状腺功能正常组降低，即CD26可能参与了HT的疾病进程机制。上述研究从甲状腺自身抗体糖基化修饰及及与自身抗体相互作用的FcγR角度，深入探讨了HT的发病机制，完善了甲状腺自身抗体参与HT发病机制的理论，为预防HT发病、延缓HT疾病进程提供了新的方向。