Disulfide linkage controls the affinity and stoichiometry of IgE Fcε3-4 binding to FcεRI
Disulfide linkage controls the affinity and stoichiometry of IgE Fcε3-4 binding to FcεRI
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DOI：
10.1074/jbc.m500965200
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发表时间：
2005-04-29
影响因子：
4.8
通讯作者：
Beavil, AJ
中科院分区：
文献类型：
--
作者：
Hunt, J;Beavil, RL;Beavil, AJ
IgE抗体导致组织肥大细胞和血液嗜碱性粒细胞对变应原诱导的交联和过敏性炎症的触发的长期敏化。IgE结合的这种持久性是由于其对受体Fc ε RI的独特的高亲和力，特别是其一旦结合后的缓慢解离速率。结合界面由两个亚位点组成，一个亚位点由IgE Fc的每个C β 3结构域以1：1复合物贡献。我们研究了C-13二硫键和糖基化对Fc亚片段（Fc-13 - 4）与可溶性受体片段（sFc-14-RI α）结合的动力学和亲和力的贡献。与去糖基化消除受体结合活性的IgG Fc相反，Fc ε 3-4中Asn-394处N-连接的碳水化合物的去除仅使结合亲和力降低4倍，主要是因为解离速率更快。去除重链间二硫键出乎意料地产生解离速率快得多的片段，并可能形成化学计量比为2：1的复合物（sFc β RI α：Fc β 3 4）。这允许确定的亲和力的一个单一的，天然折叠的C β 3结构域的第一次。低亲和力K-a约为10（5）-10（6）M-1，与先前对分离和部分折叠的C β 3结构域测定的相似，表明通过阻止两个C β 3结构域之一的接合可以实现亲和力的显著降低。最近的结构数据表明，IgE的构象变化是允许两个C β 3结构域结合所必需的，因此阻止进入第二个C β 3的变构抑制剂具有降低IgE致敏过敏效应细胞的能力的潜力。
IgE antibodies cause long-term sensitization of tissue mast cells and blood basophils toward allergen-induced cross-linking and triggering of allergic inflammation. This persistence of IgE binding is due to its uniquely high affinity for the receptor Fc epsilon RI and in particular its slow rate of dissociation once bound. The binding interface consists of two subsites, one contributed by each C epsilon 3 domain of IgE Fc in a 1: 1 complex. We have investigated the contributions of C epsilon 3 disulfide linkage and glycosylation to the kinetics and affinity of binding of an Fc subfragment (Fc epsilon 3 - 4) to a soluble receptor fragment (sFc epsilon RI alpha). In contrast to IgG Fc where deglycosylation abrogates receptor binding activity, the removal of the N-linked carbohydrate at Asn-394 in Fc epsilon 3-4 only reduces binding affinity by a factor of 4, principally because of a faster off-rate. Removal of the inter-heavy chain disulfide bond unexpectedly resulted in a fragment with a much faster off-rate and the potential to form a complex with a 2: 1 stoichiometry (sFc epsilon RI alpha: Fc epsilon 3 4). This permitted the determination of the affinity of a single, natively folded C epsilon 3 domain for the first time. The low affinity K-a approximate to 10(5)-10(6) M-1, similar to that determined previously for an isolated and partially folded C epsilon 3 domain, demonstrates that substantial reduction in affinity can be achieved by preventing the engagement of one of the two C epsilon 3 domains. Recent structural data indicate that conformational change in IgE is required to allow both C epsilon 3 domains to bind, and thus an allosteric inhibitor that prevents access to the second C epsilon 3 has the potential to reduce the ability of IgE to sensitize allergic effector cells.