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Contents lists available at ScienceDirect

The International Journal of Biochemistry

& Cell Biology
journal homepage: www.elsevier.com/locate/biocel

Molecules in focus

Complement component 5a (C5a)

Helga D. Manthey a,∗ , Trent M. Woodruff a , Stephen M. Taylor a , Peter N. Monk b
a
School of Biomedical Sciences, University of Queensland, Brisbane, Australia
b
Department of Neuroscience, School of Medicine and Biomedical Science, University of Sheffield, Sheffield, UK

a r t i c l e i n f o a b s t r a c t

Article history: The 74 amino acid glycoprotein, complement component 5a (C5a), is a potent pro-inflammatory mediator
Received 18 February 2009 cleaved enzymatically from its precursor, C5, upon activation of the complement cascade. C5a is quickly
Received in revised form 4 April 2009 metabolised by carboxypeptidases, forming the less potent C5adesArg. Acting via a classical G protein-
Accepted 6 April 2009
coupled receptor, CD88, C5a and C5adesArg exert a number of effects essential to the innate immune
Available online xxx
response, while their actions at the more recently discovered non-G protein-coupled receptor, C5L2 (or
GPR77), remain unclear. The widespread expression of C5a receptors throughout the body allows C5a to
Keywords:
elicit a broad range of effects. Thus, C5a has been found to be a significant pathogenic driver in a number
Complement system
C5a
of immuno-inflammatory diseases, making C5a inhibition an attractive therapeutic strategy.
C5a receptor antagonist © 2009 Elsevier Ltd. All rights reserved.
Inflammation
Innate immunity

1. Introduction
The complement system is composed of over 30 proteins, acti-
vated in response to tissue injury, invading pathogens or other
foreign surfaces. C5a was first described as a cleavage product
of complement factor 5 (C5) with chemotactic and anaphylatoxic
properties (Shin et al., 1968). Further characterisation revealed that
C5a is an essential part of the innate immune response and evidence
now suggests that it may also play a role in adaptive immunity
(Kohl, 2006). Although not necessarily the initiating factor, the
excessive or uncontrolled production of C5a that occurs in a number
of inflammatory diseases, suggests that C5a promotes and perpet-
uates inflammatory reactions (Guo and Ward, 2005). Thus, there is
considerable interest in the development of therapeutics to block
these pro-inflammatory effects. This review will give an outline of
current knowledge on C5a and the therapeutic strategies that have
been developed to combat its pro-inflammatory effects.
2. Structure
The precursor for C5a, C5, is a 1676 amino acid, 188 kDa protein
whose gene is located at 9q33–9q34 (Wetsel et al., 1988). Human
C5a is an ∼11 kDa, 74 amino acid glycoprotein released from the
alpha-chain of C5 by C5 convertase enzymes (Fig. 1A). The N-linked
∗ Corresponding author at: The University of Queensland, School of Biomedical
Sciences, Skerman Building No. 64, St Lucia, Queensland 4067, Australia.
Tel.: +61 7 3365 8275; fax: +61 7 3365 1766.
E-mail address: h.manthey@uq.edu.au (H.D. Manthey).
glycosylation is not essential for function. C5a has four anti-parallel
alpha helices (Fig. 1B) connected by peptide loops, stabilised by
three critical disulphide linkages (Monk et al., 2007). Mutagenesis
and antibody studies have identified several basic residues that are
involved in the interaction with receptors (reviewed in Monk et
al., 2007). However, all of the agonist activity is located in the C-
terminal region of C5a, which assumes an elongated 1.5 turn helix
(critical for receptor activation) that spans residues 69–74 and is
attached to the helical core by a four-residue loop (Monk et al.,
2007).
3. Expression and turnover
The primary source of serum C5 is the liver hepatocyte although
cells such as macrophages can independently synthesise and
secrete C5 and thus may be local sources of C5a generation (Amara
et al., 2008; Morris et al., 1982).
The complement cascade is activated via four pathways: the
classical, the alternative, the mannan-binding lectin (MBL) or the
extrinsic protease pathway (Fig. 2) (Ricklin and Lambris, 2007). The
classical and lectin pathways are activated by the formation of anti-
body complexes on the surface of pathogens and by interaction with
mannose on bacterial surfaces, respectively. Both pathways lead to
the cleavage of C4 to C4b and C4a by serine proteases. C4b binds C2
and the same proteases lead to the generation of C2a which is part
of the classical pathway C3 convertase (or C4b2a). The alternative
pathway can be activated by foreign surfaces or by ‘tickover’; the
spontaneous hydrolysis of C3 allowing binding of factor B and for-
mation of the alternative pathway C3 convertase (C3bBb), which
maintains a continuous low level of complement cascade activa-

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of the C5 convertases (C4b2aC3b or C3bBbC3b) that cleaves C5 to

yield C5a and C5b. C5b goes on to initiate the assembly of the pore-
forming membrane attack complex (MAC; C5b–9). The complement
cascade is closely regulated by a series of soluble and membrane-
bound regulatory proteins which prevent complement activation
products from targeting host tissues (Ricklin and Lambris, 2007).
However, this control can be bypassed by extrinsic pathways that
involve direct cleavage of C3 and C5 by proteases such as throm-
bin (Amara et al., 2008). Further, activated neutrophils and alveolar
macrophages can generate C5a from C5 with secreted serine pro-
teases (Amara et al., 2008).
Upon cleavage from C5, C5a is quickly metabolised by plasma
and cell surface carboxypeptidases which remove the C-terminal
arginine to form C5adesArg (Bokisch and Muller-Eberhard, 1970).
C5adesArg has reduced potency compared to C5a, in line with
a reduced binding affinity for the classical C5a receptor, CD88
(Higginbottom et al., 2005). C5a and C5adesArg are cleared quickly
from the body, with ∼50% of both cleared from the circulation
within 2–3 min, mediated partly by the binding of C5a to CD88
on leukocytes and other cells (Oppermann and Gotze, 1994). How-
ever, the second receptor, C5a receptor-like 2 (C5L2), may be
more effective at the removal of complement fragments, partic-
ularly C5adesArg by rapidly internalising C5a/C5adesArg, where it
is retained and, in some cell types, degraded (Scola et al., 2009). In
contrast, cells expressing CD88 internalise C5a but release a higher
proportion in an undegraded, possibly still active form. Plasma C5a
may also be cleared by the liver (Chenoweth and Goodman, 1983).

Fig. 1. Amino acid sequence and structure of C5a. Human C5a is a 74 amino acid gly-
coprotein (A) consisting of four alpha helices arranged in an anti-parallel orientation
(B), connected by peptide loops located at the surface of the molecule and stabilised
by three critical disulphide linkages (Cys21 -Cys47 , Cys22 -Cys54 and Cys34 -Cys55 ). The
C-terminus (red) contains a four-residue loop, critical for receptor activation.
tion to ensure a rapid response to pathogens (Ricklin and Lambris,
2007). All three pathways result in the formation of C3 conver-
tases which cleave C3 to form C3a and C3b. C3b acts to opsonise
pathogen surfaces for recognition and clearance, but also forms part
4. Biological function
C5a elicits a number of biological effects which are essen-
tial in the clearance of pathogens and for host defence, including
increased vascular permeability, chemotaxis of inflammatory cells,
respiratory burst, cytokine and chemokine release and phagocy-
tosis (Guo and Ward, 2005). However, it is now known that the
functions of C5a are not limited to the primary innate immune
response. For example, the actions of C5a are also linked with the

Fig. 2. The complement cascade. C5a is formed via activation of the complement cascade. Activation via the classical pathway (activated by the formation of antigen–antibody
complexes), alternative pathway (activated by bacteria or foreign surfaces) and the lectin pathway (activated upon binding of mannan-binding lectin to carbohydrates on
bacterial surfaces) results in the formation of the C5 convertase (C4b2a3b or C3bBb3b) which cleaves C5 to form C5a and C5b. Activation via the extrinsic pathway results in
the direct cleavage of C3 and C5 by extrinsic proteases such as thrombin.

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Table 1 4.2. C5L2

Cellular expression and biological functions of C5a.

Cell type Action of C5a

C5L2 is expressed on most of the same cell types as CD88, such
a,b
as neutrophils, monocytes, lymphocytes and macrophages, as well
Neutrophils Adhesion molecule expression
as non-myeloid cells such as vascular smooth muscle cells and
Superoxide release
Release of granule enzymes cells from tissues such as adrenal gland, heart, liver, lung spleen
Cytokine release and brain, albeit at much lower levels than CD88 under non-
↓Neutrophil apoptosis inflammatory conditions (Gao et al., 2005). The function of C5L2
remains unclear. Some experimental data suggest that C5L2 func-
Monocytesa,b Cytokine release tions as a non-signalling decoy receptor; knockout or blockade of
Chemotaxis C5L2 was found to exacerbate the inflammatory response in mice
Macrophagesa,b Enhanced phagocytosis (Gao et al., 2005; Gerard et al., 2005). This suggests C5L2 has anti-
Cytokine release inflammatory functions, possibly by reducing the C5a available to
Eosinophilsa,b Chemotaxis bind to CD88. However, C5L2 can act as a positive regulator that
Granule enzyme release is critical for C5a and C3a signalling (at least in mice) (Chen et al.,
Mast cellsb,c Chemotaxis
2007). In vitro, it was found that C5L2 is required to facilitate C5a
Histamine release signalling in neutrophils, macrophages and fibroblasts, while lack of
Induce change to prothrombotic phenotype C5L2 in vivo resulted in reduced ovalbumin-induced airway hyper-
T-lymphocytesd Co-stimulation; suppression of apoptosis responsiveness and inflammation (Chen et al., 2007). Furthermore,
in a mouse model of ‘high-grade’ sepsis (100% lethality), only block-
Thymocytesa Suppressed apoptosis
ade of both C5L2 and CD88 provided protection (Rittirsch et al.,
b,c
Endothelial cells Vasodilation 2008).
Induce tissue factor activity
Adhesion molecule expression
5. Medical applications
Hepatocytesb Stimulate liver regeneration

Vascular smooth muscle cellsb Contraction (indirectly) Excessive C5a appears to contribute to an increasing number
of pathologies, including sepsis, rheumatoid arthritis, inflamma-
Cardiomyocytesb Reduced contractility
tory bowel disease, ischemia reperfusion injury, antiphospholipid
a
Reviewed in Guo and Ward (2005). syndrome, systemic lupus erythematosis, psoriasis, neurodegener-
b
Reviewed in Monk et al. (2007).
c
ative disease, age-related macular degeneration (AMD) and cancer
Reviewed in Amara et al. (2008).
d (Markiewski et al., 2008; Ricklin and Lambris, 2007; Woodruff et
Strainic et al. (2008).
al., 2006).
The central role of C5a in the pathogenesis of such diseases
has driven the development of pharmacological agents to block its
coagulation system and adaptive immunity (Amara et al., 2008; effects. An analogue of compstatin, an inhibitor of C3 cleavage, is
Kohl, 2006). The consequence of C5a activation is dependent on currently undergoing clinical trials for AMD. Eculizumab, a mon-
the location of C5a receptors. The expression of its receptors, CD88 oclonal antibody against C5, recently received FDA approval for
and C5L2, is widespread hence C5a elicits a broad range of biological paroxysmal nocturnal hemoglobinuria (Ricklin and Lambris, 2007).
functions (Table 1). However, specific inhibition of C5a activity has the advantage
of not interfering with the other, protective actions, of comple-
ment; C3b can still be produced for opsonisation, and the formation
4.1. CD88
of the MAC for bactericidal functions. A number of inhibitors
of C5a have been developed (Woodruff et al., 2006; Ricklin and
C5a binds with similar high affinity to both CD88 and C5L2. In
Lambris, 2007). Perhaps the most promising therapeutic candidate
contrast, while the affinity of C5adesArg for C5L2 is similar to C5a
and certainly the most widely used experimentally, is the orally
(∼12 nM), the affinity for CD88 is much lower (∼660 nM) (Monk et
active cyclic hexapeptide PMX53 (or AcF-[OPdChaWR]). PMX53
al., 2007). CD88 and C5L2 share a 35% sequence homology and are
is a potent, highly selective CD88 antagonist with an in vitro
located in the same region of chromosome 19 (19q13.3–19q13.4).
potency of ∼5 nM (Woodruff et al., 2006). Despite low absolute
They are clustered together with genes for other chemoattrac-
oral bioavailability in rodents (∼5%), PMX53 effectively reduces the
tant receptors, such as the formyl peptide receptor family and
C5a-mediated inflammatory response in several animal models of
bradykinin receptors. Both are glycosylated, seven transmembrane
human disease and has exhibited safety and tolerability in Phase
spanning proteins with molecular weights of ∼45 kDa. CD88, is a G
I and IIa clinical trials (Woodruff et al., 2006). New topical formu-
protein-coupled receptor and a member of the rhodopsin gene fam-
lations of PMX53 are currently being developed for the wet form
ily (Monk et al., 2007). C5a is thought to interact with CD88 via a
of AMD (www.arana.com). Whether PMX53 or second-generation
two-site binding process, whereby binding occurs at two distinct
analogues (e.g. PMX205) have therapeutic potential remains to be
and physically separate sites. The first ‘recognition’ site, located
seen.
in the receptor’s extracellular amino terminus (N-terminus), binds
the C5a N-terminus and disulphide-linked core. The second ‘activa-
Acknowledgements
tion’ site is formed by the transmembrane domains of the receptor,
which interact with the C-terminus of C5a and results in genera- We thank Professor Bostjan Kobe for his assistance with gen-
tion of specific signal transduction pathways that are mediated by erating Fig. 1B. We apologise for not being able to cite all original
receptor coupled G proteins (Monk et al., 2007). work due to space limitations.
Cells of myeloid origin and activated mast cells express CD88
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