Amphioxus Branchiostoma Japonicum Research Articles

Identification and functional characterization of a novel cystatin in amphioxus, ancient origin of vertebrate type-2 cystatin homologues

Cystatins are well known as a vast superfamily of functional proteins participated in the reversible competitive inhibition of cysteine proteases. Currently, increasing evidences point to the extensive phylogenetic diversity and crucial immune roles of type-2 cystatins in the vertebrate species. However, no information is available regarding the homologue in cephalochordate amphioxus, the representative of most basal living chordates, whose immune regulation are still ambiguous. Here, we clearly identified the presence of type-2 cystatin gene in amphioxus Branchiostoma japonicum, termed Bjcystatin-2, which was structurally characterized by typical wedge-shaped cystatin feature. Evolutionary analyses revealed that Bjcystatin-2 is the ancestral type-2 cystatin for chordates, with gene diversity emerging through duplication events. The expression of Bjcystatin-2 showed tissue-specific profile and was inducible upon invasive pathogens. Significantly, the recombinant Bjcystatin-2 exhibited not merely cathepsin L inhibitory activity, but also the ability to bind with bacteria and their characteristic molecules. Furthermore, Bjcystatin-2 also showed the capacity to enhance the macrophage-driven bacterial phagocytosis and to attenuate the generation of pro-inflammatory cytokines within macrophages. In summary, these findings demonstrate that Bjcystatin-2 exhibits dual role acting as both a protease inhibitor and an immunoactive molecule, greatly enriching our understanding of immune defense mechanisms of type-2 cystatin within the amphioxus.

Amphioxus adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U and A-to-I deamination of DNA

Adenosine-to-inosine tRNA-editing enzyme has been identified for more than two decades, but the study on its DNA editing activity is rather scarce. We show that amphioxus (Branchiostoma japonicum) ADAT2 (BjADAT2) contains the active site ‘HxE-PCxxC’ and the key residues for target-base-binding, and amphioxus ADAT3 (BjADAT3) harbors both the N-terminal positively charged region and the C-terminal pseudo-catalytic domain important for recognition of substrates. The sequencing of BjADAT2-transformed Escherichia coli genome suggests that BjADAT2 has the potential to target E. coli DNA and can deaminate at TCG and GAA sites in the E. coli genome. Biochemical analyses further demonstrate that BjADAT2, in complex with BjADAT3, can perform A-to-I editing of tRNA and convert C-to-U and A-to-I deamination of DNA. We also show that BjADAT2 preferentially deaminates adenosines and cytidines in the loop of DNA hairpin structures of substrates, and BjADAT3 also affects the type of DNA substrate targeted by BjADAT2. Finally, we find that C89, N113, C148 and Y156 play critical roles in the DNA editing activity of BjADAT2. Collectively, our study indicates that BjADAT2/3 is the sole naturally occurring deaminase with both tRNA and DNA editing capacity identified so far in Metazoa.

Localization of trypsin, chymotrypsin and elastase in the digestive tract of amphioxus Branchiostoma japonicum with implications to the origin of vertebrate pancreas

The organ pancreas is characteristic of all vertebrates, but its evolutionary origin remains largely unknown. The serine proteinases trypsin, chymotrypsin and elastase are produced primarily in the pancreas in vertebrates. Here we clearly demonstrate by enzymatic activity assay, histochemical staining and in situ hybridization that trypsin, chymotrypsin and elastase are widely distributed in the digestive tract of the amphioxus Branchiostoma japonicum, especially in the hepatic caecum and mid-gut. This suggests that an extensive region of the amphioxus digestive tract including the hepatic caecum is homologous to the vertebrate exocrine pancreatic cells, providing a new angle for the study of the origin and evolution of vertebrate pancreas.

Amphioxus ribosomal proteins RPS15, RPS18, RPS19 and RPS30-precursor act as immune effectors via killing or agglutinating bacteria

Previous studies show that some ribosomal proteins perform immune effector functions via killing bacteria directly. However, it remains largely unknown about other effector functions of ribosomal proteins during a bacterial infection. In this study, we expressed and purified four ribosomal proteins of the amphioxus Branchiostoma japonicum, termed rBjRPS15, rBjRPS18, rBjRPS19 and rBjRPS30-precursor (rBjRPS30P). They all exhibited bactericidal activity against Gram-positive Staphylococcus aureus, and with the exception of rBjRPS19 and rBjRPS30P, were capable of killing Gram-negative Escherichia coli. Importantly, rBjRPS15, rBjRPS19 and rBjRPS30P were able to agglutinate S. aureus in the presence of Mg2+, but none of them could agglutinate E. coli even in the presence of Mg2+ or Ca2+. Moreover, the S. aureus agglutination was achieved by the binding of these three proteins to the peptidoglycan component of the bacterial cell wall. This is the first report showing that some ribosomal proteins possess bacterial agglutinating activity, and these data provide a new angle to the roles of ribosomal proteins in immune defense.

Identification of amphioxus protein disulfide isomerase as both an enzyme and an immunocompotent factor

Previous studies have shown that protein disulfide isomerase (PDI), a member of the thioredoxin (TRX) superfamily, are broadly associated with immune responses in a variety of animals. However, it remains largely unknown about the direct roles of PDIs during a bacterial infection. In this study, we identified the presence of a single pdi gene in the amphioxus Branchiostoma japonicum, Bjpdi. The deduced protein BjPDI is structurally characterized by the presence of four Trx-like domains in the order of a, b, b’ and a’ and a short acidic C-terminal tail, that are characteristic of PDIs. We demonstrated that rBjPDI displayed both thiol reductase and disulfide bond isomerase activities, indicating comparability of BjPDI with PDIs in term of enzymatic activities. We also showed that rBjPDI induced bacterial agglutination and exhibited a lectin-like activity capable of binding both bacteria (E. coli and S. aureus) and their signature molecules LPS and LTA. Furthermore, BjPDI could kill S. aureus via inducing membrane depolarization and intracellular ROS production in vitro, and treatment of amphioxus with a blocking anti-PDI antibody in vivo markedly reduced the survival rate of amphioxus following attack by S. aureus. Collectively, our study demonstrates that amphioxus protein disulfide isomerase acts as both an enzyme and an immunocompotent factor, and reports the specific function and mode of action of PDIs in immune responses.

Lectin-like and bacterial-agglutinating activities of heat shock proteins Hsp5 and Hsp90α from amphioxus Branchiostoma japonicum

Previous studies have shown that heat shock proteins (Hsps) are broadly associated in immune responses in a variety of animals. However, it remains largely unknown about the direct roles of Hsps during a bacterial infection. In this study, we have cloned and characterized the cDNAs of two Hsp genes in the amphioxus Branchiostoma japonicum, termed Bjhsp5 and Bjhsp90α, the first ones in this evolutionarily important animal. Both Bjhsp5 and Bjhsp90α showed distinct tissue expression patterns, and were inducible by challenge with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), suggesting they may be involved in anti-infectious responses. We also showed that both BjHsp5 and BjHsp90α displayed lectin-like property with affinity to both the Gram-negative and -positive bacteria as well as their signature molecules LPS and LTA, hinting they may both act as a pattern recognition receptor, capable of identifying pathogens. In addition, we found that BjHsp5 and BjHsp90α were both able to agglutinate the Gram-negative and -positive bacteria in the presence of Ca2+, suggesting they may be able to trap the invading pathogens together in vivo, avoiding them moving around and thereby protecting the host from pathogenic attack. These data provide a new angle to the roles of Hsps in immune defense.

Subtle Difference Generates Big Dissimilarity: Comparison of Enzymatic Activity in KL1 and KL2 Domains of Lancelet Klotho

Klotho, a putative aging suppressor, shares sequence similarity with members of the glycosidase family 1. It has been identified in several vertebrate species, but only mouse Klotho has so far been proven to exhibit β-glucuronidase activity. Thus, the argument that Klotho from animals other than mouse has glycosidase activity remains open. Moreover, little information is available regarding the structure-activity relationship of Klotho. Here, we demonstrate the presence of a single klotho gene in the amphioxus Branchiostoma japonicum, Bjklotho, which possesses two tandem domains named BjKL1 and BjKL2, and each of them has two glutamic acid residues that have been shown to be involved in the catalytic activity of family 1 glycosidase. Enzymatic activity assays of the recombinant proteins BjKL1 and BjKL2 revealed that only BjKL2 displayed β-glucosidase activity, but BjKL1 did not. Structural analysis showed that there existed nine consecutive but not conserved residues in the β6α6 loop, which affects the conformational form in the entrance to the catalytic pocket of BjKL1 and BjKL2, thereby leading to a subtle difference in the enzyme-substrate binding and interaction. Furthermore, the substitution of the nine residues 354QNRVDPNDT362 in BjKL1 by the residues 884EDNVVVGAA892 in BjKL2 resulted in significant increase in β-glucosidase activity in the BjKL1 mutant. Our results indicate that BjKL2 possesses β-glucosidase, the first data as such in invertebrates. We also identify, for the first time, the residues 884EDNVVVGAA892 in BjKL2 a sequence critical and indispensable for glucosidase.

BMP signaling is required for amphioxus tail regeneration.

Amphioxus, a cephalochordate, is an ideal animal in which to address questions about the evolution of regenerative ability and the mechanisms behind the invertebrate to vertebrate transition in chordates. However, the cellular and molecular basis of tail regeneration in amphioxus remains largely ill-defined. We confirmed that the tail regeneration of amphioxus Branchiostoma japonicum is a vertebrate-like epimorphosis process. We performed transcriptome analysis of tail regenerates, which provided many clues for exploring the mechanism of tail regeneration. Importantly, we showed that BMP2/4 and its related signaling pathway components are essential for the process of tail regeneration, revealing an evolutionarily conserved genetic regulatory system involved in regeneration in many metazoans. We serendipitously discovered that bmp2/4 expression is immediately inducible by general wounds and that expression of bmp2/4 can be regarded as a biomarker of wounds in amphioxus. Collectively, our results provide a framework for understanding the evolution and diversity of cellular and molecular events of tail regeneration in vertebrates.

Identification of neuroglobin as a novel player in anti-bacterial responses in amphioxus

Theoretical considerations support various functions of neuroglobin (Ngb), but further studies are required for full characterization of these functions. In this study, we identified the presence of a single Ngb gene, BjNgb, in the amphioxus Branchiostoma japonicum. BjNgb was expressed in various tissues including the notochord, gonads (ovary and testis) and gill, and up-regulated significantly in response to the challenge with LPS and LTA, suggesting involvement in immune response of amphioxus against bacterial infection. In accord, we demonstrated for the first time that recombinant BjNgb (rBjNgb) not only interacted with the Gram-positive and negative bacteria as well as their conserved surface components LPS and LTA, but also enhanced the phagocytosis of bacteria by macrophages. Collectively, these data suggest that BjNgb is a novel player in amphioxus, via functioning as a pattern recognition molecule and an opsonin.

Functional characterization of avidins in amphioxus Branchiostoma japonicum: Evidence for a dual role in biotin-binding and immune response

Avidin is well known for its high affinity to biotin and has been found in many egg-laying vertebrate species. However, little is known about avidin in invertebrate species to date. Here we clearly showed the presence of two avidin genes, Bjavidin1 and Bjavidin2, in the amphioxus Branchiostoma japonicum, the first ones in non-vertebrate animals. We also showed that the expression of both Bjavidin1 and Bjavidin2 were inducible by progesterone, LTA and LPS. Moreover, we demonstrated for the first time that in addition to biotin-binding, the recombinant proteins rBjAVIDIN1 and rBjAVIDIN2 were not only able to interact with Gram-positive and negative bacteria as well as their conserved surface components LTA and LPS but also to enhance phagocytosis of bacteria by macrophages, suggesting that BjAVIDIN1 and BjAVIDIN2 both function as pattern recognition receptors and opsonins. It is thus clear that avidin may play a dual role in biotin-binding and immune response.

Keeping amphioxus in the laboratory: an update on available husbandry methods

Cephalochordates, commonly known as amphioxus or lancelets, are small, marine animals that can be found in coastal habitats of temperate, subtropical, and tropical waters. Together with vertebrates and tunicates, the cephalochordates belong to the chordate phylum, whose members are characterized by a number of conserved morphological features, such as a dorsal nerve cord, a notochord, a pharynx, a segmented musculature as well as a post-anal tail. Due to their basal position within the phylum, cephalochordates have become essential models for studying the evolutionary origin and diversification of vertebrates. Here, we present the currently available methods for maintaining and rearing cephalochordates in a laboratory environment, focusing on five species: the European amphioxus (Branchiostoma lanceolatum), the Florida amphioxus (Branchiostoma floridae), the Chinese amphioxus (Branchiostoma belcheri), the Japanese amphioxus (Branchiostoma japonicum), and the Bahamas lancelet (Asymmetron lucayanum). In addition to reviewing the protocols for capture, transport, aquaculture, and feeding of adults, we discuss methods for controlling gonad development and spawning, as well as for growing embryos and larvae. This information is complemented by observations from our animal facility on the European amphioxus (Branchiostoma lanceolatum). In sum, this work summarizes the latest advances in cephalochordate animal husbandry and highlights challenges for improving the use of these animals as laboratory model systems.

Alpha 2 macroglobulin is a maternally-derived immune factor in amphioxus embryos: New evidence for defense roles of maternal immune components in invertebrate chordate.

In fish, a series of maternal derived immune components have been identified in their eggs or embryos at very early stages, which are proposed to provide protections to themselves against pathogenic attacks from hostile environment. The phenomenon of maternal immunity has been also recorded in several invertebrate species, however, so far, very limited information about the maternal immune molecules are available. In this study, it was demonstrated maternal alpha2 macroglobulin (A2m) protein, an important innate immune factor, exists in the fertilized eggs of amphioxus Branchiostoma japonicum, an invertebrate chordate. Maternal mRNA of A2m was also detected in amphioxus embryos at very early developing stages. In addition, it was recorded that the egg lysate prepared from the newly fertilized eggs can inhibit the growth of both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus in a concentration dependent manner. The bacteriostatic activity can be reduced notably after precipitated A2m with anti-A2m antibody. Thus maternal A2m is partly attributed to the bacteriostatic activity. It was further demonstrated that recombinant A2m can bind to E. coli cells directly. All these points come to a result that A2m is a maternal immune factor existing in eggs of invertebrate chordate, which may be involved in defense their embryos against harmful microbes' attacks.

Identification of a novel antimicrobial peptide from amphioxus Branchiostoma japonicum by in silico and functional analyses.

The emergence of multi-drug resistant (MDR) microbes leads to urgent demands for novel antibiotics exploration. We demonstrated a cDNA from amphioxus Branchiostoma japonicum, designated Bjamp1, encoded a protein with features typical of antimicrobial peptides (AMPs), which is not homologous to any AMPs currently discovered. It was found that Bjamp1 was expressed in distinct tissues, and its expression was remarkably up-regulated following challenge with LPS and LTA. Moreover, the synthesized putative mature AMP, mBjAMP1, underwent a coil-to-helix transition in the presence of TFE or SDS, agreeing well with the expectation that BjAMP1 was a potential AMP. Functional assays showed that mBjAMP1 inhibited the growth of all the bacteria tested, and induced membrane/cytoplasmic damage. ELISA indicated that mBjAMP1 was a pattern recognition molecule capable of identifying LPS and LTA. Importantly, mBjAMP1 disrupted the bacterial membranes by a membranolytic mechanism. Additionally, mBjAMP1 was non-cytotoxic to mammalian cells. Collectively, these data indicate that mBjAMP1 is a new AMP with a high bacterial membrane selectivity, rendering it a promising template for the design of novel peptide antibiotics against MDR microbes. It also shows for the first time that use of signal conserved sequence of AMPs is effective identifying potential AMPs across different animal classes.

Identification and biochemical characterization of polyamine oxidases in amphioxus: Implications for emergence of vertebrate-specific spermine and acetylpolyamine oxidases

Polyamine oxidases (PAOs) have been identified in a wide variety of animals, as well as in fungi and plant. Generally, plant PAOs oxidize spermine (Spm), spermidine (Spd) and their acetylated derivatives, N1-acetylspermine (N1-Aspm) and N1-acetylspermidine (N1-Aspd), while yeast PAOs oxidize Spm, N1-Aspm and N1-Aspd, but not Spd. By contrast, two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of Spm and N1-Aspm/N1-Aspd, respectively. However, our knowledge on the biochemical and structural characterization of PAOs remains rather limited, and their evolutionary history is still enigmatic. In this study, two amphioxus (Branchiostoma japonicum) PAO genes, named Bjpao1 and Bjpao2, were cloned and characterized. Both Bjpao1 and Bjpao2 displayed distinct tissue-specific expression patterns. Notably, rBjPAO1 oxidized both spermine and spermidine, but not N1-acetylspermine, whereas rBjPAO2 oxidizes both spermidine and N1-acetylspermine, but not spermine. To understand structure-function relationship, the enzymatic activities of mutant BjPAOs that were generated by site-directed mutagenesis and expressed in E. coli were examined, The results indicate that the residues H64, K301 and T460 in rBjPAO1, and H69, K315 and T467 in rBjPAO2 were all involved in substrate binding and enzyme catalytic activity to some extent. Based on our results and those of others, a model depicting the divergent evolution and functional specialization of vertebrate SMO and APAO genes is proposed.

Identification and functional characterization of viperin of amphioxus Branchiostoma japonicum: Implications for ancient origin of viperin-mediated antiviral response

Viperin, an antiviral protein, has been shown to be active against a wide range of DNA and RNA viruses, but no information is available regarding functional characterization of viperin in invertebrate species. In this study, we clearly demonstrate that amphioxus (Branchiostoma japonicum) viperin, BjVip, has features in common with those of vertebrate viperin, including the presence of the SAM superfamily domain with the characteristic CNYKCGFC motif, syntenic conservation, and predicted 3D structure. Bjvip exhibits a tissue-specific expression with abundant levels in the hepatic cecum, hind-gut, gill and muscle, and following challenge with the viral mimic poly I:C, its expression is significantly up-regulated, suggesting an involvement of BjVip in immune response of amphioxus against viral infection. Importantly, we show that the cells transfected with Bjvip is able to kill LCDV or inhibiting its propagation, and co-incubation of rBjVip with WSSV markedly attenuates its infectivity. Thus, we provide the first evidences that amphioxus viperin, like that of vertebrates, is capable of promoting resistance against viral infection in vitro and in vivo, indicating that viperin-mediated antiviral response already emerged in the primitive chordate. We also prove that amphioxus viperin has evolved under positive selection.

RFamide peptides in agnathans and basal chordates

Since a peptide with a C-terminal Arg-Phe-NH2 (RFamide peptide) was first identified in the ganglia of the venus clam in 1977, RFamide peptides have been found in the nervous system of both invertebrates and vertebrates. In vertebrates, the RFamide peptide family includes gonadotropin-inhibitory hormone (GnIH), neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP), pyroglutamylated RFamide peptide/26RFamide peptide (QRFP/26RFa), and kisspeptins (kiss1 and kiss2). They are involved in important functions such as the release of hormones, regulation of sexual or social behavior, pain transmission, reproduction, and feeding. In contrast to tetrapods and jawed fish, the information available on RFamide peptides in agnathans and basal chordates is limited, thus preventing further insights into the evolution of RFamide peptides in vertebrates. In this review, we focus on the previous research and recent advances in the studies on RFamide peptides in agnathans and basal chordates. In agnathans, the genes encoding GnIH, NPFF, and PrRP precursors and the mature peptides have been identified in lamprey (Petromyzon marinus) and hagfish (Paramyxine atami). Putative kiss1 and kiss2 genes have also been found in the genome database of lamprey. In basal chordates, namely, in amphioxus (Branchiostoma japonicum), a common ancestral form of GnIH and NPFF genes and their mature peptides, as well as the ortholog of the QRFP gene have been identified. The studies revealed that the number of orthologs of vertebrate RFamide peptides present in agnathans and basal chordates is greater than expected, suggesting that the vertebrate RFamide peptides might have emerged and expanded at an early stage of chordate evolution.

Identification, expression and regulation of amphioxus G6Pase gene with an emphasis on origin of liver

Vertebrate glucose-6-phosphatase (G6Pase) consists of three isozymes: G6Pase-I, G6Pase-II and G6Pase-III. Despite extensive study on G6Pases in vertebrates, information regarding expression and regulation of G6Pase genes is rather limited in invertebrates. Here we report the identification of G6Pase gene in amphioxus Branchiostoma japonicum, which is abundantly expressed in the digestive diverticulum and ovary in a tissue-specific manner. The phylogenetic and genomic structure analyses reveal that amphioxus G6Pase bears close resemblance to vertebrate G6Pase-III and represents the archetype of vertebrate G6Pase from which the vertebrate G6Pase isoforms may be originated by 2 rounds of genome duplication during vertebrate evolution. We also demonstrate that GH treatment induces a closely similar expression pattern and trend of g6pases in both zebrafish and amphioxus, and that G6Pase activity in amphioxus digestive diverticulum is subjected to regulation of feeding and fasting as observed in vertebrates. Collectively, all these provide functional evidences supporting the notion that the digestive diverticulum is the liver homologue playing a key role in maintaining the glucose homeostasis in amphioxus.

Two apextrin-like proteins mediate extracellular and intracellular bacterial recognition in amphioxus.

Animals exploit different germ-line-encoded proteins with various domain structures to detect the signature molecules of pathogenic microbes. These molecules are known as pathogen-associated molecular patterns (PAMPs), and the host proteins that react with PAMPs are called pattern recognition proteins (PRPs). Here, we present a novel type of protein domain structure capable of binding to bacterial peptidoglycan (PGN) and the minimal PGN motif muramyl dipeptide (MDP). This domain is designated as apextrin C-terminal domain (ApeC), and its presence was confirmed in several invertebrate phyla and subphyla. Two apextrin-like proteins (ALP1 and ALP2) were identified in a basal chordate, the Japanese amphioxus Branchiostoma japonicum (bj). bjALP1 is a mucosal effector secreted into the gut lumen to agglutinate the Gram-positive bacterium Staphylococcus aureus via PGN binding. Neutralization of secreted bjALP1 by anti-bjALP1 monoclonal antibodies caused serious damage to the gut epithelium and rapid death of the animals after bacterial infection. bjALP2 is an intracellular PGN sensor that binds to TNF receptor-associated factor 6 (TRAF6) and prevents TRAF6 from self-ubiquitination and hence from NF-κB activation. MDP was found to compete with TRAF6 for bjALP2, which released TRAF6 to activate the NF-κB pathway. BjALP1 and bjALP2 therefore play distinct and complementary functions in amphioxus gut mucosal immunity. In conclusion, discovery of the ApeC domain and the functional analyses of amphioxus ALP1 and ALP2 allowed us to define a previously undocumented type of PRP that is represented across different animal phyla.

Evolutionary origin of GnIH and NPFF in chordates: insights from novel amphioxus RFamide peptides.

Gonadotropin-inhibitory hormone (GnIH) is a newly identified hypothalamic neuropeptide that inhibits pituitary hormone secretion in vertebrates. GnIH has an LPXRFamide (X = L or Q) motif at the C-terminal in representative species of gnathostomes. On the other hand, neuropeptide FF (NPFF), a neuropeptide characterized as a pain-modulatory neuropeptide, in vertebrates has a PQRFamide motif similar to the C-terminal of GnIH, suggesting that GnIH and NPFF have diverged from a common ancestor. Because GnIH and NPFF belong to the RFamide peptide family in vertebrates, protochordate RFamide peptides may provide important insights into the evolutionary origin of GnIH and NPFF. In this study, we identified a novel gene encoding RFamide peptides and two genes of their putative receptors in the amphioxus Branchiostoma japonicum. Molecular phylogenetic analysis and synteny analysis indicated that these genes are closely related to the genes of GnIH and NPFF and their receptors of vertebrates. We further identified mature RFamide peptides and their receptors in protochordates. The identified amphioxus RFamide peptides inhibited forskolin induced cAMP signaling in the COS-7 cells with one of the identified amphioxus RFamide peptide receptors expressed. These results indicate that the identified protochordate RFamide peptide gene is a common ancestral form of GnIH and NPFF genes, suggesting that the origin of GnIH and NPFF may date back to the time of the emergence of early chordates. GnIH gene and NPFF gene may have diverged by whole-genome duplication in the course of vertebrate evolution.

Three in one: Identification, expression and enzymatic activity of lysozymes in amphioxus

The lysozymes identified so far in animals belong to the g-type, c-type, and i-type. Vertebrate animals possess only the former two types, i.e., g- and c-types, while all the three types have been reported in invertebrates. Here we demonstrate that (1) three cDNAs that encode g-, c-, and i-type lysozymes, respectively, were identified in a single species of the amphioxus Branchiostoma japonicum; (2) all the 3-type genes displayed distinct tissue-specific expression pattern; (3) recombinant g-, c-, and i-type lysozymes all exhibited enzymatic activities; and (4) native g-, c-, and i-type lysozymes were identified in the different tissues of amphioxus. Collectively, these results suggest the presence of all the 3-type lysozymes in a single animal species, first such data ever reported. The presence of biologically active i-type lysozyme in amphioxus also suggests that i-type lysozyme gene is retained at least in Protochordata, contrasting to the previous proposal that i-type lysozyme gene has been lost in a common ancestor of all chordates.