Robo2 contains a cryptic binding site for neural EGFL-like (NELL) protein 1/2.

The signaling pathways that are mediated by Slit ligands and their Roundabout (Robo) family of receptors play multifunctional roles in the development of the nervous system and other organs. A recent study identified neural epidermal growth factor-like (NEL)-like 2 (NELL2) as a novel ligand for Robo3. In this study, we carried out a comprehensive analysis of the interaction between NELL1 and the Robo family of receptors and demonstrated that Robo2 contains a cryptic binding site for both NELL1 and NELL2. NELL1/2 binds to the first fibronectin type III (FNIII) domain of Robo2 but not to intact Robo2. Mutation analysis revealed that several amino acids within the first FNIII domain are critical for NELL1 binding to Robo2 but not to Robo1. The Robo2 deletion mutants without the fourth immunoglobulin domain and single amino acid substitution mutants that can influence the architecture of the ectodomain facilitated binding to NELL1/2. Acidic conditions increased the binding affinity of Robo2 for NELL1. These results suggest that Robo2 functions as a receptor for NELL1/2, particularly under circumstances where Robo2 undergoes proteolytic digestion. If this is not the case, conformational changes of the ectodomain of Robo2 may unmask the binding site for NELL1/2.

Regulation of Pseudomonas aeruginosa internalization after contact lens wear in vivo and in serum-free culture by ocular surface cells.

PURPOSE: To determine the effects of contact lenses (CLs) and Pseudomonas aeruginosa (PA) infection on localization of cystic fibrosis transmembrane conductance regulator (CFTR) on corneal surface epithelial cells and the association between lipid raft formation and CFTR in mediating PA binding and internalization in ocular surface epithelium. METHODS: CFTR immunolocalization was evaluated in vivo in rabbit corneal-conjunctival epithelium (with/without CL wear) before and after PA exposure and in serum-free human corneal epithelial cell culture (hTCEpi). Lipid raft formation was visualized with Alexa555-conjugated cholera toxin beta-subunit. Lipid raft involvement in PA internalization was assayed in vivo by gentamicin survival assays after topical filipin pretreatment. Involvement of CFTR in PA binding and internalization was evaluated by blockade with CFTR peptides or LPS. RESULTS: CL wear in vivo enhanced anti-CFTR staining, but CFTR localization did not correlate with the PA binding by ocular surface cells. Conjunctival epithelial cells stained for CFTR but did not bind or internalize PA. Corneal epithelial cells in vivo did not stain for CFTR unless challenged by contact lens-induced hypoxia. PA internalization by hTCEpi was significantly inhibited by LPS (P < 0.01), but not by CFTR peptides. Remarkably, normal conjunctival epithelial cells showed lipid raft formation and CFTR staining but did not bind PA. Inhibition of raft formation by filipin blocked PA internalization in vivo after CL wear. CONCLUSIONS: CFTR is not the predominant receptor for ocular surface PA infection, and after hypoxic CL challenge, neither lipid rafts nor CFTR localization alone predicts PA binding; however, lipid rafts are critical to CL-mediated PA internalization.

Internalization of Pseudomonas aeruginosa is mediated by lipid rafts in contact lens-wearing rabbit and cultured human corneal epithelial cells.

PURPOSE: The internalization of Pseudomonas aeruginosa (PA) in nasal and tracheal epithelium has recently been shown to involve the formation of cholesterol- and sphingolipid-rich plasma membrane domains (lipid rafts). The purpose of this study was to investigate the role of lipid rafts in PA internalization by corneal epithelium in vivo, in vitro, and after contact lens wear. METHODS: Lipid raft formation was evaluated in rabbit corneas with and without contact lens wear and a human corneal epithelial (hTCEpi) cell line before and after PA infection with cornea-pathogenic strains by staining with FITC-conjugated cholera toxin beta-subunit, known to bind the lipid raft component GM1. Bacterial internalization was assessed by gentamicin survival assay. The role of lipid rafts in PA internalization was evaluated by pretreatment of hTCEpi cells with cholesterol metabolism inhibitors. The interaction of PA with lipid rafts was confirmed by flow cytometry. RESULTS: Contact lens wear in rabbits induced lipid raft formation in occasional surface corneal epithelial cells. Subsequent PA exposure showed preferential binding to lipid raft-forming cells, leading to lipid raft aggregation and PA internalization. A similar sequence of lipid raft formation and PA internalization was also observed in hTCEpi for all PA strains. Internalization of all PA strains was blocked by three cholesterol metabolism inhibitors (P < 0.01). Flow cytometry showed an association of PA with rafts. CONCLUSIONS: These findings demonstrate that contact-lens-mediated PA internalization involves lipid raft formation. Also, hTCEpi cells may be used as an experimental model for studying further the molecular mechanism(s) of PA infection in the corneal epithelium.

Prolonged hypoxia induces lipid raft formation and increases Pseudomonas internalization in vivo after contact lens wear and lid closure.

PURPOSE: To investigate the effects of hypoxia on lipid raft formation and Pseudomonas aeruginosa internalization by the corneal epithelium with and without the physical effects of contact lens wear. METHODS: One eye of each rabbit was randomly fitted with a low-Dk rigid gas-permeable contact lens (LDCTL) or closed with sutures, with the other as a control. After 1 day or 3 days, the rabbits were killed and bacterial invasion was assessed by gentamicin survival assay. Lipid rafts were identified by staining with FITC-conjugated beta subunit of cholera toxin. Corneal epithelial Bcl-2 expression was detected by Western blotting; surface epithelial cell size and thickness (epithelium and stroma) were measured by confocal microscopy. RESULTS: One-day hypoxia induced no significant changes in P. aeruginosa internalization, Bcl-2 expression, or lipid raft formation except in one of four eyelid-closed eyes. After 3 days, P. aeruginosa internalization was increased significantly (P < 0.05) in LDCTL-wearing eyes and not significantly (P = 0.10) increased in eyelid-closed eyes. Both 3-day test conditions also induced lipid raft-forming cells that bound P. aeruginosa, albeit in different regions of the cornea (peripherally in LDCTL-wearing eyes and centrally in closed eyes); did not alter epithelial thickness or surface cell size; and appeared to decrease epithelial Bcl-2 expression. CONCLUSIONS: This is the first direct comparison in vivo between two different methods inducing hypoxia on the corneal surface. Association of P. aeruginosa internalization with lipid raft formation in both conditions suggests a critical link among prolonged hypoxia, lipid raft formation, and susceptibility to P. aeruginosa infection. However, different distribution patterns of lipid raft-forming cells suggest physical effects of contact lens wear may direct localization of lipid raft-associated P. aeruginosa internalization on the corneal surface.