Profiling the Extended Cleavage Specificity of the House Dust Mite Protease Allergens Der p 1, Der p 3 and Der p 6 for the Prediction of New Cell Surface Protein Substrates.

House dust mite (HDM) protease allergens, through cleavages of critical surface proteins, drastically influence the initiation of the Th2 type immune responses. However, few human protein substrates for HDM proteases have been identified so far, mainly by applying time-consuming target-specific individual studies. Therefore, the identification of substrate repertoires for HDM proteases would represent an unprecedented key step toward a better understanding of the mechanism of HDM allergic response. In this study, phage display screenings using totally or partially randomized nonameric peptide substrate libraries were performed to characterize the extended substrate specificities (P5-P4') of the HDM proteases Der p 1, Der p 3 and Der p 6. The bioinformatics interface PoPS (Prediction of Protease Specificity) was then applied to define the proteolytic specificity profile of each protease and to predict new protein substrates within the human cell surface proteome, with a special focus on immune receptors. Specificity profiling showed that the nature of residues in P1 but also downstream the cleavage sites (P' positions) are important for effective cleavages by all three HDM proteases. Strikingly, Der p 1 and Der p 3 display partially overlapping specificities. Analysis with PoPS interface predicted 50 new targets for the HDM proteases, including 21 cell surface receptors whose extracellular domains are potentially cleaved by Der p 1, Der p 3 and/or Der p 6. Twelve protein substrate candidates were confirmed by phage ELISA (enzyme linked immunosorbent assay). This extensive study of the natural protein substrate specificities of the HDM protease allergens unveils new cell surface target receptors for a better understanding on the role of these proteases in the HDM allergic response and paves the way for the design of specific protease inhibitors for future anti-allergic treatments.

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.