Abstract
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 KD 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.
Highlights
The complement system mediates robust first line defense mechanisms against invading pathogens and participates in the elimination of cellular and humoral debris [1]
DNA sequencing revealed that the cDNA encoded neuropilin-1 (NRP1, known as CD304 and blood dendritic cell antigen-4 (BDCA4)) and expression of NRP1 was confirmed by flow cytometry (Figure 1D)
We assessed NRP1 expression on these cells and found NRP1 to be absent on monocytes, whereas it is homogenously expressed on monocytederived dendritic cells (moDCs) (Figure 1E)
Summary
The complement system mediates robust first line defense mechanisms against invading pathogens and participates in the elimination of cellular and humoral debris [1]. A signature feature of the pivotal complement split products C3b and C4b is that they covalently tag their target surfaces via a thioester-mediated transacylation onto target hydroxyl and amino groups [3] Their intrinsic thioester, formed between the side chains of nearly adjacent cysteine and glutamine residues located within the respective thiol estercontaining domain [4] of C3 and C4, is buried at a domain interface within the native molecules, but becomes accessible to target nucleophiles during the dramatic conformational changes following the primary proteolytic cleavage events of these molecules during complement pathway activation [5, 6]. The fragments C4d and C3d correspond approximately to the boundaries of the structurally-defined TED domain [9] Some of these CSPs promote immune activation and contribute to the clearance of pathogenic molecules and apoptotic cells from circulation by acting as ligands for cellular complement receptors. We succeeded in mapping the binding site for CSPs on NRP1 demonstrating that, unlike ILT4, NRP1 acts as a “classical” complement receptor for CSPs covalently deposited on target surfaces as a consequence of complement pathway activation
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