Proteomic analysis of cleavage events reveals a dynamic two-step mechanism for proteolysis of a key parasite adhesive complex

The transmembrane micronemal protein MIC2 and its partner M2AP comprise an adhesive complex that is required for rapid invasion of host cells by the obligate intracellular parasite Toxoplasma gondii. Recent studies have shown that the MIC2/M2AP complex undergoes extensive proteolytic processing on the parasite surface during invasion, including primary processing of M2AP by unknown proteases and proteolytic shedding of the complex by an anonymous protease called MPP1. While it was shown that MPP1-mediated cleavage is necessary for efficient invasion, it remained unclear whether the adhesive complex was liberated by juxtamembrane or intramembrane proteolysis. Here, using a three-phase strategy of assigning cleavage sites based on intact matrix-assisted laser desorption/ionization mass followed by confirmation by enzymatic digestion and inhibitor profiling, we demonstrate that M2AP is processed by two parasite-derived proteases called MPP2 and MPP3. We also define the substrate repertoire of MPP2 by two-dimensional differential gel electrophoresis using fluorescent tags. Finally, we use complementary mass spectrometric techniques to unequivocally show that MIC2 is shed by intramembrane cleavage within its anchoring domain. Based on the properties of this cleavage site, we conclude that the sheddase, MPP1, is likely a multipass membrane protease of the Rhomboid family. Our data support a novel two-step proteolysis model that includes primary processing of the MIC2/M2AP complex followed by secondary cleavage to shed the complex from the parasite surface during the final steps of invasion.