Detection of Active Matriptase Using a Biotinylated Chloromethyl Ketone Peptide

Abstract

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.

HAI‐2 stabilizes, inhibits and regulates SEA‐cleavage‐dependent secretory transport of matriptase

“…To examine, whether any matriptase, when co‐expressed with either of the HAI‐2 Kunitz domain 1 mutants, still reaches the plasma membrane, and whether this matriptase is SEA domain cleaved, we used a cell‐surface biotinylation approach. Using either a N‐Hydroxysulfosuccinimide ester of Biotin (NHS‐SS‐Biotin), which in this setup labels all membrane bound proteins, or a biotinylated peptide substrate‐based chloromethyl ketone (Biotin‐RQRR‐CMK), which in this setup detects active matriptase (both in the zymogen form and the Arg614 cleaved form), in combination with streptavidin pull down, SDS‐PAGE and western blotting, using an antibody against matriptase. This experiment was designed to analyze the status regarding SEA domain cleavage and not the quantity of matriptase bound to the plasma membrane.…”

“…Both NHS‐SS‐Biotin and Biotin‐RQRR‐CMK are non‐cell membrane permeable. The NHS‐SS‐Biotin labels plasma membrane proteins in a non‐specific manner, whereas the Biotin‐RQRR‐CMK specifically labels and inhibits active serine proteases, including matriptase . For each transfection a sample of the total extract (Figure , total extract), a pull down fraction of surface biotinylated proteins (Figure , pull down) and the remaining fraction (Figure , supernatant) representing the intracellular part of the cell were analyzed.…”

A Fluorescent‐Labeled Phosphono Bisbenzguanidine As an Activity‐Based Probe for Matriptase

“…Several radioactive, fluorescent and biotin labels are established reporter tags for ABPs . While fluorescent ABPs for matriptase have not been reported so far, a biotinylated chloromethyl ketone peptide has been used for the detection of active matriptase . Moreover, biotin‐labeled ABPs for trypsin‐like enzymes with a phosphonate warhead and one benzamidine moiety have been developed …”

<strong>A Short Peptide Inhibitor as an Activity-Based Probe for Matriptase-2</strong>

“…Here we present a short biotinylated peptide probe with a chloromethyl ketone (CMK) warhead as an irreversible inhibitor and activity-based probe for MT2. This probe was originally developed for the related enzyme matriptase, [6] which exhibits a similar substrate specificity [7].…”