Matriptase Complexes and Prostasin Complexes with HAI-1 and HAI-2 in Human Milk: Significant Proteolysis in Lactation

Abstract

Significant proteolysis may occur during milk synthesis and secretion, as evidenced by the presence of protease-protease inhibitor complex containing the activated form of the type 2 transmembrane serine protease matriptase and the transmembrane Kunitz-type serine protease inhibitor HAI-1. In order to identify other proteolysis events that may occur during lactation, human milk was analyzed for species containing HAI-1 and HAI-2 which is closely related to HAI-1. In addition to the previously demonstrated matriptase-HAI-1 complex, HAI-1 was also detected in complex with prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease. HAI-2 was also detected in complexes, the majority of which appear to be part of higher-order complexes, which do not bind to ionic exchange columns or immunoaffinity columns, suggesting that HAI-2 and its target proteases may be incorporated into special protein structures during lactation. The small proportion HAI-2 species that could be purified contain matriptase or prostasin. Human mammary epithelial cells are the likely cellular sources for these HAI-1 and HAI-2 complexes with matriptase and prostasin given that these protease-inhibitor complexes with the exception of prostasin-HAI-2 complex were detected in milk-derived mammary epithelial cells. The presence of these protease-inhibitor complexes in human milk provides in vivo evidence that the proteolytic activity of matriptase and prostasin are significantly elevated at least during lactation, and possibly contribute to the process of lactation, and that they are under tight control by HAI-1 and HAI-2.

Delineation of proteolytic and non-proteolytic functions of the membrane-anchored serine protease prostasin

“…Indeed, we have previously documented that, in a cell culture-based system, prostasin efficiently co-immunoprecipitates with HAI-2, and the effect of HAI-2 on matriptase activation and shedding in this system is largely prostasin dependent . Furthermore, the isolation of prostasin complexes with HAI-2 from human milk was recently reported, showing a direct interaction between the two proteins in vivo (Lai et al, 2016). Interaction of HAI-2 with catalytically inactive prostasin, as shown in our current study, suggests that HAI-2 can inhibit both proteolytic and nonproteolytic functions of prostasin and can explain why HAI-2 remains essential for development and survival in the Prss8 Ki/Ki background.…”

Selective Inhibition of Prostasin in Human Enterocytes by the Integral Membrane Kunitz-Type Serine Protease Inhibitor HAI-2

“…The hypothesis that it is excessive proteolysis caused by the loss of protease inhibitor that is responsible for the defect in these patients is supported by the observation that while matriptase, HAI-1, and HAI-2 play dispensable roles in placenta development, the defect in placental development caused by targeted deletion of HAI-1 or HAI-2 can be rescued by simultaneous deletion of matriptase [13,15]. In solution-based experiments, both HAI-1 and HAI-2 are potent inhibitors of matriptase and prostasin [11,20]. At the cellular level, however, the roles of HAIs in the control of matriptase and prostasin are much more complicated and largely depend on whether the proteins are coincided [20].…”

“…Kunitz-type serine protease inhibitors suppress the enzymatic activity of their target proteases by the formation of very stable complex, as exemplified by the loss of the gelatinolytic activity of activated matriptase when in complex with HAI-1 [31]. Furthermore, these protease Kunitz inhibitor complexes are so stable that they survive SDS-PAGE when samples are run under non-reducing conditions and are not boiled prior to electrophoresis [11,32]. It is worth noting that Kunitz inhibitors cannot form stable complexes with the zymogen form of proteases due to the rapid dissociation of any interaction between the protease zymogen and protease inhibitor, yielding a very high dissociation constant (K d are 10s of millions fold more stable than with protease zymogens.…”

“…The human prostasin mAbs YL11 and YL89 were generated using milk-derived prostasin-HAI-1 complexes as immunogen as described previously [11]. The human matriptase mAb M24 was used to detect matriptase zymogen and activated forms in complexes with protease inhibitors [25,26].…”

Matriptase and prostasin are expressed in human skin in an inverse trend over the course of differentiation and are targeted to different regions of the plasma membrane

“…Studies using HaCaT cells further reveal that the epidermal matriptase-prostasin proteolytic cascade is under tight control by hepatocyte growth factor activator inhibitor (HAI)-1, a Kunitz-type serine protease inhibitor, which rapidly inhibits not only matriptase but also prostasin, following the induction of zymogen activation of both serine proteases. The evidence for a physiologically relevant in vivo partnership between these proteases and HAI-1 has been bolstered by the purification and identification of matriptase-HAI-1 and prostasin-HAI-1 complexes from human milk and other body fluids (Lai et al, 2016; Lin et al, 1999; Wang et al, 2009). …”