The proteolytic processing of the serine protease matriptase-2: identification of the cleavage sites required for its autocatalytic release from the cell surface
Résumé
Matriptase-2 is a member of the type II transmembrane serine proteases (TTSPs), an emerging class of cell surface proteases involved in tissue homeostasis and several human disorders which exhibit a domain organization similar to other TTSPs, with a cytoplasmic N-terminus, a transmembrane domain and an extracellular C-terminus containing the non-catalytic stem region and the protease domain. To gain further insight into the biochemical functions of matriptase-2, we characterized subcellular localizations of complexed and uncomplexed forms and identified cell surface shedding as a hallmark in the proteolytic processing of matriptase-2. Using HEK293 cells stably transfected with cDNA encoding human matriptase-2, we demonstrate cell membrane localization of an inactive, single-chain zymogen. Membrane-associated matriptase-2 is highly N-glycosylated and occurs in monomeric as well as multimeric forms that are covalently linked by disulfide bonds. Furthermore, matriptase-2 undergoes shedding into the conditioned medium as an activated, two-chain form containing the catalytic domain which is cleaved at the canonical activation motif but linked to a released portion of the stem region via a conserved disulfide bond. Cleavage sites were identified by mass spectrometry, sequencing and mutational analysis. Interestingly, cell surface shedding and activation of a matriptase-2 variant bearing a mutation at the active site serine is dependent on the catalytic activity of co-expressed or co-incubated wild type matriptase-2, indicating a transactivation and transshedding mechanism.
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