5wi6

Publication Abstract from PubMed

Human beta-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic beta-tryptase inhibitors, but its unique activation mechanism is less well explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N-terminus into its 'activation pocket', indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein-protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric beta-tryptase mutant (I99C*:Y75A:Y37bA where C* is cysteinylated Cys99) cannot form a dimer or tetramer, yet is active, but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each beta-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity.

Dual functionality of beta-tryptase protomers as both proteases and cofactors in the active tetramer.,Maun HR, Liu PS, Franke Y, Eigenbrot C, Forrest WF, Schwartz LB, Lazarus RA J Biol Chem. 2018 Apr 16. pii: M117.812016. doi: 10.1074/jbc.M117.812016. PMID:29661938^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.