Purification and Characterization of PLUNC from Human Tracheobronchial Secretions

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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 g/ml) or in 50 mM NaCl but was more soluble in 75% ethanol ( 380 g/ml). PLUNC secretion dramatically increased during the sec- ond week in air-liquid interface culture and continued to in- crease 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 bio- chemical properties in secretions. A number of proteins have been identified that bind bacte- ria and mediate mammalian host responses to infectious challenge. Foremost among these are lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability- increasing protein (BPI) (1-3). Recently, genes with se- quence similarity to LBP and BPI have been identified and shown to be expressed in the upper respiratory tract. The

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