The isolation and characterization of a novel collagenolytic serine protease allergen (Der p 9) from the dust mite Dermatophagoides pteronyssinus

doi:10.1016/S0091-6749(96)70121-5

Journal of Allergy and Clinical Immunology

Volume 98, Issue 4, October 1996, Pages 739-747

https://doi.org/10.1016/S0091-6749(96)70121-5 Get rights and content

Abstract

Background: Dust mites have been shown to contain a serine protease distinct from the previously reported trypsin and chymotrypsin. The latter enzymes have been shown to be allergens, but the allergenic importance of the former is unknown. Objective: This study was performed to isolate and characterize the novel mite serine protease and determine its allergenicity. Methods: The mite serine protease was isolated from feces-enriched extracts of Dermatophagoides pteronyssinus by ion-exchange chromatography and affinity chromatography, and its physicochemical properties were determined. The allergenicity of the protease was assessed by using the RAST. Results: The protease was enzymatically similar to chymotrypsin and cathepsin G–like enzymes from a variety of sources and was shown to cleave collagen. It had a molecular mass of 23,780 d. N-terminal sequence analysis (18 residues) indicated homology with the mite tryptic allergen, Der p 3, and the chymotryptic allergen, Der p 6. RAST analyses showed that the frequencies of reactivity to the novel allergen and to Der p 1, Der p 2, Der p 3, and Der p 6 were 92%, 97%, 100%, 97%, and 65%, respectively (n = 35). RAST inhibition studies showed some cross-reactivity between the protease and Der p 3 but not Der p 6. Conclusions: A novel mite serine protease was isolated from D. pteronyssinus and found to be a major allergen. This allergen has been tentatively designated Der p 9. (J ALLERGY CLIN IMMUNOL 1996;98:739-47.)

Section snippets

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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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.
Using the terminology for Dermatophagoides pteronyssinus, IgE responses to house dust mites have been shown to be mostly directed to the serodominant Der p 1, 2 and 23 allergen components with mid -tier responses to Der p 4, 5, 7 and 21 that are made by 30–50% of subjects with titers proportional to those of the serodominant specificities. This pattern can be seen to evolve in childhood and although responses to minor allergens appear to contribute little to the total IgE they are at least markers for a greater propensity to develop disease. While Der p 23 is a component that induces prevalent IgE responses, sometimes in the absence of responses to Der p 1and 2, not all studies have found high titers so further investigation is needed. From limited knowledge adult onset IgE responses might have a different pattern that is not so centered on Der p 1 and 2. Responses that induce under 3.5 IU/ml of IgE antibody are not usually associated with disease and should be examined for cross reactivity expected from IgE responses to other allergens and antigens of infectious agents. Scabies that has 40% endemicity in some regions and is spread by immigration can give rise to high-titer binding that can be recognized by component resolved diagnosis. Recent studies with synthetic peptides representing allergens and non-allergenic HDM proteins now offer new research avenues on HDM induced immune responses, including the ability to use peptides representing the serodominant allergens as defined reagents for long overdue reproducible T-cell investigations.
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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.
Major domestic mite allergens are present in feces. We present a detailed 2D–E-MS/MS proteomic analysis of the Dermatophagoides pteronyssinus feces. Precise cultivation yielded a pure fecal extract. We detected differences in fecal allergens/digestive enzymes between D. pteronyssinus and D. farinae using 2D–E fingerprinting, including unique information on species-specific protease isoforms. Proteomic analysis was performed by 2D–E coupled with MALDI-TOF/TOF identification. The species-specific differences in the fecal extracts of the mites were attributed to trypsin-like proteases known as group 3 allergens. In D. farinae, Der f 3 exhibited high abundance with a pI similar (acidic) to that of the cysteine protease Der f 1 and the chymotrypsin protease Der f 6, whereas in D. pteronyssinus, Der p 3 was rarely detected and exhibited low abundance only at basic pI. Moreover, Der p 9 was detected at a pI of ~ 10, in contrast to Der p 1 and Der p 6, suggesting different compartmentalization in the body. Overall, in D. pteronyssinus feces, allergens of groups 1, 2, 6, and 15 were quantitatively similar to those of D. farinae with the exception of the group 3 and 9 allergens. This work provides novel insights into mite-defecated proteins/digestive enzymes, which are important allergens.
Millions of people are affected by allergy and asthma, and their number is growing. In homes, the major triggers of allergy and asthma are the house dust mites Dermatophagoides farinae and D. pteronyssinus, and a clear understanding of the development of diseases caused by these mites is needed. The major sources of mite allergens are their feces, which are deposited in the environment and are easily inhaled as part of aeroplankton. However, descriptions of and comparisons between the major fecal allergens of these two mites are lacking. This study shows that similar group 1 (cysteine protease), 2 (NPC2 family), 6 (chymotrypsin) and 15 (chitinase-like) allergens are present in the feces of these two mite species, as determined by 2D–E mapping, whereas group 3 (trypsin) and 9 (collagenolytic protease) allergens in the feces of the two species are different. The results provide unique MS/MS mapped fingerprints of mite species-specific isoforms in feces. The presence of ubiquitin in mite feces suggests that these proteins participate in the post-translational modification of fecal proteins. The findings are essential for understanding differences between D. farinae and D. pteronyssinus with respect to immunoreactivity, protease activation mechanisms, association with microbes, and food utilization.
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Mapping of B and T cell epitopes of an allergen can be utilised in the development of alternative therapeutic modalities and diagnostics. The present study was aimed to identify B and T cell epitopes of Per a 10, a major cockroach allergen, by computational tools and subsequent validation by in vitro experiments. Per a 10 three-dimensional structure was homology modelled using structure of anionic trypsin from pacific chum salmon as a template. Seven B cell epitopes (B-P1 to B-P7) were predicted by sequence and structure based methods. Three T cell epitopes (T-P8 to T-P10) were predicted by binding score and inhibitory concentration dependent prediction tools. Predicted epitopes were synthesized and biological activity was assessed by ELISA, ELISA inhibition and PBMC proliferation assays. B cell peptides B-P5, B-P6 and B-P7 showed significantly high IgE binding with pooled and individual cockroach hypersensitive patients’ sera while the T cell peptides did not show IgE binding. ELISA inhibition was performed to determine the potency of the predicted peptides. Fifty nanogram of peptide B-P7 was required for 50% IgE binding inhibition of surface bound Per a 10 whereas seventy five nanogram and ninety nanogram of B-P5 and B-P6 were required for the same respectively. Upon stimulation with T-P8 and T-P10 peptides, PBMCs from cockroach allergic patients’ (n = 15) showed significant lymphocyte proliferation and induced IL-4 and IL-5 cytokine release in the culture supernatant demonstrating Th2 dominant cell mediated response of predicted T cell peptides. In conclusion, Per a 10 3-D structure obtained by homology modelling was used to identify B and T cell epitopes, followed by in vitro validation. The identified peptides can be potentially used in designing diagnostics and therapies for cockroach allergy.
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Citation 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).
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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.

^☆☆: Supported by the Australian National Health and Medical Research Council and the Asthma Foundation of Western Australia.

^★: Reprint requests: Geoffrey A. Stewart, Department of Microbiology, University of Western Australia, Nedlands, Western Australia 6907.

^★★: 1/1/73114

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The isolation and characterization of a novel collagenolytic serine protease allergen (Der p 9) from the dust mite Dermatophagoides pteronyssinus☆,☆☆,★,★★

Abstract

Section snippets

Extracts and chemicals

Determination of protease activity

Isolation of Der p 9 from D. pteronyssinus SGM

DISCUSSION

Acknowledgements

J ALLERGY CLIN IMMUNOL

Lancet

Biochem Med

Anal Biochem

Methods Enzymol

J ALLERGY CLIN IMMUNOL

J Mol Biol

J ALLERGY CLIN IMMUNOL

Biochem Biophys Res Commun

Biochim Biophys Acta

Insect Biochem

Sequence analysis of cDNA coding for a major house dust mite allergen, Der p I. Homology with cysteine proteases

J Exp Med

The group III allergen from the house dust mite Dermatophagoides pteronyssinus is a trypsin-like enzyme

Immunology