Immunologic characterization of natural and recombinant Mal f 1 yeast allergen

doi:10.1016/S0091-6749(99)70433-1

Journal of Allergy and Clinical Immunology

Volume 103, Issue 5, May 1999, Pages 877-884

https://doi.org/10.1016/S0091-6749(99)70433-1 Get rights and content

Abstract

Background: Individuals with atopic dermatitis (AD) often have IgE antibodies against protein components of Malassezia furfur . The cDNA encoding one of these proteins (Mal f 1) has recently been cloned and sequenced. Objective: We sought to express recombinant Mal f 1 (rMal f 1) allergen in large quantities by using different expression systems. The primary aim was to characterize the IgE-binding properties of rMal f 1 in comparison with its natural counterpart in M furfur extract. Methods: We have expressed and purified Mal f 1 from prokaryotic (Escherichia coli ) and eukaryotic cells (baculovirus-infected insect cells). The rMal f 1 produced in both systems has been tested for the ability to be recognized by IgE from patients with specific serum IgE to M furfur by using immunoblotting and the Pharmacia CAP System RAST FEIA. Results: Sixty-one percent of sera from 95 patients showed positive RAST responses to the rMal f 1 produced in the baculovirus expression system and 43% to the E coli –produced rMal f 1. Both the E coli – and baculovirus-produced proteins can specifically inhibit IgE binding to a 36-kd protein band (Mal f 1) in immunoblotting, indicating that the recombinant proteins contain the majority, if not all, the IgE-binding epitopes of Mal f 1. Recombinant Mal f 1 is able to release histamine from basophils of an atopic individual. Conclusion: We have expressed and purified rMal f 1, which can bind IgE in a way resembling natural Mal f 1. The ability to produce recombinant allergens with similar properties to their native counterparts has many potential uses, such as accurately diagnosing causes of IgE-mediated allergy. (J Allergy Clin Immunol 1999;103:877-84.)

Section snippets

Yeast extract

M furfur from American Type Culture Collection (no. 42132) was cultured on glycerol monostearate/olive oil medium for 4 days at 37°C, and the extract was prepared as described earlier.¹⁷

Patient sera

Among sera from patients with AD sent to the Division of Clinical Immunology at the Karolinska Hospital (Stockholm, Sweden) for IgE antibody analysis from January 1997 to February 1998, all sera with a sufficient volume (≥1 mL) were selected on the basis of a specific IgE reactivity to M furfur . The 95 sera

In vitro translation

In the absence of microsomes (Fig 1, A, lane 1 ), the Mal f 1 containing its signal sequence is translated.

. A, In vitro translation of in vitro transcribed mRNA was carried out in the presence (+) or absence (–) of microsomes, acceptor peptide (AP) , or nonacceptor peptide (NAP) . After translation, some of the reactions were treated with proteinase K (PK) . The protein band at position a is the Mal f 1–containing signal sequence, at position b is the N-glycosylated mature Mal f 1, and at

DISCUSSION

The Mal f 1 allergen has been expressed in different expression systems in order to characterize its properties. In an in vitro translation system in the presence of microsomes, the signal sequence of Mal f 1 is cleaved off, and about 50% of the molecules become N-glycosylated. It appears that only a small proportion (even less than 50%) of Mal f 1 in its natural form is glycosylated. The reason for the molecule being only partially glycosylated is most likely to be caused by the suboptimal

Acknowledgements

We thank MIAB, Uppsala, Sweden, for serological analysis and Professor S. G. O. Johansson for helpful discussion.

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Cited by (16)

Activation profile of THP-1 derived dendritic cells stimulated by allergen Mal f 1 beyond its IgE-binding ability
2018, International Immunopharmacology
Citation Excerpt :
Mal f 1 is the first allergen cloned from M. furfur and is a cell wall protein that does not show sequence similarity to other proteins [11,12]. Serum IgEs from 43% and 61% of the 95 patients tested showed positive reactivity to rMal f 1 produced in the E. coli and baculovirus expression systems, respectively [13]. However, its role in the pathogenesis of the aforementioned diseases, though often displayed by IgE binding results, is inadequately understood.
Mal f 1, the first allergen cloned from Malassezia furfur, has positive IgE reactivity in sera from atopic dermatitis (AD) patients. The mechanism by which Mal f 1 induces the maturation of human dendritic cells (DCs) and maintains the symptoms of AD is not well understood.
The present study aims to explore the activation profile of THP-1 derived dendritic cells (TDDCs) stimulated by recombinant Mal f 1, as well as to explore the IgE-binding ability of rMal f 1 and its correlation with IgE-binding activity of complete allergens of M. furfur.
rMal f 1 was produced by expression in E. coli and purification with affinity chromatography. The ability of rMal f 1 and ImmunoCAP complete allergens of M. furfur to bind to serum specific IgE was assayed in parallel by ELISA and immunoblotting. Immature TDDCs were stimulated with rMal f 1 or an enzyme-digested product of rMal f 1. The expression levels of markers, CD83, CD80, CD86, and HLA-DR, were investigated by flow cytometry. The levels of interleukin (IL)-6, IL-10, IL-12p70 and tumor necrosis factor (TNF)-α in culture supernatants were determined by ELISA.
Eighteen patient sera were identified that reacted positively to the complete allergens of M. furfur as determined by ImmunoCAP and also showed positive responses to rMal f 1. Five patient sera were identified that had no reaction to ImmunoCAP complete allergens of M. furfur and also exhibited negative response to rMal f 1. All sera, except for one, had no reaction to the unrelated allergen Bet v 1. rMal f 1 upregulated the maturation surface marker CD83 on TDDCs. In addition, rMal f 1 also induced high levels of CD80 and CD86. Increased expression of HLA-DR, a first signal for T cell activation, was observed. Secretion of IL-6, TNF-α and IL-10 by TDDCs increased significantly (P < 0.0001 for IL-6, P < 0.01 for TNF-α and P < 0.05 for IL-10) after stimulation by rMal f 1, while the IL-12p70 level was unaltered.
We have shown that rMal f 1 has ideal IgE binding ability and good correlation with binding activity to M. furfur. Moreover, we have revealed a hitherto unknown DC activation profile after rMal f 1 stimulation whereby TNF-α, IL-6, and IL-10 were significantly increased and IL-12 was unaltered, suggesting that rMal f 1 can predispose a DC bias toward the T_H22/T_H17 pathway beyond the routine IgE-dependent T_H2 pathway, thus providing intriguing clues for clinical treatment involving both pathways.
High-level expression and purification of the major house dust mite allergen der p 2 in Escherichia coli
2016, Protein Expression and Purification
Citation Excerpt :
The use of defined allergen for allergy immune therapy improves both efficacy and reproducibility of the treatment. With the introduction of recombinant DNA technology into medicine, most of the clinically relevant allergens have been produced as pure recombinant allergens that mimic epitope spectrum of their natural counterparts [7–9]. Vaccines based on recombinant allergens were tested in several clinical trials and showed promising results in patients [10–13].
Der p 2, a major allergen derived from the house dust mite Dermatophagoides pteronyssinus, is one of the most clinically relevant allergens worldwide. Recombinant Der p 2 (rDer p 2) is useful in clinical diagnosis and disease-specific immunotherapy. However, previous studies showed that Der p 2 can only be expressed in Escherichia coli (E. coli) cells as inclusion bodies, thus protein refolding is required to obtain functional products. Here we report a new method to produce biologically active Der p 2 protein in E. coli. N-terminal hexahistidine- and trigger factor (TF)-tagged Der p 2 was expressed in soluble form in E. coli and purified using a combination of chromatography processes. This procedure produced milligram-level high purity Der p 2 per liter of bacterial culture. Moreover, far-UV region circular dichroism (CD) analysis and serum specific IgE reactivity test demonstrated that the secondary structure and IgE reactivity properties of rDer p 2 produced in our study were almost identical to those of natural Der p 2 (nDer p 2). In conclusion, the method developed in this work provides a useful tool for the production of immunologically active recombinant Der p 2 for clinical applications.
Sensitization to fungal allergens: Resolved and unresolved issues
2015, Allergology International
Citation Excerpt :
Mannan, a polysaccharide, is also known to be associated with IgE cross-reactivity beyond the genus.147 In contrast, Mala s 1, 7, 8, and 9 are considered to be specific allergens with unique sequences.129,144,148,149 Casagrande et al. investigated the frequency of sensitization to a panel of recombinant M. sympodialis allergens (rMala s 1 and 5–9) in 51 patients with atopic eczema who were positive for IgE Abs to crude M. sympodialis (ImmunoCAP m70).
Exposure and sensitization to fungal allergens can promote the development and worsening of allergic diseases. Although numerous species of fungi have been associated with allergic diseases in the literature, the significance of fungi from the genera Alternaria, Cladosporium, Penicillium, Aspergillus, and Malassezia has been well documented. However, it should be emphasized that the contribution of different fungal allergens to allergic diseases is not identical, but species-specific.
Alternaria and Cladosporium species are considered to be important outdoor allergens, and sensitization and exposure to species of these genera is related to the development of asthma and rhinitis, as well as epidemics of asthma exacerbation, including life-threatening asthma exacerbation. In contrast, xerophilic species of Penicillium and Aspergillus, excluding Aspergillus fumigatus, are implicated in allergic diseases as indoor allergens. A. fumigatus has a high capacity to colonize the bronchial tract of asthmatic patients, causing severe persistent asthma and low lung function, and sometimes leading to allergic bronchopulmonary aspergillosis. Malassezia are common commensals of healthy skin, although they are also associated with atopic dermatitis, especially on the head and neck, but not with respiratory allergies.
Despite its importance in the management of allergic diseases, precise recognition of species-specific IgE sensitization to fungal allergens is often challenging because the majority of fungal extracts exhibit broad cross-reactivity with taxonomically unrelated fungi. Recent progress in gene technology has contributed to the identification of specific and cross-reactive allergen components from different fungal sources. However, data demonstrating the clinical relevance of IgE reactivity to these allergen components are still insufficient.
Crystal Structure of the Major Malassezia sympodialis Allergen Mala s 1 Reveals a β-Propeller Fold: A Novel Fold Among Allergens
2007, Journal of Molecular Biology
Atopic eczema (AE) is a chronic inflammatory disease in which genetic predisposition and environmental factors such as microorganisms contribute to the symptoms. The yeast Malassezia Sympodialis, part of the normal human cutaneous flora, can act as an allergen eliciting specific IgE and T-cell reactivity in patients with AE. The major M. sympodialis allergen Mala s 1 is localized mainly in the yeast cell wall and exposed on the cell surface. Interestingly, Mala s 1 does not exhibit any significant sequence homology to known proteins. Here we present the crystal structure of Mala s 1 determined by single-wavelength anomalous dispersion techniques using selenomethionine-substituted Mala s 1. Mala s 1 folds into a 6-fold β-propeller, a novel fold among allergens. The putative active site of Mala s 1 overlaps structurally to putative active sites in potential homologues, Q4P4P8 and Tri 14, from the plant parasites Ustilago maydis and Gibberella zeae, respectively. This resemblance suggests that Mala s 1 and the parasite proteins may have similar functions. In addition, we show that Mala s 1 binds to the phosphoinositides (PI) PI(3)P, PI(4)P, and PI(5)P, lipids possibly playing a role in the localization of Mala s 1 to the cell surface. The crystal structure of Mala s 1 will provide insights into the role of this major allergen in the host–microbe interactions and induction of an allergic response in AE.
Fungal allergens and peptide epitopes
2000, Peptides
Fungal allergens represent a major cause of atopic disorders. Immunochemical and molecular characterization of fungal allergens has been hampered by the lack of pure proteins and to inherent variation among fungal proteins and in their poor yields. With the advent of molecular biology techniques, a number of allergens have been cloned, sequenced, and expressed from a variety of fungal species. The knowledge of the primary, secondary, and tertiary structures of these allergens, the immunodominant regions of these proteins, and their interaction with T and B-cell epitopes, results in better understanding of the molecular mechanisms of allergy and may provide avenues of immunologic intervention to treat patients. The present review deals with the current understanding of fungal allergen epitopes.
Expression of recombinant allergen, der f 1, der f 2 and der f 4 using baculovirus-insect cell systems
2018, Archives of Medical Science

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^☆: Supported by the Swedish Medical Research Council (grants no. 16X-7924 and 16X-12632), the Swedish Association against Asthma and Allergy, the Swedish Council for Work Life Research, the Swedish Foundation for Health Care Sciences and Allergy Research, the Hesselman Foundation, the Wenner-Gren Center Foundation, and the Karolinska Institute.

^☆☆: Reprint requests: Arezou Zargari, PhD, Department of Laboratory Medicine, Division of Clinical Immunology, Karolinska Hospital, S-171 76 Stockholm, Sweden.

^★: 0091-6749/99 $8.00+0 1/1/96780

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Journal of Allergy and Clinical Immunology

Abstract

Section snippets

Yeast extract

Patient sera

In vitro translation

DISCUSSION

Acknowledgements

References (23)

Lipophilic yeastlike organisms associated with tinea versicolor

J Invest Dermatol

Immune reactions to Pityrosporum ovale in adult patients with atopic and seborrhoeic dermatitis

J Am Acad Dermatol

Atopic dermatitis of the face, scalp, and neck: Type I reaction to the yeast Pityrosporum ovale ?

J Allergy Clin Immunol

Determination of the distance between the oligosaccharyl-transferase active site and the endoplasmic reticulum membrane

J Biol Chem

The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency

J Biol Chem

Induction of specific histamine release from basophils with purified natural and recombinant birch pollen allergens

J Allergy Clin Immunol

Pityrosporum orbiculare : incidence and distribution on clinically normal skin

Br J Dermatol

Quantitative variations in distribution of Pityrosporum orbiculare on clinically normal skin

Acta Derm Venereol (Stockh)

Pityrosporum folliculitis

Arch Dermatol

Pityrosporum orbiculare : A pathogenic factor in atopic dermatitis of the face, scalp and neck?

Acta Derm Venereol (Stockh)

IgE antibodies to Pityrosporum orbiculare in children with atopic diseases

Acta Paediatr Scand

Cited by (16)

Activation profile of THP-1 derived dendritic cells stimulated by allergen Mal f 1 beyond its IgE-binding ability

High-level expression and purification of the major house dust mite allergen der p 2 in Escherichia coli

Sensitization to fungal allergens: Resolved and unresolved issues

Crystal Structure of the Major Malassezia sympodialis Allergen Mala s 1 Reveals a β-Propeller Fold: A Novel Fold Among Allergens

Fungal allergens and peptide epitopes

Expression of recombinant allergen, der f 1, der f 2 and der f 4 using baculovirus-insect cell systems

Immunologic characterization of natural and recombinant Mal f 1 yeast allergen☆,☆☆,★

Abstract

Section snippets

Yeast extract

Patient sera

In vitro translation

DISCUSSION

Acknowledgements

J Invest Dermatol

J Am Acad Dermatol

J Allergy Clin Immunol

J Biol Chem

J Biol Chem

J Allergy Clin Immunol

Pityrosporum orbiculare : incidence and distribution on clinically normal skin

Br J Dermatol

Quantitative variations in distribution of Pityrosporum orbiculare on clinically normal skin

Acta Derm Venereol (Stockh)

Pityrosporum folliculitis

Arch Dermatol

Pityrosporum orbiculare : A pathogenic factor in atopic dermatitis of the face, scalp and neck?

Acta Derm Venereol (Stockh)

IgE antibodies to Pityrosporum orbiculare in children with atopic diseases

Acta Paediatr Scand