Role of the polycystic kidney disease domain in matriptase chaperone activity and localization of hepatocyte growth factor activator inhibitor-1.

Hepatocyte growth factor activator inhibitor-1 (HAI-1, also known as SPINT1) is an inhibitor of matriptase, a type-2 transmembrane protease widely expressed in epithelial cells. HAI-1 also functions as a chaperone to maintain the processing and localization of matriptase required for epithelial integrity. However, mechanisms underpinning the chaperone function remain to be elucidated. Here, we show that the first Kunitz domain (KD1) and the adjacent polycystic kidney disease (PKD) domain-like internal domain of HAI-1 are essential for the chaperone function. In HEK293T cells, which do not express endogenous HAI-1 or matriptase, forced matriptase overexpression was unsuccessful unless sufficient HAI-1 was co-expressed. Among mutant HAI-1 constructs, HAI-1 with inactivation mutation in KD1 (HAI-1mKD1) or HAI-1 lacking the PKD domain (HAI-1dPKD) was unable to support matriptase expression, and neither mutant formed a complex with activated matriptase. Matriptase did not localize to the cell surface when co-expressed with HAI-1dPKD. Moreover, HAI-1dPKD accumulated in the cytoplasm of HEK293T and HaCaT cells rather than localizing to the cell surface, presumably due to misfolding as judged by altered antibody recognition. On the other hand, activationlocked and activity-incompetent matriptase were stable and readily overexpressed and localized to the cell surface without HAI-1. Therefore, the observed matriptase instability was caused by its own catalytic activity in the absence of inhibitory HAI-1. The matriptase chaperone function of HAI-1 is thus mediated primarily by the inhibition of undesired intracellular matriptase activity, and the PKD domain is essential for the proper folding and trafficking of inhibitory HAI-1 and its chaperone function.