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Degradation of Pulmonary Surfactant Protein D by Pseudomonas aeruginosa Elastase Abrogates Innate Immune Function

2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês

10.1074/jbc.m400796200

1083-351X

John F. Alcorn, Jo Rae Wright,

S100 Proteins and Annexins

The alveolar epithelium is lined by surfactant, a lipoprotein complex that both reduces surface tension and mediates several innate immune functions including bacterial aggregation, alteration of alveolar macrophage function, and regulation of bacterial clearance. Surfactant protein-D (SP-D) participates in several of these immune functions, and specifically it enhances the clearance of the pulmonary pathogen Pseudomonas aeruginosa, a common cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa secretes a variety of virulence factors including elastase, a zinc-metalloprotease, which degrades both SP-A and SP-D. Here we show that SP-D is cleaved by elastase to produce a stable 35-kDa fragment in a time-, temperature-, and dose-dependent manner. Degradation is inhibited by divalent metal cations, a metal chelator, and the elastase inhibitor, phosphoramidon. Sequencing the SP-D degradation products localized the major cleavage sites to the C-terminal lectin domain. The SP-D fragment fails to bind or aggregate bacteria that are aggregated by intact SP-D. SP-D fragment is observed when normal rat bronchoalveolar lavage (BAL) is treated with Pseudomonas aeruginosa elastase, and SP-D fragments are present in the BAL of CF lung allograft patients. These data show that degradation of SP-D occurs in the BAL environment and that degradation eliminates many normal immune functions of SP-D. The alveolar epithelium is lined by surfactant, a lipoprotein complex that both reduces surface tension and mediates several innate immune functions including bacterial aggregation, alteration of alveolar macrophage function, and regulation of bacterial clearance. Surfactant protein-D (SP-D) participates in several of these immune functions, and specifically it enhances the clearance of the pulmonary pathogen Pseudomonas aeruginosa, a common cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa secretes a variety of virulence factors including elastase, a zinc-metalloprotease, which degrades both SP-A and SP-D. Here we show that SP-D is cleaved by elastase to produce a stable 35-kDa fragment in a time-, temperature-, and dose-dependent manner. Degradation is inhibited by divalent metal cations, a metal chelator, and the elastase inhibitor, phosphoramidon. Sequencing the SP-D degradation products localized the major cleavage sites to the C-terminal lectin domain. The SP-D fragment fails to bind or aggregate bacteria that are aggregated by intact SP-D. SP-D fragment is observed when normal rat bronchoalveolar lavage (BAL) is treated with Pseudomonas aeruginosa elastase, and SP-D fragments are present in the BAL of CF lung allograft patients. These data show that degradation of SP-D occurs in the BAL environment and that degradation eliminates many normal immune functions of SP-D. Surfactant protein D (SP-D) 1The abbreviations used are: SP, surfactant protein; CF, cystic fibrosis; BAL, bronchoalveolar lavage; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; cfu, colony-forming units; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorting; CRD, carbohydrate recognition domain. is one of the four surfactant proteins that is synthesized by alveolar type II epithelial cells as a component of the lipoprotein complex known as pulmonary surfactant. Although SP-D has not been shown to participate in the surface tension-reducing properties of surfactant, it has been demonstrated to participate in the host defense functions of surfactant (1Crouch E. Wright J.R. Annu. Rev. Physiol. 2001; 63: 521-554Crossref PubMed Scopus (559) Google Scholar). SP-D as well as SP-A belong to the collectin family of proteins, named for their N-terminal collagen region and C-terminal lectin domain. Intact SP-D consists of four trimers, which interact in their N-terminal region to form a cruciform structure (2Crouch E. Persson A. Heuser J. J. Biol. Chem. 1994; 269: 17311-17319Abstract Full Text PDF PubMed Google Scholar). The collectins are pattern recognition molecules that bind, in a calcium-dependent manner, to non-self oligosaccharides presented on the surface of many bacteria and viruses. SP-D binds to several pathogens and in many cases enhances their phagocytosis by innate immune cells. For example, SP-D binds to Gram-negative bacteria such as Pseudomonas aeruginosa, Klebsiella pneumoniae, rough strains of Escherichia coli, and Salmonella minnesota (3Lim B.L. Wang J.Y. Holmskov U. Hoppe H.J. Reid K.B. Biochem. Biophys. Res. Commun. 1994; 202: 1674-1680Crossref PubMed Scopus (100) Google Scholar). Additionally, SP-D has been shown to bind other pathogens including influenza virus, respiratory syncytial virus, and Pneumocystis carinii (4Hartshorn K.L. Crouch E.C. White M.R. Eggleton P. Tauber A.I. Chang D. Sastry K. J. Clin. Invest. 1994; 94: 311-319Crossref PubMed Scopus (284) Google Scholar, 5Hickling T.P. Bright H. Wing K. Gower D. Martin S.L. Sim R.B. Malhotra R. Eur. J. Immunol. 1999; 29: 3478-3484Crossref PubMed Scopus (109) Google Scholar, 6O'Riordan D.M. Standing J.E. Kwon K.Y. Chang D. Crouch E.C. Limper A.H. J. Clin. Invest. 1995; 95: 2699-2710Crossref PubMed Scopus (148) Google Scholar). Binding is mediated by interaction of the SP-D lectin domain with core oligosaccharides of lipopolysaccharide on Gram-negative bacteria or with surface glycoproteins on fungi and viruses. Binding of SP-D often results in organism aggregation, although SP-D does not aggregate P. aeruginosa (7Restrepo C.I. Dong Q. Savov J. Mariencheck W.I. Wright J.R. Am. J. Respir. Cell Mol. Biol. 1999; 21: 576-585Crossref PubMed Scopus (140) Google Scholar). SP-D increases uptake of P. aeruginosa and a variety of other pathogens by alveolar macrophages in vitro (7Restrepo C.I. Dong Q. Savov J. Mariencheck W.I. Wright J.R. Am. J. Respir. Cell Mol. Biol. 1999; 21: 576-585Crossref PubMed Scopus (140) Google Scholar). Furthermore, infection of SP-D null mice with either Group B Streptococcus or Haemophilus influenzae results in decreased bacterial uptake by alveolar macrophages versus wild-type controls (8LeVine A.M. Whitsett J.A. Gwozdz J.A. Richardson T.R. Fisher J.H. Burhans M.S. Korfhagen T.R. J. Immunol. 2000; 165: 3934-3940Crossref PubMed Scopus (317) Google Scholar). Furthermore, SP-D null mice fail to clear influenza virus as effectively as controls (9LeVine A.M. Whitsett J.A. Hartshorn K.L. Crouch E.C. Korfhagen T.R. J. Immunol. 2001; 167: 5868-5873Crossref PubMed Scopus (241) Google Scholar). Pseudomonas aeruginosa is a Gram-negative bacterium that is the predominant cause of morbidity and mortality in patients with cystic fibrosis (CF) (10Lyczak J.B. Cannon C.L. Pier G.B. Microb. Infect. 2000; 2: 1051-1060Crossref PubMed Scopus (995) Google Scholar). P. aeruginosa infections are characterized by a mucoid biofilm consisting mainly of secreted oligosaccharides. The lipopolysaccharide expressed by mucoid P. aeruginosa is of the rough phenotype and is less cytotoxic than that of other Gram-negative bacteria (11Liu P.V. J. Infect. Dis. 1974; 130: S94-S99Crossref PubMed Scopus (197) Google Scholar). Virulence is induced by P. aeruginosa via secretion of several enzymes, the most cytotoxic of which is exotoxin A, which directly inhibits protein synthesis (12Iglewski B.H. Kabat D. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2284-2288Crossref PubMed Scopus (371) Google Scholar). In addition to this toxin, P. aeruginosa secretes several proteases including elastase (Las B), protease IV, Las A protease, and alkaline protease (13Lazdunski A. Guzzo J. Filloux A. Bally M. Murgier M. Biochimie (Paris). 1990; 72: 147-156Crossref PubMed Scopus (43) Google Scholar, 14Engel L.S. Hill J.M. Caballero A.R. Green L.C. O'Callaghan R.J. J. Biol. Chem. 1998; 273: 16792-16797Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Surfactant protein composition is altered in cystic fibrosis patients. Several reports have shown that whereas phospholipids levels are largely unchanged, the levels of both intact SP-A and SP-D are decreased (15Postle A.D. Mander A. Reid K.B. Wang J.Y. Wright S.M. Moustaki M. Warner J.O. Am. J. Respir. Cell Mol. Biol. 1999; 20: 90-98Crossref PubMed Scopus (206) Google Scholar, 16Meyer K.C. Sharma A. Brown R. Weatherly M. Moya F.R. Lewandoski J. Zimmerman J.J. Chest. 2000; 118: 164-174Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 17Griese M. Birrer P. Demirsoy A. Eur. Respir. J. 1997; 10: 1983-1988Crossref PubMed Scopus (121) Google Scholar). Surfactant protein levels have also been shown to be altered in a rat model of P. aeruginosa infection, although SP-D levels were not measured (18Vanderzwan J. McCraig L. Mehta S. Joseph M. Whitsett J. McCormack D.G. Lewis J.F. Eur. Respir. J. 1998; 12: 1388-1396Crossref PubMed Scopus (27) Google Scholar). We have previously shown P. aeruginosa elastase degrades both SP-A and SP-D and that SP-A degradation fragments are present in the bronchoalveolar lavage (BAL) of cystic fibrosis patients after lung transplant (19Mariencheck W.I. Alcorn J.F. Palmer S.M. Wright J.R. Am. J. Respir. Cell Mol. Biol. 2003; 28: 528-537Crossref PubMed Scopus (123) Google Scholar). Detectable levels of P. aeruginosa elastase have been found in the BAL and serum of CF patients (20Doring G. Obernesser H.J. Botzenhart K. Flehmig B. Hoiby N. Hofmann A. J. Infect. Dis. 1983; 147: 744-750Crossref PubMed Scopus (68) Google Scholar). Furthermore, histologic studies have detected abnormal elastin fibers in lung alveoli of CF patients on autopsy (21Bruce M.C. Poncz L. Klinger J.D. Stern R.C. Tomashefski J.F. Dearborn D.G. Am. Rev. Respir. Dis. 1985; 132: 529-535PubMed Google Scholar), and P. aeruginosa elastase activity was detectable in sputum samples. These data suggest that SP-D is probably cleaved by elastase in vivo during the progression of CF. We propose that P. aeruginosa elastase cleaves SP-D to a stable 35-kDa fragment, which has impaired immune regulatory function. To test this hypothesis, we purified both P. aeruginosa elastase by ion exchange chromatography and its 35-kDa SP-D cleavage product by gel filtration chromatography. The cleavage site was mapped by Edman degradation. Purified SP-D fragment failed to aggregate, bind, or enhance the phagocytosis of several Gram-negative bacteria including P. aeruginosa, E. coli, and Salmonella typhimurium. These studies demonstrate that the stable, 35-kDa fragment of SP-D, produced by degradation by P. aeruginosa elastase, has altered ability to regulate innate immunity. Animals and Reagents—All experiments utilizing primary culture cells were conducted with pathogen-free male Sprague-Dawley rats (Taconic, Germantown, NY) ranging from 2 to 4 months of age. P. aeruginosa isolated from the sputum of CF patients was obtained from the Clinical Microbiology Laboratories at Duke University (S470); PA01 was kindly provided by Dr. Paul Phibbs (Eastern Carolina University, Greenville, NC); PA01 and PA01-B1 isolates were obtained from Dr. Barbara Iglewski (University of Rochester, Rochester, NY). S. typhimurium was obtained from Dr. Ken Sanderson (University of Calgary, Calgary, Alberta). E. coli HB101 was obtained from Dr. Clifford Harding (Case Western Reserve University, Cleveland, OH). All chemicals were purchased from Sigma except where noted. Purification of P. aeruginosa Elastase—Proteases secreted by P. aeruginosa were isolated following the protocol of Coin et al. (22Coin D. Louis D. Bernillon J. Guinand M. Wallach J. FEMS Immunol. Med. Microbiol. 1997; 18: 175-184PubMed Google Scholar). A mucoid clinical isolate (S470) of P. aeruginosa that was found to degrade SP-A was grown in a 2-liter culture in nutrient broth (Difco) for 18 h at 37 °C with shaking at 250 rpm. Alginate was precipitated from the cell-free culture supernatant with 0.25 m CaCl2 for 2 h at 4 °C and removed by centrifugation. The supernatant was then concentrated by ultrafiltration with tangential flow using the Minitan system (Millipore Corp., Marlborough, MA) with a 10-kDa cut-off membrane to a volume of 150 ml. The culture was then applied to a DEAE-Sepharose CL-6B column (1.6 × 50 cm) in 30 mm Tris-HCl, pH 8.3, and bound proteins were eluted with a continuous linear gradient of NaCl (0–0.5 m) in the same buffer. Pooled fractions were then analyzed by Western blot with a polyclonal anti-elastase antibody. The polyclonal rabbit anti-elastase antibody was kindly provided by Dr. Efrat Kessler (Sheba Medical Center, Tel Aviv University, Tel Aviv, Israel). Pooled fractions containing elastase were then analyzed for purity by Coomassie stain and silver stain (data not shown). The elastase preparations contained only 1 band (38 kDa) by Coomassie staining and three bands (65, 38, and 25 kDa) by silver stain; however, the predominant band was the 38-kDa elastase as determined by densitometry. Purification of Rat Recombinant SP-D—Recombinant rat SP-D was purified by maltose affinity chromatography from the media of a stably transfected Chinese hamster ovary cell line (23Dong Q. Wright J.R. Am. J. Physiol. 1998; 18: L97-L105Google Scholar). Briefly, Chinese hamster ovary cells were grown in serum-free medium for 10–12 days, and the culture supernatant was collected and dialyzed to remove glucose against maltose loading buffer containing 50 mm Tris, 150 mm NaCl, and 5 mm CaCl2 pH 7.8. The dialyzed supernatant was then incubated with maltose-Sepharose beads overnight at 4 °C. The maltose-Sepharose beads were then poured into a column and washed with maltose loading buffer to remove unbound proteins. SP-D was then eluted from the column with maltose elution buffer, 50 mm Tris, 150 mm NaCl, and 2 mm EDTA, pH 7.8, and stored at 4 °C. Alveolar Macrophage Isolation—Alveolar macrophages were obtained by BAL of pathogen-free rats. Rats were killed by intraperitoneal injection of Nembutal (Abbott) followed by exsanguination; the trachea was cannulated, and the lungs were isolated and removed from the rats. The lungs were then lavaged six times to total lung capacity with phosphate-buffered saline containing 0.2 mm EGTA previously warmed to 37 °C. Cells were then collected by centrifugation at 230 × g for 10 min. The cell pellet was then resuspended in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin, bovine serum albumin, low endotoxin (Sigma). Average cell yields were ∼6–8 × 106 cells/rat, and greater than 95% of the cells were macrophages. Degradation of Surfactant Proteins with P. aeruginosa Elastase—Rat recombinant SP-D in 50 mm Tris-buffered saline (TBS) plus 2 mm EDTA was incubated with purified elastase in 30 mm TBS at varying concentrations, times, and temperatures as noted in the figure legends. Volumes used in these assays resulted in a 4–15-fold final dilution of the EDTA containing SP-D buffer. The resulting products were resolved by 15% SDS-PAGE gel and Western blot using a well characterized anti-SP-D antibody (24McIntosh J.C. Swyers A.H. Fisher J.H. Wright J.R. Am. J. Respir. Cell Mol. Biol. 1996; 15: 509-519Crossref PubMed Scopus (122) Google Scholar). SP-A was obtained from the lavage of alveolar proteinosis patients as previously reported (25Tino M.J. Wright J.R. Am. J. Physiol. 1996; 14: L677-L688Google Scholar). Detection of SP-A degradation fragments was determined using anti-human SP-A antibody as previously reported (19Mariencheck W.I. Alcorn J.F. Palmer S.M. Wright J.R. Am. J. Respir. Cell Mol. Biol. 2003; 28: 528-537Crossref PubMed Scopus (123) Google Scholar). Cell-free, normal rat BAL was concentrated using an Apollo spin concentrator (Orbital Biosciences, Topsfield, MA), with a molecular mass cut-off of 10 kDa. Aliquots of rat BAL were then incubated with purified elastase as indicated, and degradation fragments were detected by Western blot. Analysis of Lung Transplant BAL—BAL from CF patients who had received bilateral lung allografts were analyzed for the presence of SP-D and possible degradation fragments. Samples from the CF lung transplant recipients were separated under nonreducing conditions by SDS-PAGE and transferred to nitrocellulose. Immunoreactive SP-D was detected using an anti-human SP-D antibody provided by Dr. Erika Crouch (Washington University, St. Louis, MO). SP-D Fragment Purification—SP-D (227 μg/ml) was incubated with P. aeruginosa elastase (150 μg/ml) for at least 24 h at 37 °C. The resulting solution was then centrifuged at 8000 × g for 5 min to remove any large debris and applied to a Superose 6 high pressure liquid chromatography column (Amersham Biosciences). The sample volume was 200 μl, and the column was eluted at a flow rate of 0.5 ml/min using TBS containing 2 mm EDTA, pH 7.60. Absorbance at 280 nm was monitored throughout the run. Fractions were collected every minute and were analyzed by Western blot for intact SP-D, SP-D fragment, and elastase. Elastase was shown to separate from SP-D fragment (data not shown). SP-D fragment-containing fractions were then pooled and concentrated using the Apollo spin concentrator (Orbital Biosciences) to a final volume of ∼1 ml. Protein concentration was determined by the Nano-orange protein assay (Molecular Probes, Inc., Eugene, OR). Sequence Analysis of SP-D Fragments—Recombinant rat SP-D (10 μg) was incubated with P. aeruginosa elastase (Elastin Products Co., Owensville, MO) (10 μg). The resulting solution was then analyzed via Edman degradation performed by the Harvard Microchemistry Facility (Cambridge, MA). Aggregation of Bacteria by SP-D and SP-D Fragment—Bacteria were grown overnight on Nutrient Agar plates (Difco) and collected into 1 ml of L-broth. Colony-forming units (cfu) were determined by multiplication of absorbance at 660 nm times the calculated extinction coefficients for each isolate. The extinction coefficients were determined by serial dilution and counting of live bacterial colonies. A volume equal to 108 cfu of bacteria was suspended in 1 ml of TBS containing 2 mm CaCl2, pH 7.60. SP-D and SP-D fragment were added to the bacteria at the indicated concentrations, and absorbance at 660 nm was measured every 30 min for 180 min. Aggregation is observed as a decrease in absorbance as bacterial aggregates precipitate out of solution. Binding Assays with Intact SP-D and SP-D Fragment—Bacteria (108 cfu) were pelleted by centrifugation at 8000 × g for 5 min and resuspended in 250 μl of TBS containing either 2 mm CaCl2 or 10 mm CaCl2, pH 7.60. SP-D and SP-D fragment were added at concentrations of 1 and 2 μg/ml, and binding was allowed to proceed for 1 h at 4 °C. The bacteria were then collected by centrifugation and washed twice with TBS. Fluorescein-5-isothiocyanate (FITC)-labeled anti-rat SP-D IgG was then utilized to measure SP-D binding. Anti-rat SP-D IgG was purified from rabbit serum using a Protein A-Sepharose column (Pierce). Purified IgG was then dialyzed into Na2CO3, pH 9.0. FITC labeling of the antibody was then performed by adding 10 μl of FITC (Molecular Probes) at a concentration of 10 mg/ml in dimethylformamide for 6 h at 4 °C. Excess FITC was removed by dialysis versus PBS. Labeled anti-rat SP-D was added to bacteria at a volume of 25 μl per 500 μl of bacteria in TBS plus 2 mm CaCl2 or 10 mm CaCl2, pH 7.60, for 30 min at 4 °C. Bacteria were then washed twice with TBS, and fluorescence was measured at 495-nm excitation, 530-nm emission. Binding of SP-D was expressed as the relative fluorescence units above background levels. Phagocytosis of Bacteria as Measured by FACS Analysis—Bacteria were collected from nutrient agar plates and were resuspended in Na2CO3, pH 9.0. FITC labeling of the bacteria was then performed by adding 10 μl of FITC (Molecular Probes) at a concentration of 10 mg/ml in dimethylformamide for 1 h at room temperature. Excess dye was then removed by washing of the bacteria three times with PBS. Bacterial concentration was determined by measurement of absorbance at 660 nm, utilizing known extinction coefficients. FITC-labeled bacteria were then frozen in aliquots with 10% glycerol or stored at 4 °C. Labeled bacteria were later thawed and then added to alveolar macrophages at a ratio of 5 × 107 bacteria to 5 × 105 macrophages in 250 μl of PBS plus 2 mm CaCl2 and 0.1% bovine serum albumin. Assay tubes were precoated with 1% bovine serum albumin in PBS for 1 h at 4 °C. Phagocytosis was allowed to occur for 1 h at 37 °C, and then the cells were washed twice with PBS buffer. Finally, macrophages were fixed in 300 μl of 1% formaldehyde in PBS and analyzed for fluorescence by FACS. Data Analysis—All of the data presented in the figures were subjected to unpaired Student's t test analysis assuming unequal variances. All statistics were performed using the Microsoft Excel software package. Analyses with a resultant p < 0.05 were determined significant. P. aeruginosa Elastase Degrades SP-D Dose-, Time-, and Temperature-dependently—Our laboratory has previously reported that P. aeruginosa elastase degrades SP-D (19Mariencheck W.I. Alcorn J.F. Palmer S.M. Wright J.R. Am. J. Respir. Cell Mol. Biol. 2003; 28: 528-537Crossref PubMed Scopus (123) Google Scholar). In order to characterize further the kinetics of degradation, we examined the effects of dosage, time, and temperature on elastase activity. SP-D (14 μg/ml) was incubated with increasing concentrations of elastase for 3 h at 37 °C (Fig. 1A). At the lowest dose tested, 7 μg/ml, elastase produced fragments, whereas greater degradation was observed with concentrations up to 140 μg/ml elastase. SP-D, 60 μg/ml, was then incubated with 112.5 μg/ml of elastase at 37 °C for various time points (Fig. 1B). Immunoreactive fragment was detected as early as 10 min after the addition of elastase. Finally, elastase at either 37.5 or 112.5 μg/ml was added to SP-D (60 μg/ml) for 3 h at 4, 25, and 37 °C (Fig. 1C). SP-D was poorly degraded by elastase at both 4 and 25 °C. These data show that P. aeruginosa elastase degrades SP-D dose-, time-, and temperature-dependently. P. aeruginosa Elastase Degradation of SP-A and SP-D Is Inhibited by Phosphoramidon—In order to confirm that the degradative activity observed in our elastase preparations was indeed due to elastase, we incubated SP-D (20 μg/ml) or SP-A (40 μg/ml) with elastase (75 μg/ml) in the presence or absence of increasing concentrations (0.05–5 mm) of phosphoramidon, an elastase inhibitor, for 18 h at 37 °C (Fig. 2). Phosphoramidon inhibited SP-D and SP-A degradation dose-dependently. In addition, a colorometric assay (chromozym PL) specific for the P. aeruginosa serine protease, protease IV was performed to test the purity of our elastase preparations. The P. aeruginosa elastase utilized in these studies was negative for serine protease activity (data not shown). Furthermore, a commercial preparation of elastase resulted in production of an identical SP-D fragment. Degradation by commercial elastase was also inhibited by phosphoramidon at the same concentrations described above (data not shown). Divalent Metal Cations and EDTA Inhibit Degradation of SP-D and SP-A by P. aeruginosa Elastase—SP-A has been shown to bind to divalent cations via its C-terminal lectin domain (26Haagsman H.P. Sargeant T. Hauschka P.V. Benson B.J. Hawgood S. Biochemistry. 1990; 29: 8894-8900Crossref PubMed Scopus (84) Google Scholar). Binding of SP-A and SP-D to oligosaccharides has been shown to be calcium-dependent (27Haagsman H.P. Hawgood S. Sargeant T. Buckley D. White R.T. Drickamer K. Benson B.J. J. Biol. Chem. 1987; 262: 13877-13880Abstract Full Text PDF PubMed Google Scholar, 28Persson A. Chang D. Crouch E. J. Biol. Chem. 1990; 265: 5755-5760Abstract Full Text PDF PubMed Google Scholar). SP-D binding to oligosacchrides requires either calcium, magnesium, or manganese, whereas SP-A lectin binding requires either calcium or manganese. We examined the effects of calcium, magnesium, and manganese on degradation of SP-D and SP-A by elastase. SP-D or SP-A at 20 μg/ml were incubated with 40 μg/ml elastase in the presence or absence of either 2 or 10 mm divalent metal cations (Fig. 3A). Degradation of SP-D by elastase was inhibited by manganese > calcium > magnesium. Contrastingly, SP-A degradation was inhibited by calcium and manganese but only slightly by magnesium. We then tested the ability of the metal chelator, EDTA, to inhibit elastase degradation of SP-A and SP-D. SP-D (10 μg/ml) was incubated with 30 μg/ml elastase, or SP-A (40 μg/ml) was incubated with 75 μg/ml elastase in the presence or absence of increasing EDTA concentrations for 18 h at 37 °C (Fig. 3B). Degradation of SP-A and SP-D was inhibited by the metal chelator, EDTA. However, much higher concentrations of EDTA were required to inhibit SP-D degradation as compared with SP-A degradation. Purification of SP-D Fragment by Gel Filtration Chromatography—In order to examine the functional characteristics of the SP-D 35-kDa degradation fragment, we purified the fragment by gel filtration chromatography. SP-D (230 μg/ml) was incubated with 150 μg/ml P. aeruginosa elastase for greater than 24 h at 37 °C. The degradation solution was then applied to a Superose-6 fast protein liquid chromatography column in sequential 200-μl aliquots followed by successive elutions. SP-D fragment eluted from the column at an approximate mass of 400 kDa, suggesting a multimer. As shown by the Western blot under reducing conditions in Fig. 4, the degradation assay resulted in nearly 100% conversion of intact SP-D to the 35-kDa fragment monomer which was collected, pooled, and concentrated for use in functional protein assays. Sequence Analysis of the SP-D Fragments—Previous work in our laboratory has suggested that elastase cleaves SP-D in the C-terminal lectin domain (19Mariencheck W.I. Alcorn J.F. Palmer S.M. Wright J.R. Am. J. Respir. Cell Mol. Biol. 2003; 28: 528-537Crossref PubMed Scopus (123) Google Scholar). To map the cleavage site, the SP-D degradation products were identified by Edman degradation. Sequencing identified five novel N termini generated by elastase cleavage of SP-D (Fig. 5). Cleavage of SP-D occurred at two alternate sites at the beginning of the lectin domain, Val-259 and Ala-267. In addition, two alternate cleavage sites were identified in the middle region of the lectin domain, Phe-323 and Trp-336. A fifth cleavage site was identified in the collagen domain of SP-D, Ile-112. The relevance of this cleavage was not determined. The SP-D Fragment Does Not Aggregate S. typhimurium or E. coli—Aggregation of bacteria has been shown to be an important immune function of SP-D that is mediated by the lectin domain of SP-D. In order to test the hypothesis that SP-D fragment would fail to aggregate bacteria, we incubated a clinical isolate (S470) and a laboratory strain (PA01) of P. aeruginosa, S. typhimurium, and E. coli with intact SP-D and SP-D fragment. Consistent with prior observations from our laboratory (7Restrepo C.I. Dong Q. Savov J. Mariencheck W.I. Wright J.R. Am. J. Respir. Cell Mol. Biol. 1999; 21: 576-585Crossref PubMed Scopus (140) Google Scholar), SP-D (2 μg/ml) did not aggregate P. aeruginosa (data not shown). SP-D at 1 μg/ml was effective at aggregating both S. typhimurium and E. coli, whereas the SP-D degradation fragment had no effect on either bacteria (Fig. 6). These data show that SP-D fragment no longer retains the lectin-dependent ability to aggregate bacteria. SP-D Fragment Fails to Bind to P. aeruginosa or S. typhimurium—The inability to aggregate bacteria suggests that the SP-D fragment may exhibit impaired binding to bacteria. To examine bacterial binding, we incubated SP-D or SP-D fragment at 1 or 2 μg/ml with bacteria. Binding was determined by detection of SP-D by fluorescently labeled antibody. Intact SP-D bound dose-dependently to both S. typhimurium and P. aeruginosa (Fig. 7). The SP-D fragment at 1 or 2 μg/ml failed to bind to either bacteria. Due to the fact that SP-D fragment preparations contain EDTA and are more dilute than intact SP-D stocks, we confirmed that the lack of binding was not due to EDTA inhibition by adding SP-D fragment to bacteria in 10 mm CaCl2 buffer. SP-D fragment still showed impaired binding to bacteria in high calcium buffer. These results confirm that the SP-D degradation fragment does not have a functional lectin domain. SP-D Fragment Does Not Stimulate Phagocytosis of E. coli by Alveolar Macrophages—As a consequence of deficient aggregation and binding activity by SP-D fragment, we proposed that SP-D fragment does not enhance phagocytosis of bacteria by alveolar macrophages as does intact SP-D. Alveolar macrophages were incubated with FITC-labeled E. coli for 1 h at 37 °C. Uptake of bacteria was then measured by FACS. Intact SP-D dose-dependently increased uptake of E. coli as measured by mean fluorescence and percentage of positive cells (Fig. 8). SP-D fragment had no effect on uptake of bacteria by alveolar macrophages at either dose tested. These data combined with earlier results show that degradation of SP-D by P. aeruginosa elastase abrogates SP-D immune function. SP-D Fragment Increases Uptake of P. aeruginosa Deficient in Elastase Production by Alveolar Macrophages—In order to examine the potential contribution of elastase in abrogating SP-D-mediated uptake of P. aeruginosa, we tested the effects of intact SP-D on alveolar macrophage uptake of wild-type (PA01) and elastase deficient (PA01-B1) P. aeruginosa. Alveolar macrophages were incubated with FITC-labeled P. aeruginosa for 1 h at 37 °C in the presence or absence of SP-D. Intact SP-D had no effect on the uptake of wild-type PA01; however, SP-D dose-dependently enhanced the uptake of elastase-deficient P. aeruginosa (Fig. 9). These data suggest that the absence of elastase production by isolates of P. aeruginosa may alter the ability of SP-D to enhance phagocytosis of bacteria by macrophages. Degradation of Rat BAL Results in the Production of a 35-kDa SP-D Fragment—SP-