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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01068

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Processing and activation of latent heparanase occurs in lysosomes

Anna Zetser^1,*, Flonia Levy-Adam^1,*, Victoria Kaplan¹, Svetlana Gingis-Velitski¹, Yulia Bashenko¹, Shay Schubert¹, Moshe Y. Flugelman², Israel Vlodavsky^1, and Neta Ilan¹

¹ Cancer and Vascular Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel
² Department of Cardiology, Lady Davis Carmel Medical Center, Haifa 34632, Israel

View larger version (72K): [in a new window]   Fig. 1. Characterization of antibody 733 specificity. (A) Immunoblot analysis. Lysates of heparanase-transfected HEK-293 cells were resolved by SDS-PAGE and blots were probed with antibody 1453 (left panel), 733 (middle panel) and anti-heparanase monoclonal antibody purchased from Becton-Dickinson (BD, right panel). (B) Metabolic-labeling followed by immunoprecipitation. Heparanase-transfected CHO cells were pulsed for 20 minutes with [35S]methionine (0) and then chased for the indicated time intervals in complete growth medium containing an excess of non-radioactive methionine. Equal volumes of cell lysate samples were subjected to immunoprecipitation (IP) with mAb 130 (top panel) or antibody 733 (bottom panel), as described in Materials and Methods. (C) Immunohistochemistry. Placenta sections (5 µm) were subjected to immunostaining with antibody 733, as described in Materials and Methods. Negative control, in which the primary antibody was omitted, is shown in the bottom panel. Original magnifications: top and bottom panels x20, middle panel x100.

View larger version (102K): [in a new window]   Fig. 2. Heparanase localization in cell xenografts and tumor biopsies. (A) PC3 xenograft (a,b), prostate biopsy (c,d) and (B) MCF7 xenograft (a-c) and breast cancer biopsy (d-f) sections were subjected to antigen retrieval and immunostaining with antibody 733. Negative control, in which the primary antibody was omitted, is shown for MCF7 xenograft (Bc) and breast cancer biopsy (Bf). Original magnifications: Aa,c and Ba,d x20; Ab,d and Bb,c,e,f x100.

View larger version (72K): [in a new window]   Fig. 3. Heparanase uptake, processing and localization in human MDA-435 breast carcinoma and U87 glioma cells. MDA-435 cells were left untreated (Con, top row) or incubated with the 65 kDa heparanase precursor (5 µg/ml) for 5 minutes (second row), 1 hour (third row) or 3 hours (fourth row) and stained with monoclonal anti-heparanase antibody (BD, left column, red) or with antibody 733 (middle column, green). Merged images are shown in the right column. U87 cells were incubated with heparanase for 3 hours and similarly stained (fifth row). Original magnification: x100. MDA-435 cells were also stained with monoclonal anti-cathepsin D antibody, or with antibody 733. Merged image is shown in the right panel (bottom row). (Inset: top right) Heparanase uptake and processing. MDA-435 (upper panel) and U87 glioma cells (second panel) were left untreated (0) or incubated with the 65 kDa heparanase precursor. At the indicated time points, cells were washed and total cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibody 1453 (first and second panels), or with anti-actin antibody (third panel).

View larger version (42K): [in a new window]   Fig. 4. Heparanase processing is inhibited by lysosomal proteinase inhibitors. (A-D) Chloroquine treatment. (A) Heparanase-transfected 293 (upper panel), MDA-435 breast carcinoma (second panel), C6 rat glioma (third panel) and NMU rat mammary adenocarcinoma (fourth panel) cells were left untreated (0) or incubated for 20 hours with the indicated concentrations (µM) of chloroquine. Total cell lysates were immunoblotted with anti-heparanase antibody 1453. Note a dose-response inhibition of heparanase processing and accumulation of the unprocessed 65 kDa heparanase precursor. (B) Chloroquine treatment is reversible. Heparanase-transfected C6 glioma cells were left untreated (Con) or treated with 50 µM chloroquine for 20 hours. Cells were then lysed (Chl) or washed and chased for an additional 24 hours with chloroquine-free medium (Chase). Total cell lysates were then analyzed for heparanase processing by immunoblotting as above. Note re-appearance of the processed 50 kDa heparanase form upon chloroquine removal. (C,D) Uptake studies. (C) U87 glioma cells were left untreated (Control) or pre-treated with 100 µM chloroquine (Chloroquine) for 2 hours. The latent 65 kDa heparanase protein was then added for the indicated time points and total cell lysates were analyzed for heparanase processing by immunoblotting as above. Note complete inhibition of exogenously added heparanase processing upon chloroquine pre-treatment. (D) U87 glioma cells were left untreated (Control) or incubated with chloroquine (100 µM) for 2 hours, followed by the addition of the 65 kDa heparanase protein for additional 2 hours. Cells were then fixed and immunostained with anti-heparanase monoclonal antibody (BD) and 733 anti-heparanase (733) antibodies. Merged images are shown in the third row. Negative control, in which the primary antibody was omitted, is shown in the bottom row. Note the complete absence of heparanase processing as evident by the lack of reactivity with antibody 733 upon chloroquine treatment (second panel, right), and the accumulation of the latent heparanase form in large vesicles (upper and 3rd panels, right). Original magnifications x100. (E) Heparanase-transfected C6 glioma (upper panel) and NMU (lower panel) cells were left untreated (0) or incubated with the indicated concentrations (µM) of bafilomycin A1 or chloroquine (Chl, 50 µM) for 20 hours. Total cell lysates were analyzed by immunoblotting as above. Note complete inhibition of heparanase processing upon bafilomycin treatment.

View larger version (32K): [in a new window]   Fig. 5. Processing of membrane-targeted heparanase is chloroquine sensitive. (A) Expression of membrane-targeted heparanase in stably transfected 293 and C6 cells. Control (M) and heparanase (Hepa) transfected cells were lysed and subjected to immunoblot analysis with anti-heparanase antibody 1453 (upper panel) or anti-actin antibodies (lower panel). 293 cells expressing the membrane-targeted heparanase gene construct were analyzed by FACS (B) with anti-heparanase monoclonal antibody (BD, upper panel) and anti-c-Myc epitope-tagged antibodies (lower panel), or stained by immunofluorescence (C) with anti-heparanase monoclonal antibodies (BD). Negative control, in which the primary antibody was omitted, is shown in the lower panel. Note heparanase accumulation at areas of cell-cell junctions in sparse cultures (C, top panel) and exclusive localization at the cell borders in confluent cells (C, middle panel). (D) Processing of membrane-targeted heparanase is chloroquine sensitive. 293 (left panel) and C6 (right panel) cells stably transfected with the membrane-targeted heparanase gene construct were left untreated (Con) or treated for 20 hours with 100 µM chloroquine (Chl). Total cell lysates were immunoblotted with anti-heparanase antibody 1453. Non-transfected cell lysates were included as control (M).

View larger version (22K): [in a new window]   Fig. 6. Antibody 733 inhibits heparanase enzymatic activity. (A) Purified, active recombinant heparanase (20 ng) was added to 1 ml RPMI medium and incubated with affinity-purified antibody 733 (10 µg, red symbols), or rabbit IgG (black symbols) for 1 hour on ice, followed by 1 hour incubation with 35S-labeled ECM. Heparanase activity was determined as described in Materials and Methods. (B) Heparanase-transfected 293 cells (2x105) were plated on 35S-labeled ECM in the presence of affinity-purified antibody 733 (30 µg/ml, red symbols) or control rabbit IgG (black symbols) for 2 hours. The incubation medium containing sulfate-labeled degradation fragments was subjected to gel filtration on a Sepharose CL-6B column.