Identification and characterization of an amphioxus matrix metalloproteinase homolog BbMMPL2 responding to bacteria challenge.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases mainly involved in extracellular matrix (ECM) degradation. We have cloned and identified BbMMPL2 as homolog of MMPs from adult amphioxus. Recombinant BbMMPL2 proteins underwent self-processing during refolding in vitro. The final ~23 kDa polypeptide displayed proteolytic activity against ECM components like casein, gelatin, collagen IV and fibrinogen, but not laminin, fibronectin or α1-PI. This activity could be inhibited by GM6001 and TIMP-1/2. In addition, real-time RT-PCR analysis revealed that BbMMPL2 expressed in all issues/organs in adult amphioxus we tested. Its transcription was significantly up-regulated 12 h post immune challenge by Escherichia coli in epidermis and hepatic diverticulum but only slightly increased by Staphyloccocus aureus in epidermis. Furthermore, recombinant BbMMPL2-EGFP expressed in 293T and NIH/3T3 cells showed aggregation in cytoplasm and induced cell death. Our results provided new evidence that MMP was involved in immune response which could be conserved through evolution.

The carboxylesterase/cholinesterase gene family in invertebrate deuterostomes.

Carboxylesterase/cholinesterase family members are responsible for controlling the nerve impulse, detoxification and various developmental functions, and are a major target of pesticides and chemical warfare agents. Comparative structural analysis of these enzymes is thus important. The invertebrate deuterostomes (phyla Echinodermata and Hemichordata and subphyla Urochordata and Cephalochordata) lie in the transition zone between invertebrates and vertebrates, and are thus of interest to the study of evolution. Here we have investigated the carboxylesterase/cholinesterase gene family in the sequenced genomes of Strongylocentrotus purpuratus (Echinodermata), Saccoglossus kowalevskii (Hemichordata), Ciona intestinalis (Urochordata) and Branchiostoma floridae (Cephalochordata), using sequence analysis of the catalytic apparatus and oligomerisation domains, and phylogenetic analysis. All four genomes show blurring of structural boundaries between cholinesterases and carboxylesterases, with many intermediate enzymes. Non-enzymatic proteins are well represented. The Saccoglossus and Branchiostoma genomes show evidence of extensive gene duplication and retention. There is also evidence of domain shuffling, resulting in multidomain proteins consisting either of multiple carboxylesterase domains, or of carboxylesterase/cholinesterase domains linked to other domains, including RING finger, chitin-binding, immunoglobulin, fibronectin type 3, CUB, cysteine-rich-Frizzled, caspase activation and 7tm-1, amongst others. Such gene duplication and domain shuffling in the carboxylesterase/cholinesterase family appears to be unique to the invertebrate deuterostomes, and we hypothesise that these factors may have contributed to the evolution of the morphological complexity, particularly of the nervous system and neural crest, of the vertebrates.

Identification and expression of an elastase homologue in Branchiostoma belcheri with implications to the origin of vertebrate pancreas.

Elastases have been identified in a variety of organisms ranging from bacteria to insects to mammals, yet little is known to date about them in amphioxus, a model animal for insights into the origin and evolution of vertebrates. In this study we demonstrate the presence of an elastase homologue, named BbElas, in Branchiostoma belcheri. The recombinant BbElas hydrolyses the elastase specific substrate N-succinyl-Ala-Ala-Ala p-nitroanilide, which can be inhibited by the serine proteinase inhibitor PMSF, the elastase-specific inhibitor elastatinal and the cysteine proteinase inhibitor PCMB. Phylogenetic analysis shows that BbElas represents the archetype of vertebrate elastases, hinting at the clues that the different isoforms of vertebrate elastases are originated from an ancestral gene like BbElas. Our results also suggest that the mid-gut in amphioxus is to homologous vertebrate pancreas, a novel proposal which deserves further study.

Creatine kinase is a bacteriostatic factor with a lectin-like activity.

Creatine kinase (CK), an enzyme catalyzing the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP), has been shown to be an acute phase reactant in the amphioxus Branchiostoma belcheri. However, immune and functional characterization of this protein remains lacking. Here we demonstrate clearly that the expression of B. belcheri gene, BbCK, was inducible by the challenge with LPS, and the recombinant BbCK was capable of binding to the Gram-negative bacterium Escherichia coli and inhibiting the bacterial growth. Moreover, the bacteriostatic activity of the recombinant BbCK against E. coli was able to be suppressed by some sugars including N-acetyl-d-mannosamine, N-acetyl-d-glucosamine, l-fucose, d-mannose, d-fructose and d-glucose. In contrast, BbCK exerted little effect on the growth of the Gram-positive bactterium Staphylococcus aureus. Taken together, these data suggest that CK is a bacteriostatic factor with a lectin-like activity, capable of specifically inhibiting the growth of the Gram-negative bacteria like E. coli. This is the first report exhibiting the integrative role of CK in immunity via its pleiotropic effects on recognizing the Gram-negative bacterium E. coli and inhibiting its growth.

Identification and functional characterization of legumain in amphioxus Branchiostoma belcheri.

Legumain has been reported from diverse sources such as plants, parasites (animals) and mammals, but little is known in the lower chordates. The present study reports the first characterization of legumain cDNA from the protochordate Branchiostoma belcheri. The deduced 435-amino-acid-long protein is structurally characterized by the presence of a putative N-terminal signal peptide, a peptidase_C13 superfamily domain with the conserved Lys123-Gly124-Asp125 motif and catalytic dyad His153 and Cys195 and two potential Asn-glycosylation sites at Asn85 and Asn270. Phylogenetic analysis demonstrates that B. belcheri legumain forms an independent cluster together with ascidian legumain, and is positioned at the base of vertebrate legumains, suggesting that B. belcheri legumain gene may represent the archetype of vertebrate legumain genes. Both recombinant legumain expressed in yeast and endogenous legumain are able to be converted into active protein of approximately 37 kDa via a C-terminal autocleavage at acid pH values. The recombinant legumain efficiently degrades the legumain-specific substrate Z-Ala-Ala-Asn-MCA (benzyloxycarbonyl-L-alanyl-L-alanyl-L-asparagine-4-methylcoumaryl-7-amide) at optimum pH 5.5; and the enzymatic activity is inhibited potently by iodoacetamide and N-ethylmaleimide, partially by hen's-egg white cystatin, but not by E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane], PMSF and pepstatin A. In addition, legumain is expressed in vivo in a tissue-specific manner, with main expression in the hepatic caecum and hind-gut of B. belcheri. Altogether, these results suggest that B. belcheri legumain plays a role in the degradation of macromolecules in food.

Identification, expression, function and localization of a DUF985 domain-containing hypothetical gene from amphioxus Branchiostoma belcheri.

The progress in genome sequencing has led to an increasing submission of uncharacterized hypothetical genes with the domain of unknown function, DUF985, in GenBank, and none of these genes is related to a known protein. We therefore underwent an experimental study to identify the function of a DUF985 domain-containing hypothetical gene BbDUF985 (GenBank Accession No. AY273818) isolated from amphioxus Branchiostoma belcheri (B. belcheri). BbDUF985 was successfully expressed in both prokaryotic and eukaryotic systems, and its recombinant proteins expressed in both systems definitely exhibited an activity of phosphoglucose isomerase (PGI). Both tissue-section in situ hybridization and immunohistochemistry demonstrated that BbDUF985 was expressed in a tissue-specific manner, with most abundant levels in the hepatic caecum and ovary. In CHO cells transfected with the expression plasmid pEGFP-N1/BbDUF985, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that BbDUF985 is a cytosolic protein. In contrast, Western blotting indicated that BbDUF985 was also present in amphioxus humoral fluids, suggesting that it exists as a secreted protein as well. Our study provided a framework for further understanding the biochemical properties and physiological function of DUF985-containing hypothetical proteins in other species.

Characterization, expression and localization of S-adenosylhomocysteine hydrolase from amphioxus Branchiostoma belcheri tsingtaunese.

A cDNA clone encoding AmphiSAHH [amphioxus SAHH (S-adenosylhomocysteine hydrolase)] protein was isolated from a cDNA library from the gut of Branchiostoma belcheri tsingtaunese. It contained a 1305 bp open reading frame corresponding to a deduced protein of 434 amino acid residues, with a predicted molecular mass of approx. 47.8 kDa. Phylogenetic analysis showed that AmphiSAHH and sea-urchin SAHH joined together and positioned at the base of the vertebrate SAHH clade, suggesting that both AmphiSAHH and sea-urchin SAHH might share some characteristics of the archetype of vertebrate SAHH proteins. The genomic DNA sequence of AmphiSAHH contained eight exons and seven introns, which was similar to B. floridae and sea-urchin SAHH exon/intron organization. Sequence comparison suggested the evolutionary appearance of the ten exon/nine intron organization of SAHH genes after the split of invertebrates and vertebrates, after which it has been highly conserved. AmphiSAHH has been successfully expressed in Escherichia coli and purified. Western blotting confirmed that the enzyme has a native molecular mass of approx. 48 kDa, and the catalytic activities and NAD(+)/NADH binding affinity of recombinant AmphiSAHH were measured. Immunohistochemistry analysis showed that SAHH was strongly expressed in hepatic caecum, gill, spermary and ovary of amphioxus.

Gene expansion and retention leads to a diverse tyrosine kinase superfamily in amphioxus.

Tyrosine kinase (TK) proteins play a central role in cellular behavior and development of animals. The expansion of this superfamily is regarded as a key event in the evolution of the complex signaling pathways and gene networks of metazoans and is a prominent example of how shuffling of protein modules may generate molecular novelties. Using the intron/exon structure within the TK domain (TK intron code) as a complementary tool for the assignment of orthology and paralogy, we identified and studied the 118 TK proteins of the amphioxus Branchiostoma floridae genome to elucidate TK gene family evolution in metazoans and chordates in particular. Unlike all characterized metazoans to date, amphioxus has members of all known widespread TK families, with not a single loss. Putting amphioxus TKs in an evolutionary context, including new data from the cnidarian Nematostella vectensis, the echinoderm Strongylocentrotus purpuratus, and the ascidian Ciona intestinalis, we suggest new evolutionary histories for different TK families and draw a new global picture of gene loss/gain in the different phyla. Surprisingly, our survey also detected an unprecedented expansion of a group of closely related TK families, including TIE, FGFR, PDGFR, and RET, due most probably to massive gene duplication and exon shuffling. Based on their highly similar intron/exon structure at the TK domain, we suggest that this group of TK families constitute a superfamily of TK proteins, which we termed EXpanding TK, after their seemingly unique propensity to gene duplication and exon shuffling, not only in amphioxus but also across all metazoan groups. Due to this extreme tendency to both retention and expansion of TK genes, amphioxus harbors the richest and most diverse TK repertoire among all metazoans studied so far, retaining most of the gene complement of its ancestors, but having evolved its own repertoire of genetic novelties.

Presence of sex steroids and cytochrome P450 genes in amphioxus.

The presence of sex steroids and their receptors has been demonstrated in all vertebrate groups from Agnatha to Mammalia but not in invertebrates. In genomic analyses of urochordates, cytochrome P450 (CYP) genes important for biosynthesis of sex steroids are absent. In the present study, we confirmed the presence of estrogen, androgen, and progesterone by using radioimmunoassay in gonads of amphioxus, Branchiostoma belcheri, which is considered to be evolutionarily closer to vertebrates than other invertebrates. Furthermore, CYP genes encoding CYP11A, CYP17, and CYP19 and transcripts for 17beta-hydroxysteroid dehydrogenase were cloned from amphioxus ovaries. Among invertebrates, the presence of hydroxysteroid dehydrogenase enzymes and metabolized steroids was shown in paracytic Taenia and corals. However, CYPs metabolizing sex steroids have not been demonstrated in invertebrates, nor has an attempt been made to consider the entire pathway from cholesterol to estrogen. This study is the first evidence to suggest the presence of CYP enzymes in sex steroid production in invertebrates.

Purification and characterisation of phenoloxidase from amphioxus Branchiostoma belcheri tsingtauense.

Phenoloxidase (PO) from the humoral fluid of amphioxus B. belcheri tsingtauense was purified using a sequential combination of ammonium sulphate precipitation, Sephadex G-200 chromatography and DEAE Sepharose Fast Flow chromatography. In PAGE, the purified enzyme exhibited a single band of 150 kDa under non-reducing conditions, and was resolved to three bands with molecular masses of 72, 46 and 44 kDa, respectively, under reducing conditions, suggesting that the PO in amphioxus humoral fluid seems to be a heterotrimer of three polypeptides held together by disulphide bonds. The substrate specificity and inhibition characteristics both indicate that the PO isolated from amphioxus humoral fluid is a tyrosinase-type enzyme. In addition, mouse antisera against the purified PO were prepared, and their specificity was confirmed by Western blotting, facilitating the future determination of the origin of PO in the humoral fluid and the distribution of PO-synthesising tissues in amphioxus.

Presence of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense.

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase (proPO). The optimum temperature for activation of the proPO ranged from 30 degrees C to 35 degrees C, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.

Isolation of AmphiCASP-3/7, an ancestral caspase from amphioxus (Branchiostoma floridae). Evolutionary considerations for vertebrate caspases.

Caspases are a large family of cysteine proteases that play an essential role as effectors of apoptosis in metazoans. Thirteen different caspases have been identified in vertebrates so far, and their function in apoptotic or inflammatory responses is well documented. We have taken advantage of the broadly accepted condition of amphioxus (Cephalochordata, Branchiostoma floridae) as the closest living relative to vertebrates to study the molecular evolution of caspases. Here we report for the first time the pattern of programmed cell death during development of cephalochordates. We also describe the isolation and functional characterisation of the first caspase related gene in amphioxus, which we named AmphiCASP-3/7. The amphioxus caspase is expressed throughout development, from the gastrula to larva stage. AmphiCASP-3/7 induced cell death when ectopically expressed in human HEK 293T cells, and the recombinant protein was inhibited by DEVD peptides. AmphiCASP-3/7 reflects the primitive condition of the executor vertebrates caspases -3 and -7, prior to vertebrate specific duplication. Interestingly, AmphiCASP-3/7 is functionally closer to vertebrate caspase-7, as shown by substrate specificity both in vitro and in MCF7 cells. Our phylogenetic and functional data help in drawing the evolutionary history of caspases, and illustrates an example of acquisition in vertebrates of novel functional properties after gene duplication.

Evolution of the prohormone convertases: identification of a homologue of PC6 in the protochordate amphioxus.

Many of the protein precursors traversing the secretory pathway undergo cleavage at multibasic sites to generate their bioactive forms. The proprotein convertases (PCs), a family of subtilisin-like proteases, are the major endoproteases that serve this function. Genes encoding seven distinct members of this family have so far been characterized in vertebrates: furin, PC2, PC1/PC3, PC4, PACE4, PC5/PC6 and PC7/PC8/LPC. Multiple PC genes have also been cloned from a number of invertebrates, including Drosophila melanogaster and Caenorhabditis elegans. These findings suggest that gene duplication and diversification of the PCs have occurred throughout metazoan evolution. To investigate the structural and functional changes which have occurred during vertebrate development, we have analyzed the expression of PC genes in the protochordate amphioxus. We have previously shown that amphioxus express homologous PC2 and PC1/PC3 genes [Proc. Natl. Acad. Sci. USA 92 (1995) 3591]. Here we report the characterization of amphioxus cDNAs encoding proteases with a high degree of similarity to mammalian PC6. Three cDNAs encoding three PC6 isoforms differing only in their carboxy-terminal sequences were found, derived by alternative splicing. Two isoforms appear to be soluble enzymes, whereas the third contains a transmembrane hydrophobic segment and thus is likely to be membrane-bound. All three variants contain many repeats of a cysteine-rich motif that is found in several other PC family members. Thus, amphioxus, like the vertebrates, expresses two types of PCs, e.g., PC2 and PC1/PC3 which function in the regulated secretory pathway in neuroendocrine cells, and the more widely expressed PC6 which functions mainly in the constitutive pathway.

cDNA cloning, in vitro expression, and biochemical characterization of cholinesterase 1 and cholinesterase 2 from amphioxus--comparison with cholinesterase 1 and cholinesterase 2 produced in vivo.

We have isolated cDNAs coding for the complete amino acid sequences of cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2) from amphioxus. Both ChE transcripts have the characteristics of H-type catalytic subunits, which are inserted in the membrane via an ethanolamine-glycan-phosphatidylinositol anchor. The members of the catalytic triad of ChEs, the three pairs of cysteine residues involved in intrachain disulfide bonding, a cysteine near the carboxy terminal of both sequences, which could mediate interchain disulfide bonding, and 11 of the 14 aromatic amino acids that line the catalytic gorge of AChE are conserved. A remarkable difference between the two enzymes is in the region of the acyl-binding pocket, which plays an important role in determining substrate specificity in cholinesterases. ChE2 contains a sequence that resembles the acyl pocket of invertebrate ChE, while the acyl-binding site of ChE1 is novel. There are also differences between the two enzymes in the peripheral anionic site, which mediates inhibition by certain ligands. In vitro expression in COS-7 cells demonstrates that ChE2 hydrolyzes acetylthiocholine almost exclusively, while ChE1 hydrolyzes both acetylthiocholine and butyrylthiocholine. Both enzymes are inhibited comparably by BW284c51, but ChE1 is considerably more resistant to inhibition by propidium, ethopropazine, and eserine than is ChE2. Velocity sedimentation indicates that ChE1 and ChE2 are present as amphiphilic and nonamphiphilic G2 forms in vivo and in vitro. Another molecular form, which sediments at 17 S, is also present in vivo. Nondenaturing gel electrophoresis in conjunction with digestion by phosphatidylinositol-specific phospholipase C demonstrates that the vast majority of ChE1 and ChE2 is present as ethanolamine-glycan-phosphatidylinositol-anchored G2 forms in vivo. ChE1 also possesses an ethanolamine-glycan-phosphatidylinositol-anchor in vitro; however, ChE2 produced in vitro could not be detected on nondenaturing gels.

Protein tyrosine phosphatase domains from the protochordate Styela plicata.

Protein tyrosine phosphorylation is an important regulatory mechanism in cell physiology. While the protein tyrosine kinase (PTKase) family has been extensively studied, only six protein tyrosine phosphatases (PTPases) have been described. By Southern blot analysis, genomic DNA from several different phyla were found to cross-hybridize with a cDNA probe encoding the human leukocyte-common antigen (LCA; CD45) PTPase domains. To pursue this observation further, total mRNA from the protochordate Styela plicata was used as a template to copy and amplify, using polymerase chain reaction (PCR) technology, PTPase domains. Twenty-seven distinct sequences were identified that contain hallmark residues of PTPases; two of these are similar to described mammalian PTPases. Southern blot analysis indicates that at least one other Styela sequence is highly conserved in a variety of phyla. Seven of the Styela domains have significant similarity to each other, indicating a subfamily of PTPases. However, most of the sequences are disparate. A comparison of the 27 Styela sequences with the ten known PTPase domain sequences reveals that only three residues are absolutely conserved and identifies regions that are highly divergent. The data indicate that the PTPase family will be equally as large and diverse as the PTKases. The extent and diversity of the PTPase family suggests that these enzymes are, in their own right, important regulators of cell behavior.