Functional Characterization of a Ficolin-mediatedComplement Pathway in Amphioxus*□S

Received for publication, March 30, 2011, and in revised form, July 20, 2011 Published, JBC Papers in Press, August 8, 2011, DOI 10.1074/jbc.M111.245944

Huiqing Huang‡1, Shengfeng Huang‡1, Yingcai Yu‡1, Shaochun Yuan‡, Rui Li‡, Xin Wang‡, Hongchen Zhao‡,Yanhong Yu§, Jun Li‡, Manyi Yang‡, Liqun Xu‡, Shangwu Chen‡, and Anlong Xu‡2

From the ‡Department of Biochemistry, College of Life Sciences, State Key Laboratory of Biocontrol, National Engineering ResearchCenter of South China Sea Marine Biotechnology, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China and the§Institute of Reproductive Immunology, Jinan University, Guangzhou 510632, People’s Republic of China

The ficolin-mediated complement pathway plays an impor-tant role in vertebrate immunity, but it is not clear whether thispathway exists in invertebrates. Here we identified homologsof ficolin pathway components from the cephalochordateamphioxus and investigated whether they had been co-optedinto a functional ficolin pathway. Four of these homologs, fico-lin FCN1, serine proteaseMASP1 andMASP3, and complementcomponent C3, were highly expressed in mucosal tissues andgonads, and were significantly up-regulated following bacterialinfection. Recombinant FCN1 could induce hemagglutination,discriminate among sugar components, and specifically recog-nize and aggregate several bacteria (especially Gram-positivestrains) without showing bactericidal activity. This suggestedthat FCN1 is a dedicated pattern-recognition receptor. Recom-binant serine protease MASP1/3 formed complexes with re-combinant FCN1 and facilitated the activation of native C3 pro-tein in amphioxus humoral fluid, in which C3 acted as animmune effector. We conclude that amphioxus have developeda functional ficolin-complement pathway. Because ficolin path-way components have not been reported in non-chordate spe-cies, our findings supported the idea that this pathwaymay rep-resent a chordate-specific innovation in the evolution of thecomplement system.

The jawed vertebrate complement system is amajor humoraleffector in both innate and adaptive immunity, which has threeactivation pathways (1). The alternative pathway is directlyactivated by hydrolyzed C3 and serine protease factor B (Bf),which is independent of pathogen-recognition receptors

(PRRs)3 and plays a core role in self-amplification of the com-plement reaction (2, 3). The lectin pathway is triggered bylectins (e.g. collectins and ficolins), which recognize pathogen-associated carbohydrates and subsequently activate C1/MASP-like serine proteases. Serine proteases in turn cleave C4 (a hom-olog of C3) and C2 (a homolog of Bf/C2) complexes andirreversibly activate the complement cascade (4). The classicalpathway is initiated by the special lectin family C1q (5). Unlikecollectins and ficolins, C1q proteins recognize antibody-anti-gen complexes rather than pathogen-associated carbohydratesin complement activation. Therefore, C1q and the classicalpathway connect innate with adaptive immunity.The vertebrate complement system is not only a major

humoral effector, but possibly represents a fundamental shiftfrom the antimicrobial peptide-based humoral immunitywhich is prevalent in most invertebrates (6). Hence, the originof the vertebrate complement system is not trivial. Previousefforts have dated the origin of some complement pathways tobefore the radiation of jawed vertebrates. For example, it hasbeen suggested that a functional alternative pathway is presentin a basal deuterostome, the purple sea urchin (7). The jawlessvertebrate lamprey, which lacks Ig-based antibodies, possessesa prototype of the classical pathway, in which the C1q proteinrecognizes pathogen-associated carbohydrates instead of anti-body-antigen complexes (8). In addition, functional lectin path-ways mediated by GBL (a C-type lectin without collagen) andMBL (a collectin), which can trigger the complement cascade,have been found in both urochordates and lampreys (9, 10).However, thus far no functional ficolin pathways have beenfound outside the jawed vertebrate lineage (11–13).Ficolins contain amiddle collagen (COL) region and a C-ter-

minal fibrinogen-like (FBG) domain (14). In comparison, col-lectins contain a middle COL region and a C-terminal C-typelectin (CTL) domain (15), while C1q proteins contain a middleCOL region and a C-terminal C1q-like domain (16). All threetypes of lectins useCOL regions for serine protease binding andC-terminal domains for target recognition (either pathogens orantibody-antigen complexes). The ficolin pathway is with amore recent discovery than the collectin and C1q pathways.

* This work was supported by Projects 2007CB815800 and 2011CB946101 ofthe National Basic Research Program (973), Project 2008AA092603 of theState High-Tech Development Project (863), and 2007DFA30840 (Interna-tional S&T Cooperation Program) from the Ministry of Science and Tech-nology of China; Key Project (0107) from the Ministry of Education; KeyProject (30901103) and Project (30730089) from the National Natural Sci-ence Foundation of China; and projects from the Commission of Scienceand Technology of Guangdong Province and Guangzhou City and fromSun Yet-sen University Science Foundation.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table SI and Figs. S1–S5.

1 These authors contributed equally to this work.2 Recipient of the Outstanding Young Scientist Award from the National

Nature Science Foundation of China. To whom correspondence should beaddressed: Molecular Biology and Immunology, Director of State Key Lab-oratory of Biocontrol, College of Life Sciences, Sun Yat-sen UniversityGuangzhou, P.R. China, 510275. Tel.: 86-20-39332990; Fax: 86-20-39332950; E-mail: lssxal@mail.sysu.edu.cn.

3 The abbreviations used are: PRR, pathogen-recognition receptors; FCN, fico-lin protein; FBG domain, fibrinogen-like domain; MBL, mannose-bindinglectin; GBL, glucose-binding lectin; MASP, MBL-associated serine protease;GlcNAc, N-acetylglucosamine; ORF, open-reading frame; CCP, comple-ment control protein; CUB, complement C1r/C1s; Uegf, Bmp1 module; HA,hemagglutinin activity.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 42, pp. 36739 –36748, October 21, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Ficolin was first documented as a transforming growth factor(TGF)-�1-binding protein on pig uterus membranes in 1991before their opsonic functions had been recognized (17). Thereare three human ficolins. Human ficolin1 (HmFCN1) recog-nizes Staphylococcus aureus and smooth Salmonella typhimu-rium (LT2), and recruits MASP1 and 2 to cleave C4 possiblythrough interaction with N-acetylglucosamine (GlcNAc). Italso displays a binding preference for sialyl-N-acetyllac-tosamine (SiaLacNAc) (18). HmFCN2 interacts with severalstrains of bacteria, including Escherichia coli, S. typhimuriumTV119, and S. aureus (19–22), possibly through interactionwith LTA, 1, 3-�-D-glucan and glucose/galactose rings (23–26).HmFCN2 activates the complement cascade through a Ca2�-dependent association with MASPs (22). HmFCN3 binds toAerococcus viridans (27) and a few types of ligands, includingD-fucose, galactose, and acetylated albumin (27–29). HmFCN3activates the complement system by the cleavage of C4 basedon binding toA. viridans (27). Many studies have reported thatmutations or ectopic expression of ficolins are associated withhuman diseases, including systemic lupus erythematosus (30),chronic rheumatic heart disease (31), rheumatoid arthritis (32),fever, and neutropenia (33).Amphioxus is the oldest extant chordate lineage and is

invaluable for the understanding of vertebrate origins and chor-date biology. Early studies showed that amphioxus share severalcategories of complement components with vertebrates (6, 12).However, despite thousands of vertebrate homologs in inverte-brates, notmany of them (especially non-house-keeping genes)organize and function in ways similar to their vertebrate coun-terparts. In this work, we cloned and expressed BjFCN1, acanonical ficolin gene from amphioxus Branchiostoma japoni-cum (Bj), as well as three other relevant genes includingBjMASP1, BjMASP3, andBjC3.Wedemonstrated that BjFCN1is a dedicated PRR for the amphioxus complement system,which can interact with BjMASP1/3 and therefore lead to theactivation of BjC3. Our findings not only suggest that a func-tional ficolin pathway have been established in the last commonancestor of chordates, but help to piece together the evolution-ary story of the chordate complement system.

EXPERIMENTAL PROCEDURES

AmphioxusCollection, cDNACloning, andSequenceAnalysis—Adult Chinese amphioxus (B. japonicum) were collected fromQingdao, China and reared in aerated sea water with algae.Partial cDNAs of BjFCN1, BjMASP1, BjMASP3, and BjC3wereclonedwith gene-specific primer pairs (supplemental Table SI).Full-length cDNAs were isolated by using RACE technology(Invitrogen). These cDNA sequenceswere cloned into pGEX-Teasy vector (Promega) and verified by DNA sequencing. Multi-ple alignments were created by using ClustalX and furtherrefined by using GeneDoc (34). Molecular phylogenetic analy-sis was conducted with Mega4 using Neighbor-joining methodwith pairwise deletion and 1000 bootstrap tests (35, 36).Section in Situ Hybridization—Tissue fixation and section in

situ hybridization were conducted by the method previouslydescribed (37). Digoxigenin-labeled sense and antisense probesfor BjFCN1, BjMASP1, BjMASP3, and BjC3 cDNA sequences(primers shown in supplemental Table SI) were prepared by

usingDig RNA synthesis kit (Roche). Hybridizationwas carriedout at 42 °C overnight, with probe concentration of 1 �g ml�1.After extensive washes, sections were subjected to signal detec-tion by using NBT/5-bromo-4-chloro-3-indolyl phosphatestock solution (Roche).Immune Stimulation and Real-time Quantitative PCR

Analysis—Several strains of bacteria including S. saprophyti-cus, S. hemeolyticus, S. aureus, E. faecalis, Vibrio anguillarum,Bacillus subtilis, Acinetobacter calcoaceticus, Klebsiella pneu-moniae, and E. coli were obtained from the Third AffiliatedHospital of Sun Yat-Sen University (China), and verified bysequencing their 16 S rRNAs. LPS (from E. coli 0111:B4) andLTA (from S. aureus) were purchased from Sigma. In theimmune stimulation, live S. aureus (108 cells/ml), liveV. anguillarum (108 cells/ml), LPS (1 mg/ml), and LTA (1mg/ml) were suspended/dissolved in PBS buffer (137mMNaCl,3mMKCl, 1mMNa2HPO4, 2mMKH2PO4, pH 7.4) and injectedinto the gut and coelomof amphioxus (15�l animal�1), respec-tively. The injection of PBSwas used as control. Twenty animalsof each treatmentwere collected at 1, 2, 4, 8, 12, and 24 hpostin-jection and frozen until use. Total RNA was purified by usingRNeasy Plus Mini kit (Qiagen) and then treated with DNaseI(Promega). Double-stranded cDNAs were synthesized withSYBR perfect real-time series kit (Takara). Real-time quantita-tive PCR (qRT-PCR) was conducted using SYBR PrimeScriptqRT-PCR kit (Takara) and a LightCycler 480 system (Roche).The PCR programwas set for 40 cycles, with annealing temper-ature at 60 °C and extending temperature at 70 °C. Experimentsfor each samplewere repeated for three times. Expression levelsof each gene were determined by 2��� Ct method, usingGAPDH as endogenous control.Vector Construction and Recombinant Protein Preparation—

The recombinant BjFCN1 protein (without signal peptide)fused with TRX-His tag was expressed using pET32a vector(Novagen). GST-tagged recombinant BjMASP1/3-N protein(commonN-terminal portion of BjMASP1 and 3without signalpeptide, CUB1-EGF-CUB2domain)was expressed using pGEX4T-2 vector (Promega). His-tagged recombinant BjMASP1-Cprotein (C-terminal portion of BjMASP1, CCP2-SP domain)and BjC3 protein (amino acid residues 579–755) were ex-pressed using pET21b and pET28a vectors (Novagen), respec-tively. The inclusion recombinant BjC3, BjMASP1/3-N, andBjMASP1-C proteins were denatured and renatured asdescribed (38). Proteins were purified withNi2�-chelating Sep-harose column (BjFCN1, BjC3, and BjMASP1-C) or Glutathi-one SepharoseTM 4B column (BjMASP1/3-N) in TBS buffer (50mM Tris-HCl, 150 mM NaCl, pH 7.5). Purified proteins weredesalted with a G-25 column, and then concentrated by filtra-tion through an ULTRAFREE centrifugal filter device (Milli-pore). TRX protein used as a negative control was purified bythe same method. Purified recombinant BjC3 was sent to theFourth Military Medical University, China for the preparationof monoclonal antibody. Flag-tagged full-length BjFCN1 withsignal peptide was inserted into pcDNA3.0 vector and trans-fected into HEK293T cells. Flag-tagged recombinant BjFCN1protein was then purified from the cell medium and concen-trated as described above. All proteins were examined by Bio-

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Rad protein assay with BSA (bovine serum albumin) as astandard.Hemagglutination and Sugar Binding Assays—Hemaggluti-

nation activity (HA) of His-tagged recombinant BjFCN1 wasexamined by using mouse erythrocytes as previously described(38). Calcium and EDTA were added to the working buffer(TBS buffer) to investigate the effect of calciumonBjFCN1HA.HA-based assays for BjFCN1 sugar binding activity were alsoconducted. Assessed sugar components included maltose,D-glucose, lactose, galactose, sucrose, D-mannose, fructose,GlcNAc, LPS, and LTA. The lowest dilution of each sugar com-ponent that inhibited HA of BjFCN1 was considered the mini-mal inhibitory concentration of that sugar. Purified TRX pro-tein was used as control.The binding activity of His-tagged recombinant BjFCN1 to

GlcNAc and �-lactose was also performed by co-pull-downassays. After being equilibrated with TBS buffer, GlcNAc aga-rose beads were incubatedwith rough BjFCN1 protein (150�g)containing CaCl2 or EDTA (10 mM each) for 2 h at 26 °C. Afterextensive washes, the bound proteins were eluted with 30 �l ofSDS-PAGE loading buffer (100 mM Tris-HCl, 0.01% bromphe-nol blue, 36% glycerol, 4% SDS, pH 6.5) and heated for 15min at100 °C before subjecting to SDS-PAGE. The binding activitywith lactose agarose was tested by the same method. RoughBjFCN1 protein (30 �g) alone was used as control.Bacteria Binding and Aggregation Analysis—Bacterial cells

were suspended in TBS buffer (approximately A600 � 2). His-tagged recombinant BjFCN1 protein (100 �g) was mixed withbacteria suspension in the presence of CaCl2 or EDTA (10 mM

each) and incubated at 4 °C overnight. After extensive washes,the bound proteins were dissociated from the pellets by loadingbuffer, and analyzed by SDS-PAGE andWestern blotting (withanti-His mAb from Novagen). The binding activity of am-phioxus humoral fluid (abundant in C3) to bacteria was alsoexamined by the same procedure. Anti-BjC3 mAb was used inWestern blotting. As comparison, BjFCN1 protein (15 �g) andhumoral fluid (30 �g) alone were used as a positive control,respectively. Amphioxus humoral fluid was prepared by themethod previously described (39).In aggregation analyses, bacterial cells were suspended in 1

mlTBSbuffer (approximatelyA600� 3) andmixedwith 50�l ofFITC solution (10 mg/ml, Sigma) for labeling (1 h at 26 °C withgentle agitation in darkness). After extensive washes, each typeof FITC-labeled bacteria was mixed with BjFCN1 protein (100�g), and incubated for 1 h at 26 °C in the presence of CaCl2 orEDTA (10 mM each). The agglutination effect was examinedunder fluorescencemicroscopy. Purified TRX protein was usedas a negative control.GST Pull-down Analysis—GST-tagged recombinant

BjMASP1/3-N protein and His-tagged recombinant BjFCN1protein were prepared as described, and their interaction wasinvestigated by GST pull-down assay. A mixture of the twoproteins (200 �g each) was incubated at 26 °C for 1 h with theaddition of 10 mM CaCl2. The mixture was added to glutathi-one-SepharoseTM 4B beads and incubated with gentle rotationfor 2 h at 26 °C. After extensive washes, the bound proteinswere eluted with loading buffer, and analyzed by SDS-PAGEand Western blotting with anti-His mAb and anti-GST mAb

(Novagen). GST-tagged human rhinovirus 3C protease protein(GST-3C) was used as a negative control and examined inparallel.C3 Cleavage Analysis—Amphioxus humoral fluid and His-

tagged recombinant BjMASP1-C protein were obtained asdescribed above, and a mixture of the two proteins (100 �geach) was incubated at 26 °C for 2 h with the addition of CaCl2or EDTA (10 mM each). As negative controls, the incubation ofhumoral fluid alone, humoral fluid with recombinantBjMASP1/3-N protein, and humoral fluid with BSA (100 �geach) were analyzed by the same procedure. The interactionsbetween these proteins were analyzed by Native PAGE andSDS-PAGE under non-reducing conditions and Western blot-ting using anti-BjC3 mAb. Native PAGE is modified differentlyfrom SDS-PAGE, including using 7.5% native acrylamide gelwithout SDS, which is pre-run for 30 min at 4 °C in an electro-phoresis buffer composed of an upper chamber buffer (25 mM

Tris, pH 8.4, 192 mM glycine, 1% sodium deoxycholate) and alower chamber buffer (25 mM Tris, pH 8.4, 192 mM glycine).

RESULTS

Identification of Putative Ficolin Pathway Components fromAmphioxus—An EST-encoding ficolin BjFCN1 was identifiedfrom cDNA library, and its full-length cDNA was cloned. ThiscDNA is 2583 bp long, containing an ORF of 1269 bp. Thededuced protein of BjFCN1 is a canonical ficolin, which con-tains a signal peptide of 24 aa, a middle COL region of 20 Gly-X-Y repeats, a short neck domain of 3 aa, and a C-terminalglobular FBG domain of 219 aa (Fig. 1A and supplemental Fig.S1). BjFCN1 has a long linker sequence between the signal pep-tide and the COL region compared with HmFCN2 (Fig. 1B),and all four conserved cysteines that are important for stabiliz-ing the FBG domain (Fig. 1A). The FBG domain of BjFCN1shares 36–41% amino acid identities with its vertebratehomologs (Fig. 1A and supplemental Fig. S2). In addition toBjFCN1, we identified three other complement genes, desig-nated BjMASP1 (supplemental Fig. S3), BjMASP3 (supplemen-tal Fig. S4), and BjC3 (supplemental Fig. S5), which areorthologs to human MASP1, MASP3, and C3, respectively.Expression Regulation of Four Amphioxus Putative Ficolin

Pathway Components—The tissue distribution of BjFCN1,BjMASP1, BjMASP3, and BjC3 mRNAs in adult amphioxuswas analyzed by performing section in situ hybridization (Fig.2). While only slight or no expression was detected in skin,spinal cord,muscle, and notochord, all four geneswere stronglyexpressed in ovary, gut, gill, and hepatic cecum (the equivalentof mammalian liver). These four tissues are known to be majorsites involved in amphioxus immunity. The co-expression ofthese genes suggested that their encoding proteins might phys-ically interact with each other during immune responses.Indeed, an early study reported that there was a high concen-tration of C3 protein in amphioxus ovary (40); thus our findingsimplied that ficolin and MASPs might play a role in the activa-tion of this C3 arsenal in ovary.The time course expression of these genes during immune

challenge was also monitored using qRT-PCR. Both BjFCN1and BjC3 were quickly and highly up-regulated in response tobacterial components LPS and LTA, whereas BjMASP1/3 was

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mildly up-regulated (the probe detected the expression ofBjMASP1 and 3 simultaneously because it targets the commonN-terminal region of two genes) (Fig. 3).We reason thatMASPsmay not require high expression levels because of their power-ful catalytic activity. Also, once C3 is activated, the alternativepathwaywill step in to sustain and amplify the reaction.We alsoobserved that BjFCN1 responded towhole bacteriamore slowlyand less dramatically, suggesting that purified bacterial compo-

nents are more potent immune stimulants than the whole bac-teria (Fig. 3A).Hemagglutinin and Sugar Binding Activity of BjFCN1—Flag-

tagged recombinant BjFCN1 was transiently transfected intoHEK293T cells. The abundance of this protein was detected incell culture medium after transfection, suggesting that BjFCN1is a secreted protein. The molecular mass of this secreted pro-tein is about 50 kDa, slightly larger than the predicted molecu-

FIGURE 1. A, sequence alignment of ficolins from various species. B, architecture of cephalochordate amphioxus BjFCN1 and human HmFCN2. The deducedprotein of BjFCN1 is a canonical ficolin, which consists of an N-terminal signal peptide, a middle collagen (COL) region, and a C-terminal fibrinogen-like (FBG)domain. Homo sapiens, Hm; Mus musculus, Mm; Xenopus laevis, Xe; Ascidiacea, Halocynthia roretzi, As. The sequences used in the alignment are indicated asfollows: HmFCN2 (NP_004099); HmFCN3 (NP_003656); MmFCN1 (NP_032021); XeFCN3 (NP_001079138); AsFCN1 (BAB60704); AsFCN3 (BAB60706).

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lar weight of 46 kDa, possibly due to post-translated glycosyla-tion. Electrophoresis also showed that this protein wasseparated into three bands, suggesting that, under natural con-ditions, BjFCN1 forms a dimmer and a tetramer through disul-fide bonds (Fig. 4A).We also expressed recombinantTRX-His-BjFCN1protein in

E. coli BL21 (DE3) and obtained purified soluble fusion proteinwith a Ni2�-chelating Sepharose column. The recombinantprotein was �64 kDa, corresponding to the predicted molecu-lar mass (Fig. 4B). TRX-His-tagged BjFCN1 protein inducedhemagglutination of mouse erythrocytes in the presence ofCaCl2. This activity was completely inhibited by the addition ofEDTA (Fig. 4C), suggesting that the lectin activity of BjFCN1is Ca2�-dependent. Further competition inhibition assaysshowed that 10 mM galactose, 10 mM D-glucose, 50 mM lactose,150mMD-mannose, 180mMGlcNAc, 200mM fructose, 200mM

sucrose, and 0.005 mg/ml LTA could sufficiently inhibit thehemagglutinating activity of BjFCN1 (Table 1), suggesting thatBjFCN1 has different binding specificities to these sugars.However, as much as 500mMmaltose and 2mg/ml LPS did notaffect BjFCN1’s hemagglutinating activity, suggesting that theyare not ligands for BjFCN1. Soluble TRX protein was purifiedand used as control, but showed no hemagglutinating activity.Because LTA is abundant in Gram-positive bacteria, whereasLPS is present in Gram-negative bacteria, BjFCN1 may prefer-entially target Gram-positive bacteria.GlcNAc is a part of the components ofmicrobial cell wall and

anthropoid cuticle, such as peptidoglycans and chitins. So far,

all tested vertebrate ficolins have affinity for GlcNAc, and thisaffinity is critical for human ficolins. To verify whetheramphioxus ficolins have this critical function, affinity chroma-tography technologywas used to assess the interaction betweenBjFCN1 and GlcNAc-agarose. Simultaneously, the interactionbetween BjFCN1 and �-lactose agarose was evaluated. Thebound BjFCN1 proteins were eluted and analyzed by SDS-PAGE (Fig. 4D). Results showed that BjFCN1 proteins could beconcentrated by more than 100 fold with GlcNAc and �-lac-tose. Notably, the effect for BjFCN1-GlcNAc interaction wasconsistent in both calcium and calcium-free (EDTA-added)conditions, suggesting that calcium cations affect BjFCN1’shemagglutinin activity but do not completely block its interac-tion with ligands.Differential Binding Affinities of BjFCN1 to Bacteria—From

the above assays, we inferred that BjFCN1 may serve as a PRRfor bacteria through their surface carbohydrates. Hence, weincubated His-tagged recombinant BjFCN1 protein with bac-teria and examined its binding activity by using Western blot-ting.We found that BjFCN1 protein could bind to all examinedbacteria with different affinities in the presence of 10mMCaCl2(Fig. 5A). We also noted that the binding affinity was signifi-cantly stronger for Gram-positive bacteria than for Gram-neg-ative bacteria, except for V. anguillarum. This binding activitycould be completely inhibited by adding 10 mM EDTA.BjFCN1 proteinwas incubatedwith FITC-labeled bacteria to

analyze its bacterial aggregating activity. As shown in Fig. 5B,fluorescence microscopy showed that, in the presence of cal-

FIGURE 2. Tissue distributions of amphioxus BjFCN1 (D–F), BjMASP1 (G–I), BjMASP3 (J–L), and BjC3 (M–O) detected by section in situ hybridization. Thenegative control is showed in A–C. Endostyle, e; gill, g; hepatic cecum, hc; intestine, i; muscle, m; notochord, nc; skin, s; spinal cord, sc; testis, t; ovary, o. The barindicates 200 �m.

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cium, BjFCN1 protein induced strong aggregation of S. aureusand weak aggregation of B. subtilis, S. hemeolyticus, andE. faecalis. The parallel experiments with purified TRX proteinas control generated no such effects (Fig. 5). BjFCN1 may be adual function receptor, not only recognizing bacteria but alsoaggregating them. This function is largely observedwithGram-positive bacteria.We performed a series of experiments evaluating bacteri-

cidal and inhibitive activities, but failed to detect any suchaction for BjFCN1. This is in contrast with early reports (see“Discussion”), suggesting that unlike other invertebrate FBG-

containing proteins, BjFCN1 could mainly function as a dedi-cated PRR.TheN-terminal Portion of BjMASP1/3 Forms aComplexwith

BjFCN1—In mammals, MASP1, 2, and 3 are major activatingserine proteases for the complement lectin pathway. TheseMASP proteins are normally pre-coupled with collectins orficolins (collectin/ficolin-MASP complexes) in humoral fluid.When collectins or ficolins are engaged with pathogens, thecoupled MASPs are activated to cleave C4 (a paralog of C3).Amphioxus has an ortholog of vertebrate MASP1/3 gene (41).As does its vertebrate ortholog, BjMASP1/3 can produce twoprotein isoforms (MASP1 and 3) by alternative splicing, whichhave different C-terminal serine protease domains but share acommon N-terminal domain (CUB1-EGF-CUB2) (Fig. 6A). Asstated previously, the mRNA of BjMASP1/3, BjFCN1 and BjC3

FIGURE 3. Expression profiles of BjFCN1 (A), BjC3 (B), and BjMASP1/3(C)detected by qRT-PCR after challenge with purified pathogens LPS andLTA, bacteria S. aureus and V. anguillarum. Endogenous control for quali-fication was cytoplasm GAPDH.

FIGURE 4. Purification and characterization of recombinant BjFCN1 pro-tein. A, Flag-tagged recombinant BjFCN1 protein was subjected to non-re-ducing SDS-PAGE (12%) (left panel) and immunoblotted with anti-Flag mAb(right panel). BjFCN1 formed monomer (1mer), dimer (2mer), and tetramer(4mer) with disulfide bonds based on our analysis. B, His-tagged recombinantBjFCN1 protein was subjected to SDS-PAGE (12%) (left panel) and immuno-blotted with anti-His mAb (right panel). C, hemagglutinin activity analysis ofHis-tagged recombinant BjFCN1 protein. TRX as a negative control was testedin parallel. D, binding of His-tagged recombinant BjFCN1 protein to GlcNAcand lactose agarose. Rough BjFCN1 protein (30 �g) alone as control was alsosubjected to SDS-PAGE.

TABLE 1Effects of saccharides on hemagglutinin activity of BjFCN1 protein

SaccharidesMinimal inhibitory

concentration

Maltose �500 mMD-glucose 10 mMLactose 50 mMGalactose 10 mMSucrose 200 mMD-mannose 150 mMFructose 200 mMGlcNAc 180 mMLPS from E. coli 0111:B4 �2 mg/mlLTA from S. aureus 0.005 mg/ml

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were co-localized and concentrated in the gut and the gonads,and were significantly up-regulated following bacterial infec-tion. As described, BjFCN1 recognized and aggregated bacteriawithout showing bactericidal activity, which suggested thatBjFCN1 relies on other downstream pathways to clear bacteria.The presence of a COL region suggests that BjFCN1may com-plete its function by recruiting humoral MASPs. Prompted bythis, we designed a GST pull-down assay to determine if therewas physical interaction between BjMASP1/3 and BjFCN1.

Similar to mammalian MASPs (42, 43), full-length recombi-nant BjMASP1 and 3 are large and insoluble, so we expressedthe N-terminal portion and the C-terminal domain ofBjMASP1/3 separately (Fig. 6A). The GST-tagged N-terminalportion of BjMASP1/3 (designated BjMASP1/3-N) was puri-fied and had a molecular mass of �55 kDa, correspondingto the predicted molecular mass (Fig. 6B). RecombinantBjMASP1/3-N protein was mixed with His-tagged recombi-

FIGURE 5. The interaction of His-tagged recombinant BjFCN1 protein andbacteria. A, micro-organism binding analysis of BjFCN1 protein. BjFCN1bound to A. calcoaceticus (a), E. coli (b), K. pneumoniae (c), B. subtilis (d),V. anguillarum (e), S. aureus (f), S. hemeolyticus (g), S. saprophyticus (h), andE. faecalis (i) with calcium, while 10 mM EDTA completely inhibited the activa-tion. BjFCN1 protein (15 �g) was a positive control (j). B, microbial aggrega-tion analysis of BjFCN1 protein. BjFCN1 strongly aggregated S. aureus andweakly aggregated B. subtilis, S. hemeolyticus, and E. faecalis with the pres-ence of calcium, while the effect was inhibited with EDTA. The bar on themicrographs indicates 100 �m. TRX protein (27 kDa) as a negative control wastested in parallel.

FIGURE 6. A, architecture of amphioxus BjMASP1 and BjMASP3, humanHmMASP1 and HmMASP3. B, GST-tagged recombinant BjMASP1/3-N proteinwas subjected to SDS-PAGE (12%) (left panel) and immunoblotted with anti-GST mAb (right panel). C, binding of His-tagged recombinant BjFCN1 proteinto GST-tagged recombinant BjMASP1/3-N protein. BjFCN1 protein formed acomplex with BjMASP1/3-N. D, His-tagged recombinant BjMASP1-C proteinwas subjected to SDS-PAGE (12%) (left panel) and immunoblotted with anti-His mAb (right panel).

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nant BjFCN1 protein and incubated with glutathione-Sephar-oseTM 4B beads. The bound BjFCN1 protein co-pulled withBjMASP1/3-N using the beads was analyzed by Western blot-ting. It showed that BjFCN1 can clearly form a complex withBjMASP1/3-N (Fig. 6C), GST-tagged human rhinovirus 3Cprotease protein (GST-3C) did not bind with BjFCN1.The C-terminal Protease Domain of BjMASP1 Cleaves

Humoral BjC3—The His-tagged C-terminal protease domainof BjMASP1 (designated BjMASP1-C) was expressed and puri-fied and found to be �37 kDa, corresponding to the predictedmolecular mass (Fig. 6D). As does HmC3, BjC3 contains a �chain and an � chain, which join together via disulfide bond.The � chain starts with the ANA domain, which can be cleavedoff to form the C3a and C3b fragments (the BjC3 structureshown in Fig. 7A). To assess whether BjMASP1-C catalyzes thecleavage of humoral C3 in amphioxus, we expressed a portion

of C3 protein (containing the C-terminal of the � chain and theN-terminal of the � chain) and raised a monclonal Ab (anti-BjC3 mAb) against it (Fig. 7A). The binding site for this mAbwas located in the C-terminal of the � chain (Fig. 7A). Subse-quently we extracted fresh humoral fluid, which is known tocontain a high concentration of C3 proteins, from adultamphioxus. The humoral fluid and the BjMASP1-C proteinwere mixed and incubated for 2 h in the presence of calcium orEDTA. The mixture was analyzed by Native PAGE and SDS-PAGE under non-reducing conditions, and anti-BjC3mAbwasused in Western blotting. As negative controls, the incubationof humoral fluid alone, humoral fluid with GST-taggedBjMASP1/3-N protein, and humoral fluid with BSA were con-ducted in parallel. These experiments demonstrated that in thepresence of BjMASP1-C proteins, but not in other controlledconditions, themolecule size of BjC3was altered, and the cleav-age of humoral BjC3 was accelerated (Fig. 7B). As indicated byNative PAGE, the migration of BjC3b did not correspond to itsmolecular mass and lagged behind intact BjC3 (Fig. 7B, leftpanels). Amphioxus MASP1/3 have acquired the function thatall mammalian MASP1/3 share, that is, to bridge ficolins andC3 during complement activation.Bacterial Binding Activity of Humoral BjC3—It is reported

that amphioxus humoral fluid contains high concentration ofC3 and mediates hemolytic activity for mammalian erythro-cytes (39). To provide more supporting evidence for a func-tional humoral complement pathway, amphioxus humoralfluid was incubated with several types of bacteria and the bind-ing activity of humoral BjC3 to bacteria was analyzed usingWestern blotting under reducing conditions. Humoral fluidalone as a positive control was also tested. Our results showedthat humoral BjC3 could bind to seven examined bacteria withdifferent affinities. The strongest binding was with S. hemeo-lyticus (Gram-positive bacteria) and B. subtilis (Gram-negativebacteria). This binding was calcium dependent because allbinding activity could be completely suppressed by the additionof EDTA (Fig. 7C).

DISCUSSION

In vertebrates, ficolins (containing COL-FBG domain) serveas PRRs for the complement lectin pathway without showingany bactericidal activity. Prior to the current study, an FBG-containing protein from amphioxus was reported to have bac-teriolytic activity (44). Unlike this protein, BjFCN1 containsboth COL and FBG domains, and is able to agglutinate a broadspectrum of bacteria with varying affinities and without show-ing bacteriolytic or inhibitive activity. Our findings suggestedthat BjFCN1 is a PRRmainly for Gram-positive bacteria, and iscapable of activating the complement system to destroy bacte-ria.Hence, BjFCN1 structurally and functionally resembles ver-tebrate ficolins.In vertebrates, the ficolin pathway is a combination of fico-

lins and over ten other proteins, including MASPs (MASP1, 2,3), C3-like proteins (C3, C4, C5), and several regulatory com-ponents (45). Only two unambiguous orthologs of these verte-brate genes have been found in amphioxus, includingMASP1/3and C3 (12). However, the structural and functional resem-blance between BjFCN1 and vertebrate ficolins raises the pos-

FIGURE 7. A, architecture of amphioxus BjC3 and human HmC3. �2M, �-2-macroglobulin domain; ANA, anaphylatoxin homologous domain; TE, thiol-ester_cl domain; C345C, netrin C-terminal domain. B, humoral BjC3 cleavageanalysis. The protein mixtures described were analyzed by Native PAGE (leftpanels) and SDS-PAGE (right panels) under non-reducing conditions, and anti-BjC3 mAb was used in Western blotting. The presence of BjMASP1-C proteinaltered the molecular size of humoral BjC3 under both calcium and EDTAconditions, suggesting that BjMASP1-C protein exhibited proteolytic func-tion on humoral BjC3. C, bacterial binding analysis of humoral BjC3 by West-ern blotting under reducing condition. Humoral fluid alone as a positive controlwas tested (a and k). BjC3 bound to A. calcoaceticus (b), K. pneumoniae (d), B. sub-tilis (e), V. anguillarum (f), S. aureus (g), S. hemeolyticus (h), and E. faecalis (j) undercalcium condition, and 10 mM EDTA completely inhibited the activation.

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sibility that there exists a functional ficolin pathway inamphioxus. We have expressed BjMASP1/3 recombinant pro-tein and successfully demonstrated that it is able to interactwith both BjFCN1 and BjC3, suggesting a BjFCN1-BjMASP1/3-BjC3 pathway in amphioxus (Fig. 8). Although this am-phioxus counterpart is simplified and rudimentary comparedwith the full-fledged vertebrate ficolin pathway, it suggestedthat the basic framework of the ficolin pathway had been estab-lished since the radiation of chordate phylum. Furthermore,because of the apparent lack of ficolins in basal deuterostomes(12, 46), this pathway may represent a prototype of vertebrateficolin-mediated complement pathway in the evolution of thecomplement system.The formation of the sophisticated human complement sys-

tem appears to be a slow and gradual process with intermittentleaps. The core complement component, C3-like proteins, areancient immune effectors present in most metazoans (47–49),whereas other key components seem to have arisen with deu-terostomes. Factor B is a symbol of the alternative pathway,and, to date, the most ancient factor B discovered was from thebasal deuterostome sea urchin (7, 46). The genome of the seaurchin also encodes collectins and C1q proteins, but it lackscanonical ficolins and conserved complement-related serineproteases; hence the lectin pathways are unlikely to be presentin this lineage (12). The urochordates, which represent the clos-est sister lineage of vertebrates, possess collectins, ficolins, C1qproteins, andMASP-like serine proteases (12, 50); although evi-dence that these genes have been co-opted into canonical lectinpathways is lacking.Despite the undeterminedpresence of clas-sical lectin pathways, a non-classical lectin pathway, of whichthe sensor receptor is a C-type lectin (without a COL region)instead of a collectin (with a COL region), has been identified inurochordates (9, 51). On the other hand, we know that C1qprotein and the classical pathway in jawed vertebrates bridgethe gap between innate and adaptive immunity by recognizingantibody-antigen complexes. However, C1q-like proteins wereoriginally sugar-binding lectins (50), and, in jawless vertebrates,

C1q of the classical pathway indeed acts as a direct sensor forpathogen-associated carbohydrates (8).Our works based on amphioxus have helped to piece

together this story and provide more detail. In a previousgenomic survey, we demonstrated that amphioxus possess allmajor complement components, including collectins, ficolins,C1q proteins,MASP-like serine proteases, factors B, C3, C6, etc(12). In a subsequent transcriptomic analysis, we suggested thatthese genes work together through their co-regulation patterns(6). In the current study, we demonstrated that some of thesegenes have been organized into a functional ficolin pathway.Hence, evidence leads to the hypothesis that the framework ofthe modern vertebrate complement system have been estab-lished in the most recent common chordate ancestor. Follow-ing this hypothesis, we envisage thatwith further evolution, tworounds ofwhole genomeduplication at the origin of vertebratesincreased the gene number and made this system more intri-cate; and, in the jawed vertebrate lineage, the system was fur-ther involved in adaptive immunity by contributing one type oflectin receptor (i.e. C1qs) to form the classical pathway.

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A Ficolin-mediated Complement Pathway in Amphioxus

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Anlong XuHongchen Zhao, Yanhong Yu, Jun Li, Manyi Yang, Liqun Xu, Shangwu Chen and

Huiqing Huang, Shengfeng Huang, Yingcai Yu, Shaochun Yuan, Rui Li, Xin Wang,Amphioxus

Functional Characterization of a Ficolin-mediated Complement Pathway in

doi: 10.1074/jbc.M111.245944 originally published online August 8, 20112011, 286:36739-36748.J. Biol. Chem. 

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VOLUME 284 (2009) PAGES 25593–25601DOI 10.1074/jbc.A109.025452

Angiopoietin-like 4 (ANGPTL4, fasting-induced adiposefactor) is a direct glucocorticoid receptor target andparticipates in glucocorticoid-regulated triglyceridemetabolism.Suneil K. Koliwad, Taiyi Kuo, Lauren E. Shipp, Nora E. Gray, Fredrik Backhed,Alex Yick-Lun So, Robert V. Farese, Jr., and Jen-Chywan Wang

PAGE 25599:

On the y axis in panel A of Fig. 6, the measurement for serum TGshould be mg/dL (not �g/mL), and on the y axis in panel B, the meas-urement for liver TG should be nmol/mg tissue (not mmol/mg tissue).The corrected figure is presented below.

VOLUME 286 (2011) PAGES 36739 –36748DOI 10.1074/jbc.A111.245944

Functional characterization of a ficolin-mediatedcomplement pathway in amphioxus.Huiqing Huang, Shengfeng Huang, Yingcai Yu, Shaochun Yuan, Rui Li,Xin Wang, Hongchen Zhao, Yanhong Yu, Jun Li, Manyi Yang, Liqun Xu,Shangwu Chen, and Anlong Xu

The grant information footnote should read as follows. “This workwas supported by Project 30901103 from the National Natural ScienceFoundation of China; Projects 2011CB946101 and 2007CB815800 ofthe National Basic Research Program (973), Project 2008AA092603of the State High-Tech Development Project (863), and Project2007DFA30840 of the International S&T Cooperation Program fromthe Ministry of Science and Technology of China; Key Project 0107from the Ministry of Education; and projects from the Commission ofScience and Technology of Guangdong Province and Guangzhou Cityand from the Sun Yet-sen University Science Foundation.”

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 6, p. 4394, February 3, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

4394 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 6 • FEBRUARY 3, 2012

ADDITIONS AND CORRECTIONS

Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.