Neglected but Clinically Relevant Allergens in Korea.

PURPOSE OF REVIEW: Allergy diagnostics and immunotherapeutics in Asia heavily rely on imported products from Western countries, raising concerns about the accuracy and efficacy of these products for the management of Asian allergy patients. RECENT FINDINGS: Recent advancements in allergen research have led to the identification and characterization of novel allergens from indigenous Korean species. While some allergens share homology with well-known allergens, others lack counterparts in imported allergen extracts. Classifying regional allergens in Asia into three categories based on their cross-reactivity with imported allergens offers valuable insights. Highly cross-reactive allergens, such as oak allergens Que m 1 from Quercus mongolica and Que ac 1 from Q. acutissima, can be effectively substituted with the imported allergens. Allergens with partial cross-reactivity, like the Asian needle ant allergen Pac c 3 (Antigen 5), permit limited diagnostic value by the currently available products. Unique allergens, including the Japanese hop allergen Hum j 6 (pectin methylesterase inhibitor) and the silkworm pupa allergen Bomb m 4 (30 kDa hemolymph lipoprotein) lack alternatives in the available product list. Greater attention is needed, particularly for species listed as ecologically invasive in Western regions. Additionally, allergens from domestic fruits and vegetables causing pollen food allergy syndrome require characterization for the development of improved diagnostics.

Comparative Genomics Reveals Insights into the Divergent Evolution of Astigmatic Mites and Household Pest Adaptations.

Highly diversified astigmatic mites comprise many medically important human household pests such as house dust mites causing ∼1-2% of all allergic diseases globally; however, their evolutionary origin and diverse lifestyles including reversible parasitism have not been illustrated at the genomic level, which hampers allergy prevention and our exploration of these household pests. Using six high-quality assembled and annotated genomes, this study not only refuted the monophyly of mites and ticks, but also thoroughly explored the divergence of Acariformes and the diversification of astigmatic mites. In monophyletic Acariformes, Prostigmata known as notorious plant pests first evolved, and then rapidly evolving Astigmata diverged from soil oribatid mites. Within astigmatic mites, a wide range of gene families rapidly expanded via tandem gene duplications, including ionotropic glutamate receptors, triacylglycerol lipases, serine proteases and UDP glucuronosyltransferases. Gene diversification after tandem duplications provides many genetic resources for adaptation to sensing environmental signals, digestion, and detoxification in rapidly changing household environments. Many gene decay events only occurred in the skin-burrowing parasitic mite Sarcoptes scabiei. Throughout the evolution of Acariformes, massive horizontal gene transfer events occurred in gene families such as UDP glucuronosyltransferases and several important fungal cell wall lytic enzymes, which enable detoxification and digestive functions and provide perfect drug targets for pest control. This comparative study sheds light on the divergent evolution and quick adaptation to human household environments of astigmatic mites and provides insights into the genetic adaptations and even control of human household pests.

Allergenic characterization of Bomb m 4, a 30-kDa Bombyx mori lipoprotein 6 from silkworm pupa.

BACKGROUND: Silkworm pupa (SWP) food anaphylaxis has been described frequently in Asian countries. However, false-positive reactions by skin pricks and serum IgE (sIgE) tests to the extract complicate diagnosis, requiring identification of clinically relevant major allergens. OBJECTIVES: In this study, we characterized a novel SWP allergen, Bomb m 4, a 30-kDa lipoprotein, and evaluated its diagnostic sensitivity. METHODS: Bomb m 4 was identified by a proteomic analysis. This recombinant (r)Bomb m 4 was overexpressed in Escherichia coli, and the IgE reactivity by ELISA was compared with other reported allergenic proteins: Bomb m 1 (arginine kinase), 27-kDa glycoprotein, Bomb m 3 (tropomyosin) using the serum samples from 17 SWP allergic patients and 11 asymptomatic sensitized subjects. RESULTS: rBomb m 4-specific IgE was recognized by all 17 SWP allergic patients. The 27-kDa glycoprotein and Bomb m 1 sIgE were found in 35.3% and 0%, respectively, in the SWP allergic patients. ELISA sIgE reactivity increased significantly, when 4 M urea was added in serum samples. However, only 16% inhibition of sIgE reactivity to the whole SWP extract was exhibited by rBomb m 4, whereas more than 93% of self-inhibition of rBomb m 4 sIgE was obtained, possibly due to the low abundance of Bomb m 4 in the extract. Three linear epitopes (81-95, 191-205 and 224-238 residues) of rBomb m 4 were identified. These epitopes are shown to be released by pepsin digestion. Receiver operator characteristic (ROC) analysis showed the highest diagnostic value of Bomb m 4 followed by Bomb m 1, 27-kDa glycoprotein and Bomb m 3. CONCLUSION: Bomb m 4 is the major allergen of SWP allergic patients. It has cryptic epitopes which are exposed to IgE antibodies with digestive enzymes. This recombinant Bomb m 4 allergen permits exact diagnosis of SWP allergy.

Comparison of sensitization patterns to dust mite allergens between atopic dermatitis patients and dogs, and non-specific reactivity of canine IgE to the storage mite Tyrophagus putrescentiae.

House dust mite is a common cause of atopic dermatitis (AD) both in humans and dogs. Detection of serum IgE to allergens is commonly used to diagnose allergic diseases. However, false-positive reactions due to cross-reactivity and non-specific reactivity may lead to misdiagnosis. We compared human and canine IgE reactivities to mite component allergens. Canine IgE-reactive components of Dermatophagoides farinae and Tyrophagus putrescentiae were identified by tandem mass spectrometry. Recombinant proteins were produced and IgE reactivities to component allergens were assessed by ELISA and inhibition assays using sera from AD patients and dogs. Canine IgE-reactive proteins (Der f 1, Der f 11, Tyr p 4, Tyr p 8, Tyr p 11, Tyr p 28) were identified by proteome analysis. Most patients were sensitized to Der f 1 (93.3%) and Der f 2 (86.7%). Dogs showed high sensitization to Der f 2 (94.1%) and Der f 18 (84.6%). Both patients and dogs showed low IgE binding frequency to Tyr p 8, 43.3% and 4%, respectively. The ELISA inhibition study indicated that canine IgE reactivity to T. putrescentiae is mostly due to non-specific reaction and cross-reaction with D. farinae. Different IgE sensitization patterns were shown between allergic humans and dogs with AD, especially to Der f 18, for the first time in Korea. Furthermore, non-specific canine IgE reactivity to storage mite indicates the possibility of misdiagnoses. Standardizations focused on the major canine allergen content of extracts should be developed. This will allow precision diagnosis and individuated treatments for each patient and atopic dog.

Sensitization profile to sawtooth oak component allergens and their clinical implications.

OBJECTIVE: The component allergens from sawtooth oak, which is a main cause of tree pollinosis in Korea, have not been extensively characterized except Que ac 1. This study was undertaken to characterize the allergenic components from sawtooth oak pollen and investigate the diagnostic values of each component allergen. METHODS: Transcriptomic analysis was performed to identify the birch pollen allergen homologues from sawtooth oak pollen. Recombinant Que ac 1, 2, 3, 6, 7, and 8 were produced in an E. coli expression system. IgE reactivity to each allergen was examined by ImmunoCAP and ELISA using the sera of 50 Korean tree pollinosis patients. RESULTS: Six birch pollen allergen homologues were identified using transcriptome analysis, as follows: Que ac 1 (54.8% identity to Bet v 1), Que ac 2 (79.7% to Bet v 2), Que ac 3 (24.9% to Bet v 3), 6 (71.3% to Bet v 6), Que ac 7 (80.9% to Bet v 7), and Que ac 8 (78.9% to Bet v 8). Que ac 1 sIgE was the most frequently recognized (84.0%), followed by Que ac 2 (12.0%), Que ac 3 (6.0%), and three other allergens (2.0% each). Que ac 1 was a dominant allergen affecting 83.7% of patients suffering from allergic rhinoconjunctivitis, and 92.9% of pollen food allergy syndrome patients. CONCLUSION: Five novel IgE reactive components of sawtooth oak were characterized using transcriptome analysis. Que ac 1 is the single most important component allergen of sawtooth oak pollen.

Efficacy of transdermal immunotherapy with biodegradable microneedle patches in a murine asthma model.

BACKGROUND: House dust mite (HDM) is a well-known cause of asthma. Allergen-specific immunotherapy (AIT) can only modify the natural course of the disease. Conventional routes of HDM AIT are subcutaneous or sublingual. Subcutaneous immunotherapy (SCIT) has a disadvantage of systemic hypersensitive reaction, and the sublingual immunotherapy has a disadvantage of local allergic reaction and low drug adherence. OBJECTIVE: To overcome the weak points of conventional AIT, we developed a HDM loaded biodegradable microneedle patch (MNP) for transdermal immunotherapy (TDIT). We aim to demonstrate the efficacy of TDIT in murine asthma model triggered by HDM compared with conventional SCIT. METHODS: To make HDM asthma mouse model, 5-week-old BALB/c female mice were sensitized and challenged by intranasal administration of HDM. The mice were divided into 5 groups: sham, asthma, low (10 µg) and high dose (100 µg) SCIT, and TDIT (10 µg). To make HDM loaded MNP, droplet-born air blowing method was used. Airway hyperresponsiveness and allergic inflammation markers were analysed by bronchoalveolar lavage fluid, immunohistochemistry, serum immunoglobulin (Ig) analysis, and lung cytokine assays. RESULTS: Airway hyperresponsiveness was ameliorated by TDIT. Eosinophilic inflammation in bronchoalveolar lavage was improved without adverse reactions. Reduction of Th2 (IL-4, IL-5, and IL-13) cytokines, and HDM-specific IgE, induction of Treg (IL-10, TGF-ß), Th1 (IFN-γ) cytokines were observed. Eosinophilic infiltration, goblet cell hyperplasia, and subepithelial fibrosis were also alleviated by TDIT. These changes were more significant in the TDIT group than in subcutaneous AIT group. CONCLUSION: In conclusion, HDM loaded biodegradable TDIT is a novel treatment option to treat asthma which showed more effectiveness and may have better safety profiles than conventional SCIT.

Allergen content in German cockroach extracts and sensitization profiles to a new expanded set of cockroach allergens determine in vitro extract potency for IgE reactivity.

BACKGROUND: Cockroach allergens are an important cause of IgE-mediated sensitization in inner-city asthmatic patients. However, cockroach extracts used for diagnosis and immunotherapy are not standardized. OBJECTIVE: We sought to determine the allergen content of nonstandardized German cockroach extracts and the levels of sensitization to an expanded set of cockroach allergens as determinants of in vitro extract potency for IgE reactivity. METHODS: Twelve German cockroach extracts were compared for allergen content and potency of IgE reactivity. Bla g 1, Bla g 2, and Bla g 5 were measured by using immunoassays. IgE antibody levels to 8 purified recombinant allergens from groups 1, 2, 4, 5, 6, 7, 9, and 11 were measured by using ImmunoCAP. IgE antibody binding inhibition assays were performed to assess extract in vitro potencies (concentration inhibiting 30% of the total IgE antibody-binding inhibition) relative to an arbitrarily selected reference extract in 5 patients with cockroach allergy. RESULTS: Allergen levels were highly variable. Three new major allergens (groups 6, 9, and 11), were identified among highly cockroach-sensitized subjects (CAP class ≥ 3). Sensitization profiles were unique per subject without immunodominant allergens. The sum of IgE to 8 allergen components showed a good correlation with cockroach-specific IgE levels (r = 0.88, P < .001). In vitro potencies varied among different extracts per subject and among subjects for each extract. CONCLUSIONS: The in vitro potency of German cockroach extracts for IgE reactivity depends on allergen content and allergen-specific IgE titers of patients with cockroach allergy. These factors are relevant for selection of potent extracts to be used for immunotherapy and for the design and interpretation of data from immunotherapy trials.

Comparison between Newly Developed and Commercial Inhalant Skin Prick Test Reagents Using In Vivo and In Vitro Methods.

BACKGROUND: We developed skin prick test (SPT) reagents for common inhalant allergens that reflected the real exposure in Korea. The study aim was to evaluate diagnostic usefulness and allergen potency of our inhalant SPT reagents in comparison with commercial products. METHODS: We produced eight common inhalant allergen SPT reagents using total extract (Prolagen): Dermatophagoides farinae, Dermatophagoides pteronyssinus, oak, ragweed, mugwort, Humulus japonicus pollens, as well as cat and dog allergens. We compared the newly developed reagents with three commercially available SPT reagents (Allergopharma, Hollister-Stier, Lofarma). We measured total protein concentrations, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), major allergen concentration, and biological allergen potencies measured by immunoglobulin E (IgE) immunoblotting and ImmunoCAP inhibition test. RESULTS: Diagnostic values of these SPT reagents were expressed as positivity rate and concordance rate of the results from ImmunoCAP allergen-specific IgE test in 94 allergic patients. In vitro analysis showed marked differences in protein concentrations, SDS-PAGE features, major allergen concentrations, and biological allergen potencies of four different SPT reagents. In vivo analysis showed that positive rates and concordance rates of Prolagen® SPT reagents were similar compared to the three commercial SPT reagents. CONCLUSION: The newly developed Prolagen® inhalant SPT reagents are not inferior to the commercially available SPT reagents in allergy diagnosis.

Characterization of a Major Allergen from Mongolian Oak, Quercus mongolica, a Dominant Species of Oak in Korea.

BACKGROUND: Oaks are the most common trees in Korean forests, and Mongolian oak, Quercus mongolica, is the dominant species. However, no allergen has been characterized from Mongolian oak. In this study, we tried to characterize a major allergen from Mongolian oak. METHODS: A molecule homologous to pathogenesis-related 10 (PR-10)-like protein, Que m 1, was cloned by RT-PCR. Its recombinant protein, along with Que a 1, an allergen from white oak (Q. alba), was produced. The allergenicity and diagnostic value of recombinant Que m 1, Que a 1, and Bet v 1 proteins were compared by ELISA using sera from oak-sensitized subjects. A basophil activation test was also performed using CD63 expression as an activation marker. RESULTS: Que m 1 sequence shares 57.5-96.2% amino acid sequence identity with PR-10-like allergens from various plants. Specific IgE to recombinant Que m 1, Que a 1, and Bet v 1 were detected in 92.0, 74.0, and 38.0% of 50 serum samples from Korean tree pollinosis patients. Recombinant Que m 1 was able to inhibit IgE reactivity to Que a 1 and Bet v 1, indicating its strong cross-reactivity. The activation patterns of basophils from 5 patients were similar in terms of the CD63 expression and protein concentration of challenged Bet v 1 and Que m 1. CONCLUSIONS: A major allergen, Que m 1, was cloned, and its recombinant protein was produced from Mongolian oak, a dominant species in Korea. Recombinant Que m 1 is potentially useful for the diagnosis and treatment of tree pollinosis in Korea.

IgE Reactivity of Recombinant Pac c 3 from the Asian Needle Ant (Pachycondyla chinensis).

BACKGROUND: Stings from the Asian needle ant are an important cause of anaphylaxis in East Asia. A 23-kDa protein homologous to antigen 5 is the major allergen produced by these ants. In this study, we aimed to produce a recombinant antigen 5 allergen, Pac c 3. METHODS: Recombinant Pac c 3 allergen from the Asian needle ant was expressed in Pichia pastoris and purified by ammonium sulfate precipitation and Ni affinity chromatography. IgE reactivity was demonstrated by ELISA and immunoblotting. RESULTS: The recombinant protein was recognized in 5 of 6 (83.3%) serum samples from patients with demonstrated anaphylaxis to ants. IgE reactivity to an antigen 5 allergen from Asian needle ant venom sac extract was specifically inhibited by the recombinant protein. It was also able to inhibit IgE binding to the vespid allergen Ves v 5 by ImmunoCAP analysis, indicating the presence of cross-reactivity. CONCLUSION: A recombinant Pac c 3, cross-reactive with Ves v 5, from the Asian needle ant was successfully produced in the methylotrophic yeast P. pastoris. This protein could be useful for the development of component-resolved diagnostics.

Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori.

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.

Cross-Reactivity between Oak and Birch Pollens in Korean Tree Pollinosis.

Oak and birch trees belong to Fagales order. Specific IgE to pollen allergens of both trees are frequently found in Korea pollinosis patients. Oak trees which comprise 40% of forest area are common in Korea. However, birch trees are sparse. We compared the allergenicity of pollen extracts of white oak, sawtooth and Mongolian oaks which are prevalent species in Korea, with the pollen extract of birch. The cross-reactivity of four pollen extracts was examined with pooled sera of 12 patients by ELISA, immunoblotting and CAP inhibitions. A protein of 17 kDa, putatively homologous to a major birch allergen Bet v 1, displayed strong IgE reactivity from white oak and sawtooth oak pollen extract but not from Mongolian oak pollen. Notably, a 23-kDa protein from sawtooth and white oaks showed strong IgE reactivity and inhibited by Bet v 1. IgE binding to white oak was inhibited a maximum of 94.6% by white oak, 93.4% by sawtooth oak, 83.2% by Mongolian oak, and 68.8% by birch. Furthermore, sawtooth oak, white oak, and Mongolian oak extracts were able to inhibit up to 78.5%, 76.6% and 67.3% of IgE binding to birch extract, while birch extract itself inhibited up to 94.3%. Specific IgE to Bet v 1 was inhibited a maximum of 79.1% by sawtooth oak, 77.4% by white oak, and 72.7% by Mongolian oak, while 81.5% inhibition was shown by birch. Bet v 1 was able to partially inhibit its homologous molecules from sawtooth oak and white oak in immunoblotting. Birch pollen extract was found to be cross-reactive primarily with Bet v 1-homologous allergen from oak pollens in Korea pollinosis patients. Considering the sparseness of birch tree in Korea, oak, especially sawtooth oak may be the main cause of tree pollinosis in Korea, rather than birch.

House dust mite allergen Der f 1 induces IL-8 in human basophilic cells via ROS-ERK and p38 signal pathways.

Der f 1, a major house dust mite allergen and member of the papain-like cysteine protease family, can provoke immune responses with its proteolytic activity. To understand the role of Der f 1 in inflammatory immune responses, we studied the mechanism of the regulation of interleukin (IL)-8 expressions in human basophilic cell KU812 by proteolytically active recombinant Der f 1. Not only production of IL-8 mRNA was induced but also the DNA binding activity of activator protein-1 (AP-1) and phosphorylation of NF-κB p65 were increased in Der f 1-treated KU812. Furthermore, Der f 1 induction of IL-8 expression was sensitive to pharmacological inhibition of ERK and p38 mitogen activated protein kinase (MAPK) pathways. Der f 1 also activated ERK and p38 MAPK phosphorylation and rapidly induced reactive oxygen species (ROS) production. The antioxidant N-acetyl-cysteine (NAC) inhibited phosphorylation of ERK, but not p38, suggesting that secretion of IL-8 in KU812 cells treated with Der f 1 is dependent on ROS, ERK MAPK and p38 MAPK. We describe the mechanism of Der f 1-induced IL-8 secretion from human basophilic cells, which are thought to be important for allergic inflammation independent of IgE antibodies. These findings improve our understanding of the inflammatory immune response in human basophils to protease allergens.

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.

Anti-aminoacyl-tRNA synthetase-interacting multifunctional protein-1 antibody improves airway inflammation in mice with house dust mite induced asthma.

Background: Several biologics have been developed and used to treat severe asthma. However, commercialized biologics have limitations in treating T2-low asthma because their main target is the T2 inflammation marker. Therefore, there is an unmet need for treating T2-low severe asthma. Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) is an auxiliary protein in the mammalian multi-aminoacyl-tRNA synthetase complex. AIMP1 also acts as a cytokine and induces the secretion of proinflammatory cytokines. Since anti-AIMP1 has been shown to reduce interleukin (IL)-6, tumor necrosis factor-α, and IL-17A levels in a mouse model, it could be effective in the treatment of T2-low severe asthma. Methods: Wild-type BALB/c mice were sensitized and challenged with intranasal inoculation of a crude HDM extract. Atliximab, a chimeric AIMP1 antibody, was administered once (20 µg, 40 µg, 100 µg) on Day 14. We evaluated airway hyperresponsiveness (AHR), performed cellular analyses of the bronchoalveolar lavage fluid (BALF), measured inflammatory cytokine levels, and examined peribronchial histological features. Results: Atliximab reduced AIMP1 levels in asthmatic mice in a dose-dependent manner. AHR and Inflammatory cells such as neutrophils and eosinophils in the BALF decreased in asthmatic mice treated with atliximab. The levels of IL-6, IL-13, and transforming growth factor-ß (TGF-ß) in the lung tissue decreased in asthmatic mice treated with a high dose of atliximab (100 µg). Atliximab also reduced goblet cell hyperplasia and peribronchial fibrosis. Conclusions: Atliximab improved asthmatic airway inflammation including neutrophilic inflammation in HDM-induced asthma mice. These data suggest that anti-AIMP1 plays an important role in the treatment of severe T2-low asthma.

Identification of novel allergenic components from German cockroach fecal extract by a proteomic approach.

BACKGROUND: Cockroaches produce potent allergens, and cockroach feces are known to be especially rich in allergens. In this study, we analyze the allergenic components from cockroach feces and evaluate allergenicity of recombinant α-amylase identified from fecal extract. METHODS: IgE-reactive proteins from German cockroach fecal extract were analyzed by proteomic analysis and immunoblotting. Recombinant α-amylase was produced and its allergenicity was evaluated by ELISA. RESULTS: Analysis of German cockroach fecal extracts identified 12 IgE-reactive components. Most of these allergens were found to be digestive enzymes such as α-amylase, trypsin, chymotrypsin, metalloprotease, and midgut carboxypeptidase A, but the identity of 3 IgE-reactive proteins is still unknown. Glycinin-like proteins, which were likely derived from the cockroach diet, were also identified. German cockroach α-amylase shares the highest identity with pig α-amylase (55.8%), followed by mite group 4 allergens (Blo t 4, 50.4%; Der p 4, 49.8%; Eur m 4, 47.4%). In this study, recombinant α-amylase from German cockroach was expressed, and its allergenicity was examined by ELISA. Specific IgE against recombinant amylase was detected in 41.4% (12/29) of serum samples from German cockroach-sensitized subjects. Recombinant α-amylase was able to inhibit 55% of specific IgE to German cockroach whole-body extract. CONCLUSIONS: Amylase was found to be an important novel allergen in cockroach feces. It is hoped that recombinant α-amylase will be useful for further studies and clinical applications.

Characterization of Hum j 6, a Major Allergen From Humulus japonicus Pollen, the Primary Cause of Weed Pollinosis in East Asia.

PURPOSE: Humulus japonicus (HJ) is one of the most important causes of weed pollinosis in East Asia. The 10 kDa protein with pI 10 in 2-dimensional gel has been recognized as the representative major allergen of HJ, but its major allergens have not been characterized. This study aimed to characterize the major allergen of HJ. METHODS: A major allergen in Japanese hop was detected by proteome analysis; it was purified to homogeneity and its sequence was obtained by transcriptome analysis. The recombinant proteins were produced in Escherichia coli and Pichia expression systems, and their immunoglobulin E (IgE) reactivities were compared to those of the natural counterpart. We also analyzed post-translational modifications such as glycosylation and phosphorylation. RESULTS: Pectin methylesterase inhibitor, Hum j 6, was found to be the major allergen of HJ, and in silico signal peptide prediction corresponds to a 15.1 kDa protein with a theoretical pI of 8.28. Natural Hum j 6 was recognized by IgE antibodies from 86.4% (19/22) of HJ pollinosis patients, whereas the recombinant proteins did not show strong IgE reactivity. No glycosylation was detected, while at least 15 phosphorylated amino acids, possibly causing the pI and molecular weight shift, were detected by tandem mass spectrometry analysis. CONCLUSIONS: Hum j 6 was identified as the representative major allergen of HJ and seems to be modified significantly after translation. These findings are useful for the development of component-resolved diagnosis and immunotherapy.

Sequence polymorphisms of Der f 1, Der p 1, Der f 2 and Der p 2 from Korean house dust mite isolates.

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.