Consequences of Alteration in Leucine Zipper Sequence of Melittin in Its Neutralization of Lipopolysaccharide-induced Proinflammatory Response in Macrophage Cells and Interaction with Lipopolysaccharide

Srivastava, Raghvendra M.; Srivastava, Saurabh; Singh, Manish; Bajpai, V.K.; Ghosh, Jimut Kanti

doi:10.1074/jbc.m111.302893

Cited by 38 publications

(56 citation statements)

References 64 publications

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“…To study the effects of treatments with TempL or Q3K-TempL on the physical state of LPS, we assayed the peptide-induced disaggregation of LPS by employing FITC-labeled LPS, as reported previously (25,28). FITC-LPS (0.5 g/ml) was treated with increasing concentrations of either TempL or its analog from 10 M to 100 M. The changes in the emission of FITC-LPS as a function of change in the aggregation state of LPS in 10 mM sodium phosphate buffer (pH, 6.9) were monitored at 515 nm using a fluorescence spectrometer (Perkin-Elmer LS55) with the excitation wavelength set at 488 nm and excitation and emission slits at 8 and 6 nm, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The fluorescence data for FITC-LPS at 515 nm were collected with or without peptide addition after background subtraction. Peptideinduced dissociation of the aggregated state of FITC-LPS was measured as an increase in its fluorescence (25,29).…”

Section: Methodsmentioning

confidence: 99%

“…We performed circular-dichroism (CD) experiments to study the propensity of TempL and its analog to self-assemble in LPS and aqueous environments. For this purpose, dose-dependent CD spectra of the peptides were recorded in the presence of LPS and in PBS (pH 7.4; 1.5 M NaCl) (25) by employing a Jasco J-500A spectropolarimeter, which was calibrated with (ϩ)-10-camphorsulfonic acid. Spectra of the samples were recorded at 30°C in a capped quartz optical cell with a path length of 0.2 cm in the wavelength range of 250 to 190 nm.…”

Section: Methodsmentioning

confidence: 99%

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Introduction of a Lysine Residue Promotes Aggregation of Temporin L in Lipopolysaccharides and Augmentation of Its Antiendotoxin Property

Srivastava

Ghosh

2013

Antimicrob Agents Chemother

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Temporin L (TempL) is a 13-residue frog antimicrobial peptide that shows moderate bactericidal activity and antiendotoxin properties in macrophages. We envisioned that, due to its very hydrophobic nature, the peptide might fail to show its desired biological properties. It was predicted by employing the available algorithms that the replacement of a glutamine by lysine at position 3 could appreciably reduce its aggregation propensity in an aqueous environment. In order to investigate the structural, functional, and biological consequences of replacement of glutamine by lysine at its third position, TempL and the corresponding analog, Q3K-TempL, was synthesized and characterized. Introduction of the lysine residue significantly promoted the selfassembly and oligomeric state of TempL in lipopolysaccharide (LPS). Q3K-TempL exhibited augmented binding to LPS and also dissociated LPS aggregates with greater efficacy than TempL. Further, Q3K-TempL inhibited the LPS-induced proinflammatory cytokines in rat primary macrophages in vitro and in vivo in BALB/c mice with greater efficacy than TempL. The results showed that a simple amino acid substitution in a short hydrophobic antimicrobial peptide, TempL, enhanced its antiendotoxin properties and illustrate a plausible correlation between its aggregation properties in LPS and LPS detoxification activity.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Introduction of a Lysine Residue Promotes Aggregation of Temporin L in Lipopolysaccharides and Augmentation of Its Antiendotoxin Property

Srivastava

Ghosh

2013

Antimicrob Agents Chemother

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“…It has been used as a model peptide for various purposes over the last decades (for a general review, see 16 ), and has shown therapeutic potential with antimicrobial, anti-inflammatory [17][18][19][20] , antiparasitic 21 , and anticancer [22][23] activity, as well as for treatment 24 and prevention 25 of HIV. Like many CAPs, melittin binds to membranes as an amphipathic α-helix [26][27][28] .…”

Section: Introductionmentioning

confidence: 99%

“…Melittin-21Q exhibited a reduced or similar binding to DMPC and to DMPG bilayers compared to native melittin, suggesting a favorable or a limited contribution of the cationic C-terminal portion to membrane affinity. Another investigation, using tryptophan fluorescence, revealed that melittin and its Cterminal truncated versions , and [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], all showed an increased affinity for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) 70/30 large unilamellar vesicles (LUVs) compared POPC LUVs 47 (See Supporting Information). These findings highlighted the role of attractive electrostatic interactions.…”

Section: Introductionmentioning

confidence: 99%

Role of the Cationic C-Terminal Segment of Melittin on Membrane Fragmentation

2016

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The widespread distribution of cationic antimicrobial peptides capable of membrane fragmentation in nature underlines their importance to living organisms. In the present work, we determined the impact of the electrostatic interactions associated with the cationic Cterminal segment of melittin, a 26-amino acid peptide from bee venom (net charge +6), on its binding to model membranes and on the resulting fragmentation. In order to detail the role played by the C-terminal charges, we prepared a melittin analogue for which the 4 cationic amino acids in positions 21 to 24 were substituted with the polar residue citrulline, providing a peptide with the same length and amphiphilicity but with a lower net charge (+2). We compared the peptide bilayer affinity and the membrane fragmentation for bilayers prepared from dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) mixtures. It is shown that neutralization of the C-terminal considerably increased melittin affinity for zwitterionic membranes. The unfavorable contribution associated with transferring the cationic C-terminal in a less polar environment was reduced, leaving the hydrophobic interactions, which drive the peptide insertion in bilayers, with limited counterbalancing interactions. The presence of negatively charged lipids (DPPS) in bilayers increased melittin binding by introducing attractive electrostatic interactions, the augmentation being, as expected, greater for native melittin than for its citrullinated analogue. The membrane fragmentation power of the peptide was shown to be controlled by electrostatic interactions and could be modulated by the charge carried by both the membrane and the lytic peptide. The analysis of the lipid composition of the extracted fragments from DPPC/DPPS bilayers revealed no lipid specificity. It is proposed that extended phase separations are more susceptible to lead to the extraction of a lipid species in a specific manner than a specific lipid-peptide affinity. The present work on the lipid extraction by melittin and citrullinated melittin with model membranes emphasizes the complex relation between the affinity, the lipid extraction/membrane fragmentation, and the lipid specificity.

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Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Malan

Serem

Bester

et al. 2015

Journal of Peptide Science

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Antimicrobial peptides (AMPs) are small, cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti-inflammatory and antioxidant activities. Several AMPs are known to inhibit lipopolysaccharide (LPS)-induced inflammation in vitro and to protect animals from sepsis. In this study the cellular anti-inflammatory and anti-endotoxin activities of Os and Os-C, peptides derived from the carboxy-terminal of a tick defensin (OsDef2), were investigated. Both Os and Os-C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin-binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os-C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione (GSH), a known NO scavenger. In contrast, Os-C showed no scavenging activity. Os and Os-C inhibited LPS/IFN-γ induced NO and TNF-α production in RAW 264.7 cells in a concentration-dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF-α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os-C and GSH protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os-C, both peptides have in addition anti-inflammatory and antiendotoxin properties.

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Consequences of Alteration in Leucine Zipper Sequence of Melittin in Its Neutralization of Lipopolysaccharide-induced Proinflammatory Response in Macrophage Cells and Interaction with Lipopolysaccharide

Cited by 38 publications

References 64 publications

Introduction of a Lysine Residue Promotes Aggregation of Temporin L in Lipopolysaccharides and Augmentation of Its Antiendotoxin Property

Introduction of a Lysine Residue Promotes Aggregation of Temporin L in Lipopolysaccharides and Augmentation of Its Antiendotoxin Property

Role of the Cationic C-Terminal Segment of Melittin on Membrane Fragmentation

Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

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