Der P1 JBCJuly24

Cysteine and serine proteases function via protease-activated (PARs) and mas-related Gprotein coupled receptors (Mrgprs) to contribute to allergy and inflammation. Der p1 is a cysteine protease and major allergen from the house dust mite and is associated with allergic rhinitis and allergic asthma. Der p1 activates PAR2 and induces the release of the pro-inflammatory cytokine IL-6 from cells. However, the possibility that Der p1 acts on Mrgprs has not been considered. We report here that ratiometric calcium imaging reveals that Der p1 activates the human receptor MRGPRX1 and the mouse homolog MrgprC11, implicated previously in itch. Der p1 cleavage of N-terminal receptor peptides followed by site-directed mutagenesis of the cleavage sites links receptor activation to specific amino acid residues. Der p1 also induced the release of IL-6 from heterologous cells expressing MRGPRX1. In summary, activation of Mrgprs by the allergen Der p1 may contribute to inflammation. Asthma and rhinitis are chronic widespread heterogeneous conditions. Asthma is characterized by difficulty breathing, wheezing and cough, while symptoms of rhinitis include a running and itchy nose and tearing eyes. Environmental allergens are associated and shared between these two conditions. An important source of allergen is the house dust mite Dermatophagoides pteronyssinus and a prominent allergen from the mite is the cysteine protease, Der p1(1). While it has been established that Der p1 activation of protease-activated receptor 2 (PAR2) on lung epithelial cells results in the release of pro-inflammatory cytokines(2), recent reports and the complexity of biology led us to consider the possibility that Der p1 had the capacity to activate Mrgprs. Thus, it has been reported that endogenous and exogenous cysteine proteases activate not only PARs but also members of the Mrgpr family(3) and that hexapeptides derived from serine protease cleavage of PAR2 unexpectedly activate Mrgprs to cause itch(4). In addition, Mrgprs are expressed primarily on mast cells and sensory neurons where they serve as innate sensors, contribute to allergy and mediate the nociceptive sensations pain and itch(3,5). The appreciation that epithelia, mast cells and sensory nerves together contribute to allergy, including asthma (6) provided further impetus for these studies. Here we determine that Der p1 can activate distinct members of the Mrgpr family, identify the N-terminal amino acids in Mrgprs required for cleavage, that N-terminal peptides can activate certain Mrgprs and PARs, and that Der p1 activation of a Mrgpr induces the release of the pro-inflammatory cytokine IL-6. Taken together, these data support the possibility that the house dust mite allergen Der p1 may contribute to inflammation through activation of Mrgprs. Results Der p1 activates specific Mrgprs 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M117.787887 The latest version is at JBC Papers in Press. Published on August 2, 2017 as Manuscript M117.787887 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 6, 2017 hp://w w w .jb.org/ D ow nladed from Dust mite protease activates Mrgprs As Der p1 is an allergen, we first asked if it could activate any of the human or mouse Mrgprs associated with allergy or itch. To address this question, we used ratiometric calcium imaging to determine if Der p1 could activate heterologous cells that had been transfected with cDNAs encoding the human Mrgprs, X1 X4 or the relevant mouse Mrgprs. The only Mrgprs activated by Der p1 were the human receptor MRGPRX1 and the mouse receptors MrgprC11 and MrgprA3. These receptors have each been linked to itch but not previously to allergy. Der p1 was also found to activate human PAR2 (Fig.1). Receptor activation was blocked in all cases by E-64, an established irreversible inhibitor of cysteine proteases. This result establishes that protease activity was necessary for receptor activation. The data for each receptor are shown in Table 1. Der p1 cleavage of N-terminal receptor sequences We next sought to determine the cleavage sites associated with receptor activation by Der p1. Attention was directed to MRGPRX1 as it was the only human receptor that was activated by Der p1. Attention was also directed to the mouse receptor MrgprC11 as it was activated by Der p1 and is also activated by SLIGRL, the tethered ligand and hexapeptide ligand for PAR2. PAR2 serve as a positive control. The N-terminal peptides of MRGPRX1, MrgprC11 and PAR2 were synthesized, incubated with Der p1 and sequences of the resultant series of cleavage products obtained via tandem mass spectrometry, MS/MS arranged in descending order of ion abundance (Fig. 2). It is appreciated that strict quantitative conclusions cannot be drawn from this approach but the findings establish sites of cleavage. To account for the peptides identified by MS/MS in order of abundance, the major sites for Der p1 cleavage of the N-terminus of MRGPRX1 were after E11, L22 and D2 respectively as indicated below by the large arrows. These sites were followed by cleavage before D2, L8 and L12, as indicated by the small arrows. 1 MDêPTISTLDTEêLTPINGTEETLêCY KQTLSL 30 The preferred site for Der p1 cleavage of the N-terminal peptide of MGRPRX1 was deduced to be between E11 and L12. Both large and small arrows are noted at this site to account for the two most abundant peptides and the less abundant peptides 6, 7 and 8. In contrast, the peptides TPINGTEET, TPINGTE and TPINGTEE result from cleavage after L12 but as these peptides were not abundant, no arrow is included after L12. The major sites for cleavage of the Nterminus of MrgprC11 occurred after D2, E11 and E17 as indicated below by the large arrows. These were followed by cleavage before D2 and before and after S12, as indicated by the small arrows. 1 MDêPTISSHDTEêSTPLNEêTGHPNC