Division of Immune Receptors and T Cell Activation, Center for Pathophysiology, Infectiology, and Immunology, Institute of Immunology, Medical University of Vienna, Vienna, Austria.
Department of Clinical Cell Biology and FACS Core Unit, Children's Cancer Research Institute (CCRI), Vienna, Austria.
Front Immunol. 2019 Sep 13;10:2209. doi: 10.3389/fimmu.2019.02209. eCollection 2019.
Complement split products (CSPs), such as the fragments C4d and C3d, which are generated as a consequence of complement regulatory processes, are established markers for disease activity in autoimmunity or antibody-mediated graft rejection. Since immunoglobulin-like transcript 4 (ILT4) was previously shown to interact with soluble CSPs, but not with CSPs covalently-bound to target surfaces following classical complement activation, the present study aimed to identify novel cellular receptors interacting with covalently-deposited CSPs. By applying an unbiased screening approach using a cDNA mammalian expression library generated from human monocyte-derived dendritic cells and probed with recombinant human C4d, we identified neuropilin-1 (NRP1) as a novel receptor for C4d, C3d, and iC3b. NRP1, a highly conserved type 1 transmembrane protein, plays important roles in the development of the nervous and cardiovascular system as well as in tumorigenesis through interaction with its established binding partners, such as vascular endothelial growth factor (VEGF) and semaphorin 3A (Sema3A). NRP1 is also expressed on immune cells and serves as a marker for murine Tregs. Although NRP1 contains domains homologous to ones found in some complement proteins, it has not been linked to the complement system. We demonstrate that binding of C4d to NRP1 expressing cells was dose-dependent and saturable, and had a K value of 0.71 μM. Importantly, and in contrast to ILT4, NRP1 interacted with CSPs that were covalently bound to target surfaces in the course of complement activation, therefore representing a classical complement receptor. The binding site of CSPs was mapped to the b1 domain of the coagulation factor V/VIII homology domain of NRP1. Taken together, our results demonstrate a novel role for NRP1 as a receptor for CSPs deposited on surfaces during complement activation. Further work is required to elucidate the functional consequences of the NRP1-CSP interactions in immunity.
补体分裂产物（CSPs），如片段 C4d 和 C3d，是补体调节过程的结果，是自身免疫或抗体介导的移植物排斥反应中疾病活动的既定标志物。由于免疫球蛋白样转录物 4（ILT4）先前被证明与可溶性 CSPs 相互作用，但不与经典补体激活后共价结合到靶表面的 CSPs 相互作用，因此本研究旨在鉴定与共价沉积的 CSPs 相互作用的新型细胞受体。通过应用一种无偏见的筛选方法，使用从人单核细胞衍生的树突状细胞生成的 cDNA 哺乳动物表达文库，并与重组人 C4d 进行探测，我们鉴定出神经纤毛蛋白 1（NRP1）为 C4d、C3d 和 iC3b 的新型受体。NRP1 是一种高度保守的 1 型跨膜蛋白，通过与血管内皮生长因子（VEGF）和 Sema3A 等已建立的结合伴侣相互作用，在神经系统和心血管系统的发育以及肿瘤发生中发挥重要作用。NRP1 也在免疫细胞上表达，并作为鼠 Treg 的标志物。尽管 NRP1 包含与某些补体蛋白中发现的同源结构域，但它与补体系统没有联系。我们证明 C4d 与表达 NRP1 的细胞的结合是剂量依赖性和饱和的，K 值为 0.71 μM。重要的是，与 ILT4 相反，NRP1 与在补体激活过程中共价结合到靶表面的 CSPs 相互作用，因此代表经典补体受体。CSPs 的结合位点映射到 NRP1 的凝血因子 V/VIII 同源结构域的 b1 结构域。总之，我们的结果表明 NRP1 作为补体激活过程中沉积在表面上的 CSPs 的受体具有新的作用。需要进一步的工作来阐明 NRP1-CSP 相互作用在免疫中的功能后果。
