Research

The Complement System

Introduction

The complement system is a pivotal part of innate immunity and is found in evolutionary early species such as sea anemones, corals and horseshoe crabs. Complement works as a host defense mechanism that detects and eliminates invading pathogens and and as a balancing mechanism maintaining host cell homeostasis by recognizing necrotic cells and damaged tissue. The system is a highly complex network of soluble and membrane embedded proteins and is activated upon recognition of danger- or pathogen-associated molecular patterns (DAMPs and PAMPs). The pattern-recognition molecules (PRMs) of complement are assemblies of large multimeric proteins that recognize the DAMPs/PAMPs on the activator surface. In circulation, the PRMs are associated with highly specific serine proteases that, when clustered on the activator surface, initiates a proteolytic cascade that ultimately leads to the formation of the complement convertases. Carbohydrates and immune complexes serve as the DAMPs and PAMPs of the lectin and classical pathways, respectively.

In the lectin pathway, mannose-binding lectins (MBL), ficolins and collectins constitute the PRMs that are bound to one of three MBL-associated serine proteases (MASPs). In the classical pathway, C1q recognizes the activator surface, and is found associated with a hetero-tetramer of the serine proteases C1r and C1s. The serine proteases of these two pathways cleaves two components of the complement system known as C4 and C2. This generates the classical/lectin pathway C3 convertase, C4b2a. C4b features an internal highly reactive thioester that renders C4b able to covalently deposit on the activator surface. As the name implies, the C3 convertase is able to cleave the central complement component C3, a reaction catalyzed by the serine protease domain of C2a.

Complement lectin and classical pathway activation and assembly of CP/LP C3 convertases .  The upper panel shows a model for the complex of MBL bound to MASP-1 based on SAXS data (Kjaer et al., 2015, Structure), the crystal structure of native C4 bound to a fragment of MASP-2 (Kidmose et al. 2012, PNAS) and a cryo-EM subtomogram average of the C1 complex bound to immune complexes (Diebolder et al., 2014, Science). All complexes in the upper panel are shown on the same scale. The lower panel shows the route of C4 activation and CP/LP C3 convertase assembly, with crystal structures of apo-C4 and the C4:MASP-2 complex in a different orientation as compared to the upper panel (Kidmose et al., 2012, PNAS) to the left. In the middle, the crystal structure of C4b is shown, with the thioester shown in red (Mortensen et al., 2015, Journal of Immunology). To the right, two SAXS-based models of C4b in complex with intact C2 (C3 pro-convertase) and cleaved C2a (C3 convertase) are shown (Mortensen et al., 2016, JBC). All structures in the lower panel are shown in scale. In both panels an activator surface is shown and in the upper panel an additional surface representing the outer membrane leaflet is shown.

 

Upon cleavage of C3, two fragments are formed known as C3b and the anaphylatoxin C3a. Whereas C3a acts as a proinflammatory chemoattractant, C3b covalently deposits on the activator surface similar to C4b and interacts with another complement component known as factor B. Factor B is a structural homologue of C2 and upon cleavage of factor B by a third serine protease called factor D, the serine protease domain of the resulting factor Bb is activated and the second C3 convertase of the system is formed. This pathway is known as the alternative pathway and acts as a survey system and an amplification loop of the lectin and classical pathways. As C3 is continuously cleaved by the C3 convertases, the density of C3b on the activator surface increases and through a mechanism not yet described, the C3 convertase shifts specificity towards the fifth complement component C5, forming what is known as the C5 convertases. These convertases are traditionally denoted C4b2a3b and C3bBb3b, and similar to the cleavage of C3, two fragments of C5 are generated throughout the proteolytic event exerted by these complexes. C5a like C3a, acts as a pro-inflammatory chemoattractant however with a highly increased potency compared to C3a. C5b, in contrast to C3b and C4b, does not feature the internal thioester and is instead captured by the complement components C6, C7 and C8 that allow the insertion of this complex into the target membrane of the activator. C5b-8 further attracts multiple copies of the complement component C9, which penetrates the activator membrane even further and the resulting C5b-9n forms a lytic pore, known as the membrane attack complex (MAC) that ultimately kills the targeted cell.

 


Experimental work

Through experimental setups we attempt to describe the details of this highly complex network of complement proteins and the assembly of the delicate and unstable complexes formed throughout the cascade. Since complement features opsonins, highly potent pro-inflammatory signaling molecules as well as the potential to form membrane-lytic pores, the system needs to be carefully regulated. The human genome encodes numerous regulators of the complement system and we also study the regulatory nature of these proteins in complex with complement components. Beside regulators exerting inhibitory roles on complement activation and effector mechanisms, we have also studied the only positive regulator of complement known as properdin or factor P that stabilizes the C3bBb complex on the activator surface.

As we are primarily interested in comprehending the structural details of the system, we use a combination of high resolution X-ray crystallography, small-angle X-ray scattering (SAXS) and single particle electron microscopy (EM) to illuminate the fascinating and intriguing biological questions found in this subpart of innate immunity. We are experts in protein-protein interaction and complex formation and purify our material from native as well as recombinant source, using both bacterial, yeast, mammalian and baculovirus infected insect cell systems.

We are very well aware that a rigid protein structure is not sufficient to fully characterize the processes of interest and for this reason we use several biochemical and functional techniques to enlarge our perspective of the dynamics of complement biology. Such techniques include surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), microscale thermophoresis (MST) and flow cytometry using FACS analyzers. Through collaborations, we furthermore perform bactericidal and hemolytic assays as well as time-resolved immunofluorometric assays (TRIFMAs).

In collaboration with Assistant Professors Nick Stub Laursen and Kasper Røjkjær Andersen, we recently started working with the development of nanobodies for various applications including co-crystallisation. Moreover, in collaboration with Associate Professor Edzard Spillner, we also work with other different antibody formats and these collaborations expand our working field of Structural Immunology.

If you are interested in knowing more about our research or in the possibility of starting a collaboration, do not hesitate to contact us. Otherwise take a tour around our website to ‘meet’ the lab members, scroll through our publication list or check for vacant positions if you are interested in joining the laboratory.