Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand a¤nity Oktay K. Gasymov, Adil R. Abduragimov, Taleh N. Yusifov, Ben J. Glasgow * Departments of Pathology and Ophthalmology, UCLA School of Medicine, Jules Stein Eye Institute, 100 Stein Plaza, Los Angeles, CA 90095, USA Received 22 March 1999; received in revised form 3 June 1999; accepted 3 June 1999 Abstract The principal lipid binding protein in tears, tear lipocalin (TL), binds the spin-labeled analog of lauric acid and the fluorescent fatty acid analogs, DAUDA and 16-AP at one site. The native ligands of TL compete for this binding site. A fluorescent competitive binding assay revealed that apo-TL has a high affinity for phospholipids and stearic acid (K i ) of 1.2 WM and 1.3 WM, respectively, and much less affinity for cholesterol (K i ) of 15.9 WM. For fatty acids, binding affinity correlated with the length of the hydrocarbon chain. TL binds most strongly the least soluble lipids permitting these lipids to exceed their maximum solubility in aqueous solution. These data implicate TL in solubilizing and transporting lipids in the tear film. Phenylalanine, tyrosine and cysteine were substituted for Trp 17, the only invariant residue throughout the lipocalin superfamily. Cysteine substitution resulted in some loss of secondary structure, relaxation of aromatic side chain rigidity, decreased binding affinity for DAUDA and destabilization of structure. Mutants of TL, W17Y, and W17F showed a higher binding affinity for DAUDA than wild-type TL. Comparison of the results of tryptophan 17 substitution in lipocalin with those of tryptophan 19 substitution in L-lactoglobulin revealed important differences in binding characteristics that reflect the functional heterogeneity within the lipocalin family. ß 1999 Elsevier Science B.V. All rights reserved. Keywords : Tear lipocalin ; Circular dichroism; Ligand a¤nity; Site directed mutagenesis; Von Ebner's gland protein 1. Introduction Tear lipocalin (TL) is secreted by the lacrimal glands and comprises 15^33% of the protein content of tears [1^4]. The identical protein is secreted by Von Ebner's gland and has been identi¢ed in the prostate and skin [5^7]. TL is the principal lipid binding protein in tears and binds a broad array of lipid molecules including cholesterol, fatty acids, gly- colipids and phospholipids [8]. TL, acting as a lipid carrier in tears, could potentially protect the eye in several ways. TL may scavenge lipid from the cor- neal surface and prevent dry spots from forming on the cornea. Solubilization of lipid in tears promotes optical clarity. Lipids bound to TL in the aqueous portion of tears provide a reservoir of lipid molecules that upon release, could migrate to the ¢lm surface to reduce water evaporation and inhibit microbial infection of the exposed ocular surface [8]. Several other putative functions have been identi¢ed for TL including antimicrobial activity [9], cysteine protein- ase inhibition [10], and transport of sapid molecules in saliva [5], as well as retinol in tears [6]. The relative binding a¤nity of TL for its native ligands has not 0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII:S0167-4838(99)00133-8 * Corresponding author: Fax: +1-310-794-2144; E-mail : bglasgow@pathology.medsch.ucla.edu Biochimica et Biophysica Acta 1433 (1999) 307^320 www.elsevier.com/locate/bba