Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function

Abstract

Alcohol consumption with concurrent cigarette smoking produces malondialdehyde acetaldehyde (MAA)-adducted lung proteins. Lung surfactant protein D (SPD) supports innate immunity via bacterial aggregation and lysis, as well as by enhancing macrophage-binding and phagocytosis. MAA-adducted SPD (SPD-MAA) has negative effects on lung cilia beating, macrophage function, and epithelial cell injury repair. Because changes in SPD multimer structure are known to impact SPD function, we hypothesized that MAA-adduction changes both SPD structure and function. Purified human SPD and SPD-MAA (1 mg/mL) were resolved by gel filtration using Sephadex G-200 and protein concentration of each fraction determined by Bradford assay. Fractions were immobilized onto nitrocellulose by slot blot and assayed by Western blot using antibodies to SPD and to MAA. Binding of SPD and SPD-MAA was determined fluorometrically using GFP-labeled Streptococcus pneumoniae (GFP-SP). Anti-bacterial aggregation of GFP-SP and macrophage bacterial phagocytosis were assayed by microscopy and permeability determined by bacterial phosphatase release. Viral injury was measured as LDH release in RSV-treated airway epithelial cells. Three sizes of SPD were resolved by gel chromatography as monomeric, trimeric, and multimeric forms. SPD multimer was the most prevalent, while the majority of SPD-MAA eluted as trimer and monomer. SPD dose-dependently bound to GFP-SP, but SPD-MAA binding to bacteria was significantly reduced. SPD enhanced, but MAA adduction of SPD prevented, both aggregation and macrophage phagocytosis of GFP-SP. Likewise, SPD increased bacterial permeability while SPD-MAA did not. In the presence of RSV, BEAS-2B cell viability was enhanced by SPD, but not protected by SPD-MAA. Our results demonstrate that MAA adduction changes the quaternary structure of SPD from multimer to trimer and monomer leading to a decrease in the native anti-microbial function of SPD. These findings suggest one mechanism for increased pneumonia observed in alcohol use disorders.

Keywords: adduction; alcohol; aldehydes; lung; pneumonia; surfactant.

Figure 1

Bradford protein assay with corresponding bands from the Western blot. Column fraction protein concentrations were quantified using a Bradford assay. Protein was detected in 3 fractions eluted by gel filtration. In SPD, most of the protein (multimer) was collected in Fraction 3. In SPD-MAA, almost no multimer eluted while trimer (Fraction 7) and monomer (Fraction 9) were collected. Western blots for each slot blotted fraction were probed for SPD and SPD-MAA. The SPD column fractions showed the presence of SPD in 3 fractions. SPD-MAA column fractions only contained SPD in Fractions 7 and 9. Blots probed with anti-MAA detected protein from SPD-MAA column fractions 7 and 9 (not shown).

Figure 2

Surfactant protein binding to S. pneumoniae. Plates were coated with bacteria and incubated with 0-20 µg/mL SPD, SPD-MAA, BSA (negative control), or BSA-MAA. Bound SPD was detected with antibodies by ELISA. Absorbance at 450 nm of bound SPD at the different concentrations was determined (n = 5), non-parametric Kruskal-Wallis test was performed, differences between SPD and SPD-MAA were significant at all concentrations (*p< 0.01). The data are the averages +/- SD of five experiments. The only significant difference between SPD-MAA and the BSA negative control was observed at 20 µg/mL. No binding was observed for BSA-MAA.

Figure 3

Aggregation of S. pneumoniae in the presence of surfactant protein. GFP-SP were incubated with 10 µg/ml SPD or SPD-MAA for 90 min in the presence of calcium (5 mM) and visualized by fluorescence microscopy (X 500). The average area of aggregated clumps of bacteria was measured per field of view. The data are the averages +/- SD of five experiments. In the presence of calcium, SPD significantly increased aggregation compared to SPD-MAA, *p<0.0003. No clumping or aggregation of pneumococci was observed in the absence of calcium.

Figure 4

MAA adduction of SPD decreases phagocytosis of S. pneumoniae. GFP-S. pneumoniae were incubated with the murine macrophage cell line RAW264.7. The percent of RAW264.7 cells containing internalized bacterial cells per field was determined using fluorescence microscopy (X 500) after quenching non-internalized GFP-SP with Trypan blue. The image results are a representative of 5 independent experiments summarized by graph. Bars represent averages +/- SD, *p<0.0004.

Figure 5

SPD-induced anti-bacterial permeability is decreased by MAA adduction. S. pneumoniae was incubated in the presence of either 10 µg/mL SPD or SPD-MAA for up to 30 min and supernatant media phosphatase release measured as a function of bacterial permeability. A significant reduction in permeability (*p<0.05) was observed between SPD and SPD-MAA. Bars represent averages +/- SD, n=5 experiments.

Figure 6

MAA adduction of SPD decreases cell viability in response to respiratory syncytial virus (RSV). Infection of BEAS-2B with RSV increases LDH release compared to control media (no RSV). SPD (10-100 µg/mL) significantly (p<0.0002) decreases RSV-induced LDH release vs. the absence of SPD. No such decrease is observed with either 10 µg/mL (*p<0.01) or 100 µg/mL (**p<0.006) SPD-MAA vs. equal amounts of SPD. Bars represent averages +/- SD, n=5 experiments.

Figure 7

Summary model. Covalent modification of surfactant protein D (SPD) by malondialdehyde-acetaldehyde (MAA) adduction changes the structure of the protein from multimeric form to trimeric form and reduces anti-microbial innate defense.