The proline-rich motif of the proDer p 3 allergen propeptide is crucial for protease-protease interaction.
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.
Macromolécules Biologiques, Centre for Protein Engineering, Université de Liège, Liège, Belgium.
Marie-Eve
ME
Dumez
Julie
J
Herman
Vincenzo
V
Campisi
Ahlem
A
Bouaziz
Frédéric
F
Rosu
André
A
Luxen
Isabel
I
Vandenberghe
Edwin
E
de Pauw
Jean-Marie
JM
Frère
André
A
Matagne
Andy
A
Chevigné
Moreno
M
Galleni
D000485:Allergens / Q000737:chemistry*; D020816:Amino Acid Motifs; D039741:Antigens, Dermatophagoides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D060829:Arthropod Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D009154:Mutation / Q000235:genetics; D010843:Pichia / Q000254:growth & development / Q000378:metabolism; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D004792:Enzyme Precursors; D011392:Proline; C451165:Dermatophagoides pteronyssinus antigen p 3; D012697:Serine Endopeptidases