Detection of Active Matriptase Using a Biotinylated Chloromethyl Ketone Peptide

Sine Godiksen,Lotte K Vogel,Thomas H Bugge,Christoffer Soendergaard,Jette Bornholdt,Mingdong Huang,Katiuchia Uzzun Sales,Stine Friis,Jan K Jensen

doi:10.1371/journal.pone.0077146

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.

Highlights

Matriptase is a type II transmembrane serine protease that is expressed in most epithelia and has pleiotropic roles in epithelial development and homeostasis [1,2,3,4,5]
The biotin group allows for efficient isolation of biotin-RQRR-chloromethyl ketone (CMK)-labeled proteases and endogenously biotinylated proteins by streptavidin precipitation, whereby catalytically-inactive matriptase not able to bind the substrate is segregated away [35]
We found that 50 mM biotin-RQRRCMK renders 0.2 nM matriptase serine protease domain (SPD) unable to cleave the chromogenic substrate, whereas matriptase SPD in the absence of biotin-RQRR-CMK cleaved the substrate efficiently

Summary

Introduction

Matriptase ( known as MT-SP1, epithin, TADG-15 and SNC19) is a type II transmembrane serine protease that is expressed in most epithelia and has pleiotropic roles in epithelial development and homeostasis [1,2,3,4,5]. Matriptase is converted into its active conformation by proteolytic cleavage after Arg614 within the conserved activation cleavage site R-VVGG located within the serine protease domain This cleavage has been reported to require the proteolytic activity of matriptase, as mutations in any of the residues of the catalytic triad renders matriptase unable to undergo activation site cleavage. This finding has led to a model for matriptase activation in which a weak intrinsic proteolytic activity of the SEA domain-cleaved matriptase zymogen activates neighboring SEA domain-cleaved matriptase molecules [7]. Consistent with this model, the purified SEA domain-cleaved soluble matriptase has been shown to be capable of hydrolyzing synthetic peptide substrates in solution, catalytic activity of the cell surface matriptase zymogen still needs to be demonstrated [13,14]

Methods

Results

Conclusion