Reactive oxygen species inactivation of surfactant involves structural and functional alterations to surfactant proteins SP-B and SP-C

Abstract

Exposing bovine lipid extract surfactant (BLES), a clinical surfactant, to reactive oxygen species arising from hypochlorous acid or the Fenton reaction resulted in an increase in lipid (conjugated dienes, lipid aldehydes) and protein (carbonyls) oxidation products and a reduction in surface activity. Experiments where oxidized phospholipids (PL) were mixed with BLES demonstrated that this addition hampered BLES biophysical activity. However the effects were only moderately greater than with control PL. These results imply a critical role for protein oxidation. BLES oxidation by either method resulted in alterations in surfactant proteins SP-B and SP-C, as evidenced by altered Coomassie blue and silver staining. Western blot analyses showed depressed reactivity with specific antibodies. Oxidized SP-C showed decreased palmitoylation. Reconstitution experiments employing PL, SP-B, and SP-C isolated from control or oxidized BLES demonstrated that protein oxidation was more deleterious than lipid oxidation. Furthermore, addition of control SP-B can improve samples containing oxidized SP-C, but not vice versa. We conclude that surfactant oxidation arising from reactive oxygen species generated by air pollution or leukocytes interferes with surfactant function through oxidation of surfactant PL and proteins, but that protein oxidation, in particular SP-B modification, produces the major deleterious effects.

FIGURE 1

Primary and secondary lipoperoxidation products. (A) The conjugated dienes formed during oxidation were monitored at 235 nm for 5 h (n = 3). Control samples are BLES in the absence of oxidants (C∼BLES-1, •); in the presence of H₂O₂ (C∼BLES-3, ○); and with Fe²⁺:EDTA (C∼BLES-2, ▾). Experimental samples included BLES in the presence of HOCl/⁻OCl (∇) and complete Fenton reagents (▪). (B) The presence of secondary lipoperoxidation products was tested as the content of MDA and 4HNE by using the lipoperoxidation assay kit (BIOXYTECH). Comparisons between two groups were made using paired Student t-test (*, p < 0.05; **, p < 0.001).

FIGURE 2

Protein carbonyl derivatives. Protein carbonyl levels were determined by ELISA, using the Zentech PC Test Kit (Zenith Technology). BLES values are expressed per mg BLES protein, where BLES contains 2% protein w/w. SP-B and SP-C values refer to isolated protein. Comparisons between two groups were made using paired Student t-test. a, p < 0.05 (control versus treated); b, p < 0.05 (H∼ versus F∼).

FIGURE 3

Effects of addition of oxidized PL to BLES on the initial film formation. Horizontal bar graph representing the time required for the films to achieve equilibrium surface tension. Control or oxidized POPG, POPC, PLPC, PLPG, AAPC, or AAPG were added to 50 μg/ml of BLES at 20% w/w relative to surfactant PL. All measurements were performed in triplicate at 37°C. Comparisons among the samples were conducted by ANOVA followed by Bonferroni and Tukey post-hoc tests. (*, p < 0.05 of appropriate controls).

FIGURE 4

Effects of addition of oxidized PL to BLES. Minimum (•) and maximum (○) γs reached by the samples during the 21st dynamic cycling. Samples are the same as in Fig. 3. All measurements were performed in triplicate at 37°C. Comparisons among the samples were conducted by ANOVA followed by Bonferroni and Tukey post-hoc tests (*, p < 0.05 of appropriate controls).

FIGURE 5

Effect of addition of oxidized PL to BLES, surface area reduction. The bar graph depicts the percentage of surface area compression required for the films to attain a minimum surface tension near 0 from 20 mN/m during the 21st dynamic cycling. Samples are the same as in Fig. 3. Extrapolated compressions of >100% were estimated for those films that could not attain low minima during compression. Comparisons among the samples were conducted using Tukey's studentized range (highly significant difference) test. Letters A–D represent comparisons among samples within a cycle. Means with the same letter are not significantly different for p < 0.05.

FIGURE 6

SP-B analysis. (A) Representative Coomassie blue stained gel, (B) representative western blot analysis, and (C) summary of SP-B ELISA values for control and oxidized BLES, as well as for the isolated proteins from BLES, H∼BLES, and F∼BLES. A total of 2 μg of protein as detected by Lowry were applied to each lane, as described in Experimental Procedures. Comparisons between two groups were made using paired Student t-test (*, p < 0.05; **, p < 0.01).

FIGURE 7

SP-C analysis. (A) Representative Coomassie blue gel and (B) representative silver-stained gel for control and oxidized BLES as well as for the isolated SP-C from C∼BLES, H∼BLES, and F∼BLES. A total of 2 μg of protein as detected by Lowry was applied per lane. Procedures are described in Experimental Procedures. The white arrows show the location of SP-B, whereas the black arrows show the location of SP-C. (C) Representative Western Blot confirming qualitative alterations of SP-C in the oxidized surfactants.

FIGURE 8

SP-C palmitoylation. (A) Representative autoradiography of ¹⁴C-labeled SP-Cs obtained from BLES, H∼BLES, and F∼BLES, as well as isolated C∼SP-C, H∼SP-C, and F∼SP-C. After the reacted samples were separated by Tricine-SDS-PAGE and transferred to nitrocellulose, a [¹⁴C] SP-C-labeled band corresponding to palmitoylated SP-C was detected at 3.7 kDa. (B) Bar graph summarizing the relative amount of mature SP-C as determined by quantitation of [¹⁴C] SP-C bands with the software QuantiScan for Windows, BIOSOFT 99. Comparisons between two groups were made using paired Student t-test (*, p < 0.05; **, p < 0.01).

FIGURE 9

Reconstitution studies. Effect of PL oxidation versus protein oxidation on surfactant adsorption. Horizontal bar graph illustrating the total adsorption times for C∼BLES, H∼BLES and F∼BLES before isolation and the following reconstituted samples in working buffer; control (PL:SP-B:SP-C), oxidized PL with nonoxidized proteins (H∼PL:SP-B:SP-C and F∼PL:SP-B:SP-C), both oxidized proteins with control PL (PL:H∼SP-B:H∼SP-C and PL:F∼SP-B:F∼SP-C) and all the constituents oxidized (H∼PL:H∼SP-B:H∼SP-C and F∼PL:F∼SP-B:F∼SP-C). Samples contain 500 μg PL/ml. Data are from at least three separate experimental samples. Comparisons among the samples were conducted using Tukey's studentized range test. Letters represent comparisons among samples within a cycle. Means with the same letter are not significantly different for p < 0.05.

FIGURE 10

Reconstitution studies. Effect of SP-B versus SP-C oxidation on surfactant adsorption. Horizontal bar graph illustrating the total adsorption times for controls (PL:SP-B, PL:SP-C and PL:SP-B:SP:C); samples containing oxidized SP-B (PL:H∼SP-B, PL:F∼SP-B, PL:H∼SP-B:SP-C, PL:F∼SP-B:SP-C); and samples containing oxidized SP-C, (PL:H∼SP-C, PL:F∼SP-C, PL:SP-B:H∼SP-C, and PL:SP-B:F∼SP-C). Samples are as in Fig. 9. Data are from at least three separate experimental samples. Comparisons among the samples were conducted by ANOVA followed by Bonferroni and Tukey post-hoc tests. Letters represent comparisons among samples. Means with the same letter are not significantly different for p < 0.05.

FIGURE 11

Reconstitution studies. Effect of PL oxidation versus protein oxidation on minimum and maximum γs. Minimum (•) and maximum (○) γs reached by the samples during the 21st dynamic cycling. Samples are as in Fig. 9. The data are the average of three separate experimental samples. Comparisons among the samples were conducted by ANOVA followed by Bonferroni and Tukey post-hoc tests. Letters a–f and A–G represent comparisons among samples in Figs 11 and 12. Means with the same letter are not significantly different for p < 0.05.

FIGURE 12

Reconstitution studies. Effects of SP-B versus SP-C oxidation on minimum and maximum γs. Minimum (•) and maximum (○) γs reached by the samples during the 21st dynamic cycling. Samples are as in Fig. 10. The data are the average of three separate experimental samples. Comparisons among the samples were conducted by ANOVA followed by Bonferroni and Tukey post-hoc tests. Letters a–f and A–G represent comparisons among samples. Means with the same letter are not significantly different for p < 0.05.

FIGURE 13

Reconstitution studies. Effect of PL oxidation versus protein oxidation on surface area reduction. The bar graph shows the percentage of surface area reduction required for the films to attain a minimum surface tension near 0 from 20 mN/m during the 21st dynamic cycling. Extrapolated compressions of >100% were estimated for those films that could not attain low minima during compression. Samples are as in Fig. 9. Data are the average of three independent experiments. Comparisons among the samples were conducted using Tukey's studentized range (highly significant difference) test. Letters A–E represent comparisons among samples within Figs 13 and 14. Means with the same letter are not significantly different for p < 0.05.

FIGURE 14

Reconstitution studies. Effect of SP-B and SP-C oxidation on surface area reduction. The bar graph shows the percentage of surface area reduction required for the films to attain a minimum surface tension near 0 from 20 mN/m during the 21st dynamic cycling. Extrapolated compressions of >100% were estimated for those films that could not attain low minima during compression. Samples are as in Fig. 10. The data are the average of three separate experimental samples. Comparisons among the samples were conducted using Tukey's studentized range (highly significant difference) test. Letters A–E represent comparisons among samples within a cycle. Means with the same letter are not significantly different for p < 0.05.