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Purification and Characterization of PLUNC from Human Tracheobronchial Secretions

Division of Pulmonary and Critical Care Medicine, Department of Medicine; Department of Anesthesiology; and Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida

Address correspondence to: Philip L. Whitney, Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, P.O. Box 016960 (R-47), Miami, FL 33101. E-mail: pwhitney{at}miami.edu

	Abstract

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To study proteins secreted into the airway, we used secretions from primary human airway epithelial cells, re-differentiated at the air–liquid interface, and from patients intubated during surgery. A major protein of the cultured cell secretions was ethanol soluble. This protein was purified, analyzed by Edman degradation, matrix-assisted laser-desorption ionization time-of-flight mass spectroscopy of tryptic digests, and Western blots of two-dimensional electrophoresis gels using antisera against the purified preparation. The protein was identified as palate, lung, nasal epithelium clone protein (PLUNC). The protein had multiple truncated molecules, a pattern also seen in tracheal aspirates. PLUNC was poorly soluble in water (50 µg/ml) or in 50 mM NaCl but was more soluble in 75% ethanol (> 380 µg/ml). PLUNC secretion dramatically increased during the second week in air–liquid interface culture and continued to increase over time. Immunohistochemistry showed that PLUNC was expressed in human airway epithelium and submucosal glands. Although PLUNC is in the lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein family of antibacterial host defense proteins, purified PLUNC failed to compete with LBP for the binding of LPS, whereas polymyxin B, a known inhibitor of LPS-LBP binding, did interfere with binding. This study showed that plunc gene product is expressed both in vivo and in vitro, detailed a method for its purification and provided basic information on its biochemical properties in secretions.

Abbreviations: two-dimensional gel electrophoresis, 2-DE • air–liquid interface, ALI • bactericidal/permeability-increasing protein, BPI • bovine serum albumin, BSA • enzyme-linked immunosorbent assay, ELISA • high-performance liquid chromatography, HPLC • human tracheal aspirates, HTA • lipopolysaccharide-binding protein, LBP • lipopolysaccharide, LPS • long PLUNC, LPLUNC • lung-specific X protein, LUNX • matrix-assisted laser-desorption ionization time-of-flight mass spectroscopy, MALDI-TOF • polyacrylamide gel electrophoresis, PAGE • phosphate-buffered saline, PBS • palate, lung, nasal epithelium clone protein, PLUNC • sodium dodecyl sulfate, SDS • short PLUNC, SPLUNC • secretory protein of the upper respiratory tract, SPURT • Tris-buffered saline, TBS • TBS + Tween-20, TTBS

	Introduction

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A number of proteins have been identified that bind bacteria and mediate mammalian host responses to infectious challenge. Foremost among these are lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) (1–3). Recently, genes with sequence similarity to LBP and BPI have been identified and shown to be expressed in the upper respiratory tract. The genes plunc, lunx, and spurt (4–6) were first cloned using differential expression methods from either mouse or human tissues, and, in the case of the human homologs, predict expression of an identical protein. The expression of this protein is known through analysis by two-dimensional gel electrophoresis (2-DE) (7, 8) and by Western blots and immunohistochemistry using antisera raised against synthetic peptides predicted from the transcript sequences (6, 9). However, the purification and biochemical characterization of the protein has not been reported.

Plunc, first identified in the mouse embryo (4), was shown by in situ hybridization to have expression restricted to the epithelium of the nasal structures and palate of the mouse embryo (4, 9, 10), the nasal and tracheobronchial epithelium of the adult animal (4, 10), and thymus (10). Plunc gene homologs have also been described in rat (9) and cow (11). The pattern of expression in humans, studied by Northern blots (12) and in situ hybridization (10), also showed that human plunc expression was confined to the nasopharyngeal and tracheobronchial areas. In humans, plunc has been postulated to be a member of a family of genes that encode three distinct short proteins (short [S] palate, lung, nasal epithelium clone protein [PLUNC]1-3; 256, 249, and 253 amino acids) and four distinct long proteins (long PLUNC [LPLUNC]1-4; 484, 458, 463, and 469 amino acids) (13).

The product of the human plunc, lunx, and spurt genes was predicted to have 256 amino acids and to be rich in leucine, suggesting that the protein might be hydrophobic (6, 12). Based on analysis of the predicted amino acid sequence and its similarity to other proteins that are secreted (murine parotid secreted protein and von Ebner minor salivary gland protein), it was proposed that PLUNC is secreted (4, 6, 10), a fact later confirmed by finding the protein in nasal secretions (7–9), in sputum and supernatants of cultured airway epithelial cells (6). An increase in PLUNC secretion into the airways has been suggested to occur in inflammatory airway diseases (6, 7).

All of the studies of the protein product of these genes have been directed by predictions from its nucleic acid sequence or used anti-peptide antibodies. In this report, we describe purification of PLUNC from secretions, biochemical and functional properties of PLUNC, and its immunolocalization in the airways using antibodies to the purified protein.

	Materials and Methods

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Human Tracheobronchial Cell Cultures and Secretions
Human tracheobronchial cell cultures were prepared by methods described previously (14, 15). Human tracheas and bronchi were obtained from lungs that were not deemed suitable for transplant through the Life Alliance Organ Recovery Agency of the University of Miami and approved by the local institutional review board. The mucosa from the larger airways was dissected and digested with protease, and released cells were collected by centrifugation. Cells were plated on collagen-coated culture dishes with bronchial epithelial growth medium (15) and harvested with trypsin after reaching confluence. Such de-differentiated cells were plated onto 24-mm Transwell-clear culture inserts coated with human placental collagen. The cultures were maintained in a medium containing 50% DMEM and 50% LHC basal medium (Biosource International, Camarillo, CA) supplemented with hormones and trace elements as described (14). Upon reaching confluence (after 3–7 d), the medium from the apical surface was removed, leaving the top surface exposed to air (air-liquid interface cultures, ALI). Apical surface secretions were harvested by washing the cultures with 0.5 ml phosphate-buffered saline (PBS) every 2–3 d. PBS rinses were stored at -20°C until use. Before use, thawed samples were centrifuged at 2,000 x g for 10 min. Upon thawing, some samples were treated with protease inhibitors (Protease Inhibitor Cocktail Set I; Calbiochem, San Diego, CA).

Human Tracheal Secretions
Following an IRB-approved protocol, human tracheal aspirates (HTA) were collected from patients undergoing general anesthesia for elective surgery indicated for nonpulmonary reasons. Respiratory secretions were collected by instilling 4 ml saline through a suction catheter that was advanced through an endotracheal tube into the trachea followed by immediate suctioning. The samples were centrifuged at 500 x g for 5 min to remove cells and the supernatant was centrifuged at 16,000 x g for 20 min at 4°C. The second supernatant was stored at -20°C until use.

Electrophoresis
Protein concentrations were determined using the Micro BCA Protein Assay Reagent Kit (Pierce Chemical, Rockford, IL) according to the manufacturer's instructions. Because the samples were too dilute for direct use, protein was precipitated by addition of four volumes of cold acetone; after 10–15 min, the sample was centrifuged at 16,000 x g for 5–10 min. For sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), the pellet was re-suspended in SDS-PAGE sample buffer (80 mM TrisCl pH 6.8 with 10 mM EDTA, 50 mM dithioerythritol [DTE], 3% SDS, and 10% glycerol). Before electrophoresis, the sample tubes were placed in an ultrasonic water bath for 1 min and then heated at 100°C in a sand bath for 5 min. Electrophoresis was performed with 13% polyacrylamide minigels (Duracryl, 30% total, 2.2% cross-linked; Genomic Solutions, Ann Arbor, MI) (16).

2-DE was performed following the procedures described by Gorg (17). Immobilized pH gradient (IPG) strips (13 cm pH 3–10 NL; Amersham Biosciences, Piscataway, NJ) were used for the first dimension. Acetone pellets from secretion samples were dissolved in 250 µl 8 M urea, 10% glycerol, 4% CHAPS, 0.5% IPG buffer and absorbed by the IPG strips during 12 h at 20°C at 20 V in an Ettan IPGphor isoelectric focusing system (Amersham Biosciences). Electrofocusing began at 200 V for 0.5 h and increased to 500 V for 1 h, 1,000 V for 1 h, 3,500 V for 2 h, and 4,000 V until 50–70 kVh were accumulated. Current was limited to 35 µA/strip. For the second dimension, 16 x16 cm gels of 13% Duracryl were used. Electrophoresis was performed at 20°C in a Hoefer SE600 unit (Amersham Biosciences). The gels were silver-stained using DTE as the reducing agent (18). 2-DE analysis was performed with the Phoretix 2D software (Nonlinear Dynamics, Newcastle upon Tyne, UK).

Purification and Concentration of PLUNC
Sample proteins were precipitated by adding 3 volumes of ethanol; after 10–15 min on ice the sample was centrifuged at 16,000 x g for 10 min at 4°C. PLUNC remained in the supernatant and was precipitated by adding 2.5 volumes of ice-cold acetone per volume of supernatant fluid. After 10–15 min on ice, the sample was centrifuged at 16,000 x g for 10 min at 4°C. Complete precipitation by acetone required the presence of salt, such as 20 mM NaCl.

For preparations using more than 1 ml sample, an initial acetone precipitate was dissolved in < 10% of the original volume of PBS and precipitated again with 3 volumes of ethanol. After centrifugation, 2.5 volumes of acetone were added to the supernatant fluid to precipitate the PLUNC. The last pellet was then dissolved in 75% ethanol and stored at –20°C.

In early studies, PLUNC in 75% ethanol was concentrated by evaporating the solvent with a stream of nitrogen in a warm bath, but this procedure also concentrated the low molecular weight components of the medium (salts). We found that it was better to precipitate the protein with acetone to avoid interference during electrophoresis.

High-Performance Liquid Chromatography of Purified PLUNC
PLUNC that had been purified by ethanol fractionation was analyzed and further purified by high-performance liquid chromatography (HPLC) using a reverse-phase Supelco C4 guard column (5 µm, 300 A°, 4.6 x 20 mm; Sigma, St Louis, MO) and a gradient from 10% acetonitrile, 0.05% trifluoroacetic acid (solvent A) to 75% acetonitrile, 0.05% trifluoroacetic acid (solvent B) at room temperature. A peak was obtained at 55–60% acetonitrile. The gradient consisted of a 5 min linear segment from 0 to 40% B followed by a 10-min linear segment from 40–100% B. The flow rate was 0.8 ml/min. The eluate was monitored at 215 nm and PLUNC eluted at 16 min. PLUNC would not elute from a Microsorb C4 column (5 µm, 300A°, 4.6 x 250 mm) and eluted poorly from an Aquapore C8 guard column (7 µm, 300A°, 4.6 x 30 mm).

Solubility of PLUNC
Acetone pellets of PLUNC (20–30 µg), collected from 75% ethanol, were suspended in 65 µl of a test solvent with vortex mixing and a 1-min treatment in an ultrasonic water bath at room temperature. Following centrifugation at 16,000 x g for 10 min at 4°C, 50 µl of the supernatant fluid was used for analysis by HPLC as detailed above. The concentration of solubilized protein was measured from the peak height of absorbance at 215 nm. Peak width did not vary significantly from run to run. Protein concentration was measured with the BCA assay (Pierce Chemical, Rockford, IL) and related to peak height in 0.1% SDS, 50 mM NaCl, a solvent that completely dissolved the protein.

Anti-PLUNC Antibodies
The antigen used to prepare antiserum to PLUNC was purified from ALI secretions by ethanol and acetone precipitations as described above. It was then subjected to preparative SDS-PAGE and stained with colloidal Coomassie Blue (GelCode Blue Stain Reagent; Pierce, Rockford, IL). The stained band at 21 kD was cut from the gel and sent to Covance Research Products, Inc. (Denver, PA) to immunize NZW rabbits and collect antisera. Enzyme-linked immunosorbent assay (ELISA) of serial bleeds showed increasing titers from 1:100–1:15,000.

Western Blots
For Western blots following one and two-dimensional SDS-PAGE, the proteins were electroblotted onto Immobilon P membranes (Millipore Corp, Billerica, MA) in 10 mM CAPS/NaOH buffer, pH 11, plus 10% methanol. Membranes were blocked with 1% bovine serum albumin (BSA) in Tris-buffered saline, pH 7.6 (TBS) with 0.05% Tween-20 (TTBS) for 1 h and then incubated with rabbit anti-PLUNC serum (diluted into blocking solution, 1:2,500 for HTA or 1:20,000 for ALI samples) for 1 h. After washing with TTBS for 30 min, the second antibody was added for 1 h (alkaline phosphatase-labeled goat anti-rabbit IgG affinity-purified to IgG [H + L], diluted 1:2,500 for HTA or 1:5,000 for ALI samples; Kirkegaard and Perry Laboratories, Gaithersburg, MD). Final wash was with TBS for 30 min. Color development was performed with 0.16 mg/ml 5-bromo-4-chloro-3-indoyl phosphate (BCIP) plus 0.32 mg/ml nitroblue tetrazolium (NBT) in 100 mM TrisCl (pH 9.5)/5 mM MgCl₂/100 mM NaCl.

Immunohistochemistry
Samples of tissue from human trachea and lung parenchyma were fixed with 4% paraformaldehyde in PBS and embedded in paraffin. After rehydration, slides were prepared for both alkaline phosphatase and peroxidase staining. The slides used for peroxidase staining were incubated in 3% hydrogen peroxide for 15 min to quench endogenous peroxidase activity, blocked with 100% goat serum for 1 h, and then incubated with rabbit antibodies against PLUNC (1:100 in 100% goat serum) at 4°C overnight. After washing in PBS (3x), slides were incubated with peroxidase-conjugated goat anti-rabbit immunoglobulin (ABC kit; Vector Laboratories, Burlingame, CA) at room temperature for 30 min and washed with PBS (3x). Visualization was achieved by incubating with diaminobenzidine (1 mg/ml) in PBS containing 0.01% H₂O₂ at room temperature for 8 min. Slides used for alkaline phosphatase staining followed a similar procedure except that quenching with 3% hydrogen peroxide was omitted, slides were washed with TBS instead of PBS, and the second antibody used was alkaline phosphatase–conjugated goat anti-rabbit immunoglobulin (1:500; Kirkegaard and Perry Laboratories). Visualization was achieved with BCIP and NBT as described above for Western blots. As controls, we used slides incubated with rabbit preimmune sera as first antibody at the same dilutions.

LPS-PLUNC Binding Experiments
To test if PLUNC competes with LBP for binding to LPS, we used the Endoblock test (HyCult Biotechnology, Uden, The Netherlands), following the manufacturer's instructions. Briefly, microtiter plates coated with anti-LBP antibodies were incubated with LBP (25 ng/ml) for 1 h at room temperature. The unbound LBP was removed by washing with buffer containing Tween-20. Serial dilutions of PLUNC (starting at 100 µg/ml in 8% ethanol) were preincubated with biotinylated LPS for 30 min at 37°C. Serial dilutions of polymyxin B (starting at 10 µg/ml), a known inhibitor of LBP binding to LPS, were used as a standard. After preincubation, the mixtures were added to the wells (1 h at room temperature). The excess biotinylated LPS and inhibitor were removed by washing. Next, a streptavidin–peroxidase conjugate was added to the wells (1 h at room temperature) and tetramethylbenzidine was used as substrate. The reaction was stopped with citric acid and the color was read at 450 nm. A standard curve was obtained by plotting absorption versus the log of the corresponding polymyxin B concentration, and the results were fitted by a one-site competition curve using Prism software (GraphPad, San Diego,CA).

ELISA for PLUNC
Microtiter plates (Immulon 4HBX; DYNEX Technologies, Chantilly, VA) were coated with 2 µg/ml of PLUNC in TBS (100 µl/well) overnight at 4°C; subsequent steps were performed at room temperature. The wells were washed with TTBS (3x) and then blocked with 3% BSA in TTBS (200 µl/well) for 2 h. Serial dilutions of PLUNC standard solution (6.7 µg/ml) and samples in 3% BSA in TTBS were incubated with an equal volume of rabbit antiserum against PLUNC (1:2,000 in 3% BSA in TTBS) for 1 h. After preincubation, the mixtures (100 µl/well) were added to the wells (1 h with agitation). After washing with 3% BSA in TTBS (3x), alkaline phosphatase–conjugated goat anti-rabbit immunoglobulin (diluted 1:2,000 in 3% BSA in TTBS, 100 µl/well; Kirkegaard and Perry Laboratories) was added for 1 h with agitation. Following three TTBS washes, color development was achieved using p-nitrophenyl phosphate (0.5 mg/ml, 100 µl/well; Sigma) for 30 min. The reaction was stopped with 3 N NaOH (50 µl/well) and the color was read at 405 nm. A standard curve was obtained by plotting absorption versus the log of the corresponding concentrations of known PLUNC standard and the results were fitted by a sigmoidal dose response (variable slope) curve using Prism software (GraphPad).

	Results

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Analysis of Proteins Secreted by ALI Cultures
In the course of exploring different methods for collecting secretions from the apical surface liquid of ALI cultures, we used serial concentrations of ethanol to precipitate protein. As expected, the amount of precipitated protein increased as the ethanol concentration was increased from 33 to 75%. Although 67–75% ethanol precipitated most of the proteins, one major protein band at 21–23 kD remained in the ethanol supernatant (Figure 1). Based on BCA assay results from eight experiments, the yield of this ethanol soluble protein was 10 ± 3% (mean ± SEM) of the total secreted protein.

View larger version (118K): [in this window] [in a new window]   Figure 1. Protein electrophoresis of ALI secretions. Proteins present in the ALI growth medium (M, 17 µl) and in ALI secretions (acetone precipitate [AP], 3 µg protein) were analyzed by SDS-PAGE using 13% Duracryl and were silver stained. The arrows indicate bands at 84, 71, 57, 53, and 21 kD in AP. Recombinant protein standards (Std) are in the third and last lanes. Secreted proteins were precipitated with increasing concentrations of ethanol and centrifuged to obtain the precipitate (EP) and the soluble (ES) fractions (lanes 4–11). The only band present in the supernatant fluid at high concentrations of ethanol is a 21-kD band (PLUNC). Each pair of lanes contains 3 µg of protein obtained from 14 µl sample.

Characterization of the Ethanol-Soluble Protein as PLUNC
To identify the ethanol-soluble protein, it was subjected to amino acid sequencing by Edman degradation at the University of Miami Protein Analysis Core Facility. No N-terminal sequence was detected using an estimated 200 pmol of protein, suggesting that the amino-terminal amino acid was blocked. Thus, additional sample was purified by ethanol precipitation followed by HPLC (described in MATERIALS AND METHODS), subjected to SDS-PAGE, and stained with Coomassie Blue. The main protein band at 22 kD was cut out and sent to the Interdisciplinary Center for Biotechnology Research at the University of Florida for analysis. A tryptic digest was analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectroscopy (MALDI-TOF) (Figure 2A) and the six highest peaks matched those for amino acids 95–109, 110–128, 129–138, 139–152, 157–167, and 214–232 of SPLUNC1 (Figure 2B) (13). Peptides 1–94, 153–156, 168–169, 170–172, and 173–213 were not identified because they were larger or smaller than the recorded spectrum. Peptide 233–255 has a mass within the detection range but was not detected, suggesting that the C-terminal region of the protein was truncated. A portion of this trypsin digest was also fractionated by HPLC and one peptide was analyzed for amino acid sequence by Edman degradation. The results matched amino acids 110–121 of SPLUNC1 (Figure 2B), further confirming that the ethanol soluble protein was a product of the plunc gene.

View larger version (20K): [in this window] [in a new window]   Figure 2. Identification of PLUNC by MALDI-TOF. (A) Tryptic digest of PLUNC purified by ethanol fractionation, HPLC, and SDS-PAGE was analyzed by MALDI-TOF using an Applied Biosystems Voyager-DE PRO mass spectrometer. (B) PLUNC amino acid sequence showing the six tryptic peptides identified by MALDI-TOF (underlined) and the amino acids sequenced by Edman degradation (lined above). Theoretical trypsin cleavage sites are denoted by arrowheads. (C) PLUNC (undigested) purified by ethanol fractionation (0.25 ng in 75% ethanol) was mixed with matrix and analyzed by MALDI-TOF to resolve singularly charged species.

The true molecular masses of the major proteins in the PLUNC preparation were determined by MALDI-TOF analysis (Figure 2C). The spectrum showed two peaks with molecular masses of 24.01 and 24.59 kD in a region with many other smaller peaks with lower and higher molecular masses. These results further suggested that the PLUNC preparation was a mixture of molecules that were the products of partial proteolysis.

2-DE revealed that PLUNC that had been purified by ethanol fractionation is composed of several protein components with calculated molecular masses of 20.7–22.4 kD and pIs of 4.7–5.5 (Figure 3A). After the protein was further purified by HPLC, a similar pattern of spots was obtained after 2-DE (Figure 3B). A sample of PLUNC purified by ethanol fractionation of secretions from a different ALI culture also gave a similar pattern of spots after 2-DE (Figure 3C). A Western blot of that sample also showed a similar pattern of spots (Figure 3D). The staining for the Western blot was more sensitive than silver staining and showed some tailing from the spots, perhaps due to multiple minor species of truncated protein.

View larger version (76K): [in this window] [in a new window]   Figure 3. Characteristic multiple-spot pattern of PLUNC by 2-DE and Western blot. (A) Silver stain of a 2-DE gel of 10 µg of PLUNC purified by ethanol fractionation showing a cluster of spots with molecular weights ranging from 21,800–22,500 and pI ranging from 4.7 to 5.5. (B) Silver stain of a 2-DE gel from the same sample used in A after purification by HPLC showing a similar cluster of spots. (C) Silver stain of a 2-DE gel of a different sample of PLUNC purified by ethanol fractionation (11 µg) showing a similar cluster of spots. (D) Western blot of a 2-DE gel from 1 µg of same purified PLUNC used in C. (E) Silver stain of a 2-DE gel of 100 µg of protein precipitated from a human tracheal aspirate. The arrows indicate the two spots corresponding to PLUNC. (F) Western blot of the same sample as in E. The larger two spots (arrows) correspond to the spots visible in E.

To further verify that more than one of the spots in 2-DE is due to PLUNC, another sample of PLUNC was purified by ethanol fractionation and analyzed by 2-DE with silver staining. The two darkest spots were digested with trypsin and identified as PLUNC by MALDI-TOF analysis.

An ELISA method to measure PLUNC was developed and used to measure the amount of PLUNC expressed as percentage of total protein in ALI secretions at different times after initiating the ALI (Figure 4). Little if any PLUNC was present initially, but the levels rose quickly during the second week in culture and continued to rise gradually over the next few weeks. During the second week, the volume and viscosity of the secretions also increased. Although the amount of protein secreted per culture plate increased somewhat during this period, the increase in PLUNC far outpaced the increase in total protein.

View larger version (13K): [in this window] [in a new window]   Figure 4. Quantification of PLUNC using an ELISA assay. (A) Standard curve obtained by plotting the absorptions versus the log of the corresponding concentrations of known PLUNC. (B) PLUNC in secretions from ALI cultures of human airway epithelial cells increased sharply during the second week and continued to increase gradually over time. The total protein concentration of the samples was diluted to 5 µg/ml before it was mixed with an equal volume of rabbit anti-PLUNC serum (1:2,000).

View larger version (14K): [in this window] [in a new window]   Figure 5. HPLC of purified PLUNC. PLUNC eluted at 16 min; the peak near 0 min was material that did not bind to the column. PLUNC was purified from secretions from human airway epithelial cells in ALI culture by ethanol and acetone precipitations. The protein (23 µg) was dissolved in 65 µl of 50% ethanol, and 50 µl was injected into a reverse phase C4 column and eluted with an acetonitrile gradient in 0.05% trifluoroacetic acid running at 0.8 ml/min and monitored at 215 nm.

Western blot analysis using 2-DE gels of HTA samples (Figure 3F) was more sensitive than silver staining and, similar to what was observed in ALI secretions, showed several spots of PLUNC. Although the patterns are not identical, these spots have a size and charge distribution similar to those of PLUNC purified from ALI secretions by ethanol fractionation (Figure 3D). These results suggest that partial proteolysis may occur in vivo and in culture.

Localization of PLUNC
To localize the sites where PLUNC is synthesized and secreted, immunohistochemistry of human airway tissues was performed using polyclonal antibodies from human PLUNC-immunized rabbits. Trachea showed positive labeling in the cells of the submucosal ducts (Figures 6A and 6B) and submucosal glands (Figures 6C and 6D). Using periodic-acid Schiff staining as a guiding tool, PLUNC expression was higher in serous cells compared with mucous cells of the gland (compare Figures 6C and 6E). Some cells in the surface epithelium showed weak cytoplasmic staining. In addition, PLUNC was localized along the ciliary border of the whole epithelium (better visualized with peroxidase staining [Figures 6I and 6J]). Distal lung sections (parenchyma) did not stain for PLUNC (Figure 6F). In this area, PLUNC was only seen in mucus accumulations in the airway lumen and on the ciliary border of smaller airways (Figures 6G and 6H).

View larger version (98K): [in this window] [in a new window]   Figure 6. PLUNC is localized in human surface epithelial cells, ciliary border, and serous cells of airway submucosal glands and ducts. Immunohistochemistry of human trachea and lung parenchyma paraffin sections labeled with rabbit antibodies to purified PLUNC and visualized with alkaline phosphatase (A–G) or horseradish peroxidase (H–I). (A) Section of trachea incubated in immune serum showing PLUNC staining in submucosal gland duct. Bar = 50 µm. (B) Same as A, but incubated with preimmune serum. (C) Section of trachea incubated with immune serum showing PLUNC in some surface epithelial cells and submucosal gland cells. Bar = 100 µm. (D) Same as C, but with preimmune rabbit serum (control). (E) PAS stain of tracheal section almost contiguous with sections in C and D for localization of submucosal gland mucus cells (purple) and serous cells (light blue). Note that cells stained with PLUNC in C (thick arrow) correspond to the serous cells. The opposite is seen with mucous cells (thin arrows). (F) Section of human lung parenchyma incubated with immune serum. No PLUNC is visualized. Bar = 100 µm. (G) Section of lung parenchyma incubated with immune serum showing a small peripheral airway. PLUNC is located in secreted mucus. (H) Same as G, but with preimmune rabbit serum (control). Bar = 100 µm. (I) Closer view of tracheal surface epithelium incubated with immune serum. PLUNC is seen inside some epithelial cells and in the ciliary border. Bar = 25 µm. (J) Same as I, but with preimmune rabbit serum (control).

View larger version (16K): [in this window] [in a new window]   Figure 7. PLUNC does not inhibit binding of LPS by LBP in contrast to polymyxin B. In a competition assay done in microtiter plates, polymyxin inhibited the association of biotin-labeled LPS with LBP. Similar and higher concentrations of PLUNC had no effect on this binding. Streptavidin–peroxidase conjugate was used to visualize the binding of LPS to LBP using tetramethylbenzidine as substrate.

	Discussion

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Several human genes and several splice variants of plunc have been identified (5, 6, 12). All appear to encode a PLUNC molecule with the same predicted amino acid sequence, with the exception of one amino acid (see below; also see GenBank reference sequence NM_016583.2). In addition, spurt and plunc share similar chromosomal locations (6, 12, 13). The predicted coding sequence of spurt is the same as that of lunx, and differs from the reported sequence of plunc (12) by only a single nucleotide, resulting in a glutamine in SPURT and a lysine in PLUNC at position 220. A BLAST search of plunc sequences and matched ESTs yielded 44 sequences with CAG (Glu) and only 1 with AAG (Lys) at amino acid position 220. MALDI-TOF analysis of our PLUNC preparation showed strong peaks for the tryptic peptide 214–232 with no evidence of cleavage at position 220, supporting the presence of glutamine rather than lysine in that position and consistent with similar MALDI-TOF data from nasal lavage fluid (7). Furthermore, the mass of this peptide matched the expected mass for the presence of glutamine rather than lysine, and shows that the protein that we isolated has glutamine at residue 220. The prediction of lysine at position 220 in PLUNC (12) is likely due to an experimental error in the sequence of one nucleotide or to the presence of a genetic variant. Together the data strongly suggest that, for all practical purposes, human PLUNC, LUNX, and SPURT are the same protein.

The concentration of PLUNC in secretions obtained from ALI cultures (8–10% of the total soluble protein) was higher than that reported in nasal lavage (0.05–1.6%) (7) or in samples of tracheal secretions reported here ( 0.4%). Because our gels were normalized for the amount of protein, this difference in expression could be a reflection of different environmental conditions of cells in culture versus cells in vivo. Another factor is that the contribution of plasma-derived protein to the content of tracheal aspirates is quite significant, whereas the ALI culture secretions did not appear to contain a significant amount of protein from the basal media. Consequently, the secreted PLUNC would be more dilute (mg/mg of total protein) in the tracheal aspirates.

Noteworthy is the finding of the heterogeneous nature of PLUNC. The 2-DE pattern of both crude samples from ALI cultures or HTA and purified PLUNC showed a cluster of spots that was unchanged by use of a cocktail of protease inhibitors. Several results support the conclusion that the spots are due to truncated PLUNC molecules without significant contamination by other proteins. First, PLUNC purified by ethanol fractionation eluted from a reverse-phase HPLC column as a single peak well into the second step of the gradient. Analysis of the material in that peak by 2-DE gave the same pattern of spots as the ethanol-fractionated PLUNC. Second, analysis of the 21-kD band in SDS-PAGE (which is seen as multiple spots by 2-DE) by MALDI-TOF shows that all the major peaks in the spectrum were consistent with the expected PLUNC tryptic peptides. The only missing peptides were the C-terminal region peptide and those out of the 1,000–3,000 mass range of the analysis. If contaminating proteins were present, other significant peaks should have been present in the spectrum. Finally, two spots from a 2-DE gel were analyzed by MALDI-TOF and both were identified as PLUNC. These results support the argument that all the spots in 2-DE were truncated PLUNC molecules and that ethanol fractionation is an effective purification method for PLUNC.

The rabbits were immunized with an ethanol fractionated/SDS-PAGE–purified PLUNC sample. Such purified samples give a 2-DE pattern that is highly similar to the pattern for samples purified by HPLC and analyzed by MALDI-TOF, suggesting that the antiserum is sufficiently specific for PLUNC.

MALDI-TOF analysis of PLUNC purified by ethanol fractionation showed two peaks (24.01 and 24.60 kD); these are smaller than the predicted molecular mass for full-length PLUNC (26.713 kD). The missing amino acids could have been cleaved from either end of the protein. Failure to detect peptide 233–255, located near the C-terminus, suggests that amino acids may be missing from the C-terminal region. Ghafouri and coworkers (8) reported two forms of PLUNC in 2-DE of protein from nasal lavage fluid. The C-terminal tryptic peptide was also absent in their MALDI-TOF spectrum, suggesting that PLUNC isolated from nasal secretion may also be truncated in the C-terminal region. Results from the MALDI-TOF analysis of the tryptic peptides were not helpful near the N-terminus because the N-terminal tryptic peptide (M_r = 9.303 kD) is outside the range analyzed (0.8–3.0 kD).

Prior studies using in-situ hybridization techniques have localized PLUNC in the respiratory epithelia of the upper airways of mice (4) and in human bronchial epithelium (10). Using antibodies against synthesized PLUNC sequences, PLUNC has been localized more specifically in the ciliary border of the nasal epithelium in rats (9). Using antisera raised against PLUNC purified by ethanol fractionation and SDS-PAGE, we observed that PLUNC is localized in the cytoplasm of only some epithelial cells but is present in mucus and on the ciliary border of the whole epithelia. It has also been reported that plunc (spurt) mRNA is expressed in human submucosal glands and ducts in a pattern that correlates with a serous cell distribution (6). Consistent with this observation, we have found that PLUNC is expressed in higher amounts in submucosal gland cells, mainly serous cells, suggesting that submucosal gland cells are a larger source of PLUNC production or that epithelial cells have exocytosed PLUNC before the human tissue is available for fixation. The high epithelial expression in mouse and rat surface epithelia may reflect the paucity of submucosal glands in these animals. Distal airways showed PLUNC in luminal secretions and along the epithelial border. In agreement with other reports (13), PLUNC was not found in lung parenchyma using our antisera against the purified protein.

PLUNC was predicted to have antimicrobial action (6, 9–11), as it shares a BPI-like domain (9), and was postulated to share similar three-dimensional structures to BPI (13). Our inability to confirm the hypothesis that PLUNC shares LPS binding ability suggests that PLUNC is not binding to LPS at the same site as LBP but does not rule out binding to a different site of the LPS molecule. Although it was possible that PLUNC was inactivated during the isolation process, assays performed with untreated ALI secretions containing PLUNC also failed to interfere with LBP–LPS binding, ruling out the idea that ethanol denaturation prevented PLUNC from binding to LPS. Thus, PLUNC does not bind to LPS at the same site as LBP or has a much lower binding affinity for LPS than for LBP.

The function of PLUNC has not been elucidated yet. The solubility characteristics reported here provide a purification approach that may provide an opportunity to study the possible antibacterial activities predicted from PLUNC's sequence similarity to other host defense proteins.

	Acknowledgments

 
: The authors thank Drs Matthias Salathe and Rosanna Forteza for their thoughtful advice and strong support throughout this work. This work was supported in part by grants from the International Chronic Bronchitis Center, Alpha-1 Foundation, and the NIH (HL-66125 to G.E.C.).

Received in original form April 23, 2003

Received in final form August 11, 2003

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