Carayannopoulos L, Hexham J M, Capra J D
Program in Molecular Biophysics, The Molecular Immunology Center, University of Texas Southwestern Medical Center, Dallas, 75235-9140, USA.
J Exp Med. 1996 Apr 1;183(4):1579-86. doi: 10.1084/jem.183.4.1579.
Immunoglobulin (Ig) A serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities of blood. In playing its role in humoral immunity, IgA interacts with a variety of effector molecules present both in serum and on the surfaces of immune and inflammatory cells. To study these interactions, we previously established expression of human IgA1 in insect cells using recombinant baculoviruses and showed that the expressed antibody is a structurally and functionally intact polypeptide useful for examining the molecular properties of IgA. Indeed, since the C alpha 2 N-linked glycosylation site lies near the Fab-distal pole of C alpha 2, the inability of a mutant IgA1 lacking C alpha 2 N-glycosylation to bind its cognate receptor suggested that the monocyte Fc alpha receptor (mFcalphaR) recognizes IgA at a hinge-distal site encompassing the boundary between the C alpha 2 and C alpha 3 domains. In this report, we utilize both domain-swapped IgA/IgG and point-mutated IgA chimeras to verify the above hypothesis. Using an antigen-specific rosetting assay and a mFc alpha R-expressing cell line, we show that (a) C alpha 2 and C alpha 3 together are necessary and sufficient for binding; (b) neither the IgA hinge nor the tailpiece is necessary for binding; (c) mutations away from the interdomain boundary do not affect binding; and (d) mutations located near the three-dimensional boundary between C alpha 2 and C alpha 3 completely disrupt binding. Taken together, these results localize the mFc alpha R recognition site on IgA to the boundary region between the second and third constant domains--a site analogous to that recognized by Staphylococcus aureus protein A on IgG. The use of this hinge-distal site is, to date, unique among Fc receptors of the Ig superfamily.
免疫球蛋白（Ig）A是所有黏膜表面体液防御的第一道防线，且大量存在于血液中。在体液免疫中发挥作用时，IgA与血清以及免疫和炎症细胞表面存在的多种效应分子相互作用。为了研究这些相互作用，我们之前利用重组杆状病毒在昆虫细胞中建立了人IgA1的表达，并表明所表达的抗体是一种结构和功能完整的多肽，可用于研究IgA的分子特性。实际上，由于Cα2 N-连接糖基化位点位于Cα2的Fab远端极点附近，缺乏Cα2 N-糖基化的突变型IgA1无法结合其同源受体，这表明单核细胞Fcα受体（mFcαR）在一个包含Cα2和Cα3结构域边界的铰链远端位点识别IgA。在本报告中，我们利用结构域交换的IgA/IgG和点突变的IgA嵌合体来验证上述假设。使用抗原特异性玫瑰花结试验和表达mFcαR的细胞系，我们表明：（a）Cα2和Cα3共同对于结合是必要且充分的；（b）IgA铰链区和尾段对于结合都不是必需的；（c）远离结构域间边界的突变不影响结合；（d）位于Cα2和Cα3三维边界附近的突变完全破坏结合。综上所述，这些结果将IgA上的mFcαR识别位点定位到第二和第三恒定结构域之间的边界区域——一个类似于金黄色葡萄球菌蛋白A在IgG上识别的位点。迄今为止，这种铰链远端位点的使用在Ig超家族的Fc受体中是独特的。
