Zhenghua Ren

Identification of a novel M-superfamily conotoxin with the ability to enhance tetrodotoxin sensitive sodium currents

In this work, a novel M-superfamily conotoxin, designated lt3a, was purified from the crude venom of Conus litteratus. Combined with peptide sequencing, MALDI-TOF mass spectrometry and cDNA cloning techniques, the amino acid sequence of lt3a was supposed to be DγCCγ OQWCDGACDCCS, where O is hydroxyproline and γ is carboxyglutamate. The Cys framework of lt3a (–CC–C–C–CC–) is similar to that of ψ-, μ-, κM-conotoxins, which are representatives of M-conotoxins. Peptide lt3a is categorized into M1 branch based on the number of residues in the last Cys loop. Whole cell patch-clamp study on adult rat dorsal root ganglion neurons indicated that lt3a could enhance tetrodotoxin-sensitive sodium currents. This is a previously unknown function of M-superfamily conotoxins.

Identification and characterization of a novel O‐superfamily conotoxin from Conus litteratus

A novel conotoxin named lt6c, an O‐superfamily conotoxin, was identified from the cDNA library of venom duct of Conus litteratus. The full‐length cDNA contains an open reading frame encoding a predicted 22‐residue signal peptide, a 22‐residue proregion and a mature peptide of 28 amino acids. The signal peptide sequence of lt6c is highly conserved in O‐superfamily conotoxins and the mature peptide consists of six cysteines arranged in the pattern of CCCCCC that is defined the O‐superfamily of conotoxins. The mature peptide fused with thioredoxin, 6‐His tag, and a Factor Xa cleavage site was successfully expressed in Escherichia coli. About 12 mg lt6c was purified from 1L culture. Under whole‐cell patch‐clamp mode, lt6c inhibited sodium currents on adult rat dorsal root ganglion neurons. Therefore, lt6c is a novel O‐superfamily conotoxin that is able to block sodium channels. Copyright

Ancestral genetic complexity of arachidonic acid metabolism in Metazoa.

Eicosanoids play an important role in inducing complex and crucial physiological processes in animals. Eicosanoid biosynthesis in animals is widely reported; however, eicosanoid production in invertebrate tissue is remarkably different to vertebrates and in certain respects remains elusive. We, for the first time, compared the orthologs involved in arachidonic acid (AA) metabolism in 14 species of invertebrates and 3 species of vertebrates. Based on parsimony, a complex AA-metabolic system may have existed in the common ancestor of the Metazoa, and then expanded and diversified through invertebrate lineages. A primary vertebrate-like AA-metabolic system via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) pathways was further identified in the basal chordate, amphioxus. The expression profiling of AA-metabolic enzymes and lipidomic analysis of eicosanoid production in the tissues of amphioxus supported our supposition. Thus, we proposed that the ancestral complexity of AA-metabolic network diversified with the different lineages of invertebrates, adapting with the diversity of body plans and ecological opportunity, and arriving at the vertebrate-like pattern in the basal chordate, amphioxus.

Characterizing the evolution and functions of the M-superfamily conotoxins

Conotoxins from cone snails are valuable in physiology research and therapeutic applications. Evolutionary mechanisms of conotoxins have been investigated in several superfamilies, but there is no phylogenetic analysis on M-superfamily conotoxins. In this study, we characterized identical sequences, gene structure, novel cysteine frameworks, functions and evolutionary mechanisms of M-superfamily conotoxins. Identical M-superfamily conotoxins can be found in different Conus species from the analysis of novel 467 M-superfamily conotoxin sequences and other published M-superfamily conotoxins sequences. M-superfamily conotoxin genes consist of two introns and three exons from the results of genome walking. Eighteen cysteine frameworks were identified from the M-superfamily conotoxins, and 10 of the 18 may be generated from framework III. An analysis between diet types and phylogeny of the M-superfamily conotoxins indicate that M-superfamily conotoxins might not evolve in a concerted manner but were subject to birth-and-death evolution. Codon usage analysis shows that position-specific codon conservation is not restricted to cysteines, but also to other conserved residues. By analysing primary structures and physiological functions of M-superfamily conotoxins, we proposed a hypothesis that insertions and deletions, especially insertions in the third cysteine loop, are involved in the creation of new functions and structures of the M-superfamily conotoxins.

Genome-Wide Analyses of Amphioxus MicroRNAs Reveal an Immune Regulation via miR-92d Targeting C3

Recently, amphioxus has served as a model for studying the origin and evolution of vertebrate immunity. However, little is known about how microRNAs (miRNAs) are involved in the immune defense in amphioxus. In this article, we present a systematic study of amphioxus miRNAs in the acute-phase response to bacterial infection; miR-92d was found to regulate the complement pathway in this basal chordate. We identified all 155 possible miRNAs present in the amphioxus Branchiostoma belcheri genome by bioinformatics analyses, including 57 newly identified miRNAs (called bbe-miRNAs), and characterized the miRNA expression pattern. Four miRNAs (bbe-miR-7, bbe-miR-4868a, bbe-miR-2065, and bbe-miR-34b) were upregulated and bbe-miR-92d was downregulated under the challenge of both Vibrio anguillarum and Staphylococcus aureus bacteria. We further predicted miRNA targets and identified mRNA targets of immune-related miRNA using the hybrid PCR method. We propose that miR-92d regulates the complement pathway through targeting C3 for controlling the acute immune response to bacterial infections. This study provides evidence for the complex immune regulation of miRNAs in the acute-phase response in basal chordates.

Molecular evolution and diversity of Conus peptide toxins, as revealed by gene structure and intron sequence analyses.

Cone snails, which are predatory marine gastropods, produce a cocktail of venoms used for predation, defense and competition. The major venom component, conotoxin, has received significant attention because it is useful in neuroscience research, drug development and molecular diversity studies. In this study, we report the genomic characterization of nine conotoxin gene superfamilies from 18 Conus species and investigate the relationships among conotoxin gene structure, molecular evolution and diversity. The I1, I2, M, O2, O3, P, S, and T superfamily precursors all contain three exons and two introns, while A superfamily members contain two exons and one intron. The introns are conserved within a certain gene superfamily, and also conserved across different Conus species, but divergent among different superfamilies. The intronic sequences contain many simple repeat sequences and regulatory elements that may influence conotoxin gene expression. Furthermore, due to the unique gene structure of conotoxins, the base substitution rates and the number of positively selected sites vary greatly among exons. Many more point mutations and trinucleotide indels were observed in the mature peptide exon than in the other exons. In addition, the first example of alternative splicing in conotoxin genes was found. These results suggest that the diversity of conotoxin genes has been shaped by point mutations and indels, as well as rare gene recombination or alternative splicing events, and that the unique gene structures could have made a contribution to the evolution of conotoxin genes.

The conservation and uniqueness of the caspase family in the basal chordate, amphioxus

BackgroundThe caspase family, which plays a central role in apoptosis in metazoans, has undergone an expansion in amphioxus, increasing to 45 members through domain recombination and shuffling.ResultsIn order to shed light on the conservation and uniqueness of this family in amphioxus, we cloned three representative caspase genes, designated as bbtCaspase-8, bbtCaspase-1/2 and bbtCaspase3-like, from the amphioxus Branchiostoma belcheri tsingtauense. We found that bbtCaspase-8 with conserved protein architecture is involved in the Fas-associated death domain-Caspase-8 mediated pro-apoptotic extrinsic pathway, while bbtCaspase3-like may mediate a nuclear apoptotic pathway in amphioxus. Also, bbtCaspase-1/2 can co-localize with bbtFADD2 in the nucleus, and be recruited to the cytoplasm by amphioxus apoptosis associated speck-like proteins containing a caspase recruitment domain, indicating that bbtCaspase-1/2 may serve as a switch between apoptosis and caspase-dependent innate immune response in invertebrates. Finally, amphioxus extrinsic apoptotic pathway related caspases played important roles in early embryogenesis.ConclusionsOur study not only demonstrates the conservation of bbtCaspase-8 in apoptosis, but also reveals the unique features of several amphioxus caspases with novel domain architectures arose some 500 million years ago.

Pharmacological characterization of conotoxin lt14a as a potent non-addictive analgesic.

Conotoxin lt14a is a small peptide consisting of 13 amino acids. It was originally identified from the cDNA of Conus litteratus in the South China Sea. Previous reports showed lt14a exhibited antinociceptive activity using a hot plate-induced pain mouse model and acted as an antagonist of neuronal nicotinic acetylcholine receptors. We confirmed that conotoxin lt14a administration resulted in antinociception activity using a mouse inflammatory pain model and a rat model of mechanically-induced pain. The mRNA expression of c-fos and NOS in the spinal cord of rats was suppressed by lt14a. Labeling of lt14a with an Alexa Fluor 488 ester showed that lt14a was bound to the surface of PC12 cells and that this binding was inhibited by pre-application of the nicotinic acetylcholine receptor (nAChR) antagonist tubocurarine chloride (TUB) and the nAChR blocker hexamethonium bromide (HB). These data confirm previous reports that showed lt14a binds to the surface of PC12 cells via nAChRs with patch clamp whole-cell recordings. Additional results showed that lt14a suppressed extracellular signal-regulated kinase (ERK1/2) phosphorylation in PC12 cells activated by Ach. Our results showed that lt14a did not induce drug dependence but rather suppressed morphine withdrawal symptoms. Our work suggests that lt14a is a novel antinociceptive agent that targets the nAChR receptor without inducing drug dependence.

Soluble expression, purification and functional identification of the framework XV conotoxins derived from different Conus species

The conotoxin cysteine framework XV (-C-C-CC-C-C-C-C-), which was named Lt15a, was firstly identified from the cDNA library of Conus litteratus. After that, 18 new framework XV conotoxin sequences were cloned from nine Conus species. Like other conopeptides, the XV-conotoxins have the conserved signal peptide and propeptide, and there are also some conserved residues in their mature peptide. All the framework XV conotoxins were apparently converged into two branches, because of the indel and point mutations occurred in their mature peptides. By fused with thioredoxin and 6×His tag, six XV-conotoxins were successfully expressed in Escherichia coli and purified. Different framework XV conotoxins have distinct biological activities on mice and frogs, and that may be related to the diversity of the toxin sequences. All the six XV-conotoxins had no obvious effects on the sodium currents of DRG neuron cells of Sprague-Dawley (SD) rats. The identification of this framework of conotoxins enriches our understanding of the structural and functional diversity of conotoxin.

Discovery of two P-superfamily conotoxins, lt9a and lt9b, with different modifications on voltage-sensitive sodium channels

ABSTRACT In this work, two P‐superfamily conotoxins, lt9a and lt9b, were purified and characterized from the crude venom of Conus litteratus. The amino acid sequences of lt9a and lt9b were determined by the Edman degradation method. It has been suggested that both lt9a and lt9b are produced from the precursor encoded by the gene Lt9.1. During the conotoxin maturation process, different post‐translational modifications occurred between lt9a and lt9b. Conotoxin lt9b was predicted to have two prolines that underwent hydroxylation and one glutamate that underwent carboxylation, while lt9a had no hydroxyproline and carboxyglutamate residue. The calculated mass weights of two P‐superfamily conotoxins with three proposed disulfide bonds were confirmed by matrix‐assisted laser desorption/ionization–time‐of‐flight (MALDI‐TOF) mass spectrometry after considering corresponding post‐translational modifications. These two conotoxins showed different effects on tetrodotoxin‐sensitive sodium currents. Conotoxin lt9a (300 nM) resulted in marked slowing of the tetrodotoxin‐sensitive sodium current decay, a notable increase in the peak current, and an alteration in reversal potential. However, lt9b inhibits tetrodotoxin‐sensitive sodium currents, and the inhibition showed a concentration‐dependent with the half maximal inhibitory concentration (IC50) value of 504.04 nM, but there were no change in the activation and inactivation kinetics of currents. To the best of our knowledge, this is the first investigation of two P‐superfamily conotoxins identified to act on voltage‐sensitive sodium channels with different modifications. HIGHLIGHTSConotoxins lt9a and lt9b were identified from the venom of Conus litteratus.Lt9b is the natural mutant of lt9a with posttranslational modifications.Lt9a and lt9b show different modifications on TTX‐S sodium currents.