Abstract
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. KL4 ([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-KL4 interactions behind its function, which is an inevitable knowledge to design improved therapeutic agents. To examine the phase behavior, topography, and lipid specificity of KL4/lipid systems, we aimed to study different surfactant model systems containing KL4, neutral dipalmitoylphosphatidylcholine (DPPC) and/or negatively charged dipalmitoylphosphatidylglycerol (DPPG) in the presence of Ca2+ ions. Surface pressure-area isotherms, fluorescence microscopic images, scanning force microscopy as well as time-of-flight secondary ion mass spectrometry suggest (i) that KL4 is not miscible with DPPC and therefore forms peptide aggregates in DPPC/KL4 mixtures; (ii) that KL4 specifically interacts with DPPG via electrostatic interactions and induces percolation of DPPG-rich phases; (iii) that existing DPPG-Ca2+ interactions are too strong to be overcome by KL4, 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.
Highlights
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
Surface Pressure-Area (-A) Isotherms—One approach to verify if specific interactions between KL4 and negatively charged lipids occur in lipid monolayers at an air/liquid interface is to perform film balance measurements
They display considerable differences depending on the lipid that can only be attributed to discriminative forces occurring between KL4 and DPPC or DPPG, respectively
Summary
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. 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 [21,22,23,24] 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. To obtain a more complete picture of KL4 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-offlight secondary ion mass spectrometry (TOF-SIMS). 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
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