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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
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]
Total Authors: 8
Affiliation:
[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
Total Affiliations: 2
Document type: Journal article
Source: Biochimie; v. 93, n. 2, p. 345-351, FEB 2011.
Web of Science Citations: 14
Abstract

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)

FAPESP's process: 98/14307-9 - Center for Applied Toxinology
Grantee:Hugo Aguirre Armelin
Support type: Research Grants - Research, Innovation and Dissemination Centers - RIDC