Hong Chou

Pen ch 13 allergen induces secretion of mediators and degradation of occludin protein of human lung epithelial cells.

Background:u2002 Alkaline serine proteases from six prevalent airborne Penicillium and Aspergillus species have been identified as a group of major allergens (group 13). After entering human airways, the allergens are in initial contacts with respiratory epithelial cells. The purpose of this study is to investigate interactions between the Pen ch 13 allergen from P. chrysogenum and human lung epithelial cells.

cDNA cloning and immunologic characterization of a novel EF‐1β allergen from Penicillium citrinum

Background:u2002 We have identified previously that Penicillium citrinum is the most prevalent Penicillium species in the Taipei area. It is important to delineate the whole spectrum of allergenic components of this prevalent airborne fungus. The purpose of this study was to identify novel P. citrinum allergens through molecular cloning of allergen genes using a cDNA library of P. citrinum and sera from patients with bronchial asthma.

Molecular and structural analysis of immunoglobulin E-binding epitopes of Pen ch 13, an alkaline serine protease major allergen from Penicillium chrysogenum

Background Through proteomic and genomic approaches we have previously identified and characterized an alkaline serine protease that is a major allergen (88% frequency of IgE binding) of Penicillium chrysogenum (Pen ch 13).

Homology modeling and monoclonal antibody binding of the Der f 7 dust mite allergen

The group 7 allergens are important allergenic specificities for mite‐sensitive patients and may need to be incorporated into new diagnostic and therapeutic strategies. However, little is known about their biological and structural features. Position‐specific iterative BLAST showed that they had strong ancestral homology to two related families of lipid‐binding proteins, namely, the bactericidal permeability‐increasing (BPI) proteins and the odorant‐binding protein. A three‐dimensional model of Der f 7 made with the Phyre and SWISS‐MODEL homology‐modeling servers showed a close match with the human BPI coordinates used for its construction. The binding of the monoclonal antibody HD12 known to block IgE binding could be blocked by the linear sequence (46GILDF50) with a critical role for L48 or F50. These hydrophobic residues were located on a surface loop of the model. The properties of Der f 7 that can be deduced from the model provide avenues for further characterizing these allergens, their IgE binding structures and biological properties that can enhance allergenicity.

Asp159 is a critical core amino acid of an IgE-binding and cross-reactive epitope of a dust mite allergen Der f 7.

Der f 7 and Der p 7 are important house dust mite allergens with known structure and suggested biological function recently. However, their IgE-binding determinants remain unknown. The purpose of this study is to identify the IgE-reactive epitopes of Der f 7 and the determinants of IgE-mediated cross-reactivity between Der f 7 and Der p 7. IgE-reactive determinants were identified by immunodot blot inhibition using synthetic overlapping peptides, allergen mutants, and a Der f 7 structural model. Our results showed that synthetic peptides with sequence (156)SILDP(160) on Der f 7 bind IgE in two of the 30 asthmatic serum samples tested. Recombinant Der f 7 I157A, L158A, or D159A mutants have reduced IgE-binding activity. Inhibition experiments confirmed Asp159 as a critical core residue for IgE-binding. Among Der p 7, Der f 7 and Der f 7 mutants with single substitution between residues 156 and 160, only the D159A mutant cannot inhibit significantly IgE-binding against Der p 7. Therefore, Asp159 contributes to IgE-mediated cross-reactivity between Der f 7 and Der p 7. The structural model constructed for Der f 7 suggests that the IgE-binding epitope forms a loop-like structure on the surface of the molecule. In conclusion, Asp 159 is a critical core residue of an IgE-binding and IgE-mediated cross-reactive epitope (156)SILDP(160) of Der f 7. Results obtained from this study provide more information on molecular and structural features related to allergenicity, underlying basis of IgE cross-reactivity between allergens, and in designing safer immunotherapy.

Transaldolases are novel and immunoglobulin E cross-reacting fungal allergens.

Background Mould‐induced atopic respiratory diseases are a worldwide problem. Characterization of fungal allergens is of major clinical importance.

Lys, pro and trp are critical core amino acid residues recognized by FUM20, a monoclonal antibody against serine protease pan-fungal allergens

Background: Alkaline/vacuolar serine proteases comprise a major group of pan-fungal allergens from several prevalent airborne fungal species. It is of importance to characterize antigenic determinant(s) recognized by monoclonal antibodies against these major allergens. Methods: The antigenic determinant of fungal serine proteases recognized by a monoclonal antibody, FUM20, was analyzed by dot immunoassay of synthetic peptides immobilized on cellulose membrane. Results obtained were confirmed by wild-type recombinant protease and its mutants. The epitopes were mapped to the structure of serine proteases by molecular modeling. Results: A linear epitope encompassing 9 amino acids from Pen ch 18 (6EKNAPWGLA14) binds FUM20. The corresponding peptide (5AKGAPWGLA13) from Rho m 2 also binds FUM20. Substitution of K6, P9 or W10 with alanine in this peptide resulted in drastic loss of FUM20 binding. Rho m 2 mutants with single K6A, P9A, P9G, W10A or W10F substitute showed negative immunoblot reactivity against FUM20. However, the Rho m 2 K6R mutant can bind FUM20. Three-dimensional structural models of the FUM20 antigenic determinants on serine proteases were constructed. The lysine residue critical for FUM20 interaction is on the surface of the proteases and solvent accessible. The critical core residue proline is located at the beginning of an α-helix. Conclusions: The lysine, proline and tryptophan residues located on the N-terminal region of fungal serine proteases are critical core amino acid residues recognized by FUM20, a monoclonal antibody against serine protease pan-fungal allergens. These findings advance our understanding of the antigenic structures responsible for the antigenicity of serine protease allergens.

Characterization of a novel Candida albicans 29 kDa IgE-binding protein – purification, cDNA isolation and heterologous expression of Cand a 3

Background: Candida albicans has been implicated in human allergic disorders. However, many of its immunoglobulin E (IgE)‐reacting components have not yet been identified. The purpose of the present study is to characterize a novel 29u2003kDa IgE‐binding protein from C. albicans.

The Transaldolase, a Novel Allergen of Fusarium proliferatum, Demonstrates IgE Cross-Reactivity with Its Human Analogue

Fusarium species are among airborne fungi and recognized as causative agents of human atopic disorders. However, Fusarium allergens have not been well characterized and the lack of information limits clinical diagnosis and treatment of fungal allergy. The purpose of this study is to identify and characterize important allergens of F. proliferatum. IgE-reacting F. proliferatum components were identified by immunoblot using serum samples from patients of respiratory atopic diseases. Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning, then homologous expression and immunoblot inhibition studies. We identified nine different F. proliferatum components that can be recognized by IgE antibodies in 17 (28%) of the 60 atopic sera tested. Components with molecular masses of about 43, 37.5 and 36.5 kDa with IgE-binding frequencies of about 88, 47 and 53%, respectively, were considered as important allergens of F. proliferatum. The 37.5 kDa IgE-binding component was putatively considered as a transaldolase protein of F. proliferatum. The full-length cDNA of F. proliferatum transaldolase was subsequently cloned. It encodes an open reading frame of 312 amino acids and has sequence identifies of 73 and 61%, respectively, with Cladosporium and human transaldolases. The purified recombinant F. proliferatum transaldolase can inhibit the IgE-binding against the 37.5 kDa component of F. proliferatum and the transaldolase allergen from Cladosporium cladosporioides. More importantly, the recombinant F. proliferatum transaldolase can inhibit IgE-binding against human transaldolase in a concentration-dependent manner. Thus, a novel and important F. proliferatum transaldolase allergen was identified. In addition to IgE cross-reactivity between the Fusarium and the Cladosporium transaldolase allergens, IgE cross-reactivity between the Fusarium and the human transaldolases also exists and might contribute to atopic manifestations in the absence of exogenous allergen exposure.

Lys89, Lys90, and Phe91 are critical core amino acid residues of the Pen ch 18 major fungal allergen recognized by human IgE antibodies

A vacuolar serine protease (Pen ch 18) has been identified as a major allergen of Penicillium chrysogenum. The molecular features of antigenic determinant(s) on Pen ch 18 recognized by human IgE antibodies, however, have remained unclear. Here, we show that a dominant IgE epitope on the N-terminally processed Pen ch 18 allergen was narrowed down to residues 83-91. In addition, Lys89, Lys90, and possibly Phe91 were identified as the core residues. Substitution of Lys89, Lys90, or Phe91 with alanine can significantly reduce IgE-binding to Pen ch 18. Immunoblot inhibition confirmed that Lys89 and Phe91 played a significant role in IgE-binding against Pen ch 18. Molecular modeling suggests they are located on a loop-like structure at or near the surface of the major fungal allergen.