STRUCTURE OF HUMAN LUNG MAST CELL b-TRYPTASE, A SERINE PROTEINASE INVOLVED IN ALLERGIC ASTHMA

Pedro Jose Barbosa Pereira¹, Andreas Bergner¹, Sandra Macedo-Ribeiro¹, Robert Huber¹, Gabriele Matschiner², Hans Fritz², Christian P. Sommerhoff² and Wolfram Bode¹

¹ Abt. für Strukturforschung, Max-Planck-Institut für Biochemie, Martinsried, Germany
² Abt. für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innenstadt der LMU, München, Germany

Keywords: asthma, tryptase, serine proteinase

Human tryptase (EC 3.4.21.59), the predominant protein of most human mast cells, has been implicated in the pathogenesis of asthma and other allergic and inflammatory disorders[1].

Although tryptase displays striking similarities with other serine proteinases, it has a number of unique properties (reviewed in refs. 1,2):

• it is enzymatically active only as a non-covalent heparin stabilized tetramer;
• in the absence of acidic polysaccharides (or of high salt concentrations in vitro), tryptase dissociates into inactive monomers rapidly and irreversibly, a process believed to limit its activity in vivo;
• it resists inhibition by all known endogenous proteinase inhibitors.
  

Tryptase efficiently hydrolyses a number of (neuro-)peptide substrates (in vitro) in a trypsin-like manner[3]. Unlike trypsin, however, tryptase cleaves only a few proteins, among these fibrinogen, fibronectin and high molecular weight kininogen (inactivation), and the zymogens of stromelysin-1 and u-PA (activation).

Attempts to model the structure of tryptase[4-6] are necessarily based on monomeric serine proteinases, and are thus unable to predict the tetrameric architecture. In order to define the tryptase-tetramer and to obtain a reliable model for rational drug design, we determined the X-ray crystal structure of human lung mast cell b-tryptase.

The 3 A crystal structure of human b-tryptase[7] in complex with 4-amidinophenylpyruvic acid reveals four quasi-equivalent monomers arranged in a square flat ring. The four active centres of the tetramer are directed towards an oval central pore, restricting the access for macromolecular substrates and inhibitors. Heparin chains could stabilise the complex by binding to an elongated patch of positively charged residues spanning two adjacent monomers.

Fig. 1: Overall architecture of the tryptase tetramer (front view). The four protease monomers are disposed in the corners of a square, with active sites facing the central pore.

This unique tetrameric architecture explains many of tryptaseís distinct biochemical properties and provides a basis for the rational design of mono- and multifunctional tryptase inhibitors.

Acknowledgements: We thank D. Grosse for her excellent help in crystallisation. This work was supported by scholarships PRAXIS XXI/BD/9782/96 (to P. J. B. P.) and PRAXIS XXI/BD/4050/94 (to S. M-R.) from the Fundaçäo para a Ciencia e a Tecnologia, Portugal, and by Biotech programs of the European Union, by the Sonderforschungsbereich 469 of the University of Munich, by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. P. J. B. P. is a Programa Gulbenkian de Doutoramento em Biologia e Medicina fellow.

1. Caughey, G.H. (1995) Clinical Allergy and Immunology, ed. M.A. Kaliner. 1995, Marcel Dekker, Inc.: New York.
2. Schwartz, L.B. (1994) Methods in Enzymology 244, 88-100.
3. Kam, C.M., et al. (1995) Archives of Biochemistry & Biophysics 316, 808-814.
4. Johnson, D.A. and G.J. Barton (1992) Protein Science 1, 370-377.
5. Matsumoto, R., et al. (1995) J. Biol. Chem. 270, 19524-19531.
6. Caughey, J.H. (1997) Am. J. Respir. Cell Mol. Biol. 16, 621-628.
7. Pereira, P.J.B., et al. (1998) Nature 392, 306-311.