Functional and Structural Characterization of a Potent C1q Inhibitor Targeting the Classical Pathway of the Complement System.

Nick S Laursen,Dennis V Pedersen,Alessandra Zarantonello,Steffen Thiel,Gregers R Andersen,Jens Magnus Bernth Jensen,Heidi Gytz,Annette G Hansen

doi:10.3389/fimmu.2020.01504

Nick S Laursen, Dennis V Pedersen + Show 6 more

Open Access

https://doi.org/10.3389/fimmu.2020.01504

Copy DOI

Abstract

The classical pathway of complement is important for protection against pathogens and in maintaining tissue homeostasis, but excessive or aberrant activation is directly linked to numerous pathologies. We describe the development and in vitro characterization of C1qNb75, a single domain antibody (nanobody) specific for C1q, the pattern recognition molecule of the classical pathway. C1qNb75 binds to the globular head modules of human C1q with sub-nanomolar affinity and impedes classical pathway mediated hemolysis by IgG and IgM. Crystal structure analysis revealed that C1qNb75 recognizes an epitope primarily located in the C1q B-chain that overlaps with the binding sites of IgG and IgM. Thus, C1qNb75 competitively prevents C1q from binding to IgG and IgM causing blockade of complement activation by the classical pathway. Overall, C1qNb75 represents a high-affinity nanobody-based inhibitor of IgG- and IgM-mediated activation of the classical pathway and may serve as a valuable reagent in mechanistic and functional studies of complement, and as an efficient inhibitor of complement under conditions of excessive CP activation.

Highlights

The complement system plays an important role in maintaining homeostasis by removal of apoptotic cells and pathogens [1]
After initial characterization of the nanobodies’s ability to inhibit immunoglobulin G (IgG) mediated complement activation (Figure S1A) we identified C1qNb75 as a potent classical pathway (CP) inhibitor and we decided to further characterize this Nb
Recognition of a plethora of molecular patterns by C1q induces C1r and C1s activation resulting in C4 cleavage and assembly of the CP C3 convertase [35,36,37]

Summary

Introduction

The complement system plays an important role in maintaining homeostasis by removal of apoptotic cells and pathogens [1]. The classical pathway (CP) of complement is activated by binding of the C1 complex to a number of ligands, including immunoglobulin G (IgG) and immunoglobulin M (IgM) bound to surface antigens or in immune complexes. C1q is a multimeric molecule with six heterotrimeric collagen helices made up by the A, B, and C-chains [2]. The N-terminal collagen regions in the three C1q subunits form heterotrimers that hexamerize to form intact C1q, and these collagen regions firmly hold C1r and C1s attached. The collagen helices are tightly packed at the N-terminus but diverge toward the C-terminus into six individual collagen stems that end in trimeric C-terminal globular heads (gC1q). Each chain of gC1q adopts a ten-stranded β-sandwich fold that together form the highly compact spherical structure of gC1q [2,3,4]

Methods

Results

Conclusion