Roles of CUB and LDL receptor class A domain repeats of a transmembrane serine protease matriptase in its zymogen activation.

Matriptase is a type II transmembrane serine protease containing two complement proteases C1r/C1s-urchin embryonic growth factor-bone 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.

Identification of the matriptase second CUB domain as the secondary site for interaction with hepatocyte growth factor activator inhibitor type-1.

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). Analyses using a secreted variant of recombinant matriptase comprising the entire extracellular domain (MAT), its truncated variants, and a recombinant HAI-1 variant with an entire extracellular domain (HAI-1-58K) revealed that the second CUB domain (CUB domain II, Cys(340)-Pro(452)) likely contains the site(s) of interest. We also found that MAT undergoes cleavage between Lys(379) and Val(380) within CUB domain II and that the C-terminal residues after Val(380) are responsible for facilitating the interaction with HAI-1-58K. A synthetic peptide corresponding to Val(380)-Asp(390) markedly increased the matriptase-inhibiting activity of HAI-1-58K, whereas the peptides corresponding to Val(380)-Val(389) and Phe(382)-Asp(390) 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.

The optimal activity of a pseudozymogen form of recombinant matriptase under the mildly acidic pH and low ionic strength conditions.

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.

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.

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.

Activation of a membrane-bound serine protease matriptase on the cell surface.

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.