Interaction of phospholipid transfer protein with human tear fluid mucins

Abstract

In addition to circulation, where it transfers phospholipids between lipoprotein particles, phospholipid transfer protein (PLTP) was also identified as a component of normal tear fluid. The purpose of this study was to clarify the secretion route of tear fluid PLTP and elucidate possible interactions between PLTP and other tear fluid proteins. Human lacrimal gland samples were stained with monoclonal antibodies against PLTP. Heparin-Sepharose (H-S) affinity chromatography was used for specific PLTP binding, and coeluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. Immunoprecipitation assay and blotting with specific antibodies helped to identify and characterize PLTP-mucin interaction in tear fluid. Human tear fluid PLTP is secreted from the lacrimal gland. MALDI-TOF analysis of H-S fractions identified several candidate proteins, but protein-protein interaction assays revealed only ocular mucins as PLTP interaction partners. We suggest a dual role for PLTP in human tear fluid: (1) to scavenge lipophilic substances from ocular mucins and (2) to maintain the stability of the anterior tear lipid film. PLTP may also play a role in the development of ocular surface disease.

Fig. 1.

Production of human tear PLTP in lacrimal glands. A: Histological sections of paraffin-embedded human lacrimal gland stained with hematoxylin-eosin and with MAb59 monoclonal antibody to PLTP. Basal cells of intraglandular ducts display positive staining against PLTP. B and C: Accumulation of secreted PLTP in the lumen (thick arrows) and positively stained basal cells (thin arrows) as demonstrated with strong MAb59 (B) and MAb66 (C) immunostaining. D: Nonimmunized mouse serum showed no staining of the lacrimal glands. At least 10 microscopic fields were visualized for each sample and antibody used. B, basal cell; L, lumen; PLTP, phospholipid transfer protein.

Fig. 2.

Analysis of tear fluid PLTP during heparin affinity separation. A: A two-step Heparin-Sepharose affinity chromatography was used for PLTP isolation from tear fluid (see “Experimental Procedures”). Tear fluid (400 µl) was applied to the HiTrap Heparin column. The column was washed with buffer A (25 mM Tris-HCl buffer, pH 7.4 containing 1 mM EDTA), and 0.5 ml fractions were collected at a flow rate of 0.5 ml/min. Arrows show the start of 0.5 M and 2 M NaCl elution. The dotted gray line represents 280 nm absorbance readings for the collected fractions, and the black line shows PLTP activity from the corresponding fractions. The inset shows a Western blot image using MAb59 of the pooled 20–21 fractions (lane A) and fractions from the second HiTrap Heparin column affinity chromatography where the 20–21 fraction pool was run to the column and then eluted with a linear 0–0.5 M NaCl gradient (flow rate 0.5 ml/min; fraction size 0.5 ml). Fractions that contained active PLTP (36–39) were pooled and used for the proteomic analysis of copurified proteins. PLTP activity was measured using a radiometric method described in “Experimental Procedures.” B: Silver stain of an SDS-PAGE gel separation of active PLTP-containing fractions after heparin affinity separation of human tear fluid. The pooled fractions from PLTP purification were loaded on a 4–2% Bis-Tris NuPAGE gel (Invitrogen, CA). P1 represents nonbound material from the washing of the heparin column); P2 is constituted from fractions containing active PLTP; and P3 is the fraction eluted with high-salt concentration. Arrow depicts the migration position of mucins (for immunoblot images of tear fluid mucins and PLTP see supplementary Fig. II). PLTP, phospholipid transfer protein; TF, tear fluid.

Fig. 3.

Interaction of PLTP with human tear fluid mucins. Human tear fluid (50 μl) was incubated with rabbit polyclonal anti-PLTP antibody (R290, 1 mg) in the presence of Protein G. The immunoprecipitate was recovered by low-speed centrifugation. Separation on nonreducing 5% SDS-PAGE was done for mucin. PLTP was analyzed using 12.5% SDS-PAGE under reducing conditions. Lane 1: Human tear fluid. Lane 2: Nonprecipitated supernatant fraction. Lane 3: Immunoprecipitate. A: Western blot analysis of mucin with monoclonal antibody MUC-5AC. PLTP was detected by monoclonal anti-PLTP Mab59 antibody (B). All immunoprecipitation assays were repeated at least three times. PLTP, phospholipid transfer protein.

Fig. 4.

Binding of PLTP to mammalian-derived mucin BSM but not to avian-derived soluble form of ovomucin. BSM and soluble ovomucin were incorporated with increasing concentrations (0–45 µg/well) to nitrocellulose membranes and incubated with purified PLTP (25 µg). Bound PLTP protein was detected by using monoclonal anti-PLTP Mab59 antibody. The data shown is background-corrected. Binding experiments were repeated at least three times. BSM, bovine submaxillary gland mucin; PLTP, phospholipid transfer protein.

Fig. 5.

Size exclusion chromatography of phospholipidated mucins (BSM and OVM) and PLTP. Elution profile of the mucin-PC-PLTP sample in PBS (A and C) and in the presence of Tween 20 (0.05%) (B and D). Panel E represents SEC profile for the nonlipidated OVM and PLTP comixture both in PBS and PBS-Tween. PLTP was incubated with the phospholipidated mucins (final volume of the incubation mix was 0.830 ml) for 30 min at room temperature and applied onto the Superose 6HR column. Fractions (0.5 ml) were collected with a flow rate of 0.5 ml/min. Fractions were analyzed for radioactivity and protein content. Protein data was assessed using immunological dot-blot system (described in “Experimental Procedures”) and quantified by densitometry measurements (ImageJ v1.42q software). Results are presented as percentages relative to the total measured value considered as 100%. The arrow at the apparent molecular weight position of 160 kDa depicts the elution position for active PLTP protein (15). SEC runs were repeated three times on average, and a representative run is presented. BSM, bovine submaxillary gland mucin; OVM, ovomucin; PLTP, phospholipid transfer protein; SEC, size exclusion chromatography; V₀, column void volume.