Mechanisms for the control of matriptase activity in the absence of sufficient HAI-1

Abstract

Matriptase proteolytic activity must be tightly controlled for normal placental development, epidermal function, and epithelial integrity. Although hepatocyte growth factor activator inhibitor-1 (HAI-1) represents the predominant endogenous inhibitor for matriptase and the protein molar ratio of HAI-1 to matriptase is determined to be >10 in epithelial cells and the majority of carcinoma cells, an inverse HAI-1-to-matriptase ratio is seen in some ovarian and hematopoietic cancer cells. In the current study, cells with insufficient HAI-1 are investigated for the mechanisms through which the activity of matriptase is regulated. When matriptase activation is robustly induced in these cells, activated matriptase rapidly forms two complexes of 100- and 140-kDa in addition to the canonical 120-kDa matriptase-HAI-1 complex already described. Both 100- and 140-kDa complexes contain two-chain, cleaved matriptase but are devoid of gelatinolytic activity. Further biochemical characterization shows that the 140-kDa complex is a matriptase homodimer and that the 100-kDa complexes appear to contain reversible, tight binding serine protease inhibitor(s). The formation of the 140-kDa matriptase dimer is strongly associated with matriptase activation, and its levels are inversely correlated with the ratio of HAI-1 to matriptase. Given these observations and the likelihood that autoactivation requires the interaction of two matriptase molecules, it seems plausible that this activated matriptase homodimer may represent a matriptase autoactivation intermediate and that its accumulation may serve as a mechanism to control matriptase activity when protease inhibitor levels are limiting. These data suggest that matriptase activity can be rapidly inhibited by HAI-1 and other HAI-1-like protease inhibitors and “locked” in an inactive autoactivation intermediate, all of which places matriptase under very tight control.

The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

“…HAI-1 is expressed in a high molar excess to matriptase in most epithelial cells (46). A potential caveat of the above experiment, therefore, was that residual HAI-1 could be expressed at levels that eluded detection by Western blot or by immunofluorescence, but were sufficient to allow for appropriate transport to the epithelial cell surface.…”

Matriptase and prostasin are expressed in human skin in an inverse trend over the course of differentiation and are targeted to different regions of the plasma membrane

“…The prostasin mAb YL11 was covalently coupled to Sepharose 4B at 5 mg/ml gel as described previously (Xu et al, 2012). For immunodepletion, the cell lysates (200 µl) were incubated with 15 µl of the mAb conjugated Sepharose in micro-centrifuge tubes that were rotated end over end in a cold room for 2 h. The supernatants were collected by centrifugation as the immunodepleted fraction.…”

Natural Endogenous Human Matriptase and Prostasin Undergo Zymogen Activation via Independent Mechanisms in an Uncoupled Manner

“…M24 anti-human matriptase monoclonal antibody (mAb) was used for immunoblot analyses to detect total matriptase, the use and validation of which has been previously reported [20,25,26]. The mouse mAb M19 was used for immunoblot analyses to detect HAI-1, as we have previously described [20,25,26].…”

“…The mouse mAb M19 was used for immunoblot analyses to detect HAI-1, as we have previously described [20,25,26]. The mouse mAbs YL10 and YL11 were used to detect human prostasin as previously described [10].…”

“…The mouse mAbs YL10 and YL11 were used to detect human prostasin as previously described [10]. The detection sensitivity is estimated to be around 0.1–0.2 ng for matriptase mAb [27], 0.5–1 ng for HAI-1 mAb [25], and 0.1–0.2 ng for prostasin mAbs.…”