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• W2093203710 abstract "The lung is composed of a series of branching conducting airways that terminate in grape-like clusters of delicate gas-exchanging airspaces called pulmonary alveoli. Maintenance of alveolar patency at end expiration requires pulmonary surfactant, a mixture of phospholipids and proteins that coats the epithelial surface and reduces surface tension. The surfactant lining is exposed to the highest ambient oxygen tension of any internal interface and encounters a variety of oxidizing toxicants including ozone and trace metals contained within the 10 kl of air that is respired daily. The pathophysiological consequences of surfactant oxidation in humans and experimental animals include airspace collapse, reduced lung compliance, and impaired gas exchange. We now report that the hydrophilic surfactant proteins A (SP-A) and D (SP-D) directly protect surfactant phospholipids and macrophages from oxidative damage. Both proteins block accumulation of thiobarbituric acid-reactive substances and conjugated dienes during copper-induced oxidation of surfactant lipids or low density lipoprotein particles by a mechanism that does not involve metal chelation or oxidative modification of the proteins. Low density lipoprotein oxidation is instantaneously arrested upon SP-A or SP-D addition, suggesting direct interference with free radical formation or propagation. The antioxidant activity of SP-A maps to the carboxyl-terminal domain of the protein, which, like SP-D, contains a C-type lectin carbohydrate recognition domain. These results indicate that SP-A and SP-D, which are ubiquitous among air breathing organisms, could contribute to the protection of the lung from oxidative stresses due to atmospheric or supplemental oxygen, air pollutants, and lung inflammation. The lung is composed of a series of branching conducting airways that terminate in grape-like clusters of delicate gas-exchanging airspaces called pulmonary alveoli. Maintenance of alveolar patency at end expiration requires pulmonary surfactant, a mixture of phospholipids and proteins that coats the epithelial surface and reduces surface tension. The surfactant lining is exposed to the highest ambient oxygen tension of any internal interface and encounters a variety of oxidizing toxicants including ozone and trace metals contained within the 10 kl of air that is respired daily. The pathophysiological consequences of surfactant oxidation in humans and experimental animals include airspace collapse, reduced lung compliance, and impaired gas exchange. We now report that the hydrophilic surfactant proteins A (SP-A) and D (SP-D) directly protect surfactant phospholipids and macrophages from oxidative damage. Both proteins block accumulation of thiobarbituric acid-reactive substances and conjugated dienes during copper-induced oxidation of surfactant lipids or low density lipoprotein particles by a mechanism that does not involve metal chelation or oxidative modification of the proteins. Low density lipoprotein oxidation is instantaneously arrested upon SP-A or SP-D addition, suggesting direct interference with free radical formation or propagation. The antioxidant activity of SP-A maps to the carboxyl-terminal domain of the protein, which, like SP-D, contains a C-type lectin carbohydrate recognition domain. These results indicate that SP-A and SP-D, which are ubiquitous among air breathing organisms, could contribute to the protection of the lung from oxidative stresses due to atmospheric or supplemental oxygen, air pollutants, and lung inflammation. epithelial lining fluid surfactant protein human SP-A purified from the alveolar lavage of patients with alveolar proteinosis bovine serum albumin mannose-binding protein the first component of complement C-type lectin domain low density lipoprotein tert-butylhydroperoxide, TBARS, thiobarbituric reactive substances recombinant SP-A containing a deletion of the collagen-like region recombinant SP-A containing a deletion of the N-terminal segment and the collagen-like region N187S, recombinant SP-A containing a deletion of the N-terminal segment and the collagen-like region and containing an Asn to Ser substitution at position 187 that prevents carbohydrate attachment 2,2′-azobis(2-amidinopropane)dihydrochloride Air breathing is made possible through the surface tension-lowering properties of lung surfactant, an oily film located at the boundary between the aqueous pulmonary epithelial lining fluid (ELF)1 and air in the lumen of the alveoli, the gas-exchanging units of the lung. By weight, surfactant is composed of 90% phospholipids and 10% protein, including the hydrophilic surfactant proteins A (SP-A) and D (SP-D), and the hydrophobic surfactant proteins B (SP-B) and C (SP-C) (1Goerke J. Biochim. Biophys. Acta. 1998; 1408: 79-89Crossref PubMed Scopus (582) Google Scholar). After secretion into the ELF, the components of surfactant form membranes at the air-liquid interface that spread readily and compress poorly during cyclical respiratory expansion and contraction of the alveolus. These properties of surfactant result in enhanced lung compliance during inspiration, which reduces the work of breathing, and very low alveolar surface tension at end expiration, which helps to maintain airspace patency. Exposure of surfactant to ambient oxygen and potent environmental oxidants such as ozone results in peroxidation of unsaturated phospholipids, surfactant inactivation, airspace collapse, and impaired gas exchange (2Matalon S. Holm B.A. Notter R.H. J. Appl. Physiol. 1987; 62: 756-761Crossref PubMed Scopus (89) Google Scholar). Antioxidant protection of surfactant phospholipids in the ELF has classically been attributed to low molecular mass components urate, ascorbate, and reduced glutathione and to proteinaceous antioxidants superoxide dismutase and catalase (3Macnee W. Rahman I. Am. J. Respir. Crit. Care Med. 1999; 160 (suppl.): 58-65Crossref Scopus (201) Google Scholar). The serum apolipoproteins apoE and apoAIV, which are intimately associated with lipid aggregates including chylomicrons, low density lipoproteins (LDL), and high density lipoproteins, have been reported to prevent lipid peroxidation and to protect against the development of atherosclerosis (4Qin X. Swertfeger D.K. Zheng S. Hui D.Y. Tso P. Am. J. Physiol. 1998; 274: H1836-H1840Crossref PubMed Google Scholar, 5Hayek T. Oiknine J. Brook J.G. Aviram M. Biochem. Cell Biol. 1994; 201: 1567-1574Google Scholar). The objective of this study was to determine if the surfactant-associated proteins contribute to the prevention of lipid oxidation in the airspaces of the lung. Surfactant proteins A and D are the pulmonary members of the collectin family of proteins, which also includes the serum proteins mannose-binding proteins A (MBP-A) and C (MBP-C), bovine conglutinin, and CL-43 (6Lawson P.R. Reid K.B. Immunol. Rev. 2000; 173: 66-78Crossref PubMed Scopus (126) Google Scholar). Like all collectins, SP-A and SP-D have similar basic structural organization including an N-terminal segment with one or two interchain disulfide bonds, a collagen-like domain of Gly-X-Y repeats containing hydroxylated amino acids, an amphipathic helical “neck” region, and a C-terminal, C-type lectin domain (CLD) (7Drickamer K. Curr. Opin. Struct. Biol. 1999; 9: 585-590Crossref PubMed Scopus (525) Google Scholar). Trimerization occurs by triple helix formation in the collagen-like domain and bundled α-helical coiled-coil formation in the neck region. At the level of quarternary structure, however, the pulmonary collectins diverge. Variably glycosylated subunits of SP-A (apparent molecular mass range 26–38 kDa) assemble into a hexamer of trimers that are disulfide-linked at the N terminus and laterally associated through the first portion of the collagen-like domain, forming a flower bouquet-like structure with an estimated mass of 600,000 kDa (8Voss T. Eistetter H. Schafer K.P. Engel J. J. Mol. Biol. 1988; 201: 219-227Crossref PubMed Scopus (204) Google Scholar). For SP-D, four trimers composed of glycosylated 42-kDa subunits form a 500,000-kDa cruciform-shaped oligomer joined by disulfide bonds at the N terminus (9Crouch E. Chang D. Rust K. Persson A. Heuser J. J. Biol. Chem. 1994; 269: 15808-15813Abstract Full Text PDF PubMed Google Scholar). Both proteins bind calcium ions at two or three sites within the CLD (10Haagsman H.P. Sargeant T. Hauschka P. Benson B.J. Hawgood S. Biochemistry. 1990; 29: 8894-8900Crossref PubMed Scopus (84) Google Scholar, 11Hakansson K. Lim N.K. Hoppe H.J. Reid K.B. Struct. Fold Des. 1999; 7: 255-264Abstract Full Text Full Text PDF Scopus (125) Google Scholar). Data from sedimentation, immunohistochemical, and ultrastructural analyses indicate that SP-D resides primarily in the aqueous compartment of the ELF, while SP-A is intimately associated with surfactant lipid membranes and aggregates (12Wright J.R. Physiol. Rev. 1997; 77: 931-962Crossref PubMed Scopus (502) Google Scholar). The unifying functional theme for the collectins is innate host defense, and several laboratories have reported that the pulmonary collectins aggregate and opsonize diverse microbial species and enhance the clearance of microorganisms from the lungs of mice (13Crouch E.C. Am. J. Respir. Cell Mol. Biol. 1998; 19: 177-201Crossref PubMed Scopus (322) Google Scholar). However, the pulmonary collectins also perform specialized roles in the structure and function of pulmonary surfactant. Experimental data suggest that SP-A protects the surface activity of surfactant from serum protein inhibitors (14Cockshutt A.M. Weitz J. Possmayer F. Biochemistry. 1990; 29: 8424-8429Crossref PubMed Scopus (251) Google Scholar), contributes to stability of surfactant aggregates such as tubular myelin (15Suzuki Y. Fujita Y. Kogishi K. Am. Rev. Respir. Dis. 1989; 140: 75-81Crossref PubMed Scopus (274) Google Scholar), and inhibits the activity of some phospholipases A2 (16Fisher A.B. Dodia C. Chander A. Am. J. Physiol. 1994; 267: L335-L341PubMed Google Scholar, 17Touqui L. Arbibe L. Mol. Med. Today. 1999; 5: 244-249Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), while SP-D appears to be required for the maintenance of surfactant homeostasis and lung structure (18Wert S.E. Yoshida M. LeVine A.M. Ikegami M. Jones T. Ross G.F. Fisher J.H. Korfhagen T.R. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5972-5977Crossref PubMed Scopus (360) Google Scholar, 19Botas C. Poulain F. Akiyama J. Brown C. Allen L. Goerke J. Clements J. Carlson E. Gillespie A.M. Epstein C. Hawgood S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11869-11874Crossref PubMed Scopus (348) Google Scholar). Almost all reported SP-A and SP-D ligand binding properties require calcium, but neither protein has been reported to have enzymatic activity or to be associated with metals other than calcium. Here we demonstrate that SP-A and SP-D also have potent, direct phospholipid and cellular antioxidant properties. Substrates for lipid oxidation included mixtures of natural and synthetic glycerophospholipids that are found in pulmonary surfactant and human LDL. The model surfactant lipids, composed of egg phosphatidylcholine, dipalmitoylphosphatidylcholine, cholesterol, and 1-oleoyl-2-linoleoyl-sn-glycero-3-phosphocholine (1:1:0.15:0.15, w/w/w/w, respectively) (Avanti Polar Lipids) were mixed in chloroform and dried to a film under nitrogen. Following resuspension in phosphate-buffered saline or 0.15 m NaCl, multilamellar vesicles were generated by vigorous vortexing for 5 min. LDL were isolated from the plasma of normal blood donors by density gradient ultracentrifugation (density = 1.019–1.063 g/ml), as described previously (4Qin X. Swertfeger D.K. Zheng S. Hui D.Y. Tso P. Am. J. Physiol. 1998; 274: H1836-H1840Crossref PubMed Google Scholar) and stored in saline-EDTA. Prior to use, EDTA was removed by dialysis to equilibrium against at least 2000 volumes of saline, and the concentration of LDL in mg of LDL-associated protein was determined by BCA assay (Pierce). Native SP-A and SP-D were isolated from the alveolar wash of rats that had been pretreated with intratracheal silica to enhance the collectin yield (20Dethloff L.A. Gilmore L.B. Brody A.R. Hook G.E.R. Biochem. J. 1986; 233: 111-118Crossref PubMed Scopus (77) Google Scholar). After overnight centrifugation at 40,000 × g, rat SP-D was purified from the supernatant by maltose-Sepharose affinity chromatography, and rat SP-A was purified from the pellet by NaBr flotation, butanol extraction, and mannose-Sepharose affinity chromatography (21McCormack F.X. Pattanajitvilai S. Stewart J.J. Possmayer F. Inchley K. Voelker D.R. J. Biol. Chem. 1997; 272: 27971-27979Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Wild-type and mutant recombinant rat SP-A, SP-D, and MBP were synthesized using baculovirus vectors and purified by carbohydrate-Sepharose affinity chromatography as described previously (21McCormack F.X. Pattanajitvilai S. Stewart J.J. Possmayer F. Inchley K. Voelker D.R. J. Biol. Chem. 1997; 272: 27971-27979Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 22McCormack F.X. Stewart J.J. Voelker D.R. Damodarasamy M.D. Biochemistry. 1997; 36: 13963-13971Crossref PubMed Scopus (38) Google Scholar, 23McCormack F.X. Damodarasamy M. Elhalwagi B.M. J. Biol. Chem. 1999; 274: 3173-3183Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Mouse SP-D purified from granulocyte-macrophage colony-stimulating factor/SP-A null mice by maltose-Sepharose affinity chromatography was a gift from J. Whitsett (Children's Hospital, Cincinnati, OH). Human SP-A purified from the alveolar lavage of patients with alveolar proteinosis (AP-SP-A) was isolated by repeated washings of the surfactant pellet with buffer containing 0.9% NaCl and 2 mmCa2+, followed by elution of SP-A with 4 mmEDTA, dialysis, and Ca2+-dependent adsorption to a mannose-Sepharose column (modified from Ref. 24Suwabe A. Mason R.J. Voelker D.R. Arch. Biochem. Biophys. 1996; 327: 285-291Crossref PubMed Scopus (49) Google Scholar). AP-SP-A was examined for the presence of lipid soluble antioxidants by isocratic high pressure liquid chromatography (Genox Corp.). The lower detection limits included the following: lutein, 20 ng/ml; zeaxanthin, 10 ng/ml; cryptoxanthin, 10 ng/ml; α-carotene, 10 ng/ml; β-carotene, 10 ng/ml; retinol, 60 ng/ml; retinyl-palmitate, 10 ng/ml; α-tocopherol, 1 μg/ml; δ-tocopherol, 10 ng/ml; γ-tocopherol, 250 ng/ml. The purity of all proteins was verified to be >95% by 8–16% SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Brilliant Blue. To ensure that the collectin reagents used were free of diffusable contaminants including EDTA, all proteins were dialyzed to equilibrium against at least 400 volumes of buffer (5 mm Tris for SP-A and AP-SP-A and 10 mm Tris, 150 mm NaCl for SP-D and MBP), with at least three buffer exchanges. For some experiments, rat SP-A and rat SP-D were alkylated by incubation with 0.5 m iodoacetamide at 37 °C in the dark for 1 h and then extensively dialyzed. To examine the role of metal ion chelation in the collectin antioxidant function, two methods were used to presaturate the SP-A and SP-D with copper sulfate. The collectins were concentrated and incubated with 66–70 μmcopper sulfate (CuSO4) for 1 h at 4 °C or equilibrium-dialyzed overnight against more than 10,000 volumes of a 20 μm copper sulfate, 5 mm Tris solution at 4 °C. The pretreated proteins were then added directly to solutions containing lipid substrates and 10 μm CuSO4as outlined below. Stock solutions of 10 mm CuSO4 were freshly prepared daily. Reaction mixtures composed of 1 mg/ml surfactant lipids or 150 μg/ml LDL, 10 μm CuSO4, and putative antioxidant proteins or controls were prepared in phosphate-buffered saline or 0.9% saline. The mixtures were incubated at 37 °C in a shaking water bath for 4 h for LDL and 24 h for surfactant lipids (25Gelvan D. Saltman P. Biochim. Biophys. Acta. 1990; 1035: 353-360Crossref PubMed Scopus (63) Google Scholar). Control reactions that included LDL only, copper only, or protein controls of bovine serum albumin (BSA), rat IgG, recombinant MBP, rat serum, or human complement C1q were also performed. In some experiments, alternative oxidant stimuli were used. Ferric chloride (FeCl3) (50 μm) or ferric pyrophosphate (FePPi) (1 μm) plus ascorbic acid (0.25 mm) was incubated with the model surfactant lipids for 24 h in 0.9% saline. The free radical generator, 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) (Aldrich) was incubated with surfactant lipids or LDL for 0.5–3 h. In all cases, thiobarbituric acid-reactive substances (TBARS) were measured using a method adapted from Gelvan and Saltman (25Gelvan D. Saltman P. Biochim. Biophys. Acta. 1990; 1035: 353-360Crossref PubMed Scopus (63) Google Scholar). Samples and 0–10 μm 1,1,3,3-methylmalondialdehyde standards were developed by the addition of a solution composed of 0.375% thiobarbituric acid, 15% trichloroacetic acid, and 0.25 n HCl at a volume ratio of 1:2 of sample/developer. Following incubation at 95 °C for 30 min and centrifugation at 14,000 rpm for 15 min, an aliquot was read in a spectrophotometer using a 540-nm filter. An absorption scan (500–570 nm) of both the malondialdehyde-TBA adducts and the lipid-aldehyde TBA adducts indicated that the absorbance at 540 nm was representative of the peak obtained at the absorption maximum at 532 nm (not shown). For continuous assessment of lipid oxidation, the accumulation of conjugated dienes during LDL oxidation was monitored spectrophotometrically (26Esterbauer H. Gebicki J. Puhl H. Jurgens G. Free Radical Med. Biol. 1992; 13: 341-390Crossref PubMed Scopus (2136) Google Scholar). Mixtures of 50 μg/ml LDL, 10 μm copper, and various amounts of surfactant proteins or control proteins were placed in quartz cuvettes and allowed to oxidize at room temperature over 335 min. Conjugated diene formation was assessed by measuring absorbance at a wavelength of 234 nm in a spectrophotometer. Proteins were analyzed for oxidative modification of amino acid side groups that occur during lipid oxidation (27Levine R.L. Garland D. Oliver C.N. Amici A. Climent I. Lenz A.G. Ahn B.W. Shaltiel S. Stadtman E.R. Methods Enzymol. 1990; 186: 464-478Crossref PubMed Scopus (4920) Google Scholar). Briefly, carbonyl-containing protein adducts in reaction mixtures of 150 μg/ml LDL, 10 μm CuSO4, native or iodoacetamide-treated SP-A or SP-D, and BSA were derivatized to 2,4-dinitrophenylhydrazones by reaction with 2,4-dinitrophenylhydrazine (Oxyblot; Intergen). After size fractionation by 8–16% SDS-polyacrylamide gel electrophoresis under reducing conditions, protein species were electrophorectically transferred to nitrocellulose membranes. The membrane was sequentially incubated with a rabbit anti-DNP IgG and a horseradish peroxidase-conjugated goat anti-rabbit IgG. Blots were developed by horseradish peroxidase-dependent oxidation of a chemiluminescent substrate and visualized using autoradiography. A murine macrophage cell line (RAW 264.7) was adhered to 24-well plates (2 × 104cells/well) in Ham's F-12 medium containing 10% FBS overnight at 37 °C in a 10% CO2 atmosphere. After washing, the cells were incubated with 40 μm tert-butylhydroperoxide (t-BOOH) in serum-free Ham's F-12 for 24 h in the presence of various concentrations of the surfactant proteins or BSA. Viability was assessed by exclusion of the vital dye, trypan blue. The ability of SP-A and SP-D to inhibit the copper-induced oxidation of a mixture of saturated and unsaturated lipids found in surfactant was assessed by measuring the accumulation of TBARS during a 24-h incubation at 37 °C (Fig. 1). Native SP-A isolated from rat lungs inhibited oxidation of surfactant lipids in a dose-dependent fashion that was half-maximal at a concentration of 5.1 μg/ml (IC50 = 8.4 nm, assuming a mass of 600 kDa) and complete at 10.0 μg/ml (Fig. 1 a). Native SP-D also inhibited copper-induced surfactant lipid oxidation in a dose-dependent manner with maximal protection observed at doses equal to or greater than 0.5 μg/ml (Fig. 1 a). The IC50 for protection by SP-D was 0.1 μg/ml (IC50 = 0.2 nm, assuming a mass of 500 kDa), or approximately 35-fold lower than SP-A, and 100-fold lower than the antioxidant serum lipoprotein apoAIV (IC50≈ 50 nm) (4Qin X. Swertfeger D.K. Zheng S. Hui D.Y. Tso P. Am. J. Physiol. 1998; 274: H1836-H1840Crossref PubMed Google Scholar). The inhibitory concentrations for both lung proteins were well below their physiologic ELF levels, estimated to be 300–1800 μg/ml for SP-A and 36–216 μg/ml for SP-D (12Wright J.R. Physiol. Rev. 1997; 77: 931-962Crossref PubMed Scopus (502) Google Scholar). Alkylation of SP-A with iodoacetamide completely blocked the antioxidant effects of the protein, even when the modified protein was added at concentrations that were 5-fold greater than the inhibitory level (Fig. 1 b). There was no protection from oxidation by control proteins albumin, rat serum, or the structurally similar molecule, C1q, at concentrations of 50 μg/ml (Fig. 1 b). However, BSA exhibited dose-dependent inhibition at 100, 200, and 500 μg/ml, and rat serum partially inhibited oxidation at 500 μg/ml. A BSA concentration of 200 μg/ml or rat serum concentration of 500 μg/ml was required to achieve the same level of inhibition of oxidation that occurred with about 5.0 μg/ml SP-A or 0.1–0.2 μg/ml SP-D. These data indicate that the two hydrophilic surfactant proteins, SP-A and SP-D, protect surfactant lipids from copper-induced oxidation in vitro, at physiologically relevant concentrations. Both SP-A and SP-D exhibited very similar antioxidant activity when LDL particles, which include unsaturated phospholipids, triglycerides, cholesterol, and cholesterol esters, were used as the substrates for lipid oxidation (Fig. 2). The IC50 values for inhibition of copper-induced oxidation of LDL for SP-A and SP-D were 4.8 μg/ml (7.9 nm) and 0.1 μg/ml (0.3 nm), respectively, and complete inhibition of oxidation occurred at 10 and 1 μg/ml, respectively. In contrast, 20 μg/ml of the highly homologous collectin, recombinant rat mannose-binding protein A (rMBP) did not inhibit LDL oxidation (Fig. 2 b). Rat serum, albumin, C1q, and IgG had also no effect on LDL lipid oxidation at concentrations of 50 μg/ml (Fig. 2 b). Only approximately 2.5 μg/ml SP-A or 0.05 μg/ml SP-D was required to provide the same level of antioxidant protection as 500 μg/ml of rat serum. In the absence of oxidation inhibitors, the absolute TBARS level following a 4-h incubation with 10 μm copper was over 3 times greater for LDL than for surfactant lipids. For this reason, LDL was used as the lipid substrate for kinetic experiments and mutagenesis studies of the collectin antioxidant activities. The temporal relationship between the addition of SP-A or SP-D and the inhibition of LDL oxidation was determined spectrophotometrically by continuously monitoring conjugated diene accumulation associated with exposure to copper (26Esterbauer H. Gebicki J. Puhl H. Jurgens G. Free Radical Med. Biol. 1992; 13: 341-390Crossref PubMed Scopus (2136) Google Scholar). This assay is based on the oxidation-dependent rearrangement of 1,4-pentadienyl double bonds of LDL lipids to 1,3-butadienyl double bonds, which absorb in the ultraviolet range. In the presence of 10 μm copper at room temperature, LDL particles resist oxidation for up to 100 min as endogenous antioxidants such as α-tocopherol are consumed (Fig. 3). At that point, the rate of oxidation increases in proportion to the concentration of initiating radicals, reaching a plateau when all unsaturated fatty acids are consumed. When SP-A (Fig. 3 a) or SP-D (Fig. 3 b) proteins were included at zero time, they blocked the accumulation of conjugated dienes in a dose-dependent manner. With increasing surfactant protein concentrations, the predominant change was a decrease in the slope during the rapid oxidation phase, consistent with inhibition of free radical chain initiation or with free radical chain termination. The concentrations of SP-A and SP-D that prevented conjugated diene formation were very similar to those that were required to block TBARS formation. When fully suppressive concentrations of SP-A (10 μg/ml) (Fig. 3 c) or SP-D (1 μg/ml) (Fig. 3 d) were added at various time points during the propagation phase of oxidation, conjugated diene formation was completely arrested at the point of addition. The kinetics of oxidation inhibition by the collectins were distinctly different from those of 2 mm EDTA, which blocked conjugated diene accumulation only after a significant lag period (Fig. 3 d, inset). These data indicate that SP-A and SP-D directly interfere with lipid oxidation. Exposure of cultured mammalian cells to t-BOOH promotes a variety of toxic events including depletion of glutathione, mitochondrial dysfunction, and peroxidation of membrane lipids (28Sestili P. Brambilla L. Cantoni O. FEBS Lett. 1999; 457: 139-143Crossref PubMed Scopus (13) Google Scholar). To determine if SP-A and SP-D protect cells from oxidative stress, RAW 264.7 murine macrophages were exposed to 40 μm t-BOOH for 24 h in the presence of SP-A or SP-D at concentrations from 0.01 to 50 μg/ml (Fig. 4). Cell death was assessed by staining cells with the vital dye, trypan blue. We found that both SP-A and SP-D protected RAW cells from t-BOOH-induced death in a dose-dependent fashion that was half-maximal at concentrations of 0.52 μg/ml for SP-A and 0.56 μg/ml for SP-D and that reached a plateau at a concentration of approximately 1 μg/ml for both proteins. These data indicate that both SP-A and SP-D protect macrophages from oxidant stress. To determine the domain(s) of SP-A that are responsible for protection from copper-induced oxidation of LDL and synthetic lipids, we tested the activity of mutant recombinant SP-As containing deletions in N-terminal domains in the TBARS assay (Fig. 5). Wild type recombinant SP-A inhibited copper-induced oxidation of LDL to the half-maximal point at 2.1 μg/ml and to basal levels at 5.0 μg/ml. A mutant SP-A containing a deletion of the collagen-like region (ΔG8–P80) (21McCormack F.X. Pattanajitvilai S. Stewart J.J. Possmayer F. Inchley K. Voelker D.R. J. Biol. Chem. 1997; 272: 27971-27979Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) but retaining the N-terminal segment and interchain disulfide bonds was nearly as active as the wild type recombinant protein (IC50 = 2.8 μg/ml). A non-disulfide-cross-linked trimeric construct composed solely of the neck and CLD region of the protein (ΔN1–P80) (23McCormack F.X. Damodarasamy M. Elhalwagi B.M. J. Biol. Chem. 1999; 274: 3173-3183Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) was also a potent antioxidant, with an IC50 of 6.7 μg/ml. Although carbohydrates are known to have antioxidant properties, the protection of lipids by SP-A was not attributable to the oligosaccharide attached to Asn187, since a nonglycosylated neck and CLD construct containing an N187S mutation also inhibited both LDL and surfactant lipid oxidation (ΔN1-P80,N187S) (Figs. 1 b and 2 b). We conclude that the antioxidant activity of SP-A is localized to the polypeptide sequences in the C-terminal (neck + CLD) domains of the protein. Several experiments were performed to evaluate the role of collectin-mediated copper chelation in the prevention of lipid oxidation (Figs. 6 and7). First, the ability of SP-A to inhibit the oxidation of surfactant lipids in the presence of 2 mmCa2+ was assessed. Saturation of SP-A's known metal binding site in this manner had no effect on the antioxidant properties of the protein (Fig. 6). Second, SP-A retained activity and was equally efficacious in the oxidant assay whether extensively dialyzed against either 5 mm Tris or 20 μm copper sulfate (Fig. 6). Third, presaturation of SP-A and SP-D with 66–70 μm for 1 h before the addition to the reaction mixtures containing 10.0 μm copper and model surfactant lipids also had no effect on the antioxidant activity of either protein (Fig. 6). Fourth, SP-A inhibited lipid peroxidation by alternative oxidants, including ferric chloride and ferric pyrophosphate plus ascorbic acid (Fig. 7). However, SP-A did not inhibit LDL oxidation induced by the soluble free radical generator, AAPH, at 1, 5, 10, or 25 mm concentrations (not shown). Collectively, these data indicate that the lung collectins protect lipids from oxidation by a mechanism that is neither specific to copper or dependent on protein-mediated chelation of copper.Figure 7Inhibition of iron-induced oxidation of model surfactant lipids by SP-A. Model surfactant lipids were incubated with ferric chloride or ferric pyrophosphate plus ascorbic acid in the presence of the indicated concentrations of SP-A. Lipid peroxidation was quantified by TBARS assay and expressed as a fraction of lipid oxidation in the absence of surfactant proteins. Data are means ± S.E., n = 3–4.View Large Image Figure ViewerDownload (PPT) To determine if the surfactant proteins act as sinks for reactive lipid intermediates, we assessed covalent modification of SP-A and SP-D during LDL oxidation using a Western analysis technique that detects carbonyl adducts (27Levine R.L. Garland D. Oliver C.N. Amici A. Climent I. Lenz A.G. Ahn B.W. Shaltiel S. Stadtman E.R. Methods Enzymol. 1990; 186: 464-478Crossref PubMed Scopus (4920) Google Scholar) (Fig. 8). Oxidation of LDL produced a dense high molecular weight band that corresponded to B-100, the 514-kDa protein component of LDL, and several smaller bands. Incubation with 10 μg/ml SP-A or 1 μg/ml of SP-D blocked LDL oxidation without the appearance of oxidized protein species at the expected molecular masses for SP-A or SP-" @default.
• W2093203710 created "2016-06-24" @default.
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• W2093203710 date "2000-12-01" @default.
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• W2093203710 title "Pulmonary Surfactant Proteins A and D Are Potent Endogenous Inhibitors of Lipid Peroxidation and Oxidative Cellular Injury" @default.
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