Hyperglycosylation impairs the inhibitory activity of rCdtPLI2, the first recombinant beta-phospholipase A₂ inhibitor

Author(s)

Wiezel, Gisele A. ; Oliveira, Isadora S. ; Ferreira, Isabela G. ; Bordon, Karla C. F. ; Arantes, Eliane C.

• Document type: Journal article
• Source: International Journal of Biological Macromolecules; v. 280, p. 12-pg., 2024-09-22.

Abstract

Crotoxin, a phospholipase A(2) (PLA(2)) complex and the major Crotalus venom component, is responsible for the main symptoms described in crotalic snakebite envenomings and a key target for PLA(2) inhibitors (PLIs). PLIs comprise the alpha, beta and gamma families, and, due to a lack of reports on beta-PLIs, this study aimed to heterologously express CdtPLI2 from Crotalus durissus terrificus venom gland to improve the knowledge of the neglected beta-PLI family. Thereby, recombinant CdtPLI2 (rCdtPLI2) was produced in the eukaryotic Pichia pastoris system to keep some native post-translational modifications. rCdtPLI2 (similar to 41 kDa) presents both N- and O-linked glycans. Alpha-mannosidase digested-rCdtPLI2 (1 mol) strongly inhibited (73%) CB-Cdc catalytic activity (5 moles), demonstrating that glycosylations performed by P. pastoris affect rCdtPLI2 action. Digested-rCdtPLI2 also inhibited PLA(2)s from diverse Brazilian snake venoms. Furthermore, rCdtPLI2 (1 mol) abolished the catalytic activity of Lmr-PLA(2) (5 moles) and reduced the CTx-Cdc (5 moles) enzyme activity by 65%, suppressing basic and acidic snake venom PLA(2)s. Additionally, crotalic antivenom did not recognize rCdtPLI2, suggesting a lack of neutralization by antivenom antibodies. These findings demonstrate that studying snake venom components may reveal interesting novel molecules to be studied in the snakebite treatment and help to understand these underexplored inhibitors. (AU)

• FAPESP's process: 21/11936-3 - Center for Translational Science and Biopharmaceutical Development
• Grantee: Benedito Barraviera
• Support Opportunities: Research Grants - Science Centers for Development
• FAPESP's process: 09/54098-6 - Acquisition of a mass spectrometer for the generation of images at the Mass Spectrometry Center of the University of São Paulo Ribeirão Preto School of Pharmaceutical Sciences: studies of molecular localization of biologically active substances
• Grantee: Norberto Peporine Lopes
• Support Opportunities: Multi-user Equipment Program
• FAPESP's process: 19/10173-6 - Production, modification and characterization of animal toxins with potential biotechnological application
• Grantee: Eliane Candiani Arantes Braga
• Support Opportunities: Regular Research Grants
• FAPESP's process: 20/13176-3 - Human monoclonal antibodies (scFv) discovery with cross-reactivity and pH-dependent to metalloproteases from Bothrops spp
• Grantee: Isadora Sousa de Oliveira
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 17/00586-6 - Characterization of a phospholipase A2 inhibitor from the Crotalus durissus terrificus venom gland: a possible adjuvant in the antivenom therapy
• Grantee: Gisele Adriano Wiezel
• Support Opportunities: Scholarships in Brazil - Doctorate