Detection of Active Matriptase using a Biotinylated Chloromethyl Ketone Peptide

Matriptase is a member of the family of type II transmembrane serine proteases that is essential for development and maintenance of several epithelial tissues. Matriptase is synthesized as a single-chain zymogen precursor that is processed into a two-chain disulfide-linked form dependent on its own catalytic activity leading to the hypothesis that matriptase functions at the pinnacle of several protease induced signal cascades. Matriptase is usually found in either its zymogen form or in a complex with its cognate inhibitor hepatocyte growth factor activator inhibitor 1 (HAI-1), whereas the active non-inhibited form has been difficult to detect. In this study, we have developed an assay to detect enzymatically active non-inhibitor-complexed matriptase by using a biotinylated peptide substrate-based chloromethyl ketone (CMK) inhibitor. Covalently CMK peptide-bound matriptase is detected by streptavidin pull-down and subsequent analysis by Western blotting. This study presents a novel assay for detection of enzymatically active matriptase in living human and murine cells. The assay can be applied to a variety of cell systems and species

Structural basis of synergistic neutralization of Crimean-Congo hemorrhagic fever virus by human antibodies

International audienceCrimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-borne zoonotic virus, with a 30% case fatality rate in humans. Structural information is lacking in regard to the CCHFV membrane fusion glycoprotein Gc-the main target of the host neutralizing antibody response-as well as antibody-mediated neutralization mechanisms. We describe the structure of prefusion Gc bound to the antigen-binding fragments (Fabs) of two neutralizing antibodies that display synergy when combined, as well as the structure of trimeric, postfusion Gc. The structures show the two Fabs acting in concert to block membrane fusion, with one targeting the fusion loops and the other blocking Gc trimer formation. The structures also revealed the neutralization mechanism of previously reported antibodies against CCHFV, providing the molecular underpinnings essential for developing CCHFVspecific medical countermeasures for epidemic preparedness

Arg614-cleaved matriptase is able to form complexes with HAI-1 and biotin-RQRR-CMK at pH 6.0.

<p>(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.</p

Biotin-RQRR-CMK detects both SEA domain-cleaved zymogen matriptase and Arg614-cleaved matriptase.

<p>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.</p

Biotin-RQRR-CMK efficiently inhibits matriptase even after 3 hours of pre-incubation at 37°C.

<p>(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 1^st order reaction due to the high molar excess of substrate to protease. Results shown are representative of 3 independent experiments.</p

Detection of active matriptase in cultured primary murine keratinocytes.

<p>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.</p