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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

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

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