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

Abstract

The membrane-associated serine proteases matriptase and prostasin are believed to function in close partnership. Their zymogen activation has been reported to be tightly coupled, either as a matriptase-initiated proteolytic cascade or through a mutually dependent mechanism involving the formation of a reciprocal zymogen activation complex. Here we show that this putative relationship may not apply in the context of human matriptase and prostasin. First, the tightly coupled proteolytic cascade between matriptase and prostasin might not occur when modest matriptase activation is induced by sphingosine 1-phospahte in human mammary epithelial cells. Second, prostasin is not required and/or involved in matriptase autoactivation because matriptase can undergo zymogen activation in cells that do not endogenously express prostasin. Third, matriptase is not required for and/or involved in prostasin activation, since activated prostasin can be detected in cells expressing no endogenous matriptase. Finally, matriptase and prostasin both undergo zymogen activation through an apparently un-coupled mechanism in cells endogenously expressing both proteases, such as in Caco-2 cells. In these human enterocytes, matriptase is detected primarily in the zymogen form and prostasin predominantly as the activated form, either in complexes with protease inhibitors or as the free active form. The negligible levels of prostasin zymogen with high levels of matriptase zymogen suggests that the reciprocal zymogen activation complex is likely not the mechanism for matriptase zymogen activation. Furthermore, high level prostasin activation still occurs in Caco-2 variants with reduced or absent matriptase expression, indicating that matriptase is not required and/or involved in prostasin zymogen activation. Collectively, these data suggest that any functional relationship between natural endogenous human matriptase and prostasin does not occur at the level of zymogen activation.

Hepatocyte growth factor activator inhibitor type-2 (HAI-2)/SPINT2 contributes to invasive growth of oral squamous cell carcinoma cells

“…Recent studies indicated that prostasin mediates matriptase activation both enzymatically and nonenzymatically [ 25 ], whereas this prostasin function in matriptase zymogen activation is limited to those cells with exogenous expression of matriptase and prostasin [ 26 ]. Thus, increased prostasin may contribute to matriptase activation.…”

Tissue distribution and subcellular localizations determine in vivo functional relationship among prostasin, matriptase, HAI-1, and HAI-2 in human skin

“…The proposed functional relationships include one involving a reciprocal activator-substrate interaction between matriptase and prostasin and another involving potent inhibition of matriptase and prostasin by HAI-1 and HAI-2. Our previous studies, however, suggested that though they may be correct in some contexts these proposed functional linkages can not be generalized and are somewhat tissue-selective or context-dependent [ 11 , 17 , 18 ]. In the current study, the context-dependent nature of any functional relationship is further supported by the distinct pattern of zymogen activation observed in human skin samples, in which prostasin is activated at high levels primarily in the granular layer whereas matriptase zymogen activation occurs at much lower levels predominantly in the cells of the basal layer.…”

“…In contrast, the prostasin mAb YL89 did not detect the prostasin zymogen protein band in 184 A1N4 cell lysates ( Fig 1A , lane 3), and only a very weak band was seen in this area with HaCaT cell lysates. Upon induction of matriptase and prostasin zymogen activation by transiently exposing the cells to a pH 6.0 buffer, a proportion of the prostasin zymogen was converted to activated prostasin, which forms a stable 100-kDa complex with HAI-1 [ 7 , 17 ]. The 100-kDa activated prostasin complex can be detected by the total prostasin mAb YL11 ( Fig 1 , lanes 2, indicated by *) and by the activated prostasin mAb YL89 ( Fig 1 , lanes 4, indicated by *).…”

“…The mAb DC16 for HAI-2 was generated using recombinant HAI-2 as the immunogen and characterized previously [ 11 , 27 , 28 ]. The prostasin mAbs YL11 and YL89 were generated using purified prostasin-HAI-1 complex as the immunogen and characterized previously [ 10 , 11 , 17 ].…”

“…Nevertheless, in HaCaT human keratinocytes, prostasin zymogen activation is induced simultaneously when matriptase zymogen activation is induced, and matriptase is required for the induction of prostasin zymogen activation [ 7 ]. Concomitant induction of prostasin and matriptase activation can also be observed in some other epithelial cells [ 17 ], indicating that matriptase and prostasin can function as a tightly coupled proteolytic cascade, at least, in vitro in cultured cells.…”