Hsiao-Yun Tai

Vacuolar Serine Protease Is a Major Allergen of Cladosporium cladosporioides

Background:Cladosporium is an important allergenic fungus worldwide. We report here a major allergen of C. cladosporioides.Methods: Major C. cladosporioides allergens were characterized by immunoblotting, N-terminal amino acid sequencing, protein purification and cDNA cloning. Results: Seventy-four sera (38%) from 197 bronchial asthmatic patients demonstrated IgE binding against C. cladosporioides extracts. Among these 74 sera, 41 (55%) and 38 (51%) showed IgE binding against a 36- and a 20-kDa protein of C. cladosporioides, respectively. Both IgE-reacting components reacted with FUM20, a monoclonal antibody against fungal serine proteases. N-terminal amino acid sequencing results suggest that they are vacuolar serine proteases, and the 20-kDa component is possibly a degraded product of the 36-kDa allergen. A corresponding 5′-truncated 1,425-bp cDNA fragment was isolated. The mature protein after N-terminal processing starts with an N-terminal serine that is the ninth residue encoded by the 5′-truncated cDNA. The protein sequence deduced shares 69–72% sequence identity with Penicillium vacuolar serine proteases and was designated as Cla c 9. The purified 36-kDa Cla c 9 allergen showed proteolytic activity with peptide Z-Ala-Ala-Leu-pNA as substrate. IgE cross-reactivity was detected between the purified Cla c 9 and serine protease allergens from Aspergillusfumigatus and Penicillium chrysogenum.Conclusion: We identified a vacuolar serine protease as a major allergen of C. cladosporioides (Cla c 9) and a major pan-allergen of prevalent airborne fungi. IgE cross-reactivity among these highly conserved serine protease pan-fungal allergens was also detectable.

A vacuolar serine protease (Rho m 2) is a major allergen of Rhodotorula mucilaginosa and belongs to a class of highly conserved pan-fungal allergens.

Background:Rhodotorula mucilaginosa is one of the most frequently encountered species of yeasts in our environment. We reported here a major allergen of R. mucilaginosa. Methods: A major R. mucilaginosa allergen (Rho m 2) was characterized by two-dimensional (2D) immunoblotting, protein sequencing, cDNA cloning and IgE cross-reactivity with fungal serine proteases. Results: Fourty-four sera (28%) from 157 bronchial asthmatic patients showed IgE-immunoblot reactivity against R. mucilaginosa extract. Among these 44 sera, 25 (57%) demonstrated IgE binding against a 31-kDa protein of R. mucilaginosa. Protein sequencing results suggest that it is a vacuolar serine protease. The corresponding cDNA clone encoding a mature protein of 312 residues was isolated. It shares 67–68% sequence identity with vacuolar serine protease allergens from three different Penicillium species (Pen ch 18, Pen o 18 and Pen c 18) and designated as Rho m 2 by the Allergen Nomenclature Committee. The native and recombinant Rho m 2 react with IgE antibodies and monoclonal antibody (MoAb) FUM20 against fungal serine proteases. IgE cross-reactivity between nRho m 2 and nPen ch 18 was observed. It was also detectable between rRho m 2 and rPen o 18. Conclusion: Our results suggest that R. mucilaginosa may also be a significant causative agent of human respiratory allergic disorders. We identified a vacuolar serine protease as a major allergen of R. mucilaginosa (Rho m 2) and a pan allergen of prevalent airborne fungal species. We detected IgE cross-reactivity among these highly conserved serine protease pan-fungal allergens.

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.

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.

Epitope Mapping and In Silico Characterization of Interactions between Der p 7 Allergen and MoAb WH9

Der p 7 is an important house dust mite allergen. However, antigenic determinants of Der p 7 are largely unknown. The purpose of this study is to analyze the determinants of Der p 7 and determine the structural basis of interactions between Der p 7 and WH9, an IgE-binding inhibition mouse monoclonal antibody (MoAb). IgE and WH9-reactive determinant(s) was identified by immunoblot using allergen mutants. A 3-D binary complex structure of Der p 7 and WH9 was simulated with homology modeling and docking methods. Our results obtained showed that among the five Der p 7 mutants (S156A, I157A, L158A, D159A, P160A), serum no. 1045 with IgE-binding against Der p 7 exhibited a reduced IgE immunoblot reactivity against Der p 7 L158A and D159A mutants. WH9 showed reduced immunoblot reactivity against S156A, L158A, D159A and P160A and the observation was confirmed by immunoblot inhibition. The WH9-binding determinant on Der p 7 containing S156, L158, D159 and P160 assumes a loop-like structure. The structural model of the Der p 7-WH9 complex suggests residues S156, I157, L158, D159 and P160 of Der p 7 contribute to WH9 binding via potential hydrogen bonds, electrostatic and hydrophobic interactions. In conclusion, MoAb WH9 interacts with critical residues L158 and D159 of Der p 7 and inhibits IgE-binding to Der p 7. Results obtained advance our understanding on molecular and structural bases of the antigenicity of Der p 7, its interactions with MoAb WH9 and facilitate the design of safer immunotherapy of human atopic disorders.