Activation of a Membrane-Bound Serine Protease Matriptase on the Cell Surface

Abstract

Matriptase is a type II transmembrane serine protease. The activation (i.e. conversion of the single-chain pro-form to the disulphide-linked-two-chain active form) of this enzyme is known to occur via a mechanism requiring its catalytic triad. We reported previously that the activated enzyme was produced in the conditioned medium when full-length rat matriptase was expressed in monkey kidney COS-1 cells. The present study aimed to address when and where the matriptase activation occurs. COS-1 cells expressing matriptase were labelled with a membrane-impermeable biotin derivative and then solubilized with Triton. Both activated and non-activated matriptase molecules were detected in the avidin precipitants of Triton extracts, whereas only the non-activated molecules were detected in the flow-through fraction of avidin-precipitation procedure. Single-chain matriptase has been thought to have an inherent activity. Indeed, a secreted single-chain variant of recombinant matriptase bearing mutation at the activation-cleavage site was found to exhibit the activity in hydrolyzing a synthetic peptide substrate at pH 7.5. However, the variant had little activity at pH 5.5, as found in the lumen of post-Golgi secretory vesicles. Altogether, it is concluded that the activation of matriptase may occur when the enzyme reaches the cell surface.

Requirement of the activity of hepatocyte growth factor activator inhibitor type 1 for the extracellular appearance of a transmembrane serine protease matriptase in monkey kidney COS-1 cells

“…Figure 2b shows that the medium sample from cells transfected with pcDNA-WT-matriptase and pSec-HAI66K produced a band at the 28-kDa position (right lane, indicated by closed arrowhead), whereas those from cells transfected with parental vectors and pcDNA-WT-matriptase alone did not (left and middle lanes, respectively). These results confirm our previous findings (Tsuzuki et al 2005;Miyake et al 2009) that co-expression of HAI-1 is important for the detection of WT-matriptase from conditioned media.…”

“…For convenient detection, S-tag was fused to the N-terminus of the protein (Tsuzuki et al 2005). This variant was shed from cellsurface membrane and was detected mainly as a 58-kDa band from medium samples (Miyake et al 2009). We have recently shown that HAI66K inhibits secreted variants of r-matriptase in vitro (Kojima et al 2009b).…”

“…Importantly, WT-matriptase was detected abundantly from the conditioned medium but poorly from a cell membrane-enriched fraction in the co-expression (Tsuzuki et al 2005, Miyake et al 2009). These findings suggested that most of WT-matriptase molecules are shed immediately after reaching the cell surface when expressed in this cell line.…”

“…2a), (Tsuzuki et al 2005;Miyake et al 2009). The 28-kDa band has been shown to represent the catalytic domain part of activated WT-matriptase (Satomi et al 2001;Tsuzuki et al 2005;Kojima et al 2008Kojima et al , 2009a) (refer to Fig.…”

“…The activation of this protease (i.e., conversion to disulfide-linked two-chain form via cleavage after Arg614, Fig. 2a) is known to occur via a mechanism requiring its catalytic triad (Takeuchi et al 1999;Oberst et al 2003b;Désilets et al 2008;Miyake et al 2009). The activated two-chain protease cleaves to activate a number of substrates, including pro-HGF, possibly at the cell surface (Lee et al 2000;Satomi et al 2001;Yamasaki et al 2003;Kojima et al 2009a).…”