the lectin-complement pathway – its role in innate immunity and evolution

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 Endo1

1Department of Biochemistry, Fukushima

Medical University, Fukushima, Japan, and

CREST, Japan Science and Technology Agency.2Department of Applied Biochemistry and

Institute of Glycobiology, Tokai University,

Kanagawa, Japan.

Correspondence to:

Teizo FujitaDepartment of Biochemistry

Fukushima Medical University

Fukushima 960-1295

Japan

Tel.: þ81245471146

Fax: þ81245486760

e-mail: [email protected]

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-specificimmune response characterized by phagocytosis. However, innate immun-ity has considerable specificity and is capable of discriminating betweenpathogens and self. Recognition of pathogens is mediated by a set of patternrecognition receptors, which recognize conserved pathogen-associated molecular patterns (PAMPs) shared by broad classes of micro-organisms, thereby successfully defending invertebrates and vertebratesagainst 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 lectindomain attached to collagenous region. However, they use a different lectindomain: a carbohydrate recognition domain (CRD) is responsible for MBLand a fibrinogen-like domain for ficolin. These two collagenous lectins arepattern recognition receptors, and upon recognition of the infectious agent,they trigger the activation of the lectin-complement pathway throughattached serine proteases, MBL-associated serine proteases (MASPs). Asimilar lectin-based complement system, consisting of the lectin–proteasecomplex 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 lowervertebrates, and herein we discuss the molecular evolution of these mole-cules. Based on these findings, it seems likely that the complement systemplayed a pivotal role in innate immunity before the evolution of anacquired 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 2004Vol. 198: 185–202Printed in Denmark. All rights reserved

Copyright � Blackwell Munksgaard 2004

Immunological Reviews0105-2896

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 � 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 NH3-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 NH3-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 FrogAscidian Chicken

Acquired immunityClassical pathwayC3 and factor BLectin pathwayMBLFicolinsMASPsMASP-1MASP-2MASP-3

Fig. 1. The complement system from an evolutionary perspective.Acquired immunity was established at an early stage in the evolution ofthe jawed vertebrates, illustrated with a shark in this figure. Evolutionarystudies have revealed that cartilaginous fish (shark) and higher vertebratespossess a well-developed complement system with all three activationpathways, although not all components of each pathway have beenidentified. C3, the central component of the complement system, and C2/

factor B-like sequence have been identified in a marine invertebrate, thesea urchin. In ascidian, several pivotal molecules, such as glucose-bindinglectin (GBL) homologous to mannose-binding lectin (MBL), ficolins,MBL-associated serine proteases (MASPs), C3, and C3 receptor, have beenidentified. The development of each type of MASP is also shown. Thisfigure was reprinted by permission from Nature Reviews Immunology (4)[copyright (2002) Macmillan Magazines Ltd] with modification.


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 Ca2þ 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. Theclassical pathway is initiated by the binding of the C1 complex to antibodiesbound to antigen on the surface of bacteria. The C1 complex consists of C1qand two molecules of C1r and C1s. The binding of the recognitionsubcomponent C1q to the Fc portion of immunoglobulins results inautoactivation of the serine protease C1r. C1r then cleaves and activates C1s,the enzyme that translates the activation of the C1 complex into complementactivation through the cleavage of C4 and C2 to form a C4bC2a enzymecomplex. C4bC2a acts as a C3 convertase and cleaves C3, resulting inproducts that bind to and result in the destruction of invading bacteria. Thelectin pathway is initiated by binding of either of MBL or ficolin, associatedwith 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 responsiblefor the C4 and C2 activation, leading to generation of the sameC3 convertaseas the classical pathway. MASP-1 is able to cleave C3 directly. The alternativepathway is initiated by the low-grade activation of C3 by hydrolyzed C3 [C3(H2O)] 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 C3convertase, C3bBb. Once C3b is attached to the surface, the amplificationloop consisting of the alternative pathway components is activated and theC3 convertase enzymes cleave many molecules of C3 to C3b, which bindcovalently around the site of complement activation. This figure wasreprinted by permission from Nature Reviews Immunology (4) [copyright(2002) Macmillan Magazines Ltd].



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 ofmannose-binding lectin (MBL) and ficolin.

(A) MBL and ficolin are both oligomers ofstructural subunits, each composed of threeidentical 32 kDa and 35 kDa polypeptides,respectively. Each polypeptide contains anamino-terminal cysteine-rich region, a collagen-like domain consisting of tandem repeats of Gly-X-Y triplet sequences (where X and Y representany amino acid), a neck region, and a COOH-terminal CRD in MBL, and fibrinogen-likedomain in ficolin. (B) One subunit composed ofthree polypeptides. (C) MBL forms several sizesof oligomers, and the hexametric form is shownin this figure. The tetrameric form of L-ficolinshown here, and the hexameric form of H-ficolinare proposed based on electron microscopy.



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 wasconstructed based on the alignments of the sequencesof several MBL CRDs by the neighbor-joining method(A), and the partial sequences of CRGs are shown (B).Numbers on branches are bootstrap percentagessupporting a given partitioning. In zebrafish,goldfish, and carp, a homolog of MBL was reported.Because another carp MBL was identified recently, inthis figure, we used CaGalBL for the previouslyreported MBL and CaMBL for newly identified lectinsimilar to MBL. AsGBL, ascidian GBL; BovMBL,bovine MBL; HuMBL, human MBL; MonMBL monkeyMBL; MuMBL, mouse MBL; ChMBL, chicken MBL.



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 NH3-terminal region of 13 amino acids

Table 1. Properties of the ficolin family

Ficolins Tissue expression Protein carbohydrate Binding substance (the other)

HumanL-ficolin Liver Serum GlcNAc Elastin, corticosteroidH-ficolin (Hakata) Liver and lung Serum GlcNAc/GalNAcM-ficolin Monocyte and lung Recombinant GlcNAc

MouseFicolin-A Liver and spleen Serum GlcNAc ElastinFicolin-B Bone marrow and spleen

PigFicolin-a Liver and lung Serum GlcNAc TGF-b1Ficolin-b Bone marrow

HedgehogErinacin Muscle Metalloprotease

XenopusXeFCN1 Liver, spleen, and heart Serum GlcNAc/GalNAcXeFCN2–4 Leukocyte, spleen, and heartXeFCN3

AscidianAsFCN1, 2 Hepatopancreas Plasma GlcNAcAsFCN3 Hepatopancreas Plasma GlcNAcAsFCN4 Hepatopancreas

TGF, transforming growth factor.



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 NH3-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 50UT, signal peptide, and nine

NH3-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 30UT (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 NH3-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-bremains 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-aplays 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 0.15M GlcNAc and 0.8M 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



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 NH2-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



revealed that the fibrinogen-like structure shares not only a

common fold but also has functional sites related to a g-chainof 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 ofthe entire sequences of the ficolin family (A) and thesequences of the fibrinogen-like domains (B).Numbers on branches are bootstrap percentagessupporting 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, Petromyzonmarinus fibrinogen a-2; CFIBA, chicken fibrinogen-a;HFIBA, human fibrinogen-a; RFIBA, rat fibrinogen-a; CFIBB, chicken fibrinogen-b; HFIBB, humanfibrinogen-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, Petromyzonmarinus fibrinogen-g; MFGL2, mouse fibrinogen-likeprotein 2; HFGL-2, human fibrinogen-like protein-2; HAGP-1, human angiopoietin-1; MAGP-1, mouseangiopoietin-1; BAGP-1, bovine angiopoietin-1;HAGP-2, human angiopoietin-2; BAGP-2, bovineangiopoietin-2; MAGP-2, mouse angiopoietin-2;PaFIB A, Parastichopus parvimensis fibrinogen-likeprotein 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, humanmicrofibril-associated glycoprotein-4; TL-5 A,horseshoe crab tachylectin-5 A; TL-5B, tachylectin-5B.



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 C1sconsist of six domains: two C1r/C1s/Uegf/bone morphogenetic protein1 (CUB) domains, an EGF-like domain, two CCP domains or SCRs, and aserine protease domain. Histidine (H), aspartic acid (D), and serine (S)residues are essential for the formation of the active center in serineprotease. Only MASP-1 has two additional cysteine residues in the light

chain, which forms a ‘histidine loop’ disulfide bridge as is found intrypsin and chymotrypsin. On binding of MBL and ficolin tocarbohydrate on the surface of a pathogen, the proenzyme form of MASPis cleaved between the second CCP and the protease domain, resultingin 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 50Chromosome 3q27 1p36 3q27 12p3 12p3Serine protease domainNumber of exon 6 1 1 1 1Number of cysteine 7 5 5 5 5Codon of active serine TCT AGC AGC AGT AGTSubstrate specificityC4 – þ ? – þ8 C2 þ þ ? – þC3 þ – ? – –

MASP, mannose-binding lectin-associated serine protease.



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. Theorigin of MASP genes can be traced back to ascidians (urochordates).Before the emergence of amphioxus (cephalochordates), retropositionof partially processed TCN-type MASP gene and base changes from TCNto AGY at the active site serine generated a prototype of the

MASP-1/3-type gene. All of the amphioxus/vertebrate MASP/C1r/C1sgenes evolved from this ancestral gene by gene duplication. We proposethat TCN-type L-chain-coding region has been lost in the lampreylineage, generating a prototype of the MASP-2-type gene including C1rand C1s genes.



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



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 wasincubated with fresh ascidian plasma that had been treated withphosphate 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 andtreated yeasts were subjected to the assay. The degree of phagocytosis ofuntreated yeast was defined as 100%. The antibodies to GBL and C3completely inhibited the opsonic effect of ascidian plasma (A). Whenanalyzing C3 binding by flow cytometry (B), it is clear that the degree ofphagocytosis is dependent on C3 binding to yeast, showing a lectin-basedopsonic complement system in ascidians.



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,

BacteriaLectin

MASPC3

C3 receptor

Phagocyte

C3b

BacteriaLectin

MASP

C3 C3 receptor

Phagocyte

C3b

BacteriaAb

C1

C3 receptor

Phagocyte

C3b

BacteriaC5-C9(Lytic pathway)

BacteriaC5-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 andC3 receptor are probably the minimal ancestralcomponents of the primordial complementsystem that functioned in an opsonic mannerand appeared in the ascidian lineage. Thecomplement system of the lamprey (the mostprimitive vertebrate) lacks the classical and lyticpathway and so lamprey appear to have acomplement system similar to ascidians.Therefore, the complement system developeddramatically at an early stage of vertebrateevolution into a sophisticated multifunctionalsystem. Gene duplication events seem to haveplayed a major role in this process, and severalsets of homologous complement componentsare noted, such as MBL and C1q, MASPs andC1r/C1s, C2 and factor B, and C4 and C3. Thisfigure was reprinted by permission from NatureReviews Immunology (4) [copyright (2002)Macmillan Magazines Ltd].



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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the lectin-complement pathway – its role in innate immunity and evolution

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