The mammalian chitinase family 18 consists of two members, chitotriosidase (ChT) and acidic chitinase (AMCase). Despite the enormous progress on mammalian ChT study, little information regarding ChT is available to date in lower animals. In this study, we identified a chitotriosidase-like gene from the amphioxus Branchiostoma japonicum, named BjChTl, which consisted of a signal peptide, a catalytic domain, a Ser/Thr-rich linker region and a chitin-binding domain (CB domain). Sequence comparison and phylogenetic analysis showed that BjChTl was the common ancestor of ChTs and AMCases, implicating that ChT and AMCase evolved from an ancient gene like BjBhTl via gene duplication. qRT-PCR analysis revealed that BjChTl was expressed in the hepatic caecum and hind gut in a tissue-specific fashion. Both chitin-binding and enzymatic activities as well as antifungal activity assays demonstrated that like human ChT, recombinant BjChTl was able to bind to chitin particles, to hydrolyze artificial chitin substrate 4-methylumbelliferyl-β-D-N,N',N″-triacetylchitotrioside, and to inhibit the growth of the fungus Candida albicans. Surprisingly, recombinant BjChTl-CD lacking CB domain retained partial capacity to bind to chitin, but its enzymatic activity was almost completely lost. These findings suggest that the CB domain is necessary for the execution of both enzymatic and antifungal activities of recombinant BjChTl. It is also the first study showing the presence of a ChT-like homolog with both chitinolytic activity and fungistatic activity in non-vertebrate species.

Peptidoglycan recognition proteins (PGRPs) are widely distributed in invertebrates and vertebrates, and structure-activity relationship of insect and mammalian PGRPs has been well characterized, but functional and structural insights into PGRPs in other species are rather limited. Here we identified a novel short PGRP gene from the amphioxus Branchiostoma japonicum, named pgrp-s, which possesses a domain combination of ChtBD1 domain-PGRP domain, which is unique to all known PGRPs. Amphioxus pgrp-s was predominantly expressed in the hepatic caecum, hind-gut and muscle in a tissue-specific manner. Recombinant PGRP-S, rPGRP-S, and truncated protein with ChtBD1 domain deleted, rP86/250, both showed affinity to Dap-type PGN, Lys-type PGN and chitin. Consistently, they were also able to bind to Escherichia coli, Staphylococcus aureus and Pichia pastoris. Moreover, both rPGRP-S and rP86/250 had amidase enzymatic activity, capable of hydrolyzing Dap-type and Lys-type PGNs. Like vertebrate PGRPs, rPGRP-S was directly microbicidal, capable of killing E. coli, S. aureus and P. pastoris, whereas rP86/250 only inhibited the growth of E. coli and S. aureus, and its anti-P. pastoris activity was significantly reduced. It is clear that neither the binding of amphioxus PGRP-S nor its amidase enzymatic activity depend on the N-terminal ChtBD1 domain, but its antifungal activity does. Collectively, these data suggested that amphioxus PGRP-S may function as a multivalent pattern recognition receptor, capable of recognizing PGN and chitin, a microbicidal agent, capable of killing bacteria such as E. coli and S. aureus and fungus like P. pastoris, and probably a PGN scavenger, capable of hydrolyzing PGN.

The function and mechanism of chitinases in early embryonic development remain largely unknown. We show here that recombinant chitinase-3 (rChi3) is able to hydrolyze the artificial chitin substrate, 4-methylumbelliferyl-β-D-N,N',N″-triacetylchitotrioside, and to bind to and inhibit the growth of the fungus Candida albicans, implicating that Chi3 plays a dual function in innate immunity and chitin-bearing food digestion in zebrafish. This is further corroborated by the expression profile of Chi3 in the liver and gut, which are both immune- and digestion-relevant organs. Compared with rChi3, rChi3-CD lacking CBD still retains partial capacity to bind to C. albicans, but its enzymatic and antifungal activities are significantly reduced. By contrast, rChi3-E140N with the putative catalytic residue E140 mutated shows little affinity to chitin, and its enzymatic and antifungal activities are nearly completely lost. These suggest that both enzymatic and antifungal activities of Chi3 are dependent on the presence of CBD and E140. We also clearly demonstrate that in zebrafish, both the embryo extract and the developing embryo display antifungal activity against C. albicans, and all the findings point to chitinase-3 (Chi3) being a newly-identified factor involved in the antifungal activity. Taken together, a dual function in both innate immunity and food digestion in embryo is proposed for zebrafish Chi3. It also provides a new angle to understand the immune role of chitinases in early embryonic development of animals.

Ovochymases have been shown to be present in vertebrates; little information is available at present regarding ovochymase in invertebrates. Here we isolated a cDNA encoding an ovochymase homolog from amphioxus Branchiostoma belcheri, named BbOvc. The cDNA contained a 1248bp open reading frame corresponding to a deduced protein of 415 amino acids with a predicted molecular mass of approximately 44.4kDa. Phylogenetic analysis showed that BbOvc was located at the base of its vertebrate counterparts, suggesting that it represents the archetype of vertebrate ovochymases. BbOvc is found to display a tissue- and stage-specific expression pattern, with a predominant expression in the ovary of sexually matured females and in the early stage embryos (1-16-cell embryos). The recombinant ovochymase expressed in vitro shows a trypsin-like activity capable of hydrolysing the trypsin prototypic substrate N(a)-benzoyl-l-arginine ethyl ester (60UBAEE/mg), which can be inhibited by the trypsin-specific inhibitor soybean trypsin inhibitor. It also exhibits an antibacterial activity capable of inhibiting the growth of bacteria like E. coli and V. parahaemolyticus. Taken together, these data indicate that BbOvc is a novel ovochymase with an antibacterial activity and offer first clues to its role as an immune-relevant molecule which may protect the early embryos from pathogenic attacks.

CD59, known as protectin, usually plays roles as a regulatory inhibitor of complement, but it also exhibits activities independent of its function as a complement inhibitor. This study reported the identification and characterization of an ortholog of mammalian cd59 from zebrafish Danio rerio, which is similar to known cd59 in terms of both amino acid sequence and genomic structure as well as synteny conservation. We showed that zebrafish cd59 was maternally expressed in early embryos and expressed in a tissue-specific manner, with most abundant expression in the brain. We further showed that recombinant zebrafish CD59 was capable of binding to both the Gram-negative and Gram-positive bacteria as well as the microbial signature molecules LPS and LTA. In addition we demonstrated that recombinant zebrafish CD59 displayed slight antimicrobial activity capable of inhibiting the growth of E. coli and S. aureus. All these data indicate that zebrafish CD59 can not only binds to the bacteria and their signature molecules LPS and LTA but can also inhibit their growth, a novel role assigned to CD59.

Midkine (MK) and pleiotrophin (PTN) are the only two members of heparin-binding growth factor family. MK/PTN homologues found from Drosophila to humans are shown to have antibacterial activities and their antibacterial domains are conserved during evolution. However, little is known about MK/PTN homologue in the basal chordate amphioxus, and overall, information regarding MK/PTN homologues is rather limited in invertebrates. In this study, we identified a single MK/PTN homologue in Branchiostoma japonicum, termed BjMiple, which has a novel domain structure of PTN-PTNr1-PTNr2, and represents the ancestral form of vertebrate MK/PTN family proteins. BjMiple was expressed mainly in the ovary in a tissue-dependent fashion, and its expression was remarkably up-regulated following challenge with bacteria or their signature molecules LPS and LTA, suggesting its involvement in antibacterial responses. Functional assays revealed that BjMiple had strong antimicrobial activity, capable of killing a panel of Gram-negative and Gram-positive bacteria via a membranolytic mechanism, including interaction with bacterial membrane via LPS and LTA, membrane depolarization and high intracellular levels of ROS. Importantly, strong antibacterial activity was localized in PTN42-61 and PTNr142-66. Additionally, BjMiple and its derived peptides PTN42-61 and PTNr142-66 were not cytotoxic to human RBCs and mammalian cells. Taken together, our study suggests that amphioxus Miple is the ancestral type of vertebrate MK/PTN family homologues, and can play important roles as innate peptide antibiotics, which renders it a promising template for the design of novel peptide antibiotics against multi-drug resistant bacteria.

Transforming growth factor beta (TGFβ) is a pleiotropic cytokine with important roles in mediating inflammatory response. TGFβ has been shown to be widely present in invertebrates, but little is known about its functions in immune and inflammatory responses. Moreover, structural and functional insights into TGFβ molecules in invertebrates remain completely lacking. Here we demonstrate the presence of a single TGFβ-like gene in the amphioxus Branchiostoma japonicum, Bjtgfβ, which represents the archetype of vertebrate TGFβ proteins, and displays a higher expression in the hind-gut, hepatic caecum, ovary, and gill. We also show that amphioxus TGFβ exerts both enhancing and suppressing effects on the migration of macrophages like RAW264.7, and the motif WSTD is important for TGFβ in inducing or inhibiting the migration of macrophages. Altogether, these data suggest that amphioxus TGFβ is phylogenetically and functionally similar to vertebrate TGFβ, suggesting an ancient origin of bipolar function of TGFβ proteins.