Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1

Y. Ma; G. Gadermaier; B. Bohle; S.T.H.P. Bolhaar; A.C. Knulst; Z. Markovic-Housley; H. Breiteneder; P. Briza; K. Hoffmann-Sommergruber; F. Ferreira

doi:10.1159/000089756

Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1

Y. Ma, G. Gadermaier, B. Bohle, S.T.H.P. Bolhaar, A.C. Knulst, Z. Markovic-Housley, H. Breiteneder, P. Briza, K. Hoffmann-Sommergruber, F. Ferreira

Dermatology and Allergology

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

BACKGROUND: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1.

METHODS: A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed.

RESULTS: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88-30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays.

CONCLUSIONS: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy.

Original language	English
Pages (from-to)	53-62
Number of pages	10
Journal	International Archives of Allergy and Immunology
Volume	139
Issue number	1
DOIs	https://doi.org/10.1159/000089756
Publication status	Published - 2006

Keywords

Allergens
Amino Acid Sequence
Antibody Specificity
Antigens, Plant
Binding Sites, Antibody
Epitopes
Food Hypersensitivity
Humans
Immunoglobulin E
Malus
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Plant Proteins
Point Mutation
Recombinant Proteins
Sequence Alignment

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10.1159/000089756

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Ma, Y., Gadermaier, G., Bohle, B., Bolhaar, S. T. H. P., Knulst, A. C., Markovic-Housley, Z., Breiteneder, H., Briza, P., Hoffmann-Sommergruber, K., & Ferreira, F. (2006). Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1. International Archives of Allergy and Immunology, 139(1), 53-62. https://doi.org/10.1159/000089756

@article{f434a5ce7e6942f2a44820707485d185,

title = "Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1",

abstract = "BACKGROUND: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1.METHODS: A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed.RESULTS: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88-30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays.CONCLUSIONS: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy.",

keywords = "Allergens, Amino Acid Sequence, Antibody Specificity, Antigens, Plant, Binding Sites, Antibody, Epitopes, Food Hypersensitivity, Humans, Immunoglobulin E, Malus, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Plant Proteins, Point Mutation, Recombinant Proteins, Sequence Alignment",

author = "Y. Ma and G. Gadermaier and B. Bohle and S.T.H.P. Bolhaar and A.C. Knulst and Z. Markovic-Housley and H. Breiteneder and P. Briza and K. Hoffmann-Sommergruber and F. Ferreira",

year = "2006",

doi = "10.1159/000089756",

language = "English",

volume = "139",

pages = "53--62",

journal = "International Archives of Allergy and Immunology",

issn = "1018-2438",

publisher = "S. Karger AG",

number = "1",

}

Ma, Y, Gadermaier, G, Bohle, B, Bolhaar, STHP, Knulst, AC, Markovic-Housley, Z, Breiteneder, H, Briza, P, Hoffmann-Sommergruber, K & Ferreira, F 2006, 'Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1', International Archives of Allergy and Immunology, vol. 139, no. 1, pp. 53-62. https://doi.org/10.1159/000089756

TY - JOUR

T1 - Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1

AU - Ma, Y.

AU - Gadermaier, G.

AU - Bohle, B.

AU - Bolhaar, S.T.H.P.

AU - Knulst, A.C.

AU - Markovic-Housley, Z.

AU - Breiteneder, H.

AU - Briza, P.

AU - Hoffmann-Sommergruber, K.

AU - Ferreira, F.

PY - 2006

Y1 - 2006

N2 - BACKGROUND: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1.METHODS: A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed.RESULTS: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88-30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays.CONCLUSIONS: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy.

AB - BACKGROUND: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1.METHODS: A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed.RESULTS: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88-30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays.CONCLUSIONS: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy.

KW - Allergens

KW - Amino Acid Sequence

KW - Antibody Specificity

KW - Antigens, Plant

KW - Binding Sites, Antibody

KW - Epitopes

KW - Food Hypersensitivity

KW - Humans

KW - Immunoglobulin E

KW - Malus

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Plant Proteins

KW - Point Mutation

KW - Recombinant Proteins

KW - Sequence Alignment

U2 - 10.1159/000089756

DO - 10.1159/000089756

M3 - Article

C2 - 16293967

SN - 1018-2438

VL - 139

SP - 53

EP - 62

JO - International Archives of Allergy and Immunology

JF - International Archives of Allergy and Immunology

IS - 1

ER -

Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1

Abstract

Keywords

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