Purification and characterization of PLUNC from human tracheobronchial secretions.
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.
Campos
Michael A
MA
Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Miami School of Medicine, Miami, FL 33101, USA.
Abreu
Alexandre R
AR
Nlend
Marie C
MC
Cobas
Miguel A
MA
Conner
Gregory E
GE
Whitney
Philip L
PL
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding; D020545:Respiratory Mucosa / Q000166:cytology* / Q000378:metabolism; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C485599:BPIFA1 protein, human; D006023:Glycoproteins; D008070:Lipopolysaccharides; D010750:Phosphoproteins