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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., Competing Interests: The authors declare no conflict of interest.

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element., Competing Interests: The authors declare no conflict of interest.

Background: The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade., Methods: The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfer method. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanism by mite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus., Results: All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite., Conclusions: Der p 1 in either its recombinant form or in the natural context of house dust mite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases., General Significance: This finding suggests that Der p 1 may be valuable target against mites., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence T(P1)R.