Distinct Developmental Functions of Prostasin (CAP1/PRSS8) Zymogen and Activated Prostasin

Abstract

The membrane-anchored serine prostasin (CAP1/PRSS8) is essential for barrier acquisition of the interfollicular epidermis and for normal hair follicle development. Consequently, prostasin null mice die shortly after birth. Prostasin is found in two forms in the epidermis: a one-chain zymogen and a two-chain proteolytically active form, generated by matriptase-dependent activation site cleavage. Here we used gene editing to generate mice expressing only activation site cleavage-resistant (zymogen-locked) endogenous prostasin. Interestingly, these mutant mice displayed normal interfollicular epidermal development and postnatal survival, but had defects in whisker and pelage hair formation. These findings identify two distinct in vivo functions of epidermal prostasin: a function in the interfollicular epidermis, not requiring activation site cleavage, that can be mediated by the zymogen-locked version of prostasin and a proteolysis-dependent function of activated prostasin in hair follicles, dependent on zymogen conversion by matriptase.Prostasin (also known as channel-activating protease-1, CAP1, and PRSS8) is a glycosylphosphatidylinositol-anchored trypsin-like serine protease that is widely expressed in epithelial tissues, including both the interfollicular and the follicular compartments of the epidermis. Loss-of-function genetic studies in mice have uncovered critical functions of prostasin in both the formation of epidermal barrier formation and the formation of whiskers and pelage hair (1, 2). Prostasin is synthesized as an inactive proform (zymogen) that is converted to a catalytically active protease by a single endoproteolytic cleavage in the conserved activation cleavage site (3-6). Prostasin zymogen conversion in the epidermis requires the membraneactivated serine protease matriptase (7-9). This observation, combined with the observation that both matriptase-deficient and prostasin-deficient mice display identical epidermal phenotypes, led to the formulation of the hypothesis that prostasin exerts its functions in this tissue as part of a matriptase-prostasin cell surface zymogen activation cascade (8). Several observations made during the last decade suggest, however, that prostasin may also execute biological functions independent of its own proteolytic activity. For example, in a reconstituted Xenopus oocyte system, prostasin can activate the epithelial sodium channel (ENaC) by inducing proteolytic cleavage of its ␥ subunit to release an inhibitory domain. However, this activation, which can be inhibited by the broad-spectrum serine protease inhibitor, aprotinin, can also be efficiently executed by mutant prostasin variants that lack the catalytic histidine-aspartate-serine triad (10 -12). Likewise, catalytically inactive prostasin mutants can stimulate the activation of protease-activated receptor-2 in a reconstituted mammalian cell-based system (13). Strong support for a non-proteolytic function of prostasin in vivo has been gained from the observation that mis-expressed catalytically inactive pros...

Delineation of proteolytic and non-proteolytic functions of membrane-anchored serine protease Prss8/prostasin

“…6). This is consistent with our recent report that catalytically inactive prostasin is found in complex with a zymogen-locked matriptase in cultured epithelial cells (Friis et al, 2016). …”

“…It is clear that, at least in one tissue, prostasin mediates the proteolytic activation of matriptase zymogen, as evidenced by a loss of the two-chain form of the matriptase in prostasin null placentas. However, the presence of active matriptase in the epidermis of prostasin-deficient mice suggests that additional proteases are likely to contribute to matriptase zymogen activation in other tissues and/or biological processes (Friis et al, 2016;Szabo et al, 2012). As a result, matriptase zymogen activation and the full activity of the matriptase/prostasin pathway may, depending on circumstances, exhibit complete, partial or no dependence on prostasin proteolytic function.…”

“…Third, prostasin and matriptase form protein complexes when co-expressed in epithelial cells (Friis et al, 2013). Finally, defects in epidermal development and barrier formation in prostasin null mice that express near-normal levels of the activated two-chain form of matriptase in the epidermis show that, even in its active form, matriptase is unable to perform its essential skin functions in the absence of prostasin (Friis et al, 2016;Szabo et al, 2012). Surprisingly, our recent work indicates that at least some of the reported physiological functions of prostasin during development are preserved in mice that express a catalytically inactive variant of the protease .…”

Selective Inhibition of Prostasin in Human Enterocytes by the Integral Membrane Kunitz-Type Serine Protease Inhibitor HAI-2

“…The limited resolution of SDS-PAGE and the presence of at least two prostasin species, which are likely the result of posttranslational modifications, make it very difficult to distinguish active prostasin from zymogen prostasin by immunoblot analysis [43]. Nevertheless, the specification of zymogen versus active prostasin by size difference has been also used in several previous studies [46][47][48]. In the study by Szabo et al, [46], the samples for immunoblot analysis were treated with reducing agent in order to dissociate active prostasin into the heavy chain and light chain.…”

“…doi:10.1371/journal.pone.0170944.g007 functions of prostasin zymogen identified via genetic manipulation in various model systems [39,[42][43][44][45].…”