The Ectodomain of Matriptase-2 Plays an Important Non-Proteolytic Role in Suppressing Hepcidin Expression in Mice.

: Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease, which plays a key role in suppressing hepatic hepcidin expression. MT2 is synthesized as a zymogen and undergoes autocleavage for activation. Previous studies suggest that MT2 suppresses hepcidin by cleaving hemojuvelin (HJV) and other components of the BMP-signaling pathway. However, the underlying mechanism is still debatable. Here we dissected the contributions of the non-proteolytic and proteolytic activities of Mt2 by taking advantage of Mt2 mutants and Tmprss6-/- mice. Studies of the protease-dead full-length Mt2 (Mt2S762A) and the truncated Mt2 that lacks the catalytic domain (Mt2mask) indicate that the catalytic domain, but not its proteolytic activity, was required for Mt2 to suppress hepcidin expression. This process was likely accomplished by the binding of Mt2 ectodomain to Hjv and Hfe. We found that Mt2 specifically cleaved the key components of the hepcidin induction pathway including Hjv, Alk3, ActRIIA, and Hfe when overexpressed in hepatoma cells. Nevertheless, studies of a murine IRIDA-causing mutant (Mt2I286F) in the CUB domain indicate that Mt2I286F can be activated but it displayed a largely compromised ability to suppress hepcidin expression. Co-immunoprecipitation analysis revealed that Mt2I286F, but not Mt2S762A, had reduced interactions with Hjv, ActRIIA, and Hfe. Additionally, increased expression of the serine protease inhibitor Hai-2 in the liver failed to alter hepcidin. Together these observations support the idea that the substrate interaction with Mt2 plays a determinant role, and suggest that the proteolytic activity is not an appropriate target to modulate the function of MT2 for clinical applications.