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

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) (1–6). 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) (1–6). 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 (7–9). The potential substrates, abundant expression in the epithelial cells and keratinocytes and characteristic phenotypes in matriptase gene-disrupted mice, suggest that the enzyme plays important roles in the establishment and maintenance of epithelial and epidermal barriers (1, 3, 10–12). Fig. 1 Schematic illustration of the structures of full-length rat matriptase and HAI-1, and their expression constructs. (A) Schematic illustration of the domain structures of full-length rat matriptase (Matriptase zymogen). The amino acid numbering starts ... The activation cleavage of matriptase zymogen is believed to occur through a mechanism requiring its own catalytic triad (His656, Asp711 and Ser805) (1, 2, 13–15). For instance, recombinant (r-) forms of full-length matriptase, in which any residues of the catalytic triad are changed to an alanine residue, are unable to undergo activation when expressed exogenously in BT549 breast cancer cells (13). To date, the zymogen molecules have been postulated to undergo transactivation by which a zymogen interacts with another zymogen, producing the activation cleavage of each zymogen (1, 13). If so, matriptase zymogen should exert protease activity. We have found that (i) a pseudozymogen form of r-matriptase comprising the entire extracellular domain forms a catalytic triad analogous to His–Asp–Ser of trypsin-like serine proteases and (ii) the r-pseudozymogen effectively hydrolyses a peptide substrate, acetyl-l-Lys–l-Thr–l- Lys–l-Gln–l-Leu–l-Arg–4-methyl-coumaryl-7-amide (Ac-KTKQLR-MCA), whose sequence shares some sequence similarities with the activation cleavage site of this protease (Fig. 1) (16). In addition, the r-pseudozymogen exhibited optimal Ac-KTKQLR-MCA-hydrolyzing activity under mildly acidic and low ionic strength conditions (e.g. 5 mM NaCl and pH 6.0), under which matriptase zymogen endogenously expressed in a human mammary epithelial line 184 A1N4 undergoes activation with optimal efficiency (17–19). Our findings reinforce the concepts that the zymogen molecules can activate themselves by self-catalyzed proteolysis and thus that this protease serves as an initiator molecule of a protease cascade that is involved in the maintenance of epithelial and epidermal integrity (1, 3). However, it should be noted that the zymogen activation seems to be mediated predominantly by two-chain molecules. Indeed, the Ac-KTKQLR-MCA-hydrolyzing activity was higher for the corresponding two-chain r-matriptase than for the r-pseudozymogen (16). Matriptase has an extracellular non-catalytic stem domain comprising a sea urchin sperm protein–enteropeptidase–agrin (SEA) domain, followed by two tandem repeats of complement proteases C1r/C1s–urchin embryonic growth factor–bone morphogenetic protein (CUB) domain and four tandem repeats of a low-density lipoprotein receptor class A (LDLRA) domain (Fig. 1) (1–6). Lin and coworkers have proposed the intimate involvement of the matriptase stem domain in its zymogen activation (13, 19). For instance, a site-directed mutant of full-length matriptase (Gly149Asn), which was designed to escape post-translational cleavage between Gly149 and Ser150 within the SEA domain (Fig. 1), was unable to undergo activation (13). A possible explanation for this is that, on cleavage within the SEA domain, the matriptase zymogen undergoes a conformational change or reorientation on the membrane surface that makes the protease susceptible to activation cleavage. The CUB and LDLRA repeats were also shown to affect the zymogen activation (13, 19). However, the definitive roles of these repeats in the zymogen activation remain unclear. We have shown that (i) the matriptase second CUB domain (CUB domain II) serves as an additional site for interaction with a physiological inhibitor of this protease, HGF activator inhibitor type-1 (HAI-1), which contains two protease-inhibitory Kunitz domains, and (ii) the interaction between CUB domain II and HAI-1 facilitates the primary inhibitory interaction between the enzyme and the inhibitor (i.e. interaction between the SPCD of two-chain matriptase and the first Kunitz domain) (Fig. 1) (20). This finding raises the possibility that CUB domain II may suppress zymogen activation by accelerating the rate of inhibition of the two-chain matriptase by HAI-1. The aim of this study was to gain insights into the role(s) of CUB and LDLRA repeats in the activation of matriptase zymogen. To date, the structural requirements of matriptase zymogen for its activation cleavage have been studied on transient expression of the membrane-bound forms of r-matriptase in cultured cells (13–15, 21, 22). In the context of membrane-bound forms, internal deletions and point mutations showed differential effects on the activation cleavage (13). For example, full-length r-matriptase bearing a mutation in its LDLRA repeat (tyrosine replacement of aspartic acid residues comprising the calcium cage in each domain) rarely underwent activation, whereas a truncated variant lacking the repeat did it to a similar extent as in full-length, wild-type (WT) r-matriptase (13). Expression studies using soluble, truncated forms of r-matriptase could be expected to solve contradictory conclusions regarding the roles of internal domains (CUB and LDLRA repeats). In addition, the activation behaviours of soluble variants in cellulo would provide a coherent interpretation of the in vitro results obtained using soluble, truncated, pseudozymogen forms of r-matriptase (pro-CLS-, pro-LS- and pro-S-matEK-A variants, Fig. 1) (20). For these reasons, we prepared plasmids for the expression of secreted variants of r-matriptase and investigated whether the r-matriptase variants undergo activation cleavage when expressed in CV-1 in origin and carrying the SV40 genetic material (COS-1) monkey kidney cells. Our in cellulo results provided suggestive evidence that the CUB repeat had an inhibitory effect, whereas the LDLRA repeat had a promoting effect, on zymogen activation. In vitro experiments using the pseudozymogen forms of r-matriptase showed that the LDLRA repeat increased the protease activity of matriptase zymogen. To our knowledge, this is the first report showing how CUB and LDLRA repeats of matriptase participate in its zymogen activation.