Disintegrin-like/cysteine-rich domains of the reprolysin HF3: Site-directed mutagenesis reveals essential role of specific residues

Autor(es)

Menezes, Milene C. ^[1] ; de Oliveira, Ana Karina ^[1] ; Melo, Robson L. ^[1] ; Lopes-Ferreira, Monica ^[1] ; Rioli, Vanessa ^[1] ; Balan, Andrea ^[2] ; Paes Leme, Adriana F. ^[1] ; Serrano, Solange M. T. ^[1]

Afiliação do(s) autor(es)

^[1] Inst Butantan, Lab Especial Toxinol Aplicada CAT CEPID, BR-05503900 Sao Paulo - Brazil

^[2] Ctr Pesquisa Energia & Mat, Lab Nacl Biociencias LNBio, BR-13083970 Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Biochimie; v. 93, n. 2, p. 345-351, FEB 2011.
• Citações Web of Science: 14

Resumo

Little is known about the biochemical properties of the non-catalytic domains of snake venom metalloproteinases (SVMPs). The ECD sequence of the disintegrin-like domain (D-domain) has been assigned as the disintegrin motif and, recently, the hyper-variable region (HVR) of the cysteine-rich domain (C-domain) was suggested to constitute a potential protein protein adhesive interface. Here we show that the recombinant C-domain of HF3, a hemorrhagic SVMP from Bothrops jararaca, as well as three peptides resembling its HVR, inhibit collagen-induced platelet aggregation, which indicates a role for the C-domain and its HVR in targeting HF3 to platelets. Site-directed mutagenesis was used for the first time to identify charged residues essential for the functionality of the disintegrin-like/cysteine-rich domains (DC-domains). Residues of the disintegrin loop (E467 and D469), and of the HVR (K568, K569 and K575) of HF3 were individually mutated to Ala. Interestingly, only the mutant D469A was obtained in soluble form in Escherichia coli and this single mutation caused loss of two functional activities of the DC-domains: inhibition of platelet aggregation and increase of leukocyte rolling in the microcirculation. In summary we demonstrate that the C-domain and its HVR are critical for HF3 to affect platelets and leukocytes, however, the disintegrin loop may be important for the functionality of the D-domain in the context of the C-domain. (C) 2010 Elsevier Masson SAS. All rights reserved. (AU)

• Processo FAPESP: 98/14307-9 - Center for Applied Toxinology
• Beneficiário: Hugo Aguirre Armelin
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 05/55821-2 - Papel do domínio rico em cisteínas do HF3, uma metaloproteinase do veneno de Bothrops jararaca, na inibição da agregação plaquetária e na ativação da fagocitose por macrófagos
• Beneficiário: Milene Cristina Menezes dos Santos
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 06/50059-8 - Proteômica aplicada ao estudo dos alvos celulares e plasmáticos do HF3, uma metaloproteinase do veneno da serpente Bothrops jararaca com atividades hemorrágica e inflamatória
• Beneficiário: Adriana Franco Paes Leme
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 04/15974-1 - Estudo sobre a função do domínio rico em cisteínas do fator hemorrágico 3 (HF3), metaloproteinases do veneno da serpente Bothrops jararaca, na interação com seus alvos celulares e plasmáticos
• Beneficiário: Solange Maria de Toledo Serrano
• Modalidade de apoio: Auxílio à Pesquisa - Regular