Molecular cloning, expression and modelling of cat allergen, cystatin (Fel d 3), a cysteine protease inhibitor

BACKGROUND: Cats are an important source of indoor allergens. However, only two cat allergens, Fel d 1 and albumin, have been cloned and sequenced. IgE antibodies to Fel d 1 and albumin do not fully account for IgE responses to cat and there is good immunochemical evidence that cats produce other allergens. OBJECTIVE: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using pooled serum obtained from five asthmatic patients which contained high levels of IgE antibody to cat dander. Selected cDNA clones were screened by plaque immunoassay and one cDNA clone, encoding cystatin, was expressed in E. coli. The three dimensional structure of cat cystatin was modelled using the SWISS-MODEL computer program. RESULTS: Three positive cDNA clones (A, B and C) were identified, two of which were fully sequenced. Clones A and C encoded the same 98 amino acid residue sequence which showed 79% and 75% homology with bovine and human cystatin A, respectively. The cat cystatin sequence contained the conserved cysteine protease inhibitor signature and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE ab to the expressed cystatin clones. The cysteine protease inhibitor motif was also partially conserved in dog allergen sequences, Can f 1 and Can f 2, which are lipocalins. The recombinant protein was expressed in E. coli as an 11-kDa protein, corresponding to the predicted MW of cat cystatin. The three-dimensional structure of cat cystatin was modelled on human cystatin structures. CONCLUSION: A newly identified allergen, cystatin (Fel d 3), has been cloned from cat skin and is a member of the cysteine protease inhibitor family.     

Cloning and expression of Blo t 1, a novel allergen from the dust mite Blomia tropicalis, homologous to cysteine proteases

BACKGROUND: House dust mite allergens have been shown to be a very important stimulus in the causation of asthma and triggers for the exacerbation of symptoms. Therefore, characterization of mite-derived allergens at the molecular level is an important step for the development of effective diagnostic and therapeutic approaches, as well as for epidemiological studies. OBJECTIVE: To clone, express and characterize at the molecular level the cysteine protease from Blomia tropicalis (Bt). METHODS: A full-length cDNA encoding Blo t 1 was cloned from a Bt cDNA library using a PCR and RACE-based strategy. The cDNA was PCR-amplified, sequenced and subcloned into a prokaryotic expression vector. The allergenicity of the recombinant Blo t 1 was evaluated for IgE reactivity by Western blot. RESULTS: Blo t 1 cDNA encodes a 221 amino acids polypeptide with an estimated molecular weight of 25 kDa. The recombinant protein is 35% identical to other mite cysteine proteases. Recombinant Blo t 1 (rBlo t 1) bound IgE from 62% of Bt skin test-positive serum. Dermatophagoides pteronyssinus (Dp) skin test-positive sera did not react with rBlo t 1, indicating the possible presence of unique IgE epitopes on the rBlo t 1 molecule. A three-dimensional image of Blo t 1, constructed based on predicted analysis, showed conserved secondary and tertiary structure with other cysteine proteases. CONCLUSION: We report the cloning, expression and IgE reactivity of Blo t 1, a novel allergen from the domestic mite Blomia tropicalis (Bt), highly homologous to cysteine proteases. This putative cysteine protease, designated Blo t 1, may play a major role as an immunodominant allergen involved in dust mite-specific IgE-mediated hypersensitivity.     

Cystatin A inhibits IL-8 production by keratinocytes stimulated with Der p 1 and Der f 1: biochemical skin barrier against mite cysteine proteases

BACKGROUND: Der p 1 and Der f 1 are the most immunodominant allergens produced by house dust mites and are suspected to be involved in the pathogenesis of allergy through their cysteine protease activity. However, stimulation of keratinocytes by these protease allergens and protective systems in the skin against them have not been well investigated. OBJECTIVE: We purified and identified the dominant skin-derived inhibitor against the proteolytic activities of these allergens and analyzed its effect on keratinocyte activation. METHODS: Recombinant allergens were used for the experiments. We analyzed whether human sweat inhibits the enzymatic activities of Der p 1 and Der f 1 and used sweat as the skin-derived material to isolate the inhibitor. The inhibitor was purified by means of column chromatography and subsequently identified by means of protein sequencing and immunoblotting. Keratinocytes were stimulated with the allergens in the absence or presence of the inhibitor, and the concentration of secreted IL-8 was measured. RESULTS: Sweat inhibited the proteolytic activities of Der p 1 and Der f 1. The sweat inhibitor was identified as cystatin A. The stimulation of normal human keratinocytes and the human keratinocyte cell line HaCaT with these protease allergens upregulated IL-8 secretion, and addition of cystatin A blocked this upregulation. Normal human keratinocytes secreted cystatin A into the medium. CONCLUSIONS: The proteolytic activity of Der p 1 and Der f 1 stimulates human keratinocytes in vitro. Cystatin A produced by keratinocytes is the dominant biochemical skin barrier that eliminates the enzymatic activity of these mite cysteine proteases and prevents them from stimulating keratinocytes.     

Assessment of recombinant dog allergens Can f 1 and Can f 2 for the diagnosis of dog allergy

BACKGROUND: The use of recombinant allergens for the diagnosis and immunotherapy of allergy may offer several advantages over allergen extracts. OBJECTIVE: To produce recombinant dog allergens Can f 1 and Can f 2 in Pichia pastoris yeast and to assess their suitability for the diagnosis of dog allergy. METHODS: Clinically diagnosed dog-allergic patients' and healthy non-atopic dog owners' reactivities against recombinant Can f 1 and Can f 2 and commercial dog epithelial extract were studied by a panel of methods including skin prick test (SPT), ELISA and IgE immunoblotting. RESULTS: Recombinant Can f 1 and Can f 2 were found immunologically functional: they bound dog-allergic patients' IgE in immunoblotting and inhibited specifically the binding of IgE to their natural counterparts in the dog allergen extract. Moreover, patients' IgE reactivity in immunoblotting to natural Can f 1 and their SPT with the recombinant allergen were perfectly concordant (phi coefficient 1.0, P<0.001). The concordance was slightly lower with recombinant Can f 2 (phi coefficient 0.92, P<0.001). A lower number of dog-allergic patients, 52%, reacted against Can f 1 than previously reported. About one-third of the patients reacted to Can f 2. In immunoblotting, the highest prevalence of reactivity, 60%, was directed to an 18 kDa component. Aminoterminal sequencing showed this to be a previously unidentified allergenic protein. CONCLUSIONS: The recombinant allergens can be used reliably to identify Can f 1 and Can f 2-sensitized individuals. However, the two allergens are insufficient as reagents for diagnosing dog allergy.     

Molecular and immunological characterization of Can f 4: a dog dander allergen cross‐reactive with a 23 kDa odorant‐binding protein in cow dander

Background Dog dander is an important cause of respiratory allergy but its content of allergenic components is still incompletely known. While Can f 1, 2, 3 and 5 have been studied in detail, only fragmentary information is available on the lipocalin Can f 4. Objective To purify, clone and characterize dog dander allergen Can f 4. Methods Can f 4 was purified from dog dander extract by size exclusion, ion exchange and reverse phase chromatography. A cDNA encoding Can f 4 was cloned and used to produce recombinant Can f 4 in Escherichia coli. A 23 kDa protein from cow dander, displaying cross‐reactivity with Can f 4, was purified and identified by amino acid sequencing and mass spectrometry. IgE antibody binding to dog and cow dander extract and to individual dog allergens among 37 dog allergic subjects and 44 pollen allergic controls was studied using ImmunoCAP. Results A dog genome segment containing the Can f 4 gene was bioinformatically identified and enabled the cloning of Can f 4 cDNA. Recombinant Can f 4 displayed close immunological and biochemical similarity to purified natural Can f 4 and bound IgE antibodies from 13/37 (35%) sera of dog allergic subjects. Can f 4 reactive sera showed IgE binding to a 23 kDa protein present in cow dander extract, related to a family of odorant‐binding proteins. The dog and cow proteins shared 37% sequence identity and their cross‐reactivity was demonstrated by IgE inhibition experiments. Conclusion Recombinant Can f 4 brings the panel of available dog allergens closer to completion and will be important in component‐resolved diagnostics in allergy to animal epithelial allergens. Cite this as: L. Mattsson, T. Lundgren, P. Olsson, M. Sundberg and J. Lidholm, Clinical & Experimental Allergy, 2010 (40) 1276–1287.     

The molecular basis of allergenicity: comparative analysis of the three dimensional structures of diverse allergens reveals a common structural motif.

BACKGROUND: Although a large number of allergens have been characterised, the structural, functional, and biochemical features that these molecules have in common, and that could explain their ability to elicit powerful IgE antibody responses, are still uncertain. Recently, there has been considerable interest in the role of the cysteine protease activity of the house dust mite allergen Der p 1 in biasing the immune response in favour of IgE production. AIMS:To search for remote homologues of Der p 1 with sequences similar to the 30 conserved amino acids surrounding the catalytic cysteine residue (Cys34). METHODS: Potential homologues were analysed by examining their three dimensional structures and multiple sequence alignments using the programs PROPSEARCH, ClustalW, GeneDoc, and Swiss Pdb Viewer. RESULTS: Diverse allergens (for example, the plant cysteine protease papain, the transport protein lipocalin Mus m 1, and the ragweed allergen Amb a 5) have a similar structural motif; namely, a groove resembling the substrate binding groove of Der p 1. The groove is located inside an alpha-beta motif, between an alpha helix on one side and an antiparallel beta sheet on the other side. A similar common motif (a cysteine stabilised alpha-beta fold) can also be found in some toxins and defensins. CONCLUSION: Allergens of diverse sources have a common structural motif, namely a groove located inside an alpha-beta motif, which could potentially serve as a ligand binding site.     

Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11

BACKGROUND: Allergy to grass pollen is typically associated with serum IgE antibodies to group 1 and/or group 5 allergens, and additionally often to one or several less prominent allergens. Most of the grass pollen allergens identified to date have been characterized in detail by molecular, biochemical and immunological methods, timothy grass being one of the most thoroughly studied species. However, a 20-kDa allergen frequently recognized by IgE antibodies from grass pollen allergics has so far escaped cloning and molecular characterization. OBJECTIVE: To clone and characterize the 20 kDa timothy grass pollen allergen Phl p 11. METHODS: Phl p 11 cDNA was cloned by PCR techniques, utilizing N-terminal amino acid sequence obtained from the natural allergen. Phl p 11 was expressed as a soluble fusion protein in Escherichia coli, purified to homogeneity and used for serological analysis and to study Phl p 11 specific induction of histamine release from basophils and skin reactivity in sensitized and control subjects. RESULTS: Phl p 11 cDNA defined an acidic polypeptide of 15.8 kDa with homology to pollen proteins from a variety of plant species and to soybean trypsin inhibitor. The sequence contained one potential site for N-linked glycosylation. Serological analysis revealed that recombinant Phl p 11 shared epitopes for human IgE antibodies with the natural protein and bound serum IgE from 32% of grass pollen-sensitized subjects (n = 184). Purified recombinant Phl p 11 elicited skin reactions and dose-dependent histamine release from basophils of sensitized subjects, but not in non-allergic controls. CONCLUSION: As the first representative of group 11 grass pollen allergens, Phl p 11 has been cloned and produced as a recombinant protein showing allergenic activity. One-third of grass pollen-sensitized subjects showed specific IgE reactivity to recombinant Phl p 11, corresponding in magnitude to a significant proportion of specific IgE to grass pollen extract.     

Involvement of Can f 5 in a Case of Human Seminal Plasma Allergy

Background: The existence of IgE binding to dog dander extract without IgE antibodies against the described dog allergens (Can f 1, 2, 3 and 4) implies the presence of other dog allergens yet to be identified. Recently, an IgE-binding protein was isolated from dog urine and identified as prostatic kallikrein; it has been named Can f 5. Cross-reactivity between a dog dander allergen and human prostate-specific antigen (PSA) has been described. The aim of this study was to identify the dog dander allergen that presents cross-reactivity with PSA and demonstrate its clinical relevance in our patient with human seminal plasma allergy. Methods: SDS-PAGE immunoblotting and inhibition tests were performed. Mass spectrometry was carried out to identify the protein involved in the allergy reactions. Results: SDS-PAGE immunoblotting-inhibition with an IgE-binding protein from dog prostatic secretion showed total IgE binding inhibition to a 28-kDa IgE-reactive band identified as PSA. The electroeluted protein from dog prostatic secretion was identified by mass spectrometry as Can f 5. IgE immunoblotting of human seminal plasma incubated with the serum of the patient revealed two IgE-binding bands (28 and 32.7 kDa). Both SDS-PAGE immunoblotting inhibition assays, with human seminal plasma or purified PSA in solid phase, showed complete IgE binding inhibition when the serum of the anaphylactic patient was preincubated with dog dander extract or recombinant Can f 5. Conclusions: The dog dander allergen that shows cross-reactivity with human PSA has been characterized and turns out to be the recently described Can f 5. We demonstrated the clinical relevance of this cross-reactivity in a patient.     

Exposure to an abundance of cat (Fel d 1) and dog (Can f 1) allergens in Swedish farming households

BACKGROUND: Earlier studies have shown that farmers are to a low degree sensitized to animal allergens. We have measured the amount of cat (Fel d 1) and dog (Can f 1) in farm households and examined the relationship between exposure and sensitization to cat and dog allergens. METHODS: Dust samples from the homes of 403 farmers who had participated in an epidemiologic follow-up study on respiratory symptoms were analyzed for allergen content by two-site ELISA methods. RESULTS: Fel d 1 was detected in 99.5% of the farmers' households ranging from 0.055 to 1455 microg/g dust in mattresses (GM 13.2) and to 3775 microg/g dust in living-room carpets (GM 17.1). Can f 1 was detected in 90.6% of the households from 0.2 to 116 microg/g dust in mattresses (GM 2.0) and to 504 microg/g dust in carpets (GM 4.3). Homes with pets present had the highest levels of the allergens (P<0.001). A total of 8.4% and 7.4% of the farmers were sensitized to cat and dog, respectively. A significant correlation was noted between exposure to the allergens and specific IgE to cat and dog, respectively (P<0.001). Sensitization to cat (OR = 4.9) and dog (OR = 17.8) was significantly associated with asthma. CONCLUSIONS: In spite of the abundance of Fel d 1 and Can f 1, farmers are only to a low degree sensitized to cats and dogs.     

Functional effects of the inhibition of the cysteine protease activity of the major house dust mite allergen Der p 1 by a novel peptide-based inhibitor.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is an important source of allergens, which can cause allergic conditions. The cysteine protease activity of Der p 1 may enhance the potency of this major mite allergen through cleavage of CD23 and CD25 from the surface of immune cells, IgE independent mast cell activation, increases in epithelial cell permeability and inactivation of an endogenous serine protease inhibitor. Inhibition of the enzymatic activity of Der p 1 may therefore be of therapeutic benefit. OBJECTIVE: To examine the activity of PTL11028, a newly developed Der p 1 inhibitor, in a range of assays that directly or indirectly measure Der p 1 protease activity and to compare its activity to endogenous cysteine protease inhibitors. METHODS: The proteolytic activities of purified Der p 1 or HDM extract and inhibitory properties of PTL11028 were examined through cleavage of an artificial peptidyl substrate, cleavage of CD23 from human B cells and permeability studies on primary human bronchial epithelial cells. RESULTS: PTL11028 is a highly potent and specific Der p 1 inhibitor, being effective against both purified protease and Der p 1 within HDM extract. PTL11028 can completely inhibit Der p 1-mediated CD23 cleavage from human B cells and also reduces HDM-induced human bronchial epithelial cell permeability by 50%. Der p 1 is potently inhibited by cystatin A and to a lesser extent by cystatins C and E/M. CONCLUSION: PTL11028 is a highly potent and selective irreversible inhibitor of the cysteine protease activity of Der p 1, an activity that may be modulated in vivo by some human cystatins. PTL11028 prevents the Der p 1-mediated cleavage of CD23 from human B cells and significantly reduces HDM-induced permeabilization of the epithelial barrier. PTL11028 is an important tool to examine the biological effects of Der p 1 in a range of in vitro and in vivo model systems.     

Molecular cloning of Indian jujube (Zizyphus mauritiana) allergen Ziz m 1 with sequence similarity to plant class III chitinases

Indian jujube (Zizyphus mauritiana) is a sweet fruit that is abundantly cultivated in Taiwan. We have previously identified 42 and 30 kDa allergens that are cross-reactive with latex allergen from crude Indian jujube extract. This study aimed to clone the 30 kDa Ziz m 1 Z. mauritiana allergen. The Ziz m 1 encoding cDNA was isolated from a ZAPII cDNA library constructed from Z. mauritiana mRNA, sequenced and expressed in Pichia pastoris. The protein predicted from the cDNA sequence has 330 amino acids, the first 25 of which constituted a putative signal peptide. The deduced molecular mass of the mature protein is 33.86 kDa, while its isoelectric point is estimated at 4.36. The recombinant Ziz m 1 showed chitinase activity, possessed IgE binding capacity, and had IgE cross-reactivity with the latex allergen. Moreover, anti-recombinant Ziz m 1 antibody-based ELISA was able to detect commercial skin testing latex reagent, laboratory prepared latex and Indian jujube extracts. Recombinant Ziz m 1 showed 87.5% skin reactivities on eight latex- and Indian jujube-sensitive subjects. Although no sequence similarity was found to other known allergens, Ziz m 1 was found to have amino acid sequence identity (39-45.3%) to many plant chitinases including chitinase (45.2%) of Hevea brasiliensis (hevamine), class III chitinases of Vigna angularis (45.3%), Capsicum annuum (44.7%) and Oryza sativa (41.2%). A conserved domain search revealed that Ziz m 1 belongs to the family 18 glycosyl hydrolases. The recombinant allergen may therefore be of value for diagnosis and therapeutic purposes, and the further characterization of Indian jujube allergen may help to elucidate the mechanism underlying latex-fruit syndrome.     

Fel d 4, a cat lipocalin allergen

BACKGROUND: Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin-like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals. OBJECTIVE: To analyse cDNA encoding a lipocalin allergen and the corresponding recombinant allergen at both the molecular and immunological levels. METHODS: A submandibular salivary gland cDNA expression library was constructed and screened for clones producing IgE-binding polypeptides. cDNA encoding a lipocalin allergen and its corresponding recombinant allergen were analysed. RESULTS: An IgE binding molecule with high sequence identity to the boar salivary lipocalin and the horse lipocalin Equ c 1 allergen was isolated and designated, Fel d 4. Serum from 62.96% of cat-allergic subjects examined had measurable IgE antibody to Fel d 4 but typically at low levels. Despite this in 47% of sera the anti-Fel d 4 IgE titres were higher than the anti-Fel d 1 titres. IgE binding to the lipocalin allergen could be blocked by an allergen extract from cow and to a lesser degree by extracts from horse and dog. CONCLUSION: Fel d 4 is a lipocalin allergen produced by the cat, which binds IgE at relatively high frequency in cat-sensitive individuals. The allergen provides not only a means for investigating differences in the immune response to lipocalin allergens from that found for other mammalian species but also an important reagent for the diagnosis of cat allergy.     

Characterization of recombinant cat albumin

BACKGROUND: Indoor allergens derived from animals and mites often contribute to exacerbation of skin manifestations in atopic dermatitis (AD) patients. OBJECTIVE: To produce and characterize recombinant cat albumin, a cross-reactive animal allergen. METHODS: A complete cDNA coding for cat albumin was obtained by RT-PCR amplification from cat liver RNA. Recombinant cat albumin was expressed in Escherichia coli as hexahistidine-tagged protein, purified by nickel affinity chromatography and studied for IgE reactivity with sera from cat-allergic patients by ELISA and immunoblotting. Furthermore, CD203c expression of basophils from cat-allergic patients upon exposure to recombinant cat albumin was analysed. RESULTS: Recombinant cat albumin, a cross-reactive animal allergen sharing most IgE epitopes with its natural counterpart, was produced in E. coli. It was recognized preferentially by IgE from AD patients and elicited IgE-dependent basophil activation in sensitized patients. CONCLUSIONS: Recombinant cat albumin may be used as a paradigmatic tool to analyse mechanisms of allergen-triggered exacerbation of AD, for diagnostic and, perhaps for therapeutic purposes.     

Lack of human IgE cross-reactivity between mite allergens Blo t 1 and Der p 1

BACKGROUND: The group 1 mite allergens are the most significant indoor allergens and they belong to the papain-like cysteine protease family. To date there is only one published report on the isolation and characterization of group 1 allergens from Blomia tropicalis mites. The aims of the study are to determine the cross-reactivity between group 1 allergens and to evaluate their clinical importance in allergic patients. METHODS: The full-length Blo t 1 gene was obtained by SMART RACE cDNA amplification method using gene-specific primers. The sequence alignment was performed using LOOK followed by three-dimensional homology modeling. The cDNA was expressed in Pichia pastoris as a secretory protein. Identification of native Blo t 1 in crude mite and spent mite medium extracts was done by Western immunoblot using monoclonal antibody. Allergenicity of recombinant Blo t 1 and native Der p 1 was examined by human IgE ELISA with 80 asthmatic sera. RESULTS: The cDNA sequence consists of 1105 base pairs, including 5'- and 3'-untranslating regions, encoding an open reading frame of 330 amino acid residues. The predicted molecular weight of the deduced protein was approximately 38 kDa. Blo t 1 shared 53 and 34% nucleotide and amino acid, respectively, sequence homology with Der p 1. Native Blo t 1 was detected in both crude mite and spent mite medium extracts, and its estimated molecular weight was about 26 kDa. The recombinant Blo t 1 reacted positively with IgE in 90 and 65% of sera from asthmatic children and adults, respectively, indicating that it is a major allergen. The correlation of human IgE reactivity between Blo t 1 and Der p 1 was low in these sera. CONCLUSION: The full-length cDNA encoding group 1 Blomia tropicalis mite allergen (designated as Blo t 1) has been characterized and expressed from local mites in Singapore. This fecal allergen showed high frequency of human IgE reactivity with asthmatic sera in the tropics and there was a low correlation of IgE reactivity between Blo t 1 and Der p 1.     

Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase

BACKGROUND: Commercially available assays for IgE antibody provide results in international units per milliliter for many allergen extracts, but this is not easily achieved with purified or novel allergens. OBJECTIVE: To develop assays for IgE antibody suitable for purified or novel allergens by using a commercially available immunosorbent. METHODS: Streptavidin coupled to a high-capacity immunosorbent (CAP) was used to bind biotinylated purified allergens from mite (Der p 1 and Der p 2), cat (Fel d 1), and dog (Can f 1). Assays for IgE antibody to these allergens were performed on sera from children (asthma and control) as well as adults with atopic dermatitis. RESULTS: The results were validated by serial dilution of sera with high and low levels of IgE antibody and were quantitated in international units per milliliter by using a standard curve. Values for IgE antibody to Der p 1, Der p 2, and Fel d 1 correlated with values obtained with the allergen extracts (r2 = 0.80, 0.84, and 0.95, respectively; P < .001 in each case). Furthermore, the values for IgE antibody in sera from children with high exposure to mite and cat allergens demonstrated 10-fold higher levels of IgE antibody to Der p 1 and Der p 2 than to Fel d 1 (P < .001). CONCLUSION: The streptavidin immunosorbent technique provides a new method for quantifying IgE antibody to purified proteins. The results provide evidence about the high quantities of IgE antibody to purified inhalant allergens in patients with atopic dermatitis. In addition, the results demonstrate major differences in IgE antibodies specific for mite and cat allergens among children with high exposure to both allergens.     

Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen

BACKGROUND: Previously, we have identified several Per a 1 (Cr-PII) allergens from a deltagt22A cDNA library of Periplaneta americana. This study aimed to sequence clone C42 and determine its molecular and antigenic properties. METHODS: The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21. The recombinant proteins were purified by ion-exchange and affinity chromatographies. Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE. RESULTS: The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino-acid protein (50kDa) determined. The recombinant C42 protein can bind both anti-Per a 1 monoclonal antibodies and human IgE and showed a 54.4% (12/22) skin reactivity in atopic patients. Sequence homology searches revealed a high degree of identity to two other members of the Per a 1 family, C17 and C6, and the German cockroach (Blattella germanica) Bla g Bd90K allergen. Interestingly, these allergens all contain internal repeats, and the crude B. germanica extract, Per a 1, and recombinant allergens share similar antigenic determinant(s) as defined by ELISA and IgE-binding inhibition studies. In IgE-binding epitope studies, an immunopositive C42 fragment was first identified from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzyme fragments in E. coli. The shortest peptide, C42-P560, identified by monoclonal antibodies and human specific IgE, can inhibit IgE binding to C42. CONCLUSIONS: An additional Per a 1 allergen has been defined at the molecular level and characterized and preliminary results showed that a potential IgE-reactive region is located within amino-acid residue 358-446 of C42, which is an internal repeat. The results defined the boundaries of the antigenic site and will facilitate further epitope-mapping studies.     

Hymenoptera venom protease allergens

BACKGROUND: Recent studies have shown the presence of additional allergenic proteins in honeybee and paper wasp venoms. Both venoms contain serine protease enzymes. OBJECTIVE: We isolated and obtained complete sequences of honeybee and Mediterranean paper wasp venom proteases, both of which have significant IgE binding activity. The structures are compared with bumblebee venom protease. METHODS: Venom proteases were chromatographically isolated from venoms and partial amino acid sequences determined. RT-PCR and rapid amplification of cDNA ends methods were used to clone cDNA, and complete sequences were determined for honeybee and a paper wasp venom protease. RESULTS: The venom proteases are all serine proteases of the trypsin type. The honeybee protease contains a complement, embryonic sea urchin protein, bone morphogenetic protein interaction domain as well as a linker and propeptide sequence, and a unique methionine residue near the active site. It has IgE binding activity. The paper wasp protease is a single trypsin domain and is an important allergen. The framework residues are poorly conserved among honeybee, bumblebee, and paper wasp enzymes. CONCLUSIONS: The 3 venom serine proteases have significant IgE binding activities. The structures are poorly conserved even among the Apidae , suggesting little cross-reactivity among the protein portions. The paper wasp venom proteases are important allergens.     

Animal-derived lipocalin allergens exhibit immunoglobulin E cross-reactivity

Background Although knowledge of the IgE cross‐reactivity between allergens is important for understanding the mechanisms of allergy, the regulation of the allergic immune response and the development of efficient modes of allergen immunotherapy, the cross‐reactivity of animal allergens is poorly known. Objective The aim of this study was to characterize IgE cross‐reactivities between lipocalin proteins, including five animal‐derived lipocalin allergens and one human endogenous lipocalin, tear lipocalin (TL). Methods The recombinant proteins were validated by chromatography and mass spectrometry. The IgE‐binding capacity of the allergens was confirmed by IgE. immunoblotting and IgE immunoblot inhibition. IgE ELISA was performed with sera from 42 atopic patients and 21 control subjects. The IgE cross‐reactivities between the lipocalin proteins were determined by ELISA inhibition. Results ELISA inhibition revealed IgE cross‐reactivities between Can f 1 and human TL, between Can f 1 and Can f 2, and between Equ c 1 and Mus m 1. Low levels of IgE to human TL were found in the sera of seven dog‐allergic patients of whom six were IgE‐positive for Can f 1. Conclusion Several lipocalins exhibited IgE cross‐reactivity, probably due to the sequential identity of the proteins and also due to similarities in their three‐dimensional structures. The clinical significance of the findings needs to be elucidated. Low‐level IgE cross‐reactivity can play a role in regulating immune response to lipocalin allergens.     

Cat (Fel d I), dog (Can f I), and cockroach allergens in homes of asthmatic children from three climatic zones in Sweden

We have investigated the levels of cat ( Fel d I), dog ( Can f I), and cockroach ( Per a I) allergens in dust from bedrooms, living rooms, kitchens, and bathrooms from 123 homes of asthmatic children in three zones of Sweden with varying climates. Absolute indoor humidity (AIH), relative humidity (RH), rate of ventilation in air changes per hour (ach), and number of airborne particles were also measured. Fel d I, Can f I, and Per a I allergen contents were determined by mab ELISA, and the levels were related to various environmental factors. The major cat allergen. Fel d I, was detected in all homes, and the concentrations varied between 16 ng and 28000 ng/g fine dust. The dog allergen, Can f I, was detected in 85% of the homes, and the levels varied from 60 ng to 866000 ng/g dust. Cockroach allergen was detected in only one home (40 ng/g). Fel d I and Can f I allergens were equally distributed geographically. Dust from living rooms contained significantly higher ( P < 0.05) concentrations of both Fel d I and Can f I allergens than dust from bedrooms, kitchens, and bathrooms. The levels tended to be higher in homes with poor ventilation (<0.5 ach) and in homes with wall-to-wall carpets. Significantly higher ( P < 0.01) numbers of airborne particles were found in homes with high humidity (i.e., AIH ≥ 7 g/kg or RH ≥ 45%). We conclude that pet allergens are ubiquitous in different climatic regions, being found in bedrooms, living rooms, kitchens, and bathrooms. Current or previous presence of a cat or dog, high indoor humidity, presence of wall-to-wall carpets, and poor ventilation all increase the risk for high allergen exposure. In contrast, cockroach allergens arc rarely found in a temperate climate.     

The major dog allergens, Can f 1 and Can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms.

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.