Palmitoylation of pulmonary surfactant protein SP-C is critical for its functional cooperation with SP-B to sustain compression/expansion dynamics in cholesterol-containing surfactant films

Abstract

Recent data suggest that a functional cooperation between surfactant proteins SP-B and SP-C may be required to sustain a proper compression-expansion dynamics in the presence of physiological proportions of cholesterol. SP-C is a dually palmitoylated polypeptide of 4.2 kDa, but the role of acylation in SP-C activity is not completely understood. In this work we have compared the behavior of native palmitoylated SP-C and recombinant nonpalmitoylated versions of SP-C produced in bacteria to get a detailed insight into the importance of the palmitic chains to optimize interfacial performance of cholesterol-containing surfactant films. We found that palmitoylation of SP-C is not essential for the protein to promote rapid interfacial adsorption of phospholipids to equilibrium surface tensions (∼22 mN/m), in the presence or absence of cholesterol. However, palmitoylation of SP-C is critical for cholesterol-containing films to reach surface tensions ≤1 mN/m at the highest compression rates assessed in a captive bubble surfactometer, in the presence of SP-B. Interestingly, the ability of SP-C to facilitate reinsertion of phospholipids during expansion was not impaired to the same extent in the absence of palmitoylation, suggesting the existence of palmitoylation-dependent and -independent functions of the protein. We conclude that palmitoylation is key for the functional cooperation of SP-C with SP-B that enables cholesterol-containing surfactant films to reach very low tensions under compression, which could be particularly important in the design of clinical surfactants destined to replacement therapies in pathologies such as acute respiratory distress syndrome.

Figure 1

Initial and postexpansion adsorption in the presence of SP-C. The effect of native and recombinant SP-C on the interfacial adsorption kinetics of lipid suspensions composed of DPPC/POPC/POPG (50:25:15, w/w/w) was measured in the absence (upper panels) or presence (lower panels) of 5% w/w cholesterol. (Left) Decrease of surface tension over time during the first 5 min after deposition of ∼0.5 μL of the different lipid or lipid/protein samples (10 mg/mL). (Right) Decrease in surface tension monitored during 5 min after expansion of the bubble from a volume of ∼0.045–0.15 mL. Data are means ± SD after averaging data from three independent experiments.

Figure 2

Quasistatic compression/expansion isotherms in the presence of SP-C. Films formed from DPPC/POPC/POPG (50:25:15, w/w/w) suspensions in the absence (upper panels) or presence (lower panels) of 5% (w/w) cholesterol and containing no protein (left), or 2% (w/w) of native SP-C (central left), rSP-C CC (central right), or SP-C FF (right) were subjected to quasistatic cycling. The surface-tension-versus-relative-area is plotted for the four consecutive stepwise compression-expansion isotherms.

Figure 3

Dynamic compression-expansion isotherms in the presence of SP-C. Films formed from DPPC/POPC/POPG (50:25:15, w/w/w) suspensions in the absence (upper panels) or presence (lower panels) of 5% (w/w) cholesterol and containing no protein (left), or 2% (w/w) of native SP-C (central left), rSP-C CC (central right), or rSP-C FF (right) were subjected to dynamic cycling at a rate of 20 cycles/min. The surface-tension-versus-relative-area is plotted for the first, 10th, and 20th cycle of continuous compression-expansion isotherms.

Figure 4

Initial and postexpansion adsorption in the presence of cholesterol, SP-B, and SP-C. The effect of native and recombinant SP-C on the interfacial adsorption kinetics of lipid suspensions composed of DPPC/POPC/POPG (50:25:15:10, w/w/w) plus 1% (w/w) porcine SP-B was evaluated in the presence of 5% w/w cholesterol. (Left panel) Initial adsorption (recorded during 5 min after deposition of the respective lipid samples. (Right panel) Postexpansion adsorption(decrease in surface tension during 5 min after expansion of the bubble to a volume of 0.15 mL. The samples, with 1% (w/w) of SP-B, contained no cholesterol, 5% (w/w) cholesterol, or 5% (w/w) cholesterol plus 2% (w/w) native SP-C, rSP-C CC, or rSP-C FF. Data are means ± SD after averaging data from three independent experiments.

Figure 5

Quasistatic cycling and SP-B/SP-C cooperation in cholesterol-containing surfactant films. Films formed from DPPC/POPC/POPG (50:25:15, w/w/w) suspensions containing 1% w/w SP-B and no cholesterol (top left) or containing 5% (w/w) cholesterol (right panels) were subjected to quasistatic cycling in the absence of SP-C (top), or in the presence of 2% (w/w) of native SP-C (upper center), rSP-C CC (lower center), or SP-C FF (bottom). The surface-tension-versus-relative-area is plotted for the first, second, third, and fourth cycle of four consecutive stepwise compression-expansion isotherms.

Figure 6

Dynamic cycling and SP-B/SP-C cooperation in cholesterol-containing surfactant films. Films formed from DPPC/POPC/POPG (50:25:15, w/w/w) suspensions containing 1% w/w SP-B and no cholesterol (top left) or containing 5% (w/w) cholesterol (right panels) were subjected to dynamic cycling in the absence of SP-C (top), or in the presence of 2% (w/w) of native SP-C (upper center), rSP-C CC (lower center), or rSP-C FF (bottom).The surface-tension-versus-relative-area is plotted for the first, 10th, and 20th cycle of continuous compression-expansion isotherms.

Figure 7

Model for the role of palmitoylation of SP-C in surfactant film stability.