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.

Matriptase is a transmembrane serine protease that is strongly expressed in epithelial cells. The single-chain zymogen of matriptase is considered to have inherent activity, leading to its own activation (i.e. conversion to the disulphide-linked-two-chain form by cleavage after Thr-Lys-Gln-Ala-Arg614). Also, there is growing evidence that the activation of zymogen occurs at the cell surface and in relation to the acidification and lowering of ionic strength within cell-surface microenvironments. The present study aimed to provide evidence for the involvement of zymogen activity in its activation in physiologically relevant cellular contexts. For this purpose, the activity of a pseudozymogen form of recombinant matriptase (HL-matriptase zymogen) was examined using acetyl-l-Lys-l-Thr-l-Lys-l-Gln-l-Leu-l-Arg-4-methyl-coumaryl-7-amide as a substrate. HL-matriptase zymogen exhibited optimal activity toward the substrate pH approximately 6.0. The substrate hydrolysis at the pH value was hardly detected when NaCl was present at a concentration of 145 mM. In a buffer of pH 6.0 containing 5 mM NaCl, the activity of HL-matriptase zymogen was only approximately 30-times lower than that of the respective two-chain form. These findings suggest that the in vivo activation of matriptase zymogen occurs via a mechanism involving the zymogen activity.

Matriptase is a type II transmembrane serine protease comprising 855 amino acid residues. The extracellular region of matriptase comprises a noncatalytic stem domain (containing two tandem repeats of complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein (CUB) domain) and a catalytic serine protease domain. The stem domain of matriptase contains site(s) for facilitating the interaction of this protease with the endogenous inhibitor, hepatocyte growth factor activator inhibitor type-1 (HAI-1). The present study aimed to identify these site(s had no effect. HAI-1-58K precipitated with immobilized streptavidin resins to which a synthetic peptide Val 380 -Pro 392 with a biotinylated lysine residue at its C terminus was bound, suggesting direct interaction between CUB domain II and HAI-1. These results led to the identification of the matriptase CUB domain II, which facilitates the primary inhibitory interaction between this protease and HAI-1.Matriptase (also known as epithin, membrane-type serine protease 1, SNC19, suppression of tumorigenicity 14) is a transmembrane serine protease (1-7). This protease belongs to the type II transmembrane serine protease group, which is characterized by an N-terminal cytoplasmic domain, a signal anchor transmembrane domain, and an extracellular C-terminal serine protease catalytic domain (8,9). Matriptase is first synthesized as a zymogen comprising 855 amino acid residues in the human, mouse, and rat (7,10,11). Interaction between the zymogens appears to result in the generation of disulfidelinked two-chain molecules with an N-terminal Val 615 (activated matriptase molecules) (Fig. 1A) (10, 12-16). In addition, matriptase appears to function both as a plasma membraneassociated form and as a shed (soluble) form (1, 15, 17). The two-chain matriptase purified from human milk exhibits activity with trypsin-like specificity and is known to cleave and thus activate single-chain urokinase-type plasminogen activator (scuPA) 2 and pro-hepatocyte growth factor (pro-HGF) (18). Bacterially expressed variants of the recombinant (r-) catalytic domain of matriptase also cleaved to activate sc-uPA (19) and pro-HGF (20). Another r-matriptase catalytic domain cleaved to activate the precursor form of prostasin, a glycosylphosphatidylinositol-linked serine protease (3, 21). Together with the abundant expression in epithelial cells and in keratinocytes (7,22,23), these characteristics lead to the proposition that matriptase plays a key role in the maintenance of epithelial integrity and homeostasis. Studies in genetically manipulated mice indicate the importance of matriptase for epithelial and epidermal barrier function (24 -27).In general, members of the type II transmembrane serine protease group have an extracellular noncatalytic stem domain comprising various structural motifs (8, 9). For instance, the stem domain of matriptase comprises a sea-urchin sperm protein-enteropeptidase-agrin (SEA) domain, followed by two tandem repeats of complement proteases C1r/C1s-u...

Hepatocyte growth factor activator inhibitor type I (HAI-1) is a membrane-bound, serine protease inhibitor with two protease-inhibitory domains (Kunitz domain I and II). HAI-1 is known as a physiological inhibitor of a membrane-bound serine protease, matriptase. Paradoxically, however, HAI-1 has been found to be required for the extracellular appearance of the protease in an expression system using a monkey kidney COS-1 cell line. In the present study, we show using COS-1 cells that co-expression of recombinant variants of HAI-1 with the inhibition activity toward matriptase, including a variant consisting only of Kunitz domain I (the domain responsible for inhibition of matriptase), allowed for the appearance of this protease in the conditioned medium, whereas that of the variants without the activity did not. These findings suggest that the inhibition activity toward matriptase is critical for the extracellular appearance of protease in COS-1 cells.

Matriptase is a type II transmembrane serine protease containing two complement proteases C1r/C1surchin embryonic growth factorbone morphogenetic protein domains (CUB repeat) and four low-density lipoprotein receptor class A domains (LDLRA repeat). The single-chain zymogen of matriptase has been found to exhibit substantial protease activity, possibly causing its own activation (i.e. conversion to a disulfide-linked two-chain fully active form), although the activation seems to be mediated predominantly by two-chain molecules. Our aim was to assess the roles of CUB and LDLRA repeats in zymogen activation. Transient expression studies of soluble truncated constructs of recombinant matriptase in COS-1 cells showed that the CUB repeat had an inhibitory effect on zymogen activation, possibly because it facilitated the interaction of two-chain molecules with a matriptase inhibitor, hepatocyte growth factor activator inhibitor type-1. By contrast, the LDLRA repeat had a promoting effect on zymogen activation. The effect of the LDLRA repeat seems to reflect its ability to increase zymogen activity. The proteolytic activities were higher in pseudozymogen forms of recombinant matriptase containing the LDLRA repeat than in a pseudozymogen without the repeat. Our findings provide new insights into the roles of these non-catalytic domains in the generation of active matriptase.Keywords: CUB repeat/hepatocyte growth factor activator inhibitor type-1/LDLRA repeat/matriptase/ zymogen activation.Abbreviations: Ac-KTKQLR-MCA, acetyl-L-LysLThrL-LysL-GlnL-LeuL-Arg4-methyl-coumaryl-7-amide; COS, CV-1 in origin and carrying the SV40 genetic material; CUB, complement proteases C1r/C1surchin embryonic growth factorbone morphogenetic protein; HAI-1, hepatocyte growth factor activator inhibitor type-1; HGF, hepatocyte growth factor; HRP, horseradish peroxidase; LDLRA, low-density lipoprotein receptor class A MWCO, molecular weight cut-off; r-EK, recombinant enteropeptidase; SEA, sea urchin sperm proteinenteropeptidaseagrin; SPCD, serine protease catalytic domain; WT, wild-type.Matriptase is a member of the type II transmembrane serine protease group, which is characterized by the presence of an N-terminal cytoplasmic domain followed by a signal-anchor transmembrane domain and an extracellular domain, including a C-terminal serine protease catalytic domain (SPCD) (Fig. 1) (16). Matriptase is first synthesized as a zymogen comprising 855 amino acid residues in human, mouse and rat enzymes. The matriptase zymogen undergoes cleavage between Arg614 and Val615 (activation cleavage), and the disulfide-linked two-chain fully active enzyme is generated (Fig. 1) (16). Two-chain matriptase exhibits activity with trypsin-like specificity and can cleave and activate a number of proteins, including pro-hepatocyte growth factor (HGF) and the precursor of a membrane-bound serine protease, prostasin (79). The potential substrates, abundant expression in the epithelial cells and keratinocytes and characteristic phenotypes in matriptase gene-disrupted mic...