Dissertation Defense: Nanxi Huang
Candidate: Nanxi Huang
Major: Tumor Biology
Advisor: Michael Johnson, Ph.D.
The Role of HAI-2 in the Intestine Depends on Its Correct Subcellular Targeting
The hepatocyte growth factor activator inhibitor 1 (HAI-1) and HAI-2 are two closely related type I transmembrane Kunitz type serine protease inhibitors, that consist of two homologous extracellular Kunitz-type protease inhibitor domains. They are widely co-expressed in most epithelial cells and inhibit a broad range of trypsin-like serine proteases with similar potency and specificity, like hepatocyte growth factor activator (HGFA), matriptase and prostasin in solution. However, they have distinct functions that cannot be replaced by the other. The different functions of HAI-1 and HAI-2 are due to their accessibility to different target proteases caused by their differential subcellular localization. HAI-1 proteins are mainly on the cell surface, while HAI-2 proteins stay inside cell in polarized vesicles. And studying the intracellular transportation of HAI-1 and HAI-2 can help us to understand and distinguish the differences between these two homologous proteins during physiological and pathological processes. This work proved that the intracellular domains of HAI-1 and HAI-2 are responsible for their subcellular localization. Particularly, the putative ER retention signals and the incomplete plasma membrane exportation motif contribute to the dominantly intracellular localization of HAI-2. And the complex N-glycan on the Asn-57 site is required for the correct HAI-2 protein folding and protease inhibitory activity. A lethal inherited disease of infancy called Syndromic Congenital Sodium Diarrhea (SCSD) is only caused by the loss-of-function mutation of HAI-2, but not HAI-1. Among all the mutations found in SCSD patients, the single amino acid substitutions that occur in the functional less important Kunitz domain 2 (KD2), are the most common mutations. This work discovered an unexpected result caused by these mutations. The SCSD associated mutations in KD2 can inactivate HAI-2’s prostasin inhibitory function via abnormal protein folding and N glycosylation.