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Originally published In Press as doi:10.1074/jbc.M504525200 on December 5, 2005

J. Biol. Chem., Vol. 281, Issue 11, 7515-7525, March 17, 2006
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Interaction of Surfactant Protein A with Peroxiredoxin 6 Regulates Phospholipase A2 Activity*

Yong-Zheng Wu, Yefim Manevich, James L. Baldwin, Chandra Dodia, Kevin Yu, Sheldon I. Feinstein, and Aron B. Fisher1

From the Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6068

Received for publication, April 25, 2005 , and in revised form, November 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pulmonary surfactant, a lipoprotein complex lining the lung surface, consists of phospholipids, specific proteins, and other lipid components. It is synthesized by alveolar type II epithelial cells, assembled in lamellar bodies, the intracellular surfactant storage organelle, and secreted into the alveolar space and terminal airways where it functions to reduce surface tension and stabilize alveoli (1). Dipalmitoylphosphatidylcholine (DPPC)2, the major phospholipid of surfactant, is the critical component for the surface-tension-lowering function (2). DPPC is cleared from the alveolar space predominantly through endocytosis by type II cells with a minor contribution from alveolar macrophages. Under normal physiological conditions, clearance and secretion of DPPC appear to be coordinately regulated (3).

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 A2 (PLA2) in addition to peroxidase activity (6, 8). PLA2 enzymes hydrolyze phospholipids at the sn-2 position leading to the release of lysophosphatidylcholine and a fatty acid. PLA2s have been classified as cytosolic PLA2, secreted PLA2, or calcium-independent PLA2. Because the PLA2 activity of Prdx6 is Ca2+ -independent and maximal at acidic conditions (pH 4), it has been named acidic calcium-independent PLA2 (aiPLA2). aiPLA2 activity is inhibited by the competitive transition state inhibitor MJ33 (9). The involvement of Prdx6 in lung [3H]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 aiPLA2 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 aiPLA2 activity of Prdx6. Addition of SP-A to rat lung homogenate, isolated lamellar bodies, or isolated rat alveolar type II cells inhibited aiPLA2 activity and the degradation of DPPC (3). Inhibitors of SP-A or SP-A knock-out resulted in increased lung aiPLA2 activity (3, 12). In the present study, we evaluated the effect of SP-A on aiPLA2 activity of recombinant enzyme and tested the hypothesis that SP-A regulates the PLA2 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).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Sprague-Dawley male rats weighing ~200 g were obtained from Charles River Breeding Laboratories (Kingston, NY). Male C57BL/6 mice were obtained from the Jackson Laboratories (Mt. Desert, ME). All animal use was approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Authentic lipids for preparation of liposomeswere purchased from Avanti (Birmingham, AL). 1-Palmitoyl-2-[3H]9,10-palmitoyl-sn-3-glycerophosphocholine ([3H]DPPC) was from American Radiolabeled Chemicals (St. Louis, MO). Polyclonal anti-human SP-A antibody was purchased from Chemicon (Temecula, CA). A Prdx6 monoclonal Ab (Prdx6 mAb) and a polyclonal antibody against a Prdx6 peptide (Prdx6196-211 pAb) have been described previously (6, 14). A polyclonal antibody against His-tagged recombinant Prdx6 (Prdx61-224 pAb) was generated in rabbits by a standard protocol (HTL Bio-Products, Ramona, CA). A monoclonal antibody specific for lung lamellar bodies (3C9 mAb) has been described previously (15). Anti-human glutathione S-transferase P1-1 pAb (anti- {pi}GST) was from Oxford Biochemical Research (Oxford, MI). Anti-human LAMP1 mAb was from BD Pharmingen (San Diego, CA). Anti-rabbit IgG was purchased from Amersham Biosciences. Alexa Fluor® 594-conjugated goat anti-rabbit IgG and Alexa Fluor® 488-conjugated goat anti-mouse IgG were purchased from Molecular Probes (Eugene, OR). Bovine serum albumin (BSA) was from Jackson ImmunoResearch (West Grove, PA). SDS-PAGE was performed using 12% Tris-glycine NuPage-precasted gels, apparatus, and buffers purchased from Invitrogen. Polyvinylidene difluoride membranes were from Millipore (Billerica, MA). Affi-Gel beads were purchased from Bio-Rad (Hercules, CA). Size-exclusion chromatography was performed using an Alliance 2695 separation unit, PDA UV detector, and YMC-Pack Diol column 500 x 8.0 mm (inner diameter), 120-Å pore size (Waters, Milford MA). Reagents for surface plasmon resonance experiments (acetate, regeneration, and amine coupling buffers and CM3 sensor chips) were purchased from Biacore (Piscataway, NJ). Nickel-chelated beads were from Invitrogen. Protein-A-agarose beads and Nonidet P-40 were from Roche Diagnostics (Mannheim, Germany). Protein-A acrylic, protein-G-agarose, and protein-G-Sepharose beads were purchased from Sigma-Aldrich. A mammalian co-immunoprecipitation kit was from Pierce. All other solutions were prepared in-house using chemicals from Fisher Scientific or Sigma-Aldrich.

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 Ni2+ 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 x 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.

Phospholipase A2 Assay—PLA2 activity was measured as previously described (12) using unilamellar liposomes containing DPPC:phosphatidylcholine:phosphatidylglycerol:cholesterol (0.5:0.25:0.1:0.15, mol fraction) with a trace of [3H]DPPC. Recombinant Prdx6 (2.5 µg/ml) was incubated with substrate under acidic (40 mM sodium acetate, pH 4.0, 5 mM EDTA, 1 mM GSH) or alkaline (50 mM Tris, pH 7.4 or 8.5, 1 mM EGTA, 1 mM GSH) conditions at 37 °C, generally for 1 h. The reaction was stopped by addition of CHCl3:CH3OH (1:2), lipids were extracted, components were separated by two-step TLC using hexane:ether:acetic acid, and the free fatty acid spot was scraped for measurement of dpm. In different experiments, Ca2+ (10 mM) or SP-A in varying concentration 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.

Gel Electrophoresis and Immunoblot—For gel electrophoresis, loading buffer and dithiothreitol (50 mM) were added to the proteins before boiling for 5 min at 95 °C. Protein was analyzed by SDS-PAGE (12% Tris-glycine gel) with an Xcell SurelockTM mini-cell system (Invitrogen). After electrophoresis, the proteins were transferred to Immobilon-P membrane (Millipore) and blocked for 1 h in Tris-buffered saline with 0.1% Tween 20 containing 5% fat-free milk. Membranes were probed with either anti-SP-A polyclonal antibody (1:1500) or one of the anti-Prdx6 antibodies (mAb 1:2000, pAb196-2111:3000, or pAb1-224 1:1500) followed by horseradish peroxidase-conjugated secondary antibody (1:4000). The reaction was detected by chemiluminescence using ECL detection reagents (GE Bioscience, Piscataway, NJ) and exposed to X-Omat AR-2 x-ray film (Kodak, Rochester, NY).

SP-A Binding to Agarose Beads—Affi-gel beads (500 µl) 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 H2O, equilibrated in PBS for 30 min, centrifuged for 2 min at 10,000 rpm, and resuspended in 500 µl of PBS. SP-A (2 µg) was incubated with or without Prdx6 (2 µg) for 30 min at room temperature in a final volume of 10 µl of PBS. Then 100 µl 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 µg) was incubated for 30 min with Histagged recombinant human Prdx6 (2 µg) in PBS to bring the mixture to a final volume of 10 µl. This mixture was then added to 100 µl of a slurry (~50 µl) of Ni2+ 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 (Ca2+ -dependent) carbohydrate binding protein family (19, 20). After incubation with the protein mixture, the Ni2+ 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 µg) and native SP-A (2 µg) with either anti-SP-A pAb or anti-Prdx6 pAb for 1 h at 4 °C with slow rotation. Proteins in 80 µl 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 µl) 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 Ca2+ 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 x g for 15 min at 4 °C, and the supernatant was kept at -80 °C in aliquots until use. A ProfoundTM mammalian co-immunoprecipitation kit from Pierce was used to perform ex vivo immunoprecipitation according to the manufacturer's recommendation. Briefly, 100 µl of antibody coupling gel slurry (50-µl 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 µg 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-{pi}GST pAb) to the coupling gel.


Figure 1
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  		FIGURE 1.
aiPLA2 activity of recombinant Prdx6. Prdx6 (2.5 µg/ml) was incubated at 37 °C with [3H]DPPC-labeled substrate for varying times in acidic (pH 4.0) or alkaline (pH 7.4) buffer.

 


Figure 2
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  		FIGURE 2.
Effect of native human SP-A on PLA2 activity of recombinant Prdx6. Prdx6 (2.5 µg/ml) was incubated with varying concentrations of SP-A in acidic (pH 4.0) (A) or alkaline (pH 8.5) (B) buffers in the absence or presence of 10 mM Ca2+. The concentration of SP-A is plotted as the log10. Activity in nanomoles/min/mg of protein with zero SP-A (not shown) was 106 ± 2 at pH 4.0 and 56 ± 3 at pH 8.5. Values represent the mean ± range for duplicate incubation.

 
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 x 8 mm inner diameter, 5-µm particle, and 120-Å pore sizes, all from Waters, Milford, MA). The column was calibrated (R2 = 0.99) with a standard mixture of proteins. The samples containing Prdx6 (15 µg) 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 µg) and Prdx6 (10 µg) 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 Ca2+, CaCl2 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° angles. 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 Ca2+, 0.005% Tween 20, 10 mM HEPES, pH 7.4), the carboxymethylated matrix was activated with a mixture of an N-ethyl-N'-((dimethylamino)propyl)carbodiimide and N-hydroxysuccinimide. Recombinant Prdx6 (10 µg/ml in 10 mM sodium acetate, pH 4.8) was injected at a flow of 5 µl/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 Ca2+ 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 µl/min. Dissociation of the SP-A·Prdx6 complex was performed by an injection of running buffer without protein and plus or minus Ca2+. 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% CO2 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 x 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 x 5 with PBS and 5 min x 2 with dH2O), the coverslip was mounted with Vectashield mounting medium (Vector Laboratories) and observed with a confocal microscope (Radiance 2000, Bio-Rad).


Figure 3
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  		FIGURE 3.
Concentration dependence of aiPLA2 inhibition by SP-A. Recombinant Prdx6 (2.5 µg/ml) was incubated for 30 min with varying concentration of DPPC substrate either at pH 4.0 (A) or at pH 7.4 (B) and in the absence of SP-A or with SP-A added at 10 or 20 µg/ml. The results are shown as double-reciprocal plots. C, plot of Km/Vmax versus SP-A concentration. The intercept with the horizontal axis indicates the Ki for SP-A.

 
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 x 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% OsO4. The lung pieces were dehydrated at -20 °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 x 6), samples were incubated for 1 h at room temperature with 20 nm gold-coupled goat-anti-mouse IgG (1:25), then rinsed with distilled water (5 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).


Figure 4
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  		FIGURE 4.
Effect of Prdx6 on SP-A binding to agarose beads. A, SP-A binding in the presence of increasing concentrations of Prdx6. SP-A (2 µg) was incubated with Prdx6 (0-10 µg) for 30 min followed by 10-min incubation with Affi-gel beads. The precipitate was analyzed by immunoblot using anti-SP-A pAb (upper panel). The position of the SP-A dimer and monomer is indicated. The sum of the two bands was quantitated by densitometry (lower panel). B, anti-Prdx6 pAb blocks the effect of Prdx6 on SP-A binding to agarose beads. SP-A (2 µg) was incubated with Prdx6 (2 µg) in the absence or in the presence of Prdx6 pAb (dilution 1:400) for 1 h and then incubated with agarose beads for an additional 30 min. The precipitate was analyzed as described in A. Results are expressed as mean ± S.E.; *, p < 0.05 as compared with the absence of Prdx6.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The PLA2 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 aiPLA2 activity is trivial at pH >6 (6-8), we have subsequently found activity of recombinant protein after the addition of GSH to the incubation medium.3PLA2 activity at both acidic and alkaline conditions was linear with time during a 60-min incubation; activity at pH 7.4 was ~50% 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) and pH8.5 (Fig.2). The addition of SP-A resulted in a concentration-dependent inhibition of PLA2 activity of Prdx6 by SP-A (Fig. 2). The effect showed a linear relationship to the logarithm of the SP-A concentration. Inhibition was Ca2+ -independent at pH 4.0 but was accentuated by Ca2 at pH 8.5. In the presence of 50 µg/ml SP-A, aiPLA2 activity was inhibited by 80% under acidic conditions, whereas with alkaline conditions, inhibition was 70% in the presence of Ca2+ and 50% in its absence. The inhibitory effect of SP-A was abolished by its reduction with beta-mercaptoethanol, inactivation by boiling, or alkylation with iodoacetamide (Table 1). As a control, BSA had no effect on aiPLA2 activity of recombinant Prdx6 (Table 1).


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  		TABLE 1
Effect of native human SP-A on aiPLA2 activity of recombinant Prdx6

The assay contained 2.5 µg of Prdx6 in a l-ml incubation volume. Values are mean ± range for n = 2 for each condition.

 
Kinetic parameters of the SP-A-aiPLA2 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 PLA2 activity of Prdx6 at both acidic and alkaline pH conditions (Fig. 3). Although the calculated Vmax at pH 4.0 was 2.1 times the value at pH 7.4, the apparent Km for aiPLA2 activity and the estimated Ki for SP-A were similar under the two conditions (Fig. 3 and Table 2).


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  		TABLE 2
Kinetic constants for the aiPLA2 activity of Prdx6 and its inhibition by SP-A

The kinetics were calculated from the results presented in Fig. 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-A·Prdx6 mixture before addition of agarose beads (Fig. 4B).

The interaction of SP-A with Prdx6 was evaluated further by a pulldown assay using Ni2+ -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 Ni2+ beads (Fig. 5). Incubation of SP-A and Prdx6 together with the Ni2+- 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 Ca2+ 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 Ca2+ on SP-A-Prdx6 interaction was next investigated. In the absence of Ca2+, Prdx6 was co-precipitated with SP-A by anti-SP-A pAb (Fig. 6C, lane 3). Addition of 2.5 mM Ca2+ to both the binding and washing buffers markedly increased Prdx6 co-precipitation (lane 6).

To study SP-A-Prdx6 interaction under physiological conditions, co-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 (Prdx61-224 pAb) or against a peptide fragment (Prdx6196-211 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).

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 monomer, 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).


Figure 5
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  		FIGURE 5.
Detection of SP-A·Prdx6 binding by pull-down assay. SP-A (2 µg) was incubated with human recombinant His-tagged Prdx6 (2 µg) in the presence of mannose (10 mM). After washing, the proteins that were precipitated with Ni2+ -chelating beads were analyzed by Western blot using pAbs to SP-A (upper panel) and Prdx6 (lower panel). The presence of SP-A or Prdx6 in the incubation mixture is indicated. Supernatant is indicated as S and pellet as P. The results shown are representative of two independent experiments.

 


Figure 6
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  		FIGURE 6.
In vitro co-immunoprecipitation of SP-A and Prdx6. Equal amounts (2 µg) of Prdx6 and SP-A were incubated with anti-SP-A pAb (dilution 1:2000) (A) or anti-Prdx61-224 pAb (1:2000) (B) and protein-A-agarose beads. The co-precipitates were analyzed by immunoblotting using these same pAbs. A, anti-SP-A precipitated both Prdx6 (top panel) and SP-A (bottom panel). B, anti-Prdx6 precipitated both SP-A (top panel) and Prdx6 (bottom panel). Bands corresponding to the antibody IgG are labeled Ab. C, effect of Ca2+ on co-immunoprecipitation of SP-A and Prdx6 by anti-SP-A pAb. The addition of 2.5 mM Ca2+ enhanced Prdx6 precipitation (bottom panel, lane 6) compared with that in the absence of Ca2+ (bottom panel, lane 3). The faint bands in lanes 1 and 5 indicate a low level of Prdx6 precipitation in the absence of SP-A.

 
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 ~21 min of isocratic elution (Fig. 9A, red trace) indicating a protein with an apparent molecular mass of ~650 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 ~26 and ~28 min corresponding to molecular masses of ~26 and ~52 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 Ca2+ (100 µM) enhanced the SP-A-Prdx6 interaction (not shown).

The interaction of SP-A with Prdx6 and the effects of pH and Ca2+ 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 Ca2+ enhanced the interaction between the proteins at pH 7.4 but had no effect at pH 4.0.


Figure 7
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  		FIGURE 7.
Co-immunoprecipitation of SP-A and Prdx6 from rat type II cells. The supernatant of the lysate of freshly isolated rat type II cell was incubated with rabbit anti-SP-A pAb or one of two different rabbit anti-Prdx6 pAbs, Prdx61-224 Ab against full-length protein and Prdx6196-211 Ab against a C-terminal peptide. The co-immunoprecipitates were analyzed by SDS-PAGE and immunoblot using anti-SP-A (upper panel) or anti-Prdx6 pAb (bottom panel). Addition of anti-SP-A pAb (lane 4) or anti-Prdx6 pAb (lanes 5 and 6) precipitated both SP-A (top panel) and Prdx6 (bottom panel). Neither SP-A nor Prdx6 was precipitated in control experiments. Controls were: gel not activated (lane 1), quenched gel with no antibody (lane 2), and a non-relevant Ab (anti-{pi}GST pAb, lane 3).

 


Figure 8
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  		FIGURE 8.
Detection of the interaction of SP-A and Prdx6 by cross-linking using Sulfo-SBED. The cross-linked proteins were evaluated with a silver stained gel (A), immunoblot with anti-SP-A pAb (B), or anti-Prdx61-224 pAb (C), and by streptavidin (D). For all gels, lane 1 is non-reducing and lane 2 is reducing conditions.

 
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 concentration-dependent and Ca2+ -independent (Fig. 11, A and B). At pH 7.4, Ca2+ enhanced the binding of SP-A to Prdx6 in a concentration-dependent manner and showed maximal effect at 2.5 mM Ca2+ (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 Ca2+ (Fig. 11D). The "off" reaction for the complex studied in the absence of Ca2+ showed rapid dissociation of the SP-A·Prdx6 complex (Fig. 11, A, C, and E). When the 5 mM Ca2+ 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-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).


Figure 9
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  		FIGURE 9.
Size-exclusion chromatography on a calibrated YMC-Pack Diol column of the complex formed by the interaction of SP-A and Prdx6. A, injection of SP-A alone resulted in one peak (red trace, eluted ~21 min) while Prdx6 alone produced two peaks (green trace, ~26 and 28 min). Preincubation of SP-A with Prdx6 (1 h) resulted in an increase of SP-A peak (~21 min) and a subsequent decrease of Prdx6 monomer peak (black trace, ~28 min). B, the fraction containing the SP-A multimer after interaction with Prdx6 was collected, concentrated, and subjected to SDS-PAGE, which showed prominent bands at ~63 kDa, 35 kDa, and 26 kDa (lane 1). Analysis of the SP-A peak (lane 2) showed bands on SDS-PAGE only at 63 and 35 kDa, which by immunoblot corresponded to the SP-A dimer and monomer (lane 3), while the band at 26 kDa corresponded to Prdx6 (lane 4). C, the interaction of SP-A with an anti-SP-A pAb was used as a positive control. Preincubation of SP-A with anti-SP-A pAb before loading onto the column resulted in an increase of SP-A peak and a decrease of pAb peak (black trace) compared with control (red and green traces). D, no interaction between SP-A and BSA was detected as negative control.

 


Figure 10
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  		FIGURE 10.
SP-A-Prdx6 interaction by static light scattering. SP-A and Prdx6 (10 µg of each) were applied to a BI-2000SM instrument either individually or as a mixture under acidic (pH 4.0) or alkaline (pH 7.4) conditions in the absence or presence of Ca2+. No signal was detected for Prdx6 alone and is not shown. The Stokes radius of the individual protein or the protein complex was measured at 45°, 60°, 75°, 90°, 105°, 120°, and 135° angles and then subjected to Guinier analysis with a Williams-Watts size distribution program. Values are the mean ± range for n = 2 experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 A2-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 aiPLA2 activity of Prdx6 is the major PLA2 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 aiPLA2 activity were uncontrolled. We have postulated that SP-A serves as a regulatory protein for the aiPLA2 activity of Prdx6.

Our previous studies have provided several lines of evidence that SP-A can regulate aiPLA2 activity and degradation of DPPC. First, addition of SP-A to rat lung homogenate or isolated lung lamellar bodies inhibited aiPLA2 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 aiPLA2 activity, which we attributed to reversal of an inhibitory effect of endogenous SP-A. aiPLA2 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 aiPLA2 (12). Fourth, SP-A gene-targeted mice showed increased aiPLA2 activity in lung and increased degradation of internalized DPPC (3). Fifth, the kinetic studies in the present report indicate non-competitive inhibition of the aiPLA2 activity of recombinant Prdx6 by SP-A with a Ki of 10 µg/ml (~15 nM SP-A). These diverse experiments indicate that SP-A can regulate the aiPLA2 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 PLA2 catalytic site. Against this possibility is the non-competitive kinetics of the interaction (present study) and the inhibition by SP-A of the aiPLA2-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 aiPLA2 activity is the direct interaction of these proteins. This second possibility was the focus of the present study.


Figure 11
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  		FIGURE 11.
SP-A-Prdx6 interaction kinetics detected using surface plasmon resonance. Prdx6 was immobilized on the dextran matrix of a CM3 chip. Purified native human SP-A, rabbit Prdx6 pAb, or BSA was injected, in a controlled flow of citrate buffer (10 mM sodium citrate, 300 mM NaCl, 0.005% Tween 20, pH 4.0) (A and B) or Hepes buffer (10 mM Hepes, 0.005% Tween 20, pH 7.4) (C-F). A, varying concentrations of SP-A were injected in the absence of Ca2+ under acidic condition (pH 4.0). B, same as A but with 5 mM Ca2+ in the running buffer. C, the effect of Ca2+ on the interaction of SP-A (80 µg/ml) and Prdx6 at pH 7.4. The concentration of Ca2+ for each trace is indicated. Ca2+ was omitted from buffers used for the dissociation phase. D, the SP-A-Prdx6 interaction at pH 7.4 was analyzed by injecting different concentration of SP-A in the presence of Ca2+ (5 mM). E, comparison of the interaction of Prdx6 pAb and SP-A with Prdx6. Prdx6 was immobilized on the CM3 chip. An equal protein concentration (2.5 µg/ml) of SP-A or one of two different Prdx6 pAbs (Prdx61-224 and Prdx6196-211 pAbs) was injected in the same buffer in the presence of Ca2+ (2.5 mM). F, the interaction of BSA with Prdx6 was analyzed as a negative control. An equal amount (10 µg/ml) of SP-A or BSA was injected in the presence of Ca2+ (2.5 mM). The results shown are representative of three separate experiments.

 
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 Ni2+ -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 aiPLA2 activity of Prdx6 (12). Sixth, static light scattering showed an increase of Stokes radius of the SP-A·Prdx6 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 Ca2+ dependence of the SP-A-Prdx6 interaction at alkaline pH but Ca2+ -independence at acidic pH.

The non-competitive kinetics of the effect of SP-A on PLA2 activity of Prdx6 is compatible with binding to a site removed from the site of substrate (phospholipid) binding. Because the Ki was 15 nM SP-A (10 µg/ml, molecular mass of ~650 kDa) for a Prdx6 concentration of 100 nM (2.5 µg/ml, molecular mass of 25 kDa), this indicates that 1 mol of SP-A interacts with ~7 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 PLA2 activity.


Figure 12
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  		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 (1:200) (b). Panels d-f, co-localization of 3C9, the lamellar body marker monoclonal antibody (1:250) and anti-SP-A pAb (1:200) (e). Panels g-i, co-localization of 3C9 mAb (1:250) (g) and anti-Prdx61-224 pAb (1:100) (h). Panels j-l, co-localization of anti-3C9 mAb (1:250) (j) and anti-Prdx6196-211 pAb (1:200) (k). Panels m-o, control; the cells were incubated only with Alexa Fluor® 488-conjugated goat anti-mouse IgG (1:250) (m) and Alexa Fluor® 594-conjugated goat anti-rabbit IgG (1:250) (n). The images in c, f, i, l, and o are the merged images. The scale bar is shown in panel o; all images were taken at the same magnification. B, ultrastructural localization of Prdx6 by immunogold. Mouse lungs were fixed, embedded, cut into ultrathin sections (80 nm), and incubated with primary mAb (left panel) or only blocking buffer (right panel) followed by 20 nm gold-coupled goat-anti-mouse IgG. Arrows indicate gold grains of Prdx6. 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 anti-mouse 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).

 
PLA2s 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 PLA2 of Habu snake venom, a group II PLA2, is inhibited by SP-A in vitro (28). Because several other snake venoms (both group I and group II secreted PLA2s) failed to show inhibition of PLA2 activity by SP-A, the interaction with Habu PLA2 appears to be specific. SP-A inhibition of Habu PLA2 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 is unlikely to be physiologically significant. SP-A also inhibits the mammalian secreted group IIA PLA2 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 PLA2 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 PLA2s so that a common SP-A binding site for the three proteins appears to be unlikely.

Previous studies have indicated Ca2+ -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 sPLA2 and Habu sPLA2 (28, 32). On the other hand, binding between SP-A and lipopolysaccharide was Ca2+ -independent (32). In some experiments such as the interaction of SP-A with mycoplasma membranes (33) or with DPPC (34), interactions were seen with Ca2+ -free conditions and were enhanced by the addition of Ca2+. The present study using co-immunoprecipitation, light scattering, and surface plasmon resonance techniques showed a basal interaction between SP-A and Prdx6 that was enhanced by Ca2+ at pH 7.4 while binding was Ca2+ -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 aiPLA2 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 ~10% of lamellar body protein (24), it would be present in sufficient amount to significantly inhibit aiPLA2 activity, although its "availability" for this interaction is not known. Thus, the presence of SP-A in lamellar bodies could inhibit Prdx6 PLA2 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 Ni2+ 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 aiPLA2 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.


   FOOTNOTES
 
* This work was supported by Grant HL19737 from the National Institutes of Health. The results of this study have been presented in part at EB2004 in Washington, DC and EB2005 in San Diego, CA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Institute for Environmental Medicine, University of Pennsylvania, One John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068. Tel.: 215-898-9108; Fax: 215-898-0868; E-mail: abf{at}mail.med.upenn.eduX.

2 The abbreviations used are: DPPC, dipalmitoylphosphatidylcholine; PLA2, phospholipase A2; aiPLA2, acidic calcium-independent phospholipase A2; Prdx6, peroxiredoxin 6; pAb, polyclonal antibody; mAb, monoclonal antibody; BAL, bronchoalveolar lavage; SP-A, surfactant protein A; {pi}GST, glutathione S-transferase {pi}; PBS, phosphate-buffered saline; BSA, bovine serum albumin. Back

3 Y. Manevich, C. Dodia, S. I. Feinstein, and A. B. Fisher, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Jian-Qin Tao for technical assistance with isolation of type II cells, Lu Lu for preparing recombinant protein, Dr. Steve Wrenn, Dr. Gregory Troup, and Andrew Guarino for light scattering studies, Jennifer Rossi for typing the manuscript, and Dr. Bruce S. Sachais, Ann Rux, and Cynthia Pennise (Biosensor Shared Resource Facility, University of Pennsylvania) for assistance with Biacore studies.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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