Complete inhibition of β-tryptase by tetramer dissociation and active site allostery due to a single antibody residue

Complete inhibition of β-tryptase by tetramer dissociation and active site allostery due to a single antibody residue

  • Wernersson, S. & Pejler, G. Mast cell secretory granules: armed for battle. Nat. Rev. Immunol. 14, 478–494 (2014).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Siebenhaar, F., Redegeld, F. A., Bischoff, S. C., Gibbs, B. F. & Maurer, M. Mast cells as drivers of disease and therapeutic targets. Trends Immunol. 39, 151–162 (2018).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Frossi, B., Mion, F., Tripodo, C., Colombo, M. P. & Pucillo, C. E. Rheostatic functions of mast cells in the control of innate and adaptive immune responses. Trends Immunol. 38, 648–656 (2017).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Caughey, G. H. Mast cell proteases as pharmacological targets. Eur. J. Pharm. 778, 44–55 (2016).

    Article 
    CAS 

    Google Scholar
     

  • Pejler, G., Abrink, M., Ringvall, M. & Wernersson, S. Mast cell proteases. Adv. Immunol. 95, 167–255 (2007).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Pejler, G. The emerging role of mast cell proteases in asthma. Eur. Respir. J. 54, 1900685 (2019).

  • Berger, P. et al. Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells. J. Appl Physiol. 91, 1372–1379 (2001).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Cairns, J. A. & Walls, A. F. Mast cell tryptase stimulates the synthesis of type I collagen in human lung fibroblasts. J. Clin. Invest 99, 1313–1321 (1997).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Woodman, L. et al. Mast cells promote airway smooth muscle cell differentiation via autocrine up-regulation of TGF-β1. J. Immunol. 181, 5001–5007 (2008).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Maun, H. R. et al. An allosteric anti-tryptase antibody for the treatment of mast cell-mediated severe asthma. Cell 179, 417–431.e419 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Cairns, J. A. Inhibitors of mast cell tryptase beta as therapeutics for the treatment of asthma and inflammatory disorders. Pulm. Pharm. Ther. 18, 55–66 (2005).

    Article 
    CAS 

    Google Scholar
     

  • Rawlings, N. D. et al. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res. 46, D624–D632 (2017).

    Article 

    Google Scholar
     

  • Le, Q. T. et al. Processing of human protryptase in mast cells involves cathepsins L, B, and C. J. Immunol. 187, 1912–1918 (2011).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Schwartz, L. B. & Bradford, T. R. Regulation of tryptase from human lung mast cells by heparin. Stabilization of the active tetramer. J. Biol. Chem. 261, 7372–7379 (1986).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Addington, A. K. & Johnson, D. A. Inactivation of human lung tryptase: Evidence for a re-activatable tetrameric intermediate and active monomers. Biochemistry 35, 13511–13518 (1996).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Ren, S., Sakai, K. & Schwartz, L. B. Regulation of human mast cell β-tryptase: Conversion of inactive monomer to active tetramer at acid pH. J. Immunol. 160, 4561–4569 (1998).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Schechter, N. M., Choi, E. J., Selwood, T. & McCaslin, D. R. Characterization of three distinct catalytic forms of human tryptase-β: their interrelationships and relevance. Biochemistry 46, 9615–9629 (2007).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Hallgren, J., Lindahl, S. & Pejler, G. Structural requirements and mechanism for heparin-dependent activation and tetramerization of human βI- and βII-tryptase. J. Mol. Biol. 345, 129–139 (2005).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Maun, H. R. et al. Dual functionality of β-tryptase protomers as both proteases and cofactors in the active tetramer. J. Biol. Chem. 293, 9614–9628 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Fajardo, I. & Pejler, G. Formation of active monomers from tetrameric human β-tryptase. Biochem J. 369, 603–610 (2003).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Fukuoka, Y. & Schwartz, L. B. Human β-tryptase: detection and characterization of the active monomer and prevention of tetramer reconstitution by protease inhibitors. Biochemistry 43, 10757–10764 (2004).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Fukuoka, Y. & Schwartz, L. B. Active monomers of human β-tryptase have expanded substrate specificities. Int. Immunopharmacol. 7, 1900–1908 (2007).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Franconi, G. M., Graf, P. D., Lazarus, S. C., Nadel, J. A. & Caughey, G. H. Mast cell tryptase and chymase reverse airway smooth muscle relaxation induced by vasoactive intestinal peptide in the ferret. J. Pharm. Exp. Ther. 248, 947–951 (1989).

    Article 
    CAS 

    Google Scholar
     

  • Gruber, B. L. et al. Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation. J. Clin. Invest 84, 1657–1662 (1989).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Maier, M., Spragg, J. & Schwartz, L. B. Inactivation of human high molecular weight kininogen by human mast cell tryptase. J. Immunol. 130, 2352–2356 (1983).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Schiemann, F. et al. The cathelicidin LL-37 activates human mast cells and is degraded by mast cell tryptase: counter-regulation by CXCL4. J. Immunol. 183, 2223–2231 (2009).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Schwartz, L. B., Bradford, T. R., Littman, B. H. & Wintroub, B. U. The fibrinogenolytic activity of purified tryptase from human lung mast cells. J. Immunol. 135, 2762–2767 (1985).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Pereira, P. J. et al. Human β-tryptase is a ring-like tetramer with active sites facing a central pore. Nature 392, 306–311 (1998).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Sommerhoff, C. P. et al. The structure of the human βII-tryptase tetramer: Fo(u)r better or worse. Proc. Natl. Acad. Sci. USA 96, 10984–10991 (1999).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Hallgren, J., Backstrom, S., Estrada, S., Thuveson, M. & Pejler, G. Histidines are critical for heparin-dependent activation of mast cell tryptase. J. Immunol. 173, 1868–1875 (2004).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Fukuoka, Y. & Schwartz, L. B. The B12 anti-tryptase monoclonal antibody disrupts the tetrameric structure of heparin-stabilized β-tryptase to form monomers that are inactive at neutral pH and active at acidic pH. J. Immunol. 176, 3165–3172 (2006).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Alter, S. C., Kramps, J. A., Janoff, A. & Schwartz, L. B. Interactions of human mast cell tryptase with biological protease inhibitors. Arch. Biochem Biophys. 276, 26–31 (1990).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Paesen, G. C. et al. A tick protein with a modified Kunitz fold inhibits human tryptase. J. Mol. Biol. 368, 1172–1186 (2007).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Sommerhoff, C. P. et al. A Kazal-type inhibitor of human mast cell tryptase: Isolation from the medical leech Hirudo medicinalis, characterization, and sequence analysis. Biol. Chem. Hoppe Seyler 375, 685–694 (1994).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Stubbs, M. T. et al. The three-dimensional structure of recombinant leech-derived tryptase inhibitor in complex with trypsin. Implications for the structure of human mast cell tryptase and its inhibition. J. Biol. Chem. 272, 19931–19937 (1997).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Rothemund, S., Sonnichsen, F. D. & Polte, T. Therapeutic potential of the peptide leucine arginine as a new nonplant Bowman-Birk-like serine protease inhibitor. J. Med Chem. 56, 6732–6744 (2013).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Murakami, Y. et al. Cyclotheonamide E4 and E5, new potent tryptase inhibitors from an Ircinia species of sponge. J. Nat. Prod. 65, 259–261 (2002).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Avrutina, O. et al. Head-to-tail cyclized cystine-knot peptides by a combined recombinant and chemical route of synthesis. Chembiochem 9, 33–37 (2008).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Sommerhoff, C. P. et al. Engineered cystine knot miniproteins as potent inhibitors of human mast cell tryptase β. J. Mol. Biol. 395, 167–175 (2010).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Thongyoo, P., Bonomelli, C., Leatherbarrow, R. J. & Tate, E. W. Potent inhibitors of beta-tryptase and human leukocyte elastase based on the MCoTI-II scaffold. J. Med Chem. 52, 6197–6200 (2009).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Scarpi, D., McBride, J. D. & Leatherbarrow, R. J. Inhibition of human beta-tryptase by Bowman-Birk inhibitor derived peptides: creation of a new tri-functional inhibitor. Bioorg. Med Chem. 12, 6045–6052 (2004).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • McGrath, M. E. et al. Structure-guided design of peptide-based tryptase inhibitors. Biochemistry 45, 5964–5973 (2006).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Ni, W. W., Cao, M. D., Huang, W., Meng, L. & Wei, J. F. Tryptase inhibitors: a patent review. Expert Opin. Ther. Pat. 27, 919–928 (2017).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Qian, X., Zheng, B., Burke, B., Saindane, M. T. & Kronenthal, D. R. A stereoselective synthesis of BMS-262084, an azetidinone-based tryptase inhibitor. J. Org. Chem. 67, 3595–3600 (2002).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Sutton, J. C. et al. Synthesis and SAR of 4-carboxy-2-azetidinone mechanism-based tryptase inhibitors. Bioorg. Med Chem. Lett. 12, 3229–3233 (2002).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Wright, C. D. et al. Inhibition of allergen-induced pulmonary responses by the selective tryptase inhibitor 1,5-bis-[4-[(3-carbamimidoyl-benzenesulfonylamino)-methyl]-phenoxy]-pen tane (AMG-126737). Biochem Pharm. 58, 1989–1996 (1999).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Costanzo, M. J. et al. Potent, nonpeptide inhibitors of human mast cell tryptase. Synthesis and biological evaluation of new spirocyclic piperidine amide derivatives. Bioorg. Med Chem. Lett. 18, 2114–2121 (2008).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Krishna, M. T. et al. Inhibition of mast cell tryptase by inhaled APC 366 attenuates allergen-induced late-phase airway obstruction in asthma. J. Allergy Clin. Immunol. 107, 1039–1045 (2001).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Ganesan, R., Eigenbrot, C. & Kirchhofer, D. Structural and mechanistic insight into how antibodies inhibit serine proteases. Biochem J. 430, 179–189 (2010).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Maun, H. R. et al. Bivalent antibody pliers inhibit beta-tryptase by an allosteric mechanism dependent on the IgG hinge. Nat. Commun. 11, 6435 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Rhee, H. et al. Airway tryptase levels inform the lack of clinical efficacy of the tryptase inhibitor MTPS9579A in asthma. Allergy 79, 2993–3004 (2024).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Hartley, B. S. Homologies in serine proteinases. Philos. Trans. R. Soc. Lond. B Biol. Sci. 257, 77–87 (1970).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Ereno-Orbea, J. et al. Structural basis of enhanced crystallizability induced by a molecular chaperone for antibody antigen-binding fragments. J. Mol. Biol. 430, 322–336 (2018).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Huber, R. & Bode, W. Structural basis of the activation and action of trypsin. Acc. Chem. Res 11, 114–122 (1978).

    Article 
    CAS 

    Google Scholar
     

  • Cook, K. M., McNeil, H. P. & Hogg, P. J. Allosteric control of betaII-tryptase by a redox active disulfide bond. J. Biol. Chem. 288, 34920–34929 (2013).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Bush-Pelc, L. A. et al. Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery. J. Biol. Chem. 282, 27165–27170 (2007).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Miyata, T. et al. Coagulation factor XII (Hageman factor) Washington D.C.: inactive factor XIIa results from Cys-571–Ser substitution. Proc. Natl. Acad. Sci. USA 86, 8319–8322 (1989).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Ganesan, R. et al. Unraveling the allosteric mechanism of serine protease inhibition by an antibody. Structure 17, 1614–1624 (2009).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Schaefer, M., Buchmueller, A., Dittmer, F., Strassburger, J. & Wilmen, A. Allosteric inhibition as a new mode of action for BAY 1213790, a neutralizing antibody targeting the activated form of coagulation factor XI. J. Mol. Biol. 431, 4817–4833 (2019).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Jiang, L. et al. Rezymogenation of active urokinase induced by an inhibitory antibody. Biochem J. 449, 161–166 (2013).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Katschke, K. J. Jr. et al. Inhibiting alternative pathway complement activation by targeting the factor D exosite. J. Biol. Chem. 287, 12886–12892 (2012).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Koschubs, T. et al. Allosteric antibody inhibition of human hepsin protease. Biochem J. 442, 483–494 (2012).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Chavarria-Smith, J. et al. Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis. Sci. Transl. Med 14, eabp9159 (2022).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Mossner, E. et al. Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity. Blood 115, 4393–4402 (2010).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Liang, W. C. et al. Dramatic activation of an antibody by a single amino acid change in framework. Sci. Rep. 11, 22365 (2021).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Fu, Z., Akula, S., Thorpe, M. & Hellman, L. Highly selective cleavage of TH2-promoting cytokines by the human and the mouse mast cell tryptases, indicating a potent negative feedback loop on TH2 immunity. Int. J. Mol. Sci. 20, 5147 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Gerhardy, S. et al. Allosteric inhibition of HTRA1 activity by a conformational lock mechanism to treat age-related macular degeneration. Nat. Commun. 13, 5222 (2022).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Walters, B. T., Ricciuti, A., Mayne, L. & Englander, S. W. Minimizing back exchange in the hydrogen exchange-mass spectrometry experiment. J. Am. Soc. Mass Spectrom. 23, 2132–2139 (2012).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Kan, Z. Y., Ye, X., Skinner, J. J., Mayne, L. & Englander, S. W. ExMS2: an Integrated Solution for Hydrogen-Deuterium Exchange Mass Spectrometry Data Analysis. Anal. Chem. 91, 7474–7481 (2019).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Walters, B. T., Mayne, L., Hinshaw, J. R., Sosnick, T. R. & Englander, S. W. Folding of a large protein at high structural resolution. Proc. Natl. Acad. Sci. USA 110, 18898–18903 (2013).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Walters, B. T. Empirical method to accurately determine peptide-averaged protection factors from hydrogen exchange MS data. Anal. Chem. 89, 1049–1053 (2017).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Masson, G. R. et al. Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments. Nat. Methods 16, 595–602 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article 
    PubMed 

    Google Scholar
     

  • Iudin, A. et al. EMPIAR: the electron microscopy public image archive. Nucleic Acids Res. 51, D1503–D1511 (2023).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article 
    CAS 
    PubMed 

    Google Scholar
     

  • Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D. Struct. Biol. 74, 519–530 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D. Struct. Biol. 74, 531–544 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Scholar
     

  • The PyMOL Molecular Graphics System, Version 3.1.3 Schrödinger, LLC.