PLoS ONE (Jan 2013)

The proline-rich motif of the proDer p 3 allergen propeptide is crucial for protease-protease interaction.

  • Marie-Eve Dumez,
  • Julie Herman,
  • Vincenzo Campisi,
  • Ahlem Bouaziz,
  • Frédéric Rosu,
  • André Luxen,
  • Isabel Vandenberghe,
  • Edwin de Pauw,
  • Jean-Marie Frère,
  • André Matagne,
  • Andy Chevigné,
  • Moreno Galleni

DOI
https://doi.org/10.1371/journal.pone.0068014
Journal volume & issue
Vol. 8, no. 9
p. e68014

Abstract

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The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen.