Abstract
Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.
Highlights
Membrane-bound or membrane-anchored serine proteases have lately emerged as a subfamily of 20 serine proteases that all share a conserved catalytic domain and a transmembrane domain [1]
We have limited our selection to members of the membrane-bound serine proteases family expressed in different gastro-intestinal (GI) tissues, because HAI-1, a highly conserved homolog of HAI-2 was reported as the physiological inhibitor of the membrane-bound serine protease matriptase
The short isoform of Spint2 lacking the first Kunitz domain appears more abundant than the long isoform in mouse [45]
Summary
Membrane-bound or membrane-anchored serine proteases have lately emerged as a subfamily of 20 serine proteases that all share a conserved catalytic domain and a transmembrane domain [1] They display diverse physiological and pathophysiological roles such as roles in skin and intestinal barrier integrity [2,3,4,5], processing of atrial natriuretic peptide [6], iron homeostasis [7,8,9], trophoblastic development [10], hearing [11,12] and ion homeostasis [13,14]. In cell-free in vitro systems, HAI-2 is a potent inhibitor of the membrane-bound serine proteases hepsin, prostasin, matriptase and tmprss13 [21,22,23,24]
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