Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: Cleavage profile of the HMPV F: A fluorogenic peptide mimicking the cleavage site of HMPV F was incubated with the indicated proteases and cleavage was monitored by the increase of fluorescence at 390 nm. RFU = relative fluorescence units. StDev = Standard Deviation.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Incubation, Fluorescence

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: IC50 values of all protease/peptide combinations.: Fluorogenic peptides mimicking the cleavage sites of A/CA/04/09 H1N1, A/Japan/305/1957 H2N2 HA, A/Aichi/2/68 H3N2 HA, A/Vietnam/1203/2004 H5N1 LPAI HA, A/Vietnam/1204/2004 H5N1 HPAI HA, A/Taiwan/2/2013 H6N1 HA, A/Shanghai/2/2013 H7N9 HA, A/Hong Kong/2108/2003 H9N2 HA and HMPV F were incubated with the indicated proteases and different SPINT2 concentrations. Cleavage was monitored by the increase of fluorescence at 390 nm and the resulting Vmax values were used to calculate the IC50 values as described in the “Material and Methods” section. (A) IC50 values of influenza A fluorogenic cleavage site peptide mimics. Concentrations are in nanomolar. (B) IC50 values of the HMPV F cleavage site peptide mimic. Concentrations are in picomolar. NT = Not tested.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Incubation, Fluorescence

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: SPINT2 inhibits cleavage of HA protein expressed in 293T cells. Cells were transfected with plasmids encoding for the indicated HA and allowed to express the protein for ~18 h. The recombinant proteases were incubated for 15 min with the indicated SPINT2 concentrations and subsequently added to the cells for 10 min (trypsin) or 90 min (matriptase and KLK5). Western blots were performed and the HA 1 band was quantified using ImageJ. (A) Quantification of the HA1 band comparing the signal intensity of the 0 nM SPINT2 samples against 10 nM and 500 nM SPINT2 of the respective HA/protease combination. Three independent experiments were carried out and the western blots of each experiment were analyzed. Quantifications were conducted as described in the methods section. (B – D) Western blots showing the cleavage of (B) A/CA/04/09 H1N1 HA by matriptase and KLK5 at different SPINT2 concentration, (C) A/Aichi/2/68 H3N2 HA by KLK5 at different SPINT2 concentration and (D) A/Shanghai/2/2013 H7N9 HA by matriptase and KLK5 at different SPINT2 concentrations. Statistical analysis was performed using a non-paired student's t-test comparing the samples tested with 10 nM SPINT2 against the respective sample incubated with 500 nM SPINT2. Error bars indicate standard deviation. * indicates p = < 0.05.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Transfection, Recombinant, Incubation, Western Blot, Concentration Assay, Standard Deviation

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: TMPRSS2, HAT, matriptase and KLK5 cleave HMPV F and SPINT2 is able to prevent cleavage by exogenous proteases . HMPV F was either expressed alone or co-transfected with protease and allowed to express for ~ 18 h. Cells were then metabolically starved of cysteine and methionine followed by radioactive S35 labeling of protein for 4 h in the presence of TPCK-trypsin or specified protease. SPINT2 treated proteases were incubated at room temperature for 10 min and placed onto cells for 4 h. Radioactive gels were quantified using ImageQuant software with percent cleavage equal to [ ( F 1 F 0 + F 1 ) x 100 ] . (A and B) Co-transfected proteases TMPRSS2, HAT and matriptase are able to cleave HMPV F (n = 4) while C and D) exogenous proteases KLK5 and matriptase but not KLK12 are able to cleave HMPV F (n = 5). (E and F) SPINT2 prevented cleavage of HMPV F by trypsin, KLK5 and matriptase at nm concentrations demonstrated by the loss of the F 1 cleavage product (n = 3). Statistical analysis was performed using a one-way ANOVA followed by a student's t-test with a bonferroni multiple comparisons test correction. P < 0.05 *, P < 0.005 **, P < 0.0005 ***, P < 0.0001 ****. N values represent independent replicates for each treatment group. Error bars represent SD.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Transfection, Metabolic Labelling, Labeling, Incubation, Software

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: SPINT2 inhibits HA-mediated cell-cell fusion. VERO cells were transfected with plasmids encoding for (A) A/CA/04/09 H1N1 HA or (B) A/Shanghai/2/2013 H7N9 HA and allowed to express the protein for ~18 h. Recombinant matriptase and KLK5 were incubated with different SPINT2 concentrations for 15 min and then added to the HA-expressing cells for 3 h. After 3 h the cells were briefly treated with cell fusion buffer at pH5, washed, supplemented with growth medium and returned to the incubator for 1 h to allow fusion. HA protein was detected using HA-specific primary antibodies and a secondary fluorogenic Alexa488 antibody. Nuclei were stained using DAPI. Magnification 40x. (C) VERO cells expressed A/Vietnam/1204/2004 H5N1 HA that was cleaved during its maturation process in the cell. SPINT2 was added at 0 nM or 500 nM at the time of transfection. Magnification 25x.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Transfection, Recombinant, Incubation, Expressing, Staining

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: SPINT2 reduces IAV growth in cell culture. MDCK cells were transfected with plasmids encoding for human matriptase or human TMPRSS2 and allowed to express the proteins for ~18 h. Cells expressing human matriptase (B) or human TMPRSS2 (C) were then infected with A/CA/04/09 H1N1 at a MOI of 0.1 and different SPINT2 concentration were added to each well. Non-transfected cells to which trypsin was added served as a control (A). (D) MDCK cells were infected with A/X31 H3N2 at an MOI of 0.1 and trypsin was added to assist viral propagation. Different SPINT2 concentration were added as indicated. After 48 h of infection the supernatants were collected and used for an immuno-plaque assay to determine the viral loads. Experiment was repeated three times and each dot represents the viral titer of a single experiment. Statistical analysis was performed using a non-paired student's t-test comparing the control (0 h) against the respective sample. Error bars indicate standard deviation. * indicates p = < 0.05. Extended horizontal line within the error bars represents mean value of the three independent replicates.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Cell Culture, Transfection, Expressing, Infection, Concentration Assay, Control, Plaque Assay, Standard Deviation

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: Viral titers measured in the infection studies: Table shows the average viral titers and standard deviation calculated from the 3 independent biological replicates depicted in Fig. 6 , Fig. 7 . (A) Titers and standard deviation from infection studies shown in Fig. 5 . (B) Titers and standard deviation shown in Fig. 7 .

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Infection, Standard Deviation

Journal: Journal of Virology

Article Title: Cleavage Activation of the Human-Adapted Influenza Virus Subtypes by Matriptase Reveals both Subtype and Strain Specificities

doi: 10.1128/JVI.00306-12

Figure Lengend Snippet: Cleavage of the human-adapted HA subtypes by matriptase. (A) Western blot analysis of HA cleavage from the A/PR/8/34, A/New Caledonia/20/99 (A/NC/99), A/California/04/09 (A/CA/09), A/South Carolina/1/18 (A/SC/18), A/WS/33, and A/WSN/33 H1-subtype strains by matriptase. A/WSN/33 HA in the absence of protease (mock) and in the presence of trypsin was added as a control. (B) Western blot analysis of HA cleavage from the A/Japan/305/57 H2-subtype strain by matriptase. (C) Western blot analysis of HA cleavage from the A/Aichi/2/68, A/Wyoming/3/03 (A/WY/03), and A/Wisconsin/67/05 (A/WI/05) (H3N2) H3-subtype strains by matriptase. A/Aichi/2/68 HA in the absence of protease (mock) and in the presence of trypsin was added as a control.

Article Snippet: The catalytic domain of human matriptase was purchased from R&D Systems.

Techniques: Western Blot, Control

Journal: Journal of Virology

Article Title: Cleavage Activation of the Human-Adapted Influenza Virus Subtypes by Matriptase Reveals both Subtype and Strain Specificities

doi: 10.1128/JVI.00306-12

Figure Lengend Snippet: Comparison of the H1 HA cleavage efficiency of trypsin and matriptase. Bar graph depicting the cleavage efficiency of A/PR/8/34, A/New Caledonia/20/99 (A/NC/99), A/California/04/09 (A/CA/09), A/South Carolina/1/18 (A/SC/18), A/WS/33, and A/WSN/33 H1-subtype strains by trypsin and matriptase. The percent HA cleavage was determined by densitometry of the Western blot shown in Fig. 1.

Article Snippet: The catalytic domain of human matriptase was purchased from R&D Systems.

Techniques: Comparison, Western Blot

Journal: Journal of Virology

Article Title: Cleavage Activation of the Human-Adapted Influenza Virus Subtypes by Matriptase Reveals both Subtype and Strain Specificities

doi: 10.1128/JVI.00306-12

Figure Lengend Snippet: Cleavage by matriptase produces a fusogenic HA. Immunofluorescence staining of cells expressing A/California/04/09 (A/CA/09), A/South Carolina/1/18 (A/SC/18), A/Japan/305/57, and A/Aichi/2/68 after treatment with either trypsin, no protease treatment (mock), or matriptase. The HA from each subtype was labeled with anti-influenza HA antibody and Alexa Fluor 488 secondary antibody (green), and the cell nuclei were stained with Hoechst 33258 (blue).

Article Snippet: The catalytic domain of human matriptase was purchased from R&D Systems.

Techniques: Immunofluorescence, Staining, Expressing, Labeling

Journal: Journal of Virology

Article Title: Cleavage Activation of the Human-Adapted Influenza Virus Subtypes by Matriptase Reveals both Subtype and Strain Specificities

doi: 10.1128/JVI.00306-12

Figure Lengend Snippet: Cleavage activation of A/PR/8/34 and A/WSN/33 H1N1 influenza viruses by matriptase. (A) Immunofluorescence staining of the viral nucleoprotein of infected cells after treatment with trypsin, no protease treatment (mock), and matriptase. The viral nucleoprotein was stained with anti-influenza nucleoprotein antibody and Alexa Fluor 488 secondary antibody (green), and the cell nuclei were stained with Hoechst 33258 (blue). (B) Quantitation of the percent infection from panel A.

Article Snippet: The catalytic domain of human matriptase was purchased from R&D Systems.

Techniques: Activation Assay, Immunofluorescence, Staining, Infection, Quantitation Assay

Journal: Journal of Virology

Article Title: Cleavage Activation of the Human-Adapted Influenza Virus Subtypes by Matriptase Reveals both Subtype and Strain Specificities

doi: 10.1128/JVI.00306-12

Figure Lengend Snippet: The amino acid residues neighboring the HA cleavage site Arg affect cleavage by matriptase. The cleavage rate of the fluorogenic peptides IPSIQSRGL (H1 consensus), IPSIQYRGL (A/WSN/33 cleavage site), VPQIESRGL (H2 consensus), and VPEKQTRGL (H3 consensus) after treatment with matriptase. The rate of cleavage was determined by monitoring the increase in fluorescence at 390 nm. RFU, relative fluorescence units.

Article Snippet: The catalytic domain of human matriptase was purchased from R&D Systems.

Techniques: Fluorescence

Journal: Journal of Virology

Article Title: Cleavage Activation of the Human-Adapted Influenza Virus Subtypes by Matriptase Reveals both Subtype and Strain Specificities

doi: 10.1128/JVI.00306-12

Figure Lengend Snippet: Comparison of cleavage rates of various HA-cleaving proteases with matriptase

Article Snippet: The catalytic domain of human matriptase was purchased from R&D Systems.

Techniques: Comparison

Journal: Journal of Virology

Article Title: Cleavage Activation of the Human-Adapted Influenza Virus Subtypes by Matriptase Reveals both Subtype and Strain Specificities

doi: 10.1128/JVI.00306-12

Figure Lengend Snippet: Cleavage activation of thrombolytic zymogens by matriptase. Cleavage of the fluorogenic peptide, GGGR-AMC by plasmin (1,611.7 relative fluorescence units [RFU]/min) (A), urokinase (2,855.1 RFU/min) (B), and plasma kallikrein (3,012.3 RFU/min) (C) after treatment with matriptase. Each graph includes the change in fluorescence at 440 nm of matriptase alone (squares), the zymogen alone (circles), and matriptase incubated with the zymogen (diamonds).

Article Snippet: The catalytic domain of human matriptase was purchased from R&D Systems.

Techniques: Activation Assay, Fluorescence, Clinical Proteomics, Incubation

Journal: ACS chemical biology

Article Title: Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein

doi: 10.1021/acschembio.7b00715

Figure Lengend Snippet: Matriptase biosensor design. (a) ddRFP-based biosensor schematic. Following proteolytic linker cleavage, the fluorescent “A” copy separates from the stabilizing “B” copy, resulting in loss of “A” copy fluorescence. (b) Example time course plot of biosensor mechanism; addition of matriptase cleaves the biosensor and reduces fluorescence over time (red), while absence of matriptase retains fluorescence over time (blue). (c) Matriptase cleavable linker designs and sequence information. Design name is derived from number of amino acids flanking the scissile bond. Scissile bond is highlighted in red (Arg) and blue (Val). Each sequence shown is derived from the natural pro-macrophage stimulating protein (Pro-MSP) sequence. N-term = N-terminus; K1–4 = Kringle domains 1–4; SPH = Serine Protease Homologue.

Article Snippet: A total of 5 × 10 5 cancer cells were resuspended in cold 1xPBS with 1 mg mL −1 bovine serum albumin (0.1% BPBS) solution, containing a 1:100 dilution of mouse anti-human matriptase antibody (Fisher Scientific).

Techniques: Fluorescence, Sequencing, Derivative Assay

Journal: ACS chemical biology

Article Title: Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein

doi: 10.1021/acschembio.7b00715

Figure Lengend Snippet: Biosensor kinetic measurements and characterization. (a) Time course trajectories of biosensors B3 to B10 with matriptase and (b) velocity graphs in the presence of varying concentrations of matriptase (Matr.). (c) Western blot bands correspond to three possible reaction products detected through the C-terminal hexahistidine-tag: uncleaved biosensor (top band, 59.4 kDa), **hydolytically cleaved biosensor (middle band, 40.3 kDa), and cleaved biosensor (bottom band, 32.5 kDa). Copy “A” product is not detected due to lack of a His-tag. Note, two gels were used for this experiment as indicated. (d) Velocity profile for B4, B8, and B9 in the presence of different serine protease family members, relative to matriptase activity. Experiments performed in triplicate and reports mean values with standard deviation. Data are normalized for background conditions of biosensor without matriptase.

Article Snippet: A total of 5 × 10 5 cancer cells were resuspended in cold 1xPBS with 1 mg mL −1 bovine serum albumin (0.1% BPBS) solution, containing a 1:100 dilution of mouse anti-human matriptase antibody (Fisher Scientific).

Techniques: Western Blot, Activity Assay, Standard Deviation

Journal: ACS chemical biology

Article Title: Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein

doi: 10.1021/acschembio.7b00715

Figure Lengend Snippet: Application of B4 for measuring protease activity and inhibition. (a) B4 measurement of matriptase activity expressed on human A549 lung (blue), PC3 prostate (red), and MDA-MB-231 breast (green) cancer cell lines, compared with media alone (magenta); *p < 0.01 vs control. (b) B4 measurement of matriptase inhibition by soluble KD1 inhibitor. (c) B4 measurement of matriptase inhibition by yeast-displayed wild-type KD1 (green), KD1-R260A (magenta), or noninduced yeast controls with (red) or without (blue) matriptase.

Article Snippet: A total of 5 × 10 5 cancer cells were resuspended in cold 1xPBS with 1 mg mL −1 bovine serum albumin (0.1% BPBS) solution, containing a 1:100 dilution of mouse anti-human matriptase antibody (Fisher Scientific).

Techniques: Activity Assay, Inhibition