Utilization of an Amphipathic Leucine Zipper Sequence to Design Antibacterial Peptides with Simultaneous Modulation of Toxic Activity against Human Red Blood Cells

Aqeel Ahmad,Sharada Prasad Yadav,Neeta Asthana,Kalyan Mitra,Swati Prakash Srivastava,Jimut Kanti Ghosh

doi:10.1074/jbc.m602378200

Aqeel Ahmad, Sharada Prasad Yadav + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m602378200

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Abstract

The toxicity of naturally occurring or designed antimicrobial peptides is a major barrier for converting them into drugs. To synthesize antimicrobial peptides with reduced toxicity, several amphipathic peptides were designed based on the leucine zipper sequence. The first one was a leucine zipper peptide (LZP); in others, leucine residues at the a- and/or d-position were substituted with single or double alanine residues. The results showed that LZP and its analogs exhibited appreciable and similar antibacterial activity against the tested gram-positive and gram-negative bacteria. However, the substitution of alanine progressively lowered the toxicity of LZP against human red blood cells (hRBCs). The substitution of leucine with alanine impaired the binding and localization of LZP to hRBCs, but had little effect on the peptide-induced damage of Escherichia coli cells. Although LZP and its analogs exhibited similar permeability, secondary structures, and localization in negatively charged membranes, significant differences were observed among these peptides in zwitterionic membranes. The results suggest a novel approach for designing antibacterial peptides with modulation of toxicity against hRBCs by employing the leucine zipper sequence. Also, to the best of our knowledge, the results demonstrate that this sequence could be utilized to design novel cell-selective molecules for the first time.

Highlights

Bacteria, fungi, protozoa, viruses, and even tumors, have been identified from a wide variety of animals, including humans [1,2,3,4]
Because self-assembly of an antimicrobial peptide seems to have a profound effect on its cell selectivity/toxicity [10, 17,18,19,20, 33], we envisioned that substitution only of one or two amino acids at the a- and/or d-position might affect the toxicity of the leucine zipper-based antimicrobial peptides
The results indicated that the antibacterial activity of leucine zipper peptide (LZP)

Summary

EXPERIMENTAL PROCEDURES

Materials—Rink amide-4-methylbenzhydrylamine resin (loading capacity, 0.63 mmol/g) and all of the N-␣-Fmocand side chain-protected amino acids were purchased from Novabiochem. Labeling at the N termini of peptides with a fluorescent probe, cleavage of the labeled and unlabeled peptides from the resin, and their precipitation and purification by reverse-phase HPLC were achieved by standard procedures [19, 21, 22]. Lipid vesicles prepared in Kϩ buffer (50 mM K2SO4 and 25 mM HEPES sulfate, pH 6.8) were mixed with isotonic (Kϩ-free) Naϩ buffer, followed by the addition of the potential sensitive dye 3,3Ј-dipropylthiadicarbocyanine iodide. The membrane permeability of the peptides in PC/Chol and PC/PG vesicles was determined by the peptide-induced release of calcein from calcein-entrapped lipid vesicles [21, 29]. Calcein-entrapped lipid vesicles were prepared with a self-quenching concentration (60 mM) of the dye in 10 mM HEPES at pH 7.4. The non-encapsulated calcein was removed from the suspension of lipid vesicles by gel filtration

Da min LZP

RESULTS

An initial change in morphology and damage to the bacterial

DISCUSSION

LZP after the substitution of alanine in zwitterionic membranes

Srivastava and Jimut Kanti Ghosh