Brown Spiders’ Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms

Abstract

Phospholipases-D (PLDs) found in Loxosceles spiders’ venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents—L. gaucho and L. laeta—were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced.

Prospective Use of Brown Spider Venom Toxins as Therapeutic and Biotechnological Inputs

“…Comparative analyses between the protection of animals immunized with recombinant active (rLlPLD1) or inactive (rLlPLD2) isoforms of L. laeta PLDs showed that both protocols reduced the development of dermonecrotic lesions induced by L. laeta venom at similar levels, thus reinforcing that the antigenic potential of these enzymes is not related to their activity (Catalán et al, 2011). Molecular engineering enabled the production of mutated PLDs from L. intermedia, L. laeta and L. gaucho venoms with appropriate conformational structure, but devoid of enzymatic activity, opening the possibility to apply these proteins as immunogens to the development of antivenoms or vaccines (Vuitika et al, 2016;da Silva et al, 2021). Figure 1 illustrate this potential of such toxins.…”

“…Since the purification of large amounts of venom is one of the major hitches in this process, the first efforts to use recombinant toxins as immunogens were made. As phospholipases D (PLDs) can trigger most of the toxic effects caused by crude venom, the first studies focused on these toxins to replace the venom in the antivenom production (Chaim et al, 2006;Kusma et al, 2008;Chaves-Moreira et al, 2009, 2011Gremski et al, 2014Gremski et al, , 2020da Silva et al, 2021).…”

“…These studies support the use of non-toxic recombinant proteins in vaccination protocols against loxoscelism. In this sense, the mutated and inactive recombinant PLDs of L. intermedia, L. laeta and L. gaucho mentioned above could be used as antigens in a new-generation vaccination protocol in areas where accidents with those species are endemic, as well as in people more exposed or not eligible to conventional treatments, as depicted in Figure 1 (Vuitika et al, 2016;da Silva et al, 2021).…”

“…Development of a second generation antivenom produced from mutant PLDs or engineered peptides/chimeric proteins based on PLDs(Mendes et al (2013),Vuitika et al (2016),Lima et al (2018),Calabria et al (2019), daSilva et al (2021)) 2. Development of monoclonal antibodies that cross-react with venom PLDs(Alvarenga et al (2003,Karim-Silva et al (2016),Costa et al (2020)) 3.…”

“…Development of monoclonal antibodies that cross-react with venom PLDs(Alvarenga et al (2003,Karim-Silva et al (2016),Costa et al (2020)) 3. Development of a vaccine for areas where the accidents are endemic(Lima et al (2018), daSilva et al (2021))• Biotool to be used in studies regarding tumor cell biology(Wille et al (2013), Siqueira et al (2019)) • Antimicrobial drug (Segura-Ramírez and Silva Júnior. (2018)) Serpins (44-46 kDa) • Development of anticoagulant drugs (Schemczssen-Graeff et al (2021)) • Development of drugs for cancer treatment (Schemczssen-Graeff et al (2021)) • As potential antibacterial (Costa et al (2014)) and insecticide (Clemente et al (2019)) molecules • As tools for cell biology studies regarding proliferation, migration and control for protein half-life (Marathe et al (2019)) Allergens (42-45 kDa) • Development of skin and blood allergic sensitivity test (Linhart and Valenta.…”