title: Relationship between propeptide pH unfolding and inhibitory ability during ProDer p 1 activation mechanism.
abstract: The major allergen Der p 1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The propeptide of Der p 1 exhibits a specific fold that makes it unique in the CA1 propeptide family. In this study, we investigated the activation steps involved in the maturation of the recombinant protease Der p 1 expressed in Pichia pastoris and the interaction of the full-length and truncated soluble propeptides with their parent enzyme in terms of activity inhibition and BIAcore interaction analysis. According to our results, the activation of protease Der p 1 is a multistep mechanism that is characterized by at least two intermediates. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 (K(D)=7 nM) at neutral pH. This inhibition is pH dependent. It decreases from pH 7 to pH 4 and can be related to conformational changes of the propeptide characterized by an increase of its flexibility and formation of a molten globule state. Our results indicate that activation of the zymogen at pH 4 is a compromise between activity preservation and propeptide unfolding.
authors.affiliation: Macromolécules Biologiques, Center for Protein Engineering, Université de Liège, Liège B-4000, Belgium.
authors.forename: Andy
authors.initials: A
authors.lastname: Chevigné
authors.forename: Roya
authors.initials: R
authors.lastname: Barumandzadeh
authors.forename: Sylvie
authors.initials: S
authors.lastname: Groslambert
authors.forename: Benoît
authors.initials: B
authors.lastname: Cloes
authors.forename: Dominique
authors.initials: D
authors.lastname: Dehareng
authors.forename: Patrice
authors.initials: P
authors.lastname: Filée
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Marx
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Frère
authors.forename: André
authors.initials: A
authors.lastname: Matagne
authors.forename: Alain
authors.initials: A
authors.lastname: Jacquet
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
mesh_terms: D000818:Animals; D039741:Antigens, Dermatophagoides / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D060829:Arthropod Proteins; D002942:Circular Dichroism; D003198:Computer Simulation; D003546:Cysteine Endopeptidases; D040002:Dermatophagoides pteronyssinus / Q000201:enzymology / Q000276:immunology; D004792:Enzyme Precursors; D005453:Fluorescence; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000737:chemistry* / Q000494:pharmacology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D020349:Surface Plasmon Resonance
chemical_list: D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D004792:Enzyme Precursors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1