Purification and Characterization of PLUNC from Human Tracheobronchial Secretions

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.

SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage

“…However, this hypothesis is controversial, and some investigators have found that SPLUNC1 appears to have little antimicrobial activity against Escherichia coli, Listeria monocytogenes, or Pseudomonas aeruginosa (17), and does not appear to bind lipopolysaccharide (15). However, SPLUNC1 has recently been shown to inhibit Mycoplasma pneumoniae in airway cultures (28), and bind to lipopolysaccharide and disrupt Epstein-Barr virus proliferation (29).…”

“…The original PLUNC gene, which is now called SPLUNC1, comprises up to 10% of total protein in the airway surface liquid, and can readily be detected in both nasal lavage and tracheal secretions (14)(15)(16). SPLUNC1 is expressed in both submucosal glands, the superficial epithelia and in neutrophils, and in theory, is present in the correct regions of the lung to be a volume sensing molecule, because it can be secreted onto the mucosal surface of the superficial epithelial where ENaC is expressed (17,18).…”

Characterisation and expression of SPLUNC2, the human orthologue of rodent parotid secretory protein

“…(1:300 dilution) as described (Campos et al 2004). A rabbit IgG (DAKO) was used as a negative control in duplicate slides of every case.…”

Function and Regulation of SPLUNC1 Protein in Mycoplasma Infection and Allergic Inflammation

“…Among the 10 PLUNC proteins described so far, short PLUNC1 (SPLUNC1) has been localized to the large airway epithelium in humans and mice (6). In cultured human primary airway epithelial cells, SPLUNC1 represents up to 10% of total proteins in the epithelial lining fluid (7). Although these studies suggest a pivotal role for SPLUNC1 protein in airway epithelial biology, the functions of SPLUNC1 protein in host defense against bacterial infections have not been determined.…”