Lipid oxidation and signalling in programmed cell death

The pulmonary surfactant lines as a complex monolayer of lipids and proteins the alveolar epithelial surface. The monolayer dynamically adapts the surface tension of this interface to the varying surface areas during inhalation and exhalation. Its presence in the alveoli is thus a prerequisite for a proper lung function. The lipid moiety represents about 90% of the surfactant and contains mainly dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG). The surfactant proteins involved in the surface tension adaption are called SP-A, SP-B and SP-C. The aim of the present investigation is to analyse the properties of monolayer films made from pure SP-C and from mixtures of DPPC, DPPG and SP-C in order to mimic the surfactant monolayer with minimal compositional requirement. Pressure-area diagrams were taken. Ellipsometric measurements at the air-water interface of a Langmuir film balance allowed measurement of the changes in monolayer thickness upon compression. Isotherms of pure SP-C monolayers exhibit a plateau between 22 and 25 mN/m. A further plateau is reached at higher compression. Structures of the monolayer formed during compression are reversible during expansion. Together with ellipsometric data which show a stepwise increase in film thickness (coverage) during compression, we conclude that pure SP-C films rearrange reversibly into multilayers of homogenous thickness.

Lipid monolayers collapse locally and irreversibly if films are compressed to approximately 0–4 nm²/molecule. In contrast, mixed DPPG/SP-C monolayers with less than 5 mol% protein collapse in a controlled and reversible way. The pressure-area diagrams exhibit a plateau at 20 mN/m, indicating partial demixing of SP-C and DPPG. The thickness isotherm obtained by ellipsometry indicates a transformation into multilayer structures. In DPPC/DPPG/SP-C mixtures again a reversible collapse was observed but without a drastic increase in surface layer thickness which may be due to the formation of protrusion under the surface. Thus lipid monolayers containing small amounts of SP-C may mimic the lung surfactant.     

Distribution of the surfactant-associated protein C within a lung surfactant model film investigated by near-field optical microscopy

Lung surfactant films at the air/water interface exhibit the particularity that surfactant molecules are expelled from the surface monolayer into a surface associated multilamellar phase during compression. They are able to re-enter the surface film during the following expansion. The underlying mechanism for this behavior is not fully understood yet. However, an important role is ascribed to the surfactant-associated protein C (SP-C). Here, we studied a model lung surfactant, consisting of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and SP-C, by means of scanning near-field optical microscopy (SNOM). Attaching a fluorescent dye to the protein allowed the localization of its lateral distribution at various surface pressures with high resolution. At an early stage of compression, the film appears demixed into a pure lipid phase and a protein-enriched phase. Within the latter phase, protein aggregations are revealed. They show a uniform density, having three times the fluorescence intensity of their surroundings. Across the phase boundary between the lipid phase and the protein-rich phase, there is a protein density gradient rather than an abrupt border. When the film is highly compressed, we observe the formation of multilamellar structures that are fluorescent. They are often surrounded by a slightly fluorescent monolayer. The fluorescence of the multilayer stacks (i. e., the protein content per unit area) is proportional to the height of the stacks.     

Analysis of lung surfactant model systems with time-of-flight secondary ion mass spectrometry

An often-used model lung surfactant containing dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and the surfactant protein C (SP-C) was analyzed as Langmuir-Blodgett film by spatially resolved time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly visualize the formation and composition of domains. Binary lipid and lipid/SP-C systems were probed for comparison. TOF-SIMS spectra revealed positive secondary ions (SI) characteristic for DPPC and SP-C, but not for DPPG. SI mapping results in images with domain structures in DPPC/DPPG and DPPG/SP-C, but not in DPPC/SP-C films. We are able to distinguish between the fluid and condensed areas probably due to a matrix effect. These findings correspond with other imaging techniques, fluorescence light microscopy (FLM), scanning force microscopy (SFM), and silver decoration. The ternary mixture DPPC/DPPG/SP-C transferred from the collapse region exhibited SP-C-rich domains surrounding pure lipid areas. The results obtained are in full accordance with our earlier SFM picture of layered protrusions that serve as a compressed reservoir for surfactant material during expansion. Our study demonstrates once more that SP-C plays a unique role in the respiration process.     

Effects of lung surfactant proteins, SP-B and SP-C, and palmitic acid on monolayer stability

Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome.     

Environmental tobacco smoke effects on lung surfactant film organization

Adsorption of the clinical lung surfactants (LS) Curosurf or Survanta from aqueous suspension to the air-water interface progresses from multi-bilayer aggregates through multilayer films to a coexistence between multilayer and monolayer domains. Exposure to environmental tobacco smoke (ETS) alters this progression as shown by Langmuir isotherms, fluorescence microscopy and atomic force microscopy (AFM). After 12 h of LS exposure to ETS, AFM images of Langmuir-Blodgett deposited films show that ETS reduces the amount of material near the interface and alters how surfactant is removed from the interface during compression. For Curosurf, ETS prevents refining of the film composition during cycling; this leads to higher minimum surface tensions. ETS also changes the morphology of the Curosurf film by reducing the size of condensed phase domains from 8-12 μm to ∼ 2 μm, suggesting a decrease in the line tension between the domains. The minimum surface tension and morphology of the Survanta film are less impacted by ETS exposure, although the amount of material associated with the film is reduced in a similar way to Curosurf. Fluorescence and mass spectra of Survanta dispersions containing native bovine SP-B treated with ETS indicate the oxidative degradation of protein aromatic amino acid residue side chains. Native bovine SP-C isolated from ETS exposed Survanta had changes in molecular mass consistent with deacylation of the lipoprotein. Fourier Transform Infrared Spectroscopy (FTIR) characterization of the hydrophobic proteins from ETS treated Survanta dispersions show significant changes in the conformation of SP-B and SP-C that correlate with the altered surface activity and morphology of the lipid-protein film.     

A scanning force- and fluorescence light microscopy study of the structure and function of a model pulmonary surfactant

The structure of an artificial pulmonary surfactant was studied by scanning force- and fluorescence light microscopy (SFM, and FLM, respectively). The surfactant – a mixture of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and recombinant surfactant-associated protein C (SP-C) – was prepared at the air-water interface of a Langmuir film balance and imaged by FLM under various states of compression. In order to visualize their topography by SFM, the films were transferred onto a solid mica support by the Langmuir-Blodgett (LB) technique. We found that a region of high film compressibility of the spread monolayer close to its equilibrium surface pressure (π=50 mN/m) was due to the exclusion of layered protrusions with each layer 5.5 to 6.5 nm thick. They remained associated with the monolayer and readily reinserted upon expansion of the film. Comparison with the FLM showed that the protrusions contained the protein in high concentration. The more the film was compressed, the larger was the number of layers on top of each other. The protrusions arose from regions of the monolayer with a distinct microstructure that may have been responsible for their formation. The molecular architecture of the microstructure remains to be elucidated, although some of it can be inferred from spectroscopic data in combination with the SFM topographical images. We illustrate our current understanding of the film structure with a molecular model. Received: 20 September 1996 / Accepted: 22 May 1997     

A modified squeeze-out mechanism for generating high surface pressures with pulmonary surfactant

The exact mechanism by which pulmonary surfactant films reach the very low surface tensions required to stabilize the alveoli at end expiration remains uncertain. We utilized the nanoscale sensitivity of atomic force microscopy (AFM) to examine phospholipid (PL) phase transition and multilayer formation for two Langmuir-Blodgett (LB) systems: a simple 3 PL surfactant-like mixture and the more complex bovine lipid extract surfactant (BLES). AFM height images demonstrated that both systems develop two types of liquid condensed (LC) domains (micro- and nano-sized) within a liquid expanded phase (LE). The 3 PL mixture failed to form significant multilayers at high surface pressure (π while BLES forms an extensive network of multilayer structures containing up to three bilayers. A close examination of the progression of multilayer formation reveals that multilayers start to form at the edge of the solid-like LC domains and also in the fluid-like LE phase. We used the elemental analysis capability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to show that multilayer structures are enriched in unsaturated PLs while the saturated PLs are concentrated in the remaining interfacial monolayer. This supports a modified squeeze-out model where film compression results in the hydrophobic surfactant protein-dependent formation of unsaturated PL-rich multilayers which remain functionally associated with a monolayer enriched in disaturated PL species. This allows the surface film to attain low surface tensions during compression and maintain values near equilibrium during expansion.     

Multilayer structures in lipid monolayer films containing surfactant protein C: effects of cholesterol and POPE

The influence of cholesterol and POPE on lung surfactant model systems consisting of DPPC/DPPG (80:20) and DPPC/DPPG/surfactant protein C (80:20:0.4) has been investigated. Cholesterol leads to a condensation of the monolayers, whereas the isotherms of model lung surfactant films containing POPE exhibit a slight expansion combined with an increased compressibility at medium surface pressure (10-30 mN/m). An increasing amount of liquid-expanded domains can be visualized by means of fluorescence light microscopy in lung surfactant monolayers after addition of either cholesterol or POPE. At surface pressures of 50 mN/m, protrusions are formed which differ in size and shape as a function of the content of cholesterol or POPE, but only if SP-C is present. Low amounts of cholesterol (10 mol %) lead to an increasing number of protrusions, which also grow in size. This is interpreted as a stabilizing effect of cholesterol on bilayers formed underneath the monolayer. Extreme amounts of cholesterol (30 mol %), however, cause an increased monolayer rigidity, thus preventing reversible multilayer formation. In contrast, POPE, as a nonbilayer lipid thought to stabilize the edges of protrusions, leads to more narrow protrusions. The lateral extension of the protrusions is thereby more influenced than their height.     

The structure of a model pulmonary surfactant as revealed by scanning force microscopy.

The structures formed by a pulmonary surfactant model system of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and recombinant surfactant-associated protein C (SP-C) were studied using scanning force microscopy (SFM) on Langmuir-Blodgett films. The films appeared to be phase separated, in agreement with earlier investigations by fluorescence light microscopy. There were smooth polygonal patches of mostly lipid, surrounded by a corrugated rim rich in SP-C. When the films were compressed beyond the equilibrium surface pressure, the protein-rich phase mediated the formation of layered protrusions. The height of these multilamellar structures embodied equidistant steps slightly higher than a DPPC double layer in the gel phase. At the air-water interface too, a high compressibility at low surface tension was indicative of the exclusion of matter. The exclusion process proved to be fully reversible. The present study demonstrates that some of the matter of the model pulmonary surfactant can move in and out of the active monolayer. The SFM images revealed a lipid-protein complex that was responsible for the reversible exclusion of double-layer structures. This mechanism may be important in the natural system too, to keep the surface tension of the alveolar air/water interface constantly low over the range of area encountered upon breathing.     

Surfactant protein C and lung function: new insights into the role of α-helical length and palmitoylation

Surfactant protein C (SP-C) is known to be essential for lung function and the formation of a surface confined reservoir at the alveolar interface. The structural features relevant for the peptide’s extraordinary ability to form extended three-dimensional structures were systematically investigated and are summarized in the present paper. The influence of palmitoylation was studied for full length SP-Cs as well as truncated variants with the N-terminal residues 1–17 and 1–13, respectively. The combined results from film balance measurements, fluorescence microscopy (FLM) and scanning force microscopy (SFM) reveal a fine-tuned balance between the influence of the palmitoyl chains and α-helical length. Native SP-C added to DPPC/DPPG monolayers (molar ratio 80:20) induced the formation of the surface confined reservoir independent of its palmitoylation degree. However, topographic images revealed that only bilayers and not multilayers where formed when the acyl chains were missing. The influence of palmitoylation increased when α-helical length was considerably reduced to 17 or even 13 amino acid residues. In these strongly truncated SP-C peptides palmitoyl chains increased monolayer stability and anchored the peptides in the lipid film. However, no multilayer formation was observed at all for all shortened peptides. The α-helix of SP-C seems to be a prerequisite for the formation of extended three-dimensional structures and obviously has to be able to span a lipid bilayer. Palmitoylation obviously mediates interactions between lipids and/or peptides not only within a protein/lipid film but also between neighbouring layers and induces a stacking of bilayers. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Monolayer–multilayer transitions in a lung surfactant model: IR reflection–absorption spectroscopy and atomic force microscopy

A hydrophobic pulmonary surfactant protein, SP-C, has been implicated in surface-associated activities thought to facilitate the work of breathing. Model surfactant films composed of lipids and SP-C display a reversible transition from a monolayer to surface-associated multilayers upon compression and expansion at the air/water (A/W) interface. The molecular-level mechanics of this process are not yet fully understood. The current work uses atomic force microscopy on Langmuir–Blodgett films to verify the formation of multilayers in a dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol, and SP-C model system. Isotherms of SP-C-containing films are consistent with exclusion and essentially complete respreading during compression and expansion, respectively. Multilayer formation was not detected in the absence of SP-C. Most notable are the results from IR reflection–absorption spectroscopy (IRRAS) conducted at the A/W interface, where the position and intensity of the Amide I band of SP-C reveal that the predominantly helical structure changes its orientation in monolayers versus multilayers. IRRAS measurements indicate that the helix tilt angle changed from approximately 80° in monolayers to a transmembrane orientation in multilayers. The results constitute the first quantitative measure of helix orientation in mixed monolayer/multilamellar domains at the A/W interface and provide insight into the molecular mechanism for SP-C-facilitated respreading of surfactant.     

Formation of three-dimensional protein-lipid aggregates in monolayer films induced by surfactant protein B

This study focuses on the structural organization of surfactant protein B (SP-B) containing lipid monolayers. The artificial system is composed of the saturated phospholipids dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in a molar ratio of 4:1 with 0.2 mol% SP-B. The different "squeeze-out" structures of SP-B were visualized by scanning probe microscopy and compared with structures formed by SP-C. Particularly, the morphology and material properties of mixed monolayers containing 0.2 mol% SP-B in a wide pressure range of 10 to 54 mN/m were investigated revealing that filamentous domain boundaries occur at intermediate surface pressure (15-30 mN/m), while disc-like protrusions prevail at elevated pressure (50-54 mN/m). In contrast, SP-C containing lipid monolayers exhibit large flat protrusions composed of stacked bilayers in the plateau region (app. 52 mN/m) of the pressure-area isotherm. By using different scanning probe techniques (lateral force microscopy, force modulation, phase imaging) it was shown that SP-B is dissolved in the liquid expanded rather than in the liquid condensed phase of the monolayer. Although artificial, the investigation of this system contributes to further understanding of the function of lung surfactant in the alveolus.     

Secondary structure and lipid interactions of the N-terminal segment of pulmonary surfactant SP-C in Langmuir films: IR reflection-absorption spectroscopy and surface pressure studies

Pulmonary surfactant, a thin lipid/protein film lining mammalian lungs, functions in vivo to reduce the work of breathing and to prevent alveolar collapse. Analogues of two hydrophobic surfactant proteins, SP-B and SP-C, have been incorporated into therapeutic agents for respiratory distress syndrome, a pathological condition resulting from deficiency in surfactant. To facilitate rational design of therapeutic agents, a molecular level understanding of lipid interaction with surfactant proteins or their analogues in aqueous monolayer films is necessary. The current work uses infrared reflection-absorption spectroscopy (IRRAS) to determine peptide conformation and the effects of S-palmitoylation on the lipid interactions of a synthetic 13 residue N-terminal peptide [SP-C13(palm)(2)] of SP-C, in mixtures with 1,2-dipalmitoylphosphatidylcholine (DPPC) or 1,2-dipalmitoylphosphatidylglycerol (DPPG). Two Amide I' features, at approximately 1655 and approximately 1639 cm(-1) in the peptide IRRAS spectra, are assigned to alpha-helical peptide bonds in hydrophobic and aqueous environments, respectively. In binary DPPC/SP-C13(palm)(2) films, the proportion of hydrated/hydrophobic helix increases reversibly with surface pressure (pi), suggestive of the peptide being squeezed out from hydrophobic regions of the monolayer. No such effect was observed for DPPG/peptide monolayers, indicative of stronger, probably electrostatic, interactions. Depalmitoylation produced a weakened interaction with either phospholipid as deduced from IRRAS spectra and from pi-area isotherms. S-Palmitoylation may modulate peptide hydration and conformation in the N-terminal region of SP-C and may thus permit the peptide to remain in the film at the high surface pressures present during lung compression. The unique capability of IRRAS to detect the surface pressure dependence of protein or peptide structure/interactions in a physiologically relevant model for surfactant is clearly demonstrated.     

Mimicking SP-C palmitoylation on a peptoid-based SP-B analogue markedly improves surface activity

Hydrophobic lung surfactant proteins B and C (SP-B and SP-C) are critical for normal respiration in vertebrates, and each comprises specific structural attributes that enable the surface-tension-reducing ability of the lipid-protein mixture in lung surfactant. The difficulty in obtaining pure SP-B and SP-C on a large scale has hindered efforts to develop a non-animal-derived surfactant replacement therapy for respiratory distress. Although peptide-based SP-C mimics exhibit similar activity to the natural protein, helical peptide-based mimics of SP-B benefit from dimeric structures. To determine if in vitro surface activity improvements in a mixed lipid film could be garnered without creating a dimerized structural motif, a helical and cationic peptoid-based SP-B mimic was modified by SP-C-like N-terminus alkylation with octadecylamine. “Hybridized” mono- and dialkylated peptoids significantly decreased the maximum surface tension of the lipid film during cycling on the pulsating bubble surfactometer relative to the unalkylated variant. Peptoids were localized in the fluid phase of giant unilamellar vesicle lipid bilayers, as has been described for SP-B and SP-C. Using Langmuir-Wilhelmy surface balance epifluorescence imaging (FM) and atomic force microscopy (AFM), only lipid-alkylated peptoid films revealed micro- and nanostructures closely resembling films containing SP-B. AFM images of lipid-alkylated peptoid films showed gel condensed-phase domains surrounded by a distinct phase containing “nanosilo” structures believed to enhance re-spreading of submonolayer material. N-terminus alkylation may be a simple, effective method for increasing lipid affinity and surface activity of single-helix SP-B mimics.     

More than a monolayer: relating lung surfactant structure and mechanics to composition

Survanta, a clinically used bovine lung surfactant extract, in contact with surfactant in the subphase, shows a coexistence of discrete monolayer islands of solid phase coexisting with continuous multilayer "reservoirs" of fluid phase adjacent to the air-water interface. Exchange between the monolayer, the multilayer reservoir, and the subphase determines surfactant mechanical properties such as the monolayer collapse pressure and surface viscosity by regulating solid-fluid coexistence. Grazing incidence x-ray diffraction shows that the solid phase domains consist of two-dimensional crystals similar to those formed by mixtures of dipalmitoylphosphatidylcholine and palmitic acid. The condensed domains grow as the surface pressure is increased until they coalesce, trapping protrusions of liquid matrix. At approximately 40 mN/m, a plateau exists in the isotherm at which the solid phase fraction increases from approximately 60 to 90%, at which the surface viscosity diverges. The viscosity is driven by the percolation of the solid phase domains, which depends on the solid phase area fraction of the monolayer. The high viscosity may lead to high monolayer collapse pressures, help prevent atelectasis, and minimize the flow of lung surfactant out of the alveoli due to surface tension gradients.     

Surfactant protein interactions with neutral and acidic phospholipid films

The captive bubble tensiometer was employed to study interactions of phospholipid (PL) mixtures of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) at 50 microg/ml with physiological levels of the surfactant protein (SP) A SP-B, and SP-C alone and in combination at 37 degrees C. All surfactant proteins enhanced lipid adsorption to equilibrium surface tension (gamma), with SP-C being most effective. Kinetics were consistent with the presence of two adsorption phases. Under the conditions employed, SP-A did not affect the rate of film formation in the presence of SP-B or SP-C. Little difference in gamma(min) was observed between the acidic POPG and the neutral POPC systems with SP-B or SP-C with and without SP-A. However, gamma(max) was lower with the acidic POPG system during dynamic, but not during quasi-static, cycling. Considerably lower compression ratios were required to generate low gamma(min) values with SP-B than SP-C. DPPC-POPG-SP-B was superior to the neutral POPC-SP-B system. Although SP-A had little effect on film formation with SP-B, surface activity during compression was enhanced with both PL systems. In the presence of SP-C, lower compression ratios were required with the acidic system, and with this mixture, SP-A addition adversely affected surface activity. The results suggest specific interactions between SP-B and phosphatidylglycerol, and between SP-B and SP-A. These observations are consistent with the presence of a surface-associated surfactant reservoir which is involved in generating low gamma during film compression and lipid respreading during film expansion.     

Effect of hydrophobic surfactant peptides SP-B and SP-C on binary phospholipid monolayers. I. Fluorescence and dark-field microscopy.

The influence of the hydrophobic proteins SP-B and SP-C, isolated from pulmonary surfactant, on the morphology of binary monomolecular lipid films containing phosphocholine and phosphoglycerol (DPPC and DPPG) at the air-water interface has been studied using epifluorescence and dark-field microscopy. In contrast to previously published studies, the monolayer experiments used the entire hydrophobic surfactant protein fraction (containing both the SP-B and SP-C peptides) at physiologically relevant concentrations (approximately 1 wt %). Even at such low levels, the SP-B/C peptides induce the formation of a new phase in the surface monolayer that is of lower intrinsic order than the liquid condensed (LC) phase that forms in the pure lipid mixture. This presumably leads to a higher structural flexibility of the surface monolayer at high lateral pressure. Variation of the subphase pH indicates that electrostatic interaction dominates the association of the SP-B/C peptides with the lipid monolayer. As evidenced from dark-field microscopy, monolayer material is excluded from the DPPC/DPPG surface film on compression and forms three-dimensional, surface-associated structures of micron dimensions. Such exclusion bodies formed only with SP-B/C peptides. This observation provides the first direct optical evidence for the squeeze-out of pulmonary surfactant material in situ at the air-water interface upon increasing monolayer surface pressures.     

N-terminally extended surfactant protein (SP) C isolated from SP-B-deficient children has reduced surface activity and inhibited lipopolysaccharide binding

In both humans and mice, a deficiency of surfactant protein B (SP-B) is associated with a decreased concentration of mature SP-C and accumulation of a larger SP-C peptide, denoted SP-C(i), which is not observed under normal conditions. Isolation of hydrophobic polypeptides from the lungs of children who died with two different SP-B mutations yielded pure SP-C(i) and showed only trace amounts of mature SP-C. Determination of the SP-C(i) covalent structure revealed a 12-residue N-terminal peptide segment, followed by a 35-residue segment that is identical to mature SP-C. The SP-C(i) structure determined herein is similar to that of a proposed late intermediate in the processing of proSP-C, suggesting that SP-C(i) is the immediate precursor of SP-C. In bronchoalveolar lavage fluid from transgenic mice with a focal deficiency of SP-B, SP-C(i) was detected in the biophysically active, large aggregate fraction and was associated with membrane structures that are typical for a large aggregate surfactant. However, unlike SP-C, SP-C(i) exhibited a very poor ability to promote phospholipid adsorption, gave high surface tension during cyclic film compression, and did not bind lipopolysaccharide in vitro. SP-C(i) is thus capable of associating with surfactant lipids, but its N-terminal dodecapeptide segment must be proteolytically removed to generate a biologically functional peptide. The results of this study indicate that the early postnatal fatal respiratory distress seen in SP-B-deficient children is combined with the near absence of active variants of SP-C.     

Effects of hydrophobic helix length and side chain chemistry on biomimicry in peptoid analogues of SP-C

The hydrophobic proteins of lung surfactant (LS), SP-B and SP-C, are critical constituents of an effective surfactant replacement therapy for the treatment of respiratory distress syndrome. Because of concerns and difficulties associated with animal-derived surfactants, recent investigations have focused on the creation of synthetic analogues of the LS proteins. However, creating an accurate mimic of SP-C that retains its biophysical surface activity is extraordinarily challenging given the lipopeptide's extreme hydrophobicity and propensity to misfold and aggregate. One successful approach that overcomes these difficulties is the use of poly-N-substituted glycines, or peptoids, to mimic SP-C. To develop a non-natural, bioactive mimic of SP-C and to investigate the effects of side chain chemistry and length of the helical hydrophobic region, we synthesized, purified, and performed in vitro testing of two classes of peptoid SP-C mimics: those having a rigid alpha-chiral aromatic helix and those having a biomimetic alpha-chiral aliphatic helix. The length of the two classes of mimics was also systematically altered. Circular dichroism spectroscopy gave evidence that all of the peptoid-based mimics studied here emulated SP-C's secondary structure, forming stable helical structures in solution. Langmuir-Wilhelmy surface balance, fluorescence microscopy, and pulsating bubble surfactometry experiments provide evidence that the aromatic-based SP-C peptoid mimics, in conjunction with a synthetic lipid mixture, have superior surface activity and biomimetic film morphology in comparison to the aliphatic-based mimics and that there is an increase in surface activity corresponding to increasing helical length.     

Kinetics of phospholipid insertion into monolayers containing the lung surfactant proteins SP-B or SP-C

The lung surfactant proteins SP-B and SP-C are pivotal for fast and reversible lipid insertion at the air/liquid interface, a prerequisite for functional lung activity. We used a model system consisting of a preformed monolayer at the air/liquid interface supplemented with surfactant protein SP-B or SP-C and unilamellar vesicles injected into the subphase of a film balance. The content of SP-B or SP-C was similar to that found in lung lavage. In order to elucidate distinct steps of lipid insertion, we measured the time-dependent pressure increase as a function of the initial surface pressure, the temperature and the phosphatidylglycerol content by means of surface tension measurements and scanning force microscopy (SFM). The results of the film balance study are indicative of a two-step mechanism in which initial adsorption of vesicles to the protein-containing monolayer is followed by rupture and integration of lipid material. Furthermore, we found that vesicle adsorption on a preformed monolayer supplemented with SP-B or SP-C is strongly enhanced by negatively charged lipids as provided by DPPG and the presence of Ca2+ ions in the subphase. Hence, long-range electrostatic interactions are thought to play an important role in attracting vesicles to the surface, being the initial step in replenishment of lipid material. While insertion into the monolayer is independent of the type of protein SP-B or SP-C, initial adsorption is faster in the presence of SP-B than SP-C. We propose that the preferential interaction between SP-B and negatively charged DPPG leads to accumulation of negative charges in particular regions, causing strong adhesion between DPPG-containing vesicles and the monolayer mediated by Ca2+ ions, which eventually causes flattening and rupture of attached liposomes as observed by in situ SFM.