MOESM1 of Profiling of metalloprotease activities in cerebrospinal fluids of patients with neoplastic meningitis

Additional file 1. Sketch of proteases and CSF infiltration, correlation analyses, and ELISA results

Thy-1-GPI anchor redirects PrP^C to the apical site.

<p>(A) Cells stably expressing PrP^CWT and PrP^C-GPIThy-1 were grown in Transwells for 4 to 5 days, processed for immunocytochemistry, and analyzed with confocal microscopy. YZ sections (left) and view on the membrane (right) at the level of tight junctions stained for ZO-1 (red) confirm both polarization and confluency of cells and show increased apical signal for PrP^C-GPIThy-1 (green). (B) After staining with PrP 3F4 antibody under non-permeabilizing conditions, serial Z-stacks from the bottom to the top were taken. YZ sections show transversal cut through cells at the level of the dashed line in mid. PrP^C-GPIThy-1 was found at the apical membrane when compared to PrP^CWT. Scale bars are 10 µm. (C) Cells grown in Transwells labeled with EZ-Link Sulfo-NHS-SS-Biotin either apically (a) or basolaterally (b) were processed for Western blotting for PrP^C and E-Cadherin (as control of cell polarization) in parallel. The graph (three independent experiments) shows mean percentages ± SEM of apical (a) or basolateral (b) amount of protein when compared to the total amount which is set at 100%.</p

PrP^C-GPIThy-1 is glycosylated and transported to the plasma membrane.

<p>(A) Schematic presentation of GPI-anchored PrP^CWT and the PrP^C fusion protein with the GPI-anchor of Thy-1 (PrP^C-GPIThy-1). The substitution of the GPI-anchor signal sequence (ss) of the PrP for the one of Thy-1 is indicated. (B) Western blots of PrP^CWT and PrP^C-GPIThy-1 stably expressed in MDCK cells. A clone with a similar expression level as PrP^CWT was chosen. The glycotype of di-, mono-, and non-glycosylated PrP^C-GPIThy-1 is unchanged. (C) Assessment of non-permeabilized membrane localization of PrP^CWT and PrP^C-GPIThy-1 by confocal microscopy shows plasma membrane localization of both proteins (scale bar is 10 µm). (D) Sucrose density gradient centrifugation of 1% Triton-X100 extraction at 4°C of PrP^CWT and PrP^C-GPIThy-1 cells reveal localization of both in flotillin enriched DRMs. Fractions were taken from the top (fraction 1) to the bottom (fraction 12).</p

Schematic drawing of constructs used in this study.

<p>Shown are the maps of PrP^CWT, PrP^CG1, PrP^CG2, and PrP^CG3 with N-terminal signal sequence (ss) and C-terminal GPI-anchor signal (ss GPI-anchor) (dark boxes) and the mutations introduced to delete N-gylcosylation sites.</p

Cell surface biotinylation assay confirms a role of the N-glycans in polarized sorting of PrP^C.

<p>Cells were grown in Transwells for 4–5 days until fully polarized and labelled with EZ-Link Sulfo-NHS-SS-Biotin either on the apical (a) or the basolateral (b) side. Cells were processed for PrP^C (recognized with the 3F4 antibody) and E-cadherin Western blotting in parallel. The graph indicates densitometric evaluation of Western blots of at least 3 independent experiments, expressed as mean percentages ± SEM apical (a) or basolateral (b) of total protein found, which is set at 100%.</p

N-glycosylation of PrP^C affects polar sorting in MDCK cells.

<p>MDCK cells stably expressing PrP^CWT, PrP^CG1, PrP^CG2 or PrP^CG3 were grown in Transwells for 4 to 5 days until they were fully polarized. (A) Cells were separately stained with the 3F4 antibody (green) followed by permeabilisation and staining with an antibody against ZO-1 (red), a constituent of tight junctions, indicating the cell polarity. Confocal microscopy of a Z-stack of PrP^CWT (left) at the level of tight junctions stained with ZO-1, and YZ-sections (right) of all glycomutants indicate both the integrity of the polarized monolayer and a redistribution of PrP^CG1 and PrP^CG2 to the apical compartment when compared to PrP^CWT and PrP^CG3. Localization of the apical (a) and basolateral (b) compartment is indicated. (B) After immunocytochemistry under non-permeabilising conditions with the 3F4 antibody, serial Z-stacks from the bottom to the top were taken with confocal microscopy. YZ images shows transversal cut trough cells at the mid level, marked with a dashed line. PrP^CWT and PrP^CG3 were mainly found in the basolateral compartment whereas PrP^CG1 and PrP^CG2 were mainly found in both compartments. Scale bars represent 10 µm.</p

Physiological membrane localization of PrP^C glycomutants.

<p>(A) Characterization of glycomutants (PrP^CG1, PrP^CG2, and PrP^CG3) and PrP^CWT for the study by Western blot analysis, using an antibody directed against the 3F4 epitope. Clones with similar amounts of overexpressed 3F4 tagged PrP^C as assessed by densitometric analysis of Western blots were used for these analyses (see graph). Relative expression of various PrP^C forms is shown in percentages of PrP^CWT that was set to 100%. (B) Assessment of plasma membrane (non-permeabilized) and intracellular (permeabilized) localization of PrP^C glycomutants by confocal microscopy shows presence of PrP^C at the plasma membrane and intracellularly (scale bar is 10 µm). (C) Assessment of DRMs localization of PrP^C glycomutants by Triton X-100 extraction at 4°C and sucrose density gradient centrifugation showing correct localization of PrP^C glycomutants with flotillin-positive DRM containing fractions.</p