The isolation and characterization of a novel collagenolytic serine protease allergen (Der p 9) from the dust mite Dermatophagoides pteronyssinus☆,☆☆,★,★★
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Extracts and chemicals
Extracts of D. pteronyssinus were prepared from spent growth medium (SGM) devoid of mites, as described previously.10 General chemicals and synthetic protease substrates were purchased from Sigma Chemical Company (St Louis, Mo.), unless otherwise stated.
Determination of protease activity
Chymotrypsin and trypsin activities were determined by using succinyl-ala-ala-pro-phe p-nitroanilide (SA 2PFpNA), succinyl-ala-ala-pro-leu p-nitroanilide (SA 2PLpNA), and N-benzoyl-arginine p-nitroanalide (BApNA), respectively, as described
Isolation of Der p 9 from D. pteronyssinus SGM
SGM was absorbed batchwise onto DE53 at pH 8.0, and the unbound fraction was then absorbed onto an SBTI–Sepharose 4B affinity matrix. The bound material, which eluted at acid pH, was shown to cleave SA 2PLpNA, SA 2PFpNA, and BApNA. Fractions containing the proteases were pooled and concentrated by cation-exchange chromatography at pH 4.5. The bound material was eluted with distilled water at pH 11.0, and two fractions were obtained (Fig. 1). The first, which eluted between pH 4.5 and 9.0, was
DISCUSSION
A novel, polymorphic serine protease with allergenic activity was isolated from SGM of D. pteronyssinus. It was clearly differentiated from both the Der p 3 tryptic allergen2 and the Der p 6 chymotryptic allergen3 on the basis of charge, N-terminal amino acid sequence, and substrate specificity. The protease was shown to be allergenic and was therefore designated Der p 9. This protease represents the fourth mite protease to be described, and these results and previous data1, 2, 3, 8, 9, 10
Acknowledgements
We thank Commonwealth Serum laboratories (Parkville, Australia) for providing spent growth medium from D. pteronyssinus, Dr. Martin Chapman for providing the anti-Der p 1 and anti-Der p 2 monoclonal antibodies, and Mr. Peter Boyne of Western Diagnostics for providing mite allergic sera.
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IgE and T-cell responses to house dust mite allergen components
2018, Molecular ImmunologyCitation Excerpt :Similar IgE binding to the group 1 and 2 allergens has however been reported in some studies (Ando et al., 1993; King et al., 1996). Most titers were low in the small sample of Ando et al. (1993) and the detail of King et al. (1996) reveals that the Der p 3 results were from a different study that found that twice the amount of Der p 3 was required to inhibit RAST responses compared to Der p 1 and explicitly used an impure Der p 3 preparation (Stewart et al., 1992). A recent re-examination showed that the use of an enzymatically inactive mutant of Der p 3 could detect more IgE binding than wild type Der p 3 (Bouaziz et al., 2015) but the titers were still much lower than those to Der p 1.
Detailed two-dimensional gel proteomic mapping of the feces of the house dust mite Dermatophagoides pteronyssinus and comparison with D. farinae: Reduced trypsin protease content in D. pteronyssinus and different isoforms
2017, Journal of ProteomicsCitation Excerpt :Whether or not this proves to be the case, we conclude that the protease activation cascade [39] likely functions differently in D. farinae than in D. pteronyssinus due to the presence of different protease isoforms in the feces of these two species. The collagenolytic serine protease Der p 9 with chymotrypsin and cathepsin G-like enzyme activity [42] may also constitute a difference between D. pteronyssinus and D. farinae. Support for this idea is that although allergenic information is available for Der p 9 (sensitization ~ 92%) [42], relevant information is lacking for D. farinae as Der f 9 is absent from the current IUIS list of allergens.
Immunogenic peptides: B & T Cell Epitopes of Per a 10 Allergen of Periplaneta americana
2016, Molecular ImmunologyDer f 34, a novel major house dust mite allergen belonging to a highly conserved Rid/YjgF/YER057c/UK114 family of imine deaminases
2016, Journal of Biological ChemistryCitation Excerpt :Der f 1 (cysteine protease), Der f 2 (MD2-like lipid-binding domain protein), and Der f 23 (peritrophin-like protein) are serodominant allergens in D. farinae (7, 15–17). Serine proteases from D. farinae, including Der f 3 (trypsin), Der f 6 (chymotrypsin), and Der f 9 (collagenase), act on mucosal epithelial cells to induce chemokine production that triggers the migration of inflammatory cells (13, 18–23). Tropomyosin as Der f 10 and gelsolin as Der f 16 are also important HDM allergens as pan-allergens (10, 14).
Hierarchy and molecular properties of house dust mite allergens
2015, Allergology International
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From aInstitute for Child Health Research, West Perth; bDepartment of Microbiology, University of Western Australia; cJoint Protein Structure Laboratory, Ludwig Institute for Cancer Research and The Walter and Eliza Hall Institute for Medical Research, Royal Melbourne Hospital, Parkville, Victoria; and dDepartment of Medicine, University of Western Australia, Nedlands.
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Supported by the Australian National Health and Medical Research Council and the Asthma Foundation of Western Australia.
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Reprint requests: Geoffrey A. Stewart, Department of Microbiology, University of Western Australia, Nedlands, Western Australia 6907.
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