Spiders are ecologically important predators with complex venom and extraordinarily tough silk that enables capture of large prey. Here we present the assembled genome of the social velvet spider and a draft assembly of the tarantula genome that represent two major taxonomic groups of spiders. The spider genomes are large with short exons and long introns, reminiscent of mammalian genomes. Phylogenetic analyses place spiders and ticks as sister groups supporting polyphyly of the Acari. Complex sets of venom and silk genes/proteins are identified. We find that venom genes evolved by sequential duplication, and that the toxic effect of venom is most likely activated by proteases present in the venom. The set of silk genes reveals a highly dynamic gene evolution, new types of silk genes and proteins, and a novel use of aciniform silk. These insights create new opportunities for pharmacological applications of venom and biomaterial applications of silk.

The Venus flytrap (Dionaea muscipula) is one of the most well-known carnivorous plants because of its unique ability to capture small animals, usually insects or spiders, through a unique snap-trapping mechanism. The animals are subsequently killed and digested so that the plants can assimilate nutrients, as they grow in mineral-deficient soils. We deep sequenced the cDNA from Dionaea traps to obtain transcript libraries, which were used in the mass spectrometry-based identification of the proteins secreted during digestion. The identified proteins consisted of peroxidases, nucleases, phosphatases, phospholipases, a glucanase, chitinases, and proteolytic enzymes, including four cysteine proteases, two aspartic proteases, and a serine carboxypeptidase. The majority of the most abundant proteins were categorized as pathogenesis-related proteins, suggesting that the plant's digestive system evolved from defense-related processes. This indepth characterization of a highly specialized secreted fluid from a carnivorous plant provides new information about the plant's prey digestion mechanism and the evolutionary processes driving its defense pathways and nutrient acquisition. Molecular & Cellular

During co-incubation of human inter-␣-inhibitor (I␣I) and human tumor necrosis factor-stimulated gene 6 protein (TSG-6) SDS-stable interactions are formed between the two proteins. We have analyzed the products of this reaction and characterized the mechanism of complex formation. Following the incubation seven new bands not previously identified were apparent in SDS-PAGE. Three of these bands did not contain TSG-6, including heavy chain (HC)1⅐bikunin, HC2⅐bikunin, and free bikunin. In addition high molecular weight complexes composed of the same components as I␣I, including HC1, HC2, and bikunin, were formed. The formation of these complexes was prevented by the addition of hyaluronan. The cross-links stabilizing these complexes displaying properties similar to the protein-glycosaminoglycan-protein (PGP) cross-link. The TSG-6-containing SDS-stable complexes were composed of HC1⅐TSG-6 or HC2⅐TSG-6 exclusively. Both glycosylated and non-glycosylated TSG-6 participated in the complex formation. The HC⅐TSG-6 cross-links were different from the PGP cross-link and were determined to be ester bonds between the ␣-carbonyl of the C-terminal Asp of the heavy chain and most likely a hydroxyl group containing the TSG-6 residue. The mechanism involved cleaving the PGP cross-link of I␣I during a transesterification reaction. A TSG-6 hydroxyl group reacts with the ester bond between the ␣-carbonyl of the C-terminal Asp residues of HC1 or HC2 and carbon-6 of an internal N-acetylgalactosamine of the chondroitin-4-sulfate chain. An intermediate is formed resulting in a partitioning of the reaction between HC(1 or 2)⅐TSG-6 complexes and transfer of HC(1 or 2) to the chondroitin via competing pathways.

IalphaI and TSG-6 interact to form a covalent bond between the C-terminal Asp alpha-carbon of an IalphaI heavy chain (HC) and an unknown component of TSG-6. This event disrupts the protein-glycosaminoglycan-protein (PGP) cross-link and dissociates IalphaI. In simple terms the interaction involves 5 components: (i) the IalphaI HCs, (ii) bikunin, (iii) chondroitin sulfate chain, (iv) TSG-6, and (v) divalent cations. To understand the molecular mechanism of complex formation, the effect of these were separately examined. The data show that although the mature covalent cross-link between the HCs and TSG-6 only involves the C-terminal Asp residue, the native fold of both IalphaI and TSG-6 was essential for the reaction to occur. Similarly, complex formation was prevented if the chondroitin sulfate chain was cleaved, releasing bikunin but maintaining the HC1 and HC2 PGP cross-links. In contrast, releasing the majority of the bikunin protein moiety by limited proteolysis did not prevent complex formation. An analysis of the divalent-cation requirements revealed two distinct interactions between IalphaI and TSG-6: (i) a noncovalent manganese, magnesium, or calcium-independent interaction between TSG-6 and the chondroitin sulfate chain (Kd 180 nM) and (ii) a covalent manganese, magnesium, or calcium-dependent interaction generating HC1 x TSG-6, HC2 x TSG-6, and high molecular weight (HMW) IalphaI. Significantly, both free TSG-6 and HC x TSG-6 complexes were able to bind the chondroitin sulfate chain suggesting that the sites on TSG-6 were distinct. On the basis of these findings, we propose a two-step reaction mechanism involving two putative binding sites. Initially, a cation-independent interaction between TSG-6 and the chondroitin sulfate chain is formed at site 1. Subsequently, a cation-dependent transesterification occurs, generating the covalent HC x TSG-6 cross-link at another site, site 2.

Analyses of arthropod genomes have shown that the genes in the different innate humoral immune responses are conserved. These genes encode proteins that are involved in immune signalling pathways that recognize pathogens and activate immune responses. These immune responses include phagocytosis, encapsulation of the pathogen, and production of effector molecules for pathogen elimination. So far, most studies have focused on insects leaving other major arthropod groups largely unexplored. Here we annotate the immune related genes of six arachnid genomes and present evidence for a conserved pattern of some immune genes, but also evolutionary changes in the arachnid immune system. Specifically, our results suggest that the family of recognition molecules of Beta-1,3-glucanase-related proteins (βGRPs) and the genes from the immune deficiency (IMD) signalling pathway have been lost in a common ancestor of arachnids. These findings are consistent with previous work suggesting that the humoral immune effector proteins are constitutively produced in arachnids in contrast to insects, where these have to be induced. Further functional studies are needed to verify this. We further show that the full hemolymph clotting cascade found in the horseshoe crab is retrieved in most arachnid genomes. Tetranychus lacks at least one major component, although it is possible that this cascade could still function through recruitment of a different protein. The gel-forming protein in horseshoe crabs, coagulogen, was not recovered in any of the arachnid genomes, however, it is possible that the arachnid clot consists of a related protein, spätzle, that is present in all of the genomes.

In the present study we purified the required component from human plasma and identified it as HC2. Proteins containing HC2 including I␣I, HC2⅐bikunin, and free HC2 promoted the formation of HC3⅐TSG-6 and subsequently HC3⅐hyaluronan complexes. HC1 or HC3 did not possess this activity. The presented data reveal that both HC2 and TSG-6 are required for the transesterification reactions to occur.

BackgroundSpiders are predaceous arthropods that are capable of subduing and consuming relatively large prey items compared to their own body size. For this purpose, spiders have evolved potent venoms to immobilise prey and digestive fluids that break down nutrients inside the prey’s body by means of extra-oral digestion (EOD). Both secretions contain an array of active proteins, and an overlap of some components has been anecdotally reported, but not quantified. We systematically investigated the extent of such protein overlap. As venom injection and EOD succeed each other, we further infer functional explanations, and, by comparing two spider species belonging to different clades, assess its adaptive significance for spider EOD in general.ResultsWe describe the protein composition of the digestive fluids of the mygalomorph Acanthoscurria geniculata and the araneomorph Stegodyphus mimosarum, in comparison with previously published data on a third spider species. We found a number of similar hydrolases being highly abundant in all three species. Among them, members of the family of astacin-like metalloproteases were particularly abundant. While the importance of these proteases in spider venom and digestive fluid was previously noted, we now highlight their widespread use across different spider taxa. Finally, we found species specific differences in the protein overlap between venom and digestive fluid, with the difference being significantly greater in S. mimosarum compared to A. geniculata.ConclusionsThe injection of venom precedes the injection with digestive fluid, and the overlap of proteins between venom and digestive fluid suggests an early involvement in EOD. Species specific differences in the overlap may reflect differences in ecology between our two study species. The protein composition of the digestive fluid of all the three species we compared is highly similar, suggesting that the cocktail of enzymes is highly conserved and adapted to spider EOD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3987-9) contains supplementary material, which is available to authorized users.

Data processing and analysis of proteomics data are challenging and time consuming. In this paper, we present MS Data Miner (MDM) (http://sourceforge.net/p/msdataminer), a freely available web-based software solution aimed at minimizing the time required for the analysis, validation, data comparison, and presentation of data files generated in MS software, including Mascot (Matrix Science), Mascot Distiller (Matrix Science), and ProteinPilot (AB Sciex). The program was developed to significantly decrease the time required to process large proteomic data sets for publication. This open sourced system includes a spectra validation system and an automatic screenshot generation tool for Mascot-assigned spectra. In addition, a Gene Ontology term analysis function and a tool for generating comparative Excel data reports are included. We illustrate the benefits of MDM during a proteomics study comprised of more than 200 LC-MS/MS analyses recorded on an AB Sciex TripleTOF 5600, identifying more than 3000 unique proteins and 3.5 million peptides.