Adil R. Abduragimov

Tear lipocalins bind a broad array of lipid ligands

To identify the native ligands of tear lipocalins, tear proteins were separated by size exclusion chromatography and the lipid content in the major protein fractions identified. Lipids extracted from native tears and purified tear lipocalins comigrated with fatty acids, fatty alcohols, phospholipids, glycolipids, and cholesterol on thin layer chromatograms. Abundant stearic and palmitic acids as well as cholesterol, and lesser amounts of lauric acid were specifically identified in extracts of purified lipocalins by gas chromatography-mass spectroscopy. A preliminary study of the ligand-protein interaction was carried out using nitroxide spin-labeled lipids.

Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand affinity

The principal lipid binding protein in tears, tear lipocalin (TL), binds acid and the fluorescent fatty acid analogs, DAUDA and 16-AP at one site TL compete for this binding site. A fluorescent competitive binding assay revealed that apo-TL has a high affinity for phospholipids and stearic acid (Ki) of 1.2 microM and 1.3 microM, respectively, and much less affinity for cholesterol (Ki) of 15.9 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 os 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 the tryptophan 17 substitution in lipocalin with those of tryptophan 19 substitution in beta-lactoglobulin revealed important differences in binding characteristics that reflect the functional heterogeneity within the lipocalin family.

LIPOPHILIN, A NOVEL HETERODIMERIC PROTEIN OF HUMAN TEARS

We identified a novel heterodimeric protein, lipophilin AC, in human tears. One of its components, lipophilin A (69 residues; mass, 7575.1; pI, 9.47) was homologous to the C1 and C2 components of prostatein (‘estramustine‐binding protein), the major secreted protein of rat prostate. Human lipophilin C (77 residues; mass, 8854.1; pI, 4.94) was homologous to the rat prostatein C3 component and to human mammaglobin, a protein overexpressed in some mammary carcinomas. Tear lipophilins A and C expand the roster of human uteroglobin superfamily members and provide models for exploring these typically steroid‐regulated and steroid‐binding molecules.

Structural changes in human tear lipocalins associated with lipid binding

Structural and conformational changes in tear lipocalins were detected in association with ligand binding and release. Circular dichroism measurements demonstrated that ligand binding induces beta structure formation, aromatic side chain asymmetry, and a more rigid state in tear lipocalins (TL). The exposure of the tyrosyl component is less in apo-TL than in holo-TL. The sole tryptophan residue, Trp17, is buried in both holo- and apo-TL. The steady state exposure of Trp17 is the same in holo- and apo-TL, but the dynamic exposure is two-fold greater in apo-TL. Maneuvers to unfold the protein with urea or incubation in an acidic environment resulted in increased exposure of aromatic amino acids. Electron paramagnetic resonance studies verified that lipids are liberated from TL in an acidic environment. Acidic pH promotes conformational changes in TL involving aromatic residues, particularly the conserved residue Trp17. These changes are associated with lipid release. The liberation of lipid from the cavity of TL under acidic conditions involves a molten globule state of the protein. We postulate that TL, exposed to the steep surface pH gradient that exists at lipid-aqueous interfaces, would release lipid in association with a molten globule transition. The data suggest a plausible regulatory mechanism for lipid delivery from lipocalins at the tear film surface.

Tear Lipocalin Captures Exogenous Lipid from Abnormal Corneal Surfaces

Purpose. The cornea is protected by apical hydrophilic transmembrane mucins and tears. In pathologic states the mucin barrier is disrupted, creating potential for meibomian lipids to adhere more strongly. Undisplaced lipids create an unwettable surface. The hypothesis that pathologic ocular surfaces alter lipid binding and the ability of tear proteins to remove lipids was tested. Methods. Corneas with pathologic surfaces were studied for lipid adhesion and removal by tears. Capture of fluorescence-labeled phospholipids by human tears was assessed by steady state fluorometry. Tear proteins were separated by gel filtration chromatography and analyzed for bound lipids. Results. Contact angle measurements revealed strong lipid adherence to corneas submerged in buffer. Lower contact angles are observed for lipids on completely de-epithelialized corneas compared with intact corneas (P = 0.04). Lipid removal from these surfaces is greater with whole tears than with tears depleted of tear lipocalin (P < 0.0005). Significantly fewer lipids are captured by tears from Bowmans layer than from epithelial-bearing surfaces (P < 0.025). The only tear component to bind the fluorescence-tagged lipid is tear lipocalin. The histology of a rare case of dry eye disease demonstrates the dominant features of contemporaneous bullous keratopathy. Lipid sequestration from this cornea by tear lipocalin was robust. Conclusions. Lipid is captured by tear lipocalin from corneas with bullous keratopathy and dry eye. Lipid removal is slightly abrogated by greater lipid adhesion to Bowmans layer. Reduced secretion of tear lipocalin documented in dry eye disease could hamper lipid removal and exacerbate ocular surface pathology.

Tear Lipocalin: Structure, Function and Molecular Mechanisms of Action

Human tear lipocalin (TL), or von Ebner’s gland protein, accounts for about 15–33% of the protein in tears.1–4 TL has been identified in lacrimal gland, von Ebner’s gland, prostate, nasal and tracheal mucosa and skin.5–12 TL was recognized as a member of the lipocalin family when its primary sequence was determined.7,13,14 Lipocalins form a functionally diverse group of proteins with extremely varied amino acid sequences, yet some similar structural properties (Fig. 1). Eight strands (A-H) are arranged in a β-barrel and are joined by loops between the β-strands.15–19 Many of the lipocalins, including TL, are believed to function as dimers.20,21 There are highly conserved regions that are important to ligand affinity and lipocalin stability. Most lipocalins have 1–3 disulfides bonds and one in particular is conserved and may play a role in modulating ligand binding.15,22 A completely conserved tryptophan on the A strand is implicated in ligand binding and prevention of oxidation of retinol, as well as protein stability of various lipocalins.23,24

Functional cavity dimensions of tear lipocalin

Purpose. We calibrated the cavity of tear lipocalin with a series of fluorescent labeled lipids of increasing chain length and varying diameter. Methods. Cavity length was assessed with competitive fluorescent assays in which DAUDA was displaced from apo-tear lipocalin with ligands of increasing carbon chain lengths from C12-C24. The concentrations of competitors that inhibit 50% of the binding of DAUDA (IC 50) were compared. Functional diameters of tear lipocalin and ß-lactoglobulin were estimated with fatty acids bearing fluorescent labels of various diameters. The cavity dimensions of other lipocalins were derived from their published crystal structure coordinates. Results. In tear lipocalin, the binding affinities of fatty acids increased up to a carbon chain length of 18 (22.5 Å) but remained constant from C18-C24. The cavity length of other lipocalins in crystal form were similar to tear lipocalin in solution. Tear lipocalin showed decreased binding affinities with progressively increasing ring dimensions of the ligand. In contrast to ß-lactoglobulin and retinol binding protein, tear lipocalin bound DAUDA and cholesterol in the calyx. Neither tear lipocalin nor ß-lactoglobulin bound P646 in their respective cavities. The calculated inter-sheet distances at the mouth of the crystallized lipocalins ranged from 16–22Å. Conclusions. Tear lipocalin is more promiscuous than ß-lactoglobulin or retinol binding protein because of a greater functional diameter. Differences in ligand specificity of the various lipocalins can not be explained simply by variation in cavity length or the intersheet distances at the calyx mouths as determined by crystal structure. Other factors may influence ligand specificity such as size and/or dynamic motion of loops between the ß strands.

Characterization of a lipophilin in rabbit tears.

A novel protein in human tears called lipophilin has homology to the uteroglobin superfamily.1–3 Putative functions of the members of the family include the binding of hydrophobic molecules, anti-inflammatory properties, anti-chemotactic properties, suppression of extracelluar matrix invasion by normal and cancer cells, and phospholipase A2 inhibition.4,5 Other proteins in the uteroglobin family include rat prostatem6 (a steroid binding protein), uteroglobin7,8 (a progesterone binding protein), Clara cell protein8 (a phosphatidylcholine and phosphatidylinositol binding protein), and mammaglobin9. The proteins share some structural features. In general, monomers are linked by disulfide bonds to form homo- (uteroglobin, Clara cell protein) or hetero-dimers (human lipophilin and prostatein). The crystal structure of uteroglobin reveals that the dimer is composed of two independent polypeptide chains of 70 residues linked by two disulfide bridges. Each monomer is folded into four alpha-helices. An oblong hydrophobic pocket composes the putative progesterone-binding site.10 The rat and human Clara cell protein also exists as 2 identical monomers joined together in an antiparallel manner to enclose a large internal hydrophobic cavity.8,11 The structural motif common to all members of the family is a hydrophobic tunnel created by the alpha helical monomers and bridged by multiple disulfide bonds. The disulfide bonds may be important in ligand binding and release. For uteroglobin, a reduction in the bonds induces a local unfolding of the N- and C-termini. The resulting separation of helices creates a channel to the binding site.12

Intracavitary Ligand Distribution in Tear Lipocalin by Site-Directed Tryptophan Fluorescence

Site-directed tryptophan fluorescence has been successfully used to determine the solution structure of tear lipocalin. Here, the technique is extended to measure the binding energy landscape. Single Trp mutants of tear lipocalin are bound to the native ligand and an analogue tagged with a quencher group to both populate and discriminate the excited protein states. Steady-state and time-resolved fluorescence quenching data reveal the intracavitary state of the ligand. The static components of fluorescence quenching identify the residues where nonfluorescence complexes form. An asymmetric distribution of the ligand within the cavity reflects the complex energy landscape of the excited protein states. These findings suggest that the excited protein states are not unique but consist of many substates. The roughness of the binding energy landscape is about 2.5kBT. The excited protein states originate primarily from conformational selections of loops AB and GH, a portal region. In contrast to static quenching, the dynamic components of fluorescence quenching by the ligand are relevant to both local side chain and ligand dynamics. Apparent bimolecular rate constants for collisional quenching of Trp by the nitroxide moiety are approximately 1 / 5 x 10(12) M(-1) s(-1). Estimations made for effective ligand concentrations establish actual rate constants on the order of 12 x 10(9) M(-1) s(-1). Prior to exit from the cavity of the protein, ligands explore binding sites in nanoseconds. Although microsecond fluctuations are rate-limiting processes in ligand binding for many proteins, accompanying nanosecond motion may be necessary for propagation of ligand binding.

Cation-π Interactions in Lipocalins: Structural and Functional Implications

The cation-π interaction impacts protein folding, structural stability, specificity, and molecular recognition. Cation-π interactions have been overlooked in the lipocalin family. To fill this gap, these interactions were analyzed in the 113 crystal and solution structures from the lipocalin family. The cation-π interactions link previously identified structurally conserved regions and reveal new motifs, which are beyond the reach of a sequence alignment algorithm. Functional and structural significance of the interactions were tested experimentally in human tear lipocalin (TL). TL, a prominent and promiscuous lipocalin, has a key role in lipid binding at the ocular surface. Ligand binding modulation through the loop AB at the open end of the barrel has been erroneously attributed solely to electrostatic interactions. Data revealed that the interloop cation-π interaction in the pair Phe28-Lys108 contributes significantly to stabilize the holo-conformation of the loop AB. Numerous energetically significant and conserved cation-π interactions were uncovered in TL and throughout the lipocalin family. Cation-π interactions, such as the highly conserved Trp17-Arg118 pair in TL, were educed in low temperature experiments of mutants with Trp to Tyr substitutions.