Modulation of anti-endotoxin property of Temporin L by minor amino acid substitution in identified phenylalanine zipper sequence.
A 13-residue frog antimicrobial peptide Temporin L (TempL) possesses versatile antimicrobial activities and is considered a lead molecule for the development of new antimicrobial agents. To find out the amino acid sequences that influence the anti-microbial property of TempL, a phenylalanine zipper-like sequence was identified in it which was not reported earlier. Several alanine-substituted analogs and a scrambled peptide having the same composition of TempL were designed for evaluating the role of this motif. To investigate whether leucine residues instead of phenylalanine residues at 'a' and/or 'd' position(s) of the heptad repeat sequence could alter its antimicrobial property, several TempL analogs were synthesized after replacing these phenylalanine residues with leucine residues. Replacing phenylalanine residues with alanine residues in the phenylalanine zipper sequence significantly compromised the anti-endotoxin property of TempL. This is evident from the higher production of tumor necrosis factor-α and interleukin-6 in lipopolysaccharide (LPS)-stimulated rat bone-marrow-derived macrophage cells in the presence of its alanine-substituted analogs than TempL itself. However, replacement of these phenylalanine residues with leucine residues significantly augmented anti-endotoxin property of TempL. A single alanine-substituted TempL analog (F8A-TempL) showed significantly reduced cytotoxicity but retained the antibacterial activity of TempL, while the two single leucine-substituted analogs (F5L-TempL and F8L-TempL), although exhibiting lower cytotoxicity, were able to retain the antibacterial activity of the parent peptide. The results demonstrate how minor amino acid substitutions in the identified phenylalanine zipper sequence in TempL could yield analogs with better antibacterial and/or anti-endotoxin properties with their plausible mechanism of action.
Molecular and Structural Biology Division, CSIR - Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226 031, India.
Saurabh
S
Srivastava
Molecular and Structural Biology Division, CSIR - Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226 031, India.
Amit
A
Kumar
Molecular and Structural Biology Division, CSIR - Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226 031, India.
Amit Kumar
AK
Tripathi
Molecular and Structural Biology Division, CSIR - Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226 031, India.
Anshika
A
Tandon
Molecular and Structural Biology Division, CSIR - Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226 031, India.
Jimut Kanti
JK
Ghosh
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D023181:Antimicrobial Cationic Peptides; D002478:Cells, Cultured; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D005454:Fluorescence Polarization; D057927:Hydrophobic and Hydrophilic Interactions; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship
D023181:Antimicrobial Cationic Peptides; D008070:Lipopolysaccharides; D011506:Proteins; C104501:temporin; D010649:Phenylalanine
Temporin L; anti-endotoxin activity of antimicrobial peptides; fluorescence and circular dichroism studies; peptide–LPS interaction; phenylalanine zipper sequence; synthetic peptides