Interaction of Surfactant Protein A with Peroxiredoxin 6 Regulates Phospholipase A2 Activity*

Peroxiredoxin 6 (Prdx6) is a “moonlighting” protein with both GSH peroxidase and phospholipase A2 (PLA2) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA2 activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA2 activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA2 activity) showed Km0.35 mm and Vmax 138 nmol/min/mg of protein. SP-A inhibited PLA2 activity non-competitively with Ki 10 μg/ml and was Ca2+ -independent. Activity at pH 7.4 was ∼50% less, and inhibition by SP-A was partially dependent on Ca2+. Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni2 -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca2+ dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA2 activity of Prdx6 by SP-A.

Peroxiredoxins (Prdxs) are a recently described superfamily of Se-independent peroxidases that are distributed in all phyla (4). They are classified according to the number (1 or 2) of conserved cysteine (Cys) residues directly involved in peroxidase catalysis. Of the six mammalian peroxiredoxins, Prdx6 has a single conserved Cys, whereas Prdx1-5 are 2-Cys enzymes. Prdx6 is expressed in various tissues, but is especially enriched in lung and brain (5,6). By immunocytochemistry, Prdx6 in lung is present in alveolar type II cells, alveolar macrophages, and bronchiolar epithelium (6), and subcellular fractionation of lungs has demonstrated the presence of this protein in lamellar body, lysosomal, and cytosolic fractions (7). Prdx6 functions to protect cells against oxidant stress by glutathione-dependent reduction of short-chain and phospholipid hydroperoxides into corresponding alcohols (4).
Unlike other members of the peroxiredoxin family, Prdx6 possesses phospholipase A 2 (PLA 2 ) in addition to peroxidase activity (6,8). PLA 2 enzymes hydrolyze phospholipids at the sn-2 position leading to the release of lysophosphatidylcholine and a fatty acid. PLA 2 s have been classified as cytosolic PLA 2 , secreted PLA 2 , or calcium-independent PLA 2 . Because the PLA 2 activity of Prdx6 is Ca 2ϩ -independent and maximal at acidic conditions (pH 4), it has been named acidic calciumindependent PLA 2 (aiPLA 2 ). aiPLA 2 activity is inhibited by the competitive transition state inhibitor MJ33 (9). The involvement of Prdx6 in lung [ 3 H]DPPC metabolism has been demonstrated using MJ33 in intact rats, isolated perfused rat lungs, and isolated rat alveolar epithelial type II cells (9,10) and more recently, by study of mice with "knock-out" of Prdx6 (11). Inhibited aiPLA 2 function in these models resulted in both decreased degradation of internalized DPPC as well as decreased DPPC synthesis by the reacylation pathway.
Our laboratory has provided evidence that SP-A modulates the aiPLA 2 activity of Prdx6. Addition of SP-A to rat lung homogenate, isolated lamellar bodies, or isolated rat alveolar type II cells inhibited aiPLA 2 activity and the degradation of DPPC (3). Inhibitors of SP-A or SP-A knock-out resulted in increased lung aiPLA 2 activity (3,12). In the present study, we evaluated the effect of SP-A on aiPLA 2 activity of recombinant enzyme and tested the hypothesis that SP-A regulates the PLA 2 activity of Prdx6 through direct protein-protein interaction. The effect of SP-A on Prdx6 activity and their interaction was studied at pH 4 as well as pH 7.4, because our studies have demonstrated that activity is maximal at acidic conditions compatible with a lysosomal localization for DPPC degradation in the lung (13).
Isolation of Proteins and Cells-Human SP-A was obtained and purified from bronchoalveolar lavage fluid of alveolar proteinosis patients (16,17). Recombinant human Prdx6 with or without a His tag was expressed in Escherichia coli and purified by affinity chromatography on a Ni 2ϩ column (4) or by ion-exchange and size-exclusion chromatography (14). The purity of SP-A and Prdx6 protein preparations was confirmed by SDS-PAGE and immunoblot.
Alveolar type II cells were isolated from rat lungs using elastase digestion as described previously (18). Cells were incubated for 1 h on IgGcoated plastic dishes to remove contaminating macrophages. Of the non-adherent cells, 90% were type II cells as indicated by staining with Nile red (Molecular Probes) for the presence of lamellar bodies. Cells were maintained in solution and used immediately for study. Alveolar macrophages were isolated by bronchoalveolar lavage of rat lungs with normal saline (9). The lavage fluid was centrifuged at 100 ϫ g for 120 min, and the cell pellet was resuspended in PBS. Cells were allowed to adhere to a glass coverslip by incubation at room temperature for 1 h and then washed twice with ice-cold PBS before use.
In different experiments, Ca 2ϩ (10 mM) or SP-A in varying concentra-tion was added. As controls, SP-A was boiled, reduced by 0.1 mM mercaptoethanol, or alkylated by 0.5 mM iodoacetimide prior to addition to the assay medium.
SP-A Binding to Agarose Beads-Affi-gel beads (500 l) were washed twice in PBS (pH 7.4), treated twice with ethanolamine (0.1 M) for 10 min at room temperature, then washed four times in PBS followed by three washes with ice-cold H 2 O, equilibrated in PBS for 30 min, centrifuged for 2 min at 10,000 rpm, and resuspended in 500 l of PBS. SP-A (2 g) was incubated with or without Prdx6 (2 g) for 30 min at room temperature in a final volume of 10 l of PBS. Then 100 l of beads was added to each sample, and the mixture was incubated at room temperature for 10 min with slow rotation. In some experiments, anti-Prdx6 pAb (1:400 dilution) was added to the incubation mixture. The beads were washed three times with PBS, and sample buffer with denaturing reagent was added to the pellet. The sample was analyzed by SDS-PAGE and immunoblot.
Pull-down Assay-SP-A (2 g) was incubated for 30 min with Histagged recombinant human Prdx6 (2 g) in PBS to bring the mixture to a final volume of 10 l. This mixture was then added to 100 l of a slurry (ϳ50 l) of Ni 2ϩ beads, incubated for an additional 10 min in the presence of mannose (10 mM), and washed three times with 1 ml of PBS containing 10 mM mannose. Mannose was used to decrease nonspecific binding to agarose, because SP-A belongs to the C-type (Ca 2ϩ -dependent) carbohydrate binding protein family (19,20). After incubation with the protein mixture, the Ni 2ϩ beads were washed three times with PBS. The sample was analyzed by SDS-PAGE and immunoblot.
Co-immunoprecipitation-Co-immunoprecipitation in vitro was performed by incubating recombinant Prdx6 (2 g) and native SP-A (2 g) with either anti-SP-A pAb or anti-Prdx6 pAb for 1 h at 4°C with slow rotation. Proteins in 80 l of binding buffer (50 mM Tris-Cl, 150 mM NaCl, 0.1% Nonidet P-40, and 0.2% BSA) were added to washed protein A-linked agarose beads (20 l) and incubated for 45 min at 4°C under slow rotation. After centrifugation (3,000 rpm, 1 min), the supernatant was removed and the pellet was washed three times with washing buffer (50 mM Tris-Cl, 150 mM NaCl, 0.1% Nonidet P-40). The bound proteins were analyzed by SDS-PAGE and immunoblot. In some experiments, 2.5 mM Ca 2ϩ was added to the binding and washing buffers. A similar protocol was used with protein-A linked to acrylic beads, protein-G linked to agarose beads, and protein-G linked to Sepharose to immunoprecipitate the proteins.
Antibodies to Prdx6 and SP-A were also used to immunoprecipitate proteins from isolated rat type II lung cells. Cells were lysed by incubation with Mammalian Cell Lysate buffer (Sigma) and brief sonication. The cell lysates were centrifuged at 16,000 ϫ g for 15 min at 4°C, and the supernatant was kept at Ϫ80°C in aliquots until use. A Profound TM mammalian co-immunoprecipitation kit from Pierce was used to perform ex vivo immunoprecipitation according to the manufacturer's rec-ommendation. Briefly, 100 l of antibody coupling gel slurry (50-l bed volume) was added to a spin column and washed four times with 0.4 ml of coupling buffer (0.14 M NaCl, 0.008 M sodium phosphate, 0.002 M potassium phosphate, and 0.01 M KCl, pH 7.4). After washing, 100 g of pAb (anti-SP-A or anti-Prdx6) was immobilized on the coupling gel by incubating for 4 h with gentle agitation, and then unreacted gel was quenched using quenching buffer (1 M Tris-HCl, pH 7.4). The supernatant of the type II cell lysate was added to the column, and the mixture was incubated for 2 h with slow rotation at 4°C. The column was washed three times with coupling buffer, and protein bound to agarose beads was eluted using elution buffer (ImmunoPure IgG Elution Buffer, pH 2.8) and analyzed by SDS-PAGE. Control experiments included gel, which was not activated, addition of quenching buffer instead of antibody to the coupling gel, and addition of a non-related Ab (anti-GST pAb) to the coupling gel.
Size-exclusion Chromatography-Size-exclusion chromatography was performed by using an HPLC Alliance 2695 separation unit, a photodiode array detector, and a YMC-Pack Diol column (500 ϫ 8 mm inner diameter, 5-m particle, and 120-Å pore sizes, all from Waters, Milford, MA). The column was calibrated (R 2 ϭ 0.99) with a standard mixture of proteins. The samples containing Prdx6 (15 g) or equimolar SP-A were eluted isocratically (1.0 ml/min) with 0.1 M phosphate buffer (pH 7.0) containing 0.15 M NaCl; absorbency was monitored at 280 nm.
In some experiments, SP-A was incubated with Prdx6 for 1 h at 20°C before loading onto the column. Fractions of interest were collected and analyzed by SDS-PAGE and immunoblot.
Protein Cross-linking-Prdx6 was incubated with sulfo-SBED reagent (Pierce, 1:50, mol/mol) in the dark under constant stirring for 30 min. Excess reagent was eliminated by size-exclusion chromatography (micro Bio-spin 6 column, Bio-Rad). The resulting biotinylated Prdx6 was incubated with equimolar SP-A for 1 h at room temperature with slow stirring in the dark and then illuminated at 365 nm for 15 min. The protein sample was analyzed by SDS-PAGE followed by immunoblot with alkaline phosphatase-conjugated streptavidin.
Static Light Scattering-Static light scattering experiments were performed using a BI-2000SM instrument equipped with an He-Ne, 623.8 nm, 75-milliwatt Melles Griot laser and BI-APD avalanche photodiode detector (Brookhaven Instruments Inc., Holtsville, NY). SP-A (10 g) and Prdx6 (10 g) in 2 ml of buffer were incubated at room temperature under either alkaline (40 mM PBS, pH 7.4) or acidic (40 mM sodium acetate, pH 4.0) conditions. To study the effect of Ca 2ϩ , CaCl 2 was added to the samples at a final concentration of 2.0 mM. The signal of light scattering was accrued at 45°, 60°, 75°, 90°, 105°, 120°, and 135°a ngles. These results were computed as a Stokes radius using Guinier analysis and expressed by the Williams-Watts size distribution program with the BI-2000SM instrument software.
Surface Plasmon Resonance-Studies were performed using a Biacore 3000 apparatus in the Biosensor Shared Resource Facility of the University of Pennsylvania. Prdx6 was covalently coupled via primary amino groups on a CM3 sensor chip surface according to the manufacturer's recommendation. After equilibration of the sensor chip surface dextran with the running buffer (5 mM Ca 2ϩ , 0.005% Tween 20, 10 mM HEPES, pH 7.4), the carboxymethylated matrix was activated with a mixture of an N-ethyl-NЈ-((dimethylamino)propyl)carbodiimide and N-hydroxysuccinimide. Recombinant Prdx6 (10 g/ml in 10 mM sodium acetate, pH 4.8) was injected at a flow of 5 l/min at 25°C. Unreacted groups were blocked by ethanolamine (pH 8.5) followed by washing with 50 mM glycine/HCl (pH 2.0) to remove unbound protein.
Another flow cell for use as a control was subjected to the identical immobilization procedure in the absence of protein. The association of SP-A with immobilized Prdx6 was studied at both acidic (pH 4.0) and alkaline (pH 7.4) conditions with varying concentrations of SP-A or Ca 2ϩ at 25°C in running buffer (10 mM sodium citrate, 300 mM NaCl, 0.005% Tween 20, pH 4.0, or 10 mM HEPES, 0.005% Tween 20, pH 7.4) at a flow rate of 30 l/min. Dissociation of the SP-A⅐Prdx6 complex was performed by an injection of running buffer without protein and plus or minus Ca 2ϩ . BSA was substituted for SP-A as a negative control and anti-Prdx6 pAb was used as a positive control. Data analysis was performed using BIAevaluation software 3.2 RCI (Biacore).  Immunohistochemistry-For light microscopy, freshly isolated rat alveolar type II cells were grown on coverslips for 24 h at 37°C in room air with 5% CO 2 in a humidified incubator. Cells were fixed and permeabilized in methanol/acetone (1:1, v:v) on ice for 5 min. Cells were treated with 5% BSA plus 10% normal goat serum in PBS (pH 7.4) for 1 h to decrease nonspecific antibody binding. Cells were incubated with primary antibody, either anti-SP-A pAb (1:200 dilution), anti-Prdx6 mAb (1:100 dilution), anti-Prdx6 pAb (1:100 dilution), or 3C9 mAb (1:250 dilution), in blocking solution for 3 h. In control experiments, cells were incubated with blocking buffer instead of primary antibody. After washing with PBS (5 min ϫ 5), the cells were incubated for 1 h at room temperature with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:250 dilution) and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:250 dilution) diluted in PBS containing 1% BSA and 2% normal goat serum. After a final washing (5 min ϫ 5 with PBS and 5 min ϫ 2 with dH 2 O), the coverslip was mounted with Vectashield mounting medium (Vector Laboratories) and observed with a confocal microscope (Radiance 2000, Bio-Rad).
For immunofluorescence of alveolar macrophages, cells were fixed and permeabilized with 4% paraformaldehyde containing 0.2% Triton X-100 on ice for 15 min and then were treated with 5% BSA plus 5% normal goat serum and 0.2% Triton X-100 for 1 h. Cells were then incubated with both mouse anti-LAMP1 mAb (1:100 dilution) and rabbit anti-Prdx6 pAb (1:100 dilution) in 1% BSA for 3 h. After washing with PBS, cells were incubated with the fluorescent secondary antibodies for 1 h and examined by confocal microscopy as described above.
For immunogold staining, mouse lungs were cleared of blood by perfusion and fixed by perfusion and endotracheal instillation of ice-cold buffer containing 1.25% glutaraldehyde, 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). After 15 min, lungs were cut into 1 ϫ 1 mm pieces and further fixed for 2.5 h in fixation buffer followed by an additional 1 h in ice-cold 0.1 M sodium cacodylate buffer containing 1% OsO 4 . The lung pieces were dehydrated at Ϫ20°C with 70%, 80%, and 90% acetone, embedded in LR White resin, and cut into ultrathin sections (80 nm) with a Leica Ultracut UCT (Vienna, Austria). Samples were treated with 0.2% sodium borohydride for 10 min, 0.3% glycine for 10 min, and blocking buffer (0.2% cold water fish skin gelatin and 2% Aurion BSA-c in PBS) for 1 h to block any remaining free aldehyde groups and nonspecific binding sites. Samples then were incubated with anti-Prdx6 mAb (1:100) in PBS with 1% Aurion BSA-c for 2 h at room temperature. For control, samples were incubated with PBS buffer containing only 1% Aurion BSA-c. After washing with PBS (5 min ϫ 6), samples were incubated for 1 h at room temperature with 20 nm goldcoupled goat-anti-mouse IgG (1:25), then rinsed with distilled water (5

TABLE 1 Effect of native human SP-A on aiPLA 2 activity of recombinant Prdx6
The assay contained 2.5 g of Prdx6 in a 1-ml incubation volume. Values are mean Ϯ range for n ϭ 2 for each condition.

Addition
Activity Percent of control  Prdx6 Interaction with SP-A min x 6) and stained for 10 min with 2.5% aqueous uranyl acetate. Labeled sections were observed and photographed at 60 kV in a Jeol 100CX electron microscope (Tokyo, Japan) coupled with an AMT advantage HR/HR-B charge-coupled device camera system (Advanced Microscopy Techniques Corp., Danvers, MA).

RESULTS
The PLA 2 activity of recombinant Prx6 and the effect of SP-A were evaluated under acidic (pH 4.0) or alkaline (pH 7.4 or 8.5) conditions. Although we have reported previously that aiPLA 2 activity is trivial at pH Ͼ6 (6 -8), we have subsequently found activity of recombinant protein after the addition of GSH to the incubation medium. 3 PLA 2 activity at both acidic and alkaline conditions was linear with time during a 60-min incubation; activity at pH 7.4 was ϳ50% of the activity at pH 4.0 (Fig. 1). Activity was the same (56 nmol/min/mg of protein) at pH 7.4 ( Fig.1)andpH8.5 (Fig.2).TheadditionofSP-Aresultedinaconcentrationdependent inhibition of PLA 2 activity of Prdx6 by SP-A (Fig. 2). The effect showed a linear relationship to the logarithm of the SP-A concentration. Inhibition was Ca 2ϩ -independent at pH 4.0 but was accentuated by Ca 2ϩ at pH 8.5. In the presence of 50 g/ml SP-A, aiPLA 2 activity was inhibited by 80% under acidic conditions, whereas with alkaline conditions, inhibition was 70% in the presence of Ca 2ϩ and 50% in its absence. The inhibitory effect of SP-A was abolished by its reduction with ␤-mercaptoethanol, inactivation by boiling, or alkylation with iodoacetamide ( Table 1). As a control, BSA had no effect on aiPLA 2 activity of recombinant Prdx6 (Table 1).
Kinetic parameters of the SP-A-aiPLA 2 interaction were investigated by varying both DPPC and SP-A concentrations. Double reciprocal plots showed non-competitive kinetics for the effect of SP-A on the PLA 2 activity of Prdx6 at both acidic and alkaline pH conditions (Fig. 3). Although the calculated V max at pH 4.0 was 2.1 times the value at pH 7.4, the apparent K m for aiPLA 2 activity and the estimated K i for SP-A were similar under the two conditions ( Fig. 3 and Table 2).
To test possible interaction of the two proteins, the effect of Prdx6 on the binding of SP-A to agarose was evaluated. As indicated by Western blot, incubation of SP-A with agarose beads resulted in binding of the protein to beads (Fig. 4), presumably through the SP-A carbohydrate binding domain (21). Preincubation of SP-A with Prdx6 for 1 h before its addition to agarose beads resulted in a concentration-dependent decrease of SP-A binding (Fig. 4A). This effect was reversed by addition of anti-Prdx6 pAb to the SP-A⅐Prdx6 mixture before addition of agarose beads (Fig. 4B).
The interaction of SP-A with Prdx6 was evaluated further by a pulldown assay using Ni 2ϩ -chelated agarose beads to precipitate His-tagged Prdx6. To inhibit nonspecific binding of SP-A, agarose beads were preincubated with mannose (10 mM) to saturate the SP-A binding sites. SP-A when incubated alone with beads in the presence of mannose was found in both the supernatant and pellet (Fig. 5). Recombinant Histagged Prdx6 incubated alone bound specifically to Ni 2ϩ beads (Fig. 5). Incubation of SP-A and Prdx6 together with the Ni 2ϩ -chelated beads resulted in a shift in the SP-A recovery from the supernatant to the pellet (Fig. 5). This result is compatible with a direct interaction of SP-A and Prdx6 with subsequent binding of the complex to the beads via the Prdx6 His tag. The addition of 5 mM Ca 2ϩ to the mixture had no effect on SP-A-Prdx6 interaction (not shown) indicating that the divalent cation is not necessary for Prdx6 binding to the specific binding site.
SP-A-Prdx6 interaction also was studied by co-immunoprecipitation in vitro. In control experiments, Prdx6 did not react with protein-A agarose beads either in the presence or absence of SP-A pAb (Fig. 6A,  upper panel), although nonspecific interaction of SP-A with the beads was noted (Fig. 6B, upper panel). Incubation of Prdx6 with SP-A and anti-SP-A pAb followed by addition of protein-A-agarose beads resulted in precipitation of both SP-A (Fig. 6A, bottom panel) and Prdx6 (Fig. 6A, upper panel). Both proteins also were precipitated when anti-Prdx6 pAb was substituted for anti-SP-A pAb in the incubation mixture (Fig. 6B, upper and lower panels). Similar results were found with use of protein-G-agarose, protein-G-Sepharose, and protein-A acrylic beads to bind the immunoreactive proteins (data not shown). The effect of Ca 2ϩ on SP-A-Prdx6 interaction was next investigated. In the absence of Ca 2ϩ , Prdx6 was co-precipitated with SP-A by anti-SP-A pAb (Fig.  6C, lane 3). Addition of 2.5 mM Ca 2ϩ to both the binding and washing buffers markedly increased Prdx6 co-precipitation (lane 6).
To study SP-A-Prdx6 interaction under physiological conditions, co-

activity of Prdx6 and its inhibition by SP-A
The kinetics were calculated from the results presented in Fig. 2.   MARCH 17, 2006 • VOLUME 281 • NUMBER 11 immunoprecipitation was performed using the supernatant from lysed rat type II alveolar epithelial cells (ex vivo immunoprecipitation). After incubation of cell lysate with anti-SP-A pAb-coupled gel, and washing, both Prdx6 and SP-A were present in the eluate indicating that these proteins were co-immunoprecipitated (Fig. 7, lane 4). Co-immunoprecipitation also was observed using anti-Prdx6 polyclonal antibodies prepared against whole protein (Prdx6 1-224 pAb) or against a peptide fragment (Prdx6 196 -211 pAb) (Fig. 7, lanes 5 and 6), and with anti-Prdx6 mAb (data not shown). Neither SP-A nor Prdx6 was detected in the eluate in control experiments (Fig. 7, lanes 1-3).

Prdx6 Interaction with SP-A
The SP-A interaction with Prdx6 also was examined by cross-linking experiments. The silver-stained gel (Fig. 8A) after cross-linking shows several bands under non-reducing (lane 1) and reducing (lane 2) conditions. Immunoblot using anti-SP-A pAb (Fig. 8B) or anti-Prdx6 pAb (Fig. 8C) indicates that the bands in Fig. 8A correspond to Prdx6, SP-A monomer, SP-A dimer, SP-A oligomer (lane 1) and Prdx6, SP-A mon-omer, and SP-A dimer (lane 2). Thus, SP-A exists under reducing condition in multiple forms. With streptavidin-horseradish peroxidase (Fig.  8D), Prdx6 and both the SP-A dimer and oligomer, but not the SP-A monomer, were detected under non-reducing conditions (lane 1). The SP-A oligomer was not seen with streptavidin-horseradish peroxidase under reducing conditions (lane 2).
Size-exclusion chromatography was used to further evaluate SP-A-Prdx6 interactions. After injection of native SP-A alone onto the column, only one peak was detected at ϳ21 min of isocratic elution (Fig.  9A, red trace) indicating a protein with an apparent molecular mass of ϳ650 kDa consistent with the previously described molecular mass of native SP-A (22). With injection of Prdx6 alone, two peaks were detected with retention times of ϳ26 and ϳ28 min corresponding to molecular masses of ϳ26 and ϳ52 kDa, respectively, compatible with the Prdx6 monomer and homodimer (Fig. 9A, green trace). Preincubation of SP-A with Prdx6 for 1 h prior to loading onto the column resulted in increase of the presumed SP-A oligomer peak and decrease of the presumed Prdx6 monomer peak (Fig. 9A, black trace). The peak corresponding to the Prdx6 dimer did not significantly change. Analysis of these fractions by SDS-PAGE and Western blot under reducing condition confirmed the identity of the SP-A and Prdx6 peaks and showed the appearance of Prdx6 in the SP-A peak when the two proteins were co-incubated (Fig. 9B). Thus, co-incubation resulted in a shift of the Prdx6 monomer into the SP-A oligomer peak compatible with binding of the two proteins. As a positive control, incubation of SP-A with anti-SP-A pAb also resulted in an increase of SP-A peak with a decrease in the pAb peak (Fig. 9C). As negative control, co-incubation of SP-A with BSA did not change the peaks obtained with SP-A or BSA alone (Fig.  9D). The addition of Ca 2ϩ (100 M) enhanced the SP-A-Prdx6 interaction (not shown).
The interaction of SP-A with Prdx6 and the effects of pH and Ca 2ϩ were investigated using static light scattering (Fig. 10). Guinier plots showed that incubating the two proteins together led to a higher signal compared with SP-A alone indicating an interaction between SP-A and  Prdx6. (Prdx6 alone did not give a detectable signal because of its small size.) The presence of Ca 2ϩ enhanced the interaction between the proteins at pH 7.4 but had no effect at pH 4.0.
The surface plasmon resonance technique was used to study the kinetics of the SP-A-Prdx6 interaction. Prdx6 was covalently immobilized on a CM3 sensor chip surface, and SP-A was injected as the soluble analyte. The sonograms indicate an interaction of SP-A with Prdx6 bound to the chip. At pH 4.0, the interaction was SP-A concentrationdependent and Ca 2ϩ -independent (Fig. 11, A and B). At pH 7.4, Ca 2ϩ enhanced the binding of SP-A to Prdx6 in a concentration-dependent manner and showed maximal effect at 2.5 mM Ca 2ϩ (Fig. 11C). The substrate concentration dependence was studied by injecting increasing concentrations of SP-A into the flow cell. The sonogram shows a progressive increase with increasing concentrations of SP-A when measured in the presence of 5 mM Ca 2ϩ (Fig. 11D). The "off" reaction for the complex studied in the absence of Ca 2ϩ showed rapid dissociation of the SP-A⅐Prdx6 complex (Fig. 11, A, C, and E). When the 5 mM Ca 2ϩ concentration in the buffer was maintained during the off reaction, the rate of dissociation of SP-A from immobilized Prdx6 was decreased markedly (Fig. 11, B and D). As a positive control, immobilized Prdx6 exhibited a significantly stronger interaction with anti-Prdx6 pAbs than with SP-A (Fig. 11E). BSA as analyte (negative control) had minimal interaction with immobilized Prdx6 (Fig. 11F).
Subcellular localization of SP-A and Prdx6 were studied in isolated rat epithelial type II cells by immunofluorescence (confocal microscopy) using mAb to Prdx6 and pAb to SP-A. SP-A-related immunofluorescence was observed in structures compatible with lamellar bodies, the surfactant storage and secretory organelle (Fig. 12A, panel b). Prdx6-related immunofluorescence was more diffuse (Fig. 12A, panel a) but showed a clear co-localization with SP-A (Fig. 12A, panel c). To examine further the site of co-localization within the type II cell, we utilized the Prdx6 pAb and mAb 3C9, a specific marker of lamellar bodies (23). Immunofluorescence showed labeling of lamellar bodies by 3C9 (Fig. 12A, panel d) and a clear overlap in the merged images between 3C9 and SP-A (Fig. 12A, panel f). Both Prdx6 pAbs (the anti-peptide and anti-protein Abs) labeled lamellar body-like structures (Fig. 12A, panels h and k) and an overlap in fluorescence with 3C9 and both Prdx6 pAbs was detected (Fig. 12A, panels i and l). No fluorescence was observed in the absence of the primary antibody (Fig.  12A, panels m and n). Immunogold staining with evaluation at the electron microscopic level showed the greatest concentration of grains in the lamellar bodies (Fig. 12B). There appears to be a fewer number of grains in the other cellular organellar compartments consistent with our previous observations using subcellular fractionation (7,9). We did not evaluate the sub-

Prdx6 Interaction with SP-A
cellular localization of SP-A by immunogold, but previous studies have demonstrated lamellar body enrichment of this protein (24,25). These results demonstrate that SP-A and Prdx6 are co-localized within lamellar bodies of rat type II cells, although both proteins appear to be present also in other intracellular compartments. Because the lamellar body in alveolar type II cells is a lysosome-related organelle that expresses lysosomal protein markers (26), rat alveolar macrophages were used to investigate the lysosomal localization of Prdx6. Immunofluorescence staining showed co-localization of Prdx6 with LAMP1, a specific lysosomal marker (Fig. 12C). This result demonstrates that Prdx6 is present in lysosomes, which is consistent with our previous observations using subcellular fractionation (7,9).

DISCUSSION
Alveolar DPPC, the principal phospholipid component of lung surfactant, is endocytosed from the alveolar space by epithelial type II cells and either degraded or resecreted. The major pathway for intracellular DPPC degradation is through phospholipase A 2 -mediated hydrolysis with minor contributions by other phospholipases. Use of the transition state analog inhibitor, MJ33, and studies with Prdx6 gene-targeted mice have indicated that the aiPLA 2 activity of Prdx6 is the major PLA 2 responsible for DPPC degradation by alveolar type II cells (9 -11). The presence of Prdx6 in the surfactant secretory organelles (lung lamellar bodies) and in the alveolar extracellular fluid (6,7,9) could result in degradation of the secreted product if aiPLA 2 activity were uncontrolled. We have postulated that SP-A serves as a regulatory protein for the aiPLA 2 activity of Prdx6.
Our previous studies have provided several lines of evidence that SP-A can regulate aiPLA 2 activity and degradation of DPPC. First, addition of SP-A to rat lung homogenate or isolated lung lamellar bodies inhibited aiPLA 2 activity (12) as confirmed in the present study with  recombinant Prdx6. These effects were abolished by pre-treatment of SP-A with boiling or strong reducing/alkylating agents. Second, addition of reducing/alkylating agents to isolated lung lamellar bodies led to a significant increase in aiPLA 2 activity, which we attributed to reversal of an inhibitory effect of endogenous SP-A. aiPLA 2 activity of Prdx6 is not affected directly by these agents (12). Third, addition of SP-A to type II cells in primary culture resulted in decreased degradation of endocytosed DPPC compatible with inhibition of aiPLA 2 (12). Fourth, SP-A gene-targeted mice showed increased aiPLA 2 activity in lung and increased degradation of internalized DPPC (3). Fifth, the kinetic studies in the present report indicate non-competitive inhibition of the aiPLA 2 activity of recombinant Prdx6 by SP-A with a K i of 10 g/ml (ϳ15 nM SP-A). These diverse experiments indicate that SP-A can regulate the aiPLA 2 activity both in vitro and in the intact cell. A possible explanation for this effect is SP-A binding to the phospholipid substrate (27), thus limiting its access to the PLA 2 catalytic site. Against this possibility is the non-competitive kinetics of the interaction (present study) and the inhibition by SP-A of the aiPLA 2 -mediated metabolism of 1-palmitoyl, 2-oleoyl-sn-glycerol-3-phosphocholine; this phospholipid binds poorly to SP-A as compared with DPPC (12). A second possibility for the SP-A effect on aiPLA 2 activity is the direct interaction of these proteins. This second possibility was the focus of the present study.
The results presented in this report indicate the direct binding of SP-A to Prdx6. First, the addition of Prdx6 inhibited the nonspecific binding of SP-A to agarose beads, suggesting that Prdx6 interacts with the carbohydrate binding domain of SP-A. Second, His-tagged Prdx6 bound to Ni 2ϩ -chelated beads resulted in precipitation of SP-A. Third, immunoprecipitation of Prdx6 resulted in co-precipitation of SP-A and, vice versa, immunoprecipitation of SP-A resulted in Prdx6 co-precipitation. These results were noted with binary systems in vitro as well as with lysates of isolated type II epithelial cells ex vivo. Fourth, size-exclusion chromatography of proteins indicated alteration of the elution profile compatible with a shift of Prdx6 into the SP-A peak when the two proteins were co-eluted. Fifth, using Sulfo-SBED, a tri-functional crosslinking reagent, the binding of Prdx6 to the SP-A multimeric complex was seen by streptavidin detection. Reduction of the internal disulfides of the Sulfo-SBED reagent resulted in partial biotin transfer to the SP-A oligomer and dimer but not to the monomer. The latter is consistent with our observations that reduced/alkylated SP-A does not inhibit the aiPLA 2 activity of Prdx6 (12). Sixth, static light scattering showed an increase of Stokes radius of the SP-A⅐Prdx6 complex, which exceeded that of SP-A alone. Seventh, surface plasmon resonance indicated binding of SP-A to immobilized Prdx6. The light scattering and surface plasmon resonance results show good correlation indicating partial Ca 2ϩ dependence of the SP-A-Prdx6 interaction at alkaline pH but Ca 2ϩ -independence at acidic pH.
The non-competitive kinetics of the effect of SP-A on PLA 2 activity of Prdx6 is compatible with binding to a site removed from the site of substrate (phospholipid) binding. Because the K i was 15 nM SP-A (10 g/ml, molecular mass of ϳ650 kDa) for a Prdx6 concentration of 100 nM (2.5 g/ml, molecular mass of 25 kDa), this indicates that 1 mol of SP-A interacts with ϳ7 mol of Prdx6 to produce 50% inhibition of activity. SP-A is an octadecamer consisting of 6 homotrimers (21). So, the stoichiometry of the interactions suggests binding of one Prdx6 monomer per SP-A homotrimer. However, the tertiary structure of SP-A is necessary for the interaction, because reduction of SP-A with mercaptoethanol abolished its ability to inhibit PLA 2 activity. PLA 2 s represent a large family of enzymes characterized by hydrolysis of the sn-2 fatty acid in phospholipids. SP-A has been demonstrated to inhibit the activity of two other members of this family. The PLA 2 of Habu snake venom, a group II PLA 2 , is inhibited by SP-A in vitro (28). Because several other snake venoms (both group I and group II secreted PLA 2 s) failed to show inhibition of PLA 2 activity by SP-A, the interaction with Habu PLA 2 appears to be specific. SP-A inhibition of Habu PLA 2 may represent an accidental effect based on the homology of SP-A to an endogenous inhibitor in Habu snake serum, and this role of SP-A FIGURE 12. Subcellular localization of Prdx6. A, co-localization of SP-A and Prdx6 in rat alveolar epithelial type II cells. Adherent rat type II cells on coverslips were fixed, permeabilized, and exposed to primary Ab followed by fluorescent-tagged secondary Ab. The left side legend indicates first the antigen for the mAb (green channel) and then the antigen for the pAb (red channel). Panels a-c, Prdx6/SP-A co-localization using monoclonal anti-Prdx6 Ab (1:100) (a) and polyclonal anti-SP-A Ab LB, lamellar body. C, localization of Prdx6 to lysosomes. Adherent rat alveolar macrophages on a coverslip were fixed, permeabilized, and exposed to anti-Prdx6 pAb and anti-LAMP-1 mAb followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat antimouse IgG. Fluorescence with LAMP1 pAb (green channel) and Prdx6 mAb (red channel) are shown and are largely co-localized as shown in the right panel (merged images).
is unlikely to be physiologically significant. SP-A also inhibits the mammalian secreted group IIA PLA 2 that is active principally in inflammatory exudates (2,29). This interaction could have physiological significance for lung inflammation but is unlikely to be relevant in other organs, because SP-A is present at sufficiently high concentration only in lung. The inhibition of PLA 2 activity for these two secreted enzymes appears to be through protein-protein interactions. There is no significant homology of amino acid composition between Prdx6 and the group II secreted PLA 2 s so that a common SP-A binding site for the three proteins appears to be unlikely.
Previous studies have indicated Ca 2ϩ -dependent binding of SP-A with a variety of ligands, including lipids such as DPPC/egg-PG bilayers (30) and other amphipathic lipids (glycerophospholipids, sphingophospholipids, glycosphingolipids, lipid A, and lipoglycans) (21), polysaccharides such as zymosan (31), and proteins, including type IIA sPLA 2 and Habu sPLA 2 (28,32). On the other hand, binding between SP-A and lipopolysaccharide was Ca 2ϩ -independent (32). In some experiments such as the interaction of SP-A with mycoplasma membranes (33) or with DPPC (34), interactions were seen with Ca 2ϩ -free conditions and were enhanced by the addition of Ca 2ϩ . The present study using coimmunoprecipitation, light scattering, and surface plasmon resonance techniques showed a basal interaction between SP-A and Prdx6 that was enhanced by Ca 2ϩ at pH 7.4 while binding was Ca 2ϩ -independent at pH 4.0.
Organellar localization studies using subcellular fractionation showed the presence of Prdx6 in the lamellar body fraction (7). Because the pH of these organelles is acidic (35), the stored DPPC would be susceptible to hydrolysis due to the aiPLA 2 activity of Prdx6. By immunofluorescence, Prdx6 and SP-A showed partial co-localization providing evidence that they are, at least in part, localized to the same compartment. The mAb 3C9, which recognizes a unique protein (ABC A3) of the lamellar body membrane, was used as a specific marker for type II cell lamellar bodies (15,23). Both SP-A and Prdx6 showed partial colocalization with 3C9 mAb indicating their presence in lamellar bodies. Immunogold studies confirmed the presence of Prdx6 in lamellar bodies, and previous studies have demonstrated the presence of SP-A in these organelles (24,27). Current understanding is that SP-A is secreted by type II cells through non-lamellar body mechanisms but is recycled to these organelles by endocytosis. Because SP-A represents ϳ10% of lamellar body protein (24), it would be present in sufficient amount to significantly inhibit aiPLA 2 activity, although its "availability" for this interaction is not known. Thus, the presence of SP-A in lamellar bodies could inhibit Prdx6 PLA 2 activity and thereby diminish degradation of surfactant DPPC. This conclusion raises the question of why Prdx6 is present in lamellar bodies. Our previous studies of subcellular fractionation have demonstrated the presence of Prdx6 in the lysosomal fraction (7,12), which is the predominant site of DPPC degradation in the intact alveolar epithelial cell (13). The present study has demonstrated the presence of Prdx6 in lysosomes of alveolar macrophages by its co-localization with LAMP1, a specific lysosomal marker. Although the precise origin of lamellar bodies is not clear, they appear to be modified lysosomes (26,35) that may arise through fusion of lysosomes and multivesicular bodies, a mechanism that could provide the basis for the presence of Prdx6 in the storage organelle.
In summary, studies with SP-A binding to agarose, pull-down assay with Ni 2ϩ beads, co-immunoprecipitation, cross-linking, co-elution by size-exclusion chromatography, static light scattering, and surface plasmon resonance all indicate that SP-A directly interacts with Prdx6 and provides a mechanism for inhibition of aiPLA 2 activity by SP-A. Immunofluorescence studies show partial co-localization of SP-A and Prdx6 in lung lamellar bodies. These results provide additional evidence that the direct interaction of SP-A and Prdx6 can play an important role in the regulation of lung surfactant phospholipid metabolism.