Mechanism of interaction of optimized Limulus-derived cyclic peptides with endotoxins: thermodynamic, biophysical and microbiological analysis

Abstract

On the basis of formerly investigated peptides corresponding to the endotoxin-binding domain from LALF [Limulus anti-LPS (lipopolysaccharide) factor], a protein from Limulus polyphemus, we have designed and synthesized peptides of different lengths with the aim of obtaining potential therapeutic agents against septic shock syndrome. For an understanding of the mechanisms of action, we performed a detailed physicochemical and biophysical analysis of the interaction of rough mutant LPS with these peptides by applying FTIR (Fourier-transform infrared) spectroscopy, SAXS (small-angle X-ray scattering), calorimetric techniques [DSC (differential scanning calorimetry) and ITC (isothermal titration calorimetry)] and FFTEM (freeze-fracture transmission electron microscopy). Also, the action of the peptides on bacteria of different origin in microbial assays was investigated. Using FTIR and DSC, our results indicated a strong fluidization of the lipid A acyl chains due to peptide binding, with a decrease in the endothermic melting enthalpy change of the acyl chains down to a complete disappearance in the 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. Via ITC, it was deduced that the binding is a clearly exothermic process which becomes saturated at a 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. The results obtained with SAXS indicated a drastic change of the aggregate structures of LPS into a multilamellar stack, which was visualized in electron micrographs as hundreds of lamellar layers. This can be directly correlated with the inhibition of the LPS-induced production of tumour necrosis factor alpha in human mononuclear cells, but not with the action of the peptides on bacteria.

Figure 1. Amino acid sequences of the synthesized cyclic LALF peptides

The cyclation is performed between the two cysteine residues as indicated for cLALF20.

Figure 2. DSC heating-scans of LPS R60 in the presence of different concentrations of peptides cLALF9–20

The specific excess heat capacity, C_pdiff, of the calorimetric sample cell as compared with the reference cell is plotted against temperature. The peak positions directed upwards are characteristic for endothermic chain melting processes.

Figure 3. Calorimetrically determined enthalpy change, ΔH_c, against [LPS]:[peptide] molar ratio from DSC scans of Figure 2

Figure 4. Gel-to-liquid crystalline phase behaviour of the hydrocarbon chains in the lipid A moiety of LPS in the absence and presence of cLALF9, 15, 18 and 20 in FTIR experiments

Plotted is the peak position of the symmetric stretching vibration of the methylene groups against temperature. The gel phase corresponds to wavenumber values around 2850 cm⁻¹ and the liquid crystalline phase corresponds to values around 2852.5–2853.0 cm⁻¹.

Figure 5. Isothermal calorimetric titration of 0.05 mM LPS Ra with 2 mM cLALF peptides (30×3 μl every 5 min)

(A) Original titration of LPS with cLALF20 (raw data). (B) Integrated peak areas of the single titrations according to (A) against [peptide]:[LPS] molar ratios for cLALF9, 15, 18 and 20.

Figure 6. Synchrotron radiation small-angle X-ray diffraction patterns of LPS/peptide mixtures ([LPS]:[cLALF]=2.5:1 molar ratio)

The scattering vector s=1/d=2sinθ/λ (where d is the spacing ratio, θ the scattering angle and λ the wavelength=0.15 nm) is plotted against the logarithm of the scattering intensity, log I, (A) at different temperatures and (B) at 40 °C, both for cLALF18 and 20.

Figure 7. Freeze-fracture electron micrographs of LPS from Salmonella Minnesota Re alone (A) and in the presence of cLALF18 ([LPS]:[cLALF]=3:1) (B) supernatant and (C) precipitation

Figure 8. Intercalation of peptide/LPS samples and Hepes buffer control into target cell liposomes made from PS in FRET spectroscopic measurements

Presented is the FRET signal I_D/I_A against time for cLALF9 (upper panel) and cLALF18 (lower panel). The samples were added subsequently at 50 and 100 s to the liposomes.

Figure 9. Concentration-dependent inhibition of the biological activity of LPS Ra from Salmonella Minnesota strain R60 by synthetic peptides

Peptides were incubated with 1 ng/ml LPS at the indicated peptide concentrations and used to activate human MNCs. As a marker of LPS-induced cell activation, the production of the cytokine TNFα was determined by ELISA. Activation of the cells by LPS alone represents 100%. PMB served as a control. Results presented are the means for two independent cell stimulations from two different preparations and each stimulation performed in duplicate.