A comparative study of allergenic and potentially allergenic enzymes fromDermatophagoides pteronyssinus,D. farinae andEuroglyphus maynei

The presence of the enzymatically active allergens equivalent toDer p I (cysteine protease),Der p III (serine protease) and amylase in extracts ofDermatophagoides pteronyssinus, D. farinae andEuroglyphus maynei was determined using appropriate enzymatic techniques. Biochemical equivalents of all three allergens were present in each extract studied. Studies also showed that the mite extracts contained a variety of other biochemically active enzymes including trypsin, chymotrypsin, carboxypeptidase A and B, glucoamylase and lysozyme. Marked differences in the relative concentrations of some of these enzymes in different mite extracts were observed, particularly trypsin and carboxypeptidase A. The enzymes were physicochemically similar to equivalent enzymes from vertebrate and invertebrate sources. Chromatofocusing studies of faecal extracts derived fromD. pteronyssinus andD. farinae showed that several isoforms of each enzyme were present. The data indicated that there were more trypsin isoforms, with pI over a wider range, in extracts prepared fromD. pteronyssinus. Proteases and carbohydrases were also found in extracts prepared from faecally enriched material suggesting that they were endoperitrophic and associated with mite digestion. The data suggest that not only are the group I, III and amylase allergens a consistent feature of most pyroglyphid dust mites but also that other proteases and carbohydrases present in mite faeces are allergenic.     

IgE reactivity to Acarus siro extract in Korean dust mite allergic patients

Although specific IgE to the storage mite Acarus siro is often detected, there are no detailed studies on IgE reactivity to A. siro in Korea. This study was undertaken to investigate the cross-reactivity to the mite species Dermatophagoides pteronyssinus, Dermatophagoides farinae, Tyrophagus putrescentiae, and A. siro in Korean mite allergic patients. Specific IgE values were determined for the four mite species and a competitive inhibition test was performed for mite extracts using the ImmunoCAP system. The IgE value to D. farinae was the highest among the four mite species tested. There was a strong correlation in the IgE value between house dust mites (D. pteronyssinus and D. farinae) and between storage mites (A. siro and T. putrescentiae). IgE reactivity to A. siro was inhibited by D. farinae and T. putrescentiae extract. Dermatophagoides farinae extract was the strongest inhibitor of IgE binding to A. siro extract, indicating that IgE reactivity to A. siro extract is a cross-reaction caused by sensitization to D. farinae. Strong IgE reactive components were observed in D. farinae and T. putrescentiae extract by SDS-PAGE and IgE immunoblotting. However, no strong IgE-binding component was observed for A. siro. Dermatophagoides farinae is the main source of mite allergens that cause sensitization in Korea. Serum IgE from some of the house dust mite-sensitized patients showed positive responses to storage mite allergens by cross-reaction. Therefore, it is necessary to pay special attention to the diagnosis of mite allergies.     

Enzymatic Activities of Allergen Extracts from Three Species of Dust Mites and Cockroaches Commonly Found in Korean Home

Allergen extracts from dust mites and cockroaches commonly found in Korean homes were used to evaluate their enzymatic activity as they are believed to influence allergenicity. Allergen extracts were prepared from 3 dust mite species (Dermatophagoides farinae, D. pteronyssinus, and Tyrophagus putrescentiae) and 3 cockroach species (Blattella germanica, Periplaneta americana, and P. fuliginosa) maintained in the Korea National Arthropods of Medical Importance Resource Bank. Proteins were extracted in PBS after homogenization using liquid nitrogen. The activities of various enzymes were investigated using the API Zym system. No significant difference in phosphatase, lipase, or glycosidase activity was observed among the 6 allergen extracts, but much difference was observed in protease activity. Protease activity was assessed in more detail by gelatin zymography and the EnzChek assay. Extract from T. putrescentiae showed the highest protease activity, followed by those of the cockroach extracts. Extracts from D. farinae and D. pteronyssinus showed only weak protease activity. Gelatinolytic activity was detected mainly in a 30-kDa protein in D. farinae, a 28-kDa protein in D. pteronyssinus, a > 26-kDa protein in T. putrescentiae, a > 20-kDa protein in B. germanica, and a > 23-kDa protein in P. americana and P. fuliginosa. The information on various enzymatic activities obtained in this study may be useful for future studies. In particular, the strong protease activity found in cockroach extracts could contribute to sensitization to cockroach allergens, which is known to be associated with the development of asthma.     

Age structure and dynamics of house dust mite populations

Age structure—the relative numbers of eggs, immatures and adults—in populations of the house dust mitesDermatophagoides pteronyssinus andEuroglyphus maynei was investigated in four sequential monthly samples taken from mattresses in each of eight homes in Glasgow, Scotland. Additionally, age structure ofD. pteronyssinus was determined in samples taken bimonthly for 6 months from nine quadrats of a double mattress. It was found that although age structure varied considerably with time, forD. pteronyssinus in different homes the most common structure was one in which immatures were dominant, then eggs and then adults (31% of samples). Immatures or eggs were dominant in 75% of samples. ForE. maynei the age structure was quite different: the most common structure was one in which adults were dominant, then immatures and then eggs (69% of samples). In different quadrats of a double mattress, mean age structure ofD. pteronyssinus underwent a shift towards higher proportions of immatures and then eggs during the sampling period, which reflected the increase in population density detected during this period.Life and fecundity tables were constructed forD. pteronyssinus andE. maynei using previously-available in vitro data on fecundity and survivorship rates and hypothetical values based on means derived from a number of studies. From the tables the stable age distributions were calculated and compared with the age structures of the natural populations. It was found that mean age structure of natural populations ofD. pteronyssinus was fairly close to the predicted stable age distribution, but those ofE. maynei indicated the populations were in decline during the sampling period, a fact confirmed by abundance data. The concept that the rate of increase of house dust mite populations can be estimated by determining age structure of mites isolated from dust samples was explored using the hypothetical population parameters ofD. pteronyssinus. It was predicted that quite large differences in fecundity and mortality would not drastically alter the proportions of eggs, immatures and adults in stable populations.Eggs as components of the house dust mite population are considered seriously for the first time. Those ofD. pteronyssinus andE. maynei were identified and differentiated by allometry. It is stressed that forD. pteronyssinus, during the sampling period, half or more of the mites in a dust sample may be represented as eggs, and to ignore them is to deliberately make a less accurate estimate of population density than could be otherwise achieved.     

Do the microorganisms from laboratory culture spent growth medium affect house dust mite fitness and microbiome composition?

The interaction of house dust mites(HDM)and microorganisms is the key factor in the survival of these mites in human-made environments.Spent growth medium(SPGM)provides the rest of the dict,along with dead mite bodies and microorganisms.SPGM represents a source of microorganisms for the recolonization of mite food and the mite digestive tract.An experiment was performed to observe how adding SPGM to the HDM diet affects HDM population growth,the microbiome composition and the microbial respiration in microcosms.We analyzed American house dust mite(Dermatophagoides farinae)and European house dust mite(Dermatophagoides pleronyssinus)originating from control diets and diets treated with an extract of SPGM from 1-and 3-month-old mite cultures.The microbiome was described using 16S and 18S barcode sequencing.The composition of the bacterial and fiungal microbiomes differed between the HDM species,but the SPGM treatment influenced only the bacterial profile of D.farinae.In the D.farinae microbiome of specimens on SPGM-treated dicts compared to those of the control situation,the Lactobacillus profile decreased,while the Candinium,Staphylococ-cus,Acinetobacter,and Sphingomonas profiles increased.The addition of SPGM extract decreased the microbial respiration in the microcosms with and without mites in almost all cascs.Adding SPGM did not influence the population growth of D.farinae,but it had a variable effect on D.pteronyssimus.The results indicated that the HDM are marginally influenced by the microorganisms in their feces.     

Distribution and abundance of house-dust mites (Acarina: Pyroglycphidae) in Rome,Italy

For the first time in Rome, house-dust mite infestation was studied in 90 randomly selected houses. In each house, mite infestation was assessed in three sites: mattress, bedroom and living room. In total, 87.8% of the sampled houses were positive for dust mites. In the houses infested, 11.4% showed densities of >100 mites/g of dust, 15.2% registered densities between 50 and 99, and in the remaining houses (73.4%), the densities were between 1 and 49 mites/g dust. The percentages of infested houses were positively correlated with the relative humidity (RH) values (r=0.89,P=0.02). At the lowest range of RH (between 46 and 50), the infestation was 50% and at the highest range of RH (between 73 and 78) it was 100%. The mattress was significantly the most infested (71.1%) of the tested sites. Only wool and spring mattresses were infested, and they did not show any significant differences in mite concentrations.Dermatophagoides farinae was the most abundant species (53.1%), followed byGlycyphagus domesticus (34.5%),D. pteronyssinus (5.2%), andEuroglyphus maynei (0.2%);D. farinae was also the most frequent species (56.9%). The remaining specimens (7.0%) were predator species commonly found in houses. The prevalence ofD. farinae in Rome is discussed.     

Mite species identification in the production of allergenic extracts for clinical use and in environmental samples by ribosomal DNA amplification

The identification of allergy‐causing mites is conventionally based on morphological characters. However, molecular taxonomy using ribosomal DNA (rDNA) may be particularly useful in the analysis of mite cultures and purified mite fractions in the production of allergenic extracts. Full‐length internal transcribed spacers (ITS1 and ITS2) were obtained from Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides microceras and Euroglyphus maynei (Astigmata: Pyroglyphidae), Glycyphagus domesticus and Lepidoglyphus destructor (Astigmata: Glycyphagidae), Tyrophagus fanetzhangorum, Tyrophagus putrescentiae, Tyrophagus longior, Tyrophagus neiswanderi, Acarus farris and Acarus siro (Astigmata: Acaridae), and Blomia tropicalis (Astigmata: Echymopodidae), using mite‐specific primers. Polymerase chain reaction (PCR) products were digested with HpaII and RsaI restriction enzymes in order to produce species‐specific PCR restricted fragment length polymorphism (RFLP) profiles. A semi‐nested re‐amplification step was introduced before the RFLP in order to apply the method to environmental samples. Results demonstrate that rDNA sequences can be used for the unambiguous identification of mite species. The PCR–RFLP system allows the identification of species in purified mite fractions when the availability of intact adult mite bodies for morphological identification is limited. This reliable and straightforward PCR–RFLP system and the rDNA sequences obtained can be of use in the identification of allergy‐causing mite species.     

Structural biology of mite allergens

Mite allergens contribute to a significant proportion of human allergic symptoms, including asthma and rhinitis. The development of therapies to treat and prevent these symptoms depends largely on our understanding of the properties of these allergens. Much effort has been devoted to determining the structure and organization of mite allergens, particularly of the house dust mites, toward understanding their activities and how they elicit immunological responses in humans. Here, we review the structural biology of the major allergens from two species of house dust mites, Dermatophagoides farinae and D. pteronyssinus, as well as allergens from a storage mite, Blomia tropicalis. The knowledge gained from the structural biology of these allergens will enable progress in producing novel, more effective treatments for mite allergies based on specific immunotherapy approaches.     

Population growth and allergen accumulation of <Emphasis Type="Italic">Dermatophagoides pteronyssinus</Emphasis> cultured at 20 and 25°C

The house dust mites, Dermatophagoides pteronyssinus and D. farinae are cultured commercially and in research laboratories and material is harvested from these cultures to make extracts that are used for diagnosis, immunotherapy and research. Temperature and other climatic conditions can influence population growth rates, dynamics of allergen production, and the associated endotoxin, enzyme and protein levels of the mite material harvested from these cultures. Here we determined how temperature affected these parameters. Dermatophagoides pteronyssinus was cultured at 20 and 25°C at 75% relative humidity, and at 2-week intervals the concentrations of mites, Der p 1 and Der p 2 allergens, endotoxin, and selected enzymes were determined. Mite density increased exponentially but growth rate and final population density were greater at 25°C compared to 20°C. The combined allergen (Der p 1 + Der p 2) concentrations accumulated in the cultures at about the same rate at both temperatures. However, individual Der p 1 and Der p 2 accumulation rates varied independently at the two temperatures. Der p 1 accumulated faster at 20°C whereas Der p 2 accumulated faster at 25°C. The amount of Der p 1 in whole cultures was greater than the amount of Der p 2. The concentration of allergen for washed mites harvested from the cultures was much less than for the whole cultures. Our study demonstrated that temperature is an important factor in population growth and the dynamics of allergen production in cultured mites.     

Hyposensitization with Dermatophagoides pteronyssinus Antigen: Trial in Asthma Induced by House Dust

A double-blind clinical trial of hyposensitization with aqueous extracts of Dermatophagoides pteronyssinus (the house-dust mite) and human skin scales showed a substantial improvement in symptoms in 11 asthmatics allergic to house dust treated with the D. pteronyssinus extract and a reduction in their need for other therapy. Five patients were well for a year but six relapsed. These results contrasted with the generally unfavourable course of the patients treated with the extract of human skin scales. Asthma due to house-dust allergy may be substantially improved by hyposensitization with D. pteronyssinus extract.     

The Potential for a Blood Test for Scabies

Background

Scabies afflicts millions of people worldwide, but it is very difficult to diagnose by the usual skin scrape test, and a presumptive diagnosis is often made based on clinical signs such as rash and intense itch. A sensitive and specific blood test to detect scabies would allow a physician to quickly make a correct diagnosis.

Objective

Our objective was to profile the mite-specific antibodies present in the sera of patients with ordinary scabies.

Methods

Sera of 91 patients were screened for Ig, IgD, IgE, IgG and IgM antibodies to S. scabiei, as well as to the house dust mites Dermatophagoides farinae, D. pteronyssinus and Euroglyphus maynei.

Results

45%, 27% and 2.2% of the patients had measurable amounts of mixed Ig, IgG and IgE that recognized scabies mite antigens. However, 73.6% of the scabies patients had serum IgM that recognized scabies proteins, and all except two of them also had IgM that recognized all of the three species of dust mites. No patient had serum antibody exclusively reactive to scabies mite antigens.

Conclusions

Co-sensitization or cross-reactivity between antigens from scabies and house dust mites confounds developing a blood test for scabies.     

Population dynamics of mites of the family pyroglyphidae and micromycetes in laboratory cultures

Original data on the study of the population dynamics in allergenic house dust mites of the family Pyroglyphidae (Dermatophagoides pteronyssinus (Trouessart 1897) and D. farinae Hughes 1961) during long (37 weeks) joint cultivation with the micromycetes Aspergillus penicillioides Speg. in simple periodical cultures without addition of food (SPC) with different initial population density of mites are given. The micromycete A. penicillioides Speg., dominating in laboratory cultures of pyroglyphids, was cultivated in parallel without mites. It was found that during joint cultivation of A. penicillioides and mites, population dynamics of D. pteronyssinus and D. farinae in SPC depended on the initial population density of mites, which affected the duration of mite developmental stages and the degree of the maximal population density. Cultures of D. farinae developed more rapidly than cultures of D. pteronyssinus, independently of the initial population density (50 or 200 specimens per gram of the substrate). A high degree of the initial population density in both mite species resulted in the shortening of the lag-phase, in more rapid reaching of the maximal population density, and in the higher degree of the maximal population density. Population density of the fungus A. penicillioides did not depend on the presence of both mite species. On the basis of our own data and literary analysis, we assume that A. penicillioides can affect the ability of mites to explore the trophic substrate, the rate of the population development, and the degree of their maximal population density. Mites, in their turn, did not significantly affect the development of A. penicillioides in our experiments.     

Dermatophagoides farinae Allergens Diversity Identification by Proteomics

The most important indoor allergens for humans are house dust mites (HDM). Fourteen Dermatophagoides farinae allergens (Der f 1–3, 6, 7, 10, 11, 13–18, and 22) are reported although more than 30 allergens have been estimated in D. farinae. Seventeen allergens belonging to 12 different groups were identified by a procedure of proteomics combined with two-dimensional immunoblotting from D. farina extracts. Their sequences were determined by Edman degradation, mass spectrometry analysis, and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests, immunoblots, basophil activation test, and skin prick tests. Eight of them are the first report as D. farinae allergens. The procedure of using a proteomic approach combined with a purely discovery approach using sera of patients with broad IgE reactivity profiles to mite allergens was an effective method to investigate a more complete repertoire of D. farinae allergens. The identification of eight new D. farinae allergens will be helpful for HDM allergy diagnosis and therapy, especially for patients without response for HDM major allergens. In addition, the current work significantly extendedthe repertoire of D. farinae allergens.The house dust mites (HDM)¹ are major sources of indoor allergens for humans, which induce asthma, rhinitis, dermatitis, and other allergic diseases (1). Extensive studies have been conducted to understand the biological, chemical, and structural properties of dust mite allergens. Most of the best characterized allergens are from dust mites Dermatophagoides pteronyssinus and D. farinae (Acari: Pyroglyphidae). Twenty-three groups of dust mite allergens are listed in the (IUIS) nomenclature data set, and 21 of them have been identified from Dermatophagoides spp (http://www.allergen.org/). There is an extreme diversity of dust mite allergens. Western blotting studies with human sera containing high levels of anti-mite IgE showed more than 32 bands with molecular weights ranging from 11 to greater than 100 kDa (2). Two groups of mite allergens (group 1 and 2) have been extensively studied. They are a 25-kDa cysteine protease and a 14-kDa epididymal protein, respectively. More than 80% of humans with house dust mite allergy mount an IgE response to the group 1 and more than 90% to the group 2 (3–6).The group 1 and 2 molecules are major allergens in HDMs but about 20% of patients do not have IgE antibody to the two group allergens (3). It has been found that there are also many other HDM allergens containing high IgE binding activity although these are present in low and variable concentrations in mite extracts (minor allergens), usually at less than 1% of the group 1 and 2 allergens (3). Allergens present in low amount in mite extracts, which can induce high titers of IgE, suggest that they are potent at low concentration. Another possibility is that the amount of allergen required to induce allergic responses in the airways is more than that required to induce IgE. It has been estimated that there are at least 30 allergens in the extracts of D. farinae by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) combined with autoradiography analysis (7). Two-dimensional (2-D) immunoblotting has been applied to study mapping of D. farinae mite allergens (7). Seven allergens including Der f 1, Der f 2, Der f 3, Der f 4, Der f 5, and 2 high molecular mass allergens, which share significant homologies with allergen Mag 3 from D. farinae and with a chitinase from prawn Penaeus japonicus, have been identified from the 2-D immunoblotting analysis (7). Up to now, 14 allergens from D. farinae have been named. Most of them are in the molecular weight range of 14 to 60 kDa. Given the extreme diversity of mite allergens, many investigations with novel allergen identification are still in progress or are yet to be undertaken. It is well known that many mite allergens are not identified on the basis of two possible reasons: (1) it is difficult to purify and characterize minor allergens because they present in low concentration in mite extracts; (2) some minor allergens are neglected because of their minor amount or abilities to only induced allergy to a minor population. It is necessary to develop efficient procedure with high accuracy and resolution to purify and characterize allergens from mite extracts. In this work, 17 allergens or their isoforms have been identified from the mite extracts of D. farinae by a procedure of proteomics combined with two-dimensional immunoblotting. Eight of them are the first to be reported as mite allergens.     

Molecular polymorphisms of house dust mite allergens

Previous studies from this laboratory have described the primary amino acid sequences of the group I and group II allergens fromDermatophagoides pteronyssinus andD. farinae. This report concentrates on polymorphisms of allergens within a species. Firstly, four cDNA clones ofDer fII produced by polymerase chain reaction have been sequenced and are compared to the sequences published previously by ourselves and others. Although the sequences come from different sources, Australia and Japan, the overriding conclusion is one of similarity, with only two possible non-conservative changes in the six sequences. The nucleotides were also very conserved including the 3 untranslated regions, although some non-coding differences could be found which may provide a genetic marker. Experiments are reported to help define the group IIID. pteronyssinus allergens. Previous studies have characterised the group III ofD. farinae as a Mr 29-kDa molecule which can be defined by monoclonal antibodies. A Mr 17-kDa molecule ofD. pteronyssinus has been reported with an almost identical N-terminal sequence. Here it is described thatDer fIII isolated from different preparations of spent mite media by affinity chromatography have predominantly Mr 32-, 28- and 21-kDa forms which vary in degree from batch to batch. 83% of adults and 38% of children react with the preparation by radioimmune dot-blot. The difference between the children and adults is statistically significant and reactivity can be to at least the 32- and 28-kDa form. Antisera produced in mice against theDer fIII react toD. pteronyssinus mite extract by Western blotting primarily to a 32-kDa moiety, but also 28- and 21-kDa forms in some extracts.     

Sensitivity to house dust mite allergens and prevalence of allergy-causing house dust mite species in Pothwar,Pakistan

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.     

Clinical significance of dust mite allergens

Dust mites are an important cause of allergic diseases worldwide. The traces of Dermatophagoides farinae and Dermatophagoides pteronyssinus can be found all over the world, while Blomia tropicalis are more common allergenic mites in tropical areas. A variety of allergenic components in dust mites have been found, apart from the different positive rates of IgE reactions in dust mite allergic patients, their biological characteristics, effects on the innate immune system, and especially their distribution characteristics in patients are different. Studying the relationship between dust mite allergens and clinical significance will help for diagnosis of patients and formulation of corresponding Allergen Specific Immunotherapy.

     

Identification of house-dust mites

The term house-dust mite usually refers to those species of the mite family Pyroglyphidae, that are known to commonly occur, although sometimes regionally, in the dust of human dwellings. These species belong to five genera:Dermatophagoides, Euroglyphus, Hirstia, andMalayoglyphus. Related species ofDermatophagoides have the most world-wide occurrence, the commonest being:D. farinae, D. microceras, andD. pteronyssinus. A correct taxonomic identification of house-dust mites is very important, not only from a biological stand point, but also regarding the consequences of their respective allergenical properties. Several immuno-chemical studies revealed differences between the products of two hard to distinguish sibling species. A preliminary practical taxonomic key for the most common and important house-dust mites is presented.     

Alpha-Actinin Is a New Type of House Dust Mite Allergen

Main indoor allergens for humans are from house dust mites. There are more than 30 allergens in Dermatophagoides farinae but only fourteen allergens have been identified from this mite including Der f 1–3, 6, 7, 10, 11, 13–18, and 22. A native allergen protein (Der f 24, 90 kDa) was purified from D. farinae by gel filtration and anionic exchange liquid chromatography combined with IgE immunodetection. Its primary structure was determined by Edman degradation, mass spectrometry analysis and cDNA cloning. Enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, basophil activation test (BAT) and skin prick test (SPT) were performed to evaluate the allergenicity. It was identified as an alpha (α)-actinin containing a CaM-like domain with EF-hand motifs. Der f 24 reacted to sera from 85.4% (35/41) of patients on western blot analysis. It reduced ∼20% sera IgE reactivity to D. farinae extracts on a competitive ELISA. Eighty percent (8/10) of patients with D. farinae allergy showed positive reactions to Der f 24 in skin prick test. The expression of CD63 on basophils from patients was up-regulated by Der f 24 by ∼5.4-fold. Alpha-actinin was identified as a new type of house dust mite allergen. To the best of our knowledge, this is the first report of α-actinin as an allergen.     

Der p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus

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.