TY - JOUR
T1 - The Proline-Rich Motif of the proDer p 3 Allergen Propeptide Is Crucial for Protease-Protease Interaction
AU - Dumez, Marie Eve
AU - Herman, Julie
AU - Campisi, Vincenzo
AU - Bouaziz, Ahlem
AU - Rosu, Frédéric
AU - Luxen, André
AU - Vandenberghe, Isabel
AU - de Pauw, Edwin
AU - Frère, Jean Marie
AU - Matagne, André
AU - Chevigné, Andy
AU - Galleni, Moreno
PY - 2013/9/20
Y1 - 2013/9/20
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84884492024&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0068014
DO - 10.1371/journal.pone.0068014
M3 - Article
C2 - 24073192
AN - SCOPUS:84884492024
SN - 1932-6203
VL - 8
JO - PLoS ONE
JF - PLoS ONE
IS - 9
M1 - e68014
ER -