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Interaction of Purified Tear Lipocalin with Lipid Membranes

¹From the Department of Ophthalmology, and the ⁴Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine, Biomedicum, University of Helsinki, Helsinki, Finland; the ²Department of Molecular Medicine, National Public Health Institute, Biomedicum, Helsinki, Finland; and the ³Division of Molecular Biology, Biocentre-Innsbruck Medical University, Innsbruck, Austria.

PURPOSE. The principal lipid-interacting protein in human tears has been reported to be tear lipocalin (Tlc). Tlc has been suggested to scavenge harmful lipophilic substances from the corneal epithelium and to maintain the integrity of the anterior tear film lipid layer by binding and releasing lipid(s) that are accommodated within the protein. Although lipids can be extracted from Tlc, it is still unclear whether Tlc can actually bind to lipid membranes and accept membrane lipids and whether it possesses lipid transfer activity. The purpose of this study was to explore the interaction of Tlc with neutral, anionic, and cationic lipid membranes and to assess the potential of Tlc to facilitate the transfer of either polar or neutral lipids in a lipid transfer assay.

METHODS. The binding of Tlc to lipid membranes was assessed by a monolayer technique and fluorescence spectroscopy. The polar lipid transfer activity of Tlc was assessed with a radiometric assay based on the transfer of ¹⁴C-phosphatidylcholine (PC) from PC-liposomes to HDL₃. The neutral lipid transfer activity of Tlc was assayed by measuring the transfer of radioactive cholesteryl ester from LDL to HDL₃.

RESULTS. Purified Tlc showed significant surface activity as evidenced by an increase in surface pressure at the air–buffer interface. Likewise, it interacted actively with neutral, anionic, and cationic lipid monolayers, as evidenced by an equal increase in surface pressure despite the surface charge. Enhanced quenching of the single tryptophan residue of Tlc by pyrene and I^– anion suggested that different protein domains are involved in the interaction of Tlc with oppositely charged lipid membranes. Finally, radiometric assays revealed that Tlc does not possess any neutral or polar lipid transfer activity between lipid vesicles or/and lipoproteins.

CONCLUSIONS. Tlc interacted with lipid membranes composed of neutral, cationic, or anionic membranes, which supports a role for Tlc in the maintenance of the tear film interfaces. Tlc did not show any neutral or polar lipid transfer activity whatsoever. The findings suggest that the notion of the role of Tlc as the major lipid-transferring protein in human tears should be revised.