AID/APOBEC-like cytidine deaminases are ancient innate immune mediators in invertebrates.

In the course of both innate and adaptive immunity, cytidine deaminases within the activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) family modulate immune responses by mutating specific nucleic acid sequences of hosts and pathogens. The evolutionary emergence of these mediators, however, seems to coincide precisely with the emergence of adaptive immunity in vertebrates. Here, we show a family of genes in species within two divergent invertebrate phyla-the echinoderm Strongylocentrotus purpuratus and the brachiopod Lingula anatina-that encode proteins with similarities in amino acid sequence and enzymatic activities to the vertebrate AID/APOBECs. The expression of these invertebrate factors is enriched in tissues undergoing constant, direct interactions with microbes and can be induced upon pathogen challenge. Our findings suggest that AID/APOBEC proteins, and their function in immunity, emerged far earlier than previously thought. Thus, cytidine deamination is probably an ancient innate immune mechanism that predates the protostome/deuterostome divergence.

Engineering Pichia pastoris for Efficient Production of a Novel Bifunctional Strongylocentrotus purpuratus Invertebrate-Type Lysozyme.

Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960 U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5 mg/L and purity of > 99%. Recombinant sp-iLys reached its peak lytic activity of 8560 U/mg at pH 6.0 and 30 °C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37 °C with the presence of 0.05 M Na+. In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.

The Role of Phospholipase D in Regulated Exocytosis.

There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca(2+)-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphatidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca(2+) sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se.

Essential elements for translation: the germline factor Vasa functions broadly in somatic cells.

Vasa is a conserved RNA-helicase found in the germ lines of all metazoans tested. Whereas Vasa presence is often indicated as a metric for germline determination in animals, it is also expressed in stem cells of diverse origin. Recent research suggests, however, that Vasa has a much broader function, including a significant role in cell cycle regulation. Results herein indicate that Vasa is utilized widely, and often induced transiently, during development in diverse somatic cells and adult precursor tissues. We identified that Vasa in the sea urchin is essential for: (1) general mRNA translation during embryogenesis, (2) developmental re-programming upon manipulations to the embryo and (3) larval wound healing. We also learned that Vasa interacted with mRNAs in the perinuclear area and at the spindle in an Importin-dependent manner during cell cycle progression. These results suggest that, when present, Vasa functions are essential to contributing to developmental regulation.

Deadenylase depletion protects inherited mRNAs in primordial germ cells.

A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.

DspA from Strongylocentrotus purpuratus: The first biochemically characterized haloalkane dehalogenase of non-microbial origin.

Haloalkane dehalogenases are known as bacterial enzymes cleaving a carbon-halogen bond in halogenated compounds. Here we report the first biochemically characterized non-microbial haloalkane dehalogenase DspA from Strongylocentrotus purpuratus. The enzyme shows a preference for terminally brominated hydrocarbons and enantioselectivity towards ß-brominated alkanes. Moreover, we identified other putative haloalkane dehalogenases of eukaryotic origin, representing targets for future experiments to discover dehalogenases with novel catalytic properties.

Identification of purple sea urchin telomerase RNA using a next-generation sequencing based approach.

Telomerase is a ribonucleoprotein (RNP) enzyme essential for telomere maintenance and chromosome stability. While the catalytic telomerase reverse transcriptase (TERT) protein is well conserved across eukaryotes, telomerase RNA (TR) is extensively divergent in size, sequence, and structure. This diversity prohibits TR identification from many important organisms. Here we report a novel approach for TR discovery that combines in vitro TR enrichment from total RNA, next-generation sequencing, and a computational screening pipeline. With this approach, we have successfully identified TR from Strongylocentrotus purpuratus (purple sea urchin) from the phylum Echinodermata. Reconstitution of activity in vitro confirmed that this RNA is an integral component of sea urchin telomerase. Comparative phylogenetic analysis against vertebrate TR sequences revealed that the purple sea urchin TR contains vertebrate-like template-pseudoknot and H/ACA domains. While lacking a vertebrate-like CR4/5 domain, sea urchin TR has a unique central domain critical for telomerase activity. This is the first TR identified from the previously unexplored invertebrate clade and provides the first glimpse of TR evolution in the deuterostome lineage. Moreover, our TR discovery approach is a significant step toward the comprehensive understanding of telomerase RNP evolution.

Molecular cloning, expression, and characterization of starfish DNA (cytosine-5)-methyltransferases.

To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form.

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.

Rho-kinase in sea urchin eggs and embryos.

The activation of sea urchin eggs at fertilization provides an ideal system for studying the molecular events involved in cellular activation. Rho GTPases, which are key signaling enzymes in eukaryotes, are involved in sustaining the activation of sea urchin eggs; however, their downstream effectors have not yet been characterized. In somatic cells, RhoA regulates a serine/threonine kinase known as Rho-kinase (ROCK). The activity of ROCK in early sea urchin development has been inferred, but not tested directly. A ROCK gene was identified in the sea urchin (Strongylocentrotus purpuratus) genome and the sequence of its cDNA determined. The sea urchin ROCK (SpROCK) sequence predicts a protein of 158 kDa with >72% and 45% identities with different protein orthologues of the kinase catalytic domain and the complete protein sequence, respectively. SpROCK mRNA levels are high in unfertilized eggs and decrease to 35% after 15 min postfertilization and remain low up to the 4 cell stage. Antibodies to the human ROCK-I kinase domain revealed SpROCK to be concentrated in the cortex of eggs and early embryos. Co-immunoprecipitation assays indicate that RhoA and SpROCK are physically associated. This association is destroyed by treatment with the C3 exoenzyme and with the ROCK antagonist H-1152. H-1152 also inhibited DNA synthesis in embryos. We conclude that the Rho-dependent signaling pathway, via SpROCK, is essential for early embryonic development.

A single residue in a novel ADP-ribosyl cyclase controls production of the calcium-mobilizing messengers cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate.

Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate are ubiquitous calcium-mobilizing messengers produced by the same family of multifunctional enzymes, the ADP-ribosyl cyclases. Not all ADP-ribosyl cyclases have been identified, and how production of different messengers is achieved is incompletely understood. Here, we report the cloning and characterization of a novel ADP-ribosyl cyclase (SpARC4) from the sea urchin, a key model organism for the study of calcium-signaling pathways. Like several other members of the ADP-ribosyl cyclase superfamily, SpARC4 is a glycoprotein targeted to the plasma membrane via a glycosylphosphatidylinositol anchor. However, unlike most other members, SpARC4 shows a remarkable preference for producing cyclic ADP-ribose over nicotinic acid adenine dinucleotide phosphate. Mutation of a single residue (tyrosine 142) within a noncanonical active site reversed this striking preference. Our data highlight further diversification of this unusual enzyme family, provide mechanistic insight into multifunctionality, and suggest that different ADP-ribosyl cyclases are fine-tuned to produce specific calcium-mobilizing messengers.

Genetic hypervariability in two distinct deuterostome telomerase reverse transcriptase genes and their early embryonic functions.

Functional proteins of complex eukaryotes within the same species are rather invariant. A single catalytic component of telomerase TERT is essential for an active telomerase complex that maintains telomeres. Surprisingly, we have identified two paralogous SpTERT-L and SpTERT-S genes with novel domains in Strongylocentrotus purpuratus (purple sea urchin). The SpTERT-S and SpTERT-L genes were differentially expressed throughout embryogenesis. An unusual germline nucleotide substitution and amino acid variation was evident in these TERTs. The hypervariability of SpTERT-S haplotypes among different individuals reached unprecedented levels of pi > 0.2 in exon 11 region. The majority of nucleotide changes observed led to nonsynonymous substitutions creating novel amino acids and motifs, suggesting unusual positive selection and rapid evolution. The majority of these variations were in domains involved in binding of SpTERT to its RNA component. Despite hypervariability at protein level, SpTERT-S conferred telomerase activity, and its suppression during early embryogenesis led to arrest at late mesenchymal blastula. Domain exchange and embryo rescue experiments suggested that SpTERT may have evolved functions unrelated to classic telomerase activity. We suggest that telomerase has a specific and direct function that is essential for integration of early polarity signals that lead to gastrulation. Identification of these unique hypervariable telomerases also suggests presence of a diversity generation mechanism that inculcates hypervariable telomerases and telomere lengths in germline.

Ca(2+) signaling occurs via second messenger release from intraorganelle synthesis sites.

Cyclic ADP-ribose is an important Ca(2+)-mobilizing cytosolic messenger synthesized from beta-NAD(+) by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCbeta and ARCgamma are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a beta-NAD(+) transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca(2+) wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol.

Soluble epoxide hydrolase homologs in Strongylocentrotus purpuratus suggest a gene duplication event and subsequent divergence.

The mammalian soluble epoxide hydrolase (sEH) is a multidomain enzyme composed of C- and N-terminal regions that contain active sites for epoxide hydrolase (EH) and phosphatase activities, respectively. We report the cloning of two 60 kDa multidomain enzymes from the purple sea urchin Strongylocentrotus purpuratus displaying significant sequence similarity to both the N- and C-terminal domains of the mammalian sEH. While one urchin enzyme did not exhibit EH activity, the second enzyme hydrolyzed several lipid messenger molecules metabolized by the mammalian sEH, including the epoxyeicosatrienoic acids. Neither of the urchin enzymes displayed phosphatase activity. The urchin EH was inhibited by small molecule inhibitors of the mammalian sEH and is the likely ancestor of the enzyme. Sequence comparisons suggest that the urchin sEH homologs are the result of a gene fusion event between a gene encoding for an EH and a gene for an enzyme of undetermined function. This fusion event was followed by a duplication event to produce the urchin enzymes.

Cytochrome P450 1 genes in early deuterostomes (tunicates and sea urchins) and vertebrates (chicken and frog): origin and diversification of the CYP1 gene family.

Cytochrome P450 family 1 (CYP1) proteins are important in a large number of toxicological processes. CYP1A and CYP1B genes are well known in mammals, but the evolutionary history of the CYP1 family as a whole is obscure; that history may provide insight into endogenous functions of CYP1 enzymes. Here, we identify CYP1-like genes in early deuterostomes (tunicates and echinoderms), and several new CYP1 genes in vertebrates (chicken, Gallus gallus and frog, Xenopus tropicalis). Profile hidden Markov models (HMMs) generated from vertebrate CYP1A and CYP1B protein sequences were used to identify 5 potential CYP1 homologs in the tunicate Ciona intestinalis genome. The C. intestinalis genes were cloned and sequenced, confirming the predicted sequences. Orthologs of 4 of these genes were found in the Ciona savignyi genome. Bayesian phylogenetic analyses group the tunicate genes in the CYP1 family, provisionally in 2 new subfamilies, CYP1E and CYP1F, which fall in the CYP1A and CYP1B/1C clades. Bayesian and maximum likelihood analyses predict functional divergence between the tunicate and vertebrate CYP1s, and regions within CYP substrate recognition sites were found to differ significantly in position-specific substitution rates between tunicates and vertebrates. Subsequently, 10 CYP1-like genes were found in the echinoderm Strongylocentrotus purpuratus (sea urchin) genome. Several of the tunicate and echinoderm CYP1-like genes are expressed during development. Canonical xenobiotic response elements are present in the upstream genomic sequences of most tunicate and sea urchin CYP1s, and both groups are predicted to possess an aryl hydrocarbon receptor (AHR), suggesting possible regulatory linkage of AHR and these CYPs. The CYP1 family has undergone multiple rounds of gene duplication followed by functional divergence, with at least one gene lost in mammals. This study provides new insight into the origin and evolution of CYP1 genes.

Sequence, annotation and developmental expression of the sea urchin Ca(2+) -ATPase family.

Whole genome sequence data permit the study of protein families regulating cellular homeostasis during development. Here we present a study of the sea urchin Ca(2+)-ATPases made possible by the Sea Urchin Genome Sequencing Project. This is of potential interest because adult sea urchins, their gametes and embryos live in the relatively high Ca(2+) concentration of 10 mM. Three Ca(2+)-ATPases regulate Ca(2+) levels in animal cells: plasma membrane Ca(2+)-ATPase (PMCA), sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and secretory pathway Ca(2+)-ATPase (SPCA). The primary structures of Sp-PMCA and Sp-SERCA in the sea urchin, Strongylocentrotus purpuratus (Sp), have been published. Here, we present the primary structure of Sp-SPCA, which is 912 amino acids and has 66% identity and 80% similarity to human SPCA1. Southern blots and genome analysis show that Sp-SPCA is a single copy gene. Each Sp Ca(2+)-ATPase is highly conserved when compared to its human ortholog, indicating that human and sea urchin share structurally similar energy driven Ca(2+) homeostasis mechanisms that have been maintained throughout the course of deuterostome evolution. Annotation using the assembled sea urchin genome reveals that Sp-SPCA, Sp-PMCA and Sp-SERCA have 23, 17 and 24 exons. RT-Q-PCR shows that transcripts of Sp-SPCA are at low levels compared to Sp-PMCA and Sp-SERCA. Gradual increases in Sp-PMCA and Sp-SERCA mRNA begin at the 18 hour hatched blastula stage and peak 4-5-fold higher by 25 h at the mid to late blastulae stage.

Adenylate kinase in sea urchin embryonic cilia.

Sea urchin embryos swim by ciliary movement. Hypertonic shock causes deciliation and loss of motility. Within 2-4 h, cilia regenerate and the embryos swim again. Regeneration of cilia occurs multiple times. The adenylate kinase (AK) activity of isolated cilia was studied. A 130-kDa Sp-AK isozyme, present in sperm flagella, is also present in embryonic cilia. AK activity is responsible for approximately 93% of nonmitochondrial ATP regeneration from ADP in embryonic cilia. This is unlike sea urchin sperm flagella, where approximately 31% of the nonmitochondrial ATP regeneration is from the 130-kDa Sp-AK isozyme and approximately 69% from the flagellar creatine kinase (Sp-CK). Embryos were deciliated 1-3 times and after a 2-h period of regeneration the major ciliary axonemal proteins such as the tubulins appeared constant in amount. However, a moderate decrease in ATPase activity, and a large decrease of total AK activity, were measured. The decrease in AK activity paralleled the decrease in embryo swimming velocity. Embryos were deciliated once and cilia regeneration followed for 4 h. ATPase activity recovered to control levels by 3 h, but AK activity and swimming velocity remained lower than in controls. Detergent solubility data and kinetic experiments indicate that, in addition to the 130-kDa Sp-AK, there is at least one additional AK isozyme in embryonic cilia. Analysis of the S. purpuratus genome indicates five AK isozymes in addition to the 130-kDa Sp-AK isozyme. Decreased swimming velocity of embryos with regenerated cilia suggests that regenerated cilia are not as functionally perfect as naturally grown cilia.

7,11-epi-thyrsiferol: completion of its synthesis, evaluation of its antimitotic properties, and the further development of an SAR model.

We (a) describe the completion of a total synthesis of 7,11-epi-thyrsiferol (4), (b) compare the antimitotic activities of thyrsiferol (2), Delta15,28-dehydrothyrsiferol (3), and 7,11-epi-thyrsiferol (4), (c) evaluate the synergistic behavior of the title compound and colchicine to inhibit cell proliferation, and (d) describe the results of conformational searches that provide additional insight concerning the SAR profile of the thyrsiferol family of natural products.

Plasma membrane calcium ATPase is concentrated in the head of sea urchin spermatozoa.

Plasma membrane Ca2+ATPases (PMCAs) export Ca2+ from cells in a highly regulated manner, providing fine-tuning to the maintenance of intracellular Ca2+ concentrations. There are few studies of PMCAs in spermatozoa, which is surprising considering the importance of this enzyme in all cell types. Here we describe the primary structure and localization of the PMCA of sea urchin spermatozoa (suPMCA). The suPMCA is 1,154 amino acids and has 56% identity and 76% similarity to all 4 human PMCA isoforms. The suPMCA shares the features of a typical PMCA, including domains for calmodulin binding, ATP binding, ATPase phosphorylation, and 10 putative transmembrane segments with two large cytoplasmic loops. Southern blots show that suPMCA is a single copy gene. Treatment of live sea urchin sperm with the PMCA inhibitor, 5-(-6)-carboxyeosin, results in elevations of intracellular Ca2+ and loss of flagellar motility. Immunoblotting and immunoflorescence show that suPMCA is concentrated in the sperm head plasma membrane. In previous work, we showed that a plasma membrane K+ dependent Na+/Ca2+ exchanger (suNCKX), which also keeps Ca2+ low in these cells, is concentrated in the sperm flagellum. Thus, the sperm head and flagellum localize different gene products, both functioning to keep intracellular Ca2+ low, while the sperm swims in seawater containing 10 mM Ca2+.