cDNA cloning of a mannose-binding lectin-associated serine protease (MASP) gene from hagfish (Eptatretus burgeri)

Hagfish, agnathan cyclostome, is the most primitive extant vertebrate and its complement (C) system seems to be a primordial system in comparison with a well-developed C system in gnathostome vertebrates. From a phylogenic perspective of defense mechanisms, we have isolated complement C3 from the serum of hagfish (Eptatretus burgeri). In this study, we first attempted to identify a hagfish Bf or C2 as a C3 convertase by RT-PCR using degenerative primers designed on the basis of the conserved amino acid stretches among the several kinds of serine proteases. Contrary to our expectation, homology search of cloned RT-PCR product suggested that there was a partial cDNA encoding the homologue of neither Bf nor C2 but a mannose-binding lectin-associated serine protease (MASP). Analyses of a full-length cDNA clone isolated from a hagfish liver cDNA library by using the partial cDNA as a probe indicated that this cDNA encoded hagfish MASP 1. This evidence strongly suggests that the hagfish defends itself against pathogens at least by the complement system composed of lectin pathway.     

Characterization of a C3-like cDNA in a coral: phylogenetic implications

C3, C4, and C5 are thiolester-containing proteins (TEPs) of vertebrate complement. The identification of the molecular origin of the TEP family, and more specifically the ancestor protein of complement components C3, C4, and C5, remains a fundamental question. The prevailing paradigm suggests that duplication and divergence of these proteins occurred after the deuterostome split in phylogeny. It is believed that the ancestor of thiolester-containing complement proteins was alpha-2-macroglobulin (A2M)-like, a noncomplement-related protein. Here we describe a C3-like cDNA from a gorgonian coral, Swiftia exserta. This study provides evidence for the origins of a complement-related C3-like gene in the Precambrian period, predating both protostomes and deuterostomes. Furthermore, one may speculate that complement-like opsonic reactions were evolving at the earliest stages of metazoan evolution. This calls for a reassessment of the present concepts concerning the origins and evolution of TEPs.     

The echinoderm adhesome

Although the development of sea urchin embryos has been studied extensively and clearly involves both cell adhesion and cell migration, rather little is known about the adhesion receptors and extracellular matrix molecules involved. The completion of the genome of Strongylocentrotus purpuratus allows a comprehensive survey of the complement of cell-cell and cell-matrix adhesion molecules in this organism. Furthermore, the phylogenetic position of echinoderms offers the opportunity to compare the complement of adhesion proteins between protostome and deuterostome invertebrates and between invertebrate and vertebrate deuterostomes. Many aspects of development and cell interactions differ among these different taxa and it is likely that analysis of the spectrum of adhesion receptors and extracellular matrix proteins can open up new insights into which molecules have evolved to suit particular developmental processes. In this paper, we report the results of an initial analysis along these lines. The echinoderm adhesome (complement of adhesion-related genes/proteins) is similar overall to that of other invertebrates although there are significant deuterostome-specific innovations and some interesting features previously thought to be chordate or vertebrate specific.     

C6-Like and C3-Like Molecules from the Cephalochordate, Amphioxus, Suggest a Cytolytic Complement System in Invertebrates

The mammalian immune system has cytotoxic mechanisms, both cellular and humoral, that destroy the membrane integrity of target cells. The main effector molecules of these cytolytic mechanisms—perforin, used by killer lymphocytes, and the membrane attack complex (MAC) components of the complement system—share a unique module called the MAC/perforin module. Until now, both immunological cytotoxicity and the MAC/perforin module have been reported only in jawed vertebrates. Here, we report the identification of a protein containing the MAC/perforin module from the invertebrate cephalochordate, amphioxus (Branchiostoma belcheri), using expressed sequence tag (EST) analysis of the notochord. The deduced amino acid sequence of this molecule is most similar to the primary structure of human complement component C6 and is designated AmphiC6. AmphiC6 shares a unique modular structure, including the MAC/perforin module, with human C6 and other MAC components. Another EST clone predicts the presence of a thioester-containing protein with the closest structural similarity to vertebrate C3 (therefore designated AmphiC3). AmphiC3 retains most of the functionally important residues of vertebrate C3 and is shown by phylogenetic analysis to be derived directly from the common ancestor of vertebrate C3, C4, and C5. Only opsonic activity has been assigned to the invertebrate complement system until now. Therefore, this is the first molecular evidence for complement-mediated immunological cytotoxicity in invertebrates. Received: 24 August 2001 / Accepted: 12 November 2001     

The macromolecular assembly of pathogen-recognition receptors is impelled by serine proteases, via their complement control protein modules

Although the innate immune response is triggered by the formation of a stable assembly of pathogen-recognition receptors (PRRs) onto the pathogens, the driving force that enables this PRR-PRR interaction is unknown. Here, we show that serine proteases, which are activated during infection, participate in associating with the PRRs. Inhibition of serine proteases gravely impairs the PRR assembly. Using yeast two-hybrid and pull-down methods, we found that two serine proteases in the horseshoe crab Carcinoscorpius rotundicauda are able to bind to the following three core members of PRRs: galactose-binding protein, Carcinolectin-5 and C-reactive protein. These two serine proteases are (1) Factor C, which activates the coagulation pathway, and (2) C2/Bf, a protein from the complement pathway. By systematic molecular dissection, we show that these serine proteases interact with the core “pathogen-recognition complex” via their complement control protein modules.     

Recent advances in the innate immunity of invertebrate animals

Invertebrate animals, which lack adaptive immune systems, have developed other systems of biological host defense, so called innate immunity, that respond to common antigens on the cell surfaces of potential pathogens. During the past two decades, the molecular structures and functions of various defense components that participated in innate immune systems have been established in Arthropoda, such as, insects, the horseshoe crab, freshwater crayfish, and the protochordata ascidian. These defense molecules include phenoloxidases, clotting factors, complement factors, lectins, protease inhibitors, antimicrobial peptides, Toll receptors, and other humoral factors found mainly in hemolymph plasma and hemocytes. These components, which together compose the innate immune system, defend invertebrate from invading bacterial, fungal, and viral pathogens. This review describes the present status of our knowledge concerning such defensive molecules in invertebrates.     

Alternative complement pathway of channel catfish (Ictalurus punctatus): molecular characterization, mapping and expression analysis of factors Bf/C2 and Df

The complement system is important in both innate and adaptive host defense against microbial infection in vertebrates. It contains three pathways: the classical, alternative, and lectin pathways. Complement component factors B and D are two crucial proteases in the alternative pathway. In this study, the genes of complement factors Bf/C2 and Df from channel catfish, Ictalurus punctatus were identified and characterized. Two complement factor B-related genes, Bf/C2A and Bf/C2B, and factor D gene Df were identified. Phylogenetic analysis suggested that Bf/C2A and Bf/C2B is likely orthologous to factor B and factor C2, respectively. Southern blot results suggested that these three genes are all single-copy genes in the catfish genome. The catfish Bf/C2A, Bf/C2B and Df genes were genetically mapped on linkage group 3, 20 and 29, respectively. Bf/C2A and Bf/C2B are highly expressed in liver and kidney, while Df is highly expressed in gill and spleen. After infection with Edwardsiella ictaluri, the expression of Bf/C2A, Bf/C2B and Df genes were found to be remarkably induced in the gill, liver, spleen and kidney at some sampling times, indicating that these three complement factors play a pivotal role in immune responses after the bacterial infection in catfish.     

Phylogenetic analysis of the Wnt gene family and discovery of an arthropod wnt-10 orthologue

Wnt genes encode a conserved family of secreted signaling proteins that play many roles in arthropod and vertebrate development. We have investigated both the phylogenetic history and molecular evolution of this gene family. We have identified a novel Wnt gene in a diversity of arthropods that it is likely an orthologue of the vertebrate Wnt-10 group. Wnt-10 is one of only two cases in which orthology between protostome and deuterostome genes could be consistently assigned based on our analyses. Despite difficulties in assessing orthologies, all of our trees suggest that the most recent common ancestor of protostomes and deuterostomes possessed more than the five Wnt genes known from either arthropods or nematodes. This suggests that Wnt gene loss has occurred during protostome evolution. In addition, we examined the rate of amino acid evolution in the two arthropod/deuterostome orthology groups we identified. We found little rate variation across taxa, with the exception that Drosophila Wnt-1 is evolving more rapidly than all vertebrate and most arthropod orthologues.     

补体系统的进化

补体系统中的大多数组织成员有着特征性的结构域及其特有的结构域组合形式,这使得我们可以利用基因组数据研究分析补体系统的起源与进化。综合文献报道和数据分析,发现补体系统的某些结构域存在于低等的后口动物,甚至在原口动物中也有大量分布,但哺乳动物中特有的结构域组合方式只在后口动物中存在。这些揭示了补体系统拥有比适应性免疫更早的起源,完善的补体系统可能形成在后口动物中的无脊椎动物,而更为原始的补体系统雏形则在原口动物中已经出现。就近些年的研究成果作一综述,以期系统阐明补体系统的起源与进化。     

Structure and the evolutionary implication of the triplicated complement factor B genes of a urochordate ascidian, Ciona intestinalis

To elucidate the evolution of the complement system and MHC class III region, we analyzed the complement factor B (Bf) genes of a urochordate ascidian, Ciona intestinalis. Three different cDNA species, termed CiBf-1, CiBf-2 and CiBf-3, were identified. The deduced amino-acid sequences all contained the usual domains of vertebrate Bf and, in addition, three extra domains at the N-terminus. Furthermore, the serine protease domain of these CiBfs shared unique features with vertebrate complement components C1r/s and mannose-binding lectin-associated serine protease (MASP)-2/3, the absence of the disulfide bond designated histidine loop, and the usage of the AGY codon for the catalytic serine residue. These results indicate that complement genes have evolved through extensive exon shuffling events in the early stage of chordate evolution. Overall deduced amino-acid identity between CiBf-1 and -2 was 88%, whereas CiBf-3 showed 49% identity to both CiBf-1 and CiBf-2. These three CiBf genes were located within an approximately 50-kb genomic region, and exons 3 and 5 of all the three Bf genes showed an extremely high degree of nucleotide identity, indicating that the CiBf genes experienced extensive reorganization, such as duplication and gene conversion, since its divergence from the vertebrate Bf/C2 gene. Fluorescent in situ hybridization (FISH) to the chromosomes showed that genetic loci for the CiBfs, CiC3-1 and CiC3-2 genes are present on three different chromosomes, suggesting the possibility that the linkage among the MHC class III complement genes was established in the vertebrate lineage after its divergence from urochordates.The sequences reported in this paper have been deposited in the DDBJ database (accession nos. AB180044–AB180051).     

Molecular cloning of C4 gene and identification of the class III complement region in the shark MHC

To clarify the evolutionary origin of the linkage of the MHC class III complement genes with the MHC class I and II genes, we isolated C4 cDNA from the banded hound shark (Triakis scyllium). Upon phylogenetic tree analysis, shark C4 formed a well-supported cluster with C4 of higher vertebrates, indicating that the C3/C4 gene duplication predated the divergence of cartilaginous fish from the main line of vertebrate evolution. The deduced amino acid sequence predicted the typical C4 three-subunits chain structure, but without the histidine residue catalytic for the thioester bond, suggesting the human C4A-like specificity. The linkage analysis of the complement genes, one C4 and two factor B (Bf) genes, to the shark MHC was performed using 56 siblings from two typing panels of T. scyllium and Ginglymostoma cirratum. The C4 and one of two Bf genes showed a perfect cosegregation with the class I and II genes, whereas two recombinants were identified for the other Bf gene. These results indicate that the linkage between the complement C4 and Bf genes, as well as the linkage between these complement genes and the MHC class I and II genes were established before the emergence of cartilaginous fish >460 million years ago.     

Further studies on lipopolysaccharide-sensitive serine protease zymogen (factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha-chymotrypsin, not by trypsin

An intracellular serine protease zymogen, factor C, is an initiator in the hemolymph coagulation system of horseshoe crab. We purified this zymogen from the hemocytes of the American horseshoe crab, Limulus (L.) polyphemus, the objective being to compare its properties with those of the Japanese horseshoe crab, Tachypleus (T.) tridentatus, factor C. The purified zymogen L.-factor C showed similar properties to those of T.-factor C, in terms of molecular mass (123,000), amino acid composition (1,011 residues), subunit structure (two chains), and antigenicity. Like the zymogen T.-factor C, this zymogen was also activated autocatalytically in the presence of bacterial lipopolysaccharide (LPS) and its synthetic lipid A analogue. A most interesting finding is that both protease zymogens are rapidly activated by alpha-chymotrypsin or rat mast cell chymase, but not by trypsin. The active enzyme factor C showed alpha-thrombin-like specificity toward synthetic tripeptide substrates. This factor C was also strongly inhibited by an alpha-thrombin inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. Thus, the enzymatic properties of factor C are similar to those of mammalian alpha-thrombin. On the other hand, the coagulation cascade system present in the hemocyte lysate was activated when chymotrypsin, free from LPS, was added to the lysate used to detect the endotoxins. The implication of our findings is that the chymotrypsin-catalyzed initiation of the horseshoe crab coagulation system is unique, since all known mammalian coagulation, fibrinolysis and complement systems are initiated by trypsin-like enzymes.     

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller)

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.     

Phylogenetic analysis of the 5-aminolevulinate synthase gene.

The evolution of 5-aminolevulinate synthase (ALS) was studied by acquiring sequence data and generating phylogenetic trees. Gene sequences were already available for a variety of vertebrates (which have both a housekeeping and an erythroid form of the gene), fungi, alpha-proteobacteria, and one protist and one protostome. In order to generate representative trees, ALS sequence data were acquired from various deuterostomes and protostomes. The species and tissues selected for study were beluga whale liver, hagfish blood, sea urchin gonadal tissue, cuttlefish hepatopancreas, horseshoe crab hepatopancreas, and bloodworm blood. The new sequences and those previously published were examined for the presence of heme-regulatory motifs (HRMs) and iron-responsive elements (IREs). The HRMs are present in almost all eukaryotic species, which suggests their fundamental role in the regulation of ALS. The IREs are present in all vertebrate erythroid forms of ALS, which indicates that in those animals, expression of the erythroid form of the enzyme and, hence, hemoglobin production can be influenced by the intracellular content of iron. The new sequences were aligned with previously reported ALS sequences, and phylogenetic analyses were performed. The resulting trees provided evidence regarding the timing of the gene duplication event that led to the two forms of the ALS gene in vertebrates. It appears that the housekeeping and erythroid forms of ALS probably arose before the divergence of hagfish from the deuterostome line leading to the vertebrates. The data also add to the evidence indicating that alpha-proteobacteria are the nearest contemporary relatives of mitochondria.     

Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner

Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.     

Molecular and immunochemical demonstration of a novel member of Bf/C2 homolog in amphioxus Branchiostoma belcheri: implications for involvement of hepatic cecum in acute phase response

A complement system operating via the alternative pathway (AP) similar to that of vertebrates has been demonstrated in the primitive chordate amphioxus. However, the factor B (Bf), a key specific protease in the AP, remains elusive in amphioxus to date. We demonstrate in this study the presence of a factor B-like protein in amphioxus Branchiostoma belcheri by both immunoblotting and molecular cloning. The factor B-like protein was immunohistochemically localized in the hepatic cecum. The B. belcheri factor B-like gene, BbBf/C2, encoded a mosaic protein with three complement control protein (CCP) domains, a von Willebrand factor A (vWFA) domain and a serine protease (SP) domain. Peculiarly, BbBf/C2 had an epidermal growth factor-like domain (EGF_CA) located between CCP1 and CCP2, therefore BbBf/C2 had a modular structure of CCP-EGF_CA-CCP-CCP-vWFA-SP, making it a novel member of Bf/C2 family proteins. Real-time PCR assay revealed that lipopolysaccharide (LPS) challenge resulted in a quick and continuously significant up-regulation of BbBf/C2 expression in the hepatic cecum, while BbBf/C2 was only expressed for a short time in the hind-gut following LPS challenge though the expression level was temporarily higher than that in the hepatic cecum. Similarly, immuno-dot blotting showed that challenge with LPS triggered a significant elevation of BbBf/C2 synthesis in the hepatic cecum and hind-gut, with a higher rise in the former tissue. These results indicate that both hepatic cecum and hind-gut may be involved in the immune response induced by LPS, but the hepatic cecum, like the vertebrate liver, is the primary tissue synthesizing BbBf/C2 in response to LPS challenge, thereby playing a major role in the acute phase response.     

Molecular mechanisms of the shrimp clotting system

Shrimp, like other invertebrates, relies solely on its innate immune system, to combat invading pathogens. The invertebrate immune system has ancient origins that involve cellular and humoral responses. The clotting system of the humoral immune response is the first line of defense against pathogens and also serves to prevent blood loss during injury and wound healing. Tranglutaminase and clotting protein are molecules involved in the blood clotting system of crayfish and shrimp. Studies have shown that the shrimp clotting system is linked with the activation of antimicrobial peptides, similar to that of the horseshoe crab. Unlike the horseshoe crab and crayfish blood coagulation which are well studied systems, blood clotting in shrimp remains poorly understood. Here we review the shrimp clotting system and its involvement in innate immunity.     

Role of carcinoscorpin, a haemolymph lectin of horseshoe crabCarcinoscorpius rotundacauda as humoral factor

This study demonstrated that a marine Indian horseshoe crab,Carcinoscorpious rotundacauda showed higher self defence in an experimental infection upon the induction of its circulatory lectin, carcinoscorpin. It resisted an infection with 10⁷ liveEscherichia coli per crab when the circulatory carcinoscorpin was 8–16-fold higher after administering 2-ketodeoxyoctonate (Kdo) into the live crab. The naive control with its natural level of circulatory lectin could tolerate a maximum infective dose of 10⁶ liveE. coli per crab. Bacterial killing and phagocytic uptake in association with the isolated crab amoebocytes in anex vivo system was considerably higher for the lectin opsonizedE. coli compared to unopsonized samples. Carcinoscorpin is thus functionally an opsonin in the defence of the primitive marine arthropod,C. rotundacauda, like vertebrate antibody, a humoral factor involved in the defence of the host. The natural capacity for defending an infection with 10⁶ liveE. coli per crab suggested that the crabs in the natural habitat hardly face such an infection and is possibly one of the reasons for its survival over millions of years as a living fossil.     

The Tol1 element of the medaka fish, a member of the hAT transposable element family, jumps in Caenorhabditis elegans

Tol1 is a DNA-based transposable element residing in the genome of the medaka fish Oryzias latipes, and has been proven to be transposed in various vertebrate species, including mammals. This element belongs to the hAT (hobo/Activator/Tam3) transposable element family, whose members are distributed in a wide range of organisms. It is thus possible that Tol1 is mobile in organisms other than vertebrates. We here show that transposition of this element occurs in the nematode Caenorhabditis elegans. A donor plasmid containing a Tol1 element and a helper plasmid carrying the transposase gene were delivered into gonad cells and, after several generations of culturing, were recovered from worms. PCR analysis of the donor plasmid, using primers that encompassed the Tol1 element, revealed excision of the Tol1 portion from the plasmid. Analysis of genomic DNA of the worms by the inverse PCR method provided evidence that Tol1 had been integrated into the C. elegans chromosomes. Vertebrates and C. elegans are phylogenetically distantly related organisms in that the former are deuterostomes and the latter a protostome animal. Our results indicate (1) the transposition reaction of the Tol1 element requires, besides the transposase, no factors from host cells, or (2) the host factors, even if required, are those that are common to protostomes and deuterostomes. The results also have significance for the development of a gene transfer vector and other biotechnology tools for C. elegans.     

Reconstructing the Origins of the Somatostatin and Allatostatin-C Signaling Systems Using the Accelerated Evolution of Biodiverse Cone Snail Toxins

Somatostatin and its related peptides (SSRPs) form an important family of hormones with diverse physiological roles. The ubiquitous presence of SSRPs in vertebrates and several invertebrate deuterostomes suggests an ancient origin of the SSRP signaling system. However, the existence of SSRP genes outside of deuterostomes has not been established, and the evolutionary history of this signaling system remains poorly understood. Our recent discovery of SSRP-like toxins (consomatins) in venomous marine cone snails (Conus) suggested the presence of a related signaling system in mollusks and potentially other protostomes. Here, we identify the molluscan SSRP-like signaling gene that gave rise to the consomatin family. Following recruitment into venom, consomatin genes experienced strong positive selection and repeated gene duplications resulting in the formation of a hyperdiverse family of venom peptides. Intriguingly, the largest number of consomatins was found in worm-hunting species (>400 sequences), indicating a homologous system in annelids, another large protostome phylum. Consistent with this, comprehensive sequence mining enabled the identification of SSRP-like sequences (and their corresponding orphan receptor) in annelids and several other protostome phyla. These results established the existence of SSRP-like peptides in many major branches of bilaterians and challenge the prevailing hypothesis that deuterostome SSRPs and protostome allatostatin-C are orthologous peptide families. Finally, having a large set of predator–prey SSRP sequences available, we show that although the cone snail’s signaling SSRP-like genes are under purifying selection, the venom consomatin genes experience rapid directional selection to target receptors in a changing mix of prey.