Carrier Protein Identification for Airway Secreted Antimicrobial Cholesterol Esters

The immune system fights off infectious microbes which our body encounters daily.  Key contributors to the immune system include epithelial cells that line our external and internal surfaces and secrete antimicrobial proteins as well as antimicrobial lipids, particularly cholesterol esters (CE). CEs are highly hydrophobic and it is unclear how they are transported in aqueous media like epithelial secretions.  Palate, Lung, and Nasal epithelium clone (PLUNC), a hydrophobic protein with lipid binding pockets, has been recently discovered in airways.  We hypothesize that PLUNC is a carrier for antimicrobial lipids.  To determine whether in vivo PLUNC is associated with antimicrobial CEs we aim to fractionate pig bronchial lavage (BAL), shown to be similar to human BAL, through gel-column size exclusion and by determining the lipid content of PLUNC-positive fractions.  As part of the characterization of BAL and to test whether lysozyme, another highly hydrophobic protein in BAL, serves as CE carrier, fractions will be also subjected to lysoplate assay which quantifies the peptidoglycan hydrolyzing activity of lysozyme.  While the original material of BAL showed minute amounts of lysozyme, the fractions from the size exclusion showed no lysozyme activity likely because they were too dilute.  Therefore, we will concentrate the BAL sample 20-fold before fractionation which requires us also to test the effects of NaCl on lysozyme activity as salt is also being concentrated with the sample.  Identification of carrier proteins for antimicrobial lipids could ultimately lead to new drugs for delivery of antimicrobial lipids in infectious diseases.