SP-C Palmitoylation is Crucial for Stabilizing Cholesterol-Containing Surfactant Films during Continuous Compression/Expansion Cycling

Abstract

Cholesterol is critical to maintain a dynamic lateral structure in pulmonary surfactant membranes, including a defined fluid-ordered/fluid-disordered phase equilibrium and proper lateral sorting of surfactant proteins and lipids. However, an excess of cholesterol has been linked to impaired surface activity both in surfactant models and in surfactant from injured lungs. Surfactant protein C (SP-C), the smallest and most hydrophobic of all surfactant proteins, has been shown to interact with cholesterol and dual palmitoylation of its N-terminal segment has been shown to drive association with ordered phases in model membranes. Furthermore, it has been proposed that native palmitoylated SP-C can act in concert with surfactant protein B (SP-B) to permit cholesterol-containing surfactant films to reach very low surface tensions upon compression. In the present work, we report that palmitoylation of SP-C is important for its ability to counteract deleterious effects of cholesterol on surfactant film stability under continuous expansion/compression cycling, as evaluated in a captive bubble surfactometer (CBS) setup. Presence of 5% cholesterol impairs significantly the stability under quasi static and dynamic compression of films composed of DPPC/POPC/POPG/SP-B (50:25:15:1, w/w/w/w), which are able to reach tensions below 3 mN/m with only 20% compression and almost no hysteresis in the absence of cholesterol. Incorporation in the films of 2% native palmitoylated SP-C could alleviate these deleterious effects. However, recombinant non-palmitoylated SP-C was not able to reproduce the stabilizing effect of native SP-C, confirming that palmitoylation of SP-C at its N-terminal end is crucial for its potential function of stabilizing surfactant films during the respiratory cycles in the lung.