Purification and Characterization of PLUNC from Human Tracheobronchial Secretions

Michael A Campos, Alexandre Abreu, Marie C. Nlend, Miguel Cobas, Gregory E Conner, Philip L. Whitney

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Abstract

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.

Original languageEnglish
Pages (from-to)184-192
Number of pages9
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume30
Issue number2
DOIs
StatePublished - Feb 1 2004

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ASJC Scopus subject areas

  • Cell Biology
  • Pulmonary and Respiratory Medicine
  • Molecular Biology

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