Molecular cloning and functional characterization of recombinant Loxtox from Loxosceles similis venom

• Published in: International journal of biological macromolecules Vol. 164; pp. 1112 - 1123
• Main Authors: Leal, Hortênsia Gomes, de Oliveira Mendes, Bárbara Bruna Ribeiro, Horta, Carolina Campolina Rebello, Pereira, Núbia Braga, Ferreira, Douglas Sales Medina, da Silva, Thais Soares, Biscoto, Gabriela Lago, Kalapothakis, Yan, Machado de Avila, Ricardo A., Chávez-Olórtegui, Carlos, Kalapothakis, Evanguedes
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2020
• Subjects: Animals; Cloning, Molecular; Epitopes - chemistry; Female; Hydrogen-Ion Concentration; Immune Sera - immunology; Loxosceles similis; Neutralization Tests; Phospholipase D - metabolism; Phosphoric Diester Hydrolases - genetics; Phosphoric Diester Hydrolases - metabolism; Protein Isoforms; Rabbits; Recombinant; Recombinant Proteins - metabolism; Serum; Skin - drug effects; Sphingomyelin Phosphodiesterase - metabolism; Spider Venoms - genetics; Spider Venoms - metabolism; Spiders - metabolism; Serum; Loxosceles similis; Recombinant
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2020.07.190

Loxoscelism is a recognized public health problem in Brazil, but the venom from Loxosceles similis, which is widespread in Brazil due to its adaptability to the urban environment, remains poorly characterized. Loxtox is a family of phospholipase D enzymes (PLDs), which are the major components of Loxosceles venom and are responsible for the clinical effects of loxoscelism. Loxtox toxins correspond to 15% of L. similis venom gland transcripts, but the Loxtox family of L. similis has yet to be fully described. In this study, we cloned and functionally characterized recLoxtox s1A and recLoxtox s11A. These recombinant toxins exhibited different in vitro activities depending on pH, and recLoxtox s1A had more intense effects on rabbit skin than did recLoxtox s11A in vivo. Both recombinant toxins were used in immunization protocols, and mapping of their epitopes revealed different immunological reactions for the produced immune serums. Additionally, polyclonal antibodies raised against recLoxtox s1A had greater capacity to significantly reduce the in vitro and in vivo effects of L. similis venom. In summary, we obtained and characterized two novel Loxtox isoforms from L. similis venom, which may be valuable biotechnological and immunological tools against loxoscelism. •Recombinant Loxtox s1A and s11A show high sphingomyelinase activity at pHs of 7.4 and 8, respectively.•Recombinant Loxtox s1A causes more intense damage to rabbit skin than does Loxtox s11A.•Sera raised with recLoxtox s1A better neutralize the effects of Loxosceles similis venom.•Epitopes including residues from active sites were identified in both Loxtox forms.