S-nitrosylation of surfactant protein-D controls inflammatory function

Abstract

The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D) have been implicated in the regulation of the innate immune system within the lung. In particular, SP-D appears to have both pro- and anti-inflammatory signaling functions. At present, the molecular mechanisms involved in switching between these functions remain unclear. SP-D differs in its quaternary structure from SP-A and the other members of the collectin family, such as C1q, in that it forms large multimers held together by the N-terminal domain, rather than aligning the triple helix domains in the traditional "bunch of flowers" arrangement. There are two cysteine residues within the hydrophobic N terminus of SP-D that are critical for multimer assembly and have been proposed to be involved in stabilizing disulfide bonds. Here we show that these cysteines exist within the reduced state in dodecameric SP-D and form a specific target for S-nitrosylation both in vitro and by endogenous, pulmonary derived nitric oxide (NO) within a rodent acute lung injury model. S-nitrosylation is becoming increasingly recognized as an important post-translational modification with signaling consequences. The formation of S-nitrosothiol (SNO)-SP-D both in vivo and in vitro results in a disruption of SP-D multimers such that trimers become evident. SNO-SP-D but not SP-D, either dodecameric or trimeric, is chemoattractive for macrophages and induces p38 MAPK phosphorylation. The signaling capacity of SNO-SP-D appears to be mediated by binding to calreticulin/CD91. We propose that NO controls the dichotomous nature of this pulmonary collectin and that posttranslational modification by S-nitrosylation causes quaternary structural alterations in SP-D, causing it to switch its inflammatory signaling role. This represents new insight into both the regulation of protein function by S-nitrosylation and NO's role in innate immunity.

Figure 1. SP-D Structure

(A) A model of SP-D structure. (Upper panel) The SP-D monomer (43 kDa) consists of a carbohydrate recognition domain which forms the globular head structure. This domain is connected to the collagen-like helical tail domain by a short, 30–amino acid, neck domain. At the end of the tail domain is the amino terminus in which cysteines 15 and 20 are positioned (shown as yellow projections). (Lower panel) Stylized representation of SP-D multimer assembly (note tail domains are shown shortened for ease of visualization). The head and neck domains drive the aggregation of the SP-D monomer to form a trimer of ∼130 kDa. These trimers associate to form a dodecamer (∼520 kDa). The forces holding this dodecamer together are unclear, although there is a dependency upon the amino-terminal cysteines as mutant lacking these cysteines do not form dodecamers. These dodecamers can assemble to a multimer of greater than 1 MDa. It is unclear whether the dodecamer is an essential intermediate in multimer formation. It should be noted that neither the trimer nor the dodecamer are globular proteins, due to the presence of the long collagen tail and thus under native conditions will behave as molecules with greater molecular radius. (B) Hydropathy plot of SP-D. A Kyle Doolittle hydropathy plot for the SP-D sequence was constructed using a window size of nine residues. A positive value indicates a region of hydrophobicity. The position of cysteines 15 and 20 are marked within the tail domain. As one can see, this is the most hydrophobic portion of the molecule.

Figure 2. Role of Cysteine Residues 15 and 20 in Formation of SP-D Trimers

(A) Recombinant rat SP-D (RrSP-D) or a mutant in which cysteines 15 and 20 have been mutated to serine (Ser15/20) were denatured under reducing (using mercaptoehtanol or dithiothreitol as the reductant) and non-reducing conditions. The resultant proteins were analyzed by SDS-PAGE and Western blotting with SP-D antibody. (B) RrSP-D and Ser15/20 were pre-incubated with NEM-linked to biotin at 37 °C for half an hour either with or without prior incubation with unlinked NEM. Biotin-labeling was determined by Western blotting following SDS-PAGE with anti-biotin antibody.

Figure 3. SNO-SP-D Formation In Vitro and Its Effect on Multimerization

(A) (Top) SNO-SP-D formation in BAL. BAL from normal rats either with or without treatment with L-SNOC (200 μM) was analyzed for SNO-SP-D content by biotin-switch assay. Total SP-D content was also measured by immunoblot. (Middle) Transnitrosation of recombinant SP-D. Control and SNOC treated recombinant SP-D (0.2 μM) were analyzed by biotin-wwitch assay. W/o biotin-HPDP represents the assay performed in the absence of biotin linked [N-(6-biotinamido)hexyl-1′-(2' pyridyldithio) propionamide]. (Bottom) Recombinant SP-D (0.2 μM) was transnitrosated with increasing doses of L-SNOC or exposed to 200 μM authentic NO and then analyzed by biotin-switch assay. (B) SNOC treatment alters the conformational state of SP-D. (Left panel) BAL from SP-D overexpressing mice or 0.2 μM recombinant SP-D were treated with L-SNOC and subjected to native electrophoresis and Western blot for SP-D, revealing disruption of the native multimers to dodecamers and trimers. (Upper right panel) The BAL samples in the left panel were subjected to gel-filtration. Total protein from BAL (0.75 mg) in a volume of 250 μl was resolved onto a Superdex 200 HR 10/30 column (GE Healthcare Bio-Sciences) for size-exclusion chromatography (SEC) analysis. Protein extracts were resolved at flow rate of 0.3 ml/min in 25 mM HEPES, PH 7.25, and 150 mM NaCl using an Agilent 1100 Series HLC system. Fractions (0.5 ml) were collected and concentrated with 5000 NMWL Ultrafree-MC filters (Millipore). The gel filtration column was calibrated using the following mixture of globular proteins standards: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldlase (158 kDa), albumin (67kDa), ovalbumin (43 kDa), chymotrypsin (25 kDa), and ribonuclease A (13.7 kDa). The void volume was determined from the elution migration of blue dextran (2,000 kDa). The fractions were analyzed by SDS-PAGE and Western blot for total SP-D content. The large multimers seen in control BAL are reduced in size upon SNOC treatment. (Lower right panel) Gel filtration samples containing SP-D were analyzed by native electrophoresis and Western blot, revealing that the 720-kDa fraction that arises upon SNOC treatment contains dodecamers and trimers of SP-D.

Figure 4. SNO-SP-D Induces Macrophage Chemotaxis

(A and B) BAL and recombinant SP-D were assessed for their ability to induce RAW 264.7 macrophage migration using a modified Boydin chamber, following treatment with 200 μM SNOC or cysteine. (C) Using a BAL does of 100 μg/ml, cell migration was assayed subsequent to treatment with anti-SP-D or non-immune IgG. (D) SNOC treatment of BAL from SP-D ^–/– mice did not induce macrophage chemotaxis. (asterisk represents significantly different from BAL; pound symbol represents significantly different from control or IgG; carat symbol represents significantly different from SNO SP-D ^+/+; p < 0.05)

Figure 5. Chemotactic Activity of SNO-SP-D Trimers

(A) Recombinant rat SP-D (RrSP-D) or the mutant (Ser15/20) was incubated with 200 μM l-cysteine or l-CysNO for 30 min at room temperature. SNO content was analyzed by Western blotting of non-reduced SDS-PAGE samples following use of the biotin switch assay. (B) Chemotactic activity recombinant and modified SP-Ds. 0.1 μg/ml of the treated SP-Ds in (A) were used to measure chemotaxis in a modified Boyden chamber assay with RAW 264.7 cells. Data are mean ± SEM. (asterisk represents significantly different from RrSP-D; p < 0.05)

Figure 6. Acute Lung Injury in the Rodent Produces Results in SNO-SP-D Formation and Multimer Disruption

(A) Untreated (control), saline, or bleomycin was intratracheally administered to Sprague-Dawley rats. BAL was collected at days 2, 4, 7, 14, and 21 after injection. BAL (upper) was subjected to biotin-switch assay to detect SNO-SP-D. (Lower) Total SP-D in BAL was identified by immunoblot. (B) Untreated (control), saline, or bleomycin was intratracheally injected at a dose of 8 U/kg in Sprague-Dawley rats. BAL was collected at day 4 after injection. BAL (upper) was subjected to electrophoresis for native gel to detect different fragments of SP-D. BAL (middle) was subjected to biotin-switch assay to detect SNO-SP-D. (Lower) Total SP-D in BAL was identified by immunoblot. (C) Untreated (control), saline, or bleomycin was administered to C57/BL6 mice intratracheally at a dose of 3 U/kg. BAL was collected at day 8 after treatment; total SNO content within the BAL was 0.2 ± 0.22 μM in control mice and 2.1 ± 0.88 μM in bleomycin treated. SNO-SP-D content was assessed by the biotin switch method. (D) SNO-SP-D in the BAL of bleomycin-treated wild type versus bleomycin-treated iNOS ^–/– mice (8-d post-injury) was measured by biotin-switch assay; Total input of SP-D was same between groups. (asterisk represents significantly different from wild type; p < 0.05). Representative blots show SNO-SP-D and total SP-D. Data are mean ± SEM.

Figure 7. BAL from Bleomycin-Treated Rats Induces Macrophage Chemotaxis in Part through SNO-SP-D

(A) BAL from bleomycin- and saline-treated rats were analyzed for their ability to induce RAW cell chemotaxis. Bleomycin BAL induced chemotaxis to a greater extent than saline BAL; however, pretreatment with ascorbate to remove SNO abrogated this response. The effect of ascorbate treatment on BAL SNO-SP-D content is shown in the inset where samples were analyzed by biotin-switch. (B) The importance of SP-D in this SNO-mediated increase is demonstrated by effect of SP-D immunoprecipitation. Bleomycin and saline BAL were analyzed for chemotactic effect following pretreatment with anti-SP-D or non-immune IgG. Only anti-SP-D pre-treatement reduced the increase in chemotaxis induced following bleomycin administration. (asterisk represents significantly different from saline-BAL; # represents significantly different from control or IgG; p < 0.05)

Figure 8. SNO-BAL Induces Inflammatory Signaling in RAW Cells via CRT

(A) RAW 264.7 macrophages grown on coverslips were preloaded with fura-2AM and stimulated with BAL (100 μg/ml) or SNO-BAL (100 μg/ml) from SP-D^+/+ mice. Change in [Ca²⁺]_i was record from 50–70 cells. (B) RAW 264.7 macrophages were treated with BAL or SNO-BAL from either SP-D ^+/+ or SP-D ^–/– mice for 5 min. Cell lysates were analyzed for total p38 MAPK and its phosphorylated form (P-p38) by SDS-PAGE and Western blotted with phospho-specific primary antibody. (C) RAW 264.7 cells were pretreated with 2 μg/ml of anti-SIRP-1α, anti-CRT or an isotype control for 20 min prior to addition to the Boyden chamber. Treated cells were analyzed for chemotaxis to BAL (control) or SNO-BAL (SNO). (D) RAW 264.7 cell were pretreated with anti-CRT for 20 min prior to stimulation with BAL or SNO-BAL for an additional 20 min. Cell lysates were then prepared and analyzed for p38 phosphorylation via SDS PAGE and Western blot with phospho-specific primary antibody. (asterisk represents significantly different from control; pound symbol represents significantly different from control, IgG, or anti-SIRP-1α (p < 0.05)

Figure 9. A Model of the Pro- and Anti-Inflammatory Functions of SP-D

Under non-inflammatory conditions, SP-D remains in large multimeric or dodecameric forms in which the tail domains remain buried. The head domains bind to SIRP-1α and activate the kindase SHP-1. SHP-1 activation inhibits p38 activations, potentially resulting in the blockaged of NF-κB action and the inhibition of infammatory function. Under inflammatory conditions the production of NO leads to the formation of SNO-SP-D and the disruption of the multimeric structure. The tail domains now become exposed and bind to calreticulin. This results in p38 phosphorylation via CD91, potentially leading to NF-κB activation and the production of pro-inflammatory mediators. Presumably other actions which result in disruption of SP-D multimeric structure may also be pro-inflammatory. (Image credit: Kirk Moldoff)