Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates

Abstract

Increases in expression of ADAM10 and ADAM17 genes and proteins have been evaluated, but not validated as cancer biomarkers. Specific enzyme activities better reflect enzyme cellular functions, and might be better biomarkers than enzyme genes or proteins. However, no high throughput assay is available to test this possibility. Recent studies have developed the high throughput real-time proteolytic activity matrix analysis (PrAMA) that integrates the enzymatic processing of multiple enzyme substrates with mathematical-modeling computation. The original PrAMA measures with significant accuracy the activities of individual metalloproteinases expressed on live cells. To make the biomarker assay usable in clinical practice, we modified PrAMA by testing enzymatic activities in cell and tissue lysates supplemented with broad-spectrum non-MP enzyme inhibitors, and by maximizing the assay specificity using systematic mathematical-modeling analyses. The modified PrAMA accurately measured the absence and decreases of ADAM10 sheddase activity (ADAM10sa) and ADAM17sa in ADAM10^-/- and ADAM17^-/- mouse embryonic fibroblasts (MEFs), and ADAM10- and ADAM17-siRNA transfected human cancer cells, respectively. It also measured the restoration and inhibition of ADAM10sa in ADAM10-cDNA-transfected ADAM10^-/- MEFs and GI254023X-treated human cancer cell and tissue lysates, respectively. Additionally, the modified PrAMA simultaneously quantified with significant accuracy ADAM10sa and ADAM17sa in multiple human tumor specimens, and showed the essential characteristics of a robust high throughput multiplex assay that could be broadly used in biomarker studies. Selectively measuring specific enzyme activities, this new clinically applicable assay is potentially superior to the standard protein- and gene-expression assays that do not distinguish active and inactive enzyme forms.

Keywords: ADAM10; ADAM17; Cancer biomarker; Cell lysate; Fluorogenic peptide substrates; Gene knockout; Gene restoration; Gene silence; Protease inhibitors; Proteolytic activity matrix analysis; Sheddase activity; Tissues; lysate.

Figure 1

PrAMA approach for solid tissue analysis. (A) Summary table and heat-map show 7 FRET-based polypeptide substrates with cleavage sequences (HomoPhe, homophenylalanine; Cha, 3-cyclohexylalanine), endogenous-protein protease substrates from which the peptides were designed, and the catalytic efficiencies with which various recombinant proteases cleave the substrates (for more information see ref. 35). (B) Workflow illustrates PrAMA for cell and tissue lysates. (C) Proof-of-principle is shown for PrAMA applied to known solutions, and one combination, of recombinant enzymes (abbreviations: rM2, recombinant MMP2; rA10, recombinant ADAM10; rA17, recombinant ADAM17). Normalized cleavage rates for each of the 4 solutions were measured across the 7 substrates (left), and data were interpreted using known enzyme-substrate preferences [as in (A)] to infer which recombinant enzymes were present in the mixture. Actual mixture composition (top right) and PrAMA results (bottom right) are presented. (D) Surface plots depict three-dimensional “systematic PrAMA” inference as a function of the two parameters sensitivity (Syntherror) and specificity (Sigmathreshold). Processing data of seven substrates obtained with recombinant MMP2, ADAM10, and ADAM17 were analyzed by PrAMA across varying combinations of Syntherror and Sigmathreshold parameters to reveal how these two parameters influence PrAMA sensitivity and specificity. The three rows of surface-plots correspond to the analyzed individual three recombinant enzyme solutions (rMMP2, rADAM10 and rADAM17), and the three columns of surface-plots correspond to the three individual PrAMA-inferred enzyme activities from these solutions (MMP2a, ADAM10sa and ADAM17sa). The color scale ranges from red to blue, which reflects the surface heights as labeled on the vertical axis. The three-dimensional surface plots shown in the figure depict a representation of the two-dimensional “systematic PrAMA” shown in Figs. 3C-3H. In the latter cases (Figs. 3C-H) and the rest of presented data, Syntherror is held constant (0.5) across a range of Sigmathreshold values. The experimental details follow those described for Fig. 1C.

Figure 2

Broad-spectrum Roche and Halt non-MP protease inhibitors decrease large proportions of cell-lysate enzyme activities, but do not affect rADAM10 and rADAM17 sheddases activities. Tris-based reaction buffer solutions (150 μL) of 10 μM PEPDAB005, 2 μg of H441-cell lysate (A, B), 10 ng of rADAM10 (C, D), 10 ng of rADAM17 (E, F) and indicated concentrations of Roche and Halt inhibitors were distributed into wells of 96-well LUMITRAC-200 plates. Additionally, the effects of different concentrations of DMSO on rADAM10 and rADAM17 processing of PEPDAB005 were tested (G, H). Negative-control wells contained the assay buffer supplemented only with 10 μM of PEPDAB005, and positive-control (total substrate processing) wells contained the assay buffer supplemented with 1 μM of PEPDAB005 and 1% trypsin. Plates were incubated at 37^oC for 4h. The fluorescence was measured with a TECAN infinite 200 pro fluorimeter, using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The presented data are pM means of duplicate measurements ± SD (A, C, E, G) and % inhibition (B, D, F, H) of PEPDAB005 processing at 3 h of incubation. (A, B) Roche and Halt inhibitors significantly inhibited in a dose-dependent manner H441-cell lysate-mediated processing of PEPDAB005 (p=0.002-0.0001). Halt was more effective (p=0.012 to p=0.0012). Processing of PEPDAB005 by rADAM10 (C, D) and rADAM17 (E, F) was slightly (non-significantly) inhibited with 0.25%, 0.5 or 1%, but significantly inhibited with 2% and especially 3% of the protease inhibitors (rADAM10: p=0.048 to p=0.019; rADAM17: p=0.05 to p=0.0038). Halt cocktail did so more efficiently than the Roche cocktail (p=0.026 to p=0.013). (G, H) DMSO did not inhibit rADAM10 or rADAM17 enzyme activity.

Figure 3

Combined solutions of 0.5% Roche and 0.5% Halt inhibitors do not affect recombinant ADAM10-mediated processing of PEPDAB substrates or PrAMA-ADAM10 sheddase activity. Processing of 5 substrates with rADAM10 (A) and rADAM17 (B) in the absence and presence of 0.5% or 1% combined inhibitors is presented. Data are pM means of duplicate measurements ± SD of processed substrates. The decreases of PEPDAB005 processing with rADAM10 and rADAM17 and the increases of PEPDAB010 processing with rADAM17 in the presence of 1% Roche/Halt inhibitors are significant (p=0.0049, p=0.0046 and p=0.0038, respectively). PrAMA-ADAM10sa data of rADAM10 processing of PEPDAB substrates in the absence (C) and presence of 0.5% (D) or 1% (E) combined inhibitors are presented. PrAMA-ADAM17sa data of rADAM17-processing of substrates in the absence (F) and presence of 0.5% (G) or 1% (H) combined inhibitors are presented. The experiments in (A) and (B) were performed using similar conditions to those presented in Fig. 1. PrAMA was performed using a fixed 0.5 Syntherror parameter and systematically increased Sigmathreshold parameters from 0.0 to 2.0. Data are PrAMA ADAM10sa (C, D, E) and PrAMA ADAM17sa (F, G, H) arbitrary units (AU). PrAMA standard errors were <5%.The arrow-had line in (D) indicates that the PrAMA specificity Sigmathreshold parameters ≥ 1.0 detects only true-positive rADAM10-ADAM10sa without any false-positive ADAM17sa.

Figure 4

Modified PrAMA detects ADAM10sa and ADAM17sa presence in wild-type and absence in ADAM10^-/- and ADAM17^-/- MEF lysates, respectively. ADAM10^+/-, ADAM10^-/- (A, C, E), ADAM17^+/+ and ADAM17^-/- (B, D, F) MEFs were activated with PMA/Ionomycin and lysed. The 150 μL Tris solutions of 2 μg cell lysates, 10 μM PEPDAB substrates and 0.5% Roche/Halt protease inhibitors were incubated for 4 h at 37^oC, and the developed fluorescence was recorded hourly using TECAN fluorimeter. The presented experiments are representative of 5 performed. The enzymatic activity data are shown as pM means of duplicate measurements ± SD of the processed substrates (A, B).The decreased processing of PEPDAB005, 010, 011, 014 and 022 with knockout MEF lysates is significant (ADAM10^+/- vs ADAM10^-/- MEFs and ADAM17^+/+ vs ADAM17^-/- MEFs: p<0.0001 and p<0.001, p=0.0056 and p<0.001, p=0.0016 and p=0.0025, p=0.0016 and p=0.009, and p=0.006 and p=0.004, respectively). Systematic PrAMA data using Syntherror/Sigmathreshold parameters 0.5/0.0 to 0.5/2.0 and 0.5/0.0 to 0.5/1.0 are presented as ADAM10sa AU (C) and ADAM17sa AU (D), respectively. Standard errors of PrAMA data were 2.5% to 5.8%. True-positive ADAM10sa and ADAM17sa in ADAM10^+/- and ADAM17^+/+MEFs, respectively, are presented as % of specific enzyme activities in wild-type MEFs, containing both the true-positive and false-positive activities, relative to ADAM10^-/- (E)and ADAM17^-/-(F)ME Fs containing only the false-positive activities, respectively.

Figure 5

Modified PrAMA detects ADAM10sa in ADAM10^-/- MEFs following restoration of ADAM10 gene. ADAM10^-/- MEFs were transfected with Lipofectamine/human ADAM10-cDNA or treated with Lipofectamine alone and incubated for 24 h. In some experiments, empty plasmid-transfected ADAM10^-/- MEFs were used as an additional control.(A) ADAM10^-/- MEFs transfected with human ADAM10-cDNA express human ADAM10 on cell surface. Lipofectamine treated (empty histogram) and Lipofectamine/human ADAM10-cDNA transfected (filled histogram) cells were stained with PE-conjugated anti-human ADAM10 and examined by flow cytometry. Cells were also stained with PE-conjugated anti-mouse ADAM10 or isotype-control mAbs, and their mean fluorescence intensities (MFIs) were similar to that of the cells treated with Lipofectamine alone and stained with PE-conjugated anti-human ADAM10 mAb (data not shown). Data are from one of two similar experiments performed. (B)Lysates of ADAM10^-/- MEFs transfected with human ADAM10-cDNA contain increased enzyme activities as assessed with all 7 PEPDAB substrates. Lipofectamine alone treated and Lipofectamine/human ADAM10-cDNA transfected ADAM10^-/- MEFs were lysed. The 150 μL Tris solutions containing 2 μg cell lysates, 10 μM PEPDAB substrates and 0.5% Roche/Halt inhibitors were incubated for 4 h at 37^oC, and the developing fluorescence was recorded every hour. Data are representative of three experiments performed. They are pM means of duplicate measurements ± SD of processed substrates. The increases of PEPDAB processing with the lysates of ADAM10-cDNA transfected cells are significant (PEPDAB010: p= 0.002; PEPDAB005, 008, 011, 013, 014 and 022: p<0.001 to p<0.0001). (C)PrAMA-ADAM10sa AU was obtained by analyzing the enzyme activity data at 4 h of incubation using the systematically increased Syntherror/Sigmathreshold parameters from 0.5/0.0 to 0.5/2.0. Standard errors of PrAMA data were 1.3% to 2.6%. (D) True-positive ADAM10sa restoration in ADAM10^-/- MEFs is presented as % of the specific enzyme activity in ADAM10-cDNA transfected MEFs, containing both the true-positive and false-positive activities, relative to Lipofectamine-alone treated ADAM10^-/- MEFs, containing only the false-positive activity.

Figure 6

Modified PrAMA detects decreases of ADAM10sa and ADAM17sa in human cancer cells after silencing of the corresponding enzyme genes. H441 cells were transfected with human ADAM10 siRNA, ADAM17 siRNA or scrambled siRNA or were treated with transfection reagents alone for 48 h. In some experiments, control cells were also untreated.(A, B) Cells were stained with PE-conjugated IgG control mAb, anti-human ADAM10 (A) or anti-human ADAM17 mAb (B) and analyzed by flow cytometry. Empty histograms represent MFI of H441 cells stained with isotype control mAb. Dark gray histograms represent MFI of H441 cells treated with scrambled siRNA and stained with PE-conjugated anti-ADAM10 (A) or anti-ADAM17 (B) mAbs. Light grey histograms represent MFI of H441 cells treated with ADAM10 (A) or ADAM17 (B) siRNA and stained with PE-conjugated anti-ADAM10 or anti-ADAM17 mAbs, respectively. Data are from a representative experiment of 8 similar performed (Suppl. Figs. 4A, 4B). In the presented experiment, ADAM10 and ADAM17 siRNA induced 90% and 45% decreases of ADAM10 and ADAM17 protein expression on H441 cell surface, respectively. (C)After performing transfection, 2 μg of cell lysates were tested for processing PEPDABs in the presence of 0.5% Roche/Halt protease inhibitors. Data are from one of seven similar experiments performed. They are pM means of duplicate measurements ± SD of processed substrates. Processing of PEPDAB substrates was differently decreased in H441 cells transfected with ADAM10 or ADAM17 siRNA (PEPDAB005: p=0.0023 and p=0.0077; PEPDAB010: p=0.012 and p=0.017; PEPDAB011: p=0.048 and p=0.06; and PEPDAB014: p=0.07 and p=0.0.019, respectively). The substrate processing data obtained at 4 h of incubation were analyzed using the systematically increased Syntherror/Sigmathreshold parameters from 0.5/0.0 to 0.5/2.0. The resulted PrAMA ADAM10sa (D) and ADAM17sa (E)AU are shown. PrAMA standard errors were 1.3% to 8.9%. Decreases of ADAM10sa and ADAM17sa in ADAM10 (F)and ADAM17 (G)siRNA transfected H441 cells, respectively, are presented as % of the specific enzyme activities in the siRNA transfected cells relative to transfection reagent-treated cells. Proportion-equation analysis of PrAMA-ADAM10sa and PrAMA-ADAM17sa of rADAM10 and rADAM17 vs the scrambled siRNA-transfected H441-cell lysates, respectively, showed that 10 μg of H441-cell lysate contained 17.0 ng of ADAM10sa and 1.65 ng of ADAM17sa.

Figure 7

Modified PrAMA detects decreases of ADAM10sa in human-cell and -tissue lysates exposed to ADAM10 inhibitor. H441-cell (A)and T2495-tissue (B) lysates (2 μg) were supplemented with 0.5% Roche/Halt protease inhibitors, vehicle (Control) or GI254023X (1 μM), and PEPDAB substrates (10 μM), and incubated for 4 h. The developing fluorescence was measured hourly. The presented data are pM means of duplicate measurements ± SD of processed substrates, and are representative of ten experiments performed. The GI254023X-mediated inhibitions of PEPDAB005, 010 and/or 022 processing with the lysates are significant (H441: p=0.017, 0.0041 and 0.0058; T2495: p=0.05, 0.04 and 0.08, respectively). The substrate processing data were analyzed using the systematically increased Syntherror/Sigmathreshold parameters from 0.5/0.0 to 0.5/2.0. The obtained data at 4 h incubation are presented as PrAMA ADAM10sa AU of H441-cell (C)and T2495-tissue (D)lysates. PrAMA standard errors were 1.2% to 9%. Decreases of ADAM10sa in H441-cell (E)and T2495-tissue (F)lysates are presented as % of the specific enzyme activities in the control lysates relative to the GI254023X treated lysates.

Figure 8

Modified PrAMA efficiently measures ADAM10sa and ADAM17sa in multiple human tumor-tissue specimens. (A) NSCLC tumor lysates contain less ADAM10 than ADAM17 protein. ADAM10 and ADAM17 were quantified in lysates of 5 human NSCLC tumor tissues using ELISAs. Presented data are means pg/10 μg lysates of ADAM10 and ADAM17 proteins ± SD of 5 tumor tissues. (B) NSCLC tissue lysates process high amounts of PEPDAB05, 008 and 010, moderate amounts of PEPDAB014 and 022, and low amounts of PEPDAB011 and 013. Duplicates of 150 μL of Tris-based reaction buffer supplemented with NSCLC tumor-tissue lysates (10 μg/replicate), 0.5% Roche/Halt protease inhibitors and 10 μM of PEPDABs were incubated at 37^oC, and fluorescence quantified hourly for 4 h. Data are pM means of duplicate measurements ± SD of processed substrates with the 5 tissue lysates. (C, E) PrAMA ADAM10sa and (D, F) PrAMA ADAM17sa are robust but quantitatively different. The 4 h substrate processing data were analyzed using the systematically increased Syntherror/Sigmathreshold scripts from 0.5/0.0 to 0.5/2.0. Presented data are of the individual tissue lysates. PrAMA standard errors were 2.7% to 5.4%. Proportion-equation analysis of rADAM10 and rADAM17, and the tissue lysate PrAMA-ADAM10sa and PrAMA-ADAM17sa, respectively, showed that 10 μg of these tissue lysates contained 20.0 ng of ADAM10sa and 1.25 ng of ADAM17sa.