mouse matriptase st14 catalytic domain  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: Matriptase protein and constructs. A, schematic drawing of full-length matriptase with the N-terminal transmembrane region (black), the SEA domain (green), two CUB domains (orange), four LDLRa domains (blue), and the C-terminal serine protease domain (purple). The cleaved activation loop rearranges to create the catalytically active protease. B, schematic drawing of the zymogen-locked form of matriptase characterized in this paper, consisting of only the serine protease domain with position R614A mutated to avoid activation (zSPD). C, a Coomassie-stained SDS-PAGE (10%) shows that zSPD (consisting of only the serine protease domain, as shown in B) is pure and the expected size (25–30 kDa). D, the sequence of the crystallized form of zSPD (zSPD-S805A) with an additional mutation in the active site, shown with matriptase numbering (sides) and chymotrypsin numbering (top). The mutations are color-coded: R614A (blue), N772Q (green), and S805A (blue).

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques: Construct, Activation Assay, Staining, SDS Page, Sequencing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: The zymogenicity factor of matriptase All values represent an average of at least three independent measurements.

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: The monoclonal antibodies aZ-mAb-6 and aZ-mAb-7 are not substrates for matriptase. Antibodies aZ-mAb-6 and aZ-mAb-7 were incubated with or without acSPD at a 1:1 molar ratio for 1 h at 37 °C. No cleavage of the antibodies is seen after incubation with matriptase.

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques: Bioprocessing, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: The monoclonal antibodies aZ-mAb-6 and -7 specifically inhibit human matriptase. The ability of aZ-mAb-4, -6, and -7 to inhibit human matriptase and other closely related serine proteases was tested in an activity assay. For all assays, an excess of antibody (400 nm) was preincubated with the proteases for 1 h at 37 °C before the addition of substrate. Human matriptase, acSPD (100 pm), was almost completely inhibited by aZ-mAb-6 (green line) and aZ-mAb-7 (black line), but not by aZ-mAb-4 (red line). The activity of mouse matriptase SPD (100 pm), human uPA (1 nm), hepsin (1 nm), and HGFA (5 nm) were not significantly inhibited by any of the monoclonal antibodies. The graphs shown are representative of three replicates.

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques: Bioprocessing, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: A, the monoclonal antibodies aZ-mAb-6 and -7 inhibit matriptase activity in the protein extracts of transiently transfected cells. HEK293 cells were transiently transfected with empty expression vector (mock) or expression vectors encoding WT full-length matriptase together with WT HAI-2 or mutant HAI-2 C47F, respectively. Protein extracts were preincubated for 1 h at room temperature with 500 ng of antibody (anti-uPA, aZ-mAb-6, aZ-mAb-7, or no antibody), and a peptidolytic activity assay was subsequently carried out with 300 μm chromogenic substrate S-2288. The absorbance of the reaction mixture was measured at 405 nm continuously every 5 min for 5 h, and the mean velocity of the substrate reaction (in milli-absorbance units/min) was calculated. The figure shown is representative of three replicates. B, aZ-mAb-6 and -7 inhibit matriptase-mediated pro-HGF cleavage. Pro-HGF (60 nm final concentration) was incubated for 4.5 h at 37 °C with either 200 nm zSPD or 1 nm acSPD that had been preincubated with aZ-mAb-6, aZ-mAb-7, anti-uPA, or biotin-RQRR-CMK, as indicated above the gels. The processing of pro-HGF was visualized on Western blotting using an antibody that recognizes pro-HGF as well as its α and β chains. Antibody-only samples were prepared in parallel to show the cross-reactivity of the antibodies themselves with the secondary antibody.

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques: Bioprocessing, Activity Assay, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Concentration Assay, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: The structure of zymogen matriptase, zSPD-S805A. A, the solved structure of zSPD-S805A (wheat) with the 60 loop, the 70 loop, the 170 loop, and the activation loop specified together with the residues in the catalytic triad and the S1 pocket (PDB entry 5LYO). B, the intact activation loop for zSPD-S805A (wheat) and the cleaved activation loop for activated matriptase (red). C, the zymogen triad for zSPD-S805A and activated matriptase, showing rearrangement of Asp804 (Asp194). D, alignment of zSPD-S805A (wheat) and activated matriptase (gray), where the loops undergoing conformational changes are marked in red for activated matriptase. The S1 pocket is not formed in the zymogen.

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques: Activation Assay