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J. Biol. Chem., Vol. 277, Issue 23, 20379-20385, June 7, 2002

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Human Surfactant Protein D (SP-D) Binds Mycoplasma pneumoniae by High Affinity Interactions with Lipids^*

Hirofumi Chiba, Surapon Pattanajitvilai, Amanda J. Evans, Ronald J. Harbeck, and Dennis R. Voelker

From the Program in Cell Biology, Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206

Received for publication, February 1, 2002, and in revised form, March 5, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Increasing evidence now identifies surfactant protein D (SP-D) as an important element of the innate immune system of the lung. In this study, we examined the interactions of rat and human SP-D with the human pathogen, Mycoplasma pneumoniae. Rat and human SP-D bound the organism with high affinity in a reaction that required Ca²⁺ and was inhibited by EGTA. Membranes derived from the organism bound the proteins in a similar manner, except the rat SP-D also exhibited a significant level of Ca²⁺-independent binding. Pretreatment of membranes with proteases did not alter the Ca²⁺-dependent SP-D binding of membranes by either protein. Mannose, glucose, maltose, and inositol, at millimolar concentrations, competed for human SP-D binding to the bacterial membrane. Lipids extracted from membranes and separated by two-dimensional thin layer chromatography bound human SP-D with high affinity in a Ca²⁺-dependent reaction. A tandem mutant of SP-D with E321Q and N323D substitutions, failed to bind M. pneumoniae lipids, directly implicating the carbohydrate recognition domain in the interaction. The interaction of rat and human SP-D with M. pneumoniae was unaffected by the presence of surfactant lipids and the hydrophobic surfactant proteins. These findings demonstrate that M. pneumoniae is likely to be recognized by SP-D in the alveolar environment and that primary determinants recognized on the organism are lipid components of the cell membrane.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mycoplasmas, the smallest self-replicating microorganisms, are pathogens capable of causing a wide variety of diseases. Mycoplasma pneumoniae is considered an important cause of pneumonia, tracheobronchitis, and pharyngitis. The bacteria also exacerbates other respiratory disorders such as asthma (1, 2) and chronic obstructive pulmonary disease (3). The mechanisms of intrapulmonary defense against mycoplasma are poorly understood, but current evidence suggests that the innate immune systems plays a major role in the immediate response to the organism.

Pulmonary surfactant is a mixture of lipids and proteins that acts to keep alveoli from collapsing during the expiratory phase of the respiratory cycle (4). Pulmonary surfactant protein D (SP-D)¹ is member of the C-type lectin superfamily that also includes surfactant protein A (SP-A), serum mannose-binding protein, conglutinin, and collectin 43 (5, 6). Mature monomeric SP-D is a 43-kDa glycoprotein that oligomerizes to form four trimers that are covalently associated at their N termini. The resultant ~516-kDa dodecameric protein has a cruciform shape with the carbohydrate recognition domains (CRDs) arranged peripherally at the end of long stalks (7). SP-D binds to carbohydrates such as maltose, glucose, and mannose in a Ca²⁺-dependent manner (8). The protein selectively recognizes cell wall carbohydrates of microorganisms especially rough forms of Gram-negative lipopolysaccharide via its CRD, and this constitutes one aspect of its role in innate lung defense mechanisms (9). Other ligands for the protein include phosphatidylinositol and glucosylceramide (10-12). Microbial targets for SP-D include both Gram-positive and Gram-negative respiratory pathogens, influenza, and respiratory syncytial viruses, Cryptococcus neoformans, Pneumocystis carinii, and Aspergillus fumigatus (9). Both monocytes/macrophages and neutrophils express surface receptors that can interact with SP-D (13-20). Direct interactions between SP-D and CD14 have also been characterized using purified recombinant proteins (21). The interactions between SP-D and microorganisms and in many instances immune cells promote both microbial aggregation and enhanced phagocytosis (9).

The purpose of this study was to determine: 1) the properties of the interactions between M. pneumoniae and SP-D; 2) the nature of the ligands present on the bacteria; and 3) whether binding could occur in the presence of other components of pulmonary surfactant. Our findings indicate that SP-D binds M. pneumoniae with high affinity and the principal ligands are lipids. Both the specificity and avidity of the SP-D binding indicate that microbial recognition by the protein is likely to occur in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of M. pneumoniae-- M. pneumoniae (strain FH, ATCC 15531) was cultured in polystyrene flasks containing 100 ml of SP-4 medium, at 37 °C, in an air, 5% CO₂ atmosphere, for 5 days. The polystyrene-adhering organisms were scraped from the surface with a rubber policeman, and collected by centrifugation at 8,000 × g for 15 min at 4 °C. The pellet was resuspended in phosphate-buffered saline (PBS; pH 7.4) and then washed twice by recentrifugation in PBS. The final pellet was resuspended in 2 ml of PBS and layered on a discontinuous sucrose gradient comprised of 60, 52, 48, and 40% steps. The gradients were centrifuged at 10,000 rpm (SW28 rotor) at 4 °C for 30 min. Cells were collected from the 48/52% sucrose interface, mixed with PBS, and centrifuged at 8,000 × g at 4 °C for 15 min. The pellet containing the purified mycoplasma was resuspended in PBS and used in experiments. The viability of the organism, as assessed by colony counting of samples, before and after centrifugation, was not significantly affected by the density gradient purification. We found that this purification is essential for all binding studies, since large quantities of denatured serum and medium components non-specifically sediment with the initial mycoplasma pellets and cause erroneous estimates of the bacterial protein concentration, and high levels of nonspecific binding by SP-D.

Preparation of M. pneumoniae Membrane-- Resuspended M. pneumoniae cells, recovered from sucrose gradients, in 1 ml of PBS were mixed with 3 ml of distilled water and incubated at 4 °C for 30 min. The mixture was then probe sonicated on ice for a total of 3 min, using 1-min sonication and 1-min cooling cycles. The sonicated preparation was centrifuged at 100,000 × g at 4 °C for 1 h. The pellet was resuspended in PBS, homogenized, and centrifuged at 100,000 × g at 4 °C for 1 h. The final membrane pellet was resuspended in PBS, homogenized, and used in experiments.

Preparation of Recombinant Human and Rat SP-D-- The expression of rat SP-D in CHO-K1 cells has previously been described (22) as has the construction and characterization of the E321Q/N323D mutant and the collagen domain deletion mutant (L27-P202) (22, 23). CHO-K1 cells expressing rat SP-D were grown in glutamine-free Glasgow minimum essential medium (Invitrogen, Grand Island, NY) containing 10% heat-inactivated and dialyzed fetal bovine serum. The medium was supplemented with 250 µM methionine sulfoximine for gene amplification. For protein purification, the cell lines were grown for 3 days in Glasgow minimum essential medium and then transferred to serum-free EX-CELL 301 medium (JRH Biosciences, Lenexa, KS) and incubated for 4 days. The medium was removed and four additional harvests were performed, allowing 24 h of culture between harvests. The medium containing recombinant protein was dialyzed against 5 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA. Following dialysis, the medium was adjusted to 5 mM CaCl₂, applied to a mannose-Sepharose column equilibrated with 5 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl₂, and eluted with 5 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA. The eluted protein was dialyzed against 5 mM Tris-HCl (pH 7.4), 150 mM NaCl and stored at 20 °C. Recombinant human SP-D was expressed and purified as described above for recombinant rat SP-D except the cells were grown in 25 µM methionine sulfoximine. The CHO-K1 cell line expressing SP-D was obtained from Erika Crouch, Washington University, St. Louis, MO. The SP-D preparations were judged pure by SDS-PAGE, Coomassie Blue staining, and Western blotting. Yields of all recombinant forms of SP-D ranged from 0.5 to 2.0 mg/liter of harvested media.

Preparation of HSC-- Hydrophobic surfactant components (HSC) were isolated from the bronchoalveolar lavage of Sprague-Dawley rats, 28 days after intratracheal instillation of 25 mg of silica (~125 mg/kg) (24, 25). Initially, the surfactant was purified by the method of Hawgood et al. (24) using NaBr density gradient centrifugation. The purified surfactant was extracted with butanol (25) and segregated into butanol-soluble and -insoluble material. The butanol-soluble HSC were recovered by drying under vacuum and resuspending in chloroform. The phospholipid content was determined by the method of Rouser et al. (26), and the mixture was stored at 20 °C. Prior to use, an aliquot of HSC was initially dried under N₂, and subsequently hydrated in 20 mM Tris (pH 7.4), 150 mM NaCl buffer at 37 °C for 1 h. Finally the HSC was probe-sonicated in 5-30 s bursts with 1 min cooling between bursts, to make a vesicle preparation for use in experiments.

Direct Binding of SP-D to M. pneumoniae-- M. pneumoniae cells (2 µg of total cell protein/tube) and the indicated concentration of human SP-D or rat SP-D solutions were prepared in 50 µl of buffer A (20 mM Tris, pH 7.4, 150 mM NaCl), containing either 5 mM CaCl₂ or EGTA, as indicated, with 2% w/v bovine serum albumin (BSA). The cells and the protein solutions were separately centrifuged at 10,000 rpm, at 4 °C for 10 min. The supernatant of the protein solutions (50 µl) was added to the cell pellet. The mixtures were resuspended and incubated for 1 h at 37 °C. The samples were layered on buffer A containing 3% BSA and centrifuged at 10,000 × g for 10 min at 4 °C. The cell pellets were resuspended in buffer A and again centrifuged through 3% BSA. The bound SP-D was eluted from cell pellets in buffer B (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EGTA, and 1% Triton X-100) with 2% BSA at room temperature for 30 min. The eluted protein was centrifuged at 10,000 rpm at 4 °C for 10 min. The recovered SP-D was quantified by sandwich ELISA using polyclonal antibody against either human or rat SP-D. Control conditions routinely included tubes lacking bacterial cells.

Binding of SP-D to M. pneumoniae membranes on Solid Phase-- Membranes equivalent to 1 µg of total protein were coated onto microtiter wells in 0.1 M NaHCO₃ (pH 9.6), at 4 °C overnight. Nonspecific binding was prevented with blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, either 5 mM CaCl₂ or EGTA, 2% BSA), and the indicated concentrations of human SP-D or rat SP-D were incubated at 37 °C for 2 h in buffer A containing 20 mg/ml BSA, and either 5 mM CaCl₂ or EGTA. Following the incubation period, the wells were washed three times with washing buffer, composed of buffer A containing 1 mg/ml BSA and either 5 mM CaCl₂ or EGTA as indicated. An HRP-conjugated polyclonal antibody to human or rat SP-D was used for detection of the proteins, as appropriate. Antibody was added into the wells and incubated at 37 °C for 1 h. The binding of the proteins to M. pneumoniae membranes was determined by measuring the absorbance at 492 nm, using o-phenylenediamine as a substrate for the peroxidase reaction. Control conditions routinely included wells without added membranes.

Proteinase K Treatment-- Aliquots of 2 µg of M. pneumoniae membranes were coated onto microtiter wells as described above. Proteinase K (1 mg/ml in 100 µl of PBS, 5 mM MgCl₂) was then incubated with the adsorbed M. pneumoniae membranes at 37 °C for 2 h. Digested membranes were washed with 5 mM Tris (pH 7.4), 150 mM NaCl. The final quantity of membrane attached to the wells was determined by measuring the lipid phosphorus content.

Ligand Blotting Analysis-- Five micrograms of M. pneumoniae membrane protein were electrophoresed and transferred to a nitrocellulose membrane. The nonspecific binding was prevented with a blocking buffer consisting of 20 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EGTA, and 2% BSA. The membrane was then incubated at room temperature for 3 h, with 1 µg/ml SP-D in the presence of 5 mM EGTA. Next, the membrane was washed with PBS containing 0.1% Triton X-100 and 3% powdered skim milk and incubated with anti-SP-D antibody (5 µg/ml) for 1 h, followed by incubation with horseradish peroxidase (HRP)-labeled anti-rabbit IgG (1:1,000) for 1 h. After the washing procedure, the SP-D binding was visualized by using diaminobenzidine.

Direct Binding of SP-D to M. pneumoniae Lipids on Thin-layer Chromatograms-- Lipids were extracted from M. pneumoniae membranes (27), and multiple aliquots of the preparation corresponding to 40 nmol of lipid phosphorus were separated by two-dimensional thin layer chromatography on plastic-backed SIL G plates (Macherey Nagel). The first dimension solvent contained chloroform:methanol:NH₄OH (65:35:8). The plate was air dried and neutralized in the vapor from a methanol:acetic acid (90:10) solution for 10 min. The second dimension solvent contained chloroform:methanol:acetic acid:water (50:25:8:2.5). The plate was air dried overnight, and soaked in blocking buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM CaCl₂, 2% BSA) for 1 h. Human SP-D (1 µg/ml) was added to the plate and incubated for 2 h at room temperature. The bound human SP-D was detected with polyclonal antibody to the protein and HRP-labeled anti-rabbit IgG. Human SP-D binding was visualized by using diaminobenzidine. In parallel with the plates used to detect SP-D binding, we visualized total lipids and glycolipids. The total lipids of M. pneumoniae membranes were detected with 0.2% (w/v) ANSA spray, and the glycolipids were identified with an orcinol spray (28). Control plates were also analyzed for reactivity to secondary antibody alone.

Other Methods-- Protein concentrations were determined by using the bicinchoninic acid assay (Pierce, Rockford, IL) and bovine serum albumin as a standard. Polyclonal anti-human and rat SP-D were raised in rabbits against purified recombinant human and rat SP-D. Data in the figures are shown as mean ± S.E. Significance was determined using a two-tailed Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

SP-D Binds M. pneumoniae with High Affinity-- M. pneumoniae is a human pathogen that resides in the same extracellular alveolar and bronchiolar compartment as SP-D. Our initial experiments were designed to determine whether SP-D interacts with the bacteria. We first conducted direct binding measurements between the intact organism and the purified protein. These experiments, shown in Fig. 1, demonstrate that human and rat SP-D bind the intact organism in a concentration-dependent and saturable manner that is dependent upon the presence of Ca²⁺. Analysis of the data using the method of Scatchard reveals an apparent K_d' value of 6.88 ± 2.43 nM (mean ± S.E., n = 3) for the human protein, and 19.4 ± 1.76 nM (mean ± S.E., n = 3) for the rat protein. The organism expresses ~2.17 × 10³ sites for human SP-D, and 2.75 × 10³ sites for rat SP-D.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Direct binding of SP-D to M. pneumoniae in solution. M. pneumoniae cells (2 µg of cell protein) were mixed with the indicated concentration of human SP-D (panel A) or rat SP-D (panel B) in the presence of 5 mM Ca2+ () or 5 mM EGTA () as indicated. The binding was carried out at 37 °C for 1 h. The amount of the protein cosedimented with M. pneumoniae cells was determined by sandwich ELISA as described under "Experimental Procedures." Control experiments without bacterial cells were also performed (). Values are mean ± S.E. from three experiments. The Scatchard plot analysis of the binding in panels A and B is shown in panels C and D, respectively.

Membranes Isolated from M. pneumoniae Bind SP-D-- Since membranes are often a more versatile system to use for examining biochemical interactions between proteins and their biological ligands, we developed a solid phase system for examining SP-D interactions with mycoplasma membranes. Purified membranes were isolated from the bacteria and adsorbed onto microtiter wells. Human SP-D bound to the membranes with high affinity (see Fig. 2A) and had an apparent K_d' of 10.1 ± 1.45 nM (mean ± S.E., n = 3) which is essentially equivalent to the value for intact cells. The Ca²⁺ dependence of the human SP-D interaction with membranes was the same as observed for the intact cells. The rat SP-D also bound membranes (see Fig. 2B) with an apparent K_d' of ~8.58 ± 0.61 nM (mean ± S.E., n = 3). In contrast to data obtained with intact cells, about 65% of the rat SP-D binding to isolated membranes was Ca²⁺ independent. This finding for rat SP-D is consistent with the physical process of membrane preparation revealing a cryptic Ca²⁺-independent ligand for the protein. This Ca²⁺-independent ligand may originally exist only on the inner aspect of the plasma membrane, or it may be generated by proteolytic, glycolytic, or lipolytic processes.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Binding of SP-D to M. pneumoniae membranes on solid phase. One microgram of M. pneumoniae membranes was coated onto microtiter wells at 4 °C overnight, and the indicated concentrations of human SP-D (panel A) or rat SP-D (panel B) were incubated at 37 °C for 2 h in the presence of 5 mM Ca2+ () or 5 mM EGTA (). After the incubation, the wells were washed and the binding of the proteins to M. pneumoniae membranes was detected by HRP-conjugated polyclonal antibody to human or rat SP-D as described under "Experimental Procedures." Control experiments without membranes were also examined (). Values are mean ± S.E. from three experiments.

Carbohydrates Compete for SP-D Binding to M. pneumoniae Membranes-- Previous work has demonstrated that SP-D binds simple and complex carbohydrates via its CRD in a calcium-dependent manner (8). The observation that EGTA inhibited human and rat SP-D binding to M. pneumoniae suggested a role for the CRD in these interactions. In experiments presented in Fig. 3 we tested the ability of mono- and disaccharides to inhibit the binding of human SP-D solid phase membranes. The data demonstrate that multiple saccharides compete for the SP-D binding with IC₅₀ values in the 2-7 mM range and a rank order of inhibition of maltose > inositol > glucose > mannose. Galactose was a significantly weaker inhibitor than the other sugars tested and had an IC₅₀ > 20 mM.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Carbohydrate competition for human SP-D binding to M. pneumoniae membranes. Human SP-D (2.5 µg/ml) was incubated with various concentrations of carbohydrates in the presence of 5 mM Ca2+ for 15 min at 37 °C. The SP-D:inhibitor mixture was then incubated with M. pneumoniae membranes, coated onto microtiter wells (1 µg/well), at 37 °C for 2 h. After the incubation, the wells were washed and the binding of human SP-D to M. pneumoniae membranes was detected by polyclonal antibody to human SP-D as described under "Experimental Procedures." Values are mean ± S.E. from three experiments.

Human SP-D Recognizes a Protease-insensitive Component on M. pneumoniae Membranes-- We used the solid phase membrane binding system to evaluate the biochemical characteristics of the SP-D ligand expressed by the cell. Solid phase membranes were treated with proteinase K, washed extensively, and tested for SP-D binding. The efficacy of the protease treatment was evaluated by gel electrophoresis and Coomassie staining, which revealed essentially complete degradation of all proteins. In addition, we determined that the amount of proteinase K added was sufficient to completely hydrolyze the same quantity of a control protein (BSA) as is present in total bacterial membrane proteins. The results shown in Fig. 4A demonstrate that the ligand for human SP-D is completely protease resistant. In contrast, the ligand for rat SP-D that requires Ca²⁺ for binding is resistant to protease attack; whereas the ligand that does not require Ca²⁺ and binds in the presence of EGTA is protease sensitive. These findings indicate that the Ca²⁺-independent SP-D ligand is a protein. To more directly test this idea we performed a ligand blot reaction on electrophoresed mycoplasma membrane proteins that were transferred to nitrocellulose. As shown in Fig. 5, a single protein species of ~20-kDa bound the rat SP-D in the absence of Ca²⁺ and the presence of EGTA. Similar ligand blot studies with human SP-D failed to demonstrate a positive reaction. From the results of the above experiments we conclude that human SP-D recognizes a protease-insensitive ligand on the bacteria and rat SP-D recognizes both protease-sensitive and -insensitive ligands on isolated membranes.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Binding of SP-D to M. pneumoniae membranes digested by proteinase K. M. pneumoniae membranes (2 µg of protein) were coated onto microtiter wells. Proteinase K (1 µg/ml) in 100 µl of PBS, 5 mM MgCl2 was then incubated with M. pneumoniae membranes on the microtiter wells at 37 °C for 2 h. The digested membranes were washed with buffer to remove the protease. The final amounts of the membranes remaining in the wells were determined by lipid phosphorus measurement. The binding of SP-D to digested membranes was determined by ELISA. Values are mean ± S.E. from three experiments. Asterisks indicate p < 0.05 from comparison of control plus 5 mM Ca2+ against other conditions.

View larger version (49K): [in this window] [in a new window]   Fig. 5.   Ligand blot analysis. Five micrograms of M. pneumoniae membrane protein were electrophoresed and transferred to a nitrocellulose membrane. The nonspecific binding was blocked with blocking buffer. The membrane was then incubated at room temperature for 3 h, with 1 µg/ml rat SP-D or BSA as indicated, in the presence of 5 mM EGTA. The rat SP-D binding to membrane protein was detected using polyclonal antibody to rat SP-D as described under "Experimental Procedures."

The Major Ligands for Human SP-D Present in M. pneumoniae Membranes Are Lipids-- We next sought to examine the molecular class of ligand recognized by human SP-D. Lipids were extracted from M. pneumoniae membranes and separated by two-dimensional thin layer chromatography. Four thin layer plates were analyzed in parallel. The plates were individually stained for total lipids (ANSA stain), glycolipids (orcinol stain), SP-D binding, or antibody binding. The results of the experiment are presented in Fig. 6. The staining in Fig. 6, A and B, demonstrates the presence of at least 8 resolved polar lipids and 7 glycolipids. The SP-D binding corresponds to 3 of the major glycolipids and a fourth component undetected by other methods. The control plate demonstrates there is no antibody reactivity in the absence of SP-D. From these experiments we conclude that human SP-D exhibits high affinity specific interactions with a subset of lipids present in M. pneumoniae membranes. The majority of the SP-D reactive lipids comigrate with glycolipids.

View larger version (74K): [in this window] [in a new window]   Fig. 6.   Binding of SP-D to total lipids of M. pneumoniae membranes on 2D TLC plates. Lipids were extracted from M. pneumoniae membranes and separated by two-dimensional thin layer chromatography on 4 SIL G plates. The first dimension was developed in chloroform:methanol:NH4OH (65:35:8). The second dimension was developed in chloroform:methanol:acetic acid:water (50:25:8:2.5). The plate was air-dried overnight. In panel A total lipids were detected with 0.2% (w/v) ANSA. The L1, L3, and L6 components correspond to sphingomyelin, phosphatidylcholine, and phosphatidylglycerol, respectively. By other criteria L2 comigrates with complex glycosphingolipid and L4 comigrates with trihexosyldiacylglycerol. The identity of L5, L7, and L8 are not known. In panel B glycolipids were detected with orcinol. G5, G6, and G7 comigrate with phosphatidylglycerol, dihexosyl, and monohexosyldiacylglycerol, respectively. The G3 component migrates at the expected position for trihexosyldiacylglycerol. The G1 component migrates at the position of complex glycosphingolipids. The G2 and G4 components are unknown. In panel C, 1 µg/ml human SP-D was added to the plate in the presence of a blocking buffer and incubated for 2 h at room temperature. The bound human SP-D was detected by polyclonal antibody to human SP-D and HRP-labeled anti-rabbit IgG. The SP-D reactivity corresponds to an unknown lipid (B1), trihexosyldiacylglycerol (B2), dihexosyldiacylglycerol (B4), and a lipid that comigrates with phosphatidylglycerol and a hexose containing lipid (B3). In panel D the control plate was treated identically to the SP-D immunoblot plate except that SP-D was omitted.

Definitive analysis of these data is complicated by a number of factors. The total complement of M. pneumoniae lipids is poorly defined in the literature (29-31). In addition, several polar lipids and glycolipids comigrate in these solvent systems. From the ANSA staining and analysis of polar lipid standards L1, L3, and L6 correspond to sphingomyelin, phosphatidylcholine, and phosphatidylglycerol. M. pneumoniae has been reported to contain all of the aforementioned polar lipids (30, 31). The remainder of the lipids do not correspond to other known phospholipids. From the orcinol staining, G6 and G7 correspond to dihexosyl and monohexosyl diacylglycerols, respectively, and the G3 spot has the expected migration of trihexosyldiacylglycerol. All three of these hexosyldiacylglycerols have also been described as M. pneumoniae lipids (29). The G5 spot comigrates with phosphatidylglycerol but by independent analyses we verified that phosphatidylglycerol does not produce a positive reaction with orcinol. Thus the L6/G5 region is likely to contain multiple components. The G1 spot has some of the migration and reactivity of complex glycosphingolipids that have been identified in M. pneumoniae (29). The G2 and G4 spots are unknown. The SP-D reactive lipids consist of an unknown component that fails to stain with either orcinol or ANSA (B1), dihexosyldiacylglycerol (B4), and presumed trihexosyldiacylglycerol (B2). The B3 component has migration properties of phosphatidylglycerol as well as the reactivity of a hexose containing lipid. In independent ligand blot studies we determined that SP-D does not recognize egg phosphatidylglycerol. We are currently working on the purification and chemical identification of each of these SP-D ligands.

Lipids Derived from M. pneumoniae Compete for Human SP-D Binding to Solid Phase Membranes-- Unilamellar liposomes were prepared from the total lipid extract of mycoplasma by sonication. These liposomes were tested as competitors for human SP-D binding to solid phase membranes. The amount of lipid added was quantified by phosphorus measurement. The results presented in Fig. 7 show that the mycoplasma lipid preparation is an effective competitor for SP-D interaction with membranes with an IC₅₀ of less than 40 µM (measured as lipid phosphorus). This IC₅₀ value must be an overestimate since the glycolipid fraction is likely to be only a minor fraction of the total lipid present in mycoplasma membranes. For comparison, phosphatidylinositol, a known phospholipid ligand for SP-D, and hydrophobic surfactant components, were also tested for inhibition of human SP-D binding to membranes. The phosphatidylinositol competed for human SP-D binding to membranes with an IC₅₀ of ~40 µM, and the HSC was ineffective as an inhibitor. From this data we conclude that the mycoplasma total lipids have affinity for SP-D that is at least as high as that for phosphatidylinositol. Phosphatidylinositol is not present in M. pneumoniae membranes (29). These results also clearly demonstrate that surfactant lipids do not interfere with human SP-D binding to mycoplasma. The above findings make it likely that SP-D binds to mycoplasma in the bronchoalveolar space of the lung.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Inhibitory effects of M. pneumoniae membrane lipids or HSC for human SP-D binding to solid phase M. pneumoniae membranes. M. pneumoniae membrane lipids were hydrated in 20 mM Tris (pH 7.4), 150 mM NaCl buffer at 37 °C for 1 h. The lipids were probe-sonicated to make unilamellar vesicles. Human SP-D (0.2 µg) was incubated with the indicated amounts of lipid in the presence of 5 mM Ca2+, at 37 °C for 30 min. The mixtures were added to M. pneumoniae membranes (1 µg/well) coated onto microtiter wells, and incubated at 37 °C for 2 h. Human SP-D bound to membranes was detected with HRP-conjugated polyclonal antibody. Values are mean ± S.E. from three experiments.

Carbohydrate Binding Specificity Is Required for SP-D Recognition of M. pneumoniae-- The above data addressing the requirements for human and rat SP-D binding to intact bacteria, and human SP-D binding to isolated membranes, are consistent with the CRD domain of SP-D participating directly in the binding reaction. We investigated this issue in more detail by performing binding studies using structural variants of SP-D generated by site-directed mutagenesis (22, 23). In studies with intact cells (Fig. 8A) we determined that the tandem mutant (E321Q/N323D) was unable to bind the bacteria. The E321Q/N323D mutant is unable to bind mannosyl and glucosyl residues, but retains very low affinity for galactosyl moieties. The protein also retains ~50% of its phosphatidylinositol binding (22), and we verified that the protein used in these experiments also retains phosphatidylinositol binding (Fig. 8B). In contrast to the findings with the (E321Q/N323D) mutant, a collagen domain deletion mutant (L27-P202) retained ~50% of the binding found for wild type SP-D. We tested two other structural variants that display dramatically enhanced binding to rough lipopolysaccharide, T268K and T285A,² but their activity was comparable with the wild type protein. From these data we conclude that carbohydrate binding specificity is a major determinant of the interaction of SP-D with M. pneumoniae ligands expressed on the surface of the intact cell.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Binding of structural variants of rat SP-D to intact M. pneumoniae cells in solution. Panel A, recombinant rat SP-D mutants were compared with the wild type protein (WT) for binding to M. pneumoniae. The E321Q/N323D substitutions occur in the CRD and confer altered carbohydrate recognition properties. The L27-P202 mutant lacks the collagen domain and the N-linked oligosaccharide, but retains a functional CRD. The T286K or T285A mutations alter recognition of Gram-negative bacterial lipopolysaccharide. M. pneumoniae cells (2 µg of protein) were mixed with 0.2 µg of the SP-D proteins in the presence of 5 mM Ca2+. The binding was carried out at 37 °C for 1 h. The amount of the protein cosedimented with M. pneumoniae cells was determined by sandwich ELISA as described under "Experimental Procedures." Values are mean ± S.E. from three experiments. Asterisks indicate p < 0.03 when compared with control (WT). In panel B, the binding of the E321Q/N323D mutant was compared with the wild type protein using dot blot analysis. Phosphatidylinositol (PI) (15 nmol) or M. pneumoniae lipids (15 nmol of phosphorus) were applied to a thin layer plate. Nonspecific binding was blocked as described under "Experimental Procedures" and the TLC plate was incubated with the different SP-D variants. Bound SP-D was detected using polyclonal anti-rat SP-D antibodies and anti-rabbit IgG conjugated with HRP.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we investigated the binding of SP-D to M. pneumoniae, a pathogen that accounts for 20-30% of all pneumonias, causes airway inflammation (32), and exacerbates other respiratory disorders such as asthma (1) and chronic obstructive pulmonary disease (3). There is increasing evidence that SP-D plays an important, immediate role in antibody independent host defense in the lung (9, 13, 18, 19, 33). The actions of SP-D and the closely related SP-A are multifaceted and involve recognition of both pathogens and immune effector cells. The recognition of pathogens may cause aggregation and facilitate phagocytosis, or enhance the production of proinflammatory cytokines (15, 34, 35). The recognition of macrophages and other phagocytes by surfactant proteins can either enhance or depress the inflammatory response, depending both on the microbial agent and the state of activation of the immune cell (36-38).

This report provides clear evidence that the binding of SP-D to M. pneumoniae is a specific and high affinity interaction. Saturable binding of SP-D to the mycoplasma occurs at levels of the collectin that are well within the physiological range, estimated at 50-90 µg/ml (100-180 nM) (39). The apparent K_d' values for the rat and human proteins are in the range of 5-20 nM and the calculated binding sites are 2-3 × 10³/cell. Several features of the binding reaction implicate the CRD of the protein in the attachment to the bacteria. The requirement for Ca²⁺ in collectin binding is a hallmark of CRD involvement. In addition, the binding of human SP-D to mycoplasma membranes is effectively inhibited by carbohydrates that are recognized by the protein. Finally, the loss of rat SP-D binding to intact cells occurs with the tandem mutation (E321Q/N323D) that confers loss of carbohydrate binding specificity. Our interpretation of these findings is that the CRD domain of SP-D primarily interacts with a glycoconjugate expressed on the surface of M. pneumoniae.

Examination of the binding between intact M. pneumoniae and the collagen deletion mutant of rat SP-D also reveals additional important characteristics of the interaction. The L27-P202 mutant lacks the entire collagen domain but still retains 50% of the binding found for the wild type protein. This indicates that the collagen domain is not required for the binding reaction. The loss of some of the binding is likely to be a consequence of the loss of higher order oligomerization of the protein, and consequent reduction of affinity. In previous work we have shown that the collagen deletion mutant behaves as a trimer in solution (23). In contrast, mature wild type SP-D is a cruciform dodecamer. In principle, 2 trimeric CRDs of the wild type dodecamer can interact with a single surface. Since the collagen deletion mutant behaves as a single trimer, the avidity of binding will be reduced relative to the wild type protein. The N-linked oligosaccharide of SP-D is also located within the collagen domain (40) and consequently this post-translational modification is lost in the L27-P202 mutant. Thus, the data also demonstrate that the oligosaccharide of SP-D is not essential for mycoplasma binding.

We were able to simplify the binding assay by adapting the method for intact cells to membranes isolated from M. pneumoniae. For human SP-D, the binding characteristics between intact cells and solid phase membranes appear nearly identical. The binding of human SP-D was completely resistant to protease treatment of the membranes. Examination of the lipid components of the membranes demonstrated that essentially all of the human SP-D binding activity resided in this fraction. In addition, liposomes prepared from the lipid extract of mycoplasma were very effective inhibitors of human SP-D binding to solid phase membranes. In contrast to the mycoplasma lipids, the interaction between human SP-D and its mycoplasma membrane ligand is unaffected by the lipid components of pulmonary surfactant. This latter situation indicates that the lipids present in the alveolar environment are unlikely to interfere with human SP-D recognition of the microorganism. The lipids isolated from mycoplasma were further subfractionated to examine their interaction with human SP-D. After separating the lipid components of mycoplasma by two-dimensional thin layer chromatography, we were able to show that the major ligands for SP-D comigrate with 3 prominent glycolipids with characteristics of di- and trihexosyldiacylglycerol. These finding are consistent with glycolipid components of M. pneumoniae functioning as the predominant bacterial receptors for SP-D. We are currently working to definitively identify the structures of these glycolipids.

Analysis of the mycoplasma membrane binding by rat SP-D presents a more complicated picture of the interaction. Unlike the rat SP-D binding observed for the intact bacterial cells, the membranes exhibit both Ca²⁺-dependent and -independent interactions. The Ca²⁺-independent binding is also protease sensitive. Furthermore, in the absence of Ca²⁺ and in the presence of EGTA, rat SP-D shows specific binding to a 20-kDa protein present in mycoplasma membranes. Our interpretation of this data is that the Ca²⁺-independent ligand for rat SP-D is unavailable in the intact cell, but becomes accessible in the membrane preparation. We favor an explanation in which the ligand is restricted to the cytoplasmic face of the cell membrane and (subsequently becomes accessible to SP-D when membranes are prepared). The access of rat SP-D to both sides of the bilayer could occur as a consequence of the bacterial membranes forming either randomly oriented vesicles or open sheets. As described in earlier parts of the "Discussion," the Ca²⁺ dependent binding activity for rat SP-D has all the properties of a lipid glycoconjugate expressed on the exoplasmic face of the cell membrane. The utility of the recognition by rat SP-D, of exposed and cryptic ligands present in M. pneumoniae is not clear at present. It is possible that the recognition of multiple classes of ligands by rat SP-D confers greater resistance to the organism than that exhibited in humans.

In summary, our findings demonstrate high affinity interactions between SP-D and M. pneumoniae. The properties of the binding reaction implicate the CRD as the protein domain that interacts with the organism. Glycolipids constitute a major class of microbial ligand that interacts with the protein. Together these findings identify SP-D as an important component of host recognition of this significant human pathogen.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HL 45286 and ALA-ARC 95.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Medicine, National Jewish Medical Research Center, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1300; Fax: 303-398-1806; E-mail: voelkerd@njc.org.

Published, JBC Papers in Press, March 26, 2002, DOI 10.1074/jbc.M201089200

² K. E. Greene, S. Pattanajitvilai, and D. R. Voelker, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: SP-D, surfactant protein D; CRD, carbohydrate recognition domains; PBS, phosphate-buffered saline; HSC, hydrophobic surfactant components; ANSA, 8-anilino-1-naphthalene sulfonic acid; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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