Pamela A. Zobel-Thropp

Molecular Evolution, Functional Variation, and Proposed Nomenclature of the Gene Family That Includes Sphingomyelinase D in Sicariid Spider Venoms

The venom enzyme sphingomyelinase D (SMase D) in the spider family Sicariidae (brown or fiddleback spiders [Loxosceles] and six-eyed sand spiders [Sicarius]) causes dermonecrosis in mammals. SMase D is in a gene family with multiple venom-expressed members that vary in functional specificity. We analyze molecular evolution of this family and variation in SMase D activity among crude venoms using a data set that represents the phylogenetic breadth of Loxosceles and Sicarius. We isolated a total of 190 nonredundant nucleotide sequences encoding 168 nonredundant amino acid sequences of SMase D homologs from 21 species. Bayesian phylogenies support two major clades that we name alpha and beta, within which we define seven and three subclades, respectively. Sequences in the alpha clade are exclusively from New World Loxosceles and Loxosceles rufescens and include published genes for which expression products have SMase D and dermonecrotic activity. The beta clade includes paralogs from New World Loxosceles that have no, or reduced, SMase D and no dermonecrotic activity and also paralogs from Sicarius and African Loxosceles of unknown activity. Gene duplications are frequent, consistent with a birth-and-death model, and there is evidence of purifying selection with episodic positive directional selection. Despite having venom-expressed SMase D homologs, venoms from New World Sicarius have reduced, or no, detectable SMase D activity, and Loxosceles in the Southern African spinulosa group have low SMase D activity. Sequence conservation mapping shows >98% conservation of proposed catalytic residues of the active site and around a plug motif at the opposite end of the TIM barrel, but alpha and beta clades differ in conservation of key residues surrounding the apparent substrate binding pocket. Based on these combined results, we propose an inclusive nomenclature for the gene family, renaming it SicTox, and discuss emerging patterns of functional diversification.

Ltv1 Is Required for Efficient Nuclear Export of the Ribosomal Small Subunit in Saccharomyces cerevisiae

In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus and exported to the cytoplasm independently of one another. Nuclear export of the 60S requires the adapter protein Nmd3, but no analogous adapter has been identified for the 40S. Ltv1 is a nonessential, nonribosomal protein that is required for 40S subunit biogenesis in yeast. Cells lacking LTV1 grow slowly, are hypersensitive to inhibitors of protein synthesis, and produce about half as many 40S subunits as do wild-type cells. Ltv1 interacts with Crm1, co-sediments in sucrose gradients with 43S/40S subunits, and copurifies with late 43S particles. Here we show that Ltv1 shuttles between nucleus and cytoplasm in a Crm1-dependent manner and that it contains a functional NES that is sufficient to direct the export of an NLS-containing reporter. Small subunit export is reduced in Δltv1 mutants, as judged by the altered distribution of the 5′-ITS1 rRNA and the 40S ribosomal protein RpS3. Finally, we show a genetic interaction between LTV1 and YRB2, a gene that encodes a Ran-GTP-, Crm1-binding protein that facilitates the small subunit export. We propose that Ltv1 functions as one of several possible adapter proteins that link the nuclear export machinery to the small subunit.

Weaponization of a hormone: convergent recruitment of hyperglycemic hormone into the venom of arthropod predators

Arthropod venoms consist primarily of peptide toxins that are injected into their prey with devastating consequences. Venom proteins are thought to be recruited from endogenous body proteins and mutated to yield neofunctionalized toxins with remarkable affinity for specific subtypes of ion channels and receptors. However, the evolutionary history of venom peptides remains poorly understood. Here we show that a neuropeptide hormone has been convergently recruited into the venom of spiders and centipedes and evolved into a highly stable toxin through divergent modification of the ancestral gene. High-resolution structures of representative hormone-derived toxins revealed they possess a unique structure and disulfide framework and that the key structural adaptation in weaponization of the ancestral hormone was loss of a C-terminal α helix, an adaptation that occurred independently in spiders and centipedes. Our results raise a new paradigm for toxin evolution and highlight the value of structural information in providing insight into protein evolution.

Sphingomyelinase D in sicariid spider venom is a potent insecticidal toxin.

Spider venoms have evolved over hundreds of millions of years with a primary role of immobilizing prey. Sphingomyelinase D (SMase D) and homologs in the SicTox gene family are the most abundantly expressed toxic protein in venoms of Loxosceles and Sicarius spiders (Sicariidae). While SMase D is well known to cause dermonecrotic lesions in mammals, little work has investigated the bioactivity of this enzyme in its presumed natural role of immobilizing insect prey. We expressed and purified recombinant SMase D from Loxosceles arizonica (Laz-SMase D) and compared its enzymatic and insecticidal activity to that of crude venom. SMase D enzymatic activities of purified protein and crude venom from the same species were indistinguishable. In addition, SMase D and crude venom have comparable and high potency in immobilization assays on crickets. These data indicate that SMase D is a potent insecticidal toxin, the role for which it presumably evolved.

Spit and Venom from Scytodes Spiders: A Diverse and Distinct Cocktail

Spiders from the family Scytodidae have a unique prey capturing technique: they spit a zig-zagged silken glue to tether prey to a surface. Effectiveness of this sticky mixture is based on a combination of contraction and adhesion, trapping prey until the spider immobilizes it by envenomation and then feeds. We identify components expressed in Scytodes thoracica venom glands using combined transcriptomic and proteomic analyses. These include homologues of toxic proteins astacin metalloproteases and potentially toxic proteins including venom allergen, longistatin, and translationally controlled tumor protein (TCTP). We classify 19 distinct groups of candidate peptide toxins; 13 of these were detected in the venom, making up 35% of the proteome. Six have significant similarity to toxins from spider species spanning mygalomorph and nonhaplogyne araneomorph lineages, suggesting their expression in venom is phylogenetically widespread. Twelve peptide toxin groups have homologues in venom gland transcriptomes of other haplogynes. Of the transcripts, approximately 50% encode glycine-rich peptides that may contribute to sticky fibers in Scytodes spit. Fifty-one percent of the identified venom proteome is a family of proteins that is homologous to sequences from Drosophila sp. and Latrodectus hesperus with uncharacterized function. Characterization of these components holds promise for discovering new functional activity.

Phospholipase D Toxins of Brown Spider Venom Convert Lysophosphatidylcholine and Sphingomyelin to Cyclic Phosphates

Venoms of brown spiders in the genus Loxosceles contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These toxins cleave the substrates sphingomyelin and lysophosphatidylcholine in mammalian tissues, releasing the choline head group. The other products of substrate cleavage have previously been reported to be monoester phospholipids, which would result from substrate hydrolysis. Using 31P NMR and mass spectrometry we demonstrate that recombinant toxins, as well as whole venoms from diverse Loxosceles species, exclusively catalyze transphosphatidylation rather than hydrolysis, forming cyclic phosphate products from both major substrates. Cyclic phosphates have vastly different biological properties from their monoester counterparts, and they may be relevant to the pathology of brown spider envenomation.

Comparative analyses of venoms from American and African Sicarius spiders that differ in sphingomyelinase D activity.

Spider venoms are cocktails of toxic proteins and peptides, whose composition varies at many levels. Understanding patterns of variation in chemistry and bioactivity is fundamental for understanding factors influencing variation. The venom toxin sphingomyelinase D (SMase D) in sicariid spider venom (Loxosceles and Sicarius) causes dermonecrotic lesions in mammals. Multiple forms of venom-expressed genes with homology to SMase D are expressed in venoms of both genera. SMase D activity levels differ among major clades with American Sicarius vastly reduced relative to all Loxosceles and African Sicarius despite expression of SMase D homologs in venoms of American Sicarius. Here we report comparative analyses of protein composition and insecticidal activity of crude venoms from three Sicarius species, two from South Africa and one from Central America. Comparative 2-dimensional electrophoresis shows dense regions of proteins in the size range of SMase D in all three species, but there are differences in sizes and isoelectric points (pIs). Few proteins strictly co-migrate and there are clusters of proteins with similar pIs and molecular weights whose patterns of similarity do not necessarily reflect phylogenetic relatedness. In addition, PD(50) estimates on crickets indicate a small though significant decrease in potency of South American Sicarius venoms relative to African species.

Not as docile as it looks? Loxosceles venom variation and loxoscelism in the Mediterranean Basin and the Canary Islands

The medical importance of Loxosceles spiders has promoted extensive research on different aspects of their venoms. Most of the reported cases of loxoscelism have occurred in the Americas, and thus, much work has focused on North and South American Loxosceles species. Interestingly, loxoscelism cases are rare in the Mediterranean Basin although Loxosceles rufescens, endemic to the Mediterranean, is an abundant spider even in human-altered areas. Thus, it has been suggested that the venom of L. rufescens could be of less medical relevance than that of its congeners. In this study, we challenge this hypothesis by using multiple approaches to study venom variation in selected species and lineages from the Mediterranean Basin and the Canary Islands. We found that SMase D activity, the key bioactive component of Loxosceles venom, is comparable to American species that are confirmed to have medically relevant bites. The venom protein composition using SDS-PAGE presents some differences among regional Loxosceles taxa in banding pattern and intensity, mostly between the Canarian and L. rufescens lineages. Differences between these species also exist in the expression of different paralogs of the SicTox gene family, with the Canarian species being less diverse. In conclusion, our results do not support the challenged hypothesis, and suggest that venom of these species may indeed be as potent as other Loxosceles species. Pending confirmation of loxoscelism with direct evidence of Loxosceles bites with species identification by professionals, Loxosceles in the Mediterranean region should conservatively be considered medically relevant taxa.

Variable substrate preference among phospholipase D toxins from sicariid spiders

Background: Phospholipase D toxins from brown spider venoms can cause disease in humans. Results: Different toxin family members show specificity for lipid substrates with choline or ethanolamine headgroups or can be ambiguous. Conclusion: Spider phospholipase D toxins have evolved diverse substrate preferences. Significance: The diverse substrate preference may be significant for predation and the mammalian toxicity of venom. Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.

Solution Structures of Two Homologous Venom Peptides from Sicarius dolichocephalus

We present solution-state NMR structures for two putative venom peptides from Sicarius dolichocephalus. These peptides were identified from cDNA libraries created from venom gland mRNA and then recombinantly expressed. They are the first structures from any species of Sicarius spiders, and the first peptide structures for any haplogyne spiders. These peptides are homologous to one another, and while they have at most only 20% sequence identity with known venom peptides their structures follow the inhibitor cystine knot motif that has been found in a broad range of venom peptides.