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

Date Published: January 26, 2017

Publisher: Public Library of Science

Author(s): Frank Shiao, Li-Ching O. Liu, Nanxi Huang, Ying-Jung J. Lai, Robert J. Barndt, Chun-Che Tseng, Jehng-Kang Wang, Bailing Jia, Michael D. Johnson, Chen-Yong Lin, Qing-Xiang Amy Sang.


Mutations of hepatocyte growth factor activator inhibitor (HAI)-2 in humans cause sodium loss in the gastrointestinal (GI) tract in patients with syndromic congenital sodium diarrhea (SCSD). Aberrant regulation of HAI-2 target protease(s) was proposed as the cause of the disease. Here functional linkage of HAI-2 with two membrane-associated serine proteases, matriptase and prostasin was analyzed in Caco-2 cells and the human GI tract. Immunodepletion-immunoblot analysis showed that significant proportion of HAI-2 is in complex with activated prostasin but not matriptase. Unexpectedly, prostasin is expressed predominantly in activated forms and was also detected in complex with HAI-1, a Kunitz inhibitor highly related to HAI-2. Immunohistochemistry showed a similar tissue distribution of prostasin and HAI-2 immunoreactivity with the most intense labeling near the brush borders of villus epithelial cells. In contrast, matriptase was detected primarily at the lateral plasma membrane, where HAI-1 was also detected. The tissue distribution profiles of immunoreactivity against these proteins, when paired with the species detected suggests that prostasin is under tight control by both HAI-1 and HAI-2 and matriptase by HAI-1 in human enterocytes. Furthermore, HAI-1 is a general inhibitor of prostasin in a variety of epithelial cells. In contrast, HAI-2 was not found to be a significant inhibitor for prostasin in mammary epithelial cells or keratinocytes. The high levels of constitutive prostasin zymogen activation and the selective prostasin inhibition by HAI-2 in enterocytes suggest that dysregulated prostasin proteolysis may be particularly important in the GI tract when HAI-2 function is lost and/or dysregulated.

Partial Text

Autosomal-recessive congenital sodium diarrhea (CSD) is a rare, inherited diarrhea of infancy, characterized by watery diarrhea with extraordinarily high fecal sodium loss [1,2]. Studies of the clinical phenotype and the molecular basis of the condition using the positional candidate approach has identified a subtype of CSD, known as the syndromic form of CSD (SCSD), which differs from classic CSD by its association with choanal or anal atresia, hypertelorism, and corneal erosions not found with classic CSD [3,4]. Five distinct mutations of SPINT2 have been identified in SCSD but none in classic CSD [3]. SPINT2 encodes an integral membrane, Kunitz-type serine protease inhibitor, known as hepatocyte growth factor activator inhibitor (HAI)-2 or placenta bikunin [5,6]. The mutations associated with SCSD appear to result in either loss of protein synthesis or reduced anti-protease activity of HAI-2, both of which theoretically result in the increased proteolytic activity of HAI-2 target proteases in the affected tissues. Alternatively, the loss of HAI-2 function may result in suppression of the synthesis, intracellular trafficking, and even zymogen activation of target proteases, resulting in a paradoxical reduction in the proteolytic activity of HAI-2 target proteases. This scenario has been observed with the type 2 transmembrane serine protease matriptase, whose expression, intracellular trafficking, and zymogen activation depends on the inhibitor HAI-1 in some systems [7,8]. Whether one of these or some other scenario is involved, the underlying mechanism by which HAI-2 mutation causes sodium loss in SCSD remains elusive because it remains unclear which serine protease(s) are impacted by HAI-2 mutation in this context.

The aberrant regulation of HAI-2 target protease(s) may be the underlying mechanism responsible for the sodium loss that seems to be at the heart of the primary symptom of patients with SCSD [3]. The detection of activated prostasin in complex with HAI-2 in lysates prepared from human intestine tissue and from Caco-2 cells identifies prostasin as the predominant physiologically relevant target protease of HAI-2 in human enterocytes. In addition, vast majority of prostasin present in activated form in complex with HAI-1 or HAI-2 in intestinal tissue indicates prostasin proteolysis is constitutively active and under tight control. The loss of HAI-2 function in patients with SCSD would, therefore, be expected to result in aberrant regulation of prostasin, which may subsequently contribute to the sodium loss seen in these patients, although the underlying mechanism remains to be further investigated. Furthermore, the high zymogen activation state of prostasin and the selective importance of the inhibition of prostasin by HAI-2 observed in enterocytes may explain why this cell type is more susceptible than other epithelial tissues to loss of function mutations of HAI-2. Given that HAI-2 contains two Kunitz domains. Kunitz domain 1 is responsible for the binding and inhibition of active prostasin. Kunitz domain 2 could also have its own target protease. Indeed, a 110-kDa HAI-2 complex was also detected in Caco-2 cells (Figs 1 and 2), which is greater than the 65-kDa prostasin-HAI-2 complex and may contain an as yet unidentified protease. Whether and how this and other putative HAI-2 target proteases are dysregulated by HAI-2 mutations and subsequently contribute to pathogenesis of SCSD can be determined only after they are identified. Although we have established the relationship between HAI-2 and prostasin, the possibility remains that the impact of HAI-2 mutation on SCSD is mediated by altered activity against a different client protease.