Studies of different fragments and mutants of SP-B suggest that the function related structural and compositional characteristics in SP-B are its positive charges with intermittent hydrophobic domains. KL 4 ([lysine-(leucine)4]4-lysine) is a synthetic peptide based on SP-B structure and is the major constituent of Surfaxin®, a potential therapeutic agent for respiratory distress syndrome in premature infants. There is, however, no clear understanding about the possible lipid-KL 4 interactions behind its function, which is an inevitable knowledge to design improved therapeutic agents. To examine the phase behavior, topography, and lipid specificity of KL 4/lipid systems, we aimed to study different surfactant model systems containing KL 4, neutral dipalmitoylphosphatidylcholine (DPPC) and/or negatively charged dipalmitoylphosphatidylglycerol (DPPG) in the presence of Ca 2+ ions. Surface pressure-area isotherms, fluorescence microscopic images, scanning force microscopy as well as time-of-flight secondary ion mass spectrometry suggest (i) that KL 4 is not miscible with DPPC and therefore forms peptide aggregates in DPPC/KL 4 mixtures; (ii) that KL 4 specifically interacts with DPPG via electrostatic interactions and induces percolation of DPPG-rich phases; (iii) that existing DPPG-Ca 2+ interactions are too strong to be overcome by KL 4, the reason why the peptide remains excluded from condensed DPPG domains and passively colocalizes with DPPC in a demixed fluid phase; and (iv) that the presence of negatively charged lipid is necessary for the formation of bilayer protrusions. These results indicate that the capability of the peptide to induce the formation of a defined surface-confined reservoir depends on the lipid environment, especially on the presence of anionic lipids.
Pulmonary surfactant, a thin lipid-protein film lining the alveolar/air interface of the vertebrate lung, functions in vivo to lower surface tension, thereby reducing the work of breathing. A significant amount of investigations has been reported dealing with the physiological importance of lung surfactant and the implication of its absence or inability to function in premature neonates and adults. Over a long period, a lot of biophysical research has been undertaken using some of the major components of lung surfactant to better understand the means by which it is delivered to the air/liquid interface and promotes alveolar stability. The main phospholipid constituent of pulmonary surfactant is phosphatidylcholine, especially dipalmitoylphosphatidylcholine (DPPC)2. Particular emphasis has been placed on the important role of this major disaturated phospholipid component in reducing surface tension to very low values and thus protecting the alveolus against collapse. There also has been a strong interest in other major lipid components, such as anionic phosphatidylglycerol, and phospholipids containing unsaturated acyl chains. Besides phospholipids, four proteins, designated as SP-A, SP-B, SP-C, and SP-D, have been found in association with lung surfactant, among which SP-A and SP-D are hydrophilic. These two proteins are believed to be related to the storage and transport of lung surfactant as well as to participate in host defense. On the other hand, the hydrophobic proteins SP-B and SP-C are thought to play an important role in promoting the adsorption and spreading of monolayers containing large amounts of DPPC, which by itself only adsorbs slowly and respreads poorly at air/liquid interfaces.
Neonatal respiratory distress syndrome is caused by lung immaturity with a deficiency of surfactant in the alveolar spaces and is a major cause of morbidity and mortality in preterm infants. Studies on replacement therapy on respiratory distress syndrome indicate that SP-B and SP-C are essential constituents of exogenous surfactants. Because of microbiological, immunological, economic and purity concerns, many efforts have been made to develop synthetic surfactant replacement formulations, which involve a combination of synthetic lipids with either synthetic or recombinant peptides. To understand the role of SP-B in such replacement materials, different spectroscopic techniques have been used to study the interaction between SP-B and phospholipids. Studies of different fragments and mutants of SP-B suggest that the function-related structural and compositional characteristics in SP-B are its positive charges with intermittent hydrophobic domains. Based on these structural characteristics a SP-B model peptide with 21 amino acids containing the hydrophobic amino acid leucine (Leu) and cationic lysine (Lys) in the sequence ([lysine-(leucine)4]4-lysine) was synthesized. KL 4 proved to be a potent mimic of SP-B not only in vitro but also in vivo as it improved lung function of premature human infants with respiratory distress syndrome.
Although KL 4-based replacement surfactants such as Surfaxin® seem to be effective therapeutic agents the molecular mechanism by which the peptide unfolds its effect in lipid mixtures remains unclear. KL 4 displays a rather complex behavior when mixed with lipids. In fluid model systems, at low peptide concentrations and/or in the presence of negatively charged lipids KL 4 adopts a predominantly α-helical secondary structure. In more rigid membranes, at higher peptide contents and/or in the absence of anionic lipids, KL 4 converts to a β-sheet. The conformational transition from α-helix to β-sheet was also found to be surface pressure dependent leading to a mainly β-sheet secondary structure in DPPC and DPPC/DPPG monolayers at surface pressures >40 mN/m. Only in the presence of pure DPPG did KL 4 form an α-helix over the whole pressure range. It is possible that this conformational flexibility is important for the peptides effectiveness in medical treatment, especially because it is assumed that during the breathing process an enrichment of negatively charged lipids in the multilayers formed at higher surface pressures occurs. A peptide capable of adapting its conformation to the changing lipid composition during inhalation and exhalation is then thought to represent an effective therapeutic agent.
To obtain a more complete picture of KL 4 function and phase behavior in surfactant model systems we performed a systematic monolayer study applying different well established and elaborate techniques such as film balance, fluorescence light microscopy, scanning force microscopy (SFM), and time-of-flight secondary ion mass spectrometry (TOF-SIMS). We employed DPPC and DPPG as lipid components for our model system because they represent main constituents of native surfactant. The aim of this study was to systematically analyze the phase behavior of KL 4 in DPPC, DPPG, as well as DPPC/DPPG (4:1, mol ratio) monolayers in the presence of Ca 2+ ions. So far, all monolayer studies published on KL 4/lipid systems were performed in the absence of Ca 2+ ions even though this cation is present in the alveolar fluid at a concentration of about ∼1.8 m m. It was our intention to study the ability of the peptide to induce the formation of multilayers by means of SFM and to identify specific lipid interactions via TOF-SIMS imaging of LB films.
The lipids used in this study, namely 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). 2-(4,4-Difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (β-BODIPY® 500/510 C 12-HPC, BODIPY-PC) was obtained from Molecular Probes (Eugene, OR). All lipids were used without further purification. Chloroform, methanol, and hexane were high pressure liquid chromatography grade and purchased from Roth (Karlsruhe, Germany). Water was purified and deionized by a multicartridge system (MilliPore, Billerica, MA) and had a resistivity >18 MΩ·m. HEPES was obtained from Sigma and CaCl 2 from Merck (Darmstadt, Germany).
The SP-B model peptide KL 4 with the sequence KLLLLKLLLLKLLLLKLLLLK was obtained from Richard Mendelsohn, Rutgers University. The concentration of the peptide was estimated by fluorescamine assays. Lipids and peptides were dissolved in chloroform/methanol solution (1:1, v/v).
All the film balance experiments were performed on an analytical Wilhelmy film balance (Riegler and Kirstein, Mainz, Germany) with an operational area of 144 cm 2. All surface pressure-area measurements were performed on a buffered subphase (25 m m HEPES, 3 m m CaCl 2, pH 7, 20 °C). Lipid/peptide monolayers were composed of DPPC, DPPG, or DPPC/DPPG (4:1, mol ratio) and were supplemented with various concentrations of KL 4. The lipid/protein mixtures were prepared in a chloroform/methanol solution (1:1, v/v) and spread onto the subphase. After an equilibration time of 10-15 min the monolayers were compressed at a rate of 5.8 cm 2/min.
All lipid/peptide systems were doped with 0.5 mol % BODIPY-PC. As described in Ref. 31 a setup consisting of an epifluorescence microscope (Olympus STM5-MJS, Olympus, Hamburg, Germany) equipped with a xy-stage and connected to a CCD camera (Hamamatsu, Herrsching, Germany) was used to obtain fluorescence micrographs of lipid/peptide mixtures at the air/water interface at certain pressures by stopping the barrier. All the measurements were performed on a subphase containing 25 m m HEPES and 3 m m CaCl 2, pH 7, at 20 °C.
Preparation of gold supports was done as described in Ref. 32. Glass slides were cleaned by bath sonication at 70 °C, three times alternately in detergent and water. Immediately before evaporation the slides were dried in a nitrogen stream and further treated with argon plasma in a plasma cleaner (PDC 32G-2, Harrick, Ossining, NY) for 3 min. First, 1 nm of chromium was deposited on the surface of the slide, serving as an adhesive layer, onto which 200 nm of gold were sublimed at a rate of 0.01 nm/s. The gold-covered slides were cleaned by rectification for 8 h in a Soxhlet apparatus using n-hexane. They were then dried and used as substrates for the Langmuir-Blodgett transfers.
For scanning force microscopy investigations mica-supported phospholipid monolayers were prepared by Langmuir-Blodgett transfer. First, a freshly cleaved mica sheet (Electron Microscopy Science, Munich, Germany) was dipped into the subphase. Then the lipid/peptide mixture was spread from chloroform/methanol (1:1, v/v) solutions onto a buffered subphase of a Wilhelmy film balance (Riegler and Kirstein) with an operational area of 39 cm 2 at a temperature of 20 °C. After an equilibration period of 10 min the film was compressed with a velocity of 1.5 cm 2/min until a surface pressure of 50 mN/m was reached. The monolayer was then equilibrated for another 25 min at this surface pressure before transferring the film onto the mica sheet with a velocity of 0.7 mm/min. For TOF-SIMS the samples were transferred onto gold supports using the same procedure as above.
Scanning force microscopy images of the LB films transferred onto mica sheets were obtained at ambient conditions (20 °C) using a Dimension 3000 scanning force microscope with a Nanoscope IIIa controller from Digital Instruments (Santa Barbara, CA) operating in contact mode. Silicon nitride tips (Budget Sensors, Sofia, Bulgaria) with a spring constant of 40 N/m. A detailed section height analysis was performed for all the images taken using WsXM/Nanoscope SFM software. The number of protrusions found in a defined height range was plotted as “counts” against the protrusion height. The obtained statistical histograms of section heights were analyzed using Gaussian functions to determine the average protrusion heights.
TOF-SIMS measurements of surface films were transferred onto gold supports obtained on a TOF-SIMS IV (IONTOF, Muenster, Germany) using Bi+3 as the primary ion at 25 keV. Spectra were taken in bunched mode (focus 3-5 μm) with a mass resolution of 5000-10,000. Cycling time of the instrument was set to 200 μs, allowing the acquisition of spectra up to a mass to charge ratio of 1800. Mass-resolved images were taken at nominal mass resolution (burst alignment mode, focus 300 nm). A surface of 80 2 μm 2 was rastered with 256 × 256 pixels (pixel size 312 nm). The primary ion dose did not exceed 8 × 10 12 ions/cm 2. In line with observations of Biesinger et al.(33), at this primary ion dose no change or inversion of contrast could be detected in any of the measurements performed.
One approach to verify if specific interactions between KL 4 a
