Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system
- 449 Downloads
Snake venom metalloproteinases (SVMPs) participate in snakebite pathology such as hemorrhage, inflammation, and necrosis. They are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Recombinant expression of the precursor of P-III class SVMPs has failed due to the instability of the multi-domain polypeptide structure. Conversely, functional recombinant non-catalytic domains were obtained by prokaryotic expression systems. Here, we show for the first time the recombinant expression of the precursor of HF3, a highly hemorrhagic SVMP from Bothrops jararaca, and its non-catalytic domains, using an E. coli-based cell-free synthesis system. The precursor of HF3, composed of pro-, metalloproteinase-, disintegrin-like-, and cysteine-rich domains, and containing 38 Cys residues, was successfully expressed and purified. A protein composed of the disintegrin-like and cysteine-rich domains (DC protein) and the cysteine-rich domain alone (C protein) were expressed in vitro individually and purified. Both proteins were shown to be functional in assays monitoring the interaction with matrix proteins and in modulating the cleavage of fibrinogen by HF3. These data indicate that recombinant expression using prokaryotic-based cell-free synthesis emerges as an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues.
KeywordsCell-free protein synthesis Cys-rich proteins Disintegrin-like/cysteine-rich Recombinant protein expression Snake venom metalloproteinase
This work used the Cell Free Expression platform (Lionel Imbert) of the Grenoble center Tutorial (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with media from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank Ismael Feitosa Lima for excellent technical help. This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (1214/2011, 7737/14-9), Fundação de Amparo à Pesquisa do Estado de São Paulo (2013/07467-1) and from a dedicated grant from the Direction des Sciences du Vivant of the CEA.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.
- Hanna SL, Sherman NE, Kinter MT, Goldberg JB (2000) Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography-tandem mass spectrometry. Microbiology 146(Pt 10):2495–2508. doi: 10.1099/00221287-146-10-2495 CrossRefPubMedGoogle Scholar
- Menezes MC, Paes Leme AF, Melo RL, Silva CA, Della Casa M, Bruni FM, Lima C, Lopes-Ferreira M, Camargo AC, Fox JW, Serrano SM (2008) Activation of leukocyte rolling by the cysteine-rich domain and the hyper-variable region of HF3, a snake venom hemorrhagic metalloproteinase. FEBS Lett 582:3915–3921. doi: 10.1016/j.febslet.2008.10.034 CrossRefPubMedGoogle Scholar
- Menezes MC, de Oliveira AK, Melo RL, Lopes-Ferreira M, Rioli V, Balan A, Paes Leme AF, Serrano SM (2011) Disintegrin-like/cysteine-rich domains of the reprolysin HF3: site-directed mutagenesis reveals essential role of specific residues. Biochimie 93:345–351. doi: 10.1016/j.biochi.2010.10.007 CrossRefPubMedGoogle Scholar
- Moura-da-Silva AM, Serrano SMT, Fox JW, Gutiérrez JM (2009) Snake venom metalloproteinases: structure, function and effects on snake bite pathology. In: Lima ME, Pimenta AMC, Martin-Eauclaire MF, Zingali R, Rochat H (org) Animal toxins: state of the art. Perspectives in health and biotechnology, Editora UFMG, Belo Horizonte, pp 525–546Google Scholar
- Oliveira AK, Paes Leme AF, Assakura MT, Menezes MC, Zelanis A, Tashima AK, Lopes-Ferreira M, Lima C, Camargo AC, Fox JW, Serrano SM (2009) Simplified procedures for the isolation of HF3, bothropasin, disintegrin-like/cysteine-rich protein and a novel P-I metalloproteinase from Bothrops jararaca venom. Toxicon 53:797–801. doi: 10.1016/j.toxicon.2009.02.019 CrossRefGoogle Scholar
- Portes-Junior JA, Yamanouye N, Carneiro SM, Knittel PS, Sant’Anna SS, Nogueira FC, Junqueira M, Magalhães GS, Domont GB, Moura-da-Silva AM (2014) Unraveling the processing and activation of snake venom metalloproteinases. J Proteome Res 13:3338–3348. doi: 10.1021/pr500185a CrossRefPubMedGoogle Scholar
- Serrano SMT, Kim J, Wang D, Dragulev B, Shannon JD, Mann HH, Veit G, Wagener R, Koch M, Fox JW (2006) The cysteine-rich domain of snake venom metalloproteinases is a ligand for von Willebrand factor A domains: role in substrate targeting. J Biol Chem 281:39746–39756. doi: 10.1074/jbc.M604855200 CrossRefPubMedGoogle Scholar
- Serrano SMT, Wang D, Shannon JD, Pinto AFM, Polanowska-Grabowska RK, Fox JW (2007) Interaction of the cysteine-rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes site-specific proteolysis of von Willebrand factor and inhibition of von Willebrand factor-mediated platelet aggregation. FEBS J 274:3611–3621. doi: 10.1111/j.1742-4658.2007.05895.x CrossRefPubMedGoogle Scholar
- Silva CA, Zuliani JP, Assakura MT, Mentele R, Camargo AC, Teixeira CF, Serrano SM (2004) Activation of alpha(M)beta(2)-mediated phagocytosis by HF3, a P-III class metalloproteinase isolated from the venom of Bothrops jararaca. Biochem Biophys Res Commun 322:950–956. doi: 10.1016/j.bbrc.2004.08.012 CrossRefPubMedGoogle Scholar
- Stöcker W, Grams F, Baumann U, Reinemer P, Gomis-Rüth FX, McKay DB, Bode W (1995) The metzincins—topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases. Protein Sci 4(5):823–840. doi: 10.1002/pro.5560040502 CrossRefPubMedPubMedCentralGoogle Scholar
- Tanjoni I, Evangelista K, Della-Casa MS, Butera D, Magalhães GS, Baldo C, Clissa PB, Fernandes I, Eble J, Moura-da-Silva AM (2010) Different regions of the class P-III snake venom metalloproteinase jararhagin are involved in binding to alpha2beta1 integrin and collagen. Toxicon 55:1093–1099. doi: 10.1016/j.toxicon.2009.12.010 CrossRefPubMedGoogle Scholar