Activation of Epithelial Sodium Channels by Mouse Channel Activating Proteases (mCAP) Expressed in Xenopus Oocytes Requires Catalytic Activity of mCAP3 and mCAP2 but not mCAP1

Abstract

Mouse channel activating proteases 1, 2, and 3 (mCAP1, mCAP2, and mCAP3) were described recently as regulators of the epithelial sodium channel (ENaC). The mCAP are membrane-bound serine proteases that are synthesized as inactive proenzymes. To mature into active proteases, they undergo intramolecular cleavage by auto-and/or heterocatalytic processing. Specific antibodies against each mCAP were developed to distinguish between proenzyme and active protease by Western blot analysis. Various point mutations were introduced in the catalytic or protein-protein interacting domains of mCAP and wild-type and mutant enzymes were expressed in the Xenopus oocyte expression system to test for ability to activate ENaC. In mCAP3, an intact catalytic triad was necessary for activation of ENaC but not for intramolecular cleavage of the protease. This suggests a heterocatalytic mechanism. Mutating the catalytic triad of mCAP2 not only abolished ENaC activation completely but also impeded cleavage of the protease. Processing of mCAP2 therefore seems to be autocatalytic. Furthermore, mutations in conserved residues of mCAP2 located in two protein-protein interacting domains significantly modulated ENaC activation. Surprisingly, mCAP1 catalytically inactive mutants were still able to fully activate ENaC, and no evidence of mCAP1 intramolecular cleavage was seen. The presence of an intact glycosylphosphatidylinositol anchor, however, was required. It is concluded that auto-and heterocatalytic requirements are specific for each CAP and that endogenous partners are a necessity for activation of ENaC by mCAP in the Xenopus oocyte expression system.

Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis

“…2A). After digestion with PNGase, both prostasin molecular species migrated faster, consistent with the previous reports of prostasin glycosylation (2,38,39,45). However, differential glycosylation does not account for the difference between the 37-and 40-kDa prostasin bands, because both molecular species were present following N-glycan digestion.…”

“…In support of the theory that prostasin activates ENaC through proteolytic cleavage, prostasin fails to activate ENaC when the prostasin cleavage site on ␥ENaC is mutated or the protease activity is inhibited by aprotinin (4, 13). Interestingly, Andreasen et al (2) reported that when the catalytic triad of prostasin is mutated, the protease maintains its ability to activate the channel, suggesting that perhaps prostasin's proteolytic activity is not required for channel activation. Furthermore, the Na ϩ current of oocytes expressing ENaC is increased by prostasin coexpression despite the biochemical absence of proteolytically processed prostasin and under neutral pH conditions where the catalytic activity of the protease is dramatically decreased (2,48).…”

“…Prostasin is synthesized as a zymogen and as such requires proteolytic processing to be active (2). Therefore, we questioned whether the 37-kDa band in HAEC represents a proteolytically processed form of prostasin.…”

“…In CF epithelia, these regulatory mechanisms are altered, leading to increased expression of processed prostasin on the luminal airway surface. Prostasin is synthesized as an inactive zymogen and has not been shown to be capable of autocatalysis (2,39); activation requires its cleavage by an upstream protease, which is believed to be matriptase (10,25,26,37). Protease nexin-1 (PN-1), an associated inhibitor of prostasin (9), inhibited the amiloride-sensitive Na ϩ current by formation of an inactive prostasin complex and by preventing the processing of prostasin zymogen to active enzyme.…”

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity

“…However, in a cell-based assay, we recently found that catalytically inactive prostasin, was capable of stimulating both matriptase auto-activation and cleavage of a physiological matriptase substrate (11). Furthermore, in a reconstituted Xenopus oocyte system, catalytically inactive prostasin has been reported to stimulate the activation of the epithelial sodium channel by inducing proteolytic cleavage of the epithelial sodium channel ␥-chain (20,21). These findings, in conjunction with the in vivo observations presented herein, lend support to the hypothesis that an allosteric interaction of prostasin with another membrane-bound serine protease, matriptase, may be required for normal epidermal barrier formation.…”