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1.
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.  相似文献   

2.
3.
Presentation and CD4(+) T cell responses to Ag in the context of MHC class II molecules require processing of native proteins into short peptide fragments. Within this pathway, IFN-gamma-inducible lysosomal thiol reductase (GILT) functions to catalyze thiol bond reduction, thus unfolding native protein Ag and facilitating further processing via cellular proteases. In contrast with professional APCs such as B cells, class II-positive human melanomas expressed relatively little to no GILT protein or mRNA. Tumor cell GILT expression was partially restored with IFN-gamma treatment but unlike other genes required for class II Ag presentation, GILT was not regulated by CIITA. Rather, studies revealed STAT1 plays a direct role in IFN-gamma-inducible GILT expression. These results define a molecular mechanism for the uncoupled regulation of MHC class II genes and the processing enzyme GILT in human melanomas.  相似文献   

4.

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.  相似文献   

5.
Ag processing and presentation via MHC class II is essential for activation of CD4(+) T lymphocytes. gamma-IFN-inducible lysosomal thiol reductase (GILT) is present in the MHC class II loading compartment and has been shown to facilitate class II Ag processing and recall responses to Ags containing disulfide bonds such as hen egg lysozyme (HEL). Reduction of proteins within the MHC class II loading compartment is hypothesized to expose residues for class II binding and protease trimming. In vitro analysis has shown that the active site of GILT involves Cys(46) and Cys(49), present in a CXXC motif that shares similarity with the thioredoxin family. To define the functional requirements for GILT in MHC class II Ag processing, a GILT-deficient murine B cell lymphoma line was generated and stably transduced with wild-type and cysteine mutants of GILT. Intracellular flow cytometric, immunoblotting, and immunofluorescence analyses demonstrated that wild-type and mutant GILT were expressed and maintained lysosomal localization. Transduction with wild-type GILT reconstituted MHC class II processing of a GILT-dependent HEL epitope. Mutation of either Cys(46) or Cys(49) abrogated MHC class II processing of a GILT-dependent HEL epitope. In addition, biochemical analysis of these mutants suggested that the active site facilitates processing of precursor GILT to the mature form. Precursor forms of GILT-bearing mutations in Cys(200) or Cys(211), previously found to display thiol reductase activity in vitro, could not mediate Ag processing. These studies demonstrate that the thiol reductase activity of GILT is its essential function in MHC class II-restricted Ag processing.  相似文献   

6.
Cloned human CD4+ T cell lines specific for the house dust mite Dermatophagoides pteronyssinus were used to map minimal T cell activation-inducing epitopes on the Group I allergen in D. pteronyssinus extracts (Der p I) molecule. Most of these Der p I-specific T cell clones expressed different TCR V alpha and V beta gene products. Using recombinant deletion proteins, three T cell epitopes were identified on the Der p I molecule; p45-67 and p117-143 were recognized by HLA-DR7-restricted T cells, whereas p94-104 was recognized in the context of HLA-DR2, DRw11 (DR5), and -DR8 molecules. This degenerate class II MHC restriction appears to be due to shared Phe and Asp residues at positions 67 and 70, respectively, in the third variable domain of the HLA-DR beta chain. All three T cell epitopes induced Th2-like cytokine production profiles by the Der p I-specific T cell clones, which were characterized by the production of very high levels of IL-4 and IL-5, as compared with those secreted by tetanus toxin-specific T cell clones derived from the same patients, but no or low amounts of IL-2 and IFN-gamma. This Th2-like production profile was, however, not an intrinsic property of the Der p I-specific T cells, but was dependent upon their mode of activation. Stimulation with Con A also induced very low or no measurable levels of IL-2 and IFN-gamma, whereas activation with TPA and the calcium ionophore A23187 resulted in the production of high levels of IL-4, IL-5, IL-2, and IFN-gamma. These results indicate that Der p I-specific T cell clones are not defective in their capacity to produce high levels of Th1 cytokines.  相似文献   

7.
NK cell clones obtained from three different donors were tested for their ability to present soluble proteins to Ag-specific T cell clones. All NK clones were CD2+CD3-CD56+, whereas the expression of CD16 varied from clone to clone. The NK cell clones were able to process and present tetanus toxoid (TT) to TT-specific T cell clones in a class II HLA restricted manner. The capacity of NK cell clones to function as APC was also observed using the house dust mite allergen Der p I and the Der p I-derived peptide Val89-Cys117. As with EBV-transformed B cell line, NK cell clones could present the peptide 3-13 derived from the 65-kDa heat shock protein of Mycobacterium leprae, but they were unable to present the whole M. leprae Ag. Freshly isolated NK cells, IL-2-activated NK cells, and NK cell lines expanded in vitro could also process and present TT. The ability of the different NK populations to act as accessory cells correlated with their levels of class II HLA expression. These data demonstrate that NK cell clones can efficiently function as APC, however they may be restricted in the types of Ag that they can process.  相似文献   

8.
An enzyme linked immunosorbent assay system using a monoclonal antibody, 15E11, specific for a major allergen Der f II in house dust mite, was developed. This system detected only Der f II in the presence of Der p II and other allergens. The Der f II contents in several house dust samples significantly correlated with the numbers of the mites in the same house dust samples (n = 14, r = 0.88, p < 0.001). These data showed that this system was useful for specific measurement of Der f II in house dusts.  相似文献   

9.
Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 A. The active site is located in a large cleft on the surface of the molecule. The 80-aa pro-peptide adopts a unique fold that interacts with the active site cleft and a substantial adjacent area on the mature region, excluding access to the cleft and the active site. Studies performed using crossed-line immunoelectrophoresis and IgE inhibition experiments indicated that several epitopes are covered by the pro-peptide and that the epitopes on the recombinant mature molecule are indistinguishable from those on the natural one. The structure confirms previous results suggesting a preference for aliphatic residues in the important P2 position in substrates. Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1.  相似文献   

10.
House dust mites produce inhalant allergens of importance to allergic patients. We measured the major group 1 allergens, Der p 1 and Der f 1, from the house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farina, respectively in 100 randomly selected domestic homes from Cheonan, Korea. Dust samples were collected by vacuuming from the living room floor and 1 mattress in each home. Der p 1 and Der f 1 were measured by double monoclonal ELISA. Der p 1 levels were very low, with geometric mean levels for floors and mattresses being 0.11 microgram/g (range: 0.01-4.05) and 0.14 microgram/g (range: 0.01-30.0), respectively. Corresponding levels of Der f 1 were higher, 7.46 microgram/g (range: 0.01-262.9) and 10.2 microgram/g (range: 0.01-230.9) for floors and mattresses, respectively. D. farinae appears to be the dominant house dust mite in Cheonan.  相似文献   

11.
BACKGROUND AND OBJECTIVE: T1DM and asthma are mediated by opposite arms of the cellular immune system namely T helper (Th)1 and Th2 CD4(+) cells, respectively. Our aim was to characterize the Th1/Th2 cytokine balance in patients with both T1DM and asthma. METHODS: Forty-four patients, mean age 19 years were matched by gender and age, to 4 paired groups: T1DM and asthma, asthma only, T1DM only and healthy controls. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with disease-specific recombinant antigens; glutamic acid decarboxylase and house dust mite (Der p1 antigen) for T1DM and asthma, respectively, and non-specific mitogens; phytohemaglutinin (PHA), tetanus toxin and anti-CD3 mAb. ELISPOT and ELISA technique were used to determine INF-gamma, IL-2, IL-4, IL-13 and IL-10 expression. RESULTS: Patients with T1DM and asthma demonstrated a similar cytokine pattern but lower Th1/Th2 ratio compared to patients with T1DM only. The Th2 cytokines response to Der p1 was enhanced in patients with both diseases compared to controls. The IL-10 overall secretion was higher in patients with both diseases compared to one disease only. CONCLUSION: The Th1 and Th2 secretory pattern of patients with T1DM and asthma combines features of both diseases suggesting a unique Th1/Th2 balance.  相似文献   

12.
In previous studies, we demonstrated that allergenic house dust mite proteases are potent inducers of proinflammatory cytokines from the respiratory epithelium, although the precise mechanisms involved were unclear. In this study, we investigated whether this was achieved through activation of protease-activated receptor (PAR)-1 or -2. Pretreatment of A549 respiratory epithelial cells with the clinically important cysteine protease allergen, Der p 1, ablated subsequent PAR-1, but not PAR-2 agonist peptide-induced IL-6 and IL-8 release. HeLa cells transfected with the plasmid coding for PAR-2, in contrast to PAR-1, released significant concentration of IL-6 after exposure to Der p 1. Exposure of HeLa cells transfected with either PAR-1/enhanced yellow fusion protein or PAR-2/enhanced yellow fusion protein to Der p 1 caused receptor internalization in the latter cells only, as judged by confocal microscopy with re-expression of the receptor within 120-min postenzyme exposure. Der p 1-induced cytokine release from both A549 and transfected HeLa cells was accompanied by changes in intracellular Ca(2+) concentrations. Desensitization studies showed that Der p 1 pretreatment of the A549 cells resulted in the abolition of both trypsin- and PAR-2 agonist peptide-induced Ca(2+) release, but not that induced by subsequent exposure to either thrombin or PAR-1 agonist peptide. These data indicate for the first time that the house dust mite allergen Der p 1-induced cytokine release from respiratory epithelial cells is, in part, mediated by activation of PAR-2, but not PAR-1.  相似文献   

13.
Amino acid sequence variations have possible influences on the allergenicity of allergens and may be important factors in allergen standardization. This study was undertaken to investigate the sequence polymorphisms of group 1 and 2 allergens from Korean isolates of the house dust mites Dermatophagoides farinae and D. pteronyssinus. cDNA sequences encoding group 1 and 2 allergens were amplified by RT-PCR and compared the deduced amino acid sequences. Der f 1.0101, which appeared in 64.0 % of the 50 sequences analyzed, was found to be predominant. Among the Der p 1 sequences, Der p 1.0102 and 1.0105 were predominant (58 %). Among the Der f 2 sequences, Der f 2.0102 (40.7 %) and a new variant with Gly at position 42 (27.8 %) were predominant. The deduced amino acid sequences of 60 Der p 2 clones were examined, and 28 variants with 1-5 amino acid substitutions were found. Interestingly, all of the Der p 2 sequences had Thr instead of Lys at position 49. Two variants (Leu40, Thr49, and Asn114 (26.6 %); Val40, Thr49, and Asn114 (20.0 %)) were found to be the most predominant forms of Der p 2. Der p 1 has a high rate of sporadic substitutions and the group 2 allergens show a more regular pattern with orderly associations of amino acid substitutions. Der f 1 and Der p 2 from Korean mite isolates have unique amino acid sequence polymorphisms. These findings provide important data for house dust mite allergen standardization.  相似文献   

14.
This study is the first report on the epidemiological status of house dust mite (HDM) allergy in Pothwar, Pakistan. Allergy data of 2087 symptomatic patients were obtained, of whom 1706 (81.7%) patients were skin-prick-test positive for HDM allergens. This percentage was significantly higher than for pollen and food allergens. In the results of this study Dermatophagoides farinae (61%) and D. pteronyssinus (29%) were the predominant species in the study area. Besides these pyroglyphids, predatory Cheyletus sp. (10%) and an oribatid mite sp. (1%) were also observed. Random and patients’ houses showed 87.4 and 87.1% positive mite infestation, respectively. Mean (±?SEM) D. farinae counts per g of dust in random samples was 235.4 ± 7.93 compared to 274.7 ± 10.78 from patients’ homes. Mean D. pteronyssinus counts from random houses compared to patients’ houses were 115.0 ± 4.57 and 124.6 ± 5.76, respectively. Mite counts depicted seasonal variation, with peaks during monsoon season. ELISA results of dust samples demonstrated that of the dust samples with?>?10 µg/g of dust, the threshold value described as a risk factor for developing asthma, 57.6% had Der f1 and 20% Der p1 allergen load. Mean Der f1 burden was significantly higher than Der p1, with maximum levels during monsoon and autumn seasons. This research established a better awareness about the epidemiological status of HDM allergy and prevalence of allergy causing HDM species in Pakistan.  相似文献   

15.
屋尘螨Ⅰ类变应原Der p1的体内定位   总被引:8,自引:4,他引:4  
对过敏性疾病主要过敏原屋尘螨Dermatophagoides pteronyssinusⅠ类变应原(Der p1)进行了抗原定位研究。选用经表达和纯化的Der p1抗原蛋白,按常规方法免疫小鼠,分离血清获抗重组Der p1的抗体(Ⅰ抗),用抗鼠IgG荧光抗体为Ⅱ抗,屋尘螨经石蜡切片,在荧光显微镜下对其特异性变应原的定位进行观察。HE染色显示屋尘螨消化系统占据大部分体腔。组织免疫荧光发现屋尘螨Ⅰ类变应原主要定位于螨的中肠组织及肠内容物,而螨的生殖系统、排泄系统等脏器以及几丁质甲壳均呈阴性反应。据此认为屋尘螨Ⅰ类变应原存在于螨的肠内容物和中肠组织中。  相似文献   

16.
As an alternative approach to conventional allergen-specific immunotherapy, transgenic rice seed expressing a major house dust mite (HDM) allergen, Der p 1, was developed as an edible vaccine. The C-terminal KDEL-tagged Der p 1 allergen specifically accumulated in seed endosperm tissue under the control of the endosperm-specific GluB1 promoter. Der p 1 reached a maximum concentration of 58 μg/grain and was deposited in the endoplasmic reticulum (ER)-derived protein body I (PB-I). Plant-derived Der p 1 was posttranslationally modified with high-mannose-type glycan structures. Glycosylated Der p 1 displayed reduced IgE binding capacity in comparison with its unglycosylated counterpart in vitro. Our results indicate that transgenic Der p 1 rice seeds are a safe, potential oral delivery vaccine for the treatment of HDM allergy.  相似文献   

17.
In rodents, airway dendritic cells (DCs) capture inhaled Ag, undergo maturation, and migrate to the draining mediastinal lymph nodes (MLN) to initiate the Ag-specific T cell response. However, the role of human DCs in the pathogenesis of the Th2 cell-mediated disease asthma remains to be clarified. Here, by using SCID mice engrafted with T cells from either house dust mite (HDM)-allergic patients or healthy donors, we show that DCs pulsed with Der p 1, one of the major allergens of HDM, and injected intratracheally into naive animals migrated into the MLN. In the MLN, Der p 1-pulsed DCs from allergic patients induced the proliferation of IL-4-producing CD4(+) T cells, whereas those from healthy donors induced IFN-gamma-secreting cells. In reconstituted human PBMC-reconstituted SCID mice primed with pulsed DCs from allergic patients, repeated exposure to aerosols of HDM induced 1) a strong pulmonary inflammatory reaction rich in T cells and eosinophils, 2) an increase in IL-4 and IL-5 production in the lung lavage fluid, and 3) increased IgE production compared with that in mice primed with unpulsed DCs. All these effects were reduced following in vivo neutralization of the CCR7 ligand secondary lymphoid tissue chemokine. These data in human PBMC-reconstituted SCID mice show that monocyte-derived DCs might play a key role in the pathogenesis of the pulmonary allergic response by inducing Th2 effector function following migration to the MLN.  相似文献   

18.
The mechanisms that cause T cell recruitment into inflamed airways of asthmatic individuals are poorly understood. It has been shown previously that both natural exposure to allergen and challenge in the laboratory induce T cell accumulation in the bronchial mucosa of sensitized asthmatics. To study the mechanisms involved in this process, we have used an explant model in which bronchial biopsies taken from mild atopic asthmatic volunteers during fiberoptic bronchoscopy were stimulated in culture for 24 h by the common aeroallergen house dust mite (Dermatophagoides pteronyssinus (Der p)). Analysis of culture supernatants showed that stimulation with Der p significantly enhanced both the generation of T cell chemotactic activity by the mucosal tissue, as assayed in microchemotaxis chambers, and the production of IL-16 and RANTES. Neutralization experiments showed that IL-16 contributed more to the chemotactic activity than RANTES. The fusion protein CTLA-4-Ig, blocking B7:CD28 costimulation, and dexamethasone both significantly reduced the ex vivo production of chemotactic activity and release of IL-16 and RANTES. The proteasome inhibitor Cbz-Ile-Glu(OtBu)-Ala-leucinal also had a significant inhibitory effect on T cell chemotactic activity and IL-16 but not RANTES generation, indicating a role for nuclear factor NF kappa B activation. These results indicate that allergen stimulates cells within the bronchial mucosa to increase IL-16 and RANTES release, both of which contribute to T cell accumulation in asthmatic airways. The allergen-induced chemotactic activity is dependent on cell activation via CD28 and involves, at least partly, NF-kappa B.  相似文献   

19.
House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophagoides pteronyssinus, were produced. Four monoclonal antibodies produced were species-specific and did not cross-react to the D. farinae crude extract. Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides. A (aa residues 1-49), B (44-93), and C fragment (84-126). Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p 2 in house dust. The sensitivity limit was 4 ng/ml with rDer p 2 and 8 micrograms/ml with the D. pteronyssinus crude extract. The result suggested that the assay using monoclonal antibodies against rDer p 2 could be useful for the environmental studies and for the standardization of mite allergen extracts.  相似文献   

20.
Chaperone-like activity and surface hydrophobicity of 70S ribosome   总被引:2,自引:0,他引:2  
Singh R  Rao ChM 《FEBS letters》2002,516(1-3):234-238
Allergen from the house dust mite (Dermatophagoides sp.) is a major trigger factor of allergic disorders, and its characterization is crucial for the development of specific diagnosis or immunotherapy. Here we report the identification of a novel dust mite (Dermatophagoides farinae) antigen whose primary structure belongs to the gelsolin family, a group of actin cytoskeleton-regulatory proteins. Isolated mite cDNA, termed Der f 16, encodes 480 amino acids comprising a four-repeated gelsolin-like segmental structure, which is not seen in conventional gelsolin family members. Enzyme immunoassay indicated that recombinant Der f 16 protein, prepared using an Escherichia coli expression system, bound IgE from mite-allergic patients at 47% (8/17) frequency. This is the first evidence that the gelsolin family represents a new class of allergen recognizable by atopic patient IgE.  相似文献   

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