Dierks S E, Bartlett W C, Edmeades R L, Gould H J, Rao M, Conrad D H
Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond 23298.
J Immunol. 1993 Mar 15;150(6):2372-82.
The low affinity receptor for IgE (Fc epsilon RII/CD23) is a type II integral membrane protein with an extracellular C-terminal region homologous to C-type animal lectins. Immediately adjacent to this lectin homology region is a sequence that is predicted to form an alpha-helical coiled-coil stalk leading to dimer or trimer formation. This provides an explanation for the known self-associative capacity for the Fc epsilon RII. In this study the self-association to a trimer or tetramer is shown with rFc epsilon RII by chemical cross-linking and affinity purification on IgE columns. The data indicate that only the oligomeric form of Fc epsilon RII has sufficient affinity/avidity to bind to an IgE adsorbent. In contrast, Fc epsilon RII that is purified using anti-Fc epsilon RII mAb adsorbents has largely lost its capacity to bind IgE, as well as its capacity to self-associate, indicating that IgE recognizes the oligomeric form of the Fc epsilon RII. This phenomenon was further examined by performing detailed binding analysis of the mouse IgE/Fc epsilon RII interaction. A biphasic binding curve with high (2-7 x 10(7) M-1) and low (2-7 x 10(6) M-1) affinity binding was seen. Fc epsilon RII mutants were prepared that lack one or more of the 21 amino acid homologous repeat domains in the stalk region of the molecule. These mutant Fc epsilon RII molecules bound IgE with only a single low affinity (5-10 x 10(6) M-1). In addition, cross-linking analysis of one of these mutants demonstrated that it does not exhibit the receptor self-association seen for the intact Fc epsilon RII. Two chimeric Fc epsilon RII molecules were prepared having the mouse Fc epsilon RII lectin homology (carboxyl-terminal) region and the stalk region of either the related human Fc epsilon RII or the corresponding domain of Ly-49. Chimeric molecules using the former (alpha-helical coiled-coil) stalk supported normal binding of IgE although the Ly-49/Fc epsilon RII chimera failed to bind IgE. Taken together, the results indicate that high (approximately 10(8) M-1) affinity IgE binding results from interaction of multiple lectin domains with (presumably) symmetrical sites on the IgE molecule. Specificity for IgE is determined by the lectin domain although the binding avidity is determined by oligomerization through the coiled coil stalk.
IgE的低亲和力受体（FcεRII/CD23）是一种II型整合膜蛋白，其细胞外C末端区域与C型动物凝集素同源。紧邻该凝集素同源区域的是一个预计会形成α-螺旋卷曲螺旋柄的序列，该序列会导致二聚体或三聚体形成。这就解释了FcεRII已知的自我缔合能力。在本研究中，通过化学交联和在IgE柱上的亲和纯化，显示重组FcεRII可自我缔合形成三聚体或四聚体。数据表明，只有FcεRII的寡聚形式具有足够的亲和力/亲合力来结合IgE吸附剂。相比之下，使用抗FcεRII单克隆抗体吸附剂纯化的FcεRII在很大程度上丧失了结合IgE的能力以及自我缔合的能力，这表明IgE识别FcεRII的寡聚形式。通过对小鼠IgE/FcεRII相互作用进行详细的结合分析，进一步研究了这一现象。观察到一条具有高亲和力（2 - 7×10⁷ M⁻¹）和低亲和力（2 - 7×10⁶ M⁻¹）的双相结合曲线。制备了分子柄区域缺少一个或多个21个氨基酸同源重复结构域的FcεRII突变体。这些突变的FcεRII分子仅以单一的低亲和力（5 - 10×10⁶ M⁻¹）结合IgE。此外，对其中一个突变体的交联分析表明，它不表现出完整FcεRII所具有的受体自我缔合现象。制备了两个嵌合FcεRII分子，它们具有小鼠FcεRII凝集素同源（羧基末端）区域以及相关人类FcεRII的柄区域或Ly - 49的相应结构域。使用前者（α-螺旋卷曲螺旋）柄的嵌合分子支持IgE的正常结合，尽管Ly - 49/FcεRII嵌合体无法结合IgE。综上所述，结果表明高亲和力（约10⁸ M⁻¹）的IgE结合是由多个凝集素结构域与IgE分子上（大概）对称位点的相互作用导致的。对IgE的特异性由凝集素结构域决定，尽管结合亲合力由通过卷曲螺旋柄的寡聚化决定。
