Chimeric proteins containing MAP-1 and functional domains of C4b-binding protein reveal strong complement inhibitory capacities

Cecilie Elkjær Hertz,Rafael Bayarri-Olmos,Nikolaj Kirketerp-Møller,Sander Van Putten,Katrine Pilely,Mikkel-Ole Skjoedt,Peter Garred

doi:10.1016/j.molimm.2018.06.029

Abstract

The complement system is a tightly regulated network of proteins involved in defense against pathogens, inflammatory processes, and coordination of the innate and adaptive immune responses. Dysregulation of the complement cascade is associated with many inflammatory disorders. Thus, inhibition of the complement system has emerged as an option for treatment of a range of different inflammatory diseases. MAP-1 is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway of the complement system, whereas C4b-binding protein (C4BP) regulates both the classical and lectin pathways. In this study we generated chimeric proteins consisting of MAP-1 and the first five domains of human C4BP (C4BP1-5) in order to develop a targeted inhibitor acting at different levels of the complement cascade. Two different constructs were designed and expressed in CHO cells where MAP-1 was fused with C4BP1-5 in either the C- or N-terminus. The functionality of the chimeric proteins was assessed using different in vitro complement activation assays. Both chimeric proteins displayed the characteristic Ca2+-dependent dimerization and binding to PRMs of native MAP-1, as well as the co-factor activity of native C4BP. In ELISA-based complement activation assays they could effectively inhibit the lectin and classical pathways. Notably, MAP-1:C4BP1-5 was five times more effective than rMAP-1 and rC4BP1-5 applied at the same time, emphasizing the advantage of a single inhibitor containing both functional domains. The MAP-1/C4BP chimeras exert unique complement inhibitory properties and represent a novel therapeutic approach targeting both upstream and central complement activation.

Highlights

The complement system constitutes a central effector arm of the vertebrate immune system occupying a pivotal position as an early danger sensor and mediator of immunological and inflammatory processes [1, 2]
MAP-1-containing chimeric proteins were purified by antibody affinity chromatography and C4BP1-5 was purified via immobilized metal ion affinity chromatography on a HisTrap excel column
The chimeric proteins migrated as a single band with an apparent molecular weight of ∼80 kDa, equivalent to the sum of rMAP-1 and rC4BP1−5 molecular weights (i.e., ∼44 kDa and ∼37 kDa respectively; Figure 1B)

Summary

Introduction

The complement system constitutes a central effector arm of the vertebrate immune system occupying a pivotal position as an early danger sensor and mediator of immunological and inflammatory processes [1, 2]. Initiation of CP is mediated mainly by the binding of C1q to immune-complexes, and to conserved pathogen-specific structures, altered self-antigens or interaction with the pentraxins [7] and leads to the activation of the serine proteases C1r and C1s [8, 9]. The LP is initiated by two groups of pattern recognition molecules (PRM): c-type lectins, such as mannosebinding lectin (MBL), collectin-10 (CL-10, CL-L1), and collectin (CL-11, CL-K1) and ficolins, i.e., ficolin-1 (M-ficolin), ficolin (L-ficolin), and ficolin-3 (H-ficolin or Hakata antigen). Binding of PRMs to ligands activates the MBL-associated serine proteases (MASPs), causing cleavage and activation of C2 and C4 [10]. Whether the AP can be activated via a similar PRM-dependent mechanism involving properdin remains controversial [13, 14]

Objectives

Results

Conclusion