Phillips M L, Tao M H, Morrison S L, Schumaker V N
Department of Chemistry, University of California, LA 90024.
Mol Immunol. 1994 Oct;31(15):1201-10. doi: 10.1016/0161-5890(94)90034-5.
The unique structure of the human IgG3 constant region with its greatly extended hinge can clearly be seen in electron micrographs, which compare a series of recombinant proteins with the same murine anti-dansyl variable domain but constant domains from human IgG1, IgG2, IgG3 and IgG4. The hinge region of IgG3 was found to be very long, with some measurements extending to 100 A. It exhibited considerable flexibility allowing the Fc to be displaced far toward either side. Upon addition of bivalent hapten, all of the monoclonal antibodies formed complexes. IgG1, IgG3 and IgG4 formed circular dimers, composed of two antibodies forming a ring-shaped complex, presumably through the binding of two bivalent haptens. IgG2, on the other hand, showed a distribution of complexes which was noticeably different from the other subclasses. Some circular dimers, some linear dimers and a large amount of monomer were seen. This was interpreted in terms of an energy barrier to ring closure arising from the orientation of the Fab arms of IgG2 probably leading to linear dimers as the predominate complex seen with the analytical ultracentrifuge. A substantial number of these dimers probably dissociated upon dilution for examination in the electron microscope. The distribution of the angles between the Fab arms of the monoclonal antibodies forming the circular dimers has been measured for the different subclasses. Most were open at wide angles (> 100 degrees) but some formed very shallow angles, with the Fab arms being nearly parallel to each other. The free energy for this transition was calculated from the ratio of open/closed angles, and it was found to be proportional to the length of the upper hinge of the monoclonal antibody, in agreement with previous nanosecond depolarization results (Dangl et al., Eur. molec. Biol. Org. J. 7, 1989-1994, 1988).
在电子显微镜照片中，可以清楚地看到人IgG3恒定区独特的结构，其铰链区大大延长。这些照片比较了一系列重组蛋白，它们具有相同的鼠抗丹磺酰可变区，但恒定区分别来自人IgG1、IgG2、IgG3和IgG4。发现IgG3的铰链区非常长，有些测量结果显示其长度延伸至100埃。它表现出相当大的灵活性，使得Fc段能够向两侧大幅度位移。加入二价半抗原后，所有单克隆抗体都形成了复合物。IgG1、IgG3和IgG4形成环状二聚体，由两个抗体形成环形复合物，推测是通过两个二价半抗原的结合。另一方面，IgG2显示出的复合物分布与其他亚类明显不同。观察到一些环状二聚体、一些线性二聚体和大量单体。这可以用IgG2的Fab臂的方向导致环闭合存在能量障碍来解释，这可能导致线性二聚体成为分析超速离心机中观察到的主要复合物。在稀释用于电子显微镜检查时，这些二聚体中的大量可能会解离。已经测量了形成环状二聚体的单克隆抗体的Fab臂之间角度的分布情况，不同亚类的情况各不相同。大多数角度较大（>100度），但有些形成非常小的角度，Fab臂几乎相互平行。根据开放/闭合角度的比例计算了这种转变的自由能，发现它与单克隆抗体上铰链区的长度成正比，这与之前纳秒去极化结果一致（Dangl等人，欧洲分子生物学组织杂志7，1989 - 1994，1988）。
