Purification and characterization of PLUNC from human tracheobronchial secretions

Am J Respir Cell Mol Biol. 2004 Feb;30(2):184-92. doi: 10.1165/rcmb.2003-0142OC. Epub 2003 Aug 14.

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 microg/ml) or in 50 mM NaCl but was more soluble in 75% ethanol (> 380 microg/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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cells, Cultured
  • Electrophoresis, Gel, Two-Dimensional
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism*
  • Glycoproteins / genetics
  • Glycoproteins / isolation & purification*
  • Glycoproteins / metabolism*
  • Humans
  • Lipopolysaccharides / metabolism
  • Molecular Sequence Data
  • Phosphoproteins / genetics
  • Phosphoproteins / isolation & purification*
  • Phosphoproteins / metabolism*
  • Protein Binding
  • Respiratory Mucosa / cytology*
  • Respiratory Mucosa / metabolism
  • Solubility
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • BPIFA1 protein, human
  • Glycoproteins
  • Lipopolysaccharides
  • Phosphoproteins