Surfactant protein A (SP-A) affects pulmonary surfactant morphology and biophysical properties

Abstract

Surfactant protein A (SP-A) is necessary in the formation of tubular myelin, the precursor to pulmonary surfactant (PS) films at the alveolar fluid-air interface. However, the exact role of SP-A in the physicochemical properties of PS films is not understood. Epifluorescence and electron microscopy were used to investigate the interaction of SP-A, and its structural homolog, C1q, with PS. Molecular films of PS lipid extract (PSLE) containing 1 mol% fluorescent probe (NBD-PC) were spread onto a saline subphase (145 mM NaCI, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, 0.16 or 0.20 μg/mL SP-A as well as 0, 1.64 or 5 mM CaCI2 in the subphase. Monolayers were compressed at a slow rate (the initial rate was 0.0089 A² /molecule/second) and film features were recorded. No differences were noted in PSLE monolayers in the absence or presence of Ca²⁺. Circular probe-excluded (dark) domains were observed against a fluorescent green background at low surface pressures (π ~ 5 mN/m). The domains grew in size with increasing π until π ≥ 25 mN/m, then diminishing domain size was observed. The amount of dark phase was maximized at π ~ 25 mN/m (at 18% of the initial film area), then decreased to ~3% when π reached ~40 mN/m. The addition of 0.16 or 0.20 μg/mL SP-A with 0 or 1.64 mM Ca²⁺ in the subphase caused a reorganization of dark domains into a loose network, and the maximum amount of dark phase was increased (25%) between π 10 - 28 mN/m. Monolayer features in the presence of 5 mM Ca²⁺ and SP-A were not significantly different from that of PSLE monolayers alone. This effect may be due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca²⁺. PSLE films were spread onto a subphase containing 0.16 μg/mL Texas-Red SP-A (TR-SP-A), a fluorescent analog of SP-A, in the presence or absence of 5 mM Ca ²⁺ to determine the location of the protein in the monolayer. In the absence of Ca²⁺, TR-SP-A associated with the reorganized dark phase lipid (probe-excluded domains). The presence of 5 mM Ca ²⁺ resulted in an aggregation of TR-SP-A in the fluid phase and fluid/gel phase boundaries of the monolayer. The interaction of PSLE monolayers with C1q was investigated by adding 0.16 μg/mL of this protein to the subphase in the presence or absence of 1.64 mM Ca ²⁺. Monolayer surface texture was reorganized in a manner similar to that when SP-A was present, but the total amount of dark phase recorded (~12% at π between 15-25 mN/m) was less than that observed in PSLE films in the presence of SP-A. The effect of C1q on the surface texture of PSLE films was similar to that of SP-A, hence the oligomeric structure of these C-type lectin proteins may have some impact on PSLE film organization. This study suggests that SP-A associates with PSLE monolayers, particularly gel-phase lipid, and results in some reorganization of gel-phase lipid in surfactant monolayers.