Dynamic Surface Activity of a Fully Synthetic Phospholipase-Resistant Lipid/Peptide Lung Surfactant

This study examines the surface activity and resistance to phospholipase degradation of a fully-synthetic lung surfactant containing a novel diether phosphonolipid (DEPN-8) plus a 34 amino acid peptide (Mini-B) related to native surfactant protein (SP)-B. Activity studies used adsorption, pulsating bubble, and captive bubble methods to assess a range of surface behaviors, supplemented by molecular studies using Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD), and plasmon resonance. Calf lung surfactant extract (CLSE) was used as a positive control.DEPN-8+1.5% (by wt.) Mini-B was fully resistant to degradation by phospholipase A(2) (PLA(2)) in vitro, while CLSE was severely degraded by this enzyme. Mini-B interacted with DEPN-8 at the molecular level based on FTIR spectroscopy, and had significant plasmon resonance binding affinity for DEPN-8. DEPN-8+1.5% Mini-B had greatly increased adsorption compared to DEPN-8 alone, but did not fully equal the very high adsorption of CLSE. In pulsating bubble studies at a low phospholipid concentration of 0.5 mg/ml, DEPN-8+1.5% Mini-B and CLSE both reached minimum surface tensions <1 mN/m after 10 min of cycling. DEPN-8 (2.5 mg/ml)+1.5% Mini-B and CLSE (2.5 mg/ml) also reached minimum surface tensions <1 mN/m at 10 min of pulsation in the presence of serum albumin (3 mg/ml) on the pulsating bubble. In captive bubble studies, DEPN-8+1.5% Mini-B and CLSE both generated minimum surface tensions <1 mN/m on 10 successive cycles of compression/expansion at quasi-static and dynamic rates.These results show that DEPN-8 and 1.5% Mini-B form an interactive binary molecular mixture with very high surface activity and the ability to resist degradation by phospholipases in inflammatory lung injury. These characteristics are promising for the development of related fully-synthetic lipid/peptide exogenous surfactants for treating diseases of surfactant deficiency or dysfunction

Critical Structural and Functional Roles for the N-Terminal Insertion Sequence in Surfactant Protein B Analogs

Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., approximately residues 8-25 and 63-78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity.FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary alpha-helix and secondary beta-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a "saposin-like" fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B.Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B

Resistance of DEPN-8+1.5% by weight Mini-B to degradation by phospholipase A₂ (PLA₂) compared to calf lung surfactant extract (CLSE).

<p>Data are mean±SEM for n = 3. DEPN-8+1.5% by weight Mini-B was incubated <i>in vitro</i> with PLA₂ (0.1 Units/ml) for 30 min at 37°C, and degradation was assessed by measuring lipid classes in weight percent based on phosphate analysis of bands on thin layer chromatography. Results for CLSE in the presence and absence of PLA₂ utilized identical methods as reported previously by Wang et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001039#pone.0001039-Wang3" target="_blank">[30]</a>.</p

Mean association and dissociation kinetic rate constants (k_on, k_off) and equilibrium dissociation constant KD calculated from plasmon resonance measurements for liposomes of DEPN-8 or DPPC flowing past a chip-linked Mini-B monolayer.

*<p>Liposomes of DEPN-8 or DPPC in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20, pH 7.4) were flowed past a monolayer of Mini-B linked via Cys4 and Cys27 to a C5M sensor chip in a Biacore 3000 system (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001039#s4" target="_blank"><u>Methods</u></a>). Mean kinetic rate constants (k_on, k_off) and the equilibrium dissociation constant (KD = k_off/k_on) were determined from curve fitting analyses of plasmon resonance results at six different lipid concentrations (0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 µg/ml for each lipid).</p

Dynamic surface activity of DEPN-8+1.5% or 3% Mini-B compared to CLSE on the captive bubble surfactometer.

<p>Minimum and maximum surface tensions are shown for DEPN-8+1.5% or 3% by weight Mini-B and CLSE on a captive bubble surfactometer during 10 cycles of rapid compression (20 cycles/min) following slow compression as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001039#pone-0001039-g005" target="_blank">Figure 5</a>. Surface tension values are Mean±SEM for at least three separate experiments. See text for details.</p

Dynamic surface activity of DEPN-8+1.5% (by wt) Mini-B compared to CLSE on the pulsating bubble surfactometer.

<p><u>Panel A</u>: 0.5 mg/ml phosphonolipid (phospholipid); <u>Panel B</u>: 2.5 mg/ml phosphonolipid (phospholipid). Surface tension at minimum bubble radius (minimum surface tension) for DEPN-8+1.5% Mini-B and CLSE is graphed as a function of time on a pulsating bubble surfactometer (37°C, 20 cycles/min, 50% area compression). Data are Mean±SEM for n = 3–5. See text for details.</p

Adsorption of DEPN-8 with and without 1.5% (by wt) Mini-B compared to calf lung surfactant extract (CLSE).

<p>Adsorption surface tensions are plotted following the addition of a bolus of DEPN-8, DEPN-8+1.5% Mini-B, or CLSE to a stirred subphase (10 mM HEPES with 0.15M NaCl and 1.5 mM CaCl₂ at pH 7.0) in a Teflon® dish at time zero. Final subphase surfactant concentration was uniform at 0.0625 mg lipid/ml. Data are Mean±SEM for n = 3–5. See text for details.</p

Mean proportions of different aspects of secondary structure for Mini-B in structure-promoting TFE solvent or in deuterium-hydrated DEPN-8 multilayers based on CD and FTIR spectroscopic analysis.

*<p>CD spectra for Mini-B in TFE were analyzed for secondary structure using the methods of Sreerama et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001039#pone.0001039-Sreerama1" target="_blank">[28]</a>, and FTIR spectra were analyzed for secondary conformation based on deconvolution of the amide I band (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001039#s4" target="_blank"><u>Methods</u></a>). FTIR spectra for Mini-B in deuterium-hydrated DEPN-8 multilayers were done at a molar ratio of 10∶1 lipid∶peptide. Tabulated results are means from four closely-reproduced separate determinations for each condition and spectral type.</p

Spectroscopic behavior of Mini-B and DEPN-8.

<p><u>Panel A</u>: CD spectrum for Mini-B in trifluoroethanol (TFE); <u>Panel B</u>: FTIR spectrum for DEPN-8; <u>Panel C</u>: FTIR spectral differences for Mini-B in DEPN-8 (dashed line) compared to Mini-B in TFE (solid line). In Panel A, mean residue ellipticity (MRE) averaged over eight scans is plotted against wavelength for Mini-B in 4∶6 (v:v) TFE:10 mM phosphate buffer, pH 7.4. The double minimum at ∼208 and 222 nm is indicative of a high α-helical content. In Panel B, the spectrum for DEPN-8 multilayers (100 µg lipid, arbitrary absorbance units) has a “C-O-C” ether linkage-associated absorption band centered at a wavenumber of 1072 cm⁻¹. In Panel C, the IR spectrum of Mini-B in TFE (solid line) has a peak at 1655 cm⁻¹ indicating high α-helix levels, while the peak at 1658 cm⁻¹ and high-field shoulder at 1678 cm⁻¹ for Mini-B in DEPN-8 (dashed line) indicates an increase in turn/bend conformation with a decreased but still prominent α-helix content. See text for discussion.</p