Figure 1.
Biotin-RQRR-CMK efficiently inhibits matriptase even after 3 hours of pre-incubation at 37°C.
(A) Schematic structure of the biotin-RQRR-CMK peptide inhibitor. (B) The reactivity of biotin-RQRR-CMK was tested after 180 min of pre-incubation at 37°C (diamonds) or without preincubation (crosses). 0.2 µM matriptase SPD was incubated for 10 min at 37°C with (diamonds and crosses) or without (squares) 50 µM biotin-RQRR-CMK before addition of the chromogenic substrate to a final concentration of 300 µM. (C) The stability of 5 nM biotin-RQRR-CMK was further tested after the time points 0 (crosses), 60 (circles), 120 (stars), and 180 min (diamonds) of pre-incubation at 37°C and compared to a control not containing biotin-RQRR-CMK (squares). As described above, 0.2 nM matriptase SPD was added to each sample and incubated for 10 min at 37°C followed by addition of the chromogenic substrate to a final concentration of 300 µM. In all cases, the enzymatic activity of SPD was monitored by conversion of the chromogenic substrate (S2288). Each plot shows the change in optical density at 405 nm of the reaction mixture as a function of reaction time. The presence of active protease results in a continued release of a yellow cleavage product resulting in a linear color development in agreement with a pseudo 1st order reaction due to the high molar excess of substrate to protease. Results shown are representative of 3 independent experiments.
Figure 2.
Biotin-RRQR-CMK reacts with a subset of matriptase molecules on the surface of Caco-2 cells.
(A) Eleven days post-confluent Caco-2 cells grown on Transwell filters were labeled with 50 µM biotin-RQRR-CMK from the basolateral side for the times indicated (2–180 min) at 37°C (lanes 3–6) or for 180 min at 4°C (lane 7). As a measure of the steady state level of matriptase, membrane proteins on the basolateral plasma membrane of filter-grown Caco-2 cells were labeled by incubation with S-NHS-SS-biotin at 4°C for 30 min (lane 1). As a negative control, cells were labeled from the basolateral side with 50 µM control peptide; biotin-RQRR (lane 2). All cells were lysed and biotinylated proteins were precipitated using streptavidin-coated resin and were analyzed by non-reducing SDS-PAGE and Western blotting using the monoclonal matriptase antibody; M32. A tenth of the surface biotinylated sample was loaded (lane 1); whereas total sample volume was loaded for the other samples (lanes 2-7). (B) Caco-2 cells grown on Transwell filters were labeled with the biotin-RQRR-CMK peptide inhibitor from either the apical (lanes 1, 4, and 7) or the basolateral (lanes 2, 5, and 8) side for 180 min at either 4°C or 37°C. An overexposure of lanes 1–3 is shown in lanes 7–9. As a negative control, cells were labeled from the basolateral side with a peptide corresponding to the inhibitory peptide but lacking the CMK moiety (CTRL, lanes 3, 6, and 9). Cells were lysed and the lysates of multiple filters were pooled. Biotinylated proteins were precipitated using streptavidin-coated resin and the streptavidin pull downs were released by boiling in SDS-PAGE samples buffer and analyzed by SDS-PAGE and Western blotting using the monoclonal M32 antibody. Positions of the molecular weight markers (kDa) are indicated on the left. Results shown are representative of 3 independent experiments.
Figure 3.
Arg614-cleaved matriptase is able to form complexes with HAI-1 and biotin-RQRR-CMK at pH 6.0.
(A) Eleven days post-confluent filter-grown Caco-2 cells were treated with either a physiologically phosphate buffer pH 6.0 for 20 min (lanes 2, 4, 6, and 8) from both the apical and the basolateral side or left untreated (lanes 1, 3, 5, and 7). Cells were lysed and lysates were analyzed by Western blotting using antibodies against total matriptase (M24; lanes 1 and 2), matriptase SPD (IM1014; lanes 5 and 6), matriptase-HAI-1 complex (M69; lanes 3 and 4) and HAI-1 (lanes 7 and 8). Samples in lanes 1–4, 7, and 8 were not boiled to avoid dissociation of matriptase-HAI-1 complexes, while samples in lanes 5 and 6 were boiled and reduced to dissociate the S-S bridged SPD from the stem domain of activated matriptase in order to distinguish between the SEA domain-cleaved form (70 kDa) and the Arg614 cleaved form (25–30 kDa). Treatment with phosphate buffer pH 6.0 and DTT is indicated by +/−. Positions of the molecular weight markers (kDa) are indicated on the left. (B) A solution of 0.2 µM SPD was incubated for 10 min at 37°C with (crosses) or without (squares) 50 µM biotin-RQRR-CMK before addition the chromogenic substrate to a final concentration of 300 µM. All experiments were performed in 20 mM citric acid buffer pH 6.0, 140 mM NaCl and 0.1% BSA at 37°C. Results shown are representative of 3 independent experiments.
Figure 4.
Biotin-RQRR-CMK does not react with matriptase-HAI-1 complexes.
Eleven days post-confluent Caco-2 cells grown on Transwell filters were labeled with 50 µM biotin-RQRR-CMK at pH 7.4 (lanes 3 and 7), in physiological phosphate buffer pH 6.0 (lanes 4 and 8), or at pH 7.4 with a 30 min pre-incubation treatment with physiological phosphate buffer pH 6.0 (lanes 5 and 9) for 30 min at 37°C. All cells were lysed and samples of lysates were analyzed under non-boiled and non-reducing conditions (lanes 1–5). Labeled proteases in the lysates were precipitated using streptavidin-coated resin and released from the beads by boiling (lanes 7–9). The streptavidin pull downs were analyzed by SDS-PAGE and Western blotting (lanes 6–9). As a negative control, lysate of cells treated with only physiological phosphate buffer pH 6.0 for 30 min was also streptavidin-precipitated and analyzed (CTRL, lanes 6). All lanes were analyzed using the M32 antibody. Positions of the molecular weight markers (kDa) are indicated on the left. Results shown are representative of 3 independent experiments.
Figure 5.
Biotin-RQRR-CMK detects both SEA domain-cleaved zymogen matriptase and Arg614-cleaved matriptase.
Eleven days post-confluent Caco-2 cells grown on Transwell filters were labeled with 50 µM biotin-RQRR-CMK from the basolateral side for 180 min at 37°C. As a negative control, cells were labeled from the basolateral side with 50 µM control peptide; biotin-RQRR (CTRL), under the same conditions. Labeled proteases were precipitated using streptavidin-coated resin and the streptavidin pull downs were analyzed by reducing SDS-PAGE and Western blotting using the IM1014 antibody raised against matriptase SPD. Positions of the molecular weight markers (kDa) are indicated on the left and position of SEA domain-cleaved zymogen matriptase and matriptase SPD is indicated on the right. Results shown are representative of 3 independent experiments.
Figure 6.
Detection of active matriptase in cultured primary murine keratinocytes.
Murine keratinocytes were isolated from newborn wildtype (WT) or matriptase-deficient pups (KO) and cultured on collagen-coated plastic. The cells were grown until sub-confluent and then labeled with S-NHS-SS-biotin (lanes 1, 4, 7, and 10), or with 50 µM biotin-RQRR-CMK (lanes 2, 5, 8, and 11), or with 50 µM control peptide; biotin-RQRR (lanes 3, 6, 9, and 12). Cells were lysed and analyzed on Western blot (lanes 7–12). The remaining lysates were precipitated using streptavidin-coated resin, released from the beads by boiling under non-reducing conditions and lysates were analyzed by SDS-PAGE and Western blotting using the matriptase antibody AF3946. Results shown are representative of 2 independent experiments.