Proteolytic processing of the serine protease matriptase-2: identification of the cleavage sites required for its autocatalytic release from the cell surface
| Autor: | Stefan Frank, Angelika Horstmeyer, Kai Prager, Tobias Bald, Eva Maurer, Jochen Walter, Sonja Kolp, Patrick Wunderlich, Michael Gütschow, Andreas Janzer, Marit Stirnberg, Katharina Arenz |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Proteases
medicine.medical_treatment Biology Transfection Biochemistry Catalysis Cell Line Serine Cell membrane Catalytic Domain medicine Humans Molecular Biology Serine protease Enzyme Precursors Protease Cell Membrane Serine Endopeptidases HEK 293 cells Membrane Proteins Cell Biology Transmembrane protein Cell biology Enzyme Activation Transmembrane domain medicine.anatomical_structure Culture Media Conditioned Mutation biology.protein Extracellular Space Protein Binding |
| Zdroj: | Biochemical Journal. 430:87-95 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20091565 |
| Popis: | Matriptase-2 is a member of the TTSPs (type II transmembrane serine proteases), an emerging class of cell surface proteases involved in tissue homoeostasis and several human disorders. Matriptase-2 exhibits 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 the subcellular localization of the monomeric and multimeric form and identified cell surface shedding as a defining point in its proteolytic processing. Using HEK (human embryonic kidney)-293 cells, stably transfected with cDNA encoding human matriptase-2, we demonstrate a cell membrane localization for the inactive single-chain zymogen. Membrane-associated matriptase-2 is highly N-glycosylated and occurs in monomeric, as well as multimeric, forms 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 is linked to a released portion of the stem region via a conserved disulfide bond. Cleavage sites were identified by MS, sequencing and mutational analysis. Interestingly, cell surface shedding and activation of a matriptase-2 variant bearing a mutation at the active-site serine residue is dependent on the catalytic activity of co-expressed or co-incubated wild-type matriptase-2, indicating a transactivation and trans-shedding mechanism. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|