REVIEW doi: 10.1111/sji.

12070
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Humoral Pattern Recognition and the Complement

System
S. E. Degn & S. Thiel

Department of Biomedicine, Aarhus University,
Aarhus, Denmark
Received 10 May 2013; Accepted in revised
form 13 May 2013
Correspondence to: S. E. Degn, Department of
Biomedicine, Aarhus University, The Bartholin
Building, Wilhelm Meyers Alle 4, 8000 Aarhus C,
Denmark. E-mail: sdegn@microbiology.au.dk
Abstract
In the context of immunity, pattern recognition is the art of discriminating
friend from foe and innocuous from noxious. The basis of discrimination is the
existence of evolutionarily conserved patterns on microorganisms, which are
intrinsic to these microorganisms and necessary for their function and existence.
Such immutable or slowly evolving patterns are ideal handles for recognition and
have been targeted by early cellular immune defence mechanisms such as Toll-
like receptors, NOD-like receptors, RIG-I-like receptors, C-type lectin receptors
and by humoral defence mechanisms such as the complement system.
Complement is a proteolytic cascade system comprising around 35 different
soluble and membrane-bound proteins. It constitutes a central part of the innate
immune system, mediating several major innate effector functions and
modulating adaptive immune responses. The complement cascade proceeds via
controlled, limited proteolysis and conformational changes of constituent
proteins through three activation pathways: the classical pathway, the alternative
pathway and the lectin pathway, which converge in common effector functions.
Here, we review the nature of the pattern recognition molecules involved in
complement activation, as well as their close relatives with no or unknown
capacity for activating complement. We proceed to examine the composition of
the pattern recognition complexes involved in complement activation, focusing
on those of the lectin pathway, and arrive at a new model for their mechanism of
operation, supported by recently emerging evidence.

response is detrimental. According to the danger theory,

Fundamentals of innate immunity and pattern
recognition
The immune system is a double-edged sword: one edge
holds the power to efficiently eliminate any unwelcome
intruder; the other edge poses the risk of wreaking havoc
on the bearer. The balance between the two rests on (1) the
ability to precisely discriminate self from non-self; (2) the
ability to determine when and where to mount a response;
and (3) the ability to control the magnitude of the response
and to dampen it once the threat is eliminated.
The fundamental immunological basis for discriminat-
ing self from non-self is the presence of recognizable
differences between foreign entities and the host. The
innate immune system relies on recognition of evolution-
arily conserved pathogen-associated molecular patterns
(PAMPs) by innate pattern recognition molecules and
receptors (PRMs and PRRs) [1], whereas the adaptive
immune system is principally trained to recognize every-
thing, except what is intrinsic to the host. The decision to
mount a response rests on whether not mounting a
the read-out for such detriment is danger- or damage-
associated molecular patterns (DAMPs), signifying host
cell and tissue damage [2]. Responses are localized by
PAMPs and DAMPs (from endogenous sources, i.e. tissue-
resident cells) and subsequently by localization of inflam-
matory mediators resulting from the response itself (from
tissue-exogenous sources, i.e. immigrant immune cells).
The magnitude of the response is similarly encoded in the
amount of DAMPs and PAMPs present. The mechanisms
governing dampening of the response after elimination of
the initiator may be attributed to clearance of PAMPs,
DAMPs and inflammatory mediators, depletion of effector
mechanisms and desensitization of immune cells.
Thus, immune responses are initiated through the
concomitant recognition of PAMPs and DAMPs. The
simultaneous recognition of danger patterns contextual-
izes the recognition of a pathogen, acting as a safeguard
against untoward responses. A further dimension of pattern
recognition in immunity is the recognition of so-called
host-associated molecular patterns (HAMPs), serving again

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182 Pattern Recognition and Complement Activation
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as a safeguard against untoward responses, by providing an
inverted means of recognition of non-self or altered self,
through the recognition of the absence of self-patterns. As
we shall describe below, the two different modes of
operation are exemplified in complement, where the
classical and lectin pathways are initiated upon recognition
of DAMPs and PAMPs, while the basis of alternative
pathway function is the recognition, or absence of
recognition, of self, by potent regulatory molecules.
While classical biochemistry has tended to operate with
simple one-to-one interactions, describing how one mol-
ecule binds to one ligand, the unifying feature of detection
of pathogens, danger and self is that of pattern recognition.
Pattern recognition is made possible through multivalent
soluble protein complexes, multivalent receptors or mono-
valent receptors requiring clustering for signalling. Each
recognition subunit possesses low affinity for the individual
molecular subunits of the pattern, but high-affinity
binding is achieved upon concomitant binding of all
recognition subunits to their cognate target subunits. The
pattern is either defined based on a certain spacing and
conformational arrangement or a certain density of the
target subunits, affording a much higher degree of
specificity than would otherwise be possible through
high-affinity one-to-one interactions. The phenomenon of
pattern recognition is not limited to the immune system,
but is gaining particular interest in this area. In this
context, it is important to remember the duality of the
immune system as both a guardian of homoeostasis and as
an anti-microbial defence mechanism, a duality, which is
also demonstrated by the complement system.
To limit infection, the mammalian host uses a wide
armamentarium of pattern recognition molecules [3].
Importantly, through redundant mechanisms, multiple
PRMs are engaged simultaneously in the course of
microbial infection and the final results of the stimuli are
highly complex [4]. While much attention has been
focused on the cellular side of pattern recognition,
following the identification of Toll-like receptors, NOD-
like receptors, RIG-I-like receptors and C-type lectin
receptors, this is only part of the story. An intricate
network of humoral PRMs is found in the blood, and the
complement system is a central part of this. This review
will focus on soluble PRMs that can collectively be
designated complement-activating pattern recognition
molecules. These can be seen as a subset of a larger and
more diverse family of PRMs, sometimes referred to as
collagen defence molecules, their unifying features being
multimeric macromolecular complexes composed of mono-
mers containing collagenous stalks and some sort of
recognition domain. In this family, one may include a
number of collectins (collagenous stalks with C-type lectin
recognition domains), the ficolins (collagenous stalks with
fibrinogen-like recognition domains) and C1q (collagenous
stalk with a globular C1q (gC1q) recognition domain).
S. E. Degn & S. Thiel
Complement a humoral pattern recognition and
proteolytic cascade system
The complement system is a humoral proteolytic cascade
based defence mechanism, comprising around 35 different
soluble and membrane-bound proteins. It is part of the
innate immune system and proceeds via controlled, limited
proteolysis of constituent proteins through three activation
pathways: the classical pathway, the alternative pathway
and the lectin pathway (Fig. 1).
The classical pathway recognizing patterns of
immunoglobulins and more
Activation of the classical pathway occurs through binding
of immune complexes or aggregates containing IgG or
IgM, as well as direct recognition of some microorganisms
and C-reactive protein (CRP), by C1q [5].
C1q, the recognition subunit of C1, is the archetypal
multimeric humoral pattern recognition molecule; yet, it is
somewhat of an odd one out among the collagen defence
molecules. C1q is a 460-kDa protein complex with the
overall shape of a bouquet of tulips. It is composed of three
different polypeptide chains (A, B and C) that form six
heterotrimeric (each containing an A-, a B- and a C-chain)
collagen-like triple helices that associate in their
N-terminal halves to form a stalk, then diverge to form
individual stems, each terminating in a C-terminal
heterotrimeric globular domain [6] (Fig. 1). The insertion
of a (Gly-X-Y-Z) element into the (Gly-X-Y) triplet
pattern in the A-chain of C1q combined with the
substitution of one glycine for an alanine in the C-chain
determines a structure that forces the triple helix to form a
kink at this position with a fixed angle of 60 [7]. The
C-terminal globular region (gC1q domain) fold is also
found in a variety of non-complement proteins [8].
Furthermore, there is a structural and evolutionary link
between tumour necrosis factor (TNF) and gC1q-contain-
ing proteins, defining a C1q and TNF superfamily (Fig. 2).
The ligands bound by the globular domains of C1q can
be immune complexes containing IgG or IgM, but not
those formed by IgA, IgD or IgE [9]. C1q has low affinity
for the Fc region of monomeric IgG, but due to avidity
effects, the binding strength is increased dramatically for
multiple Fc sites. The binding of arrays of IgG to the
surface of microorganisms can be said to generate an
acquired pattern. With regard to IgM, which is already
multivalent, it is the exposure of cryptic-binding sites in
the Fc regions upon interaction with large antigens, which
allows tight C1q binding to take place [10]. C1q is
traditionally known for its ability to bind antibodies, but
an amazing variety of other ligands have been suggested.
These include certain bacteria, viruses, parasites and
mycoplasma, indicating a role as an antibody-independent
defence protein. C1q has also been reported to bind to CRP
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S. E. Degn & S. Thiel Pattern Recognition and Complement Activation 183
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Figure 1 Overview of the three complement activation pathways. The figure summarizes the events leading to the formation of their respective C3
convertases, while for simplicity, the formation of the terminal membrane attack complex and the regulatory mechanisms are omitted. Components and
enzymatic activities of the classical, the lectin and the alternative pathways are represented in red, blue and green, respectively. Common components are
shown in dark blue, and associations/dissociations of protein components are represented with grey arrows. Active proteases are underlined. The acronyms
of participating proteins are used (see text for full names). The classical pathway is activated by C1q in complex with C1s and C1r upon binding of
antibodyantigen complexes. The lectin pathway is activated when MBL or ficolins, in complex with MASPs, recognize foreign patterns of carbohydrate or
acetyl groups, respectively. The alternative pathway is constitutively activated by the formation of C3(H2O)B complexes and inhibited on self-surfaces, but
is allowed to propagate on foreign surfaces. The three pathways converge on C3 and C5 and the common terminal pathway leading to formation of the
membrane attack complex.

Complement-related innate humoral pattern recognition molecules

FREPs C-type lectin domain superfamily
(fibrinogen-related proteins)

C1q/TNF superfamily
C-type lectins NK-cell receptor family

Bovine conglutinin
Collectins
CL-43
TNF family gC1q family CL-46

Ficolins Tachylectins SP-D

MBL-A CL-P1 CL-L1

SP-A2
C1q H-ficolin L-ficolin M-ficolin MBL-C CL-K1 SP-A1

Complement-activating pattern recognition molecules

Collagen defence molecules

Figure 2 Evolutionary relationships and overview of the complement-related innate humoral pattern recognition molecules. The set of complement-
related humoral pattern recognition molecules present in humans is indicated. They hail from three different families: the C1q/TNF superfamily (C1q), the
fibrinogen-related proteins (H-, L- and M-ficolin) and the C-type lectin-domain superfamily (MBL and potentially CL-K1 are able to activate complement,
whereas the others are not or have unknown capacity, see text). Some molecules not present in humans are also shown in grey, namely the tachylectins
(found in horse-shoe crab and related organisms), MBL-A and finally conglutinin, CL-43 and CL-46, all three only found in the bovidae. Although far from
exhaustive, some related branches have been included for reference (TNF family, NK-cell receptor family).

when this is complexed with exposed phosphocholine C1q lacks enzymatic activity, but is associated with
residues on bacteria, providing a further means of host two serine protease proenzymes, C1r and C1s, in the
defence [11]. heteropentameric C1 complex, C1qr2s2. Binding of C1q is

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184 Pattern Recognition and Complement Activation
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believed to induce a conformational change leading to
auto-activation of the associated C1r, which then cleaves
and activates C1s (Fig. 1) [12]. The conformational
change also facilitates interaction with C4, which is
cleaved by C1s into C4a and C4b. C4b has the ability to
bind covalently to hydroxyl or amino groups through a
reactive thioester and may thus bind to the surface
recognized by C1q [13]. Cleavage of C4 also exposes a
cryptic C2-binding site on C4b. Upon binding, C2 is
cleaved into C2a and C2b by C1s. C2a remains bound to
C4b, and the complex thus generated acts as a C3
convertase, recruiting C3 and cleaving it into C3a and
C3b. Release of the small C3a anaphylatoxin induces a
conformational change in C3b, exposing a previously
buried thioester [14]. Like for C4b, this allows C3b to
become covalently bound to the target that elicited
complement activation. Association of the C4b2a complex
with C3b forms a C5 convertase, which initiates the
common terminal pathway.
The lectin pathway is highly parallel to the classical
pathway (see below), and quite probably, at some stage in
the evolution of the classical pathway, C1q recognized
carbohydrate structures on immunoglobulins [15]. This
puts into perspective both a reported recognition of
glycosylations on immunoglobulins by mannan-binding
lectin (MBL), one of the collagen defence molecules of the
lectin pathway, and the reported activation of the classical
pathway by direct binding of C1q to PAMPs. Thus, the
primordial C1q pathway may have been very lectin
pathway like. This is further reinforced by the observation
that lamprey C1q acts as a lectin [16] that binds serine
proteases and activates the C3/C4-like thioester-containing
protein of the lamprey complement system [15], in a
manner similar to that illustrated in Fig. 1.
The alternative pathway recognizing patterns of
sialic acids
The course of activation of the alternative pathway, on the
other hand, is distinct from that of the two other activation
pathways until the generation of a C5 convertase and
initiation of the common effector mechanisms. The
principle behind the pathway also differs from that of the
other two, in that the classical and lectin pathways are
activated upon recognition of non-self, whereas the alter-
native pathway is constantly auto-activated and then
inhibited on self-surfaces, but allowed to propagate on
non-self-surfaces. Thus, the alternative pathway primarily
makes use of what has been termed reverse recognition,
based not on the recognition of PAMPs, but rather the
recognition of host-associated molecular patterns
(HAMPs). The key host-pattern recognition molecule
appears to be factor H, predominantly recognizing HAMPs
composed of polyanionic surface structures, for example
sialic acids [17].
S. E. Degn & S. Thiel
C3 is continuously activated at a low rate in the fluid
phase through either cleavage by serum proteases to C3a
and C3b, reaction of the thioester with small nucleophiles
or water, or non-specific perturbations leading to confor-
mational changes and the exposure and hydrolysis of the
thioester. This is known as the tick-over mechanism.
Intact C3 that has a hydrolysed thioester without being
cleaved is referred to as C3i or C3(H2O) (when the
nucleophile is water). It has a molecular conformation like
C3b and is able to associate with factor B (see Fig. 1). The
C3(H2O)B complex is cleaved by factor D to form the
alternative pathway initiator, the C3-convertase C3(H2O)
Bb. These processes operate at low levels and are inhibited
in various ways by self. On host-surfaces, deposited C3b
forms a complex with the soluble molecule factor H, which
is drawn to the surface by a pattern of high density of sialic
acids and serves as cofactor for the regulatory serine
protease factor I in the breakdown of C3b to iC3b
(inactivated C3b). Also the membrane proteins comple-
ment receptor 1 (CR1), membrane cofactor protein (MCP)
and decay-accelerating factor (DAF) interact with the C3
convertase and promote its degradation or dissociation.
Factor H and factor I also act on the fluid-phase
C3-convertase, C3(H2O)Bb. However, if C3b deposition
occurs on an activating, that is, non-inhibitory, surface, it
can serve as a seed for an explosive positive amplification
loop (Fig. 1). Importantly, the alternative pathway serves
not only as an independent pathway, but also as an
amplifier of the two other pathways once these have been
initiated and have generated the C3-convertases specific for
these pathways (see above and below) [18, 19].
The lectin pathway recognizing patterns of
carbohydrates and acetyl groups
The lectin pathway parallels the classical pathway, the
difference being at the initial step of target recognition and
subsequent activation. Evolutionarily, it is arguably the
most ancient of the activation pathways [20, 21]. Activa-
tion of the lectin pathway occurs through direct recogni-
tion of carbohydrate or acetylated PAMPs by MBL and
ficolins, respectively, in association with MBL-associated
serine proteases (MASPs). Upon binding of MBL or ficolins
to their target, the associated MASPs are activated,
presumably in a manner analogous to that of the C1
complex. MASP-2 is able to cleave both C4 and C2 in
principle rendering it sufficient for lectin pathway activa-
tion, but under physiological conditions, MASP-1 plays an
important role by transactivating MASP-2 (see Fig. 1) [22,
23]. Furthermore, convertase formation appears to be
accelerated by MASP-1 through auxiliary C2 cleavage [24,
25]. The importance of MASP-1 may partly lie in its
relative abundance (10 lg/ml), as compared to MASP-2
(0.5 lg/ml) [26]. Recently, MASP-1 has also been impli-
cated in the alternative pathway, with the demonstration of
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its requirement for activation of pro-fD to active fD in the
MASP1 knock-out mouse [27]. However, in a human
MASP1-deficient patient, the alternative pathway was
found to function normally [22].
Collectins recognizing patterns of carbohydrates
Lectins are carbohydrate-binding proteins. The animal
lectins have been subdivided into a number of families.
One of these is a family of proteins containing a C-type
lectin-like domain (CTLD). The mammalian members of
the CTLD family can be further divided into 17 subgroups
based on phylogenetic relationships and domain structures
[28]. Despite having a CTLD, not all members actually
bind carbohydrate structures, but many are Ca2+-depen-
dent carbohydrate-binding proteins and thus true C-type
lectins. Within the CTLD, the highly conserved Glu-
Pro-Asn (EPN) and Gln-Pro-Asp (QPD) motifs determine
specificity for mannose- or galactose-containing ligands,
respectively [29].
One of the subgroups of the CTLD family is the
collectins. Similarly to C1q, these contain collagen-like
regions and usually assemble into large oligomeric com-
plexes, allowing high-avidity binding based on multiple
low-affinity interactions of their CTLDs [30]. This enables
collectins to discriminate not only specific carbohydrate
moieties but also specific patterns of these, often charac-
teristic to pathogens. MBL is the first discovered and most
well-known collectin. It was originally named mannan-
binding protein, but has later become known as mannose-
binding lectin or mannan-binding lectin. Lung surfactant
was also shown to contain two collectins, surfactant protein
A (SP-A) (of which two similar forms are found in humans,
SP-A1 and SP-A2) and SP-D. Furthermore, genome
analyses revealed two related human collectins, collectin
liver 1 (CL-L1) and CL placenta 1 (CL-P1), and a third, CL
kidney 1 (CL-K1), was recently cloned [31].
Mannan-binding lectin is a plasma protein of hepatic
origin. The polypeptide chain of MBL has the character-
istics of the collectins: a short N-terminal region contain-
ing cysteines that mediate disulphide bridging between
structural subunits or monomers of the same subunit [32];
a collagen-like region with variable repeats of Gly-X-Y
triplets (where X and Y may be any amino acid) stabilized
by the presence of hydroxyprolines and glycosylated
hydroxylysines [33]; a small neck region that forms an
a-helical coiled-coil; and a C-terminal globular CTLD,
dictating the ligand specificity. Three such polypeptide
chains assemble to form homotrimeric structural subunits
(Fig. 3), which then associate into higher-oligomeric forms
(Fig. 4), ranging from dimers to hexamers and even higher
oligomers [34]. In human MBL, an interruption in the
collagenous sequence between the first 7 and the ensuing
12 collagen repeats is assumed to give rise to a flexible joint
in the collagenous region of MBL, also known as the kink
(a similar kink is found in SP-A, and these are analogous to

Figure 3 Subunit structures of the humoral

collectins and ficolins. Lengths of collagen-like
regions are calculated according to a
translation of 0.286 nm per residue in the
triple helix [95], corresponding to a triplet
pitch of a type I collagen helix of 0.85 nm
[96]. The a-helical coiled-coil length of
~3 nm and the domain size of the C-type
lectin-like domains (CTLDs) of approximately
3 9 4 nm were derived from X-ray diffraction
[97], and the domain sizes for FBGs are about
the same size, also based on X-ray diffraction
[98, 99]. The FBGs are marginally larger than
the CTLDs based on spherical volume
calculations as described in the Supporting
Information, and they are depicted as such in
the figure. The figure is drawn to scale.

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186 Pattern Recognition and Complement Activation S. E. Degn & S. Thiel
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Figure 4 Schematic drawings of the ultrastructures of the multimeric humoral pattern recognition molecules. Based on [75, 76] (C1q), [77] (SP-A), [100]
(SP-D), [39] (MBL), [101, 102] (IgM), [78] (L-/M-ficolin), [51] (H-ficolin). The drawings are to scale. Some structure dimensions were corrected according
to the calculations in the Supporting Information.

the kink in C1q described above). The binding site for

MASPs has been localized around lysine 55 [35] and
suggested to be the motif, Hyp-Gly-Lys-X-Gly-Pro/Tyr
(where Hyp is hydroxyproline), which is conserved between
MBL and ficolins of various species, as well as C1q [36, 37]
(Fig. 5). In MBL and ficolins (see below), this motif has
been associated with binding of MASPs and hence
complement activation via C4 and C2. Whereas the overall
structure of MBL resembles that of C1q, MBL has a more
open or extended conformation (Fig. 4). While the arms of
C1q are joined in a sizeable stalk, restricting flexibility
somewhat, MBL seems to be joined N-terminally in a
so-called hub [38, 39].
The PAMPs recognized by MBL comprise various
Figure 5 Alignment of the collagen-like sequence around the putative
simple and complex carbohydrate motifs. MBL selectively
MASP-binding motif of MBL, the ficolins, the chains of C1q, collectin-L1,
binds not only mannose or its multimers, as the often used collectin-K1 and SP-D. SP-A1 and SP-A2 lack this motif entirely.
name mannose-binding lectin implies, but rather in general X = any amino acid. : entirely conserved. . highly conserved. Prolines in
recognizes sugars with 3- and 4-OH groups placed in the third position in the Gly-X-Y triplet are likely hydroxyprolines. The
equatorial plane of the sugar ring structure, for example, putative MASP-binding motif in the collagen sequence is indicated in
blue. Residues involved in binding are given in bold, and the essential
mannose, N-acetylmannosamine, glucose, N-acetylglucos-
lysine (55 in MBL) is indicated with an asterisk.
amine and L-fucose [40]. High-avidity binding of MBL
requires appropriate spacing of ligand sugars, allowing polymorphisms in the promotor region and in exon 1
concomitant binding of multiple CTLDs. [41]. Three naturally occurring mutations in the
The level of MBL in plasma varies interindividually by N-terminal part of the collagenous region cause decreased
more than three orders of magnitude (2 ng10,000 ng) MBL levels, low levels of oligomerization of the subunits
and is to a large degree genetically determined by and decreased binding of MASPs [34]. MBL deficiency,

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S. E. Degn & S. Thiel
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caused either by low MBL levels or defective oligomeri-
zation, is widely recognized as one of the most common
human immunodeficiencies.
CL-L1 was cloned from liver and has been reported to be
present mainly in liver as a cytosolic protein and at low
levels in placenta [42]. CL-L1 has 24 Gly-X-Y repeats, not
interrupted by a kink, and lacks the first hydroxyproline of
the traditional binding motif for MASPs and C1r/C1s
(Figs. 3 and 5). Evolutionarily, CL-L1 appears to be
restricted to mammals and birds. Studies on CL-L1 are
lacking, and no physiological role has been established.
CL-P1 was isolated from placenta [43]. It is a type II
membrane protein, which has a coiled-coil region, a
collagen-like domain and a CTLD. It appears to be a
scavenger receptor found on vascular endothelial cells and
able to bind both to bacteria and yeast, as well as oxidized
low-density lipoprotein [43, 44].
CL-K1 was discovered in kidney [31], but is expressed
also in the adrenal glands and the liver [45] and other tissues.
The mean level in plasma was found to be 2.1 lg/ml in 10
individuals. CL-K1 possesses an uninterrupted collagen-like
region (24 repeats) (Fig. 3), which lacks the first hydroxy-
proline in the proposed protease-binding motif: Hyp-
Gly-Lys-X-Gly-Pro/Tyr (Fig. 5) found in MBL, ficolins
and C1q [46]. However, MASP-1 was found to copurify with
CL-K1 indicating that the motif is not absolutely required
and that variants are allowed [45]. The other MASPs were
not identified in the study. The protein seems to have direct
antimicrobial activities, and another study suggests an
ability to activate the lectin pathway [47].
Phylogenetic analyses indicate that the collectins can be
divided into five distinct subgroups. MBLs group together,
SP-A1 and SP-A2 group together, SP-D forms a group
with bovine conglutinin and collectin-43 and collectin-46,
CL-P1 groups alone and finally CL-L1 and CL-K1 group
together [31] (Fig. 2).
Ficolins recognizing patterns of acetyl groups
The lectin pathway can also be activated by the ficolins. They
are structurally and functionally related to the collectins, the
defining difference being that they have fibrinogen-like
(FBG) domains in place of CTLDs [48] (Fig. 3). Similar to
the collectins, the ficolins form structural subunits through a
collagen helix (Fig. 3) and these associate into larger
oligomeric structures (Fig. 4). Three ficolins are found in
humans: H-ficolin, L-ficolin and M-ficolin, while two are
found in rodents (Ficolin A and Ficolin B, homologues of L-
and M-ficolin, respectively) [49]. The ligands for the ficolins
were initially suggested to be acetylated monosaccharides
like GlcNAc and GalNAc [50, 51], in line with their
evolutionary relationship to the tachylectins. However, it is
now known that the motifs recognized are more generally
acetylated compounds, including non-sugars such as N-
acetyl-glycine, N-acetyl-cysteine and acetylcholine [52].
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Pattern Recognition and Complement Activation 187
Based on this selectivity, the ficolins are not easily defined
as lectins, rendering the term lectin pathway somewhat of a
misnomer, but we shall use this name indiscriminately in
this review, as is also the case in the literature in general.
H-ficolin, also referred to as Ficolin-3, was initially
noted as an auto-antigen in patients with systemic lupus
erythematosus [53] and was subsequently characterized and
recognized as a ficolin based on structural and functional
characterization [51, 54]. Meanwhile, human L-ficolin
(Ficolin-2), initially termed P35, had been discovered,
isolated from plasma and characterized [5557]. M-ficolin
was identified by cloning of cDNA encoding a molecule
resembling L-ficolin [58, 59] and was also referred to as
P35-related protein or Ficolin-1.
H-ficolin is produced by bile duct epithelial cells and
hepatocytes, as well as in the lung by ciliated bronchial
epithelial cells and type II alveolar epithelial cells [60].
From these sites of production, H-ficolin is secreted into
serum, bile and bronchoalveolar fluid. The median serum
H-ficolin level has been determined at 18.4 lg/ml,
ranging from 11.2 to 33.8 lg/ml [61], by far making it
the most abundant of the activators of the lectin pathway.
Upon gel permeation chromatography, serum H-ficolin
runs at 650 kDa [54]. The ligand specificity of H-ficolin is
reportedly N-acetyl-galactosamine (GalNAc) and N-acetyl-
glucosamine (GlcNAc), and H-ficolin was found to
agglutinate human erythrocytes coated with LPS from
Salmonella and Escherichia [51]. H-ficolin was also found to
bind a polysaccharide from Aerococcus viridans [62]. Perhaps
more biologically relevant, this finding was supported by
binding to whole bacteria [61] and higher serum concen-
trations of H-ficolin were reportedly linked with inhibition
of A. viridans growth [63]. Moreover, H-ficolin was also
reported to activate the lectin pathway of complement [61,
64].
L-ficolin is hepatically produced and the median
concentration in serum has been established at 3.3 lg/
ml, ranging from 1.8 to 9.0 lg/ml [61]. It is found in
serum in two forms, at 650 and at 150 kDa, and it binds
acetylated compounds, including GlcNAc, GalNAc,
N-acetyl-mannosamine, N-acetyl-glycine, N-acetyl-cyste-
ine and acetylcholine [52]. L-ficolin has also been found to
bind some encapsulated bacteria [57, 61], to associate with
MASPs and MAps in a calcium-dependent manner [65] and
to activate complement [66].
M-ficolin is found on the surface of monocytes and
granulocytes [67, 68], although it lacks a transmembrane
domain and also in secretory granules in the cytoplasm of
neutrophils and monocytes [69]. The expression of
M-ficolin was reported to cease when the monocytes
differentiated into macrophages [68, 70]. M-ficolin was
found to bind acetylated patterns, associate with MASPs,
and activate complement [67, 69]. Although M-ficolin was
initially not thought to be found in serum, a mean
concentration of 1.07 lg/ml, ranging from 0.28 to
188 Pattern Recognition and Complement Activation
..................................................................................................................................................................
4.05 lg/ml, has been reported [71]. On GPC of serum, M-
ficolin is found in a complex of ~900 kDa [71], much
larger than what has previously been seen for H-ficolin [54]
and L-ficolin [52]. The cell-surface association of M-ficolin
in the absence of a transmembrane motif was recently
reported to be caused by tethering through recognition of
sialic acid by the fibrinogen-like domain [72]. The
preference of M-ficolin for sialic acid was supported by
studies based on glycan array screening [73]. Such
recognition would appear to be a risky mechanism,
potentially triggering complement activation on self-
surfaces. It is curious that such binding to patterns of
sialic acids may be counteracted by the affinity of factor H
of the alternative pathway for sialic acids in combination
with C3-convertases (see above for the alternative
pathway).
Ultrastructures of the multimeric humoral PRMs
Although the defining feature of innate PRMs and PRRs is
that their specificities are germ-line encoded and thus fixed,
in contrast to the somatically recombined B- and T cell
receptors, taken together, even within single species, the
total pool of innate PRMs and PRRs exhibit an extremely
broad repertoire of recognition capabilities. This repertoire
is enhanced by natural antibodies of the IgM isotype,
produced at tightly regulated levels and in the complete
absence of external antigenic stimulation [74]. IgM and the
bona fide innate PRMs also resemble each other somewhat
in terms of their ultrastructures.
The humoral recognition molecules commonly attain
high-avidity recognition capabilities by being polymeric,
for example, IgM is pentameric or hexameric, and C1q is
hexameric, while ficolins and collectins are found in a
number of multimeric forms (Fig. 4). It is important to
note, however, that the interactions may be even higher
order than the oligomeric nature suggests. In the case of
MBL, each subunit has three CTLD heads, meaning that a
hexamer of subunits in reality has 18 recognition sites. The
large size of the multimeric PRMs also gives them an
advantage in the agglutination of large and often mutually
repulsive particles. Electron microscopy of rat SP-D
preparations demonstrated a highly homogeneous popula-
tion of molecules with four identical rod-like arms (46 nm
in length) that emanated from a central hub in two pairs
that closely paralleled each other for their first 10 nm.
Higher-order oligomers were also observed, in which four
such dimers were arranged so that each rod-like arm
constituted the spoke of a wheel-like structure (Fig. 4).
The diametrally opposed rod-like arms and their
recognition domains together span an impressive
100 nm, that is, the diameter of small viruses! SP-A was
found to have a structure much like that reported for C1q
[75, 76]. Collagen arms with a length of around 19.6 nm
and a diameter of 1.5 nm were observed, terminated by
S. E. Degn & S. Thiel
globular heads with a diameter of around 5.3 nm [77]
(Fig. 4).
The structure of MBL has recently been characterized by
atomic force microscopy [39], and the observations were
well in agreement with previous studies using electron
microscopy and with the estimated dimensions of the
collagen-like region. While the kink in C1q is generated
by two different interruptions in two of three chains in the
collagen-like helix, the kinks in MBL and SP-A are based
on the same interruption in three identical chains. This was
interpreted to mean that the kink in C1q is probably more
rigid, whereas the kink in MBL and SP-A are more flexible
[77]. There is also a difference in the kink between MBL
and SP-A, as in the former, the interruption is the lack of a
Y residue in a triplet, whereas in the latter, a 4 aa motif is
inserted into the collagen-like sequence. L- and M-ficolin,
but not H-ficolin, also possess a kink, at the very beginning
of their collagen-like regions (Fig. 3), and in both cases,
this is due to the insertion of a 6 aa sequence. The
ultrastructures of H-ficolin [51] and L-/M-ficolin (equiv-
alent to the porcine ficolin a and/or b in the figure) [78]
have also been studied by electron microscopy, revealing
overall structures resembling that of MBL (Fig. 4).
Nature and composition of the complement-
activating complexes
The nature of the complexes of the serine proteases and
the collectins (MASPs) and C1q (C1r and C1s) must be
tied intimately to the mechanism of activation. In the
case of C1, the complex consists of one C1q with a C1s-
C1r-C1r-C1s tetramer, and the model of activation is that
the C1rs trans-autoactivate, followed by their transacti-
vation of the C1ss. The scenario for the complexes of the
lectin pathway is inordinately more complex, with at least
four PRMs (MBL, H-, L- and M-ficolin) interacting with
five MASP/MAps (MASP-1, -2, -3, MAp19 and MAp44)
[79]. A fundamental prerequisite to the goal of under-
standing complement activation through the lectin
pathway is to define the nature and composition of the
complexes.
The three MASPs are multidomain modular proteases
with the same overall architecture as C1r and C1s of the
classical pathway, that is, they are composed of well-
described domains in the order: CUB1-EGF-CUB2-CCP1-
CCP2-SP, the latter being the serine protease domain. The
CUB1 and EGF domains are responsible for their calcium-
dependent binding to C1q (for C1r, C1s) and MBL and the
ficolins (for MASPs) [80, 81]. Studies using truncated
proteins have shown that MASP-1 and MASP-2 bind to
MBL in a Ca2+-dependent manner through interactions
involving the CUB and EGF-like domains [82, 83].
Fragments encompassing these regions are Ca2+-indepen-
dent homodimers that are stabilized by interactions
involving the two N-terminal domains, with the CUB1
Scandinavian Journal of Immunology, 2013, 78, 181193
S. E. Degn & S. Thiel
..................................................................................................................................................................
domain being essential [84]. We have recently found that
while MASPs and MAp44 are able to form heterodimers,
only homodimers (e.g. MASP-3/MASP-3) are found in
serum [85]. The three N-terminal domains of each MASP
are necessary and sufficient to reproduce the MBL-binding
properties of the full-length proteins [84]. The contiguous
CCPs of C1r, C1s, and the MASPs, especially CCP2, have
been implicated in the binding of macromolecular sub-
strates [86, 87]. Finally, the SP domain is the catalytically
active unit and defines them as S1A family, chymotrypsin-
like, proteases. CCP2 and the SP domain are connected by a
somewhat flexible linker or activation peptide, which lies
just upstream of the R-I cleavage site and contains the
recognition motif for activating proteases.
A study employing site-directed mutagenesis in rat
MBL and assessing sequence conservation between mam-
malian MBLs suggested that the site of complex formation
with the MASPs and MAps is likely located C-terminally
to the break in the collagenous sequence [37] and lysine55
in this position of human MBL was found to be essential
for binding to MASPs [35]. The same binding motif is
found in the ficolins [36] (Fig. 5). Surprisingly, CL-K1 was
recently reported to associate with MASP-1, even though
CL-K1 lacks the first hydroxyproline in the otherwise
conserved binding motif, perhaps indicating that the
requirements for this motif are not as stringent as initially
believed [45].
In humans, MBL is found in oligomeric forms ranging
from dimers to octamers [34]. The most prominent
oligomer contains four subunits, that of three subunits is
the second most abundant, closely followed by that of five
subunits and decreasing presence of larger oligomers [39].
Analyses of CUB-EGF-CUB fragments indicated that each
MASP dimer contains binding sites for two MBL subunits
and both sites must be occupied by subunits from a single
MBL oligomer to form a stable complex. Thus, the smallest
functional unit for complement activation has been
proposed to consist of MBL dimers bound to MASP-1 or
MASP-2 homodimers [84]. This raises the possibility that
two or more homodimers could associate in a single
complex with a higher-oligomeric PRM. Conversely,
others have suggested that MASP dimers have four separate
binding sites for MBL stalks, one for each stalk of a MBL
tetramer [88] and that trimeric and tetrameric MBL may
bind only a single MASP homodimer [89]. Nonetheless, we
recently demonstrated that single complexes with MBL
could harbour more than one dimer of MASP [22], but
naturally, this might be due to association with higher-
order oligomers of MBL. However, we have observed that
such heterocomplexes can also be formed through associ-
ation with purified H-ficolin or L-ficolin, and while
H-ficolin is present in a range of oligomeric forms,
L-ficolin from serum is predominantly tetrameric, again
suggesting that tetramers may bind two MASP dimers
[85].
2013 John Wiley & Sons Ltd
Pattern Recognition and Complement Activation 189
Mechanism of activation
The nature of the PRM complexes is tied intimately to the
mechanism of activation, and the main question remains
how binding of PRM to a ligand surface triggers MASP
activation. MBL binds microorganisms through the
C-terminal CTLDs, which are far from the reported
binding site of MASPs and MAps C-terminally to the
kink in the collagenous sequence [37]. It has been
suggested that binding to the surface of a microorganism
could induce conformational changes within individual
MBL subunits, leading to MASP activation, but crystal-
lographic data show that no structural changes occur in the
individual carbohydrate recognition domains upon sugar
binding [40].
The presently favoured model for complement activa-
tion maintains that binding to the surface of a microor-
ganism causes a global change in the structure of MBL,
resulting in the displacement of the relative positions of
subunits, translating from the CTLDs into the stalks.
Indeed, recent studies have demonstrated a great flexibility
of MBL and large conformational changes upon binding to
artificial ligand surfaces [38, 39]. Furthermore, the nature
of the recent structure of the CUB-EGF-CUB domain of
MASP-1/-3/MAp44 with the part of the collagen stalk of
MBL harbouring the proposed MASP-binding motif was
interpreted as evidence for such a model [90].
Yet, another, simpler, possibility remains, namely that
MBL and ficolins serve merely as carriers of MASPs and
that their clustering upon binding to appropriately spaced
ligand patterns on microorganisms causes the juxtaposition
of MASPs, allowing intercomplex transactivation. We
favour this mechanism for a number of reasons:
Firstly, it is supported by the observation of concentra-
tion-dependent auto-activation of MASP-1 and MASP-2
[91, 92]. This implies that there is no (absolute) require-
ment for a sterical strain for catalytic activity of the
MASPs, as has otherwise been suggested for the initiating
protease of the classical pathway, C1r [12].
Secondly, until recently, it was believed that while two
activation steps (C1r and C1s) are required for C1
activation, strictly only one step (auto-activation of
MASP-2) is required for MBL-MASP activation. However,
as mentioned earlier, recent data suggest that under
physiological conditions, MASP-1 is important in trans-
activating MASP-2 [22, 23]. Indeed, MASP-1 can be
viewed as the trigger; in that zymogen MASP-1 has a
significant activity for either zymogen MASP-1 or MASP-
2, allowing it to initiate a ping-pong transactivation
(Fig. 6). Despite our recent demonstration that MASP-1
and MASP-2 may be found together in the same PRM
complex [22, 85], this may only account for a minor
proportion of PRM/MASP complexes. The function of
lower-order oligomers of MBL or ficolins with single
MASP homodimers would be unaccounted for, unless such
190 Pattern Recognition and Complement Activation
..................................................................................................................................................................
Figure 6 Potential scenarios for concentration- or juxtaposition-dependent activation and transactivation of MASPs upon PRM binding. See text for
discussion of this figure. The modelled scenarios are not mutually exclusive and may even co-operate on the same target surface.
oligomers are able to co-operate upon binding to the same
target surface.
Thirdly, recent structures of the CCP-CCP-SP frag-
ments of MASPs (e.g. MASP-1 [93]) and MAp44
(representing CUB-EGF-CUB-CCP of MASP-1) [94],
allow the in silico assembly of intact MASPs. This gives
rise to an estimated length of the dimer of more than
30 nm, which is similar to (or longer than) the longest
dimension of MBL! This would allow the MASPs to
protrude significantly from the PRM, a prerequisite for
such co-operation.
Finally, it is hard to imagine enough structural
flexibility in MASPs to allow for the two antiparallel
MASPs in a MASP homodimer to curl up on themselves,
in order for the two serine protease domains to sequentially
activate each other. Furthermore, this mechanism would
only allow transactivation of MASP-1 on MASP-1 or
MASP-2 on MASP-2, not MASP-1 on MASP-2, because as
mentioned earlier, heterodimers are not found [85]. While
we have demonstrated that two MASP dimers may be
found on the same PRM, potentially addressing this
problem, the function of lower-order oligomers of MBL or
ficolins with single MASP homodimers would again be
unaccounted for.
Importantly, the intercomplex transactivation model is
not mutually exclusive with that of intracomplex transac-
tivation in complexes harbouring two MASP dimers (or
S. E. Degn & S. Thiel
potentially more). Indeed, such higher-order oligomers with
multiple MASPs may still be functionally important, both
by virtue of the intrinsic colocalization of, for example
MASP-1 and MASP-2, and the higher-oligomeric nature of
the MBL, allowing high-avidity binding. The intercomplex
transactivation model rather extends the scope, allowing for
intricate pattern recognition-encoding directing diverse
physiological responses to discriminate self versus non-self,
as we have previously alluded to [22]. Of note, this would
allow not only for co-operation of distinct MBL/MASP
complexes, but also distinct PRM/MASP complexes, such as
MBL/MASP-2 with H-ficolin MASP-1. We have presented
an overview of the proposed modes of operation in Fig. 6.
Future experiments should address the questions raised here,
such as the possibility of PRM/MASP co-operation on
ligand surfaces, as well as the structural aspects in terms of
placement and orientation of the MASPs in the activating
complexes when bound to their targets.
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Supporting Information
Additional supporting information may be found in the
online version of this article:
Data S1 The supporting information describes calcula-
tions used to correct the dimensions derived from the
electron microscopy, and the appropriate references for
these calculations. This forms part of the basis of Figures 3
and 4, which show schematic drawings of the subunits and
ultrastructures of the oligomeric molecules, drawn to scale.