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Identification of a precursor processing protease from the spider Cupiennius salei essential for venom neurotoxin maturation.

ABSTRACT: Spider venom neurotoxins and cytolytic peptides are expressed as elongated precursor peptides, which are post-translationally processed by proteases to yield the active mature peptides. The recognition motifs for these processing proteases, first published more than 10 years ago, include the processing quadruplet motif (PQM) and the inverted processing quadruplet motif (iPQM). However, the identification of the relevant proteases was still pending. Here we describe the purification of a neurotoxin precursor processing protease from the venom of the spider Cupiennius salei The chymotrypsin-like serine protease is a 28-kDa heterodimer with optimum activity at venom's pH of 6.0. We designed multiple synthetic peptides mimicking the predicted cleavage sites of neurotoxin precursors. Using these peptides as substrates, we confirm the biochemical activity of the protease in propeptide removal from neurotoxin precursors by cleavage C-terminal of the PQM. Furthermore, the PQM protease also cleaves the iPQM relevant for heterodimerization of a subgroup of neurotoxins. An involvement in the maturing of cytolytic peptides is very likely, due to high similarity of present protease recognition motifs. Finally, bioinformatics analysis, identifying sequences of homolog proteins from 18 spiders of 9 families, demonstrate the wide distribution and importance of the isolated enzyme for spiders. In summary, we establish the first example of a PQM protease, essential for maturing of spider venom neurotoxins. In the future, the here described protease may be established as a powerful tool for production strategies of recombinant toxic peptides, adapted to the maturing of spider venom toxins.

SUBMITTER: Langenegger N

PROVIDER: S-EPMC5808768 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis.Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-?-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.

Project description:BackgroundThe spider Cupiennius salei (Keyserling 1877) has become an important study organism in evolutionary and developmental biology. However, the available staging system for its embryonic development is difficult to apply to modern studies, with strong bias towards the earliest developmental stages. Furthermore, important embryonic events are poorly understood. We address these problems, providing a new description of the embryonic development of C. salei. The paper also discusses various observations that will improve our understanding of spider development.ResultsConspicuous developmental events were used to define numbered stages 1 to 21. Stages 1 to 9 follow the existing staging system for the spider Achaearanea tepidariorum, and stages 10 to 21 provide a high-resolution description of later development. Live-embryo imaging shows cell movements during the earliest formation of embryonic tissue in C. salei. The imaging procedure also elucidates the encircling border between the cell-dense embryo hemisphere and the hemisphere with much lower cell density (a structure termed 'equator' in earlier studies). This border results from subsurface migration of primordial mesendodermal cells from their invagination site at the blastopore. Furthermore, our detailed successive sequence shows: 1) early differentiation of the precheliceral neuroectoderm; 2) the morphogenetic process of inversion and 3) initial invaginations of the opisthosomal epithelium for the respiratory system.ConclusionsOur improved staging system of development in C. salei development should be of considerable value to future comparative studies of animal development. A dense germ disc is not evident during development in C. salei, but we show that the gastrulation process is similar to that in spider species that do have a dense germ disc. In the opisthosoma, the order of appearance of precursor epithelial invaginations provides evidence for the non-homology of the tracheal and book lung respiratory systems.

Project description:The biogenic amines octopamine (OA), tyramine (TA), dopamine (DA), serotonin (5-HT), and histamine (HA) affect diverse physiological and behavioral processes in invertebrates, but recent findings indicate that an additional adrenergic system exists in at least some invertebrates. Transcriptome analysis has made it possible to identify biogenic amine receptor genes in a wide variety of species whose genomes have not yet been sequenced. This approach provides new sequences for research into the evolutionary history of biogenic amine receptors and allows them to be studied in experimentally accessible animal models. The Central American Wandering spider, Cupiennius salei, is an experimental model for neurophysiological, developmental and behavioral research. We identified ten different biogenic amine receptors in C. salei transcriptomes. Phylogenetic analysis indicated that, in addition to the typical receptors for OA, TA, DA, and 5-HT in protostome invertebrates, spiders also have α1- and α2-adrenergic receptors, but lack TAR2 receptors and one invertebrate specific DA receptor type. In situ hybridization revealed four types of biogenic amine receptors expressed in C. salei mechanosensory neurons. We used intracellular electrophysiological experiments and pharmacological tools to determine how each receptor type contributes to modulation of these neurons. We show that arachnids have similar groups of biogenic amine receptors to other protostome invertebrates, but they lack two clades. We also clarify that arachnids and many other invertebrates have both α1- and α2-adrenergic, likely OA receptors. Our results indicate that in addition to an OAβ-receptor that regulates rapid and large changes in sensitivity via a Gs-protein activating a cAMP mediated pathway, the C. salei mechanosensory neurons have a constitutively active TAR1 and/or α2-adrenergic receptor type that adjusts the baseline sensitivity to a level appropriate for the behavioral state of the animal by a Gq-protein that mobilizes Ca2+.

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Identification of a precursor processing protease from the spider Cupiennius salei essential for venom neurotoxin maturation.

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