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JM Tiffany, SM Gouveia; Human Tear Lipocalin Is Monomeric . Invest. Ophthalmol. Vis. Sci. 2002;43(13):69.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Human tear lipocalin (tear-specific prealbumin) is a major component of human tears and has lipid-binding properties with associated conformational changes. Tear lipocalin (TL) is thought to be important in stabilizing the tear film and contributing to its viscosity. It has frequently been claimed that TL exists as a dimer in its native form, though evidence for this is indirect. Bovine b-lactoglobulin (BLG), another member of the lipocalin family, has been used as a model for TL. We investigated the self-association of both lipocalins with and without bound lipid. Methods: TL and BLG were studied at tear concentration (1.2 mg/ml) both unbound and bound to cholesterol, palmitic acid, sphingomyelin, ceramides, or a mixture of tear lipids. Viscosity of protein solutions was measured in a Contraves Low-Shear 30 rheometer. Polyacrylamide gel electrophoresis was carried out under reducing and non-reducing conditions, and after protection of free thiol groups by iodoacetylation, or chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS3). Analytical ultracentrifugation was carried out in a Beckman Optima XL-A instrument, with equilibrium distributions analysed with the non-linear curve-fitting algorithm Nonlin. Results: Viscosity-shear rate curves for both proteins showed conversion from Newtonian to non-Newtonian viscosity due to binding of lipids. PAGE separations showed no dimer bands for TL or BLG under reducing or non-reducing conditions without bound lipid, even in the absence of iodoacetylation. However, when bound to lipids, both proteins showed faint dimer bands when incubated with BS3, but with much stronger bands at the monomer weight. Analytical ultracentrifugation showed that unbound TL was entirely monomeric, while unbound BLG formed a monomer-dimer equilibrium. Similar results were seen for both proteins when bound to lipid. Conclusion: Unbound TL, even at high concentrations, is purely monomeric, whereas BLG forms a monomer-dimer equilibrium. When bound to lipid, BLG is unchanged, but a very small proportion of TL may dimerize. TL is therefore unlikely to contribute to the observed non-Newtonian rheology of human tears when complexed with lipids, but may do so through a yet undefined interaction with other tear proteins.
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