Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom.

Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom.

Molecular cloning, heterologous expression and functional characterization of a novel translationally-controlled tumor protein (TCTP) family member from Loxosceles intermedia (brown spider) venom.

Envenoming with brown spiders (Loxosceles genus) is common throughout the world. Cutaneous symptoms following spider bite accidents include dermonecrosis, erythema, itching and pain. In some cases, accidents can cause hypersensibility or even allergic reactions. These responses could be associated with histaminergic events, such as an increase in vascular permeability and vasodilatation. A protein that may be related to the effects of spider venom was identified from a previously obtained cDNA library of the L. intermedia venom gland. The amino acid sequence of this protein is homologous to proteins from the TCTP (translationally-controlled tumor protein) family, which are extracellular histamine-releasing factors (HRF) that are associated with the allergic reactions to parasites. Herein, we described the cloning, heterologous expression, purification and functional characterization of a novel member of the TCTP family from the Loxosceles intermedia venom gland. This recombinant protein, named LiRecTCTP, causes edema, enhances vascular permeability and is likely related to the inflammatory activity of the venom. Moreover, LiRecTCTP presents an immunological relationship with mammalian TCTPs.

Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: endothelial cell membrane phospholipids as targets for toxicity.

Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. The mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids.

Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles).

Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom.

Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles)

Brown spiders have a worldwide distribution, and their venom has a complex composition containingmany different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacinlike toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins inthe venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showedgelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers weresequenced, and their deduced amino acid sequences confirmed they were members of the astacin familywith the family signatures(HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequencecomparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are relatedto the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom.

Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase-D (dermonecrotic toxin) from brown spider venom.

Brown spiders have world-wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase-D, causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose-dependent and time-dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP-phospholipase-D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin-treated erythrocytes reacted with annexin-V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase-D, which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine-protease inhibitor) that had no effect on hemolysis. By site-directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase-D triggers direct human blood cell hemolysis in a catalytic-dependent manner.

Structural and functional characterization of a P-III metalloproteinase, leucurolysin-B, from Bothrops leucurus venom.

Leucurolysin-B (leuc-B) is an hemorrhagic metalloproteinase found in the venom of Bothrops leucurus (white-tailed-jararaca) snake. By means of liquid chromatography consisting of gel filtration on Sephracryl S-200, S-300 and ion-exchange on DEAE Sepharose, leuc-B was purified to homogeneity. The proteinase has an apparent molecular mass of 55kDa as revealed by the reduced SDS-PAGE, and represents approximately 1.2% of the total protein in B. leucurus venom. The partial amino acid sequence of leuc-B was determined by automated Edman sequencing of peptides derived from digests of the S-reduced and alkylated protein with trypsin. Leuc-B exhibits the characteristic motif of metalloproteinases, HEXXHXXGXXH and a methionine-containing turn of similar conformation ("Met-turn"), which forms a hydrophobic basis for the zinc ions and the three histidine residues involved as ligands. Leuc-B has been characterized as a P-III metalloproteinase and possesses a multidomain structure including a metalloproteinase, a disintegrin-like (ECD sequence instead of the typical RGD motif) and a cysteine-rich C-terminal domain. Leuc-B contains three potential sites of N-glycosylation. The enzyme only cleaves the Ala14-Leu15 peptide bond of the oxidized insulin B-chain and preferentially hydrolyzes the Aalpha-chain of fibrinogen and the alpha-chain of fibrin. Its proteolytic activity was completely inhibited by metal chelating agents but not by other typical proteinase inhibitors. In addition, its enzymatic activity was stimulated by the divalent cations Ca2+ and Mg2+ but inhibited by Zn2+ and Cu2+. The catalytic activity of leuc-B on extracellular matrix proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites (MHD=30ng in rabbit), with alterations in platelet function. In summary, here we report the isolation and the structure-function relationship of a P-III snake venom metalloproteinase.