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 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.

The Dual Prey-Inactivation Strategy of Spiders—In-Depth Venomic Analysis of Cupiennius salei

“…Recently, we purified the venom gland-specific serine protease VSP1 that specifically cleaves PQMs. According to its target motif, we named this enzyme PQM protease [25]. The active PQM protease exhibits a heterodimeric structure and is responsible for the specific cleavage of the pro-peptide to activate mature peptide toxins in the venom.…”

“…VSP1_b isoforms differ from VSP1_a in an additional Asn residue at position 205, and 16 further non-silent mutations. Besides two silent mutations, the most remarkable difference between the VSP1_b isoforms is a point mutation resulting in amino acid exchange C219R, because the mutation affects the disulfide bond C5-C6 [25]. This may influence the three-dimensional structure and the substrate specificity of the protease.…”

New Insights into the Type II Toxins from the Sea Anemone Heteractis crispa

“…RTX-VI appears to be the result of a previously undescribed post-translational modification that occurs in the sea anemone rather than during isolation of the peptide. The processing quadruplet motif (PQM) protease participating in the maturation of two-chain spider toxins and performing cleavage of the C-termini of Arg was identified [42]. However, there is no processing site of this protease in the sequence of RTX-III.…”

Venomics Approach Reveals a High Proportion of Lactrodectus-Like Toxins in the Venom of the Noble False Widow Spider Steatoda nobilis

“…However, they are not known to be toxic to insects or mammals in their purified form [33]. Other proteins detected in the venom that target neurones include CRISP/Allergen/PR-1 which block Ca 2+ channels [34], U21-ctenitoxin-Pn1a which has a protease activity through the hydrolysis of peptide bonds [35], as well as a putative neurotoxin LTDF 06-01, and U3-theritoxin-Lm1 which have a neurotoxin-like activity. Collectively, these toxins act simultaneously to facilitate targeting and disrupting various aspects of normal nerve function.…”