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.
Highlights
The isolated protease was identified to be heterodimeric by liquid chromatography-mass spectrometry (LC-MS) of the reduced and alkylated protein (Fig. 2B)
Isolation and characterization of spider venom enzymes can be challenging due to: 1) their often up to 1000-fold lower venom concentration compared with the main toxins (e.g. for C. salei: venom hyaluronidase, 1.3–9.3 M [16] versus main neurotoxin CsTx-1, 1.4 –3.3 mM [13]), and 2) potential loss of activity during the purification
The heterodimeric structure is likely to be a result of proteolytic cleavage of a monomeric precursor C-terminal of Arg35
Summary
Ple metalloproteases and some serine proteases with unclear functions have been identified in spider venoms [3]. Different proteins have been hypothesized to be involved in propeptide processing of spider venom peptides. The involvement in the maturation process of venom neurotoxins has so far not been experimentally confirmed for any protease isolated from spider venom. We report the isolation and characterization of a PQM processing protease from the venom of the Central American spider Cupiennius salei (Ctenidae). We demonstrate the specificity of the PQM protease for propeptide removal from spider venom neurotoxin precursors. We provide evidence for the involvement of this protease in the generation of the two heterodimeric neurotoxins CsTx-13 (C. salei, P83919) [11] and -agatoxin-1A (Agelenopsis aperta, P15969) [12]
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