The membrane-anchored serine protease prostasin (CAP1/PRSS8) supports epidermal development and postnatal homeostasis independent of its enzymatic activity.

The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8(-/-)) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8(Cat-/Cat-) mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8(-/-) and Prss8(Cat-/Cat-) mice born within the same litter. Furthermore, Prss8(Cat-/Cat-) mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane-anchored serine protease.

A matriptase-prostasin reciprocal zymogen activation complex with unique features: prostasin as a non-enzymatic co-factor for matriptase activation.

Matriptase and prostasin are part of a cell surface proteolytic pathway critical for epithelial development and homeostasis. Here we have used a reconstituted cell-based system and transgenic mice to investigate the mechanistic interrelationship between the two proteases. We show that matriptase and prostasin form a reciprocal zymogen activation complex with unique features. Prostasin serves as a critical co-factor for matriptase activation. Unexpectedly, however, prostasin-induced matriptase activation requires neither prostasin zymogen conversion nor prostasin catalytic activity. Prostasin zymogen conversion to active prostasin is dependent on matriptase but does not require matriptase zymogen conversion. Consistent with these findings, wild type prostasin, activation cleavage site-mutated prostasin, and catalytically inactive prostasin all were biologically active in vivo when overexpressed in the epidermis of transgenic mice, giving rise to a severe skin phenotype. Our finding of non-enzymatic stimulation of matriptase activation by prostasin and activation of prostasin by the matriptase zymogen provides a tentative mechanistic explanation for several hitherto unaccounted for genetic and biochemical observations regarding these two membrane-anchored serine proteases and their downstream targets.

Reduced prostasin (CAP1/PRSS8) activity eliminates HAI-1 and HAI-2 deficiency-associated developmental defects by preventing matriptase activation.

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1-deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2-deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.