1. Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming
- Author
-
Naan Rodrigues Gonçalves, Fernando B. Zanchi, Marcos B. Luiz, Carla F. C. Fernandes, Juliana P. Zuliani, Anderson M. Kayano, Cléberson de Freitas Fernandes, Nidiane D. R. Prado, Soraya dos Santos Pereira, André Lopes Fuly, Andreimar Martins Soares, Juliana C. Sobrinho, Leandro S. Moreira-Dill, and Rodrigo G. Stábeli
- Subjects
Male ,0301 basic medicine ,Phage display ,Health, Toxicology and Mutagenesis ,Myotoxin ,Snake Bites ,lcsh:Medicine ,VHH ,Crotoxina ,Biopanning ,Toxicology ,Immunoglobulin light chain ,Article ,Mice ,03 medical and health sciences ,Muscular Diseases ,Western blot ,crotoxin ,CB ,Crotalus durissus terrificus ,Escherichia coli ,medicine ,Animals ,biology ,medicine.diagnostic_test ,Chemistry ,lcsh:R ,Crotalus ,Single-Domain Antibodies ,Crotoxin ,biology.organism_classification ,Molecular biology ,In vitro ,Molecular Docking Simulation ,030104 developmental biology ,biology.protein ,Crotalus cascavella ,Antibody ,Camelids, New World - Abstract
Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A2 (CB) and a subunit with no toxic activity (CA). Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB (KF498602, KF498603, KF498604, and KF498605) and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB throughWestern blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment.
- Published
- 2018