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.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: II. Monolayers of pulmonary surfactant protein SP-C and phospholipids.

The interaction of the hydrophobic pulmonary surfactant protein SP-C with dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and DPPC:DPPG (7:3, mol:mol) in spread monolayers at the air-water interface has been studied. At low concentrations of SP-C (about 0.5 mol% or 3 weight%protein) the protein-lipid films collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At initial protein concentrations higher than 0.8 mol%, or 4 weight%, the isotherms displayed kinks at surface pressures of about 50 mN.m-1 in addition to the collapse plateaux at the higher pressures. The presence of less than 6 mol%, or 27 weight%, of SP-C in the protein-lipid monolayers gave a positive deviation from ideal behavior of the mean areas in the films. Analyses of the mean areas in the protein-lipid films as functions of the monolayer composition and surface pressure showed that SP-C, associated with some phospholipid (about 8-10 lipid molecules per molecule of SP-C), was squeezed out from the monolayers at surface pressures of about 55 mN.m-1. The results suggest a potential role for SP-C to modify the composition of the monolayer at the air-water interface in the alveoli.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: I. Monolayers of pulmonary surfactant protein SP-B and phospholipids.

The effects of pulmonary surfactant protein SP-B on the properties of monolayers of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), and a mixture of DPPC:DPPG (7:3, mol:mol) were studied using spread films at the air-water interface. The addition of SP-B to the phospholipid monolayers gave positive deviations from additivity of the mean areas in the films. At low protein concentrations (less than 45% amino acid residues which corresponds to 0.5 mol% or 10 weight% SP-B) monolayers of SP-B/DPPC, SP-B/DPPG and SP-B/(DPPC:DPPG) collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At higher concentrations of SP-B in the protein-lipid monolayers, kink points appeared in the isotherms at about 40-45 mN.m-1, implying possible exclusion of material from the films, hence, changes in the original monolayer compositions. Calculated analyses of the monolayer compositions as a function of surface pressure indicated that nearly pure SP-B, associated with small amounts of phospholipid (2-3 lipid molecules per SP-B dimer), was lost from SP-B/DPPC, SP-B/DPPG, and SP-B/(DPPC:DPPG) films at surface pressures higher than 40-45 mN.m-1. The results are consistent with a low effectiveness of SP-B in removing saturated phospholipids, DPPC or DPPG, from the spread SP-B/phospholipid films.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: III. Proteins SP-B plus SP-C with phospholipids in spread monolayers.

Spread binary monolayers of surfactant-associated proteins SP-B and SP-C were formed at the air-water interface. Surface pressure measurements showed no interactions between the hydrophobic proteins. The effects of a mixture of SP-B plus SP-C (2:1, w/w) on the properties of monolayers of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and DPPC:DPPG (7:3, mol:mol) were studied. During compression of ternary and quaternary films, containing less than 0.4 mol% or 5 weight% total protein, the proteins were not squeezed out and appeared to remain associated with the film until collapse at surface pressures of about 65-70 mN.m-1. At initial concentrations of total protein of about 0.9 mol% or 10 weight%, exclusion of protein-lipid complexes was observed at 40-50 mN.m-1. Larger amounts of phospholipid were removed by proteins from (SP-B:SP-C)/DPPG films than from (SP-B:SP-C)/DPPC ones. Separate squeeze-out of SP-B (or SP-B plus DPPC) at about 40 mN.m-1, followed by exclusion of SP-C (or SP-C plus DPPC) at about 50 mN.m-1, was observed in (SP-B:SP-C)/DPPC films. This led to a conclusion that there was independent behavior of SP-B and SP-C in (SP-B:SP-C)/DPPC monolayers. The quaternary (SP-B:SP-C)/(DPPC:DPPG) films showed qualitatively similar process of squeeze-out of the proteins. In the ternary mixtures of SP-B plus SP-C with DPPG separate exclusion of SP-B was not detected; rather, the data was consistent with exclusion of a (SP-B:SP-C)/DPPG complex at about 50 mN.m-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the hydrophobic surfactant proteins on the surface activity of spread films in the captive bubble surfactometer

The main function of pulmonary surfactant, a mixture of lipids and proteins, is to reduce the surface tension at the air/liquid interface of the lung. The hydrophobic surfactant proteins SP-B and SP-C are required for this process. When testing their activity in spread films in a captive bubble surfactometer, both SP-B and SP-C showed concentration dependence for lipid insertion as well as for lipid film refinement. Higher activity in DPPC refinement of the monolayer was observed for SP-B compared with SP-C. Further differences between both proteins were found, when subphase phospholipid vesicles, able to create a monolayer-attached lipid reservoir, were omitted. SP-C containing monolayers showed gradually increasing minimum surface tensions upon cycling, indicating that a lipid reservoir is required to prevent loss of material from the monolayer. Despite reversible cycling dynamics, SP-B containing monolayers failed to reach near-zero minimum surface tensions, indicating that the reservoir is required for stable films.     

Effect of hydrophobic surfactant proteins SP-B and SP-C on binary phospholipid monolayers: II. Infrared external reflectance-absorption spectroscopy

In situ external reflection infrared spectroscopy at the air-water interface was used to study the influence on phospholipid structure of an endogenous mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, which are thought to play pivotal roles in the adsorption and function of pulmonary surfactant. Mixtures studied were 1:1, 2:1, and 7:1 (mol:mol) DPPC-d(62):DPPG, and 7:1 DPPC-d(62):DOPG, alone and in the presence of 0.5-10 wt % mixed SP-B/C purified chromatographically from calf lung surfactant extract. Perdeuteration of DPPC produced a shift in vibrational frequencies so that it could be differentiated spectroscopically from the phosphoglycerol component in the surface monolayer. CH(2) antisymmetric and symmetric stretching bands ( approximately 2920 and 2852 cm(-1)) along with the analogous CD(2) stretching bands ( approximately 2194 and 2089 cm(-1)) were analyzed, and band heights and peak wavenumber positions were assessed as a function of monolayer surface pressure. Small, near-physiological contents of 1-2 wt % SP-B/C typically produced the maximum observed spectroscopic effects, which were abolished at high protein contents of 10 wt %. Analysis of CH(2) and CD(2) stretching bands and C-H/C-D band height ratios indicated that SP-B/C affected PC and PG lipids differently within the surface monolayer. SP-B/C had preferential interactions with DPPG in 1:1, 2:1, and 7:1 DPPC-d(62):DPPG films that increased its acyl chain order. SP-B/C also interacted specifically with DOPG in 7:1 DPPC-d(62):DOPG monolayers, but in this case an increase in CH(2) band heights and peak wavenumber positions indicated a further disordering of the already fluid DOPG acyl chains. CD(2) band height and peak wavenumber analysis indicated that SP-B/C had no significant effect on the structure of DPPC-d(62) chains in 7:1 films with DPPG or DOPG, and had only a slight tendency to increase the acyl chain order in 1:1 films of DPPC-d(62):DPPG. SP-B/C had no significant effect on DPPC-d(62) structure in films with DOPG. Infrared results also indicated that interactions involving SP-B/C and lipids led to exclusion of PC and PG lipids from the compressed interfacial monolayer, in agreement with our previous report on the phase morphology of lipid monolayers containing 1 wt % SP-B/C.     

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.     

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.     

Interaction of lung surfactant proteins with anionic phospholipids.

Langmuir isotherms, fluorescence microscopy, and atomic force microscopy were used to study lung surfactant specific proteins SP-B and SP-C in monolayers of dipalmitoylphosphatidylglycerol (DPPG) and palmitoyloleoylphosphatidylglycerol (POPG), which are representative of the anionic lipids in native and replacement lung surfactants. Both SP-B and SP-C eliminate squeeze-out of POPG from mixed DPPG/POPG monolayers by inducing a two- to three-dimensional transformation of the fluid-phase fraction of the monolayer. SP-B induces a reversible folding transition at monolayer collapse, allowing all components of surfactant to remain at the interface during respreading. The folds remain attached to the monolayer, are identical in composition and morphology to the unfolded monolayer, and are reincorporated reversibly into the monolayer upon expansion. In the absence of SP-B or SP-C, the unsaturated lipids are irreversibly lost at high surface pressures. These morphological transitions are identical to those in other lipid mixtures and hence appear to be independent of the detailed lipid composition of the monolayer. Instead they depend on the more general phenomena of coexistence between a liquid-expanded and liquid-condensed phase. These three-dimensional monolayer transitions reconcile how lung surfactant can achieve both low surface tensions upon compression and rapid respreading upon expansion and may have important implications toward the optimal design of replacement surfactants. The overlap of function between SP-B and SP-C helps explain why replacement surfactants lacking in one or the other proteins often have beneficial effects.     

Effects of cholesterol on surface activity and surface topography of spread surfactant films

Pulmonary surfactant forms a monolayer of lipids and proteins at the alveolar air/liquid interface. Although cholesterol is a natural component of surfactant, its function in surface dynamics is unclear. To further elucidate the role of cholesterol in surfactant, we used a captive bubble surfactometer (CBS) to measure surface activity of spread films containing dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylglycerol (DPPC/POPC/POPG, 50/30/20 molar percentages), surfactant protein B (SP-B, 0.75 mol %), and/or surfactant protein C (SP-C, 3 mol %) with up to 20 mol % cholesterol. A cholesterol concentration of 10 mol % was optimal for reaching and maintaining low surface tensions in SP-B-containing films but led to an increase in maximum surface tension in films containing SP-C. No effect of cholesterol on surface activity was found in films containing both SP-B and SP-C. Atomic force microscopy (AFM) was used, for the first time, to visualize the effect of cholesterol on topography of SP-B- and/or SP-C-containing films compressed to a surface tension of 22 mN/m. The protrusions found in the presence of cholesterol were homogeneously dispersed over the film, whereas in the absence of cholesterol the protrusions tended to be more clustered into network structures. A more homogeneous dispersion of surfactant lipid components may facilitate lipid insertion into the surfactant monolayer. Our data provide additional evidence that natural surfactant, containing SP-B and SP-C, is superior to surfactants lacking one of the components, and furthermore, this raises the possibility that the cholesterol found in surfactant of warm-blooded mammals does not have a function in surface activity.     

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.     

Fluorescently labeled pulmonary surfactant protein C in spread phospholipid monolayers.

Pulmonary surfactant, a lipid-protein complex, secreted into the fluid lining of lungs prevents alveolar collapse at low lung volumes. Pulmonary surfactant protein C (SP-C), an acylated, hydrophobic, alpha-helical peptide, enhances the surface activity of pulmonary surfactant lipids. Fluorescein-labeled SP-C (F-SP-C) (3, 6, 12 wt%) in dipalmitoylphosphatidylcholine (DPPC), and DPPC:dipalmitoylphosphatidylglycerol (DPPG) [DPPC:DPPG 7:3 mol/mol] in spread monolayers was studied by epifluorescence microscopy. Mass spectometry of F-SP-C indicated that the protein is partially deacylated and labeled with 1 mol fluorescein/1 mol protein. The protein partitioned into the fluid, or liquid expanded, phase. Increasing amounts of F-SP-C in DPPC or DPPC:DPPG monolayers decreased the size and total amounts of the condensed phase at all surface pressures. Calcium (1.6 mM) increased the amount of the condensed phase in monolayers of DPPC:DPPG but not of DPPC alone, and such monolayers were also perturbed by F-SP-C. The study indicates that SP-C perturbs the packing of neutral and anionic phospholipid monolayers even when the latter systems are condensed by calcium, indicating that interactions between SP-C and the lipids are predominantly hydrophobic in nature.     

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.     

Surface activity in vitro: role of surfactant proteins

Pattle, who provided some of the initial direct evidence for the presence of pulmonary surfactant in the lung, was also the first to show surfactant was susceptible to proteases such as trypsin. Pattle concluded surfactant was a lipoprotein. Our group has investigated the roles of the surfactant proteins (SP-) SP-A, SP-B, and SP-C using a captive bubble tensiometer. These studies show that SP-C>SP-B>SP-A in enhancing surfactant lipid adsorption (film formation) to the equilibrium surface tension of approximately 22-25 mN/m from the 70 mN/m of saline at 37 degrees C. In addition to enhancing adsorption, surfactant proteins can stabilize surfactant films so that lateral compression induced through surface area reduction results in the lowering of surface tension (gamma) from approximately 25 mN/m (equilibrium) to values near 0 mN/m. These low tensions, which are required to stabilize alveoli during expiration, are thought to arise through exclusion of fluid phospholipids from the surface monolayer, resulting in an enrichment in the gel phase component dipalmitoylphosphatidylcholine (DPPC). The results are consistent with DPPC enrichment occurring through two mechanisms, selective DPPC adsorption and preferential squeeze-out of fluid components such as unsaturated phosphatidylcholine (PC) and phosphatidylglycerol (PG) from the monolayer. Evidence for selective DPPC adsorption arises from experiments showing that the surface area reductions required to achieve gamma near 0 mN/m with DPPC/PG samples containing SP-B or SP-A plus SP-B films were less than those predicted for a pure squeeze-out mechanism. Surface activity improves during quasi-static or dynamic compression-expansion cycles, indicating the squeeze-out mechanism also occurs. Although SP-C was not as effective as SP-B in promoting selective DPPC adsorption, this protein is more effective in promoting the reinsertion of lipids forced out of the surface monolayer following overcompression at low gamma values. Addition of SP-A to samples containing SP-B but not SP-C limits the increase in gamma(max) during expansion. It is concluded that the surfactant apoproteins possess distinct overlapping functions. SP-B is effective in selective DPPC insertion during monolayer formation and in PG squeeze-out during monolayer compression. SP-A can promote adsorption during film formation, particularly in the presence of SP-B. SP-C appears to have a superior role to SP-B in formation of the surfactant reservoir and in reinsertion of collapse phase lipids.     

Effect of Pulmonary Surfactant Protein SP-B on the Micro- and Nanostructure of Phospholipid Films

Monolayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3, w/w) in the absence or in the presence of 2, 5, 10, or 20 weight percent of porcine surfactant protein SP-B were spread at the air-liquid interface of a surface balance, compressed up to surface pressures in the liquid-expanded/liquid-condensed (LE-LC) plateau of the isotherm, transferred onto mica supports, and analyzed by scanning force microscopy. In the absence of protein, the films showed micrometer-sized condensed domains with morphology and size that were analogous to those observed in situ at the air-liquid interface by epifluorescence microscopy. Scanning force microscopy permits examination of the coexisting phases at a higher resolution than previously achieved with fluorescent microscopy. Both LE and LC regions of DPPC films were heterogeneous in nature. LC microdomains contained numerous expanded-like islands whereas regions apparently liquid-expanded were covered by a condensed-like framework of interconnected nanodomains. Presence of increasing amounts of pulmonary surfactant protein SP-B affected the distribution of the LE and LC regions of DPPC and DPPC/DPPG films both at the microscopic and the nanoscopic level. The condensed microdomains became more numerous but their size decreased, resulting in an overall reduction of the amount of total LC phase in both DPPC and DPPC/DPPG films. At the nanoscopic level, SP-B also caused a marked reduction of the size of the condensed-like nanodomains in the LE phase and an increase in the length of the LE/LC interface. SP-B promotes a fine nanoscopic framework of lipid and lipid-protein nanodomains that is associated with a substantial mechanical resistance to film deformation and rupture as observed during film transference and manipulation. The effect of SP-B on the nanoscopic structure of the lipid films was greater in DPPC/DPPG than in pure DPPC films, indicating additional contributions of electrostatic lipid-protein interactions. The alterations of the nanoscopic structures of phospholipid films by SP-B provide the structural framework for the protein simultaneously sustaining structural stability as well as dynamical flexibility in surfactant films at the extreme conditions imposed by the respiratory mechanics. SP-B also formed segregated two-dimensional clusters that were associated with the boundaries between LC microdomains and the LE regions of DPPC and DPPC/DPPG films. The presence of these clusters at protein-to-lipid proportions above 2% by weight suggests that the concentration of SP-B in the surfactant lipid-protein complexes may be close to the solubility limit of the protein in the lipid films.     

Folding of lipid monolayers containing lung surfactant proteins SP-B1-25 and SP-C studied via coarse-grained molecular dynamics simulations

To explore the role of lung surfactant proteins SP-B and SP-C in storing and redelivering lipid from lipid monolayers during the compression and re-expansion occurring in lungs during breathing, we simulate the folding of lipid monolayers with and without these proteins. We utilize the MARTINI coarse-grained force field to simulate monolayers containing pure dipalmitoylphosphatidylcholine (DPPC) and DPPC mixed with palmitoyloleoylphosphatidylglycerol (POPG), palmitic acid (PA), and/or peptides. The peptides considered include the 25-residue N-terminal fragment of SP-B (SP-B_1-25), SP-C, and several SP-B_1-25 mutants in which charged and hydrophilic residues are replaced by hydrophobic ones, or vice-versa. We observe two folding mechanisms: folding by the amplification of undulations and folding by nucleation about a defect. The first mechanism is observed in monolayers containing either POPG or peptides, while the second mechanism is observed only with peptides present, and involves the lipid-mediated aggregation of the peptides into a defect, from which the fold can nucleate. Fold nucleation from a defect displays a dependence on the hydrophobic character of the peptides; if the number of hydrophobic residues is decreased significantly, monolayer folding does not occur. The addition of POPG or peptides to the DPPC monolayer has a fluidizing effect, which assists monolayer folding. In contrast, the addition of PA has a charge-dependent condensing affect on DPPC monolayers containing SP-C. The peptides appear to play a significant role in the folding process, and provide a larger driving force for folding than POPG. In addition to promoting fold formation, the peptides also display fusogenic behavior, which can lead to surface refining.     

External reflection absorption infrared spectroscopy study of lung surfactant proteins SP-B and SP-C in phospholipid monolayers at the air/water interface.

The interactions of the hydrophobic pulmonary surfactant proteins SP-B and SP-C with 1,2-dipalmitoylphosphatidylcholine in mixed, spread monolayer films have been studied in situ at the air/water interface with the technique of external reflection absorption infrared spectroscopy (IRRAS). SP-C has a mostly alpha-helical secondary structure both in the pure state and in the presence of lipids, whereas SP-B secondary structure is a mixture of alpha-helical and disordered forms. When films of SP-B/1,2-dipalmitoylphosphatidylcholine are compressed to surface pressures (pi) greater than approximately 40-43 mN/m, the protein is partially (15-35%) excluded from the surface, as measured by intensity ratios of the peptide bond amide l/lipid C==O stretching vibrations. The extent of exclusion increases as the protein/lipid ratio in the film increases. In contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids. The amide l peak of SP-C becomes asymmetric as a result of the formation of intermolecular sheet structures (1615-1630 cm-1) suggestive of peptide aggregation. The power of the IRRAS experiment for determination of film composition and molecular structure, i.e., as a direct test of the squeeze-out hypothesis of pulmonary surfactant function, is evident from this work.     

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.     

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.     

Effect of acylation on the interaction of the N-Terminal segment of pulmonary surfactant protein SP-C with phospholipid membranes

SP-C, the smallest pulmonary surfactant protein, is required for the formation and stability of surface-active films at the air-liquid interface in the lung. The protein consists of a hydrophobic transmembrane α-helix and a cationic N-terminal segment containing palmitoylated cysteines. Recent evidence suggests that the N-terminal segment is of critical importance for SP-C function. In the present work, the role of palmitoylation in modulating the lipid-protein interactions of the N-terminal segment of SP-C has been studied by analyzing the effect of palmitoylated and non-palmitoylated synthetic peptides designed to mimic the N-terminal segment on the dynamic properties of phospholipid bilayers, recorded by spin-label electron spin resonance (ESR) spectroscopy. Both palmitoylated and non-palmitoylated peptides decrease the mobility of phosphatidylcholine (5-PCSL) and phosphatidylglycerol (5-PGSL) spin probes in dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) bilayers. In zwitterionic DPPC membranes, both peptides have a greater effect at temperatures below than above the main gel-to-liquid-crystalline phase transition, the palmitoylated peptide inducing greater immobilisation of the lipid than does the non-palmitoylated form. In anionic DPPG membranes, both palmitoylated and non-palmitoylated peptides have similar immobilizing effects, probably dominated by electrostatic interactions. Both palmitoylated and non-palmitoylated peptides have effects comparable to whole native SP-C, as regards improving the gel phase solubility of phospholipid spin probes and increasing the polarity of the bilayer surface monitored by pK shifts of fatty acid spin probes. This indicates that a significant part of the perturbing properties of SP-C in phospholipid bilayers is mediated by interactions of the N-terminal segment. The effect of SP-C N-terminal peptides on the chain flexibility gradient of DPPC and DPPG bilayers is consistent with the existence of a peptide-promoted interdigitated phase at temperatures below the main gel-to-liquid-crystalline phase transition. The palmitoylated peptide, but not the non-palmitoylated version, is able to stably segregate interdigitated and non-interdigitated populations of phospholipids in DPPC bilayers. This feature suggests that the palmitoylated N-terminal segment stabilizes ordered domains such as those containing interdigitated lipids. We propose that palmitoylation may be important to promote and facilitate association of SP-C and SP-C-containing membranes with ordered lipid structures such as those potentially existing in highly compressed states of the interfacial surfactant film.