The lectin-complement pathway – its

role in innate immunity and
evolution
Teizo Fujita
Misao Matsushita
Yuichi Endo
Authors’ addresses
Teizo Fujita
1
, Misao Matsushita
2
, Yuichi Endo
1
1
Department of Biochemistry, Fukushima
Medical University, Fukushima, Japan, and
CREST, Japan Science and Technology Agency.
2
Department of Applied Biochemistry and
Institute of Glycobiology, Tokai University,
Kanagawa, Japan.
Correspondence to:
Teizo Fujita
Department of Biochemistry
Fukushima Medical University
Fukushima 960-1295
Japan
Tel.: þ81245471146
Fax: þ81245486760
e-mail: tfujita@fmu.ac.jp
Acknowledgements
This work was supported by Grant-in-Aid for Scientific
Research (12470079 and 13143204) from the Ministry
of Education, Science, Sports, and Culture of Japan.
Summary: Innate immunity was formerly thought to be a non-specific
immune response characterized by phagocytosis. However, innate immun-
ity has considerable specificity and is capable of discriminating between
pathogens and self. Recognition of pathogens is mediated by a set of pattern
recognition receptors, which recognize conserved pathogen-
associated molecular patterns (PAMPs) shared by broad classes of micro-
organisms, thereby successfully defending invertebrates and vertebrates
against infection. Lectins, carbohydrate-binding proteins, play an import-
ant role in innate immunity by recognizing a wide range of pathogens.
Mannose-binding lectin (MBL) and ficolin are lectins composed of a lectin
domain attached to collagenous region. However, they use a different lectin
domain: a carbohydrate recognition domain (CRD) is responsible for MBL
and a fibrinogen-like domain for ficolin. These two collagenous lectins are
pattern recognition receptors, and upon recognition of the infectious agent,
they trigger the activation of the lectin-complement pathway through
attached serine proteases, MBL-associated serine proteases (MASPs). A
similar lectin-based complement system, consisting of the lectin–protease
complex and C3, is present in ascidians, our closest invertebrate relatives,
and functions in an opsonic manner. We isolated several lectins homo-
logous to MBLs and ficolins and several MASPs in invertebrates and lower
vertebrates, and herein we discuss the molecular evolution of these mole-
cules. Based on these findings, it seems likely that the complement system
played a pivotal role in innate immunity before the evolution of an
acquired immune system in jawed vertebrates.
Introduction
Immunity to infection is mediated by two general systems:
acquired (or adaptive) and innate (or natural). Acquired
immunity arose early in vertebrate evolution, between the
divergence of cyclostomes (lampreys) and cartilaginous fish
(sharks). Genes encoding several pivotal molecules in acquired
immunity, including immunoglobulin (Ig), T-cell receptor
(TCR), major histocompatibility complex (MHC) class I and
II, and recombination-activating gene (RAG), have been
identified only in sharks and higher vertebrates. The innate
immune system is an evolutionarily ancient form, and it is
crucial for the first line of defense before the acquired immune
Immunological Reviews 2004
Vol. 198: 185–202
Printed in Denmark. All rights reserved
Copyright ß Blackwell Munksgaard 2004
Immunological Reviews
0105-2896
185
system comes into play (1). Innate immunity was formerly
thought to be a non-specific immune response characterized
by phagocytosis. However, innate immunity has considerable
specificity and is capable of discriminating between pathogens
and self, as proposed in the concept of pattern recognition
receptors of host. These receptors recognize conserved pathogen-
associated molecular patterns (PAMPs) shared by large group of
microorganisms, thereby successfully defending invertebrates and
vertebrates against infection (2).
Complement was first described in the 1890s as a heat-labile
protein in serum that ‘complemented’ heat-stable antibodies
in the killing of bacteria. Fifty years later, it was proposed that
complement could be activated by bacterial surfaces through
an antibody-independent pathway, the alternative pathway
that was not easily accepted at that time. Recently, the third
pathway, the lectin pathway, was found (3). The complement
system, which consists of three activation pathways, is
engaged in both acquired and innate immunity (4–6). The
classical pathway is activated by antibody–antigen complexes
and is a major effector of antibody-mediated immunity. The
other two, the lectin and alternative, pathways function in
innate immune defense. The lectin pathway involves carbohy-
drate recognition by mannose-binding lectins (MBLs) and
ficolins (7–9) and the subsequent activation of associated
unique enzymes, MBL-associated serine proteases (MASPs)
(10, 11). The alternative pathway does not involve specific
recognition molecules.
In animals, lectins (carbohydrate-binding proteins) serve as
weapons against pathogens by aggregating and opsonizing
them. These are primitive strategies of innate immunity
found in both invertebrates and vertebrates. The evolutionary
pressure, however, has afforded lectins a more powerful
ability to eliminate pathogens from the host. This is evidenced
by a link between lectins and the complement system. One of
the outstanding advances in recent complement research is
the discovery of the lectin pathway. In the lectin pathway,
MBL and ficolin act as the recognition molecules and activate
complement in association with MASPs, C1r/C1s-like serine
proteases that are capable of cleaving the complement com-
ponents C4, C2, and C3. Recent identification of several
components of the lectin pathway from ascidians reveals that
the primitive complement system is one of the most highly
organized innate immune systems in invertebrates. Because the
lectin–protease complex is structurally and functionally
equivalent to C1, the first component of the classical com-
plement pathway, the presence of an ancient lectin-based
complement system suggests that the lectin pathway evolved
to the classical pathway, as shown in Fig. 1. Therefore, it is
possible that the complement system plays a pivotal role in
innate immunity before the evolution of an adaptive
immune system in vertebrates, which means that the comple-
ment system links innate immunity to acquired immunity
(4, 12, 13).
In this review, we focus on the lectin pathway and its role in
innate immunity defense. We have isolated several lectins
homologous to MBLs and ficolins and also several MASPs
in invertebrates and lower vertebrates, and we discuss the
molecular evolution of these molecules.
Activation of the complement system
Once the complement system is activated, a chain reaction
that involves proteolysis and assembly occurs, which results
in cleavage of the third component of complement (C3).
The cascade that leads to the cleavage of C3 is called the
activation pathway. It is followed by the lytic pathway,
during which the membrane attack complex (MAC) is
formed. As mentioned above, there are three activation
pathways: the classical, lectin, and alternative pathways.
Activation of the complement system promotes the three
main biological activities: opsonization of pathogens, che-
motaxis and activation of leukocytes, and direct killing of
pathogens. Recently, accumulating evidence shows that the
complement system acts as an adjuvant, enhancing and
directing the adaptive immune response, and also functions
to dispose of apoptotic cells (5, 6).
As illustrated in Fig. 2, the classical activation pathway is
triggered by binding of the recognition subcomponent C1q
to the antibody that in turn is translated into activation of the
serine proteases C1r and C1s (14). Likewise, binding of the
lectin pathway recognition molecules (i.e. MBLs or ficolins) to
microbial carbohydrates activates the MASPs, MASP-1 (15–
17), MASP-2 (18), and MASP-3 (19), which are specific for
the lectin pathway (20). MASP-2 is the enzyme component
that, like C1s in the classical pathway, cleaves the complement
components C4 and C2 to form the C3 convertase C4b2a,
common for activation of both the lectin and the classical
pathways. Alternatively, MASP-1 is capable of cleaving C3
directly (19, 21, 22), resulting in activation of the alternative
pathway (21). The alternative pathway is initiated by the
covalent binding of a small amount of C3 to hydroxyl or
amine groups on cell surface molecules of microorganisms
and does not involve specific recognition molecules. This
pathway also functions to amplify C3 activation (amplification
loop) (5, 6).
Fujita et al Á Evolution of the lectin-complement pathway
186 Immunological Reviews 198/2004
Overall structure of recognition molecules in
complement activation
In the classical pathway, C1q, a subcomponent of the first
component of complement (C1), recognizes the Fc region of
immunoglobulins that are bound to antigen. C1q has an
unusual modular structure consisting of six globular heads,
each connected by a strand to a central fibril-like region,
composed of collagen-like triple-helical structure (23). The
overall structure of C1q is similar to that of two types of
lectins, MBLs and ficolins (9).
MBLs and ficolins are oligomers of structural subunits, each
of which is composed of three identical 32-kDa and 35-kDa
polypeptide chains, respectively, whereas that of C1q is com-
posed of three different chains. As shown in Fig. 3A, one
polypeptide chain contains an NH
3
-terminal region rich in
cysteine, a collagen-like domain consisting of tandem repeats
of Gly-X-Y triplet sequences (where X and Y represent any
amino acid), a neck region, and a COOH-terminal region. In a
COOH-terminal domain, MBLs, ficolins, and C1q have a car-
bohydrate recognition domain (CRD), a fibrinogen-like
domain, and a globular head (gC1q) region, respectively.
Through the collagen-like domain, these collagenous proteins
are associated with serine proteases, and in the case of MBLs
and ficolins, through the COOH-terminal domain, they bind
carbohydrates. Trimerization of the polypeptide chain is
achieved through the collagenous triple-helical region,
thereby resulting in the formation of one subunit (Fig. 3B).
The NH
3
-terminal cysteine-rich region is involved in the
covalent interaction between the three polypeptide chains of
the subunit and is also responsible for the covalent binding of
several subunits into the oligomeric structure. In short, three
polypeptides fold together to form the structural subunit, and
three to six of these subunits join to form a mature protein,
which has an apparent molecular mass of approximately
Sea urchin Lamprey Carp Snake Mammals
Shark Frog Ascidian Chicken
Acquired immunity
Classical pathway
C3 and factor B
Lectin pathway
MBL
Ficolins
MASPs
MASP-1
MASP-2
MASP-3
Fig. 1. The complement system from an evolutionary perspective.
Acquired immunity was established at an early stage in the evolution of
the jawed vertebrates, illustrated with a shark in this figure. Evolutionary
studies have revealed that cartilaginous fish (shark) and higher vertebrates
possess a well-developed complement system with all three activation
pathways, although not all components of each pathway have been
identified. C3, the central component of the complement system, and C2/
factor B-like sequence have been identified in a marine invertebrate, the
sea urchin. In ascidian, several pivotal molecules, such as glucose-binding
lectin (GBL) homologous to mannose-binding lectin (MBL), ficolins,
MBL-associated serine proteases (MASPs), C3, and C3 receptor, have been
identified. The development of each type of MASP is also shown. This
figure was reprinted by permission from Nature Reviews Immunology (4)
[copyright (2002) Macmillan Magazines Ltd] with modification.
Fujita et al Á Evolution of the lectin-complement pathway
Immunological Reviews 198/2004 187
300–650 kDa. Overall structures of these molecules are shown
in Fig. 3C. Human MBL exists in several oligomeric forms, such
as trimers, tetramers, and pentamers, in contrast with the
hexametric form of C1q.
Structure, function, and phylogeny of MBL
Mannose-binding lectin is a C-type lectin that plays a crucial
role in the first line of host defense (24–27). The importance
of this molecule is underlined by a number of clinical studies
linking MBL deficiency with increased susceptibility to a vari-
ety of infectious diseases (28–31). MBL belongs to the collec-
tin family of proteins that consists of a collagen-like domain
and a CRD (32). Through its CRD, MBL binds carbohydrates
with 3- and 4-hydroxyl groups in the pyranose ring in the
presence of Ca

through the five conserved residues (Glu184,
Asn186, Glu191, Asn205, and Asp206) in the MBL CRD (33,
34). Prominent ligands for MBL are, thus, D-mannose and
N-acetyl-D-glucosamine (GlcNAc), whereas carbohydrates
that do not fit this steric requirement, i.e. D-galactose and sialic
acid that usually decorate the mammalian glycoprotein, have
an undetectable affinity for MBL. This steric selectivity of MBL
Fig. 2. Activation of the classical, lectin, and alternative pathways. The
classical pathway is initiated by the binding of the C1 complex to antibodies
bound to antigen on the surface of bacteria. The C1 complex consists of C1q
and two molecules of C1r and C1s. The binding of the recognition
subcomponent C1q to the Fc portion of immunoglobulins results in
autoactivation of the serine protease C1r. C1r then cleaves and activates C1s,
the enzyme that translates the activation of the C1 complex into complement
activation through the cleavage of C4 and C2 to form a C4bC2a enzyme
complex. C4bC2a acts as a C3 convertase and cleaves C3, resulting in
products that bind to and result in the destruction of invading bacteria. The
lectin pathway is initiated by binding of either of MBL or ficolin, associated
with MASP-1, MASP-2, MASP-3, and sMAP to an array of carbohydrate
groups on the surface of a bacterial cell. As with C1s, MASP-2 is responsible
for the C4 and C2 activation, leading to generation of the same C3 convertase
as the classical pathway. MASP-1 is able to cleave C3 directly. The alternative
pathway is initiated by the low-grade activation of C3 by hydrolyzed C3 [C3
(H
2
O)] and activated factor B (Bb). The activated C3b binds factor B (B)
which is cleaved into Bb by factor D (D) to form the alternative pathway C3
convertase, C3bBb. Once C3b is attached to the surface, the amplification
loop consisting of the alternative pathway components is activated and the
C3 convertase enzymes cleave many molecules of C3 to C3b, which bind
covalently around the site of complement activation. This figure was
reprinted by permission from Nature Reviews Immunology (4) [copyright
(2002) Macmillan Magazines Ltd].
Fujita et al Á Evolution of the lectin-complement pathway
188 Immunological Reviews 198/2004
along with differences in the spatial organization of the ligands
allows for the specific recognition of carbohydrates on patho-
genic microorganisms, including bacteria, fungi, parasitic
protozoans, and viruses, but avoids recognition of self (9).
Human MBL displays only one form (35), while MBL occurs
in two distinct forms, MBL-A and MBL-C in rodents (36) and
other animals, such as the rhesus monkey (37). Human MBL
shares structural and functional properties with both forms of
rodent MBL. Mouse MBL-A and MBL-C were purified from
mouse serum, and both forms of MBL were able to activate
the lectin-complement pathway (38). Although there is a
tendency in the literature to regard MBL-A as the closest
homolog of human MBL, the phylogenetic analysis indicates
that human MBL originated from a common MBL-C ancestor,
and a pseudogene corresponding to the closest human MBL-A
gene has been characterized (39). As shown in Fig. 4A, the
phylogenetic tree clearly shows that mammal MBLs form a
tight cluster; therefore, the gene duplication leading to two
different MBL forms in mammals occurred after the split from
birds and reptiles.
As mentioned above, sequence analysis of CRDs in compari-
son with monosaccharide specificity revealed that Glu185 and
Asn187 (EPN type) are highly conserved in CRDs that bind
mannose/glucose. Galactose-binding CRDs have Gln185 and
A
B
C
Fig. 3. Domain and oligomeric structure of
mannose-binding lectin (MBL) and ficolin.
(A) MBL and ficolin are both oligomers of
structural subunits, each composed of three
identical 32 kDa and 35 kDa polypeptides,
respectively. Each polypeptide contains an
amino-terminal cysteine-rich region, a collagen-
like domain consisting of tandem repeats of Gly-
X-Y triplet sequences (where X and Y represent
any amino acid), a neck region, and a COOH-
terminal CRD in MBL, and fibrinogen-like
domain in ficolin. (B) One subunit composed of
three polypeptides. (C) MBL forms several sizes
of oligomers, and the hexametric form is shown
in this figure. The tetrameric form of L-ficolin
shown here, and the hexameric form of H-ficolin
are proposed based on electron microscopy.
Fujita et al Á Evolution of the lectin-complement pathway
Immunological Reviews 198/2004 189
Asp187 (QPD type) at these critical positions (33), and site-
directed mutagenesis has shown that mannose specificity can
be changed to galactose specificity by replacing Glu185 and
Asn187 (EPN type) with Gln185 and Asp187 (QPD type),
respectively (34). In addition to mammalian and chicken
MBLs, several lectins in bony fish were characterized (40,
41). The deduced primary structure of these lectins in carp,
zebrafish, and goldfish indicates selectivity for galactose, hav-
ing QPD type. Recently, another carp MBL with specificity
for mannose (EPN type) was purified (M Nakao, personal
communication). To avoid the confusion, we used galactose-
binding lectin (galBL) for QPD type. The amino acid sequences
of several animals are show in Fig. 4B.
We recently purified and cloned MBL-like lectin from a
urochordate, the solitary ascidian Halocynthia roretzi (42). The
purified lectin binds specifically to glucose but not to mannose
or GlcNAc, and it was designated glucose-binding lectin
(GBL). Sequence analysis of GBL reveals that the COOH-
terminal half of the ascidian lectin contains a CRD that is
homologous to C-type lectin, but a collagen-like domain was
replaced by the other sequence that has an a-helix structure
similar to the configuration of Gly-X-Y repeats. As mentioned
above, MBL is reported to bind carbohydrates with 3- and
4-hydroxyl groups in the pyranose ring through the five
conserved residues in the MBL CRD. These residues are
completely conserved in GBL and the other species’ MBLs,
except for the three bony fish galBLs (Fig. 4B). The structural
difference between mannose or GlcNAc and glucose is at the
site of the 2-hydroxyl group of the pyranose ring. Therefore, it
is possible that residues other than the five conserved ones in
GBL may be involved in recognizing the 2-hydroxyl group of
glucose.
A
B
Fig. 4. Phylogenetic trees and partial sequence of
carbohydrate recognition domains (CRDs) of
mannose-binding lectin (MBL). (A). The tree was
constructed based on the alignments of the sequences
of several MBL CRDs by the neighbor-joining method
(A), and the partial sequences of CRGs are shown (B).
Numbers on branches are bootstrap percentages
supporting a given partitioning. In zebrafish,
goldfish, and carp, a homolog of MBL was reported.
Because another carp MBL was identified recently, in
this figure, we used CaGalBL for the previously
reported MBL and CaMBL for newly identified lectin
similar to MBL. AsGBL, ascidian GBL; BovMBL,
bovine MBL; HuMBL, human MBL; MonMBL monkey
MBL; MuMBL, mouse MBL; ChMBL, chicken MBL.
Fujita et al Á Evolution of the lectin-complement pathway
190 Immunological Reviews 198/2004
The above findings raise the possibility that GBL evolved
early as a prototype of MBL. During evolution, GBL might have
acquired the broad binding specificity for carbohydrates and
the collagen structure characteristic of MBL. To prove this
hypothesis, we also purified the lectin associated with MASP
in lamprey, one of the most primitive vertebrates. The
deduced amino acid sequence shows that this lectin has a
collagenous region and a typical EPN-type CRD (Fig. 4B).
Therefore, in conjunction with the phylogenetic analysis, it
seems likely that the lamprey lectin is an ortholog of the
mammalian MBL (manuscript in preparation).
Ficolin family
Ficolins, like MBLs, are a group of proteins that contain a
collagen-like stem structure. Unlike MBLs, however, they
have a fibrinogen-like domain that is responsible for carbo-
hydrate binding (Fig. 3). They were originally identified as
transforming growth factor (TGF)-b1-binding proteins on
porcine uterus membranes (43). As shown in Table 1, ficolins
have been identified in vertebrates including human (44–49),
rodent (50), pig, hedgehog (51), and Xenopus (52) and in the
invertebrate ascidian (53). Serum ficolins from human,
mouse, pig, Xenopus and ascidian are lectins with a common
binding specificity for GlcNAc. In human serum, two types of
ficolin, named L-ficolin (ficolin L) and H-ficolin (Hakata
antigen, ficolin-H), have been identified, and both of them
have lectin activity. Another ficolin, termed M-ficolin or
P35-related protein, whose mRNA is found in leukocytes
and lung, is not considered to be a serum protein (46, 47).
L-ficolin acts as an opsonin and enhances phagocytosis of
Salmonella typhimurium by neutrophils (54). Recently, we have
reported that both L-ficolin and H-ficolin activate the lectin-
complement pathway, in association with MASPs (55, 56).
The association of L-ficolin with MASPs was confirmed by
recombinant MASP-1, MASP-2, and their fragments (57). It
is not known whether M-ficolin activates the lectin pathway.
The functions of the fibrinogen-like domains of the proteins
are not fully understood. However, accumulating evidence has
shown that one of the important functions of the fibrinogen-
like domain is to bind carbohydrates, as is seen in ficolins.
A fibrinogen-like domain of human L-ficolin shows similarity
to the COOH-terminal halves of fibrinogen-b and -g chains.
The lectins of horseshoe crab, Tachypleus tridentatus, tachylectin
5A and 5B, which have similar fibrinogen-like structures but
lack collagen-like domains, also recognize the N-acetyl group.
The structure of tachylectin 5A in complex with GlcNAc has
been solved by X-ray analysis, and the binding mechanism has
been clarified (58). These results show that the fibrinogen-like
domain of several lectins has a similar function as the CRD of
C-type lectin, and therefore, the ficolins and related lectins also
function as the pattern recognition receptors.
Human ficolins
L-ficolin is a multimeric protein consisting of 35-kDa subunits
(45). As mentioned above, the subunit of L-ficolin consists of
four domains: an NH
3
-terminal region of 13 amino acids
Table 1. Properties of the ficolin family
Ficolins Tissue expression Protein carbohydrate Binding substance (the other)
Human
L-ficolin Liver Serum GlcNAc Elastin, corticosteroid
H-ficolin (Hakata) Liver and lung Serum GlcNAc/GalNAc
M-ficolin Monocyte and lung Recombinant GlcNAc
Mouse
Ficolin-A Liver and spleen Serum GlcNAc Elastin
Ficolin-B Bone marrow and spleen
Pig
Ficolin-a Liver and lung Serum GlcNAc TGF-b1
Ficolin-b Bone marrow
Hedgehog
Erinacin Muscle Metalloprotease
Xenopus
XeFCN1 Liver, spleen, and heart Serum GlcNAc/GalNAc
XeFCN2–4 Leukocyte, spleen, and heart
XeFCN3
Ascidian
AsFCN1, 2 Hepatopancreas Plasma GlcNAc
AsFCN3 Hepatopancreas Plasma GlcNAc
AsFCN4 Hepatopancreas
TGF, transforming growth factor.
Fujita et al Á Evolution of the lectin-complement pathway
Immunological Reviews 198/2004 191
having a cysteine residue, a collagen-like sequence with two
Gly-X-Y triplets followed by a gap of six amino acid residues
and 15 Gly-X-Y repeats, a short segment (neck domain) of
nine amino acids, and a COOH-terminus of 209 amino acids
constituting a fibrinogen-like domain. The oligomeric struc-
ture of L-ficolin is formed presumably by crosslinking of three
subunits via disulfide bridges in the NH
3
-terminal half. The
fibrinogen-like domain forms a globular structure like a CRD,
and the overall structure of L-ficolin looks like a bouquet
and a tetramer consisting of four triple helices composed of
12 subunits (Fig. 3). The L-ficolin gene contains eight exons.
The first exon encodes the 5
0
UT, signal peptide, and nine
NH
3
-terminal amino acids. The second and third exons encode
the collagen-like domain. The fourth exon encodes the neck
domain. The fifth to seventh exons encode the upstream
portion of the fibrinogen-like domain. The last exon encodes
the remaining fibrinogen-like domain and 3
0
UT (47).
L-ficolin, like MBL, has a lectin activity for GlcNAc. How-
ever, its binding specificity differs from MBL, in that L-ficolin
binds to the GlcNAc residue next to galactose at the non-
reducing terminal of the complex-type oligosaccharide and
that it does not bind to mannose. L-ficolin binds to an Ra
chemotype strain (TV119) of S. typhimurium whose polysac-
charide has a GlcNAc residue at the non-reducing termini
and also binds to Escherichia coli (59). The fibrinogen-like
domain is responsible for the GlcNAc-binding activity (60).
H-ficolin was first identified as a serum antigen recognized
by an autoantibody present in patients with systemic lupus
erythematosus (SLE) (7). The pathophysiological relevance of
the presence of autoantibody to H-ficolin in patients with SLE
remains unknown. H-ficolin forms oligomers consisting of
34-kDa subunits linked by disulfide bonds, like L-ficolin,
and shows more than 10 ladder bands on sodium dodecyl
sulfate polyacrylamide gel electrophoresis under non-reducing
conditions (61). A hexameric form of H-ficolin is proposed,
based on electron microscopy. Recent cDNA cloning revealed
that H-ficolin belongs to the ficolin family (49), because it
consists of an NH
3
-terminal region of 24 amino acids, a
collagen-like domain with 11 Gly-X-Y repeats, a neck domain
of 12 amino acids, and a fibrinogen-like domain of 207 amino
acids. H-ficolin mRNA is found in liver and lung. In the liver,
H-ficolin is produced by bile duct epithelial cells and hepato-
cytes and is also secreted into the bile duct (62). In the lung,
H-ficolin is produced by ciliated bronchial epithelial cells and
type II alveolar epithelial cells and is secreted into the bronchus
and alveolus. In addition, we recently reported that a glioma
cell line produced H-ficolin (63). H-ficolin is a lectin and
binds GlcNAc and GalNAc but not mannose and lactose.
It agglutinates human erythrocytes coated with lipopolysac-
charides (LPS) derived from S. typhimurium, S. minnesota, and E. coli
(O111). The agglutination of erythrocytes coated with LPS
from S. typhimurium is inhibited by GlcNAc, GalNAc, and
fucose, indicating that the binding of H-ficolin to LPS is
mediated by its lectin activity.
M-ficolin, the third type of human ficolin, is not considered
to be serum lectin, and its mRNA is expressed in monocytes,
lung, and spleen (46, 47). The exon organization of M-ficolin
resembles that of L-ficolin (47). However, the deduced amino
acid sequence of M-ficolin has an extra exon encoding an
additional segment of four Gly-X-Y repeats. M-ficolin protein
was reported to express on the surfaces of peripheral blood
monocytes and promonocytic U937 cells. It has been demon-
strated that the recombinant fibrinogen-like domain of
M-ficolin had an affinity for GlcNAc and that antibody against
the recombinant protein inhibited phagocytosis of E. coli by
U937 cells (54). These results suggest that M-ficolin might be
a lectin and play a role in innate immunity by acting as a
phagocytic receptor for pathogens.
Ficolins in non-human species
Ficolins were first discovered as a TGF-b1-binding protein on
the uterus membranes in pigs, and cDNAs encoding two types
of ficolin, named ficolin a and ficolin b that have 83%
identity have been reported (43). However, the physiological
importance of the interaction of porcine ficolins with TGF-b
remains unsolved. Ohashi and Erickson (64) reported that the
expression of mRNA of ficolin-a is high in lung, liver, and
bone marrow and is low in uterus, suggesting that ficolin-a
plays important roles in tissues other than uterus. On the other
hand, ficolin-b messages are slightly expressed in bone
marrow and are not expressed in uterus. On a GlcNAc affinity
column, two types of protein with different molecular sizes
were purified from porcine plasma (65). These GlcNAc-
binding lectins, which are named little ficolin and big ficolin
and which are reactive with anti-ficolin-a, were eluted from
the GlcNAc column with 0.15 M GlcNAc and 0.8 M GlcNAc,
respectively. Little ficolin is a tetramer with 12 subunits, while
big ficolin consists of 24 subunits with a dimer of little ficolin.
In mice, two types of ficolins, ficolin-A and ficolin-B, have
been identified. Messages of ficolin-A are highly expressed in
liver and spleen. Ficolin-A, present in plasma, has binding
activities to GlcNAc and elastin (49). Its structure resembles
human L-ficolin, in that it is a tetramer with 12 subunits.
Ficolin-B is expressed in bone marrow and spleen and shows
60% identity to ficolin-A at the protein level (64). In
Fujita et al Á Evolution of the lectin-complement pathway
192 Immunological Reviews 198/2004
addition, Omori-Satoh and coworkers (51) reported that
erinacin, an anti-hemorrhagic factor from muscle extracts of
the European hedgehog (Erinaceus europaeus), is a multimeric
protein with molecular weight of approximately 100 kDa
and consists of two types of subunits in a ratio of 1 : 2 having
structural characteristics of ficolins. They also demonstrated
that erinacin inhibits metalloprotease in the venom of Bothrops
jararaca.
Recently, we cloned four cDNAs from Xenopus laevis (52) and
from the solitary ascidian H. roretzi (53), termed Xenopus ficolin
1–4 (XeFCN1–4) and ascidian ficolin1–4 (AsFCN1–4),
respectively. The deduced amino acid sequences of these
ficolins revealed the conserved collagen- and fibrinogen-like
domains. The fibrinogen-like domains of XeFCNs and AsFCNs
show 47–62% identity with mammalian ficolins. Northern
blots showed that XeFCN1 was expressed mainly in liver,
spleen, and heart, and XeFCN2 and XeFCN4 was expressed
mainly in peripheral blood leukocytes, lung, and spleen.
XeFCN1, identified as a serum lectin, has a binding activity
for GlcNAc and GalNAc. In the ascidian, two types of GlcNAc-
binding lectin were purified from the body fluids and cloned
from the hepatopancreas cDNA (AsFCN1/2 and AsFCN3). In
addition to these ficolins, cDNAs encoding another ficolin
have been cloned from the hepatopancreas (AsFCN4). It is of
particular interest to note that when compared with mamma-
lian ficolins, all AsFCNs and XeFCN3 contain short collagen-
like domains with five and seven Gly-X-Y repeats, respectively,
and they have long segments between the collagen-like
domain and the fibrinogen-like domain.
Many proteins that possess a fibrinogen-like domain but
lack a collagen-like domain have been reported. Tenascin
(66) and microfibril-associated glycoprotein 4 (MFAP4)
(67), which are involved in cell adhesion, are representative
examples. The functions of the fibrinogen-like domain of the
proteins are not fully understood. However, accumulating
evidence has shown that one of the important functions of
the fibrinogen-like domain is to bind carbohydrates, as is
seen in ficolins. The lectins recognizing the acetyl group
in hemolymph plasma of the horseshoe crab, named tachy-
lectins 5A and 5B, consist of an NH
2
-terminal Cys-containing
segment and a COOH-terminal fibrinogen-like domain with
the highest sequence identity to that of ficolins. Tachylectin
5A agglutinated Gram-negative and Gram-positive bacteria.
It also enhanced the anti-microbial activity of a horseshoe
crab-derived big defensin. These findings indicate that
tachylectin 5A, having a fibrinogen-like domain, recognizes
microorganisms as a lectin and plays a role in innate
immunity.
Phylogeny of the ficolin family
The phylogenetic relationships among the ficolin family and
the related proteins were analyzed by neighbor-joining trees
constructed based on the sequences of the entire amino acid
and of the fibrinogen-like domains (Fig. 5). In the tree of the
entire amino acid sequences, each of four Xenopus and ascidian
ficolins formed a respective tight cluster, supported by high
bootstrap percentages (Fig. 5A). Similarly, each group of fico-
lins in the same species, such as human L- and M-ficolins and
pig ficolins a and b independently formed monophyletic
clusters in this tree. These results suggest that each ficolin
might have diverged after the emergence of the respective
lineage by gene duplication. Human H-ficolin originated
from a root of the mammalian/amphibian ficolins, indicating
that it has an ancient origin going back to an evolutionary
stage prior to the emergence of Xenopus (Amphibia). In the tree
of the sequences of the fibrinogen-like domains, mammalian
ficolins and XeFCNs had a very similar branching to that in the
tree of the entire sequence (Fig. 5B), in which human H-ficolin
originated from the root of this branch, again suggesting its
ancient origin. The ascidian ficolins have a shorter collagen-
like domain (five Gly-X-Y repeats), when compared with those
of mammalian ficolins (11–19 repeats). This feature is similar
to XeFCN3, which has a collagen-like domain as short as seven
Gly-X-Y repeats, suggesting that AsFCNs might be a prototype
of the ficolin family and that during evolution they acquired
the long collagen-like domain.
Proteins with the fibrinogen-like domains, such as tenas-
cins, angiopoietins, and horseshoe crab tachylectins, seem to
be closely related to the ficolin family, although these families
are different in their entire domain structures. As shown in
Fig. 5B, the amino acid sequences of the fibrinogen-like
domains formed a large cluster, independent of the primary
fibrinogens. This finding suggests that all of the fibrinogen-
like domains might have evolved from a common ancestor
that was derived from the authentic fibrinogen. Among the
fibrinogen-like domains, those of the tenascin family seem to
be the closest to those of the ficolin family, although the
domain structure of tenascins is quite different from that of
ficolins. This finding suggests that these families evolved by
distinct exon shuffling, using a common ancestral fibrinogen-
like sequence. Interestingly, the fibrinogen-like domains of
tachylectins 5A and 5B, which are nonself-recognizing lectins,
isolated from the horseshoe crab (68), are also related to those
of the ficolin family. The clustering of ficolins, tenascins, and
tachylectins is supported by a relatively high bootstrap percent-
age of 69%. Recently, the crystal structure of tachylectin 5A
Fujita et al Á Evolution of the lectin-complement pathway
Immunological Reviews 198/2004 193
revealed that the fibrinogen-like structure shares not only a
common fold but also has functional sites related to a g-chain
of mammalian fibrinogen (58). This report could provide evi-
dence for the common origin of the innate immunity and blood
coagulation systems.
MBL-associated serine-protease
MASPs, a new member of the serine protease superfamily, are
proteolytic enzymes responsible for activation of the lectin
pathway (11). MBLs and ficolins have been found associated
with MASP-1 (15–17), MASP-2 (18), and MASP-3 (19), and a
non-protease, small MBL-associated proteins (sMAP or
MAP19, truncated form of MASP-2) (69, 70). The overall
structure of MASPs resembles that of two proteolytic compon-
ents of the first complement in the classical pathway, C1r and
C1s. The MASP family members consist of six domains, such as
two C1r/C1s/Uegf/bone morphogenetic protein 1 (CUB), an
epidermal growth factor (EGF)-like protein, two complement
control proteins (CCPs) or short consensus repeats (SCRs), and
a serine protease domain (Fig. 6).
When MBL and ficolins bind to carbohydrates on the surface
of microbes, the proenzyme form of MASP is cleaved between
the second CCP and protease domain, resulting in the active
form consisting of two polypeptides, called heavy (H)- and
light (L)-chains or A and B chains, thus acquiring proteolytic
A
B
Fig. 5. Phylogenetic trees of ficolins, fibrinogen-
like domain-bearing proteins, and fibrinogens.
The tree was constructed based on the alignments of
the entire sequences of the ficolin family (A) and the
sequences of the fibrinogen-like domains (B).
Numbers on branches are bootstrap percentages
supporting a given partitioning. HFCNL/P35,
Human L-ficolin; HFCNM/P35r, human M-ficolin;
HFCNH/Hakata Ag, human H-ficolin; PoFCN-a,
porcine ficolin-a; PoFCN-b, porcine ficolin-b;
MFCNB, mouse ficolin B; MFCNA, mouse ficolin A;
RFCNA, rat ficolin A; XeFCN1–4, Xenopus ficolin 1–4;
AsFCN1–4, ascidian ficolin 1–4; PeFIB2, Petromyzon
marinus fibrinogen a-2; CFIBA, chicken fibrinogen-a;
HFIBA, human fibrinogen-a; RFIBA, rat fibrinogen-
a; CFIBB, chicken fibrinogen-b; HFIBB, human
fibrinogen-b; RFIBB, rat fibrinogen-b; BFIBB,
bovine fibrinogen-b; BFIBG, bovine fibrinogen-g;
HFIBG, human fibrinogen-g; RFIBG, rat fibrinogen-
g; XFIBG, Xenopus fibrinogen-g; PeFIBG, Petromyzon
marinus fibrinogen-g; MFGL2, mouse fibrinogen-like
protein 2; HFGL-2, human fibrinogen-like protein-
2; HAGP-1, human angiopoietin-1; MAGP-1, mouse
angiopoietin-1; BAGP-1, bovine angiopoietin-1;
HAGP-2, human angiopoietin-2; BAGP-2, bovine
angiopoietin-2; MAGP-2, mouse angiopoietin-2;
PaFIB A, Parastichopus parvimensis fibrinogen-like
protein A; CTN C, chicken tenascin C; HTN C,
human tenascin C; PoTN C, porcine tenascin C;
ZTN, zebrafish tenascin; HTN X, human tenascin X;
PoTN X, porcine tenascin X; HMFA-4, human
microfibril-associated glycoprotein-4; TL-5 A,
horseshoe crab tachylectin-5 A; TL-5B, tachylectin-5B.
Fujita et al Á Evolution of the lectin-complement pathway
194 Immunological Reviews 198/2004
activities against complement components. MASP-1 cleaves C3
and C2, while MASP-2 cleaves C4 and C2 (71). These results
are confirmed by the functional analysis of recombinant
MASP-2 (72–75). The functions of MASP-3 and sMAP in the
complex remain unknown, although sMAP is associated with
MASP-1. MASP seems to be associated with MBL through its
collagenous region (76), and the activation mechanism of
MASP-2 interacted with MBL (77, 78). In addition, the case
of human MASP-2 deficiency with SLE-like syndrome was
reported, showing no activation of the lectin pathway (79).
The properties in five members of the human MASP family are
summarized in Table 2. MASP-1, MASP-2, MASP-3, and sMAP
are derived from two genes. sMAP is a truncated form of
MASP-2 (69), and MASP-3 is produced from the MASP-1/3
gene by alternative splicing (19). The MASP-1/3 gene has an
H-chain-coding region common to MASP-1 and MASP-3,
followed by tandem repeats of protease domain-coding
regions specific for MASP-3 and MASP-1. Thus, the MASP-1/3
gene is very unique in having the double protease-coding
regions among the serine protease superfamily.
Molecular evolution of the MASP family
The cDNA sequences of the members of the MASP family have
been cloned from various species of vertebrates and from two
species of invertebrates, amphioxus Branchiostoma belcheri (cepha-
lochordates), and ascidian H. roretzi (urochordates) (80–82).
Based on the primary structures and exon organization of the
genes, the protease domain of the MASP family can be divided
into two phylogenetic lineages: TCN type and AGY type. The
TCN type, including MASP-1, ascidian MASPa, and MASPb,
has of the following feature in the protease domain: the TCN
codon (where N denotes A, G, C, or T) at active-site serine, the
presence of the histidine-loop disulfide bridge, and split
exons. This feature is common in most of the chymotrypsin-
like serine-protease family. By contrast, the AGY type,
including MASP-2, MASP-3, lamprey MASP-A/B, shark
(cartilaginous fish) MASP, carp (bony fish) MASP, and C1r/
C1s, is characterized by the AGY codon (where Y denotes C or
T) at the active-site serine, the absence of histidine-loop, and a
single exon. The AGY type is very unique in having intronless
protease-coding region. From an evolutionary point of view, it
is suggested that the AGY type diverged from the TCN type
before the emergence of primitive vertebrates. From another
view for gene organization, however, the MASP gene is divided
into three types: MASP1, MASP1/3, and MASP2 including C1r/
C1s. MASP1 type was only found as the ancestral gene coding
simply H-chain and the TCN type light chain, as seen in
Fig. 6. Domain structure of the MASP family. MASPs, C1r, and C1s
consist of six domains: two C1r/C1s/Uegf/bone morphogenetic protein
1 (CUB) domains, an EGF-like domain, two CCP domains or SCRs, and a
serine protease domain. Histidine (H), aspartic acid (D), and serine (S)
residues are essential for the formation of the active center in serine
protease. Only MASP-1 has two additional cysteine residues in the light
chain, which forms a ‘histidine loop’ disulfide bridge as is found in
trypsin and chymotrypsin. On binding of MBL and ficolin to
carbohydrate on the surface of a pathogen, the proenzyme form of MASP
is cleaved between the second CCP and the protease domain, resulting
in the active form comprising of two polypeptides, heavy (H) and light
(L) chains.
Table 2. Human MASP/C1r/C1s family
MASP-1 MASP-2 MASP-3 C1r C1s
Concentration (mg/ml) 6 0.5 ? 50 50
Chromosome 3q27 1p36 3q27 12p3 12p3
Serine protease domain
Number of exon 6 1 1 1 1
Number of cysteine 7 5 5 5 5
Codon of active serine TCT AGC AGC AGT AGT
Substrate specificity
C4 – þ ? – þ
8 C2 þ þ ? – þ
C3 þ – ? – –
MASP, mannose-binding lectin-associated serine protease.
Fujita et al Á Evolution of the lectin-complement pathway
Immunological Reviews 198/2004 195
ascidian. As described above, the MASP1/3 gene has an
H-chain-coding region common to MASP-1 and MASP-3,
followed by tandem repeats of the AGY type and TCN type
protease domain specific for respective MASP-3 and MASP-1.
Therefore, it is clear that the AGY-type protease domain was
inserted upstream of the original TCN-type domain, generat-
ing an MASP-1/3-type gene. The generation of the MASP2 gene
and the C1r/C1s gene is considered to be later event, resulting
in the four genes found in human.
The origin of the MASP gene can be traced back to the
ascidian (urochordate) lineage, which has two MASP-1-type
genes. Amphioxus is one of the highly organized invertebrates
and the closest relative of vertebrates; it occupies a critical
position between lamprey and ascidian in the phylogeny.
This animal has a MASP1/3-like gene and at least two MASPs,
termed amphioxus MASP-1 and MASP-3, which may be the
orthologs of mammalian MASP-1 and MASP-3 (83). These
results suggest that a processed intronless region might have
been inserted between the regions encoding the H-chain and
TCN-type L-chain of a prototype gene (MASP-1 type). This
event should occur in the invertebrate lineage after the diver-
gence of ascidian but before the divergence of amphioxus.
Surprisingly, however, amphioxus MASP-1 has an AGC
codon for active serine with histidine-loop and slit exons,
showing the base change, and MASP-3 has an AGC codon for
active serine with histidine-loop and single exon. If these
structural features of two amphioxus MASPs are an intermedi-
ate form, it can be speculated that the conversion from the
TCN type to the AGY type occurred in at least three steps: the
base change from TCN to AGY, the loss of an intron, and then
the loss of a histidine-loop disulfide bridge. In any case, it is of
importance to note that the intronless protease region was
inserted by retrotransposition or partial gene duplication
(Fig. 7).
Lamprey Lampetra japonica (cyclostome), which is one of the
most primitive vertebrates and considered to be lacking
acquired immunity, has at least three kinds of MASPs, termed
lamprey MASP-1, MASP-A, and MASP-B (82). The structure of
lamprey MASP-1 is similar to that of ascidian MASPs, showing
the presence of MASP1-type gene in lamprey. Lamprey MASP-A
and MASP-B are closely related sequences to mammalian/
amphibian MASP-3, but their H-chain-coding regions are
Fig. 7. A model for the evolution of the MASP/C1r/C1s gene. The
origin of MASP genes can be traced back to ascidians (urochordates).
Before the emergence of amphioxus (cephalochordates), retroposition
of partially processed TCN-type MASP gene and base changes from TCN
to AGY at the active site serine generated a prototype of the
MASP-1/3-type gene. All of the amphioxus/vertebrate MASP/C1r/C1s
genes evolved from this ancestral gene by gene duplication. We propose
that TCN-type L-chain-coding region has been lost in the lamprey
lineage, generating a prototype of the MASP-2-type gene including C1r
and C1s genes.
Fujita et al Á Evolution of the lectin-complement pathway
196 Immunological Reviews 198/2004
different from that of lamprey MASP-1. We have not success-
fully cloned the MASP-1/3 type gene in lamprey. Therefore, it
is possible that in lamprey, unlike in mammals and
amphioxus, MASP-1 and MASP-3 are produced from distinct
genes. A similar question should be asked whether the
MASP-1/3 gene is present in shark (cartilaginous fish) and
carp (bony fish), because the homolog of MASP-3 was
found but that of MASP-1 was not in these species (Fig. 1).
Like mammals, such as humans and mice, we found that X.
laevis (amphibian) have two types of genes: MASP1/3 and
MASP2, which code MASP-1, MASP-2, and MASP-3 in com-
mon (83). This finding suggests a similar system of the
lectin pathway in these species.
It is likely that all the amphioxus/vertebrate MASP/C1r/C1s
genes evolved from an ancestral MASP-1/3-type gene by gene
duplication, because the amphioxus/vertebrate MASP/C1r/C1s
genes have an intronless exon encoding an AGY-type L-chain
in common. It is known that the human MASP2 gene lacks the
TCN-type L-chain-encoding region, which is replaced by an
unrelated gene (84). Thus, the absence of the TCN-type
L-chain-encoding region in some genes, such as the human
MASP2 and human C1r and C1s genes, might be explained by
the loss of a TCN-type-encoding region during evolution. It is
possible that this event occurs at early stage, when we
expected. As mentioned above, we could not find the TCN-
type L-chain-encoding region in downstream of the AGY-type
L-chain-coding region in lamprey MASP-A, showing a similar
structure of the MASP2 gene. Therefore, we propose that
lamprey MASP-A appears as the prototype of MASP2 gene in
this lineage. In other words, the prototype of MASP2 gene is a
combination of MASP-1/3 type H-chain and AGY type
L-chain, as shown in Fig. 7, because the L-chain of lamprey
MASP-A is similar to MASP-3. Our hypothesis is strongly
supported by the observation that lamprey MASP-A, associated
with lamprey MBL and lamprey C1q, is able to cleave C3, as
described below. The order of appearance of MASP2, C1r, and
C1s genes is not obvious from their gene structure alone. If our
assumption is the case, the origin of MASP-2 seems to precede
that of a C1r/C1s, although C1r/C1s-like gene is traced back at
least to bony fish lineage (85), but a definitive MASP2 gene is
traced to Xenopus. Again, it is of great interest to study the MASP
gene in shark and carp.
The phylogenetic study established that the MASP1/3 gene
has an ancient origin that can be traced back at least to the
amphioxus (cephalochordate) lineage. The origin of MASP-1
may be traced further back to the ascidian (urochordate) lin-
eage, although the view that two ascidian MASPs are the ortho-
logs of vertebrate/amphioxus MASP-1 is still controversial.
The lectin pathway seems to have developed step by step into
a sophisticated system, involving retrotransposition (or partial
gene duplication) to generate the MASP1/3 gene and gene
duplication to generate the MASP2 gene. We proposed that the
latter event occurs at an early stage, i.e. in the lamprey lineage.
The alternative processing of MASP-2 mRNA to produce the
truncated form, sMAP (69), would occur much later than this
lineage.
The primitive complement system
The complement system has a more ancient origin in evolu-
tion than acquired immunity. The central components of the
complement system, the C3 protein on which the three acti-
vation pathways converge (Fig. 2), have been identified in
jawless vertebrates, the lamprey and hagfish, as well as in
deuterostome invertebrates, ascidian, amphioxus, and sea
urchin (echinoderm). The origin of the complement system
can be traced back at least to echinoderms, because C3 and
C2/factor B-like component have been identified in sea urchin
(86–88). Sea squirts (ascidians) occupy a pivotal intermediary
position between invertebrates and vertebrates. H. roretzi is a
large solitary ascidian, native to the coastal waters of Japan.
Two lectins corresponding to mammalian MBLs and ficolins
(42, 53), two MASPs (81), C3 (89), C2/factor (90), and C3
receptor (91) have been identified in the ascidians (Fig. 1).
Therefore, the primitive complement system seems to have
been established in the deuterostome lineage; the classical
pathway of activation was then acquired in the jawed verte-
brate lineage, at the time acquired immunity arose (4, 90).
The alternative pathway has been regarded as the original
complement pathway, because it does not require the partici-
pation of the adaptive immune system. However, accumulat-
ing evidence indicates that complement originates as a lectin-
based opsonized system. As described above, MBL-like 36-kDa
lectin, GBL, was purified as a major protein from the ascidian
body fluid. Sequence analysis of GBL cDNA revealed that the
COOH-terminal half of the ascidian lectin contains a carbohy-
drate recognition domain that is homologous to C-type lectin,
but a collagen-like domain was replaced by the other
sequence. Although the structure and binding specificity of
GBL is different from mammalian MBL, GBL associated with
two ascidian MASPs, and GBL–MASP complexes activate
ascidian C3, like human MBL–MASP-1 complexes (42). In
addition, we have isolated ascidian ficolins as GlcNAc-binding
lectins that have characteristic features of mammalian ficolins
(53). Although it is presently unknown whether these ficolins
associated with MASPs and activate complement, these
Fujita et al Á Evolution of the lectin-complement pathway
Immunological Reviews 198/2004 197
observations indicate that ficolins, as well as GBL, probably act
as the recognition molecules of the primitive ascidian comple-
ment system in a similar manner to the mammalian lectin
pathway. Although C3 and C2/factor B were identified in sea
urchin and ascidian, the sophisticated recognition mechanism
of the alternative pathway to recognize a broad spectrum of
pathogens seems to have developed more recently. However,
the possibility of a simple role of C2/factor B-like protein, as
an amplifier of C3 deposition, cannot be excluded completely.
C3 was identified as the principal opsonic factor in ascidian
plasma (89), and a C3 receptor was also identified on ascidian
hemocytes as the homolog of mammalian complement
receptor type 3 or 4 (CR3 or CR4) (91). Usually, 20–30%
of ascidian leukocytes (hemocytes) ingested at least one non-
coated yeast cell, and in the case of yeast treated with ascidian
plasma, 40–60%of hemocytes ingested more than one yeast.
As reported previously, this opsonic activity is derived from C3
(89). In the experiments shown in Fig. 8, we found that the
opsonic effect of ascidian plasma was eliminated in GBL- and
C3-depleted plasma. When C3 binding is analyzed by flow
cytometry, it is clear that the degree of phagocytosis is depend-
ent on C3 binding to yeast. In addition, antibody against C3
receptor completely inhibited enhancement of phagocytosis of
yeast by plasma (91). These results indicate that complement-
mediated phagocytosis is a central part of the physiological
function of this primitive complement system. Furthermore,
yeast treated with purified GBL–MASP complex and C3
enhanced the phagocytosis by hemocytes (42). These observa-
tions strongly suggest that lectin–protease (MASP) complex,
C3, and its receptor may have developed as the minimal
ancestral components of the primordial complement system
in the ascidian lineage, as shown in Fig. 9. Therefore, the
ascidian complement system, which has similar activating
and functional mechanisms to those of mammals, remains
unchanged since its appearance at least 600 million years
ago, well ahead of the emergence of adaptive immunity.
The classical and lytic pathways of the complement system
seem to have emerged at the cartilaginous fish stage, coinci-
dent with the emergence of adaptive immunity (90). The
complement system of lamprey, the most primitive vertebrate,
also lacks the classical and lytic pathway, suggesting that
lamprey has a similar complement system to the ascidian.
Recently, we purified two lectins from lamprey serum using
GlcNAc-agarose: one was eluted with mannose, and the other
with GlcNAc (manuscripts in preparation). According to
cDNA cloning, the former was identified as lamprey MBL, as
described above, and surprisingly, the latter is a homolog of
C1q. Both lectins were associated with MASP-A, a serine
protease of the MASP family, which exhibits a proteolytic
activity against lamprey C3. The deduced amino acid sequence
Fig. 8. Phagocytosis assay and binding of C3 to yeast. Yeast was
incubated with fresh ascidian plasma that had been treated with
phosphate buffered saline (PBS), ethylenediaminetetraacetic acid (EDTA),
rabbit immunoglobulin (Ig)G, rabbit anti-GBL IgG, or rabbit anti-C3 IgG,
and then with protein A-Sepharose as shown in (C). The untreated and
treated yeasts were subjected to the assay. The degree of phagocytosis of
untreated yeast was defined as 100%. The antibodies to GBL and C3
completely inhibited the opsonic effect of ascidian plasma (A). When
analyzing C3 binding by flow cytometry (B), it is clear that the degree of
phagocytosis is dependent on C3 binding to yeast, showing a lectin-based
opsonic complement system in ascidians.
Fujita et al Á Evolution of the lectin-complement pathway
198 Immunological Reviews 198/2004
of lamprey C1q cDNA revealed that it consists of a collagen-
like domain and antibody recognition domain, gC1q domain,
found in a variety of proteins including mammalian C1q. A
phylogenetic tree of the gC1q domains of proteins shows that
lamprey C1q and mammalian C1q form a cluster. These
observations strongly suggest that C1q may have emerged as
a lectin and functioned as an initial recognition molecule of
the complement system before the establishment of acquired
immunity, such as immunoglobulins in the cartilaginous fish.
Although the molecular composition of the lectin pathway
in cartilaginous and bony fish has not been fully clarified, the
C1r and C1s components of C1 are clearly derived from the
MASP lineage. C1q is closely related to MBL or ficolins with
the substitution of antibody recognition domains for the CRDs
or fibrinogen-like domain. From an evolutionary point of
view, the primitive lectin pathway in innate immunity appears
to have developed into the more sophisticated, multifunctional
complement system of the classical pathway through gene
duplication, to serve as an effector system of acquired immun-
ity (Fig. 9). A strong link between the innate immune systems
of invertebrates and acquired immunity in vertebrates is there-
fore established.
Conclusion
Lectins play an important role in innate immunity by
recognizing a wide range of pathogens. Two classes of
collagenous lectins, MBLs and ficolins, are very similar, in
that both are hybrid proteins containing a collagen-like
domain and a lectin domain, although their lectin domains
are quite different (Fig. 3). In humans, in addition to MBL
serum ficolins, the MASP-associated L-ficolin and H-ficolin,
Bacteria
Lectin
MASP
C3
C3 receptor
Phagocyte
C3b
Bacteria
Lectin
MASP
C3
C3 receptor
Phagocyte
C3b
Bacteria
Ab
C1
C3 receptor
Phagocyte
C3b
Bacteria
C5-C9
(Lytic pathway)
Bacteria
C5-C9
(Lytic pathway)
C4
C2
C4
C2
C3
Lectin pathway
(innate immunity)
Classical pathway
(acquired immunity)
The complement system from cartilaginous fish to mammals
Ancient lectin-based complement system
Fig. 9. Putative model of an ancient lectin-
based complement system and its evolution.
In the lectin–protease (MASP) complex, C3 and
C3 receptor are probably the minimal ancestral
components of the primordial complement
system that functioned in an opsonic manner
and appeared in the ascidian lineage. The
complement system of the lamprey (the most
primitive vertebrate) lacks the classical and lytic
pathway and so lamprey appear to have a
complement system similar to ascidians.
Therefore, the complement system developed
dramatically at an early stage of vertebrate
evolution into a sophisticated multifunctional
system. Gene duplication events seem to have
played a major role in this process, and several
sets of homologous complement components
are noted, such as MBL and C1q, MASPs and
C1r/C1s, C2 and factor B, and C4 and C3. This
figure was reprinted by permission from Nature
Reviews Immunology (4) [copyright (2002)
Macmillan Magazines Ltd].
Fujita et al Á Evolution of the lectin-complement pathway
Immunological Reviews 198/2004 199
act as the recognition molecules of the lectin pathway. This
finding expands the concept of the lectin pathway. Serum
MBLs and ficolins recognize specific pathogens and eliminate
them by acting as an opsonin, presumably through their
collagen-like domain and by activating complement, thereby
playing a role in innate immunity. In addition, ficolins present
in other organs, not just serum, may also have roles in host
defense.
The lectin pathway and the classical pathway are closely
related with respect to the structures and functions of compon-
ents involved. The classical pathway is activated by binding of
C1q, followed by activation of C1r and C1s, while the lectin
pathway is activated by recognition of carbohydrates on
pathogens via MBLs and ficolins, associated with novel serine
proteases, MASPs. MASPs share domain structure and several
functions with the classical pathway proteases C1r and C1s. The
ascidian complement system, consisting of MBL-like lectin,
ficolins, two MASPs, C3 and C3 receptor, functions in an
opsonic manner, and it constitutes a primordial complement
system corresponding to the mammalian lectin pathway. In
addition, we purified two lectins corresponding to MBL and
C1q, associated with lamprey MASP-A, which cleaved C3. From
an evolutionary point of view, it is clear that the primitive lectin
pathway in innate immunity has evolved into the
classical pathway to serve as an effector system of acquired
immunity.
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