Introduction

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Pneumocystis carinii remains an opportunistic fungus that causes severe pneumonia in immunosuppressed patients, including those with acquired immunodeficiency syndrome, malignancies, and after organ transplantation (1, 2). The mechanisms by which P. carinii establishes infection in such patients and the mediators of host defense in different stages of disease are not fully understood. However, accumulating evidence indicates that both innate defenses involving macrophages and host proteins, and adaptive immune processes requiring lymphocytic responses are necessary for effective host elimination of this organism from the lung (3).

In severe P. carinii-induced pneumonitis, the alveolar spaces are filled with organisms, inflammatory cells, components of exuded serum such as fibronectin and vitronectin, and abnormal amounts of surfactant components, including surfactant protein (SP)-A and SP-D (7). Binding of these host proteins to the organism alters the interaction of P. carinii with cells of the lower respiratory tract. For instance, the interaction of fibronectin and vitronectin with P. carinii facilitates organism attachment to alveolar epithelial cells, a process that promotes P. carinii proliferation (11). Our recent work demonstrates that SP-D also binds to P. carinii (8). SP-D is known to significantly accumulate in the lung during P. carinii pneumonia, to bind to the mannose- and glucose-rich major surface glycoprotein complex of the organism (gpA), and to augment adherence of the organisms to alveolar macrophages (8).

SP-D is a collectin that exhibits structural similarity to SP-A, mannose binding protein, conglutinin, and CL-43 (8). As a group, the collectins are collagenous carbohydrate binding proteins (collagenous C-type lectins) with proposed functions in innate immune processes against microorganisms (14, 15). The basis for many of the reported interactions of collectins with microbial pathogens involves calcium-dependent recognition of sugar surface molecules on the organism's cell wall. Each collectin exhibits differential preferences for specific sugar residues, providing a potential explanation for the existence of a wide variety of collectins. SP-D has been shown to bind to a number of bacteria, viruses, and other fungi. SP-D also mediates the agglutination of microorganisms and alters their relative infectivity (15).

Alveolar macrophages recognize P. carinii through interactions of various plasma membrane receptors with different components of the P. carinii cell wall. The major antigenic glycoprotein on the surface of P. carinii, gpA, is known to directly bind to macrophage mannose receptor, stimulating phagocytosis (20). Molecular studies indicate that gpA is encoded by a family of genes and contains relatively conserved cysteine-rich regions. N-linked carbohydrate rich in mannose, glucose, and N-acetyl-glucosamine residues represents roughly one-tenth of the mass of gpA (21). Interestingly, although SP-D binds to gpA and facilitates P. carinii attachment to the macrophage, the interaction of SP-D with the organism does not facilitate phagocytosis (8). Instead, P. carinii aggregates remain attached to the surface of the macrophage and appear masked from phagocytic uptake. As such, the interaction of SP-D with P. carinii may represent a means by which the organism escapes elimination by these macrophages.

Rat SP-D monomer is a 43-kD peptide with four major structural domains (22). The noncollagenous amino-terminal domain contains two conserved cysteine residues (Cys-15 and Cys-20) involved in interchain disulfide bonding. The uninterrupted collagenous domain contains 59 Gly-X-Y repeats and is connected to the carboxyl-terminal carbohydrate recognition domain (CRD) by a short linking (neck) sequence. A single N-linked glycosylation site at Asn-70 exists within the collagenous domain, which contains a sialylated sugar. Natural and recombinant SP-D are predominantly assembled as dodecamers consisting of four homotrimeric subunits with long triple helical arms (26). Dodecamers can self-associate at their amino-termini to form more highly ordered multimers of dodecameric forms with peripheral arrays of trimeric CRDs (26). Much of what is known about the biochemical mechanisms of SP-D interactions has been derived from binding experiments using neoglycoproteins, such as maltosyl-bovine serum albumin (BSA) (29). Few studies are available defining the mechanisms of SP-D binding to fungal ligands such as gpA.

Because the interaction of P. carinii with SP-D represents a potent component of host-organism interactions, this current study was performed to elucidate the biochemical nature of the interaction of SP-D with P. carinii gpA. In light of earlier biochemical characterizations that reveal the P. carinii gpA complex is rich in mannose and glucose residues (21, 30), we hypothesized that the CRD of SP-D mediates these interactions by binding to these sugar residues on the organisms. In addition, we further postulated that SP-D interactions with P. carinii gpA would be facilitated by dodecameric and higher order forms of SP-D containing multiple independent CRD motifs compared with homotrimeric SP-D subunits. Such a detailed binding analysis would permit us to understand the biochemical basis of SP-D interactions with gpA, a natural biologically important ligand on the surface of P. carinii.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Reagents

All reagents were of analytical or molecular grade and were obtained from Sigma Chemical Co. (St. Louis, MO) or from GIBCO-BRL (Gaithersburg, MD) unless otherwise stated. Mannosylated and glucosylated BSA were purchased from EY Laboratories, Inc. (San Mateo, CA). The homobifunctional cross-linking agent bis-(sulfosuccinimidyl)-suberate (BS³) was obtained from Pierce Chemical Co. (Rockford, IL). The original rat SP-D complementary DNA (cDNA) construct was kindly provided by Dr. James H. Fisher, University of Colorado (Denver, CO) (24, 28). Ciprofloxacin was the generous gift of Dr. Barbara Painter, Bayer Pharmaceuticals, Inc. (West Haven, CT). Rats used in these studies were Harlan Sprague-Dawley rats (HSD Colony 231) purchased from Harlan, Inc. (Dublin, VA).

Isolation of the P. carinii gpA Surface Glycoprotein Complex

All animal studies were approved by the Mayo Institutional Animal Care and Utilization Committee and conducted according to standard laboratory practices. P. carinii pneumonia was induced in Harlan Sprague-Dawley rats as previously described (31, 32). Rats were freely provided with drinking water containing dexamethasone (2 mg/liter), tetracycline (500 mg/liter), and nystatin (200,000 U/liter). On a weekly basis, the animals also received oral ciprofloxacin (0.45 g/liter) for two consecutive days to further reduce the risk of bacterial infections. After 5 d of immunosuppression, rats were transtracheally inoculated with P. carinii (~ 500,000 cysts) and immunosuppressed for an additional 6 to 8 wk before being killed. P. carinii were purified by lung homogenization and differential filtration in which the homogenates were exhaustively filtered through 10-µM filters that retain lung cells but allow passage of P. carinii. The filtrates were collected, centrifuged (1,500 × g for 30 min), and the pellets resuspended in 5 ml of Hanks' balanced salt solution. If other microorganisms were noted on microbiologic examination, the material was discarded.

Freshly isolated P. carinii derived from these rats were used for the preparation of gpA (20, 33). P. carinii were solubilized in 125 mM Tris, 4% sodium dodecyl sulfate (SDS), 4% 2-mercaptoethanol, 0.002 bromophenol blue, and 20% glycerol, pH 7.4. From this extract, gpA was purified by continuous flow gel electrophoresis on 10% polyacrylamide preparative tube gel (Prep Cell Apparatus; Bio-Rad, Hercules, CA) as we previously reported (20, 33). Fractions containing the 120-kD band visible on silver-stained, 4 to 15% gradient resolving gels (Phast Gel System; Pharmacia LKB Biotechnology, Piscataway, NJ) were pooled, dialyzed, and concentrated. Immunoblotting of the purified protein with monoclonal antibody 5E12 verified this material as authentic gpA (33, 34). We have previously reported the purity and characteristics of these preparations (8, 20). Scanning densitometry of three silver-stained preparations revealed the preparations to contain 97.5 ± 0.8% of the 120-kD material (20). Quantitative immunoprecipitation studies indicate that the material collected in the 120-kD fraction was 91.6 ± 0.1% reactive with monoclonal antibody 5E12, indicating that the gpA complex has been isolated to high purity (20).

Solid-Phase Binding Assay of Rat SP-D to P. carinii gpA

A solid-phase binding assay, modified from that previously reported, was developed to specifically characterize interactions of rat SP-D with the mannose- and glucose-rich gpA complex derived from P. carinii (29). To accomplish this, enzyme-linked immunosorbent assay plates (96-wells; Corning Glass Works, Corning, NY) were coated overnight with 50 µg/ml gpA in coupling buffer (0.1 M NaHCO₃, pH 8.3) at 4°C. The wells were washed free of nonbound protein with Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 0.05% Tween-20 and 2 mM CaCl₂ (TBS-T/Ca). The binding assays were performed at room temperature as previously described for binding of SP-D to maltosyl-BSA (29). Wells were first blocked with 1% BSA in TBS-T/Ca for 2 h and washed. Next, SP-D was added to the wells with or without competitors or inhibitors at the indicated concentrations. After 1 h of additional incubation, nonbound SP-D was washed away, and the bound proteins were cross-linked with 0.125% glutaraldehyde in phosphate-buffered saline for 15 min, after which unreacted cross-linking sites were reacted with 1 M ethanolamine. An affinity-purified rabbit antirat SP-D/CRD polyclonal antibody was then added (1 µg/ml) and incubated for 1 h. The plates were washed and a commercial secondary goat antirabbit immunoglobulin conjugated to horseradish peroxidase (1:4,000 dilution; Southern Biotechnology Associates., Inc., Birmingham, AL) was placed on the wells for an additional hour. The wells were washed and o-phenylene diamine substrate added (Sigma). Absorbance was quantified at 450 nm after 20 min of reaction on a multichannel plate reader. In additional studies, the binding of SP-D to gpA was compared with SP-D binding to the neoglycoproteins mannosyl-BSA and glucosyl-BSA. Assays were performed in an identical fashion with these glycoconjugates as substrates. The coating efficiencies of gpA, mannosyl-BSA, and glucosyl-BSA to the plastic plates were determined by ¹²⁵I radiolabeling the proteins and directly measuring binding efficiencies. The coating efficiencies were found to be comparable (gpA, 8.6 ± 0.6; mannosyl-BSA, 8.4 ± 0.6; glucosyl-BSA, 8.5 ± 0.9% adsorbed to the plate; P = 0.9, not significantly different).

In competition SP-D binding assays, carbohydrates or ethylenediaminetetraacetic acid (EDTA) were added to gpA-coated wells together with SP-D at the indicated concentrations. When assessing the divalent cation requirement for SP-D binding to gpA, the binding medium was depleted of metals by passage over Chelex 100 (Bio-Rad). The SP-D used in these experiments was also extensively dialyzed against the metal-free buffer. The pH requirements for calcium-dependent binding to gpA was tested in the wells prewashed with buffers at different pHs before addition of SP-D at the corresponding pH (25).

Preparation of Natural Rat SP-D

Natural rat SP-D was isolated from the 10,000 × g supernatant of bronchoalveolar lavage fluid obtained from rats with either silica-induced alveolar lipoproteinosis or P. carinii-induced pneumonia. SP-D was purified by affinity chromatography on maltosyl-agarose as previously reported (35, 36). The purity of SP-D preparations was verified by SDS-polyacrylamide gel electrophoresis (PAGE) and silver staining. Natural recombinant dodecameric SP-D (rSP-D) preparations demonstrated a single 43-kD band under reducing conditions. Previously published studies reveal that the majority of natural SP-D obtained from normal or silica-treated rats is in the dodecameric form, with less than 5 to 10% of the material representing higher order multimers of dodecameric forms (26).

Preparation of Recombinant Rat SP-D Molecules

To evaluate the specific binding of component domains of SP-D, as well as the role of multiple CRD motifs in facilitating binding of SP-D to P. carinii gpA, a series of recombinant rat SP-D proteins was tested as follows. First, a recombinant truncated rat SP-D CRD molecule (rSP-D/CRD) was expressed as a fusion construct in Escherichia coli. Prior studies of bovine and human SP-D have revealed that the neck region mediates trimerization of the CRD (37, 38). Thus, the CRD and the contiguous neck region of rat SP-D (653 to 1,147 bp), representing glycine²¹⁰ through phenylalanine³⁷⁵ of the mature rat SP-D amino acid sequence, were amplified from a full-length rat SP-D cDNA by polymerase chain reaction (PCR), with the addition of NcoI and NheI restriction enzyme sites (24). This product was cloned into the pET-25b(+) expression vector with an inducible lac promoter (Novagen). The BL21(DE3) E. coli host with low stringency T7 polymerase was transformed, and protein expression induced with 10 mM isopropylthiogalactopyranoside (IPTG) for 3 h at 30°C. The induced cells were collected by centrifugation and stored at 70°C. Recombinant proteins were isolated from the cell pellets by resuspending in TBS/Ca²⁺ containing 100 µg/ml lysozyme and 0.1% Triton X-100. The suspensions were sonicated and centrifuged at 39,000 × g for 20 min to separate soluble from insoluble proteins. The soluble protein fraction in TBS/Ca was loaded onto 30 ml maltosyl-toyopearl affinity chromatography column. These affinity columns were prepared by coupling maltose to AF-Amino Toyopearl 650M resin (100 µm maltose per gram of dry resin; TosoHaas, Montgomeryville, PA) by reductive amination method, according to the manufacturer's instructions. Maltose-bound fractions were EDTA eluted, pooled, and dialyzed against TBS/ Ca²⁺, and concentrated. Amino-acid determination and amino-terminal sequence analysis of the final product confirmed the expected peptide composition.

A full-length rat rSP-D was also tested for its ability to bind gpA. This construct was derived from rat SP-D cDNA stably expressed in chinese hamster ovary K1 cells and isolated as dodecameric SP-D as previously described by Crouch and coworkers (28). The protein comigrated with natural rat SP-D on SDS-PAGE under reducing and nonreducing conditions, bound efficiently to maltosyl-agarose, and predominantly coeluted with natural rat SP-D dodecamers in nondenaturing gel filtration over 4% agarose (28).

To assess the binding of full-length SP-D monomers that assemble into homotrimers, but do not form dodecamers, a full-length mutant was evaluated that contained serine substitutions for the cysteines which mediate disulfide cross-linking in the generation of dodecamers (rSP-D-ser15/20), as originally reported by Brown-Augsburger and colleagues (39). This construct was generated by site-directed mutagenesis performed on the full-length rat SP-D cDNA clone, using the PCR overlap extension method, and primers for substituting serine for Cys-15 and Cys-20 of the mature protein. The mutated protein bound to maltosyl-agarose but, unlike unmutated recombinant molecule, was assembled exclusively as trimers in comparison to rSP-D on gel filtration (39).

Characterization of the Degree of Protein Oligomerization in SP-D Preparations

To gain additional information as to the state of oligomerization of various SP-D preparations, samples of SP-D material were also studied by gel filtration chromatography as previously described (29). In brief, a 1.5 × 100 cm BioGel A15 sizing column (Bio-Rad) was prepared. The column was equilibrated with buffer containing 50 mM Tris, 150 mM NaCl, 10 mM EDTA, pH 7.4, and known molecular size proteins (1,350 to 670,000 D, Bio-Rad standards) were applied and eluted in the same buffer to derive a standardized size elution profile. Subsequently, samples of natural SP-D derived from P. carinii-infected rats, SP-D derived from silica-treated rats, and recombinant SP-D CRD were applied and eluted with continuous monitoring of the eluant at A280.

Generation of Polyclonal Antirat rSP-D/CRD Antibody

For SP-D detection in the solid-phase binding assays, a polyclonal antirat rSP-D/CRD was generated in rabbits according to standard methods. Two New Zealand white female rabbits were prebled for the generation of pre-immune sera and a day later injected subcutaneously at multiple sites with 500 µg of recombinant rat SP-D/CRD in complete Freund's adjuvant. Booster injections using incomplete Freund's adjuvant were administered every 4 to 6 wk intramuscularly. Animals were bled 10 to 12 d after the antigen injection. Serum was separated and affinity-purified on a column of rSP-D/CRD coupled to CNBr-activated sepharose 4B, yielding a stock of antibody at 476 µg/ml.

Data Analysis

Differences in the extent of SP-D binding under different conditions and in the presence or absence of various competitors or inhibitors were assessed using the two-tailed Student's t test. Statistical testing was performed using the Statview II statistical package (Abacus Concepts, Berkeley, CA) with P  0.05 defining a statistically significant difference.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

SP-D Binds Directly to P. carinii gpA in Solid Phase

Ligand blot analyses performed in our laboratory indicate that gpA represents a major molecular target recognized by SP-D (8). We next sought to determine the mechanisms of SP-D interactions with this major P. carinii surface antigen. Earlier studies demonstrate that P. carinii gpA contains approximately 10% N-linked carbohydrate by weight and is particularly rich in glucosyl and mannosyl residues, both of which may bind to SP-D (21, 30). Accordingly, we hypothesized that P. carinii gpA binds to the CRD of SP-D through N-linked carbohydrates on gpA. To further define these interactions, we studied the binding of natural and recombinant forms of rat SP-D to purified gpA using a solid-phase binding assay modified from that described by Persson and associates (29) for SP-D binding to neoglycoproteins.

Natural rat SP-D isolated from silicotic rats demonstrated concentration-dependent binding to immobilized gpA (Figure 1). SP-D concentrations as low as 1 ng/ml bound efficiently to gpA in solid phase (P = 0.038 comparing total with nonspecific binding measured in the presence of EDTA). Furthermore, concentration-dependent increases in SP-D binding to gpA were noted over the range of 0 to 5 µg SP-D/ml. The binding of SP-D to gpA was also saturable with no significant difference occurring between binding at 5 and 10 µg SP-D/ml (P = 0.375, not significantly different; Figure 2). The gpA preparations were assayed to contain less than 0.125 U/ml of endotoxin activity using a sensitive and specific Limulus amebocyte lysate assay (8, 20). This is equivalent to less than 0.125 × 10³ µg/ml of lipopolysaccharide (LPS). In separate control experiments, direct binding assay of 0.125 × 10⁶ µg/ml of LPS to plastic was well below the limit of detection for SP-D binding (10 µg/ml) in the solid-phase assay. Thus, LPS contamination did not account, in any significant manner, to the observed binding of SP-D to gpA in these experiments.

View larger version (16K): [in this window] [in a new window]   Figure 1.   Natural SP-D binds to immobilized P. carinii gpA. Increasing concentrations of purified natural SP-D were added to gpA immobilized to plastic. Shown is binding of SP-D (mean ± SEM) to gpA in the absence (solid columns) or in the presence of 10 mM EDTA (gray columns). SP-D significantly binds to gpA in solid phase at all SP-D concentrations tested. EDTA significantly abolished SP-D binding over the entire range of concentrations. Shown are the mean ± SD of triplicate determinations of a representative experiment repeated on eight occasions.

View larger version (18K): [in this window] [in a new window]   Figure 2.   Comparison of the binding of SP-D and neoglycoproteins to immobilized P. carinii gpA. Natural SP-D derived from silicotic rats was permitted to bind to immobilized purified gpA (open squares), mannosyl-BSA (solid diamonds), or glucosylated-BSA (solid squares) (each prepared by allowing 50 µg/ml of the binding substrate to coat the plastic). Shown is SP-D binding to each of these substrates (mean ± SEM). SP-D binding to gpA was significantly higher than was binding to neoglycoproteins at all concentrations greater than or equal to 0.1 µg/ml (P < 0.05 comparing binding of SP-D to the neoglycoproteins). Shown are the mean ± SD of triplicate determinations of a representative experiment repeated on three occasions.

In addition, interaction of SP-D with gpA also required the presence of divalent cations. The addition of EDTA (20 mM) inhibited the binding to less than 18% of the maximal binding throughout the range of SP-D concentrations tested (P = 0.011 comparing binding of 10 µg SP-D in the presence and absence of EDTA; Figure 1). Interestingly, the capacity of gpA to interact with SP-D in this binding assay was shown to be approximately five times higher than that of the synthetic neoglycoproteins mannosyl-BSA and glucosyl-BSA (~ 30 mol sugar/mol BSA). This increased binding was not due to enhanced coating of gpA compared with the neoglycoproteins, as ¹²⁵I labeling of the proteins documented comparable coating efficiencies of gpA and the two neoglycoproteins examined. This enhanced binding to gpA may be due to either a higher glycosylation state of gpA or to a higher binding efficiency of SP-D to this molecule (Figure 2). Taken together, these data indicate that rat SP-D binds efficiently to immobilized purified P. carinii gpA in a concentration-dependent, saturable manner requiring divalent cations.

SP-D Binding to gpA Requires Calcium and Is Optimal at Physiologic pH

As noted previously, the lectin activity of SP-D binding to glycoconjugates has been derived primarily from binding assays performed using neoglycoproteins (29). To date, no studies of the divalent cation or pH requirements of SP-D binding to microbial ligands such as gpA have been reported. Accordingly, we next sought to determine whether the interactions of SP-D with purified gpA was dependent specifically on the presence of calcium ions as opposed to other divalent cations. To accomplish this, the binding of SP-D (2.5 µg/ml, extensively dialyzed against cation-free buffer) to immobilized gpA was studied in the absence of exogenous cations or in the presence of increasing concentrations of CaCl₂, MgCl₂, or MnCl₂. The addition of as little as 0.3125 mM calcium significantly increased SP-D binding to gpA (P = 0.03 comparing binding with and without calcium; triplicate determinations on two separate experiments). The addition of calcium concentrations greater than 2.5 mM did not further enhance binding. In striking contrast, the addition of increasing concentrations of magnesium or manganese cations did not support SP-D binding to gpA in comparison to the presence of no added cations. These findings indicate that SP-D interactions with gpA specifically require calcium cations at physiologic concentrations, consistent with collectin-type binding through the CRD of SP-D.

In a similar manner, we examined the binding of SP-D to gpA over a wide range of pH conditions. In these assays, wells coated with gpA were washed with pH-controlled buffers before the addition of SP-D at the corresponding pH. Appropriate wash and incubation buffers were prepared by titration of TBS to the indicated pH with 1 N NaOH or HCl. Maximal SP-D binding occurred at the physiologic pH of 7.4 but was relatively stable over a wide range of pH. For instance, decreasing the pH of SP-D/gpA interaction to 5.0 or increasing the pH to 8.0 still permitted 70% of the maximal SP-D binding, which occurred at pH 7.4. Only at a pH of 4.0 was SP-D binding to gpA strongly inhibited, with all SP-D concentrations tested. Binding of SP-D (5 µg/ml) at pH 4.0 was inhibited by 94.7 ± 1.3% compared with binding at pH 7.4 (P < 0.05; triplicate determinations representative of three separate experiments). To exclude the possibility that washing the plates with low pH might have caused dissociation of gpA from the plastic, in additional experiments gpA was immobilized on the wells and washed twice with buffers at either pH 4.0 or pH 7.4. Subsequently, the remaining bound gpA was detected by ELISA using the LM-1 monoclonal antibody recognizing gpA, as we previously described (4). Washing the plate with a buffer of pH 4.0 resulted in no discernable dissociation of gpA from the plate compared with washing the plate with a buffer of pH 7.4. Thus, low pH conditions appear to be directly inhibitory of the interaction of P. carinii gpA with SP-D.

Specific Sugars Inhibit the Binding of SP-D with P. carinii gpA

To better define the specificity of SP-D/gpA interactions, we determined the ability of different sugars to competitively interfere with SP-D binding to gpA in the solid phase (Figure 3). Maltose, a glucosyl-glucose disaccharide, was most effective, reducing SP-D binding to gpA to less than 50% of maximal with concentrations as low as 0.391 mM. Glucose was less effective than maltose, requiring 0.6 mM of the sugar to reduce the binding to less than 50% of maximal. Mannose was further less effective, with a 2-mM concentration reducing the binding to less than 50% maximal. Finally, N-acetyl-glucosamine was the least effective inhibitor, with 6 mM being necessary to observe a greater than 50% reduction in binding. These results demonstrate that SP-D binds to gpA, a natural biologically important ligand, in a manner compatible with CRD sugar ligand preferences observed with neoglycoproteins such as maltosyl-BSA (29).

View larger version (19K): [in this window] [in a new window]   Figure 3.   The binding of natural SP-D to immobilized gpA is selectively inhibited by specific sugars. Natural SP-D (2.5 µg/ml) was permitted to bind to immobilized gpA in the presence of increasing concentrations of competing sugars. The following sugars were evaluated: maltose (open squares), glucose (solid diamonds), mannose (solid squares), and N-acetyl-glucosamine (open diamonds). Competitive inhibition of SP-D binding to gpA displayed the following inhibitory potencies: maltose>glucose>mannose>>N-acetyl-glucosamine. Each data point represents the mean ± SD of triplicate determinations representative of at least five separate experiments.

Generation of a Recombinant Rat rSP-D/CRD

To further study the role of the SP-D/CRD in mediating interactions with P. carinii gpA, a rSP-D/CRD was expressed in E. coli. The CRD and contiguous neck portions of rat SP-D DNA were expressed as a histidine-tagged fusion protein construct in E. coli as described previously (Figure 4). The rSP-D/CRD protein was isolated from the soluble fraction of IPTG-induced E. coli cell lysates and purified to homogeneity by maltose affinity chromatography. Immunoblot analysis of the purified rSP-D/CRD protein eluted from the maltosyl-toyopearl affinity column was performed with an existing whole molecule rabbit antirat SP-D antibody as previously reported, verifying the immune reactivity of the purified rSP-D/CRD material (8). Automated amino-acid analysis and N-terminal amino-acid sequencing of the rSP-D/CRD confirmed the expected primary structure of recombinant protein and molecular mass of 23.5 kD. The expressed rSP-D/CRD encoded a predicted SP-D related peptide with a relative molecular mass of 20.8 kD. The recombinant protein migrated at a molecular weight of 23.5 kD, representing the additional HSV-Tag segment and six histidine residues at the carboxyterminus of the expressed fusion protein, provided by the pET-25b(+) vector.

View larger version (29K): [in this window] [in a new window]   Figure 4.   Expression of rSP-D/CRD in E. coli. A recombinant fusion protein construct encoding the CRD and neck region of rat SP-D cDNA was generated in the pET-25b(+) expression vector with an IPTG-inducible lac promoter. E. coli was transformed and selected for this construct. Equal volumes of lysates from uninduced E. coli (lanes 1 and 2) and IPTG-induced (lanes 3 and 4) E. coli cultures were separated by 12% SDS-PAGE under reducing conditions and immunoblotted with polyclonal rabbit antirat SP-D (11). Lanes 1 and 3 represent the insoluble protein fractions from these lysates, whereas lanes 2 and 4 contain soluble proteins. The majority of the recombinant protein is present in soluble protein fraction of the IPTG-induced E. coli (arrow). The rabbit polyclonal antibody generated using complete Freund's adjuvant recognizes some additional E. coli proteins in both the induced and uninduced cell lysates. Shown is a representative blot, repeated on at least three separate experiments.

Prior studies of bovine SP-D have demonstrated that the neck region is necessary for oligimerization of the CRD into trimers (37). Thus, to begin to evaluate the degree of oligomerization state (monomeric, dimeric, or trimeric SP-D) contained in our recombinant rat CRD preparation (rSP-D/CRD), the expressed protein was cross-linked in situ with the homobifunctional cross-linking agent BS³ and analyzed by SDS-PAGE under nonreducing conditions (Figure 5). Under reducing SDS-PAGE conditions, most of the rSP-D/CRD migrates as monomeric (24 kD) protein, with only trace amounts of 48-kD dimers and 72-kD trimers. In contrast, BS³ cross-linked rSP-D/CRD migrated in three major forms, as cross-linked trimeric, dimeric, and monomeric forms. This finding strongly suggests that recombinant SP-D CRD and neck regions have the ability to self-associate into oligomers containing trimeric CRD regions, even in the absence of the remainder of the SP-D collagenous domain. Gel filtration chromatography further revealed that the rSP-D/CRD material contains 60% trimers, 18% dimers, and 22% monomers, with no detectable higher ordered forms.

View larger version (51K): [in this window] [in a new window]   Figure 5.   Structural analysis of rSP-D/CRD. To determine the relative amounts of rSP-D/CRD present in monomeric, dimeric, and trimeric forms, the recombinant protein was cross-linked in situ with BS3, a homobifunctional cross-linker. (Lane A) Molecular weight standards. (Lane B) Noncross-linked rSP-D/CRD (2 µg) examined on 12% SDS polyacrylamide gel (silver staining). (Lane C) Shown is rSP-D/CRD cross-linked with BS3 and separated on 12% SDS polyacrylamide gel (silver staining). Cross-linking analysis demonstrates that rSP-D/CRD contains cross-linked monomeric, dimeric, and trimeric forms. The samples in lanes A and B were run in the presence of -mercaptoethanol reduction. Because of the presence of the cross-linking agent, the sample in lane C was run under nonreducing conditions. The apparent reduced relative molecular mass (Mr) of the monomer in lane C is likely related to cross-linking the protein into a more compact conformation under nonreducing conditions, resulting in more rapid migration.

This purified rSP-D/CRD protein was further used to generate a specific anti-rSP-D/CRD antibody. Immunoblot analysis of the purified rSP-D/CRD with affinity-purified rabbit polyclonal antirat rSP-D/CRD is shown in Figure 6. This specific polyclonal rabbit antirat SP-D/CRD antibody was used for all data shown in Figures 1-3 and 6-8.

View larger version (57K): [in this window] [in a new window]   Figure 6.   Immunoblot analysis of rSP-D/CRD with a specific antirat rSP-D/CRD antibody. To further verify the identity of the rSP-D/CRD, the expressed protein (2 µg) was electrophoresed on 12% SDS-polyacrylamide gel under reducing conditions and immunoblotted with antirat SP-D/ CRD antibody (1 µg/ml). (Lane A) Molecular weight standards. (Lane B) The rSP-D/CRD strongly reacted specifically with the specific antirat SP-D/CRD antibody. Under these denaturing and reducing conditions, the protein was predominantly visualized in the monomeric form. Shown is a representative blot, repeated on at least three occasions.

View larger version (16K): [in this window] [in a new window]   Figure 7.   The binding capacity of various recombinant forms of SP-D to immobilized P. carinii gpA varies with the number of SP-D CRDs present in each molecular form. Recombinant SP-D forms were permitted to bind to immobilized gpA. The following forms were evaluated: dodecameric rSP-D (open squares), trimeric rSP-D-ser15/20 (solid diamonds), and rSP-D/CRD (closed squares), which at least in part represents trimers. The binding capacity of the recombinant SP-D forms was greatest for the dodecameric rSP-D, intermediate for trimeric rSP-D-ser15/20, and least for the rSP-D/CRD form. Significant binding of all three forms was observed at concentrations  5 µg/ml of the SP-D forms (P < 0.05 compared with nonspecific binding control). Shown are the mean ± SD of a representative experiment from two separate experimental runs performed in triplicate.

View larger version (16K): [in this window] [in a new window]   Figure 8.   The binding capacity of natural SP-D forms isolated from P. carinii and silica-induced rats are greater than the binding of recombinant SP-D forms. Natural SP-D isolated from P. carinii-infected and silica-induced rats were permitted to bind to immobilized gpA and compared with the binding of the various recombinant forms of SP-D. The following forms were evaluated: P. carinii-induced natural SP-D (open squares), the silica-induced natural SP-D (solid circles), dodecameric rSP-D (solid squares), trimeric rSP-D-ser15/20 (open circles), and rSP-D/CRD (open triangles). The binding capacity of SP-D isolated from silica-induced rats representing dodecameric and higher order forms of SP-D was greater than the binding of recombinant SP-D molecules. Furthermore, the binding of SP-D isolated from P. carinii-infected animals was significantly greater than the binding capacity of the SP-D derived from silica-induced rats (P = 0.0071 comparing binding at 5 µg/ml of the SP-D form). Thus, SP-D forms present in the lungs of diseased animals have enhanced capacity to bind to gpA present on the surface of the organisms. Shown are the mean ± SD of a representative experiment from two separate experimental runs performed in triplicate.

Binding of Natural and Recombinant Rat SP-D to P. carinii gpA Is Related to the Physical Form of the SP-D Molecule

It has been reported that the ability of SP-D to aggregate microorganisms and to protect phagocytes from deactivation increases with a higher polymerization state of the collectin (27, 31, 36). Complete oligomerization into dodecamers is also important for effective binding of SP-D to neoglycoproteins such as mannosyl-BSA (40). Accordingly, we next sought to define the ability of various physical forms of SP-D, including SP-D trimers, SP-D dodecamers, and higher order SP-D complexes (multimers of dodecameric forms) to interact with gpA, a significant biologically active ligand. To study this, gpA binding of rSP-D/CRD was compared with the binding of full-length trimeric SP-D molecules that contain serine substitutions for the cysteines required for dodecamer formation (rSP-D-ser15/20) (39). In parallel, we assessed the binding of recombinant full-length SP-D preparations comprised predominantly of dodecamers (rSP-D), prepared as previously reported (28). rSP-D showed the greatest observed binding, whereas cysteine-mutant trimers (rSP-D-ser15/20) bound to gpA to a lesser degree (Figure 7; P = 0.023 comparing the binding of 5 µg/ml of recombinant dodecameric and cysteine mutant SP-D preparations). Furthermore, although the rSP-D/CRD truncated molecule, containing a majority of CRD trimers, showed significant binding at the higher concentrations tested, it exhibited the least ability to bind P. carinii gpA under these conditions. Thus, the overall ability of SP-D to bind immobilized gpA was strongly associated with the physical form of the SP-D molecule studied, with greater binding attributed to dodecameric forms than to trimeric or lower order forms of SP-D.

Finally, the interactions of these recombinant SP-D forms to P. carinii gpA were compared to the binding of natural rat SP-D derived from silicotic rats (identical to material used in Figures 1-4) and natural rat SP-D derived from P. carinii-infected animals (Figure 8). Although the majority of SP-D isolated from silica-treated rats is in the dodecameric form, a fraction of this material is present as higher order multimers of dodecamers (26, 36, 41). Such natural SP-D preparations from silica-induced SP-D, which have been previously reported to contain a fraction of these higher order SP-D forms, also exhibited substantially higher binding compared with recombinant dodecamers and the other simpler recombinant SP-D molecules over the entire range of SP-D concentrations tested. Interestingly, SP-D isolated from P. carinii-infected animals demonstrated even more effective binding to immobilized gpA than did SP-D preparations derived from silica-treated rats (P = 0.0071 comparing SP-D binding at 5 µg/ml). To further verify that natural SP-D preparations did not exhibit increased adherence to the plates and thus give higher background binding to the plate compared with recombinant SP-D preparations with no gpA present on the plate, we also directly assessed binding of natural SP-D and dodecameric rSP-D to the plastic plate in the absence of gpA coating. We observed equal binding of the natural SP-D and recombinant SP-D to the plastic surface in the absence of gpA. Thus, the increased binding of natural SP-D to the gpA-coated plates was related to specific interaction of SP-D with the ligand rather than increased background binding to the plastic.

We further questioned whether the affinity-purified antibody used in the solid-phase binding assay might also exhibit differential recognition of various forms of SP-D and hence exhibit differential binding on that basis. To address this, various forms of SP-D (silica-induced natural rat SP-D, P. carinii-induced natural SP-D, dodecameric rSP-D, rSP-D-ser15/20, and rSP-D/CRD) were each coated onto plastic wells (10 µg/ml) and directly tested for their binding to the anti-SP-D antibody at a variety of antibody concentrations (10, 5, 1, and 0.5 µg/ml). At every concentration tested, antibody binding to each of the natural and recombinant SP-D forms was entirely equivalent. Thus, we conclude that the observed differences in binding are not the result of differential recognition of various SP-D forms by the antibody. Instead, we believe that the observed differences in binding represent the increased presence of dodecameric polymers and higher order SP-D forms in certain natural SP-D isolates.

To further address the extent to which the presence of such higher order SP-D forms in natural SP-D preparation might influence the binding of SP-D to gpA, gel filtration chromatography was performed on samples of natural SP-D derived from rats infected with P. carinii and rats treated with silica. SP-D preparations derived from P. carinii-infected rats contained 9.1% higher order SP-D forms, with the remainder representing dodecameric forms. In contrast, SP-D isolated from silicotic rats contained 4.0% of the higher order SP-D. As noted previously, the SP-D derived from P. carinii-infected animals and containing the increased amounts of higher order SP-D aggregates exhibited higher binding to gpA than did the SP-D derived from silicotic rats. Both the natural SP-D preparations bound to a greater extent than did the dodecameric recombinant SP-D (Figure 8). These data suggest that the extent of higher order multimers present in SP-D isolates positively influences the binding of SP-D to natural ligands such as gpA. These observations further suggest that during pathologic conditions such as P. carinii pneumonia, SP-D may exist to a greater extent in higher order forms that are more effective in their interactions with the organism.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our earlier studies have demonstrated that SP-D, a lung collectin, accumulates significantly in the lung during P. carinii pneumonia, binds in abundant amounts to the surface of the organisms, and modulates interactions of the organisms with alveolar macrophages (8). Previous ligand blot analyses further indicate that gpA represents a major protein target that interacts with SP-D (8). Our current study provides strong biochemical evidence that the CRD of SP-D is principally responsible for mediating interactions with P. carinii gpA.

Biochemical parameters supporting lectin binding of SP-D have not been extensively studied using biologically relevant fungal ligands such as gpA. The binding of SP-D to purified gpA requires the presence of divalent cations (8). More specifically, the interactions of SP-D with immobilized gpA were found to be dependent on the presence of calcium ions because other divalent cations such as magnesium and manganese did not significantly support SP-D binding. Furthermore, the interaction of SP-D with purified solid-phase gpA exhibited concentration-dependent, saturable binding optimally occurring over a rather broad pH range between pH 5 and 8. These findings are parallel with CRD-dependent binding of SP-D to neoglycoproteins (29, 40, 41). Interestingly, however, SP-A has been reported to retain its calcium-dependent binding activity down to pH 5.0 (42). Differences in glycoconjugate binding of SP-A and SP-D at lower pH may have important implications for the stability of collectin-ligand complexes within intracellular compartments.

Further evidence for the role of the CRD of SP-D in mediating interactions with P. carinii gpA is provided by the specific inhibitory capacities of various sugars. Using site-directed mutagenesis of conserved residues, Ogasawara and Voelker (40) have demonstrated that the CRD of SP-D uses a mannose-type saccharide binding site. Additional studies by Persson and coworkers (29) have demonstrated that SP-D CRD binding to neoglycoproteins is specifically inhibited by maltose>glucose>mannose>N-acetyl glucosamine. P. carinii gpA is a surface-expressed glycoprotein complex encoded by a family of genes. Biochemical analysis of purified gpA indicates that a significant fraction of this complex is N-linked carbohydrate chain rich in glucose, mannose, and N-acetyl-glucosamine (21, 30). Accordingly, we tested the inhibitory capacities of these sugars cognate to P. carinii gpA and found the inhibitory potency to also exhibit the order of maltose>glucose>mannose>N-acetyl-glucosamine, again supporting the conclusion that the CRD of the SP-D molecule is responsible for gpA interactions. The inhibitory potencies of these various competitive sugars for SP-D binding to gpA were parallel but required substantially lower concentrations than those previously observed for neoglycoproteins (29). This might reflect the relative availability of specific sugar residues on the surface of gpA.

Interestingly, our data further indicate that the physical form of SP-D strongly influences its ability to bind to P. carinii gpA. A recombinant molecule comprised of the SP-D CRD and neck regions (rSP-D/CRD) exhibited weak, but significant, binding to immobilized gpA, at a level comparable to a recombinant full-length trimeric SP-D (rSP-D-ser15/20). The minor difference in binding abilities between these two molecules likely represents that rSP-D-ser15/20 consists exclusively of trimers, whereas rSP-D/ CRD contains a mixture of trimers, dimers, and monomeric forms. Prior studies indicate that the lack of a full collagenous domain in the rSP-D/CRD molecule is not likely to be responsible for the lesser binding. Consistent with our findings, Ogasawara and Voelker (40) compared two molecules that lack collagenous domain and also found that the degree of oligomerization is important in rat SP-D binding to the sugars in solid phase. In their study, a collagen domain deletion mutant, present mostly as trimers, bound mannosyl-BSA significantly, whereas a collagenase-resistant fragment, present in monomeric state, did not bind to the sugar substrate at all. Significant binding to glycoproteins of both the rSP-D/CRD in our current study and the collagen domain deletion mutant in the prior study by Ogasawara and Voelker (40) strongly indicate that collagenous domain of SP-D is not essential for this interaction.

Of further note, comparison of the binding of oligomeric SP-D (rSP-D/CRD and SP-D-ser15/20) to recombinant dodecamers (rSP-D) revealed that the observed binding of SP-D polymers (dodecamers) to gpA was qualitatively higher than when the simpler recombinant forms of the SP-D molecule were tested. Current concepts suggest that trimeric CRDs interact with substrates containing saccharide in a planar fashion. We postulate that enhanced binding may be related to increased numbers of CRD binding sites that are available in such forms of SP-D. Thus, the presence of multiple trimeric CRD regions on a given SP-D form might permit multiple and, hence, collectively more avid interactions with the ligand. Keeping this in mind, the greatest binding was achieved with the two naturally derived SP-D preparations, from silica-treated and P. carinii-infected rats. Silica-derived SP-D preparations are known to contain a significant portion of higher order multimers of dodecameric SP-D forms (26, 41). SP-D derived from P. carinii-infected lungs exhibited even greater degrees of observed binding to the gpA ligand of the organisms. Thus, the enhanced binding of natural SP-D in the setting of P. carinii infection also appears to correlate with increased amounts of higher order SP-D multimers under these conditions. One might further hypothesize that forms of SP-D with greater abilities to bind biological ligands may be differentially expressed under various conditions or that the inflammatory milieu may favor SP-D aggregation into higher order forms, which further promotes SP-D binding to host ligands. This remains unproven, and further studies will be necessary to quantitatively address these postulates.

The biological consequences of SP-D binding to P. carinii gpA are only partially understood. Our prior investigations demonstrate that SP-D strongly mediates adherence of P. carinii to the surface of alveolar macrophages. However, SP-D does not promote phagocytosis by these cells. Indeed, recent investigations suggest that SP-D significantly promotes P. carinii self-association, resulting in large aggregates of organisms that may exhibit impaired uptake by macrophages (19). As such, SP-D binding to the P. carinii surface may represent a means by which the organisms evade host elimination.

The binding of SP-D to gpA may represent the first of a number of interactions of P. carinii with this collectin. For instance, recent studies have revealed that SP-D additionally binds to fungal -glucans present on cystic forms of the organism (43). Such -glucans are potent stimulators of macrophage inflammatory activation, particularly tumor necrosis factor  release (44). SP-D interactions with P. carinii -glucans may additionally modulate these host inflammatory functions to the further benefit of the organism. In addition, P. carinii has also been previously noted to have other lectin binding activity (13). This raises the further possibility that a surface lectin on P. carinii may also interact with N-linked glycosylation of SP-D and other collectins. For example, SP-A appears to interact with the influenza A virus through such a microbial lectin binding to glycoconjugates on the collectin (45). Clearly, additional studies will be required to further define such potential interactions and consequences of SP-D binding to P. carinii.

In summary, we have shown that SP-D strongly interacts with gpA, a major glycoprotein antigen on the surface of P. carinii. The interaction of SP-D with P. carinii gpA is principally mediated by the CRD of the collectin. Furthermore, SP-D interactions with P. carinii gpA are markedly enhanced in dodecameric and higher order forms of SP-D. Additional studies of the consequences of SP-D/gpA interactions are ongoing to define the pathogenic role of this major host-organism interaction during the development of P. carinii pneumonia.