Novel venom peptides from the cone snail Conus pulicarius discovered through next-generation sequencing of its venom duct transcriptome

ManuscriptThe venom peptides (i.e., conotoxins or conopeptides) that species in the genus Conus collectively produce are remarkably diverse, estimated to be around 50,000 to 140,000, but the pace of discovery and characterization of these peptides have been rather slow. To date, only a minor fraction have been identified and studied. However, the advent of next generation DNA sequencing technologies has opened up opportunities for expediting the exploration of this diversity. The whole transcriptome of a venom duct from the vermivorous marine snail C. pulicarius was sequenced using the 454 sequencing technology. Analysis of the data set resulted in the identification of over eighty unique putative conopeptide sequences, the highest number discovered so far from a Conus venom duct transcriptome. More importantly, majority of the sequences are potentially novel, many with unexpected structural features, hinting at the vastness of the diversity of Conus venom peptides that remains to be explored. The sequences can be classified into at least 14 major superfamilies/types (disulfide- and non-disulfide-rich), indicating the structural and functional diversity of conotoxins in the venom of C. pulicarius. In addition, the contryphans were surprisingly more diverse than what is currently known. Comparative analysis of the O-superfamily sequences also revealed insights into the complexity of the processes that drive the evolution and diversification of conotoxins

Native homing endonucleases can target conserved genes in humans and in animal models

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays

Conotoxin Diversity in Chelyconus ermineus (Born, 1778) and the Convergent Origin of Piscivory in the Atlantic and Indo-Pacific Cones

The transcriptome of the venom duct of the Atlantic piscivorous cone species Chelyconus ermineus (Born, 1778) was determined. The venom repertoire of this species includes at least 378 conotoxin precursors, which could be ascribed to 33 known and 22 new (unassigned) protein superfamilies, respectively.Most abundant superfamilies were T,W, O1, M, O2, and Z, accounting for 57% of all detected diversity. A total of three individuals were sequenced showing considerable intraspecific variation: each individual had many exclusive conotoxin precursors, and only 20% of all inferred mature peptides were common to all individuals. Three different regions (distal, medium, and proximal with respect to the venom bulb) of the venom duct were analyzed independently. Diversity (in terms of number of distinct members) of conotoxin precursor superfamilies increased toward the distal region whereas transcripts detected toward the proximal region showed higher expression levels. Only the superfamilies A and I3 showed statistically significant differential expression across regions of the venom duct. Sequences belonging to the alpha (motor cabal) and kappa (lightning-strike cabal) subfamilies of the superfamily A were mainly detected in the proximal region of the venom duct. The mature peptides of the alpha subfamily had the a4/4 cysteine spacing pattern, which has been shown to selectively target muscle nicotinic-acetylcholine receptors, ultimately producing paralysis. This function is performed by mature peptides having a a3/5 cysteine spacing pattern in piscivorous cone species from the Indo-Pacific region, thereby supporting a convergent evolution of piscivory in cones

Cloning and characterization of genes involved in carbohydrate metabolism in the marine red alga Gracilaria gracilis

The molecular biology of carbohydrate metabolism in red algae is poorly known. Enzymological studies are few, and no gene for the biosynthesis of sugar nucleotides and polysaccharides has so far been characterized. To isolate genes involved in carbohydrate metabolism in Gracilaria gracilis, genomic libraries were screened with homologous probes prepared either by PCR with degenerate primers, or from cDNAs previously isolated for generating expressed sequence tags (ESTs) from G. gracilis. Genes involved in carbohydrate metabolism, photosynthesis, protein synthesis and degradation, amino acid metabolism, and stress response were among those tagged by the ESTs. Three genes were characterized. These encode galactose-1-phosphate uridylyltransferase (GALT, named GgGALT1), a key enzyme for scD-galactose metabolism; UDPglucose pyrophosphorylase (UGPase; GgUGP), a key enzyme for sugar nucleotide synthesis; and starch branching enzyme (SBE; GgSBE1), which helps determine the structure of floridean starch. The three genes are devoid of introns. Each possesses a polyadenylation signal, TAAA, which occurs in all G. gracilis genes so far characterized, as well as a potential TATA box. Southern hybridization experiments indicate that the three genes are single-copy, but that other genes related to GgGALT1 and GgSBE1 exist. GgGALT1 and GgUGP are each located close to another gene, hinting that occurrence of closely-spaced genes, atypical in eukaryotic genomes, may not be uncommon in the G. gracilis genome. The deduced proteins show high sequence similarity with their homologs in other organisms, but intriguing differences, such as nonconservative substitutions at functionally important sites, were observed. The protein encoded by GgSBE1 lacks an N-terminal portion that could contain a possible target peptide, consistent with the cytosolic localization of floridean starch synthesis. The GgUGP and GgSBE1 proteins are as phylogenetically related to plant as they are to their animal and fungal homologs.Doctor of Philosoph

Characterization of the UDP-glucose pyrophosphorylase gene from the marine red alga Gracilaria gracilis

UDP-glucose pyrophosphorylase (UGPase) is a key enzyme in carbohydrate metabolism, particularly polysaccharide biosynthesis, in red algae. In this report, we characterize at the genomic and cDNA levels the putative UGPase gene of the agarophytic red alga Gracilaria gracilis. The gene is single-copy, devoid of introns, and produces two kinds of transcripts that differ in size by 332 basepairs. The large and small transcripts appear to utilize distinct polyadenylation signals. The putative protein has 495 amino acids, and is about 50% identical in sequence to its homplogs in plants, animals and fungi. Sequencing of the,genomic clone revealed that another gene, potentially encoding a DNA helicase and containing a 76 bp-intron near its 3' end, occurs 376 bp downstream of the UGPase gene

Characterization of a galactose-1-phosphate uridylyltransferase gene from the marine red alga Graeilaria gracilis

The metabolism of D-galactose is a major feature of red-algal physiology. We have cloned and sequenced a gene from the red alga Gracilaria gracilis that encodes a key enzyme of D-galactose metabolism, galactose-l-phosphate uridylyltransferase (GALT). This gene, designated GgGALT1, is apparently devoid of introns. A potential TATA box, four potential CAAT boxes, and a repeated sequence occur in the 5'-flanking region. The predicted 369-aa peptide shares significant sequence similarity with GALTs from other organisms (human, 47%; Saccharomyces cerevisiae, 49%; Solanum tuberosum, 49%). Southern-hybridization analysis reveals two related, but apparently not identical, GALT genes in the nuclear genome of G. gracilis. Sequence analysis indicates that the GgGALT1 enzyme lacks a rubredoxin "knuckle" motif, which in bacterial and fungal GALTs is involved in binding zinc. An open reading frame encoding a potential peptidyl tRNA hydrolase occurs 179 bp downstream from the GgGALT1 gene

Cloning and characterization of a nuclear gene encoding a starch branching enzyme from the marine red alga Gracilaria gracilis

The biosynthesis of starch in red algae occurs in the cytosol, in contrast to green plants where it takes place in the plastid. We have cloned a nuclear gene from the red alga Gracilaria gracilis that encodes a homolog of starch-branching enzymes (SBEs); this gene, which is apparently intron-free, was designated as GgSBE1. A potential TATA box, CAAT boxes, and other potential regula tory elements were observed in its 5' flanking region. The encoded 766-aa peptide shares significant sequence similarity with SBEs from green plants (at least 40%), and with glycogen-branching enzymes (GBEs) from human (46%) and Saccharomyces cerevisiae (45%). Southern-hybridization analysis indicates that the gene is single-copy, although weaker signals suggest that related genes exist in the genome of G. gracilis. Phylogenetic analyses indicate that GgSBE1 groups within the eukaryote branching enzymes (BEs) and not with eubacterial GBEs, suggesting that its gene has not been derived directly from an endosymbiotic cyanobacterium, but instead is ancestrally eukaryotic