Abstract
This study investigates the importance of thioester-linked acyl groups in lung surfactant protein C (SP-C) in facilitating interactions with phospholipids that yield functionally important surface active behaviors. Native SP-C, palmitoylated at cysteine residues at positions 5 and 6, was isolated from bovine lung surfactant by liquid chromatography. Deacylated SP-C (dSP-C), unchanged in composition and sequence from SP-C but having a decreased alpha-helical content in films with dipalmitoyl phosphatidylcholine (DPPC) of 52 versus 70%, was obtained by treatment with 0.1 M sodium carbonate buffer at pH 10. Surface activity was studied for SP-C and dSP-C combined with column-purified phospholipids (PPL) from calf lung surfactant or with synthetic phospholipids (DPPC or a synthetic phospholipid mixture (SPL) containing 50:35:15, DPPC:egg phosphatidylcholine:egg phosphatidylglycerol). Interfacial measurements included surface pressure time adsorption isotherms for dispersed surfactants with diffusion minimized, dynamic surface pressure area isotherms and respreading for films in the Wilhelmy balance, and overall surface tension lowering at physiologic cycling rate in oscillating bubble experiments. Dispersions of PPL:SP-C and SPL:SP-C rapidly adsorbed to high equilibrium surface pressures of 47-48 mN/m, significantly better than corresponding dispersions containing dSP-C. The adsorption of PPL:dSP-C was essentially unchanged from that of PPL alone, and the adsorption of SPL:dSP-C was improved only slightly over SPL alone. In Wilhelmy balance studies, dynamic respreading was significantly improved over phospholipids alone in films of SP-C plus PPL, SPL, or DPPC. Respreading was improved less markedly by dSP-C in corresponding films with SPL or DPPC and not at all in films with PPL. Maximum surface pressures were also higher in cycled films of SP-C versus dSP-C combined with PPL or SPL. In bubble experiments (37 degrees C, 20 cycles/min), dispersions of PPL:SP-C and SPL:SP-C reached low minimum surface tensions of <1 and 5 mN/m, respectively, whereas PPL:dSP-C and SPL:dSP-C only reached minima of approximately 20 mN/m as did PPL and SPL alone. Acylation in SP-C is crucial for its interactions with phospholipids over the full spectrum of adsorption and dynamic surface behaviors important for lung surfactant.
Highlights
IntroductionA complex mixture of phospholipids with smaller amounts of specific proteins and neutral lipids, is essential for normal lung function [1, 2]
Pulmonary surfactant, a complex mixture of phospholipids with smaller amounts of specific proteins and neutral lipids, is essential for normal lung function [1, 2]
The equilibrium surface pressure reached by dispersions of PPL:deacylated bovine SP-C (dSP-C) was essentially unchanged from PPL alone (Fig. 1A)
Summary
A complex mixture of phospholipids with smaller amounts of specific proteins and neutral lipids, is essential for normal lung function [1, 2]. Phospholipid:apoprotein mixtures are studied for the time course of adsorption following dispersion in the aqueous phase, for dynamic respreading and surface pressure area (-A) isotherm in spread films cycled in the Wilhelmy balance, and for overall surface tension lowering ability of dispersions pulsated at rapid (physiologic) rate in an oscillating bubble apparatus. For this entire set of surface behaviors, deacylation is shown either to substantially reduce or completely remove the beneficial effects of SP-C on phospholipid surface activity, indicating that acylation is essential for the surface active function of this hydrophobic surfactant protein
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