Delain E, Barray M, Tapon-Bretaudiere J, Pochon F, Marynen P, Cassiman J J, Van den Berghe H, Van Leuven F
Laboratoire de Microscopie Cellulaire et Moléculaire, Institut Gustave-Roussy, Villejuif, France.
J Biol Chem. 1988 Feb 25;263(6):2981-9.
To study the three-dimensional organization of alpha 2-macroglobulin (alpha 2M) from human plasma, immunoelectron microscopy of negatively stained specimens was used. A panel of monoclonal antibodies (mAb) with specificities typical for the two major conformers of alpha 2M (native and protease-transformed) was explored. The mAb have been selected and were classified biochemically as specific for either native or transformed alpha 2M or as reactive with both conformers. Furthermore, among the mAb that were specific for the proteinase-transformed form of alpha 2M, those reacting with the 20-kDa receptor-binding domain were considered a fourth category. Immunoelectron microscopy with these 20-kDa receptor-binding domain-specific mAb yielded the most typical result: predominantly, individual H-like alpha 2M-chymotrypsin molecules were complexed with two IgG molecules, each one bound to the extremities of two arms of the H-like figure. The resulting planar complex has the appearance of a dumbbell. Since this was observed with eight different mAb of this specificity, the result is interpreted to mean that the 20-kDa receptor-binding domain is compact and constitutes the outermost domain at the extremes of the arms of the H-like transformed alpha 2M. The mAb which are specific for the transformed state of alpha 2M but which do not react with the 20-kDa receptor-binding domain, also bound at the arms of the H-like figure, but at nonterminal positions. Moreover, these mAb produced mostly linear, chain-like immune complexes of numerous H-like alpha 2M molecules cross-linked by the IgG. The large category of mAb that reacted with both conformers of alpha 2M (native and proteinase complex) were observed to make various types of immune complexes with intra- and intermolecular cross-linking by the IgG. The observations of reaction of these mAb with Cd2+-induced dimers (half-molecules of alpha 2M), either native or transformed, proved helpful and, for certain mAb, essential to understand the organization of the alpha 2M-IgG complexes. Combined, the observations allow us to propose new models for the three-dimensional organization of native and chymotrypsin-transformed dimeric and tetrameric human alpha 2M.
为研究人血浆中α2-巨球蛋白（α2M）的三维结构，我们采用了对负染标本进行免疫电子显微镜观察的方法。我们研究了一组单克隆抗体（mAb），这些抗体对α2M的两种主要构象（天然型和蛋白酶转化型）具有典型的特异性。这些单克隆抗体已被筛选出来，并根据生化特性分为对天然型或转化型α2M具有特异性，或与两种构象都有反应的类型。此外，在对蛋白酶转化型α2M具有特异性的单克隆抗体中，那些与20 kDa受体结合结构域反应的抗体被归为第四类。用这些针对20 kDa受体结合结构域的单克隆抗体进行免疫电子显微镜观察，得到了最典型的结果：主要是单个H型α2M-胰凝乳蛋白酶分子与两个IgG分子结合，每个IgG分子结合在H型结构两个臂的末端。形成的平面复合物呈哑铃状。由于用八种具有这种特异性的不同单克隆抗体都观察到了这种情况，因此该结果被解释为意味着20 kDa受体结合结构域是紧密的，并且构成了H型转化α2M臂末端的最外层结构域。对α2M转化状态具有特异性但不与20 kDa受体结合结构域反应的单克隆抗体，也结合在H型结构的臂上，但在非末端位置。此外，这些单克隆抗体大多产生由IgG交联的众多H型α2M分子组成的线性链状免疫复合物。观察到与α2M的两种构象（天然型和蛋白酶复合物）都有反应的一大类单克隆抗体，通过IgG形成了具有分子内和分子间交联的各种类型的免疫复合物。观察这些单克隆抗体与Cd2 +诱导的二聚体（α2M的半分子）（无论是天然型还是转化型）的反应，被证明是有帮助的，并且对于某些单克隆抗体来说，对于理解α2M-IgG复合物的结构至关重要。综合这些观察结果，我们能够提出天然型和胰凝乳蛋白酶转化型二聚体和四聚体人α2M三维结构的新模型。
