Promiscuous processing of human α/β-protryptases (86.13)

Abstract

Abstract Human β-tryptase is stored in secretory granules of human mast cells as a heparin-stabilized homotetramer. Although β-protryptase is sequentially processed in vitro by autocatalysis at Arg-3 followed by cathepsin (CTS)-C proteolysis to the mature enzyme, CTSC-deficient mice effectively process the murine pro form of mast cell protease-6, a mouse tryptase. Tryptase processing activity(ies) distinct from CTSC were recognized in and partially purified from human HMC-1 cell extracts by ion exchange and gel filtration chromatography, and identified by LC-tandem mass spectroscopy to include CTSB and CTSL. Importantly, CTSB and CTSL could process α-protryptase (Q-3) and mutated β-protryptase (R-3Q) as well as β-protryptase, indicating no need for autocatalytic cleavage at Arg-3, whereas CTSC-dependent processing required the autocatalytic step. Heparin was required for processing and for tryptase tetramer formation, and also protected tryptase from degradation by CTSB and CTSL. Processed protryptases were tetrameric by gel filtration and mature by N-terminal sequence analysis. CTSL, CTSB and β-tryptase were co-localized to skin mast cell secretory granules based on their co-release upon degranulation, indicating that they traffic together within the mast cells. Thus, CTSL and CTSB may be central to the processing of both α and β protryptases in mast cells of human and perhaps other species, and are potential targets for attenuating production of mature tryptase.