Hepatocyte growth factor activator inhibitor-2 prevents shedding of matriptase

Abstract

Hepatocyte growth factor activator inhibitor-2 (HAI-2) is an inhibitor of many proteases in vitro, including the membrane-bound serine protease, matriptase. Studies of knock-out mice have shown that HAI-2 is essential for placental development only in mice expressing matriptase, suggesting that HAI-2 is important for regulation of matriptase. Previous studies have shown that recombinant expression of matriptase was unsuccessful unless co-expressed with another HAI, HAI-1. In the present study we show that when human matriptase is recombinantly expressed alone in the canine cell line MDCK, then human matriptase mRNA can be detected and the human matriptase ectodomain is shed to the media, suggesting that matriptase expressed alone is rapidly transported through the secretory pathway and shed. Whereas matriptase expressed together with HAI-1 or HAI-2 accumulates on the plasma membrane where it is activated, as judged by cleavage at Arg614 and increased peptidolytic activity of the cell extracts. Mutagenesis of Kunitz domain 1 but not Kunitz domain 2 abolished this function of HAI-2. HAI-2 seems to carry out its

The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

“…Currently available antibodies do not allow for specific detection of endogenous HAI-2 (data not shown). However, previous studies have shown that an epitopetagged HAI-2 variant resides predominantly in an intracellular compartment when overexpressed in epithelial cells (33).…”

“…These cells were chosen because of their intestinal epithelial origin, because they express matriptase, prostasin, and HAI-2, and because they form matriptase and prostasin-dependent tight junctions with well-defined apical and basolateral membrane domains when cultured to confluency (33,42,49). Furthermore the Caco-2 cells allow easy detection of the various forms of matriptase in both lysates and conditioned media (Fig.…”

“…Thus, HAI-1 is reported to interact with the matriptase zymogen already within the biosynthetic pathway to prevent its premature activation, to facilitate its transport to the cell surface, and even to induce its activation once located on the plasma membrane (6, 29 -32). Likewise, HAI-2 was recently suggested to be critical for the retention of active matriptase on the plasma membrane (33). A potentially confounding factor in these studies, however, is the frequent reliance on cell-based overexpression systems to dissect the mechanistic interactions of matriptase with HAI-1 and HAI-2.…”

“…A potentially confounding factor in these studies, however, is the frequent reliance on cell-based overexpression systems to dissect the mechanistic interactions of matriptase with HAI-1 and HAI-2. Moreover, discrepancies have been reported as to the necessity of HAI-1 for proper expression of matriptase, even within the same cell-based model system (33)(34)(35).…”

Detection of Active Matriptase Using a Biotinylated Chloromethyl Ketone Peptide

“…The activation of matriptase zymogen (also termed zymogen conversion) is extraordinarily complex and still incompletely understood at the mechanistic level. Matriptase activation involves two sequential endoproteolytic cleavages, and may also require transient interactions with its cognate inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1 [7], [8] [reviewed in [9], [10]] or HAI-2 [11]. Full-length matriptase is first hydrolysed at the Gly149-Ser150 peptide bond, which is located in a conserved GSVIA motif within the SEA domain, whereby the SEA domain-cleaved zymogen form is generated.…”