Salivary Lipid Binding by SPLUNC1

Objectives: The purpose of this study was to determine whether short palate lung and nasal epithelial clone 1 protein (SPLUNC1) binds salivary lipids.  Previous work has demonstrated that salivary lipids consist of a mixture of cholesterol (3.9%), fatty acids (27.6%), triglycerides (32.1%), wax esters ((26.3%), cholesterol esters (1.9%) and squalene (10.3%) (Brasser et al.  Archs Oral Biol 56:588-591, 2011).

Methods:

Five ml portions of unstimulated whole saliva were collected from volunteers.    To each sample 15 ml of absolute ethanol was added.  This precipitates all protein except SPLUNC1 (Campos et al.  Am J Respir Cell Mol Biol 30:184-192, 2004).   After centrifugation at 16,000 x g for 15 min, supernatants containing SPLUNC1 were concentrated to dryness via rotary evaporator.  A synthetic lipid mixture approximating the composition of human salivary lipids (see above) was added to each of two tubes.  SPLUNC1 was reconstituted in 5 ml distilled water and added to one tube.  Five ml distilled water was added to the second tube.  After sonication, the tubes were incubated at 37^o for 1 hr.  Samples were again centrifuged at 16,000 x g for 15 min.  Supernatants were lyophilized, and the dried residues were extracted with chloroform:methanol.  Recovered lipids were analyzed by thin-layer chromatography.

Results: Lipids suspended in distilled water completely sedimented during centrifugation.  All of the lipids in the synthetic mixture bound to SPLUNC1 to considerable extents; however, there was a higher binding affinity for cholesterol and cholesterol esters.

Conclusions: SPLUNC1 can bind all of the neutral salivary lipids.  It preferentially binds cholesterol and cholesterol esters.