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.

Ligand binding complexes in lipocalins: Underestimation of the stoichiometry parameter (n)

“…Fractionations of proteins, including tears, synthetic dimers and ligand binding complexes, were performed by gel filtration liquid chromatography using the AKTA purifier Versatile FPLC with a HiLoad Superdex 75 column (GE Healthcare, Piscataway, NJ). In general, one ml column fractions were collected with elution in 50 mM Tris-HCl, 100 mM NaCl pH 8.4 run at 1 ml/min as previously described [37]. For purification of tear lipocalin monomers peak fractions from gel filtration of 1 ml of tears were pooled and applied to an anion exchange column using DEAE-Sephadex A-25.…”

“…Redl et al calculated 0.3 for the ratio of retinol to tear lipocalin at saturation [21]. A recent study with ceramide revealed stoichiometric ratios of TL:ceramide ligand from n = 0.32–0.67, accompanied by a very low dissociation constant [37]. Several explanations are possible including the formation of a multimeric complex with 2 lipocalin molecules flanking the 2 alkyl chains of the ceramide or a binding site pre-occupied by other ligands.…”

Human Meibum Cholesteryl and Wax Ester Variability With Age, Sex, and Meibomian Gland Dysfunction

“…A thin layer of lipid1,2 on the tear film surface could influence the stability of tears 3–8. Most of the tear film lipid (TFL) comes from meibomian gland, but other sources such as sebaceous9 and lacrimal glands10 could contribute a minor portion of lipid to the TFL layer. Relationships between TFL layer composition, structure, and function could provide insight into the etiology of dry eye.…”

Methods toward simplification of time resolved fluorescence anisotropy in proteins labeled with NBD (4-chloro-7-nitrobenzofurazan) adducts

“…Concomitantly, there is a rise in fluorescence at about 530 nm, which is the emission peak of C6-ceramide when protein bound [13]. The tear lipocalin dimer mirrors the ceramide binding function of the monomer to bind lipid in the calyx.…”