Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (Branchiostoma belcheri, Chordata).

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher's) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name "B. belcheri EAS/AOS" (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.

The family of amphioxus chitin synthases offers insight into the evolution of chitin formation in chordates.

Chitin is a very important and widely-used biopolymer in fungi and lower metazoans, but mysteriously disappears in mammals. Recent studies reveal that at least lower vertebrates have chitin synthases (CS) and use them to synthesize endogenous chitin. Amphioxus, a basal chordate, therefore becomes critical to understand the evolution of CS, as it occupies the transitional position from invertebrates to vertebrates, and is considered as a good proxy to the chordate ancestor. Here, by exploiting multiple genome assemblies, high-depth RNA-seq data and synteny relations, we identify 11-12 CS genes for each amphioxus species. It represents the largest CS gene pool ever found in eukaryotes so far. As comparison, most metazoans have one or two CSs. Amphioxus is the only chordate that has both the very ancient type-I CS family and the more broadly distributed type-II CS family. Specifically, amphioxus has only one type-II CS but 10-11 type-I CSs, which means that amphioxus is the only metazoan with a greatly expanded type-I CS family. Further analysis suggests that the chordate ancestor have at least one type-II CS and an expanded of type-I CS family. We hypothesize that: these ancient CSs are mostly retained in amphioxus; but the whole type-I CS family was lost in urochordates and vertebrates; the type-II CS was later duplicated into two lineages in vertebrates and followed by stochastic losses, till all type-II CSs were eventually lost in birds and mammals. Finally, our expression profiling and preliminary gene knockout analysis suggest that amphioxus CSs could have highly diverse but mildly overlapping functions in various tissues and organs. Taken together, these findings not only provide insights into the evolution of chordate CSs, lay a foundation for further functional study of the chordate CSs. After all, it is mysterious that our chordate ancestor needed so many isoenzymes for chitin formation.

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 ß-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 ß-glucosidase activity, but BjKL1 did not. Structural analysis showed that there existed nine consecutive but not conserved residues in the ß6α6 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 ß-glucosidase activity in the BjKL1 mutant. Our results indicate that BjKL2 possesses ß-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.

Transposon molecular domestication and the evolution of the RAG recombinase.

Domestication of a transposon (a DNA sequence that can change its position in a genome) to give rise to the RAG1-RAG2 recombinase (RAG) and V(D)J recombination, which produces the diverse repertoire of antibodies and T cell receptors, was a pivotal event in the evolution of the adaptive immune system of jawed vertebrates. The evolutionary adaptations that transformed the ancestral RAG transposase into a RAG recombinase with appropriately regulated DNA cleavage and transposition activities are not understood. Here, beginning with cryo-electron microscopy structures of the amphioxus ProtoRAG transposase (an evolutionary relative of RAG), we identify amino acid residues and domains the acquisition or loss of which underpins the propensity of RAG for coupled cleavage, its preference for asymmetric DNA substrates and its inability to perform transposition in cells. In particular, we identify two adaptations specific to jawed-vertebrates-arginine 848 in RAG1 and an acidic region in RAG2-that together suppress RAG-mediated transposition more than 1,000-fold. Our findings reveal a two-tiered mechanism for the suppression of RAG-mediated transposition, illuminate the evolution of V(D)J recombination and provide insight into the principles that govern the molecular domestication of transposons.

Characterisation of amphioxus protein kinase C-δ/θ reveals a unique proto-V3 domain suggesting an evolutionary mechanism for PKC-θ unique V3.

A primitive adaptive immune system has recently been suggested to be present in a basal chordate amphioxus (Branchiostoma belcheri, Bb), making it an ideal model for studying the origin of adaptive immune. The novel protein kinase C isoform PKC-θ, but not its closest isoform PKC-δ, plays a critical role for mammalian T-cell activation via translocation to immunological synapse (IS) mediated by a unique PKC-θ V3 domain containing one PxxP motif. To understand the evolution of this unique PKC-θ V3 domain and the primitive adaptive immune system in amphioxus, we comparatively studied the orthologs of PKC-δ and -θ from amphioxus and other species. Phylogenetic analysis showed BbPKC-δ/θ to be the common ancestor of vertebrate PKC-δ and PKC-θ, with a V3 domain containing two PxxP motifs. One motif is conserved in both zebrafish and mammalian PKC-θ but is absent in PKC-δ V3 domain of these species, and has already emerged in drosophila PKC-δ. The other non-conserved motif emerged in BbPKC-δ/θ, and only retained in Danio rerio PKC-δ (DrPKC-δ) but lost in mammalian PKC-δ and -θ. Comparative analyses of the sequence and function of BbPKC-δ/θ, DrPKC-δ, DrPKC-θ and Homo sapiens PKC-θ (HsPKC-θ) in IS translocation and T-cell receptor (TCR)-induced NF-κB activation revealed that retention of the conserved PxxP motif and loss of the non-conserved PxxP motif in mammalian PKC-θ and loss of both PxxP motifs in mammalian PKC-δ accomplish the unique function of PKC-θ in T cells. Together, this study suggests an evolutionary mechanism for PKC-θ unique V3 and reveals BbPKC-δ/θ is the common ancestor of PKC-δ and -θ with a functional proto-V3 domain, supplying new evidence for the existence of primitive adaptive immune system in amphioxus.

Cloning, expression and antioxidant activity of a thioredoxin peroxidase from Branchiostoma belcheri tsingtaunese.

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes that catalyze the thioredoxin- dependent reduction of hydroperoxides. In this study, a novel thioredoxin peroxidase (Bbt-TPx1), a member of the peroxiredoxin superfamily, was found by EST sequence analysis of a cDNA library of Branchiostoma belcheri tsingtaunese ovary. The sequence of a full-length cDNA clone contained an open reading frame encoding a polypeptide of 198 amino acid residues, with a calculated molecular weight of 22,150 Da. The expression patterns of the protein at different developmental stages and adult amphioxus tissues indicate that this enzyme may play important roles in anti-oxidation and innate immunity. The recombinant Bbt-TPx1 protein was expressed with a polyhistidine-tag in Escherichia coli and purified using Ni chromatography followed by SP cation exchange chromatography. The rBbt-TPx1 protein existed as a dimer under non-reducing conditions, and was dissociated into monomers by dithiothreitol (DTT); it might predominantly exist in oligomeric form. The rBbt-TPx1 protein showed a significant thiol-dependent peroxidase activity, removing hydrogen peroxide in the presence of dithiothreitol (DTT), but not glutathione (GSH). Protection of plasmid DNA and the thiol-protein from damage by metal-catalyzed oxidation (MCO) in vitro was also revealed.

Lineage-specific duplication of amphioxus retinoic acid degrading enzymes (CYP26) resulted in sub-functionalization of patterning and homeostatic roles.

BACKGROUND: During embryogenesis, tight regulation of retinoic acid (RA) availability is fundamental for normal development. In parallel to RA synthesis, a negative feedback loop controlled by RA catabolizing enzymes of the cytochrome P450 subfamily 26 (CYP26) is crucial. In vertebrates, the functions of the three CYP26 enzymes (CYP26A1, CYP26B1, and CYP26C1) have been well characterized. By contrast, outside vertebrates, little is known about CYP26 complements and their biological roles. In an effort to characterize the evolutionary diversification of RA catabolism, we studied the CYP26 genes of the cephalochordate amphioxus (Branchiostoma lanceolatum), a basal chordate with a vertebrate-like genome that has not undergone the massive, large-scale duplications of vertebrates. RESULTS: In the present study, we found that amphioxus also possess three CYP26 genes (CYP26-1, CYP26-2, and CYP26-3) that are clustered in the genome and originated by lineage-specific duplication. The amphioxus CYP26 cluster thus represents a useful model to assess adaptive evolutionary changes of the RA signaling system following gene duplication. The characterization of amphioxus CYP26 expression, function, and regulation by RA signaling demonstrated that, despite the independent origins of CYP26 duplicates in amphioxus and vertebrates, they convergently assume two main roles during development: RA-dependent patterning and protection against fluctuations of RA levels. Our analysis suggested that in amphioxus RA-dependent patterning is sustained by CYP26-2, while RA homeostasis is mediated by CYP26-1 and CYP26-3. Furthermore, comparisons of the regulatory regions of CYP26 genes of different bilaterian animals indicated that a CYP26-driven negative feedback system was present in the last common ancestor of deuterostomes, but not in that of bilaterians. CONCLUSIONS: Altogether, this work reveals the evolutionary origins of the RA-dependent regulation of CYP26 genes and highlights convergent functions for CYP26 enzymes that originated by independent duplication events, hence establishing a novel selective mechanism for the genomic retention of gene duplicates.

CYP26 function is required for the tissue-specific modulation of retinoic acid signaling during amphioxus development.

During development, morphogens, such as retinoic acid (RA), act as mediators of intercellular communication systems to control patterning and cell fate specification processes. In vertebrates, the tightly regulated production and degradation of RA creates an anterior-posterior (A-P) morphogen gradient that is required for regional patterning of the embryo. RA catabolism in particular, mediated by members of the cytochrome P450 subfamily 26 (CYP26), has been highlighted as a key regulatory component for the formation of this gradient. RA-dependent developmental patterning is now widely recognized as a shared feature of all chordate groups (i.e. of vertebrates, tunicates, and cephalochordates). However, the evolutionary origin of the RA morphogen gradient still remains elusive. Thus, in the present study, we used pharmacological approaches to assess the roles of CYP26 enzymes in tissue-specific patterning processes in embryos and larvae of the cephalochordate amphioxus (Branchiostoma lanceolatum). Marker gene analyses revealed selective requirements for CYP26 activity in anterior endoderm, general ectoderm as well as central nervous system (CNS), but not in mesoderm. Furthermore, comparisons of the effects induced by CYP26 inhibition with those obtained by the pharmacological upregulation or downregulation of global RA signaling levels yielded evidence for a role of CYP26 in establishing an A-P RA gradient in the amphioxus embryo, important at least for patterning the CNS. Altogether, this work hence highlights the involvement of CYP26 in tissue-specific modulations of RA signaling activity in the amphioxus embryo and suggests that a RA morphogen gradient already functioned in the last common ancestor of all chordates.

Evolutionary functional elaboration of the Elovl2/5 gene family in chordates.

The biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) provides an intriguing example on how multi-enzymatic cascades evolve. Essential LC-PUFA, such as arachidonic, eicosapentaenoic, and docosahexaenoic acids (DHA), can be acquired from the diet but are also endogenously retailored from C18 precursors through consecutive elongations and desaturations catalyzed, respectively, by fatty acyl elongase and desaturase enzymes. The molecular wiring of this enzymatic pathway defines the ability of a species to biosynthesize LC-PUFA. Exactly when and how in animal evolution a functional LC-PUFA pathway emerged is still elusive. Here we examine key components of the LC-PUFA cascade, the Elovl2/Elovl5 elongases, from amphioxus, an invertebrate chordate, the sea lamprey, a representative of agnathans, and the elephant shark, a basal jawed vertebrate. We show that Elovl2 and Elovl5 emerged from genome duplications in vertebrate ancestry. The single Elovl2/5 from amphioxus efficiently elongates C18 and C20 and, to a marked lesser extent, C22 LC-PUFA. Lamprey is incapable of elongating C22 substrates. The elephant shark Elovl2 showed that the ability to efficiently elongate C22 PUFA and thus to synthesize DHA through the Sprecher pathway, emerged in the jawed vertebrate ancestor. Our findings illustrate how non-integrated "metabolic islands" evolve into fully wired pathways upon duplication and neofunctionalization.

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, N(1)-acetylspermine (N(1)-Aspm) and N(1)-acetylspermidine (N(1)-Aspd), while yeast PAOs oxidize Spm, N(1)-Aspm and N(1)-Aspd, but not Spd. By contrast, two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of Spm and N(1)-Aspm/N(1)-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 N(1)-acetylspermine, whereas rBjPAO2 oxidizes both spermidine and N(1)-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, 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.

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.

Identification, expression and bioactivity of hexokinase in amphioxus: insights into evolution of vertebrate hexokinase genes.

Hexokinase family includes hexokinases I, II, III and IV, that catalyze the phosphorylation of glucose to produce glucose 6-phosphate. Hexokinase IV, also known as glucokinase, is only half size of the other types of hexokinases that contain two hexokinase domains. Despite the enormous progress in the study of hexokinases, the evolutionary relationship between glucokinase and other hexokinases is still uncertain, and the molecular processes leading to the emergence of hexokinases in vertebrates remain controversial. Here we clearly demonstrated the presence of a single hexokinase-like gene in the amphioxus Branchiostoma japonicum, Bjhk, which shows a tissue-specific expression pattern, with the most abundant expression in the hepatic caecum, testis and ovary. The phylogenetic and synteny analyses both reveal that BjHK is the archetype of vertebrate hexokinases IV, i.e. glucokinases. We also found for the first time that recombinant BjHK showed functional enzyme activity resembling vertebrate hexokinases I, II, III and IV. In addition, a native glucokinase activity was detected in the hepatic caecum. Finally, glucokinase activity in the hepatic caecum was markedly reduced by fasting, whereas it was considerably increased by feeding. Altogether, these suggest that Bjhk represents the archetype of glucokinases, from which vertebrate hexokinase gene family was evolved by gene duplication, and that the hepatic caecum plays a role in the control of glucose homeostasis in amphioxus, in favor of the notion that the hepatic caecum is a tissue homologous to liver.

Expression of carbonic anhydrase, cystic fibrosis transmembrane regulator (CFTR) and V-H(+)-ATPase in the lancelet Branchiostoma lanceolatum (Pallas, 1774).

Sequencing of the amphioxus genome revealed that it contains a basic set of chordate genes involved in development and cell signaling. Despite the availability of genomic data, up till now no studies have been addressed on the comprehension of the amphioxus osmoregulation. Using primers designed on Branchiostoma floridae carbonic anhydrase (CA) II, cystic fibrosis transmembrane regulator (CFTR) and V-H(+)-ATPase, a 100bp long region, containing the protein region recognized by the respective antibodies, has been amplified and sequenced in B. lanceolatum indicating the presence of hortologous V-ATPase, CFTR and carbonic anhydrase II genes in Branchiostoma lanceolatum. Immunohistochemical results showed that all three transporting proteins are expressed in almost 90% of epithelial cells of the skin in B. lanceolatum adults with a different degree of positivity in different regions of body wall and with a different localization in the cells. The comparison of results between young and adult lancelets showed that the distribution of these transporters is quite different. Indeed, in the young specimens the expression pattern of all tested molecules appears concentrated at the gut level, whereas in adult the gut loses its key role that is mostly supported by skin.