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Blood, Vol. 110, Issue 2, 544-552, July 15, 2007
Natural killer cell–derived human granzyme H induces an alternative, caspase-independent cell-death program
Blood Fellows et al.
110: 544
Supplemental materials for Fellows et al, Vol. 110, Issue 2, 544-552
Files in this Data Supplement:
- Document 1. Supplemental methods (PDF, 73.3 KB)
- Figure S1. Chymase specificity of recombinant GzmH (JPG, 67.3 KB)
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(A) Recombinant, nonglycosylated human GzmH and the point-mutated, inactive GzmHS195A were expressed in E. coli and refolded from inclusion bodies. Conversion efficiency and purity of the proform (Pro-GzmH), the mature form (GzmH), and the mature inactive form (GzmHS195A) were assessed by silver staining after SDS PAGE (lane 1 to 3). (B) GzmH (20 nM) had chymase-like activity. GzmH cleaved the thiobenzylester Suc-Phe-Leu-Phe-SBzl (300 µM) like cathepsin G (20 nM) whereas GzmHS195A lacked any activity. (C) GzmH (20 nM) was efficiently inhibited by DCI (100 nM). (D) GzmH and cathepsin G (200 nM), but not GzmB, cleaved the peptide substrates PTSY-AMC and ATSY-AMC (250 µM). The data points in B to C and columns in D with error bars represent the average of triplicates (± SD). Each experiment was repeated at least 3 times.
- Figure S2. Specific detection of GzmH by immunoblotting and ELISA (JPG, 83.9 KB)
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(A) Purified human granzymes prepared in our lab (lanes 2 to 6) along with a commercial preparation of recombinant pro-GzmH (lane 1) were separated by SDS PAGE and immunoblotted (left panel). The mouse monoclonal antibody MAB1377 specifically recognized pro-GzmH and GzmH (A, lanes 1 and 2). The N-terminally tagged Pro-GzmH generated in NS0 cells was glycosylated and migrated as several bands with apparent molecular masses between 38 and 42 kDa. The secreted GzmH pro-form, moreover, carried 20 artificial glutamate residues at the N-terminus and was, therefore, larger than the bacterially expressed recombinant granzymes. Equal amounts of recombinant granzymes were loaded (100 ng per lane) and immunoblotted after SDS-PAGE. The exquisite specificity of the anti-GzmH antibody was also tested against all human granzymes in a solid phase ELISA (A, right panel). Granzymes were coated on microtiter plates and incubated with either MAB1377 or an isotype control antibody. The means shown were calculated from duplicate measurements. Expression of GzmB (B, middle panel) and GzmH (lower panel) in leukocyte subsets of human peripheral blood and tumor cell lines was assessed by immunoblotting after SDS-PAGE. Loading of cellular lysates was controlled by immunostaining of actin after antibody stripping (B upper panel). Purified recombinant granzymes (50 ng left panels; 20 ng right panels) and lysates of different human leukocyte populations or cell lines (10 µg) were probed against GzmB (using monoclonal antibody 2C5) or GzmH (MAB1377). GzmB blots also showed some high molecular mass bands between 60 and 70 kDa. These bands may represent covalently linked GzmB complexes that were artificially formed between the GzmB and its serpin inhibitor, PI-9, in the lysates.
- Figure S3. Nuclear fragmentation of K562 cells treated with GzmH or GzmB and sublytic SLO for 12 hours (JPG, 62.2 KB)
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(A) Cells were stained either with the Hoechst dye 33342 alone (40× magnification) or (B) in combination with annexin V-FITC (63× magnification). After fixation, fluorescent signals were analyzed by microscopy. Contrary to treatment with GzmB (7.5 µg/mL) and GzmH (15 µg/mL), cells treated with SLO alone or in conjunction with GzmHS195A (15 µg/mL), (A-B, left) did not show fragmented nuclei with condensed chromatin or phosphatidylserine externalization. Hoechst stainings are representative of 5 independent experiments.
- Figure S4. GzmH did not activate caspase-activated DNase via ICAD cleavage (JPG, 68.9 KB)
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(A) K562 cells were exposed to GzmB or GzmH and analyzed by immunoblotting 6, 9 and 12 hours after exposure. Actin was used to control loading. (B) DNA fragmentation was monitored by the 3H-thymidine release assay after 4, 12 and 24 hours in K562 cells that had been treated with SLO and the indicated proteases. zVAD-fmk (50 µM) only partially inhibited both proteases after 12 and 24 hours. The data points represent the average of triplicate measurements (± SD).
- Figure S5. Cytotoxic activity of GzmH did not depend on caspases (JPG, 96.5 KB)
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(A) Full time-lapse immunofluorescence analysis of the 10-hour time point experiments summarized in Figure 4A. The curves represent the average fluorescence signals of individual, triplicate measurements taken at 5-minute intervals (1 hour intervals presented). Each graph is representative of 3 independent experiments. (B) GzmH did not cleave recombinant inactive procaspase 3C285A and procasapase 7C285A. GzmH or GzmB was incubated with the respective procaspases (5 µM) at 37°C for 1 hour. Samples were analyzed by silver staining after SDS-PAGE. At an enzyme to substrate ratio of 1:1000, procaspase 3C285A and procasapase 7C285A were efficiently cleaved by GzmB into their dimer fragments, p22/p10 and p24/p11, respectively, as indicated by arrowheads. GzmH, however, did not cleave recombinant caspases. (C) Recombinant Bid was also not processed by GzmH to the proapoptotic truncated form (t-Bid). Granzymes were incubated with recombinant Bid for 1 hour at 37°C. Bid degradation was assessed by SDS-PAGE and silver staining. The disappearance of the 24-kDa band representing full length Bid indicated cleavage by GzmB. At a ratio of 1:100, GzmB completely cleaved Bid, while Bid remained intact even at high GzmH concentrations and an enzyme to substrate ratio of 1:10.
- Figure S6. GzmH homologs from different placental mammals (JPG, 389 KB)
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Alignment of known and predicted amino acid sequences for GzmH homologs; hs, homo sapiens; pt, pan troglodytes, mul, macaca mulatta, oc, oryctolagus cuniculus, cf, canis familiaris, bt, bos taurus, rn, rattus norvegicus, mm, mus musculus. The numbering is according to bovine chymotrypsinogen A (btCtra) shown at the bottom. Conserved critical residues that determine the substrate specificity of GzmH at the first (P1) and second position (P2) N-terminal to the cleaved peptide bond are boxed.
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