BARYONIC DARK MATTER ?

Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio

Characterization of rDM64 protein purified from yeast medium.

<p>(A) Purification of rDM64 from the culture medium supernatant by affinity chromatography with immobilized myotoxin II. (B) After elution, the bound fraction (indicated by an asterisk) was further purified by anion-exchange chromatography using a Mono Q column. The chromatographic fractions were analyzed by 12% SDS-PAGE under reducing conditions and were stained with Coomassie blue (C) and periodic acid-Schiff reagent (D) or by immunoblotting with polyclonal antibodies raised against DM64 (E). Lane 1, native DM64 (5 μg). A typical degradation product ~ 45 kDa in size, generated by sample manipulation, can also be observed; lane 2, crude culture medium. Asterisks indicate the chromatographic fractions and are shown in panels A and B (5 μg/lane). NG, non-glycosylated protein (soybean trypsin inhibitor) used as negative control for the periodic acid-Schiff staining. MM, molecular mass markers.</p

Time-course profiles of rDM64 secretion in <i>Pichia pastoris</i> culture during fermentation.

<p><i>P</i>. <i>pastoris</i> was grown at 30°C and 250 rpm for 288 hours. 0–24 hours: glycerol batch phase. 24–288 hours: methanol induction (fed-batch phase); 1% methanol was added every 24 hours, for 264 hours. Dry cell weight/biomass (circle) and rDM64 concentration (square) were determined in the absence <b>(A)</b> or presence <b>(B)</b> of 0.2 mM Pefabloc. The concentration of rDM64 was measured by immunoassay in the culture medium and was performed in triplicate. Error bars represent the standard deviation of the mean.</p

Screening for target toxins of the antiophidic protein DM64 through a gel-based interactomics approach

DM64 is a glycosylated protein with antivenomactivity isolated fromthe serum of the opossumDidelphis aurita. It binds non-covalently to myotoxins I (Asp49) and II (Lys49) from Bothrops asper venom and inhibits their myotoxic effect. In this study, an affinity columnwith immobilizedDM64 as baitwas used to fish potential target toxins. All ten isolated myotoxins tested were able to effectively bind to the DM64 column. To better access the specificity of the inhibitor, crude venoms from Bothrops (8 species), Crotalus (2 species) and Naja naja atra were submitted to the affinity purification. Venom fractions bound and nonbound to the DM64 columnwere analyzed by two-dimensional gel electrophoresis andMALDI-TOF/TOFMS. Although venomfractions bound to the column were mainly composed of basic PLA2, a few spots corresponding to acidic PLA2 were also observed. Some unexpected protein spotswere also identified: C-type lectins and CRISPmay represent putative new targets forDM64, whereas the presence of serine peptidases in the venom bound fraction is likely a consequence of nonspecific binding to the column matrix. The present results contribute to better delineate the inhibitory potential of DM64, providing a framework for the development of more specific antivenom therapies. Biological significance: Local tissue damage induced bymyotoxic PLA2 remains a serious consequence of snake envenomation, since it is only partially neutralized by traditional antivenom serotherapy. Myotoxin inhibition by highly specific molecules offers great promise in the treatment of snakebites, a health problemlargely neglected by governments and pharmaceutical industries. Bioactive compounds such as DM64 can represent a valuable source of scaffolds for drug development in this area. The present study has systematically profiled the binding specificity of DM64 toward a variety of snake venom toxin classes and therefore can lead to a better understanding of the structure-function relationship of this important antivenom protein.Fundação Oswaldo Cruz:uma instituição a serviço da visto/[RPT02A]/FIOCRUZ/BrasilFundação Oswaldo Cruz:uma instituição a serviço da visto/[RPT02B]/FIOCRUZ/BrasilConselho Nacional de Desenvolvimento Científico e Tecnológico/[400147 / 2014-4]/CNPq/BrasilFundação de Amparo à Pesquisa do Estado do Rio de Janeiro/[E-26 / 102.807 / 2012-BBP]/FAPERJ/BrasilCoordenação de Aperfeiçoamento de Pessoal de Nível Superior/[23038.006270 / 2011-63]/CAPES/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum

Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human α1B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood.Conselho Nacional de Desenvolvimento Científico e Tecnológico//CNPq/BrasilFundação de Amparo à Pesquisa do Estado do Rio de Janeiro//FAPERJ/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo//FAPESP/BrasilFundação Oswaldo Cruz//Fiocruz/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Molecular architecture of the antiophidic protein DM64 and its binding specificity to myotoxin II from Bothrops asper venom

DM64 is a toxin-neutralizing serum glycoprotein isolated from Didelphis aurita, an ophiophagous marsupial naturally resistant to snake envenomation. This 64 kDa antitoxin targets myotoxic phospholipases A2, which account for most local tissue damage of viperid snakebites. We investigated the noncovalent complex formed between native DM64 and myotoxin II, a myotoxic phospholipase-like protein from Bothrops asper venom. Analytical ultracentrifugation (AUC) and size exclusion chromatography indicated that DM64 is monomeric in solution and binds equimolar amounts of the toxin. Attempts to crystallize native DM64 for X-ray diffraction were unsuccessful. Obtaining recombinant protein to pursue structural studies was also challenging. Classical molecular modeling techniques were impaired by the lack of templates with more than 25% sequence identity with DM64. An integrative structural biology approach was then applied to generate a three-dimensional model of the inhibitor bound to myotoxin II. I-TASSER individually modeled the five immunoglobulin-like domains of DM64. Distance constraints generated by cross-linking mass spectrometry of the complex guided the docking of DM64 domains to the crystal structure of myotoxin II, using Rosetta. AUC, small-angle X-ray scattering (SAXS), molecular modeling, and molecular dynamics simulations indicated that the DM64-myotoxin II complex is structured, shows flexibility, and has an anisotropic shape. Inter-protein cross-links and limited hydrolysis analyses shed light on the inhibitor’s regions involved with toxin interaction, revealing the critical participation of the first, third, and fifth domains of DM64. Our data showed that the fifth domain of DM64 binds to myotoxin II amino-terminal and beta-wing regions. The third domain of the inhibitor acts in a complementary way to the fifth domain. Their binding to these toxin regions presumably precludes dimerization, thus interfering with toxicity, which is related to the quaternary structure of the toxin. The first domain of DM64 interacts with the functional site of the toxin putatively associated with membrane anchorage. We propose that both mechanisms concur to inhibit myotoxin II toxicity by DM64 binding. The present topological characterization of this toxin-antitoxin complex constitutes an essential step toward the rational design of novel peptide-based antivenom therapies targeting snake venom myotoxins.Fundação Oswaldo Cruz/[INOVA GC VPPCB-007-FIO-18-2-9]/Fiocruz/BrasilFundação de Amparo à Pesquisa do Estado do Rio de Janeiro/[APQ1 E-6/010.001929/2019]/FAPERJ/BrasilConselho Nacional de Desenvolvimento Científico e Tecnológico/[Universal 426290/2018-6]/CNPq/BrasilNational Institutes of Health/[GM120600]/NIH/Estados UnidosNational Science Foundation/[NSF-ACI-1339649]/NSF/Estados UnidosSan Diego Supercomputer Center/[TG-MCB070039N]/SDSC/Estados UnidosTexas Advanced Computing Center/[TG457201]/TACC/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region has been found in the N-terminal R0 region of the protein. Herein, we describe the antiplasmodial activity of anti-GLURP antibodies present in the sera from individuals naturally exposed to malaria in a Brazilian malaria-endemic area. The anti-R0 antibodies showed a potent inhibitory effect on the growth of P. falciparum in vitro, both in the presence (ADCI) and absence (GI) of monocytes. The inhibitory effect on parasite growth was comparable to the effect of IgGs purified from pooled sera from hyperimmune African individuals. Interestingly, in the ADCI test, higher levels of tumour necrosis factor alpha (TNF-&#945;) were observed in the supernatant from cultures with higher parasitemias. Our data suggest that the antibody response induced by GLURP-R0 in naturally exposed individuals may have an important role in controlling parasitemia because these antibodies are able to inhibit the in vitro growth of P. falciparum with or without the cooperation from monocytes. Our results also indicate that TNF-&#945; may not be relevant for the inhibitory effect on P. falciparum in vitro growth