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The ancient origin of the complement system

2004; Springer Nature; Volume: 24; Issue: 2 Linguagem: Inglês

10.1038/sj.emboj.7600533

1460-2075

Yong Zhu, Saravanan Thangamani, Bow Ho, Jeak Ling Ding,

Aquaculture disease management and microbiota

Article23 December 2004free access The ancient origin of the complement system Yong Zhu Yong Zhu Department of Biological Science, National University of Singapore, Singapore, Singapore Search for more papers by this author Saravanan Thangamani Saravanan Thangamani Department of Biological Science, National University of Singapore, Singapore, Singapore Search for more papers by this author Bow Ho Bow Ho Department of Microbiology, National University of Singapore, Singapore, Singapore Search for more papers by this author Jeak Ling Ding Corresponding Author Jeak Ling Ding Department of Biological Science, National University of Singapore, Singapore, Singapore Search for more papers by this author Yong Zhu Yong Zhu Department of Biological Science, National University of Singapore, Singapore, Singapore Search for more papers by this author Saravanan Thangamani Saravanan Thangamani Department of Biological Science, National University of Singapore, Singapore, Singapore Search for more papers by this author Bow Ho Bow Ho Department of Microbiology, National University of Singapore, Singapore, Singapore Search for more papers by this author Jeak Ling Ding Corresponding Author Jeak Ling Ding Department of Biological Science, National University of Singapore, Singapore, Singapore Search for more papers by this author Author Information Yong Zhu1, Saravanan Thangamani1, Bow Ho2 and Jeak Ling Ding 1 1Department of Biological Science, National University of Singapore, Singapore, Singapore 2Department of Microbiology, National University of Singapore, Singapore, Singapore *Corresponding author. Department of Biological Science, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore. Tel.: +65 6874 2776; Fax: +65 6779 2486; E-mail: [email protected] The EMBO Journal (2005)24:382-394https://doi.org/10.1038/sj.emboj.7600533 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The complement system has been thought to originate exclusively in the deuterostomes. Here, we show that the central complement components already existed in the primitive protostome lineage. A functional homolog of vertebrate complement 3, CrC3, has been isolated from a ‘living fossil’, the horseshoe crab (Carcinoscorpius rotundicauda). CrC3 resembles human C3 and shows closest homology to C3 sequences of lower deuterostomes. CrC3 and plasma lectins bind a wide range of microbes, forming the frontline innate immune defense system. Additionally, we identified CrC2/Bf, a homolog of vertebrate C2 and Bf that participates in C3 activation, and a C3 receptor-like sequence. Furthermore, complement-mediated phagocytosis of bacteria by the hemocytes of horseshoe crab was also observed. Thus, a primitive yet complex opsonic complement defense system is revealed in the horseshoe crab, a protostome species. Our findings demonstrate an ancient origin of the critical complement components and the opsonic defense mechanism in the Precambrian ancestor of bilateral animals. Introduction Comparative studies have suggested common ancestries of innate defense mechanisms in both vertebrates and invertebrates (Hoffmann et al, 1999; Kimbrell and Beutler, 2001). It was proposed that germline-encoded pattern recognition receptors (PRRs) conduct non-self recognition (Medzhitov and Janeway, 2000, 2002; Gordon, 2002) by binding unique microbial cell surface components, which are popularly referred to as pathogen-associated molecular patterns (PAMPs). Examples of PAMPs are endotoxin or lipopolysaccharide (LPS) of Gram-negative bacteria, lipoteichoic acid (LTA) of Gram-positive bacteria, and β-glucan of fungi. Recognition of PAMPs initiates various innate immune responses, such as the activation of opsonic and lytic complement pathways in vertebrates, the coagulation cascade in arthropods and, more generally, the phagocytosis of the pathogen, and the lysis of the pathogen by antimicrobial peptides. To date, known PRRs include membrane-integrated Toll receptors in Drosophila and Toll-like receptors in mammals, and many carbohydrate-binding lectins that circulate in the blood or hemolymph. However, the concept of PAMP and PRR was deemed to be inaccurate, while specific individual molecules rather than the ‘molecular patterns’ are recognized in most instances (Beutler, 2004). As an arthropod, the horseshoe crab is a unique ‘living fossil’ that has been in continuous existence for up to 550 million years (Twenhofel and Shrock, 1935; Størmer, 1952). A comparative study of its innate immune defense mechanisms would contribute to further understanding of the evolution of innate immunity in both invertebrates and vertebrates. LPS- or β-D-glucan-induced coagulation cascade represents a potential defense mechanism in the horseshoe crab (Iwanaga, 2002), and has been employed as a standard sensitive method of endotoxin detection for decades (Ding and Ho, 2001). However, all the coagulation cascade components, known antimicrobial peptides, and most agglutinating lectins are confined within the horseshoe crab hemocytes. How the hemocytes are activated to release these defense molecules in vivo in response to various pathogen invasions remains largely unknown. Although early studies have shown that both the plasma and hemocytes are required for the bactericidal activity (Furman and Pistole, 1976; Pistole and Britko, 1978), the molecular mechanism of the pathogen scavenging pathway in horseshoe crabs remains a mystery to be solved (Kawabata and Tsuda, 2002). In order to define the frontline defense molecules in the horseshoe crab, we used live microbes as affinity matrix to isolate innate immune molecules from the cell-free hemolymph. Serendipitously, a vertebrate C3-characteristic protein, CrC3 (Carcinoscorpius rotundicauda C3), was identified together with several plasma lectins. Similar overall profiles of the ensemble of CrC3 and plasma lectins were found to bind all the representative microbes studied. In addition, we identified CrC2/Bf, a homolog of vertebrate complement 2 (C2) and factor B (Bf), which are trypsin-like serine proteases (Tryp_SP) that take part in proteolytic activation of C3. Furthermore, we show evidence for the production of a putative anaphylactic peptide upon activation of CrC3 by various pathogens. Together with the identification of a vertebrate C3 receptor homolog, a complement defense system homologous to that of deuterostomes is discovered in this ancient protostome lineage. Complement system with opsonic and lytic effector pathways had been thought to exist exclusively in vertebrates. However, the identification of vertebrate C3 homologs with opsonic activity and potential C3 convertases in sea urchins and tunicates suggests an earlier development in the lower deuterostomes of complement system with only opsonic effector pathway, which plays an important role in innate immunity (for recent reviews, see Nonaka and Yoshizaki, 2004a, 2004b). Thus, the finding of homologs of the key components of vertebrate complement system in the horseshoe crab demonstrates an earlier origin of the complement system, in the ancestor of both protostomes and deuterostomes. Results Identification of bacteria-binding proteins using live bacteria as affinity matrix To define the frontline recognition and defense molecules in the horseshoe crab, live Staphylococcus aureus served as ‘bacterial beads’ to adsorb bacteria-binding proteins from the plasma. The plasma proteins that were associated with the bacteria were extracted with different buffers and analyzed by SDS–PAGE (Figure 1A and B). Urea effected higher extraction efficiency than acidic or alkaline buffers. The observed major protein bands of interest from urea extracts were in-gel digested with trypsin and further analyzed by mass spectrometry. Figure 1.Isolation and characterization of CrC3. Bacteria-binding proteins from horseshoe crab plasma were recovered by elution from S. aureus cells incubated with the horseshoe crab cell-free plasma (‘−’, control S. aureus treated in saline; ‘+’, S. aureus incubated with plasma), under different buffer conditions as indicated. (A) Profile of plasma proteins extracted from the bacteria at indicated conditions. Protein sample derived from ∼0.25 ml each of bacteria/plasma was loaded per lane (see Materials and methods). (B) Protein profiles of bacteria after extraction (∼0.25 ml of bacteria per lane). (C) Comparison of total protein profiles of S. aureus cells (‘−’, untreated bacterial cells; ‘+’, bacterial cells incubated with plasma), treated with triethanolamine (pH 11.5 (B)), that were solubilized in nonreducing condition (−2-ME) and reducing condition (+2-ME). Protein extract derived from ∼0.25 ml each of bacterial cells was loaded per lane. (D) 2D SDS–PAGE (nonreducing 1st D and reducing 2nd D) analysis of protein profile of S. aureus cells (treated with plasma) that were solubilized in nonreducing condition. CrC3 of ∼250 kDa (p250) observed in the 1st D was resolved into p75, p50, p36(CrC3), and p34 after reduction. Download figure Download PowerPoint The proteins identified by either peptide mass fingerprint (PMF) and/or MS-MS sequencing are summarized in Table I. p36 and p40 were revealed to be the C. rotundicauda homologs of tachylectin 5a and 5b (TL5a and TL5b), respectively, of the horseshoe crab Tachypleus tridentatus (Iwanaga, 2002). These are the major agglutinating plasma lectins in horseshoe crabs. Thus, p36 and p40 were named as carcinolectins CL5a and CL5b, respectively. p28 matched the T. tridentatus plasma lectin 1, TPL1 (Chen et al, 2001). In addition to these known immune molecules, p34 and p75 (Figure 1) were found to contain peptide fragments homologous to vertebrate C3, while no significant match was found for p50 (Table I). Vertebrate C3 is a large protein of ∼200 kDa, with two chains bridged by disulfide bond and is further cleaved upon pathogen-mediated activation. To test if p34 and p75 are fragments from a similar precursor protein, we reanalyzed the SDS-extracted proteins from plasma-incubated S. aureus cells that were treated with triethanolamine, where most CL5s were removed. The protein profiles were compared by SDS–PAGE under the nonreducing and reducing conditions (Figure 1C), and further examined by 2D SDS–PAGE (Figure 1D, nonreducing 1st D and reducing 2nd D). The results clearly show that p34, p50, and p75 are derived from a protein with an apparent molecular weight ∼250 kDa (under nonreducing condition). In addition, another fragment of CrC3, p36(CrC3), which previously comigrated with p36 (CL5a), was also displayed in the 2D gel. Table 1. Identification of bacteria-binding proteins by MS PMF hit Fragment sequences Short nearly exact match MS-BLAST hit p28 TPL1/TL1 ND N/A N/A p34 No reliable hit SVSFPVVPLK Vertebrate C3s No reliable hit All othersa No HSP p36 TL5a/TL5b EFWLGNDR TL5a/TL5b/ficolin A TL5s, ficolin, fibrinogen MDNDNGGWTLL Fibrinogen beta chain All others (not shown) No HSP p40 TL5b/TL5a ND N/A N/A p50 No reliable hit TGVGGDLGAELAAAPR Hypothetical protein No reliable hit LAAVGGGASAGFLDAAK Hypothetical protein All othersa No HSP p70S Hemocyanin G ND N/A N/A p70L Hemocyanin HR6 YDELGN(LF)/(FL)PGGK Hemocyanin subunits Hemocyanin subunit HR6 All others (not shown) No HSP p75 ND LGLLAVDEAVYLLR Vertebrate C3s Vertebrate C3s All othersa No HSP HSP, high-score pairing; ND, not determined; N/A, not applicable; PMF, peptide mass fingerprint; TL, tachylectin. It should be noted that L is either leucine or isoleucine in the sequences interpreted from mass spectra. a Peptide sequences not shown in the table for p34, p50, and p75 are as follows: p34: NEKVELKATVDK, ELME(EM)ELVK, VTFKFPLNLKGGS(SG)AR, ETWLFDDVYVGPK, ASVAG(GA)NGGSAV(ASV, SVA, VSA, TGV)K, LLSLQLDPTNQGAK, HLYLLK, VGEFPVR, LNVVPEGA(AG)K (bold fragments were used as template for the design of degenerate primers for RT–PCR); p50: FGPVVETALENTEK, AAAYLELK, LLELDEK, EELQQLVDPR, PM(MP)PVSVLADR, LLSMATFQADK, TVGPHSTG(SA)LAAVGGGASAGFLDAAK, YNVPVPPEK,CLVSVLADR; and p75: TGDLLLLGTVPPPK, LGLLAVDEAVYLLR, LDA(GE)KETLSVLLVSGGK, LVVPHVYK, ETLLLVNPR, FGPVVETALF, AGNWFHK, LNLVEAT(TA)K. Characterization of CrC3, a vertebrate C3 homolog Based on the peptide sequences of p34 determined by mass spectrometry, degenerate primers were designed for RT–PCR to amplify a cDNA fragment of p250. Thereafter, 5′ and 3′ RACE was performed to isolate the full-length cDNA sequence (6069 bases, GenBank accession number AF517564), coding for a C3-characteristic precursor protein of 1737 amino-acid residues including a predicted secretion leader peptide of 21 amino acids (Supplementary Figure 1). BLAST homology search yielded closest matches of this sequence with C3 and C4 of vertebrates, C3 of lower deuterostome species, and α-2 macroglobulin (α2M) or thioester protein (TEP) of arthropods and worms. Thus, this protein was named CrC3 (C. rotundicauda C3). The CrC3 protein sequence matches most closely, at 35% similarity, the C3 of lancelet (BAB47146). Consistently, conserved domain search indeed revealed a vertebrate C3/C4/C5-characteristic modular architecture of CrC3 (Figure 2A), which contains a central complement C3/C4/C5-specific anaphylatoxin domain (ANATO) and an extreme C-terminal C345C domain, in addition to the N-terminal A2M-N domain and C-terminal A2M domain shared by the whole TEP superfamily. Figure 2B shows the alignment of ANATO region of CrC3 with the most similar ANATO sequences found in the databases. The ANATO domain of CrC3 shows high sequence similarity to vertebrate C3/C4/C5 ANATOs, and contains all the six cysteine residues critical for C3/C4/C5 anaphylatoxin peptide structure. Figure 2.CrC3 is a homolog of vertebrate C3. (A) Schematic modular organization of CrC3, a standard modular structure of vertebrate C3/C4/C5. The scheme is drawn as revealed by conserved domain search at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. Conserved domain database references: A2M_N (pfam01835.11), N-terminal region of the TEP super family; ANATO (pfam01821.11; smart00104.10), anaphylatoxin-like domain; A2M (pfam00207.11), the C-terminal region of the α2M/TEP/C3/C4/C5 family; and C345C, an additional module at the extreme C-terminus of C3/C4/C5 (pfam01759.11, smart00643.10). (B) Alignment of C3/C4/C5-characteristic ANATO region of CrC3 with the most homologous sequences in frog C4 (BAA11188.1), hagfish C3 (P98094), human C4 (P01028), lamprey C3 (Q00685), mouse C4 (P01029), mouse C5 (P06684), pig C5 (1C5A), and snake C3 (Q01833). The six most-conserved cysteine residues critical for maintaining the structure of C345a peptides through three pairs of disulfide bonds are double-underlined. (C) Phylogeny of CrC3 and related TEPs. The scale bar corresponds to 0.1 estimated amino-acid substitutions per site. The confidence scores (in %) of a bootstrap test of 1000 replicates are indicated in red for major branching nodes. Selected proteins are: aTEP, Anopheles TEP-I; dTEPI, Drosophila TEP1; dTEPII, Drosophila TEP2; dTEPIII, Drosophila TEP3; dTEPIV, Drosophila TEP4; cTEP, Caenorhabditis TEP2; SeC3, cnidaria C3-like protein; BbC3, Amphioxus C3; SpC3, sea urchin C3; HrC3, Halocynthia C3; CiC3, Ciona C3; HaC3, hagfish C3; LaC3, lamprey C3; TrC3, trout C3; CaC3, carp C3-H1; XeC3, Xenopus C3; ChC3, chicken C3; CoC3, cobra C3; CVF, cobra venom factor; HuC3, human C3; GpC3, guinea pig C3; RaC3, rat C3; MoC3, mouse C3; CaC4, carp C4; ShC4, shark C4; ChC4, chicken C4; XeC4, Xenopus C4; MoC4, mouse C4; MoSPL, mouse sex limited protein (M21576); HuC4A, human C4A (K02403); HuC4B, human C4B (U24578); CaC5, carp C5; MoC5, mouse C5; HuC5, human C5; LiA2M, horseshoe crab α2M; LaA2M, lamprey α2M; ChOvo, chicken ovostatin; RaA1I, rat alpha1-inhibitor; HuPZP, human pregnancy zone protein; GpA2M, guinea pig α2M; HuA2M, human α2M; RaA2M, rat α2M; RaA1M, rat alpha1-macroglobulin; MoA2M, mouse α2M; CiTEP, Ciona intestinalis TEP; OmA2M, Ornithodoros (soft tick) α2M. For further information including database accession numbers, see Supplementary Table 1. (D) HA inhibits the attachment of CrC3 to bacteria. HA was added into the plasma to a final concentration of 0.2 or 1.0 M. S. aureus cells were incubated with naïve plasma or plasma supplemented with HA (see Materials and methods) at room temperature for ∼15 min. Washed bacterial cells were lysed in SDS–PAGE loading buffer and analyzed by nonreducing SDS–PAGE. ‘−’, untreated bacterial cells; ‘+’, bacterial cells incubated with horseshoe crab plasma with/without HA supplemented; protein samples derived from ∼50 μl bacteria (OD600 nm ∼0.5) or bacteria/plasma were loaded per lane. Download figure Download PowerPoint Vertebrate complement C3/C4/C5, pan-protease inhibitor α2Ms in both vertebrates and invertebrates, and nonclassical α2M-type TEPs including insect and worm TEPs (Azumi et al, 2003) form a TEP superfamily, and possibly share a common ancient ancestor. C3 is deemed to be the ortholog of C3/C4/C5, while gene duplication brought about the appearance of paralog C4 and C5 in vertebrates (Zarkadis et al, 2001). A phylogenetic tree constituting CrC3 and evolutionarily related TEPs was constructed using the neighbor-joining method (Saitou and Nei, 1987). For comparison, all C3/C4/C5 and α2M/TEP in a recent phylogenetic analysis (Levashina et al, 2001), and 10 more sequences including newly identified C3-like sequences (cnidaria, amphiox, sea urchin), were used for the phylogenetic analysis (Supplementary Table 1). Figure 2C shows an unrooted tree constructed with correction for multiple substitutions. The topologies of all the previously analyzed proteins in the phylogenetic tree appear identical as in the published trees (Levashina et al, 2001; Azumi et al, 2003), except that the C4 subgroup appears to divert from C3 before C5 subgroup. However, the confidence score of C5-diverging node is very low (38%). Without correction for multiple substitutions, the unrooted tree shows that C5 diverges before C4 (Supplementary Figure 2). This difference made by the correction most likely indicates the different evolutionary constraint and molecular evolution rate of C5 and C4 from that of C3, due to the functional divergence. In the unrooted trees, the CrC3 forms a clade with C3s of the lower deuterostome lineage (the amphiox C3 and the sea urchin C3), and the cnidaria C3-like protein (AAN86548.1), which was recently deposited into database (Dishaw et al, unpublished). The phylogenetic analysis clearly shows that CrC3 shares a common ancestor with deuterostome complement proteins. In other words, C3 diverged very early from α2M/TEP, before the protostome–deuterostome split. The C3-like sequence of cnidaria, a primitive animal with radial symmetry (Radiata), may represent the ancient Precambrian C3 ancestor, from which deuterostome C3 and protostome C3 are derived. To test whether CrC3 binds to the bacterial surface through covalent crosslink (ester bond or amide bond) like that of vertebrate C3, we studied the effect of hydroxylamine (HA) on the CrC3 attachment. HA is a small nucleophilic molecule that can penetrate into proteins and deactivate the buried thioester. The addition of HA significantly inhibited the attachment of CrC3 (p250) to bacteria (Figure 2D). This indicates that the thioester of native CrC3 is very important for its binding to bacterial surface, possibly through covalent ester bond linkage. In addition, the previously visible minimal attached fragment, p50 (vertebrate C3dg counterpart), also became undetectable when the HA concentration increased from 0.2 to 1.0 M. Thus, CrC3 is a homolog of vertebrate C3, in both sequence and function. Southern and Northern blot analysis was also carried out to further characterize CrC3. Widespread spatial expression of CrC3 shown by Northern blot analysis (Supplementary Figure 3) implies the importance of CrC3 to the innate immune defense against pathogen infection. Interestingly, Southern genomic blot analysis suggests more than one copy of the CrC3 gene in the horseshoe crab genome (Supplementary Figure 3B). Screening the hepatopancreas cDNA library showed isoforms of the CrC3 cDNAs (unpublished data). Multiplicity of C3 seems to be an evolutionary strategy used by many organisms, especially bony fish where the C3 isoforms were shown to have different reactivities to various activating surfaces (Sunyer et al, 1998). CrC3 exhibits characteristic features of human C3 To date, human C3/C4/C5 has been studied in great detail. Thus, we show a full-sequence alignment of CrC3 and human C3/C4B/C5 (Figure 3). A C3/C4/C5-characteristic α/β joint process site, RKKR (666–669), is located upstream of the ANATO domain. A canonical thioester motif, GCGEQ (1024–1028), and the conserved catalytic His1137 were found to align perfectly. All of the vertebrate species studied to date have both C3 and C4 with a catalytic His residue, while most α2M/TEPs have an Asn at the ‘catalytic’ position of C3 and C4 (Dodds and Law, 1998). C5, which functions as an initiator of lytic membrane attack complex (MAC) formation, and does not require covalent binding to the target pathogen surface, has lost its thioester site in evolution. It is observed that with the exception of one pair of Cys residues at the extreme C-terminus (Cys1637, Cys1646), all Cys in the human C3 are conserved in CrC3, suggesting that CrC3 has a similar disulfide bond bridging pattern as that of human C3. However, the vertebrate C3 convertase-cleavage site, LXR (Nonaka and Yoshizaki, 2004a), is not found in CrC3. Instead, a potential site, EGR, which is very similar to the QGR in the ascidian C3, is observed. A three-chain structure of mature CrC3 is predicted based on the observation of two α/γ cleavage site-like motifs; the α chain was further cleaved to p34 and p50 upon activation, with the observation of a four-chain structure of CrC3 recovered from bacteria. The calculated distances predict that CrC3 is closer to human C3 than to C4 and C5, supporting the orthology of C3 in C3/C4/C5. Figure 3.Sequence comparison of CrC3 and the human C3, C4, and C5. The multiple sequence alignment of CrC3 with human C3 (P01024)/C4B (U24578)/C5 (P01031) was produced with ClustalX. All the cysteine residues of human C3 (except that in the thioester site GCGEQ) and conserved cysteines in the other sequences are highlighted. Other characteristic sites, including proteolytic cleavage sites, thioester sites, catalytic His sites, are also annotated accordingly. The inferred regions of p75, p50, and p34 in the CrC3 sequence are marked. Download figure Download PowerPoint Binding of CrC3 and lectins (CL5s) to representative microbes To test whether the binding of CrC3 and plasma lectins to S. aureus represents a general mechanism of recognition and opsonization of microbes in the horseshoe crab, we compared profiles of plasma proteins associated to Escherichia coli and Kluyveromyces marxianus to the profile of proteins associated with S. aureus. The bound proteins were directly extracted from the microbial cells and analyzed by SDS–PAGE under reducing and nonreducing conditions (Figure 4A). Indeed, similar overall patterns of adsorbed proteins were observed with S. aureus, E. coli, and K. marxianus. The major proteins observed are CrC3 (p250), CL5a, and CL5b. Figure 4.Similar profiles of plasma proteins bound to representative microbes. The major proteins that bind Gram-positive bacteria S. aureus, Gram-negative bacteria E. coli, and fungus K. marxianus are CL5a, CL5b, and CrC3. CrC3 bound to all microbes is fragmented into p75, p50, p36(CrC3), and p34. (A) Comparison of protein profiles of three microbes (untreated or treated with horseshoe crab plasma) under reducing and nonreducing conditions of solubilization (±2-ME). Protein samples derived from ∼0.2 ml each of microbial cells/plasma were loaded per lane. (B) 2D SDS–PAGE (nonreducing 1st D and reducing 2nd D) analysis of protein extracts from E. coli and K. marxianus that were incubated with horseshoe crab plasma. Download figure Download PowerPoint Under nonreducing condition, CL5a and CL5b were mostly in dimeric forms, while reducing condition monomerized them, as reported for TL5a and TL5b (Gokudan et al, 1999). CL5a and CL5b appear to function synergistically in the recognition of various pathogens, which is similar to the report of widespread agglutination spectrum of TL5s. We have identified various isoforms of CL5a and CL5b by molecular cloning (unpublished results), and differential ligand specificity of these isoforms is speculated. The covalent dimerization of CL5a and CL5b, and the fragmentation of CrC3 (p250) were further confirmed by the 2D gel analysis (Figure 4B). The CrC3 attached to all the tested microbes were resolved into four fragments (p75, p50, p36, p34) under reducing condition (Figure 4B). The cleavage of CrC3 and dissociation of some of its fragments from the pathogen surface is also evidenced by the frequent observation of a greater intensity of p50, the minimal attached fragment, over that of p34 and p75 in the reducing SDS–PAGE analysis of total protein extracts or eluents from the microbes. In conclusion, the attachment of CL5s and CrC3 to the surfaces of different groups of invading microbes and subsequent proteolytic cleavage of CrC3 represent the frontline recognition/opsonization mechanism. Identification of CrC2/Bf and PAMP-triggered Tryp_SP activity in the horseshoe crab plasma Among the cDNA sequences identified by subtractive hybridization of the cDNAs of hepatopancreas from naïve and bacteria-challenged horseshoe crab (Ding et al, in preparation), a cDNA fragment with sequence homology to vertebrate Bf and C2 was isolated. Thereafter, 3′ and 5′ RACE was carried out to obtain the full-length C2/Bf homologous sequence, which is referred to as CrC2/Bf. The complete cDNA of CrC2/Bf is 3093 bp (GenBank accession number AY647279), coding for a polypeptide of 889 amino-acid residues including a secretion signal peptide (Supplementary Figure 4). Database search of homologs showed its highest homology to the sea urchin C2/Bf. Sequence alignment of CrC2/Bf with the homologs in the sea urchin, lamprey, Xenopus, and human shows the putative cleavage site for factor D like that in vertebrate C2/Bf, the Mg-binding motif (Tuckwell et al, 1997), and the catalytic triad residues involved in the serine protease domain are well conserved (Figure 5). Figure 5.Multiple alignment of CrC2/Bf and related proteins selected. The deduced amino-acid sequence of CrC2/Bf was aligned with C2 and/or Bf from Xenopus, human, lamprey, and sea urchin. For the database accession numbers of the selected sequences and information on various domains (CCP, VWF, and Tryp_SP), refer to the legend of Figure 6. The predicted active sites for serine protease activity are shown in yellow boxes, and factor D cleavage site is shown by a downward arrow. Consensus Mg2+-binding sites are marked (*). Download figure Download PowerPoint The conserved domain architecture search of this protein indeed shows a C2/Bf-like structure. C2 is the serine protease part of C3-activating convertase complex in both the lectin pathway and classical pathway, while Bf is the serine protease counterpart in the alternative pathway in mammals. Bf and C2 are very similar and are believed to diverge in the jawed vertebrates and often referred to as C2/Bf (or Bf/C2). Ancestral C2/Bf possibly performs the functions of both C2 and Bf of mammals (Zarkadis et al, 2001). In contrast to vertebrate C2/Bf, which contains three complement control protein (CCP) modules, the CrC2/Bf harbors five CCP modules (Figure 6A) like that of C2/Bf of lower deuterostomes (ascidian and erchinoderm), which is regarded as the ancient form of C2/Bf (Smith et al, 1999, 2001). The CCP modules mediate complement binding (Hourcade et al, 1995). An unrooted phylogenetic tree constituting CrC2/Bf and proteins from CrC2/Bf family was constructed (Figure 6B). CrC2/Bf forms a clade with C2/Bf of the ascidian and the echinoderm. Presence of additional CCP modules at the N-terminus of the invertebrate C2/Bf proteins is clearly reflected in the phylogenetic tree, where the protein members harboring additional modules form a separate branch including CrC2/Bf. The similarity in sequence and structure organization of CrC2/Bf to that of the family of vertebrate C2/Bf proteins indicates that CrC2/Bf shares a common ancestor with that of deuterostomes. Figure 6.Identification of CrC2/Bf and PAMP molecule-triggered Tryp_SP activity. (A) Conserved domain architecture of CrC2/Bf revealed by database search at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. Database references: complement control protein module (CCP), smart00033, pfam00084; von Willebrand factor type A domain (VWF), smart00327, pfam00092; trypsin-like serine protease (Tryp_SP), smart00020, pfam00089. (B) Unrooted phylogenetic tree of CrC2/Bf with other C2s and Bfs. The scale bar corresponds to 0.1 estimated amino-acid substitutions per site. The bootstrap test scores (in %) of 1000 replicates are indicated at the major branch nodes. Database accession numbers of the compared sequences are: human C2, AAB97607; human Bf, CAA51389; mouse C2, AAA37381; mouse Bf, P04186; Xenopus Bf, BAA06179; zebra fish Bf, AAC05096; carp C2/Bf, BAA34707; medaka fish, BAA12207; shark Bf, BAB63203; puffer fish C2/Bf, CAD21938; sea urchin Bf, AAC79682; ascidian C2, AAK00631