Fig. 1: ADAM17 cleaves the CA IX ectodomain and deletion of amino acids 393–402 in the CA IX stalk region prevents the ECD shedding.

a Verification of the cleavage activity of the recombinant human TACE/ADAM17 (rhADAM17) towards the fluorogenic peptide Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Ser-Arg-NH2. The peptide was used at the final concentration of 10 μM in a total of 100 μL reaction mixture with 10 ng of the rhADAM17. Time-related increase of the fluorescence emitted from the pro-TNF-α-derived peptide proves that rhADAM17 was active. b Recombinant CA IX-SBP fusion protein attached to a streptavidin-sepharose matrix was cleaved by rhADAM17, eluted and detected by ELISA using CA IX-specific monoclonal antibodies. c Cells transiently expressing FL-CA IX were treated with ADAM17/10 inhibitor for 24 h and CA IX ECD was detected in culture media by ELISA. Results in b and c support the role of ADAM17 in cleavage of CA IX. d Schematic illustration of the CA IX domain structure and positions of deletions in the mutants. Scissors indicates extracellular domain cleavage region. Missing amino-acid residues of individual deletion variants are represented by numbers on the right side. SP signal peptide, PG proteoglycan-like domain, CA catalytic domain, TM transmembrane region, IC intracellular tail. e Immunofluorescence analysis of CHO-wt, CHO-M2 and CHO-M2-TACE cells transiently expressing the FL-CA IX and two stalk deletion variants. The cells were fixed with methanol, incubated with M75 antibody followed by ALEXA Fluor488-secondary antibody and nuclei were stained with DAPI. Deletion of the cleavage site did not affect the CA IX localisation. f, g ELISA analysis of the CA IX deletion variants for the ECD shedding. The plasmids encoding FL-CA IX and its two deletion mutants were transiently transfected to CHO-wt, CHO-M2 (ADAM17-defective) and CHO-M2-TACE (human ADAM17 expressing) cell lines. 48 h after transfection, the cells were cultivated for 3 h in equal medium volumes in the presence or absence of PMA. Undiluted conditioned media (f) and cell lysates diluted 1:10 (g) were collected and examined by ELISA using V/10 antibody as a capture and mixture of biotinylated MAbs M75 and IV/18 as a detector. h Biochemical evidence that ADAM17 can cleave FL-CA IX, but not the NS mutant was obtained by treatment of CHO cell variants expressing FL and NS, respectively, with recombinant rhADAM17 added to medium at a concentration of 50 μg/ml for 24 h. Collected media were analysed by ELISA. i ADAM17 suppression resulting from infection by lentiviruses expressing ADAM17-specific shRNA led to a decreased CA IX shedding from C33a-FL cells, but not from C33a-NS cells proving the involvement of ADAM17 in the CA IX ECD cleavage. The results (mean ± SD) represent the mean of two measurements in triplicates. (*P < 0.05, ***P < 0.005, ns non-significant).