| Autor: |
Luiz MB; Fundação Oswaldo Cruz, Fiocruz Rondnia, Porto Velho, 76812-245 Rondnia, Brazil. marcos.barros@ifro.edu.br., Pereira SS; Fundação Oswaldo Cruz, Fiocruz Rondnia, Porto Velho, 76812-245 Rondnia, Brazil. soraya.santos@fiocruz.br., Prado NDR; Fundação Oswaldo Cruz, Fiocruz Rondnia, Porto Velho, 76812-245 Rondnia, Brazil. nidi_reis@hotmail.com., Gonçalves NR; Fundação Oswaldo Cruz, Fiocruz Rondnia, Porto Velho, 76812-245 Rondnia, Brazil. naancontato@hotmail.com., Kayano AM; Fundação Oswaldo Cruz, Fiocruz Rondnia, Porto Velho, 76812-245 Rondnia, Brazil. akayano@gmail.com., Moreira-Dill LS; Fundação Oswaldo Cruz, Fiocruz Rondnia, Porto Velho, 76812-245 Rondnia, Brazil. leandrosoaresmoreira@gmail.com., Sobrinho JC; Fundação Oswaldo Cruz, Fiocruz Rondnia, Porto Velho, 76812-245 Rondnia, Brazil. jusbbio@gmail.com., Zanchi FB; Fundação Oswaldo Cruz, Fiocruz Rondnia, Porto Velho, 76812-245 Rondnia, Brazil. fernando.zanchi@fiocruz.br., Fuly AL, Fernandes CF, Zuliani JP; Fundação Oswaldo Cruz, Fiocruz Rondnia, Porto Velho, 76812-245 Rondnia, Brazil. juliana.zuliani@fiocruz.br., Soares AM; Fundação Oswaldo Cruz, Fiocruz Rondnia, Porto Velho, 76812-245 Rondnia, Brazil. andreimar.soares@fiocruz.br., Stabeli RG, Fernandes CFC; Fundação Oswaldo Cruz, Fiocruz Rondnia, Porto Velho, 76812-245 Rondnia, Brazil. carla.celedonio@fiocruz.br. |
| Abstrakt: |
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 through Western 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. |
