Structure-function studies on jaburetox, a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease.
BACKGROUND
Ureases are metalloenzymes involved in defense mechanisms in plants. The insecticidal activity of Canavalia ensiformis (jack bean) ureases relies partially on an internal 10kDa peptide generated by enzymatic hydrolysis of the protein within susceptible insects. A recombinant version of this peptide, jaburetox, exhibits insecticidal, antifungal and membrane-disruptive properties. Molecular modeling of jaburetox revealed a prominent β-hairpin motif consistent with either neurotoxicity or pore formation.


METHODS
Aiming to identify structural motifs involved in its effects, mutated versions of jaburetox were built: 1) a peptide lacking the β-hairpin motif (residues 61-74), JbtxΔ-β; 2) a peptide corresponding the N-terminal half (residues 1-44), Jbtx N-ter, and 3) a peptide corresponding the C-terminal half (residues 45-93), Jbtx C-ter.


RESULTS
1) JbtxΔ-β disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the β-hairpin motif is not a determinant of these biological activities; 2) both Jbtx C-ter and Jbtx N-ter disrupted liposomes, the C-terminal peptide being the most active; and 3) while Jbtx N-ter persisted to be biologically active, Jbtx C-ter was less active when tested on different insect preparations. Molecular modeling and dynamics were applied to the urease-derived peptides to complement the structure-function analysis.


CONCLUSIONS
The N-terminal portion of the Jbtx carries the most important entomotoxic domain which is fully active in the absence of the β-hairpin motif. Although the β-hairpin contributes to some extent, probably by interaction with insect membranes, it is not essential for the entomotoxic properties of Jbtx.


CONCLUSIONS
Jbtx represents a new type of insecticidal and membrane-active peptide.
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; Department of Biophysics, Institute of Biosciences, UFRGS, Brazil.
Anne H S
AH
Martinelli
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.
Karine
K
Kappaun
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.
Rodrigo
R
Ligabue-Braun
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.
Marina S
MS
Defferrari
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.
Angela R
AR
Piovesan
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; Department of Molecular Biology and Biotechnology, UFRGS, Porto Alegre, RS, Brazil.
Fernanda
F
Stanisçuaski
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.
Diogo R
DR
Demartini
Interdisciplinary Centre of Biotechnological Research, Universidade Federal do Pampa, UNIPAMPA, São Gabriel, RS, Brazil.
Chariston A
CA
Dal Belo
Interdisciplinary Centre of Biotechnological Research, Universidade Federal do Pampa, UNIPAMPA, São Gabriel, RS, Brazil.
Carlos G M
CG
Almeida
Department Physical Chemistry, Institute of Chemistry, Universidade Federal do Rio de Janeiro, RJ, Brazil.
Cristian
C
Follmer
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; Department of Molecular Biology and Biotechnology, UFRGS, Porto Alegre, RS, Brazil.
Hugo
H
Verli
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; Department of Biophysics, Institute of Biosciences, UFRGS, Brazil; Instituto do Cérebro (InsCer), Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil. Electronic address: ccarlini@ufrgs.br.
Celia R
CR
Carlini
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil; Department of Molecular Biology and Biotechnology, UFRGS, Porto Alegre, RS, Brazil. Electronic address: pasquali@cbiot.ufrgs.br.
Giancarlo
G
Pasquali
D000595:Amino Acid Sequence; D000818:Animals; D032449:Canavalia / Q000201:enzymology*; D003058:Cockroaches; D007306:Insecticides / Q000494:pharmacology*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009469:Neuromuscular Junction / Q000187:drug effects; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014510:Urease / Q000737:chemistry / Q000494:pharmacology*
D007306:Insecticides; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014510:Urease; C525847:jaburetox-2Ec, Canavalia ensiformis
C-terminal domain of Jbtx; CD; Insect; Jbtx; Jbtx C-ter; Jbtx N-ter; Jbtx-2Ec; JbtxΔ-β; LUV; MD; Membrane-disturbing; Molecular modeling; N-terminal domain of Jbtx; RMSD; Site-directed mutagenesis; Urease-derived peptide; a version of Jbtx containing a V5 epitope; circular dichroism; jaburetox; large unilamellar vesicle; molecular dynamics; root mean square deviation; β-hairpin; β-hairpin deleted version of Jbtx