Structure-activity relationship of human liver-expressed antimicrobial peptide 2.
Liver-expressed antimicrobial peptide 2 (LEAP-2) is a 40-residue cationic peptide originally purified from human blood ultrafiltrate. The native peptide contains two disulfide bonds and is unique regarding its primary structure. Its biological role is not known but a previous study showed that chemically synthesized LEAP-2 exhibited in vitro antimicrobial activities against several Gram-positive bacteria. In order to determine its antimicrobial mode of action, we expressed human recombinant LEAP-2 in Escherichia coli. Circular dichroism spectroscopy and nuclear magnetic resonance analyses showed that the structure of the recombinant peptide was identical to that of the chemically synthesized and oxidized LEAP-2, with two disulfide bonds between Cys residues in relative 1-3 and 2-4 positions. Minimal inhibitory concentration (MIC) of the recombinant human LEAP-2 was determined by a conventional broth dilution assay. It was found to be bactericidal against Bacillus megaterium at a 200microM concentration. Interestingly, the linear LEAP-2 had a greater antimicrobial activity with a MIC value of 12.5microM, which was comparable to that of magainin2. SYTOX Green uptake was used to assess bacterial membrane integrity. Linear LEAP-2 and magainin2 permeabilized B. megaterium membranes with the same efficiency, whereas oxidized LEAP-2 did not induce stain uptake. Binding of the peptides to plasmid DNA was evaluated by gel retardation assays. The DNA-binding efficacy of linear LEAP-2 was three times higher than that of the peptide-containing disulfide bridges. Altogether, these results show that the secondary structure of human LEAP-2 has a profound impact on its antibacterial activity.
Hocquellet
Agnès
A
Université de Bordeaux, EA 4135, Bordeaux, France.
Odaert
Benoit
B
Cabanne
Charlotte
C
Noubhani
Abdelmajid
A
Dieryck
Wilfrid
W
Joucla
Gilles
G
Le Senechal
Caroline
C
Milenkov
Milen
M
Chaignepain
Stéphane
S
Schmitter
Jean-Marie
JM
Claverol
Stéphane
S
Santarelli
Xavier
X
Dufourc
Erick J
EJ
Bonneu
Marc
M
Garbay
Bertrand
B
Costaglioli
Patricia
P
D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001410:Bacillus megaterium / Q000187:drug effects; D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D008826:Microbial Sensitivity Tests; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D004220:Disulfides; D011994:Recombinant Proteins; C475606:liver-expressed antimicrobial peptide 2, human; D004247:DNA