Micrographs showing immunohistochemical staining of an epiretinal PVR membrane section with biotinylated recombinant Galectin-3 (Gal-3) (A), anti-CD147 (B), and anti-integrin-β1 (C).

<p>Corresponding phase contrast images are also shown. Nuclei of cellular elements are stained with DAPI. Magnification 40x.</p

Gal-3 associates specifically with CD147 and integrin-β1 in cultured RPE cells.

<p>Whole cellular protein lysates were prepared by treatment of RPE cells with 1% Triton X-100 in PBS. (<b>A</b>) CD147 containing immunocomplexes were precipitated from 375 µg of proteins using 2 µg of a monoclonal anti-human CD147 antibody (<i>lane 2</i>), or a non-related isotype control (<i>lane 3</i>). Equal amounts of immunoprecipitation eluates, or whole cellular lysate (<i>lane 1</i>) were separated by SDS-PAGE and analyzed by immunoblotting using a monoclonal anti-Gal-3 antibody. (<b>B</b>, <b>C</b>) Cell lysates were processed as described for CD147 and precipitated using 2 µg of a monoclonal antibody recognizing specifically the integrin-α3 (B, <i>lane 2</i>) and integrin-β1 (C, <i>lane 2</i>) subunit, respectively. Note that the eluates precipitated with anti-CD147 (A; <i>lane 2</i>) and anti-integrin-β1 (C; <i>lane 2</i>) as well as the total cell extract (A,B; <i>lane 1</i>) contain the anti-Gal-3 reactive band at 30 kDa. In contrast, no anti-Gal-3 reactive component was recovered from cell extracts precipitated with anti-integrin-α3 (B; <i>lane 2</i>) and with the isotype control (A,B,C; <i>lane 3</i>). Experiments have been repeated at least eight times using cultured human RPE cells from different donors. Representative blots are shown.</p

Isolation of RPE cell proteins with high affinity binding to Gal-3.

<p>Isolation of RPE cell proteins with high affinity binding to Gal-3.</p

Immunohistochemical analysis of co-localization between Gal-3, CD147, integrin-α3, and integrin-β1 in dedifferentiated human RPE cells.

<p><i>Left panels:</i> Cultured human RPE cells without prior exposure to exogenous Gal-3 show immunoreactivity for endogenous Gal-3 (<b>A</b>) throughout the cytoplasm including the perinuclear region and only weak staining of the plasma membrane, while reactivity for CD147 (<b>B</b>,<b>C</b>) and integrin-α3 (<b>D</b>,<b>E</b>,<b>F</b>) is diffusely distributed over the plasma membrane with complete co-localization (<b>E</b>, <i>white</i>) of integrin-α3 and CD147. <i>Middle and right panels:</i> Exposure to Gal-3 induces redistribution of CD147, integrin-β1 and integrin-α3. RPE cells were treated with 40 µg/mL Gal-3 for 15 minutes, fixed and stained with the same antisera. Exogenous Gal-3 binds to the RPE cell surface in a distinct punctuate pattern (<b>G</b>,<b>M</b>) and results in a marked redistribution of CD147 (<b>H</b>,<b>N</b>), integrin-α3 (<b>J</b>), und integrin-β1 (<b>P</b>) subunits into a punctuate pattern, indicative of cell membrane receptor clustering. Exogenous Gal-3 shows complete co-localization with CD147 (<b>I</b>,<b>O</b>; <i>yellow</i>) and integrin-β1 (<b>R</b>; <i>yellow</i>), while co-localization between integrin-α3 and Gal-3 (<b>L</b>; <i>yellow,</i> for better visibility purple fluorescence of integrin-α3 was converted to green) is less pronounced. The merged image of CD147 and integrin-β1 RPE cells exposed to Gal-3 also suggests a strong overlay of the two signals (<b>Q</b>, <i>white</i>). In contrast, the co-localization of CD147 and integrin-α3 (<b>K</b>, <i>white</i>) in Gal-3 treated cells was only partial. The insets are an enlarged image of the boxed regions showing discrete yellow or white spots, respectively, indicating areas of co-localization. A negative control, where the primary antibody was omitted showed no fluorescence signal (data not shown).</p

Knockdown of Mgat5 expression in cultured human RPE cells attenuates binding of Gal-3 to RPE cells.

<p>(A) Western blot analysis of Mgat5 expression in RPE cells transfected with siRNA containing the same nucleotides as Mgat5 siRNA in random order (sc siRNA), and the same cell line transfected with 50 pmol and 100 pmol of double stranded siRNA complementary to Mgat5 (Mgat5 siRNA), respectively. Lysates containing approximately equal amounts of protein were separated by SDS-PAGE and blotted for immunochemical detection of Mgat5 content. Experiments were repeated at least three times. MW; molecular weight. (B) Quantification of Mgat5 gene silencing. Values are normalized to expression of tubulin. (C) Fluorescence micrographs of Gal-3 binding to the RPE cell surface. Ninety-six hours after transfection cells were treated with 60 μg/mL biotinylated Gal-3. Cells were then fixed and stained with a fluorescent streptavidin conjugate. Nuclei were counterstained with DAPI. Localization of Gal-3 binding was visualized by fluorescence microscopy at a 40 fold magnification. Scale bars represent 100 μm. Untreated cells exposed to streptavidin conjugate alone served as negative controls and exhibited no fluorescence signal (data not shown). (D-E) The target sequence derived from the genomic sequence of Mgat5 was inserted into a CRISPR-Cas9 nuclease expressing lentiviral vector and ARPE19 cells were transfected using Lipfectamine 2000 Plus reagent. (D) Western blot analysis of Mgat5 expression in ARPE19 cells transduced with guide RNA leading to specific knockdown of the Mgat5 (gMgat5), or cells tranduced with a CRISPR-Cas9 lentiviral vector encoding for an none-coding filler RNA (LV), or wild-type ARPE-19 cells. Lysates containing approximately equal amounts of protein were separated by SDS-PAGE and blotted for immunochemical detection of Mgat5 content. (E) Flow cytometric analysis of Gal-3 binding in Mgat5-knockout cells. CRISPR-Cas9-mediated Mgat5 knockdown of cultured RPE cells reduces cells surface binding of Gal-3, when compared to cells transfected with a non-coding control vector alone (LV). Histograms represent the number of counted cells versus relative fluorescence intensity. Transduction experiments have been repeated three times.</p

Interaction of Gal-3 with the RPE cell surface is mediated by carbohydrate-dependent binding to CD147 and integrin-β1, but not to integrin-α3.

<p>For monitoring the binding of recombinant Gal-3 to the RPE cell surface RPE cells were incubated with 40 ug/mL biotinylated Gal-3 and the cell surface binding was evaluated in a flow cytometer. The expression of CD147, integrin-β1 and integrin-α3 on the RPE cell surfaces was evaluated by staining with the respective antibodies, followed by measuring the relative fluorescence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070011#s2" target="_blank">materials and methods</a> section. (<b>A</b>) Staining with biotinylated recombinant Gal-3 and anti-CD147 confirms the presence of both proteins on the RPE cell surface. The interaction of Gal-3 with the RPE cell surface is mediated via its carbohydrate-binding domain, as it could be inhibited by adding 100 mM β-lactose but not by sucrose (<i>right panel</i>). (<b>B</b>) Pre-treatment of cells with a neutralizing anti-CD147 antibody reduces cell surface binding of Gal-3 to background levels (<i>left panel</i>). Incubation of cells with an isotype control IgG has no influence on Gal-3 cell surface binding (<i>right panel</i>). (<b>C</b>) Blocking of integrin-β1 partially reduces Gal-3 binding (<i>left panel</i>) while blocking of integrin-α3 binding sites fails to reduce Gal-3 binding to RPE cells (<i>right panel</i>). Relative fluorescence level of unstained cells incubated with streptavidin-FITC alone is also shown and represents the level of background fluorescence (shaded). (<b>D</b>) Fluorescence microscopy illustrates the absence of Gal-3 binding to the RPE cell surface when CD147 is blocked by treatment with a neutralizing antibody prior to the addition of Gal-3. The data are representative of three independent experiments with different RPE cell lines.</p

CD147, integrin-β1 and integrin-α3 in dedifferentiated human RPE cells contain β1,6-GlcNAc-branched N-glycans.

<p>Total cellular lysates were incubated with agarose-bound Concanavalin-A (ConA), a plant lectin specific for high mannose type oligosaccharides, or Phaseus vulgaris leukoagglutinin (PHA- L), which is specific for β1,6-branched N-glycans, and precipitated glycoprotein complexes were immunoblotted with either anti-CD147 (<b>A</b>), or anti-integrin-α3 (<b>B</b>), or anti-integrin-β1 (<b>C</b>) antibodies as indicated. Sepharose beads alone were used as negative control. High abundance of CD147 (<b>A</b>) and integrin-β1 (<b>C</b>) in the PHA-L-bound (specificity: β1,6-GlcNAc-N-glycans) fraction, but complete absence in eluates from ConA. (<b>B</b>) Integrin-α3 carries high-mannose type and β1,6-GlcNAc-branched N-glycans. No reactive compounds were detected in cell extracts incubated with agarose beads alone (Neg. Co; negative control). Molecular weight in kDa is indicated on the left (MW). Representative blots from four experiments are shown.</p

Native RPE cells exhibit little binding of Gal-3.

<p>(A) Flow cytometric analysis of Gal-3 binding to native and myofibroblastic RPE cells. Binding of Gal-3 (<i>gray</i>) to native RPE cells was only slightly above background, whereas Gal-3 binding to myofibroblastic RPE cells was evident. ConA binding was markedly above background in both cell populations. Histograms represent the number of cells versus relative fluorescence intensity. Experiments have been repeated two times. (B) Lectin histochemistry of human RPE cells in situ. Human cadaver eyes were incubated with biotinylated lectins as indicated on the left and binding was visualized by incubation with streptavidin-coupled peroxidase and Vector VIP substrate™ (purple color). In control sections with the VIP substrate alone the RPE can be easily discerned by the characteristic brownish pigment (third panel from the top). RPE cells and extracellular matrix reacted strongly with ConA <i>(top panel</i>), whereas PHA-L (<i>second panel</i> from the top) did not recognize native RPE cells and exhibited a staining pattern comparable to that of substrate alone. Biotinylated Gal-3 (purple color, <i>fourth panel</i>) did not bind to native RPE in situ and staining patterns resembled closely the situation in untreated negative control eyes. RPE, retinal pigment epithelium; BM, Bruch´s membrane; CH, choriocapillaris.</p

Glycomic profiling of native versus cultured, myofibroblastic RPE by lectin blot analysis reveals alterations in the glycan expression pattern upon EMT in vitro.

<p>Equal amounts of whole cellular protein lysates (7.5 μg) derived from native (N) and cultured, myofibroblastic (M) human RPE cells of passage 3 were separated by SDS page. Blots were incubated with the biotin-coupled plant lectins as indicated followed by peroxidase-coupled streptavidin (<i>large boxes</i>, <i>above</i>). Normalization to GAPDH (<i>small boxes</i>, <i>below</i>) shows equal total protein amounts of native RPE cells (N) and passaged RPE cells (M) were loaded in both slots. Molecular weight in kDa is indicated on the left (MW). The experiment was repeated at least three times with protein lysates from different donors of different age groups and different primary RPE cell lines of passage 3–7. A representative blot is shown.</p