Interaction of ceramides and tear lipocalin

Abstract

The distribution of lipids in tears is critical to their function. Lipids in human tears may retard evaporation by forming a surface barrier at the air interface. Lipids complexed with the major lipid binding protein in tears, tear lipocalin, reside in the bulk (aqueous) and may have functions unrelated to the surface. Many new lipids species have been revealed through recent mass spectrometric studies. Their association with lipid binding proteins has not been studied. Squalene, (O-acyl) omega-hydroxy fatty acids (OAHFA) and ceramides are examples. Even well-known lipids such as wax and cholesteryl esters are only presumed to be unbound because extracts of protein fractions of tears were devoid of these lipids. Our purpose was to determine by direct binding assays if the aforementioned lipids can bind tear lipocalin. Lipids were screened for ability to displace DAUDA from tear lipocalin in a fluorescence displacement assay. Di- and tri-glycerides, squalene, OAHFA, wax and cholesterol esters did not displace DAUDA from tear lipocalin. However, ceramides displaced DAUDA. Apparent dissociation constants for ceramide-tear lipocalin complexes using fluorescent analogs were measured consistently in the submicromolar range with 3 methods, linear spectral summation, high speed centrifugal precipitation and standard fluorescence assays. At the relatively small concentrations in tears, all ceramides were complexed to tear lipocalin. The lack of binding of di- and tri-glycerides, squalene, OAHFA, as well as wax and cholesterol esters to tear lipocalin is consonant with residence of these lipids near the air interface.

Keywords: (O-acyl) omega-hydroxy fatty acids (OAHFA); Ceramide; Cholesterol esters; DAUDA; Di- and tri-acylglycerols; Dry eye; LCN1; Linear spectral summation; Lipid binding; Lipocalin-1; Squalene; Tear lipocalin; Tears; Wax esters.

Figure 1

DAUDA displacement assay for tear lipids. DAUDA bound to tear lipocalin displays fluorescence. Unbound DAUDA has exiguous fluorescence. Decreased fluorescence indicates displacement of DAUDA from the tear lipocalin binding site by the tested lipids. Fluorescence of tear lipocalin and DAUDA after addition of ceramide ( ), stearic acid ( ), cholesteryl stearate ( ), cholesteryl oleate ( ), stearyl behenate ( ), behenyl stearate (-x-), behenyl oleate ( ), squalene ( ), (O-oleoyl)-16-hydroxypalmitic acid ( ) 1,2-distearin ( ), 1,3-distearin ( ), tristearin ( ).

Figure 2

NBD6-Ceramide and NBD12-Ceramide show fluorescence only when bound to tear lipocalin. Fluorescence spectra of free 1 μM C6-NBD ceramide ( ), 1 μM C12-NBD ceramide ( ), 1 μM C6-NBD ceramide bound to 1 μM tear lipocalin( ) and 1 μM C12-NBD ceramide 1 μM tear lipocalin ( ).

Figure 3

High speed centrifugal precipitation assay to test binding of C6-NBD ceramide ( ) and C12-NBD ceramide ( ) to 10 μM or 20 μM tear lipocalin, respectively. Concentration of bound ligand-protein complex (supernatant) was determined by absorption spectra after centrifugal separation from unbound insoluble precipitant. Curve fit to a hyperbola (—) K_d= 0.32 μM, n=0.44 for C6-NBD ceramide and K_d= 1.23 μM and n=0.42 for C12-NBD ceramide; and the Hill equation ( ) K_d= 0.29 μM, n=0.45 for C6-NBD ceramide and K_d= 1.06 μM and n=0.39 for C12-NBD ceramide. Inset: concentration of total versus bound ligand concentration. Error bars shows the range from 3 experiments.

Figure 4

Comparison of high speed centrifugal precipitation of C6- NBD ceramide at 25° C and 34° C for a single concentration. Bars show the range from 3 experiments.

Figure 5

Binding curve of C6-NBD ceramide to tear lipocalin 10 μM by linear spectral summation ( ). Absorbance composite spectra of mixtures were fit to the sum of varying pure free and pure bound spectra. Curve was fit to hyperbola (—) K_d= 0.06 μM, n=0.45, and to Hill equation ( ) K_d= 0.07 μM, n=0.47. Inset, concentration dependent absorption of C6-NBD ceramide suspended in 10 mM sodium phosphate, pH 7.3.

Figure 6

Binding curve of C6-NBD ceramide ( ) and C12-NBD ceramide ( ) to tear lipocalin by fluorescence. Curve fit to hyperbola (—) K_d= 0.08 μM and n=0.32 for C6-NBD ceramide binding and K_d= 0.13 μM and n=0.67 for C12-NBD ceramide, and to the Hill equation ( ) K_d= 0.08 μM and n=0.32 for C6-NBD ceramide and K_d= 0.1 μM and n=0.21 for c12-NBD ceramide.

Figure 7

Swiss Dock image of C18 ceramide complexed to tear lipocalin shows pose with the highest full fitness score (see Table 4).