Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming
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 cha...
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| Format: | Article |
| Language: | English |
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MDPI AG
2018-03-01
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| Series: | Toxins |
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| Online Access: | http://www.mdpi.com/2072-6651/10/4/142 |
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| author | Marcos B. Luiz Soraya S. Pereira Nidiane D. R. Prado Naan R. Gonçalves Anderson M. Kayano Leandro S. Moreira-Dill Juliana C. Sobrinho Fernando B. Zanchi André L. Fuly Cleberson F. Fernandes Juliana P. Zuliani Andreimar M. Soares Rodrigo G. Stabeli Carla F. C. Fernandes |
| author_facet | Marcos B. Luiz Soraya S. Pereira Nidiane D. R. Prado Naan R. Gonçalves Anderson M. Kayano Leandro S. Moreira-Dill Juliana C. Sobrinho Fernando B. Zanchi André L. Fuly Cleberson F. Fernandes Juliana P. Zuliani Andreimar M. Soares Rodrigo G. Stabeli Carla F. C. Fernandes |
| author_sort | Marcos B. Luiz |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T22:49:54Z |
| format | Article |
| id | doaj.art-aa1816cdb4c34f45972efafae83339d9 |
| institution | Directory Open Access Journal |
| issn | 2072-6651 |
| language | English |
| last_indexed | 2024-04-11T22:49:54Z |
| publishDate | 2018-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Toxins |
| spelling | doaj.art-aa1816cdb4c34f45972efafae83339d92022-12-22T03:58:37ZengMDPI AGToxins2072-66512018-03-0110414210.3390/toxins10040142toxins10040142Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic EnvenomingMarcos B. Luiz0Soraya S. Pereira1Nidiane D. R. Prado2Naan R. Gonçalves3Anderson M. Kayano4Leandro S. Moreira-Dill5Juliana C. Sobrinho6Fernando B. Zanchi7André L. Fuly8Cleberson F. Fernandes9Juliana P. Zuliani10Andreimar M. Soares11Rodrigo G. Stabeli12Carla F. C. Fernandes13Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, BrazilFundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, BrazilFundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, BrazilFundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, BrazilFundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, BrazilFundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, BrazilFundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, BrazilFundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, BrazilUniversidade Federal Fluminense, UFF, Rio de Janeiro, 24220-900 Rio de Janeiro, BrazilEmbrapa Agroindústria Tropical, Fortaleza, 60020-181 Ceará, BrazilFundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, BrazilFundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, BrazilPlataforma Bi-institucional de Pesquisa em Medicina Translacional, Fiocruz-USP, Ribeirão Preto, 14049-900 São Paulo, BrazilFundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, BrazilToxic 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.http://www.mdpi.com/2072-6651/10/4/142crotoxinCBVHHCrotalus durissus terrificus |
| spellingShingle | Marcos B. Luiz Soraya S. Pereira Nidiane D. R. Prado Naan R. Gonçalves Anderson M. Kayano Leandro S. Moreira-Dill Juliana C. Sobrinho Fernando B. Zanchi André L. Fuly Cleberson F. Fernandes Juliana P. Zuliani Andreimar M. Soares Rodrigo G. Stabeli Carla F. C. Fernandes Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming Toxins crotoxin CB VHH Crotalus durissus terrificus |
| title | Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming |
| title_full | Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming |
| title_fullStr | Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming |
| title_full_unstemmed | Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming |
| title_short | Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming |
| title_sort | camelid single domain antibodies vhhs against crotoxin a basis for developing modular building blocks for the enhancement of treatment or diagnosis of crotalic envenoming |
| topic | crotoxin CB VHH Crotalus durissus terrificus |
| url | http://www.mdpi.com/2072-6651/10/4/142 |
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