HAI‐2 stabilizes, inhibits and regulates SEA‐cleavage‐dependent secretory transport of matriptase

Abstract

It has recently been shown that hepatocyte growth factor activator inhibitor-2 (HAI-2) is able to suppress carcinogenesis induced by overexpression of matriptase, as well as cause regression of individual established tumors in a mouse model system. However, the role of HAI-2 is poorly understood. In this study, we describe 3 mutations in the binding loop of the HAI-2 Kunitz domain 1 (K42N, C47F and R48L) that cause a delay in the SEA domain cleavage of matriptase, leading to accumulation of non-SEA domain cleaved matriptase in the endoplasmic reticulum (ER). We suggest that, like other known SEA domains, the matriptase SEA domain auto-cleaves and reflects that correct oligomerization, maturation, and/or folding has been obtained. Our results suggest that the HAI-2 Kunitz domain 1 mutants influence the flux of matriptase to the plasma membrane by affecting the oligomerization, maturation and/or folding of matriptase, and as a result the SEA domain cleavage of matriptase. Two of the HAI-2 Kunitz domain 1 mutants investigated (C47F, R48L and C47F/R48L) also displayed a reduced ability to proteolytically silence matriptase. Hence, HAI-2 separately stabilizes matriptase, regulates the secretory transport, possibly via maturation/oligomerization and inhibits the proteolytic activity of matriptase in the ER, and possible throughout the secretory pathway.

Hepatocyte growth factor activator inhibitors (HAI‐1 and HAI‐2): Emerging key players in epithelial integrity and cancer

“…c). Interestingly, the type of HAI required for the proper matriptase trafficking is cell‐type dependent . For example, whereas co‐expression of matriptase with either HAI‐1/SPINT1 or HAI‐2/SPINT2 is sufficient for the cell surface localization of matriptase in HEK293 cells (Fig.…”

Membrane-Anchored Serine Proteases and Protease-Activated Receptor-2–Mediated Signaling: Co-Conspirators in Cancer Progression

“…89). The ratio of matriptase to its inhibitors, or the protease-inhibitor balance, is important: loss of or decreased endogenous HAI-1 or HAI-2 enables increased matriptase activity and this has been shown to promote in vitro tumorigenesis in several studies (90)(91)(92)(93)(94)(95)(96). In human SCCs, increased matriptase expression is correlated with diminished expression of matriptase-HAI-1 complexes and with reduced PAR-2 expression (97), possibly due to PAR-2 overactivation induced by deregulated matriptase activity.…”

Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

“…B, schematic drawing of the zymogen-locked form of matriptase characterized in this paper, consisting of only the serine protease domain with position R614A mutated to avoid activation (zSPD). C, a Coomassie-stained SDS-PAGE (10%) shows that zSPD (consisting of only the serine protease domain, as shown in B) is pure and the expected size (25)(26)(27)(28)(29)(30). D, the sequence of the crystallized form of zSPD (zSPD-S805A) with an additional mutation in the active site, shown with matriptase numbering (sides) and chymotrypsin numbering (top).…”

“…Matriptase needs co-expression with the inhibitors HAI-1 or HAI-2 to obtain significant levels of plasma membrane-bound matriptase. Either WT HAI-2 or a HAI-2 variant with compromised inhibitory activity (HAI-2 C47F) due to a mutation in the predicted P2 position of the inhibitory loop of Kunitz domain 1 was used (29). Transiently transfected cells were lysed, and the peptidolytic activity of the cell lysates was assayed with or without the antibodies aZ-mAb-6 and aZ-mAb-7 or a control antibody (anti-uPA) Inhibition of zymogen matriptase activity ( Fig.…”

“…HEK293 cells were transiently transfected with empty expression vector (mock) or expression vectors encoding matriptase together with WT HAI-2 or mutant HAI-2 C47F, respectively, using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol. The constructs encoding full-length human WT matriptase and HAI-2 were inserted into pcDNA3.1 plasmid vectors (Thermo Fisher Scientific) as described previously by Nonboe et al (29). HAI-2 was co-expressed to ensure stable matriptase expression, and the HAI-2 C47F mutant was included to allow measurable matriptase activity.…”