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Am J Physiol Lung Cell Mol Physiol 291: L436-L446, 2006. First published March 23, 2006; doi:10.1152/ajplung.00415.2005
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Identification and characterization of p63 (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein A binding protein, on type II pneumocytes

Nisha Gupta, Yefim Manevich, Altaf S. Kazi, Jian-Qin Tao, Aron B. Fisher, and Sandra R. Bates

Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Submitted 28 September 2005 ; accepted in final form 20 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Surfactant protein A (SP-A) binds to alveolar type II cells through a specific high-affinity cell membrane receptor, although the molecular nature of this receptor is unclear. In the present study, we have identified and characterized an SP-A cell surface binding protein by utilizing two chemical cross-linkers: profound sulfo-SBED protein-protein interaction reagent and dithiobis(succinimidylpropionate) (DSP). Sulfo-SBED-biotinylated SP-A was cross-linked to the plasma membranes isolated from rat type II cells, and the biotin label was transferred from SP-A to its receptor by reduction. The biotinylated SP-A-binding protein was identified on blots by using streptavidin-labeled horseradish peroxidase. By using DSP, we cross-linked SP-A to intact mouse type II cells and immunoprecipitated the SP-A-receptor complex using anti-SP-A antibody. Both of the cross-linking approaches showed a major band of 63 kDa under reduced conditions that was identified as the rat homolog of the human type II transmembrane protein p63 (CKAP4/ERGIC-63/CLIMP-63) by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of tryptic fragments. Thereafter, we confirmed the presence of p63 protein in the cross-linked SP-A-receptor complex by immunoprobing with p63 antibody. Coimmunoprecipitation experiments and functional assays confirmed specific interaction between SP-A and p63. Antibody to p63 could block SP-A-mediated inhibition of ATP-stimulated phospholipid secretion. Both intracellular and membrane localized pools of p63 were detected on type II cells by immunofluorescence and immunobloting. p63 colocalized with SP-A in early endosomes. Thus p63 closely interacts with SP-A and may play a role in the trafficking or the biological function of the surfactant protein.

lung; surfactant secretion; cross-linking; immunolocalization

PULMONARY SURFACTANT IS A complex mixture of phospholipids and proteins. The major protein component, surfactant protein-A (SP-A), is a 34- to 36-kDa member of the calcium-dependent lectin family of proteins that facilitates the surface tension-lowering properties of surfactant phospholipids in the alveolus; regulates surfactant phospholipid synthesis, secretion, and recycling; and also plays an important role in pulmonary host defense (18). The mechanism of SP-A binding to lung type II cells is not completely understood, yet it appears to be critical for the cell-associated functions of SP-A. Binding of SP-A to type II cells was found to be both saturable and specific, suggesting that this interaction is mediated through a receptor on the cell surface (24, 55).

Thus far, investigators have identified SP-A receptor(s) by utilizing either anti-idiotypic antibodies (43, 46) or SP-A affinity column chromatography (9, 21). Using an idiotypic approach, one group has identified three proteins with apparent molecular mass values of 30, 52, and 60 kDa (44), and the 30-kDa protein was shown to be associated with the regulation of secretagogue-stimulated surfactant secretion (45, 46). On the other hand, another group using similar strategy identified a 55-kDa protein (43) shown to be involved in surfactant endocytosis by type II cells (53, 55). Using ligand affinity chromatography, three proteins with apparent molecular mass values of 65, 55, and 50 kDa were identified by a third group on the type II cell surface, and the 50-kDa protein was shown to be involved in phospholipid uptake (21). A fourth group described a SP-A receptor from U937 macrophages with a molecular mass of 210 kDa using SP-A affinity column chromatography (9). This receptor was detected in both alveolar macrophages and type II epithelial cells and was found to block the SP-A-mediated inhibition of phospholipid secretion by type II cells. From the these studies, it seems that the SP-A receptor(s) on the type II cell surface consists of 30-, 50- to 55-, or 60- to 65-kDa polypeptides. The cDNA and deduced amino acid sequence of the 30-kDa protein are described elsewhere (44, 46); however, the molecular nature of the other components of the receptor(s) is still not clear. Thus far, it is also not completely understood how these proteins are related, if they are separate entities and/or components of a receptor complex.

The present investigation identifies a 63-kDa type II transmembrane protein as a functional SP-A receptor on type II cells. Unlike using an indirect idiotypic approach or chromatography, we directly cross-linked SP-A to isolated plasma membranes as well as intact type II cells. We were able to successfully reproduce our cross-linking results by using two different cross-linkers and identified the SP-A binding protein as p63/ERGIC-63 by liquid chromatography-mass spectrometry/mass spectrometry analysis and National Center for Biotechnology Information database search. Coimmunoprecipitation experiments and functional assays provided firm evidence that reflected specific interaction between SP-A and p63. This is the first study that has utilized direct cross-linking of SP-A to isolated plasma membranes and intact type II cells and demonstrates direct involvement of p63 in SP-A-mediated phospholipid homeostasis in type II pneumocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of pneumocytes. Type II cells were isolated from adult male Sprague-Dawley rat lungs according to the procedure of Dobbs et al. (11) as previously described (7, 8). Briefly, after perfusion via the pulmonary artery and lavage through a tracheal cannula, the lungs were digested with elastase and minced in the presence of DNase (Sigma-Aldrich) and fetal bovine serum (ICN Biochemicals). The cells were separated by filtration and enriched for type II cells by plating on rat IgG (Sigma-Aldrich)-coated petri dishes that served to remove most contaminating macrophages. After overnight culture and removal of nonadhered cells, the purity of the type II cell preparation was 90–98%.

Type II cells were isolated from C57BL/6 mouse lungs using dispase as described previously (6, 50, 52). Briefly, perfused lungs were digested with dispase, treated with DNase I, and filtered through nylon mesh (100-, 40-, and 25-µm size). Macrophages were removed from the cell preparation by plating on mouse IgG-coated petri dishes. The nonadherent cells were seeded on 100-mm cell culture dishes in 10% fetal bovine serum at 37°C for 1 h twice to remove fibroblasts. The final cell isolates were seeded on type I collagen-coated 35-mm dishes in Ham's F-12 culture medium supplemented with 15 mM HEPES, 0.8 mM CaCl2, 0.25% BSA, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selinite, and 2% mouse serum. After 24 h, >95% of the isolated cells were type II cells.

Alveolar macrophages were isolated by bronchoalveolar lavage and centrifugation. The resultant preparation was >98% macrophages (3).

Preparation of cell lysates. Type II cells or macrophages were lysed on ice in phosphate-buffered saline, pH 7.4, containing 0.1% Triton X-100 and protease inhibitor cocktail (Roche Diagnostics), for 10 min, and they were sonicated on ice for two 15-s cycles. Thereafter, cell lysates were stored at –20°C until use. Cell protein content was measured by the method of Lowry et al. (26) using BSA as a standard.

Isolation of plasma membrane proteins. Plasma membranes were isolated as described before (7, 15). Briefly, rat type II cells were suspended in 0.32 M sucrose in cold HEPES-Tris buffer, pH 7.4, followed by three 15-s sonication cycles on ice at an interval of 5 min. Thereafter, cell lysates were layered over a discontinuous sucrose gradient containing 0.5, 0.7, 0.9, and 1.2 M sucrose and centrifuged at 40,000 g for 1 h at 4°C. Plasma membrane fraction was collected from the 0.9–1.2 M sucrose interface, diluted to 0.32 M sucrose with HEPES-Tris buffer, and centrifuged at 95,000 g for 30 min (7, 15). The plasma membrane pellet was resuspended in PBS, pH 7.4, containing all above protease/phosphatase inhibitors and 0.1% Triton X-100 followed by vortexing. Aliquots were stored at –80°C until use.

Chemical cross-linking and immunoprecipitation. Chemical cross-linking using profound label transfer sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido)hexanoamido]ethyl-1,3'-dithiopropionate (SBED) protein-protein interaction reagent: all steps were performed under dark conditions, and 0.6 mM SP-A and 6 mM sulfo-SBED (Pierce Biotechnology) predissolved in DMSO were incubated in dark for 30 min under constant stirring. BSA was used as a control protein for labeling. The reaction mixture was loaded on a desalting column (Bio-Spin P-6, Bio-Rad) and quick spun for 5 s. Collected filtrate was mixed with 100 µg of plasma membrane proteins followed by illumination at 365 nm in the presence of 1.75 mM CaCl2 for 20 min.

Cross-linked samples were analyzed thereafter by SDS-PAGE on 3–8% Tris-acetate or 4–12% Bis-Tris gradient gels (Invitrogen) under reduced (100 mM dithiothreitol) and nonreduced conditions for both test and control (BSA) reactions followed by detection of biotinylated samples on Western blots using horseradish peroxidase (HRP)-labeled streptavidin.

Chemical cross-linking using dithiobis(succinimidylpropionate). Mouse type II cells cultured for 36 h in 35-mm dishes (1.5 x 106 cells, ~120 µg protein/dish) were washed three times with PBS, pH 7.4. SP-A (1 or 5 µg) and CaCl2 (2 mM) were added to the washed cells, and cells were incubated at 37°C for 1 h. After another round of washing with PBS, pH 7.4, cross-linking was performed by incubating with dithiobis(succinimidylpropionate (DSP; Pierce Biotechnology; 1 or 2 mM) predissolved in DMSO at room temperature for 30 min.

Preliminary experiments showed that both concentrations of SP-A and/or DSP were equally as effective. Thus 1 µg SP-A and 1 mM DSP were used in subsequent studies. After being washed with PBS, cells were scraped in situ with 500 µl of radioimmune precipitation buffer containing 20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail (Roche). The cell lysate was incubated with rabbit anti-human SP-A antibody overnight at 4°C under gentle rotation. Thereafter, the incubation mixture was centrifuged at 13,000 rpm for 5 min at 4°C, and protein A-immobilized agarose beads (Sigma) were added to the supernatant and incubated for 6 h in cold. After the beads were washed three times with radioimmune precipitation buffer, the bound proteins were eluted from the agarose beads by incubation and boiling with Laemmli (25) sample buffer in the presence 2-mercaptoethanol. The eluted proteins were loaded onto 4–12% Bis-Tris gradient SDS-PAGE gels (Invitrogen) for electrophoresis followed by Western blot analysis.

To investigate the interaction of p63 and SP-A in solution, coimmunoprecipitation experiments were performed as described above using protein A-agarose beads, rabbit anti-human SP-A (Chemicon), rabbit anti-human p63, or mouse anti-human p63 (40) antibodies.

SDS-PAGE and Western blot analysis. Proteins from the cell lysates or cross-linked samples were resolved on 3–8% Tris-acetate or 4–12% Bis-Tris gradient gels (Invitrogen) by SDS-PAGE under reducing or nonreducing conditions (25). Proteins were electrophoretically transferred to a nitrocellulose membrane at room temperature overnight (47, 48). The membrane was transiently stained with Ponceau S to monitor the transfer efficiency of the proteins and was blocked with 4% Carnation nonfat dry milk in Tris-buffered saline (TBS) at room temperature for 1 h on a shaking platform. The nitrocellulose membrane was then incubated with primary antibodies. After being washed with TBST (TBS with 0.1% Tween 20), blots were incubated with appropriate HRP-labeled secondary antibodies (Amersham Biosciences). After being washed three times with TBST, two times with TBS, and a final rinse with deionized water, blots were incubated with ECL chemiluminescence reagent (Amersham Biosciences) for 1 min and developed on Hyperfilm ECL X-ray films (Amersham Biosciences). To reprobe the same membrane, blots were stripped by incubation at 55°C for 30 min in a buffer that contained 62.5 mM Tris, 2% SDS, and 100 mM 2-mercaptoethanol. After being washed four times with TBST, blots were blocked with 4% milk and were reprobed with another antibody.

Western blot analysis by Odyssey was performed according to the manufacturer’s instruction (LI-COR Bioscience, Lincoln, NE). Briefly, nitrocellulose membranes were blocked in 1:1 diluted Odyssey blocking buffer at room temperature for 1 h. The blots were incubated at 4°C overnight with p63 polyclonal antibody at 1:1,000 dilution in blocking buffer containing 0.1% Tween 20. The membranes were washed four times in TBST and were incubated with IRDye 800-conjugated goat anti-rabbit antibody (Rockland Immunochemicals, Gilbertsville, PA) for 1 h at room temperature at a dilution of 1:5,000. Membranes were scanned on the Odyssey infrared scanner after five washes in TBST buffer.

Mass spectrometry and database search. SDS-PAGE gels were stained with mass spectrometry compatible silver stain (Silver Quest from Invitrogen) and the bands were excised. The gel pieces were washed, reduced, alkylated, and digested in situ with trypsin. Peptides thus generated were injected into a nanocapillary reverse-phase HPLC coupled to a nanoelectrospray ionization source of ThermoFinnigan LCQ quadrupole ion trap mass spectrometer. This mass spectrometer measured peptide masses and then fragmented individual peptides to produce mass spectrometry/mass spectrometry spectra of fragments that reflected the peptide sequence. The mass spectrometry/mass spectrometry spectra were run against National Center for Biological Information nonredundant database. The resulting mass spectrometry/mass spectrometry matches and sequences were then reviewed for the fidelity to the matched spectra. This analysis was performed at the Wistar Institute Proteomics Facility of the University of Pennsylvania.

Purification of SP-A. Bronchoalveolar lavage fluid was obtained from lungs of alveolar proteinosis patients after therapeutic lavage at the Hospital of the University of Pennsylvania. Cellular material was removed by centrifugation, and the surfactant was purified by density gradient centrifugation, followed by dialysis and lyophilization as described previously (2). We obtained SP-A according to the method of Hawgood et al. (16) using 1-butanol and D-glucopyranoside extraction, dialysis, and microconcentration as previously described (2). The purity of the SP-A preparation was monitored by SDS-PAGE (25).

Preparation of liposomes. Lipids were obtained from Avanti Polar Lipids (Birmingham, AL). Unilamellar liposomes were prepared from dipalmitoyl phosphatidylcholine (DPPC), egg phosphatidylcholine (PC), egg phosphatidylglycerol, and cholesterol (molar ratio, 10:5:2:3) using "The Extruder" (Lipex Biomembranes) according to the directions of the manufacturer.

PC secretion. After isolation, type II cells were incubated overnight with 0.5 µCi/dish of [methyl-3H]choline (Amersham, Arlington Heights, IL) in MEM containing 10% fetal calf serum to label cellular phospholipids. Cells were washed extensively and incubated for 30 min in MEM. One set of cells was harvested served as control for phospholipid secretion associated with the medium change, as described previously (4, 44). The remaining cells were preincubated with or without nonimmune IgG or monoclonal antibody (mAb) p63 (100 µg/ml, 15 min), followed by an incubation without or with SP-A (0.1 µg/ml, 15 min), followed by the addition of ATP (1 mM, Sigma, St. Louis, MO) for 2 h. The media were removed and centrifuged to remove detached cells. Methanol was added to the cell monolayer, and the cells were scraped from the dish. The cells and the media were extracted using the Bligh and Dyer method (5). The amount of phospholipid secretion was calculated as the percentage of lipid counts per minute in the medium relative to the total counts per minute lipid present in the cells plus the medium. All experiments were performed in duplicate or triplicate, and the values were averaged.

Immunofluorescence confocal microscopy. The distribution of p63 was investigated in intact and permeabilized type II cells cultured for 24 h on glass coverslips as well as in mouse lung cryosections. Intact rat type II cells were rinsed with PBS and stained with wheat germ agglutinin Alexa 594 at 1 µg/ml for 15 min at room temperature and washed three times with PBS. After this, cells were fixed with 2% paraformaldehyde for 20 min, washed, and incubated with p63 antibody overnight at 4°C. The next day, the cells were washed and incubated for 1 h with the secondary antibody labeled with Alexa 488, after which the cells were washed, mounted, and viewed by confocal microscopy.

Other preparations of type II cells were permeabilized by fixation with either cold methanol-acetone (1:1 in volume) for 5 min or 4% paraformaldehyde for 30 min at 4°C. After being washed and treated with sodium borohydride, the samples were blocked with a mixture of BSA and normal goat serum for 1 h at room temp followed by incubation with nonimmune mouse or rabbit IgG (control) or primary antibody at room temp for 2 h. The primary antibodies used were rabbit anti-human SP-A (14); rabbit polyclonal antibody to p63 (kindly supplied by Dr. Jack Rohrer, Univ. of Zurich, Zurich, Switzerland); mouse MAb antibodies to p63 [Alexis Biochemicals, San Diego, CA (40)]; mouse anti-rat ABCA3 [MAb 3C9, labels lamellar body membranes in type II cells (29)], and anti-rat EEA-1 [early endosome marker (34)]. The samples were washed three times with PBS and then incubated with Alexa 488 (green)- and/or Alexa 594 (red)-labeled goat anti-mouse or -rabbit IgG (Molecular Probes) at room temperature for 1 h. Thereafter, samples were observed under a confocal microscope (Bio-Rad).

Statistics. Data are reported as means ± SE. Statistical comparisons were performed with SigmaStat (Jandel Scientific) using a standard t-test. Results were reported as statistically significant differences at P values <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of SP-A receptor by chemical cross-linking. Chemical cross-linking followed by mass spectrometric analysis of the cross-linked species has been utilized successfully to elucidate ligand-receptor interactions (17, 50, 51). We cross-linked SP-A to type II cell plasma membranes to identify putative SP-A receptor proteins. Sulfo-SBED is a commercially available trifunctional cross-linking reagent that allows affinity-based enrichment of cross-linked species. Sulfonated N-hydroxysuccinimide ester on this reagent reacted with primary amines on SP-A and, thereby, biotinylated SP-A in the first step. During the second step, photosensitive aryl azide moiety linked by a disulfide bond on this cross-linker captured the putative receptor from the plasma membrane lysate because of the receptor's affinity and the closeness (within a distance of 10 A) to SP-A. After reduction of the disulfide bond with 100 mM dithiothreitol, biotin label was transferred to the captured protein(s) and revealed the presence of a protein with a molecular mass of 63 kDa and minor amounts of two proteins of 90 and 35 kDa (Fig. 1, left). By reprobing the same blots with anti-rat SP-A antibody, we confirmed that the 63- and 35-kDa bands did not represent the monomer or dimer of SP-A (Fig. 1, right).


Figure 1
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  		Fig. 1. Cross-linking of surfactant protein A (SP-A) with plasma membrane proteins isolated from rat type II cells using sulfo-SBED reagent; 4–12% Bis-Tris polyacrylamide gels and reducing conditions were used. Left: biotinylated samples were detected using horseradish peroxidase (HRP)-labeled streptavidin. Right: same blot stripped and reprobed using anti-SP-A antibody.

 
Another cross-linking agent, DSP, was used to investigate an SP-A binding protein in intact mouse type II cells. This agent can be added directly to the tissue culture dishes and does not need UV illumination for cross-linking (50, 51). DSP, or Lomant's reagent, is a homobifunctional cross-linker that has two N-hydroxysuccinimide esters and a disulfide bond. Mouse cells were used because the mouse genome is characterized, facilitating the identity of proteins that might cross-link to SP-A. SP-A was cross-linked with intact mouse type II cells cultured on plastic dishes in the presence of calcium using DSP. The complex of SP-A and bound protein(s) was immunoprecipitated with rabbit anti-human SP-A and separated using gel electrophoretic procedures. Subsequent analysis of silver-stained gels resulted in the identification of three proteins of 30, 50, and 63 kDa (Fig. 2A, lane 2). Western blots of similar gels probed with anti-SP-A antibody confirmed the 50-kDa band as the rabbit anti-SP-A IgG antibody as the band reacted with the goat anti-rabbit IgG secondary antibody (Fig. 2B, lanes 1 and 2, solid arrow), whereas the 30- and 63-kDa silver-stained bands (Fig. 2A, lane 2) were not labeled by the anti-SP-A antibody (Fig. 2B, lane 2). This identification of the 50-kDa band as rabbit IgG was further confirmed by separate immunoprecipitation experiments (Fig. 3A, bottom and middle lanes, solid arrow). The presence of a 63-kDa protein in the results of both cross-linking experiments indicated the specific interaction of this protein with SP-A. Mass spectrometry of the tryptic digest of the 63-kDa band and database search confirmed the presence of the transmembrane protein p63, which is the mouse homolog of endoplasmic reticulum (ER) and Golgi intermediate compartment 63 (ERGIC-63; Table 1). The six peptide sequences from a single database entry showed 100% match to the mass spectrometry/mass spectrometry spectra and confirmed the protein identity at a high confidence level. The 63-kDa protein band in the cross-linked complex was labeled with the polyclonal anti-p63 antibody (Fig. 2C, lane 2), thus confirming the identity of the protein as p63/ERGIC-63.


Figure 2
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  		Fig. 2. Cross-linking of SP-A with intact mouse type II cells using dithiobis(succinimidylpropionate) (DSP) reagent and immunoprecipitation (IP) of the cross-linked proteins with anti-SP-A antibody; 4–12% Bis-Tris polyacrylamide gels and reducing conditions were used. Lane 1, protein A beads + SP-A + anti-SP-A antibody. Lane 2, protein A beads + cross-linked SP-A with type II cell lysate + anti-SP-A antibody. Lane 3, protein A beads alone. A: silver-stained gel. B: immunoblot of gels in A probed with anti-SP-A antibody. C: immunoblot in B stripped and reprobed with polyclonal anti-p63 antibody. WB, Western blot. Open arrowhead, 63 kDa; shaded arrowhead, SP-A (~34, 60, and 90 kDa); solid arrowhead, unknown (~30 kDa); solid arrows, IgG (~50 kDa).

 

Figure 3
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  		Fig. 3. Interaction of p63 and SP-A by coimmunoprecipitation. A: immunoprecipitation using rabbit anti-human SP-A antibody (Ab). First lane, protein A beads alone. Second lane, protein A beads + rabbit anti-human SP-A antibody + rat type II cell (T II) lysate. Third lane, human SP-A. Top: blot probed with mouse anti-human p63 showing presence of p63 (open arrow) in rat type II cells. Bottom: same blot stripped and reprobed with anti-human SP-A showing IgG (solid arrow) and human SP-A (shaded arrows). B: immunoprecipitation using mouse monoclonal anti-human p63 antibody. First lane, protein A beads + mouse anti-human p63 antibody + rat type II cell plasma membrane (PM) lysates. Second lane, protein A beads + mouse anti-human P63 + rat type II whole cell lysates. Third lane, human SP-A. Top: blot probed with anti-human SP-A showing SP-A (shaded arrows). Bottom: blot stripped and reprobed with anti-human p63 antibody showing IgG (solid arrows) and p63 (open arrow). Protein A beads alone showed no bands.

 

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  		Table 1. Identification of p63

 
Coimmunoprecipitation of p63 and SP-A. Protein-protein interactions between p63 and SP-A in vitro were studied by coimmunoprecipitation using rabbit polyclonal antibody to SP-A or mouse monoclonal antibody to p63. Anti-SP-A antibody immunoprecipitated p63 from rat type II cell lysates (Fig. 3A, top). Reprobing of the same blot with SP-A antibody showed a very strong 50- to 60-kDa band in the type II cell lysate, likely a mixture of IgG and SP-A dimer (Fig. 3A, bottom, second lane). Bands of 90, 120, and 150 kDa may be higher molecular mass oligomers of SP-A, as shown in lanes containing isolated human SP-A (Fig. 3A, bottom, third lane, and especially in Fig. 3B, top, third lane). p63 monoclonal antibody immunoprecipitated SP-A from both isolated plasma membranes and whole type II cells (Fig. 3B, top), and reprobing of the same blot confirmed the presence of p63 in the immunoprecipitate (Fig. 3B, bottom).

Identification and localization of p63 in type II cells. Immunoblots of lysates from lung cells probed with polyclonal antibody to p63 revealed that p63 is expressed in type II cells but absent from alveolar macrophages (Fig. 4). Reducing agents did not alter the mobility of the protein ruling out the presence of lower molecular mass disulfide-linked protein components.


Figure 4
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  		Fig. 4. p63 is found in type II cells and absent from macrophages (MØ). WBs show of type II and alveolar macrophage cell lysates run under nonreduced (left) and reduced (middle) conditions on 4–12% Bis-Tris gels (right). Plasma membrane fraction was from type II cell lysates (PM) run under reduced conditions. Blots are probed with mouse anti-human p63 polyclonal antibody.

 
In order for p63 to interact with SP-A in the alveolar space of the intact lung, p63 should be on the cell surface. As shown in Figs. 4, right, and 5A, \. plasma membranes isolated from type II cells demonstrated the presence of p63. Further experiments demonstrated that p63 also was present in the plasma membranes of A549 cells, a human lung adenocarcinoma with some type II cell characteristics (Fig. 5B). Caveolin-1 is a protein enriched in the plasma membrane and is commonly used to validate the identity of this fraction. Although type II cells do not have caveolin, the plasma membrane fraction from the A549 cells contained caveolin (Fig. 5B). Finally, p63 was demonstrated in the plasma membrane of pneumocytes by immunocytochemical techniques. Type II cells grown on glass slides were incubated with wheat germ agglutinin to label the plasma membrane, fixed, and incubated with anti-p63 antibody. Figure 5, C–F, demonstrates the colocalization of p63 and wheat germ agglutinin on the outer surface of type II cells.


Figure 5
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  		Fig. 5. p63 in the plasma membrane of type II cells. A: WB shows type II cell PM fractions probed with antibodies to p63. Experiments used 18 µg protein/lane. B: A549 cell PM fractions probed with antibodies to p63 or caveolin-1. Experiments used 50 µg protein/lane. Two bands are {alpha}- and beta-subunits of caveolin-1. C–F: immunocytochemistry of type II cells grown on glass coverslips and fixed but not permeabilized. Cells were stained with anti-p63 antibody (C, green) and wheat germ agglutinin (D, red) as a plasma membrane marker. E: colocalization of p63 and wheat germ agglutinin (orange-yellow). F: phase of C–E. MW, molecular mass.

 
Permeabilized rat type II cells showed an ER-like distribution of p63, which occasionally colocalized with ABCA3 a protein marker for lamellar body membranes (Fig. 6, A–C). \. Because p63 is a putative receptor for SP-A, there should be intracellular compartments that are shared by both SP-A and p63. Double labeling of type II cells using antibodies against SP-A and p63 showed colocalization of the two on the outside of some vesicular structures (Fig. 6, D–F). After uptake by type II cells, SP-A has been shown to transport toward early endosomal compartments (52). Using an antibody to EEA-1, a protein marker for early endosomal vesicles, we confirmed that SP-A colocalized with EEA-1 (Fig. 7, D–F). \. In addition, p63 was partially colocalized in the same early endosome intracellular compartment (Fig. 7, A–C).


Figure 6
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  		Fig. 6. Immunolocalization of p63 in rat type II cells. Isolated type II cells after 24 h of culture were permeabilized with Triton X-100 and double labeled with anti-p63 antibody (A and D) and either anti-ATP-binding cassette transporter A1 (ABCA3) antibody (B) or anti SP-A antibody (E). C: merged view of A and B. F: merged view of D and E. G and H: control cells treated similarly to cells in A–F but incubated with only secondary antibodies. G and H: fluorescence image. I: phase of G. Images show intracellular expression of p63, minimal colocalization (yellow) with ABCA3 within the cell (C), and significant colocalization with SP-A (arrows, F). DIC, differential interference constrast (phase). Bars = 10 µm.

 

Figure 7
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  		Fig. 7. Partial colocalization of p63 and SP-A with the early endosome compartment of type II cells. Rat type II cells were cultured for 24 h and immunolabeled with EEA-1, a marker for early endosomes and with either antibodies against p63 or SP-A. A: anti-P63 antibody. B and E: anti-EEA-1 antibody. C: merged view of A and B and phase. D: anti-SP-A antibody. F: merged view of D and E and phase. Yellow indicates colocalization. A–C are x40; D–F are x60.

 
Expression and distribution of p63 in the lung was investigated by immunofluorescence and confocal microscopy in mouse whole lung cryosections. MAb 3C9, which labels the lamellar body membrane ATP-binding cassette transporter A3 (ABCA3), was used as a marker of type II cells (29, 37). Type II cells, identified as ABCA3 positive, and other cells that did not stain for ABCA3, possibly type I cells, showed high intracellular expression of p63 (Fig. 8, A–F). \.


Figure 8
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  		Fig. 8. Immunolocalization of p63 in mouse lung. Lung cryosections were double labeled with anti-p63 antibody and anti-ABCA3 antibody. ABCA3 is a protein specific for type II cell lamellar body membranes and serves as a marker for type II cells. A and D: anti-p63 antibody. B and E: anti-ABCA3 antibody. C and F: merged. A–C and D–F represent 2 different fields of the same lung.

 
Antibody to p63 inhibited SP-A function. SP-A has been shown to inhibit PC secretion through interaction with a specific cell surface receptor (22, 28). To determine whether a MAb to p63 (mAb p63) would block the interaction of SP-A with its receptor, we examined the ability of the anti-p63 MAb to interfere with one of the biological activities of SP-A, the inhibition of ATP-stimulated PC secretion from type II cells. Neither mAb p63 (Fig. 9, A and B) nor nonimmune IgG (Fig. 9B) affected basal or ATP-stimulated PC secretion when incubated with type II cells in the absence of added SP-A. p63 mAb inhibited the ability of SP-A to block secretagogue-stimulated PC secretion in a dose-dependent manner (Fig. 9A). SP-A (0.1 µg/ml) reduced ATP-stimulated PC secretion by 67%. Addition of mAb p63 resulted in a concentration-dependent reversal of the effect of SP-A on stimulated secretion (Fig. 9A). At 100 µg mAb/ml, the reversal of the SP-A affect was 50%, whereas IgG had no effect (Fig. 9B).


Figure 9
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  		Fig. 9. Anti-p63 monoclonal antibody inhibits the biological activity of SP-A. Type II cell phospholipids were labeled by overnight incubation with [3H]choline. A: labeled cells were incubated without additions (no ATP, {triangleup}), with ATP (1 mM) alone ({blacktriangleup}), or together with SP-A (0.1 µg/ml, bullet; or 0.25 µg/ml, {circ}). Samples were incubated without or with increasing concentrations of monoclonal antibody (MAb) to p63. Antibodies were added for 15 min followed by SP-A for 15 min. Finally, ATP was added, and the experiment continued for 2 h (see MATERIALS AND METHODS). Data are means ± range or ± SE of duplicate or triplicate samples from 1 to 3 experiments and are expressed as %phospholipid [phosphatidylcholine (PC)] secretion. B: [3H]phospholipid-labeled type II cells were incubated with ATP (1 mM), monoclonal antibody to p63 (100 µg/ml) or nonimmune IgG (100 µg/ml) without or with SP-A (0.1 µg/ml), and PC secretion was measured over a 2-h period. Data are means ± SE of duplicate or triplicate samples from 3 experiments. aSignificantly different from no SP-A, P < 0.05. bSignificantly different from no antibodies or IgG, P < 0.05.

 

   DISCUSSION
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Earlier investigators have identified receptor(s) for SP-A using various approaches. The molecular nature and the functional roles played by this receptor, however, remain only partially understood. Unavailability of a highly specific antibody to the receptor and the size and stickiness of SP-A itself are some practical hindrances in designing an efficient experimental approach to reveal the molecular nature of the receptor. Among the different methods used to unravel ligand-receptor interactions, chemical cross-linking followed by identification of the cross-linked peptides by mass spectrometry has proven especially useful in dynamic and complex systems (10, 48, 51, 52, 55). Using this approach, we identified p63 as an SP-A binding protein on type II cells. Several lines of evidence support this conclusion. First, the cross-linking reagent sulfo-SBED linked SP-A to a 63-kDa protein in a preparation of isolated rat type II cell plasma membranes. Second, immunoprecipitation after cross-linking of SP-A to intact mouse type II cells in culture identified a 63-kDa protein in a silver-stained gel. Third, mass spectrometry of tryptic digests of the 63-kDa protein eluted from the gel followed by a database search positively identified the peptides as segment components of p63/ERGIC-63 protein. Fourth, the immunoprecipitate of the SP-A-mouse type II cell cross-linked preparation reacted with anti-p63 antibody confirming the identity of the cross-linked protein as p63/ERGIC-63. Fifth, treatment of type II cell lysates with anti-p63 antibody coprecipitated SP-A. Sixth, by immunohistchemical analysis, SP-A and p63 were colocalized and both proteins are found in the early endosome compartment of type II cells. Finally, antibodies to p63 interfered with one of the biological activities of SP-A, the ability of SP-A to block secretagogue-stimulated DPPC secretion from type II cells. Because human SP-A was used with rodent type II cells in this study, possible affects due to species variation may influence the results. However, to date, human and rat SP-A interactions with rat type II cells believed to involve an SP-A receptor have shown no differences between the two species of SP-A. Thus neither the binding characteristics of SP-A to type II cells (55) nor to the ability of SP-A to stimulate the uptake of phospholipid liposomes into type II cells (23) differed between SP-A isolated from humans or rats.

The p63/ERGIC-63 protein is a nonglycosylated, reversibly palmitoylated type II transmembrane protein. Transmembrane proteins of type II have the NH2 terminus exposed to the cytoplasm, whereas the COOH terminus is in the lumen of the endomembrane compartment. p63 is also known as cytoskeleton-associated protein 4 the Human Genome Organization gene name) and cytoskeleton-linking membrane protein (20). At its NH2 terminus, p63 has a 106-amino acid-long cytosolic tail, a single transmembrane domain, and a large extracytoplasmic domain of 474 amino acids (38, 41). It was originally suggested that p63 is a resident protein of a membrane network interposed between the rough ER and Golgi apparatus or the ERGIC compartments (38, 39). Antibodies against ERGIC-63/p63 were first generated to elucidate the structural organization of the ER-to-Golgi pathway and to study the dynamics of its membrane elements (38, 40). The distribution of p63 was shown to overlap with the ER-Golgi intermediate compartment as confirmed by another marker of ERGIC (ERGIC-53). Although original studies of p63 described it as an ER resident protein (40) and subsequently as an ER-microtubule linking protein (20), it is now known also to be expressed on the surface of vascular smooth muscle cells (35). The presence of normally endoplasmic reticulum-resident proteins at the plasma membrane is not unusual, as illustrated by the integral membrane proteins kinectin (48, 49) and calnexin (32). Our results from immunocytochemical studies indicate that p63 is localized both intracellularly and on the surface of type II cells. Plasma membrane localization for p63 is supported by anti-p63 antibody labeling (by Western blots) of p63 in the plasma membrane fraction isolated from type II cell lysates; immunofluorescent staining of nonpermeabilized type II cells with anti-p63 antibody and colocalization with wheat germ agglutinin, a marker of plasma membranes; cross-linking of SP-A and p63 in plasma membrane preparations as well as to p63 on the surface of live cells; and immunoprecipitation of p63 with anti-p63 from plasma membrane preparations. The inhibitory effect of an anti-p63 monoclonal antibody on SP-A-mediated inhibition of PC secretion provides further evidence toward establishing that there are pools of p63 expressed on plasma membrane that can serve as specific binding sites for SP-A.

The cross-linking procedures utilized in the present report also identified a 30- to 32-kDa protein that complexed with SP-A. Previously, using anti-idiotypic antibody A2R, Strayer et al. (44, 46, 47) identified SP-A binding proteins of 30 kDa as well as 56 and 60 kDa. The cDNA for the 30-kDa protein was identified and sequenced (44, 46). Using ligand blots of type II cell plasma membranes, we showed that SP-A and A2R bound to proteins of ~30 and 60 kDa. Studies in our laboratory have demonstrated that the bulk of the proteins were intracellular and were transported to the cell surface with secretagogue treatment (7). What remains to be established is whether or not the 30- and 60-kDa protein in those studies are the same as the 30-kDa unknown protein and p63 described in the present work. One contradictory point is that p63 does not seem to be a subunit of a larger protein complex because it remains at 63 kDa in both reduced and nonreduced gels. In our laboratory's previous work using ligand blots with labeled A2R and SP-A, the bands of 30 and 60 kDa were labeled in reduced gels, whereas a 210-kDa protein was labeled in nonreduced gels (7), indicating that the smaller proteins were components of a larger disulfide-bonded complex.

It has been demonstrated that although all three domains of the p63 protein are required to achieve complete intracellular retention, the cytoplasmic domain plays a dominant role. A truncation mutant of p63, which lacks this domain, {Delta}2–101AA has been shown to traffic exclusively to plasma membrane (40). COS-1 cells transfected with this mutant led to an increase in tissue plasminogen activator-catalyzed plasminogen activation and thus served as the functional binding site for tissue plasminogen activator on the surface of vascular smooth muscle cells (35).

The pathway whereby p63 reaches the plasma membrane is unclear; previous studies on trafficking and localization have shown subdomain-specific localization of p63 in the ER mediated by its luminal {alpha}-helical segment (20) and its possible role in the positioning of the rough ER along microtubules (13, 19). As discussed elsewhere, it is indeed possible that a small pool of p63, when overexpressed, loses its association with microtubules and escapes its subdomain-specific localization, freeing it to reach the plasma membrane (35). Another possibility is that p63 reaches the plasma membrane due to interactions with adaptor proteins such as 14-3-3. The 14-3-3 family of proteins consists of highly conserved acidic proteins of 30–33 kDa that are known to bind to nearly 200 target proteins in a phosphoserine/phosphothreonine-dependent manner and are involved in an impressively diverse range of functions, including receptor function and targeting of proteins to the plasma membrane (27, 30). Most of the 14-3-3 proteins bind to target proteins via specific motifs, and motif II [RXRXX(pS/pT)] is found in p63. Because p63 is located in the ER, of interest is the ability of 14-3-3 to override ER retention. In the case of many potassium channel proteins such as KCNK3 and KCNK9 (31), the diarginine motifs present on the NH2 terminus end binds to beta-COP, a component of the coat protein complex I (COPI) (36). The forward transport of proteins retained in the ER by beta-COP can be initiated by Ser/Thr phosphorylation of the protein's 14-3-3 binding motif, resulting in the subsequent binding of 14-3-3 to the protein and causing the release of beta-COP by an unknown mechanism. This process has been demonstrated for channel proteins like KCNK3 and may be the case with p63, in view of the fact that several diarginine (RR/RXR) motifs are present in p63, including one in the cytoplasmic amino terminus. Whether or not 14-3-3 accompanies p63 to the cell surface and/or has additional functions at that site is under investigation (12). However, it is intriguing that, in our p63-SP-A cross-linking studies, we identified an additional unknown protein of molecular mass of 30 kDa that cross-linked to SP-A at the type II cell surface. By Western blotting techniques, 14-3-3 was found to be present in type II cells (data not shown). The importance of the cytoplasmic tail of p63 for retention in the ER was demonstrated by Schweizer et al. (40). Deletion of the first 100 amino acid residues of p63 resulted in constitutive migration of p63 to the plasma membrane, indicating that the diarginine motif may be functionally important in retention of p63 in the ER (40). Thus we hypothesize that 14-3-3 may play a role in the binding, transport or stability of p63 in the plasma membrane. Since p63 is found in cells that are not exposed to SP-A in vivo, p63 may have multiple, cell-specific functions. 14-3-3 and p63 may be part of a receptor complex specific for SP-A and functioning only in type II cells.

It is attractive to speculate that, although p63 is normally an ER-resident protein, under specialized conditions of type II cells expressing SP-A or vascular smooth muscle cells expressing tPA, p63 would act not as an SP-A binding protein but as a chaperone that binds to a receptor complex and helps in its transport to or from the cell surface. p63 becomes reversibly palmitoylated in the presence of brefeldin A (protein transport blocker) (42), making palmitoylation a possible mechanism for protein transport by p63 as reported for transferrin receptors (1). It has been shown before that endocytosed SP-A is taken up in early endosomes (52, 54) that are located in the periplasm of the cells. Whatever the role of p63, our results clearly demonstrate the presence of both SP-A and p63 in early endosomes. Therefore, it is quite possible that p63 compartments are involved both in the internalization of secreted SP-A and in the exocytosis of newly synthesized SP-A that initially bypasses the lamellar body compartments but is eventually taken up from the extracellular space and assembled into the secretory organelle (6, 33).

To conclude, we have shown that cross-linking of SP-A to intact type II cells or isolated plasma membranes results in binding of the surfactant protein to a transmembrane protein called p63. SP-A and p63 interact in vitro and in situ as revealed by coimmunoprecipitation and effects on PC secretion. Type II cells express p63 on the cell surface as well as in intracellular compartments where it colocalizes with SP-A. Thus p63 may play a role in SP-A recycling through transport of SP-A from the ER to the plasma membrane and/or in SP-A binding at the plasma membrane and subsequent internalization.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-19737.


   ACKNOWLEDGMENTS
 
We sincerely thank Dr. Michael Koval for supplying EEA-1 antibodies and Drs. Jack Rohrer and Anja Schweizer for the gift of the polyclonal anti-p63 antibody. Drs. Sheldon Feinstein and David Spiecher were very helpful in suggesting experimental protocols for this project.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. R. Bates, Institute for Environmental Medicine, Univ. of Pennsylvania School of Medicine, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104 (e-mail: batekenn{at}mail.med.upenn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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