GzmB binding and uptake by cell surface heparan sulfate. (A-D) K562 cells were stained with an FITC-labeled monoclonal antibody against heparan sulfate and GzmB-Bio/SA-PE. (A) Staining without the GzmB-Bio and anti HS-FITC antibody. (B) Staining with anti HS-FITC and SA-PE, omitting GzmB-Bio. (C) Single staining with GzmB-Bio and SA-PE, omitting anti HS-FITC. (D) Double staining with GzmB-Bio/SA-PE and anti HS-FITC. Note the clear correlation between GzmB-Bio binding and heparan-sulfate expression. (E) Binding of GzmB and HS expression of K562, HL-60, and U937 cells. (Left) FACS representation of anti–HS-FITC versus GzmB-Bio/SA-PE staining of HL-60 and U937 cells. (Right) The diagram shows the MFI of GzmB-Bio binding at the indicated GzmB-Bio concentrations. (F) Sodium chlorate treatment diminishes granzyme binding to cell surfaces. HL-60 cells were treated with the indicated sodium chlorate concentrations, and binding of the indicated proteins was investigated. (G) HS-bound GzmB is rapidly internalized. GzmB^Alexa633 (40 μg/mL) was bound to HL-60 cells for 1 hour at 4°C (left). Thereafter, cells were washed twice and incubated at 37°C for 15 (middle) or 45 minutes (right). Shown is an analysis by confocal fluorescence microscopy of permeabilized HL-60 cells. Merged images of 2 cytoplasmic planes of z series are shown. GzmB^Alexa633 is shown in red, and the lysosomal antigen LAMP-1 (CD107a) is shown in green. Image processing was identical for all images, and control primary antibody staining for this marker appeared black under these conditions. Already after 15 minutes, a clear colocalization (in yellow) between GzmB and lysosomes can be observed. Scale bar = 20 μm.