The Proline-Rich Motif of the proDer p 3 Allergen Propeptide Is Crucial for Protease-Protease Interaction

Abstract

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.

Development of recombinant stable house dust mite allergen Der p 3 molecules for component‐resolved diagnosis and specific immunotherapy

“…1a). 1b) [33]. The intrinsic fluorescence and far UV-CD analysis showed that rDer p 3 and its inactive counterpart rDer p 3-S196A exhibit a similar tertiary and secondary structure content, respectively (Figs 1b and c).…”

“…Such tion of trypsin [33]. Therefore, in the context of immunotherapy with HDM extracts, it would be of interest to stabilize nDer p 3 protein using specific serine protease inhibitors treatment.…”

“…Unglycosylated proDer p 1 and proDer p 3 and the corresponding catalytically inactive molecules proDer p 1C34A and proDer p 3-S196A, respectively, were expressed in P. pastoris, purified and activated into mature rDer p 1 and rDer p 3 and their inactive counterparts, rDer p 1-C34A and rDer p 3-S196A, respectively, as previously described [28,[31][32][33]. Natural Der p 3 (nDer p 3) was purified to homogeneity from HDM extracts (see below).…”

“…Allergens were analysed by SDS-PAGE, and Der p 3 or Der p 1 activities were measured in 10fold diluted aliquots using 150 lM QAR-AMC as substrate on a Perkin Elmer LS 50 B spectrofluorimeter as previously described [28,33]. Allergens were analysed by SDS-PAGE, and Der p 3 or Der p 1 activities were measured in 10fold diluted aliquots using 150 lM QAR-AMC as substrate on a Perkin Elmer LS 50 B spectrofluorimeter as previously described [28,33].…”

“…Although the secondary structures of nDer p 3 (24.9 kDa) and proDer p 3 (26.4 kDa) were previously shown to be similar [28], the intrinsic fluorescence spectra of these proteins, which are characteristic of the proteins' tertiary structures, are different [33]. Here, we describe the immunological characterization of recombinant proDer p 3 as a hypoallergen of nDer p 3.…”