Jensen Rasmus K, Plum Melanie, Tjerrild Luna, Jakob Thilo, Spillner Edzard, Andersen Gregers Rom
Department of Molecular Biology and Genetics, Aarhus University, Gustav Wiedsvej 10C, 8000 Aarhus, Denmark.
Allergy Research Group, Department of Dermatology, University Freiburg Medical Center, Hauptstrasse 7, 79104 Freiburg, Germany.
Acta Crystallogr F Struct Biol Commun. 2015 Apr;71(Pt 4):419-26. doi: 10.1107/S2053230X15004100. Epub 2015 Mar 20.
Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor FcℇRI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc Cℇ2-4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Å were obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity.
奥马珠单抗是一种人源化抗IgE抗体，它可抑制IgE与其在肥大细胞和嗜碱性粒细胞上的受体结合，从而阻断这些细胞中IgE介导的炎症介质释放。奥马珠单抗在高亲和力IgE受体FcℇRI的结合位点附近与IgE的Fc结构域结合，但表位以及识别的机制和构象仍不清楚。为了阐明其抗IgE活性的分子机制，目的是分析奥马珠单抗与人IgE的相互作用。因此，在哺乳动物HEK-293细胞中重组产生了IgE Fc Cℇ2-4。通过ELISA和介质释放试验证明了IgE Fc的功能。用木瓜蛋白酶裂解奥马珠单抗IgG，通过离子交换色谱法纯化得到的Fab。通过尺寸排阻色谱法制备IgE Fc与奥马珠单抗的复合物。然而，未获得含有该复合物的晶体，这表明结晶过程有利于两种蛋白质的解离。相反，获得了分辨率最高为1.9 Å和3.0 Å的两种奥马珠单抗Fab结构。这些结构揭示了互补决定区（CDR）的排列以及先前功能研究中已知的参与IgE结合的奥马珠单抗残基的位置。因此，奥马珠单抗的结构为理解其功能提供了结构基础，有助于优化复合物结晶的程序，并提出了关于抗IgE活性构象要求的问题。
