Stability of the Bet v 1 cross-reactive allergens Api g 1 and Dau c 1 : a biophysical approach

M.A. Bollen

Research output: Thesisinternal PhD, WU

Abstract

The allergen Bet v 1 is known as the primary sensitizer for birch pollen-related food allergy and is responsible for IgE cross-reactivity to pathogenesis-related 10 (PR-10) proteins from, in particular, fruits from the Rosaceae and vegetables from the Apiaceae families. The allergenic potential of PR-10 proteins is mainly characterized for specific recombinantly produced isoforms, which are used for research and diagnostic purposes. However, in natural food sources these allergens are often present as isoform mixtures. The first aim of this research was to purify and characterize PR-10 allergens as natural isoform mixtures to determine whether differences could be observed between natural and recombinant allergens and between plant families. The second aim was to find a relationship between the physico-chemical stability of PR-10 proteins and structural characteristics to explain differences in IgE binding potential and cross-reactivity. The PR-10 allergens Bet v 1 from birch, Api g 1 from celery, and Dau c 1 from carrot were purified under mild conditions following a standardized protocol. Different allergen isoforms were determined and circular dichorism (CD) analyses of the allergen mixtures showed a similar secondary structure composition as observed for other PR-10 proteins. The allergen mixtures and recombinant allergens were characterized by stability studies to pH, temperature and denaturant where CD was used to detect structural changes. Minor differences were observed in stability between natural isoform mixtures and between the recombinant isoforms, although recombinant Dau c 1 was likely destabilized by its attached His-tag. A general trend was observed for allergen stability, structural differences and their relationship to the IgE binding capacity in aqueous solutions. The allergenic potential decreases in the following order: Bet v 1, the primary allergen of birch pollen-related allergies, Mal d 1, Api g 1 and Dau c 1, in accordance with their amino acid sequence identity. Bet v 1 cross-reactive IgE antibodies preferably bind to the charged and polar residues of Mal d 1 for which the positive charge can be increased by the physiological pH of fruit. Api g 1 appears to be more stable than Dau c 1 as the result of a tighter hydrophobic packing. However, the thermodynamic stability of Api g 1 is similar to that of Bet v 1, but the higher proportion of hydrophobic residues and the reduced proportion of charged residues are responsible for the lower IgE binding capacity. Furthermore, the IgE binding capacity is not severely affected, as long as the protein is able to refold. The implications of these findings are discussed in the context of the development of allergic symptoms upon exposure to these PR-10 proteins.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • van Boekel, Tiny, Promotor
  • Savelkoul, Huub, Promotor
  • Wichers, Harry, Promotor
  • Helsper, Hans, Co-promotor
Award date20 May 2009
Place of Publication[S.l.]
Publisher
Print ISBNs9789085853763
Publication statusPublished - 2009

Fingerprint

Allergens
Immunoglobulin E
Protein Isoforms
Allergies
Proteins
Fruits
Chemical stability
Vegetables
Thermodynamic stability
Amino Acids
Antibodies

Keywords

  • allergens
  • food allergies
  • cross reaction
  • betula
  • daucus carota
  • carrots
  • celery
  • apium graveolens
  • pollen allergy

Cite this

@phdthesis{e6070c0847214fccafb5838c123cf69e,
title = "Stability of the Bet v 1 cross-reactive allergens Api g 1 and Dau c 1 : a biophysical approach",
abstract = "The allergen Bet v 1 is known as the primary sensitizer for birch pollen-related food allergy and is responsible for IgE cross-reactivity to pathogenesis-related 10 (PR-10) proteins from, in particular, fruits from the Rosaceae and vegetables from the Apiaceae families. The allergenic potential of PR-10 proteins is mainly characterized for specific recombinantly produced isoforms, which are used for research and diagnostic purposes. However, in natural food sources these allergens are often present as isoform mixtures. The first aim of this research was to purify and characterize PR-10 allergens as natural isoform mixtures to determine whether differences could be observed between natural and recombinant allergens and between plant families. The second aim was to find a relationship between the physico-chemical stability of PR-10 proteins and structural characteristics to explain differences in IgE binding potential and cross-reactivity. The PR-10 allergens Bet v 1 from birch, Api g 1 from celery, and Dau c 1 from carrot were purified under mild conditions following a standardized protocol. Different allergen isoforms were determined and circular dichorism (CD) analyses of the allergen mixtures showed a similar secondary structure composition as observed for other PR-10 proteins. The allergen mixtures and recombinant allergens were characterized by stability studies to pH, temperature and denaturant where CD was used to detect structural changes. Minor differences were observed in stability between natural isoform mixtures and between the recombinant isoforms, although recombinant Dau c 1 was likely destabilized by its attached His-tag. A general trend was observed for allergen stability, structural differences and their relationship to the IgE binding capacity in aqueous solutions. The allergenic potential decreases in the following order: Bet v 1, the primary allergen of birch pollen-related allergies, Mal d 1, Api g 1 and Dau c 1, in accordance with their amino acid sequence identity. Bet v 1 cross-reactive IgE antibodies preferably bind to the charged and polar residues of Mal d 1 for which the positive charge can be increased by the physiological pH of fruit. Api g 1 appears to be more stable than Dau c 1 as the result of a tighter hydrophobic packing. However, the thermodynamic stability of Api g 1 is similar to that of Bet v 1, but the higher proportion of hydrophobic residues and the reduced proportion of charged residues are responsible for the lower IgE binding capacity. Furthermore, the IgE binding capacity is not severely affected, as long as the protein is able to refold. The implications of these findings are discussed in the context of the development of allergic symptoms upon exposure to these PR-10 proteins.",
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author = "M.A. Bollen",
note = "WU thesis, no. 4625",
year = "2009",
language = "English",
isbn = "9789085853763",
publisher = "S.n.",
school = "Wageningen University",

}

Bollen, MA 2009, 'Stability of the Bet v 1 cross-reactive allergens Api g 1 and Dau c 1 : a biophysical approach', Doctor of Philosophy, Wageningen University, [S.l.].

Stability of the Bet v 1 cross-reactive allergens Api g 1 and Dau c 1 : a biophysical approach. / Bollen, M.A.

[S.l.] : S.n., 2009. 160 p.

Research output: Thesisinternal PhD, WU

TY - THES

T1 - Stability of the Bet v 1 cross-reactive allergens Api g 1 and Dau c 1 : a biophysical approach

AU - Bollen, M.A.

N1 - WU thesis, no. 4625

PY - 2009

Y1 - 2009

N2 - The allergen Bet v 1 is known as the primary sensitizer for birch pollen-related food allergy and is responsible for IgE cross-reactivity to pathogenesis-related 10 (PR-10) proteins from, in particular, fruits from the Rosaceae and vegetables from the Apiaceae families. The allergenic potential of PR-10 proteins is mainly characterized for specific recombinantly produced isoforms, which are used for research and diagnostic purposes. However, in natural food sources these allergens are often present as isoform mixtures. The first aim of this research was to purify and characterize PR-10 allergens as natural isoform mixtures to determine whether differences could be observed between natural and recombinant allergens and between plant families. The second aim was to find a relationship between the physico-chemical stability of PR-10 proteins and structural characteristics to explain differences in IgE binding potential and cross-reactivity. The PR-10 allergens Bet v 1 from birch, Api g 1 from celery, and Dau c 1 from carrot were purified under mild conditions following a standardized protocol. Different allergen isoforms were determined and circular dichorism (CD) analyses of the allergen mixtures showed a similar secondary structure composition as observed for other PR-10 proteins. The allergen mixtures and recombinant allergens were characterized by stability studies to pH, temperature and denaturant where CD was used to detect structural changes. Minor differences were observed in stability between natural isoform mixtures and between the recombinant isoforms, although recombinant Dau c 1 was likely destabilized by its attached His-tag. A general trend was observed for allergen stability, structural differences and their relationship to the IgE binding capacity in aqueous solutions. The allergenic potential decreases in the following order: Bet v 1, the primary allergen of birch pollen-related allergies, Mal d 1, Api g 1 and Dau c 1, in accordance with their amino acid sequence identity. Bet v 1 cross-reactive IgE antibodies preferably bind to the charged and polar residues of Mal d 1 for which the positive charge can be increased by the physiological pH of fruit. Api g 1 appears to be more stable than Dau c 1 as the result of a tighter hydrophobic packing. However, the thermodynamic stability of Api g 1 is similar to that of Bet v 1, but the higher proportion of hydrophobic residues and the reduced proportion of charged residues are responsible for the lower IgE binding capacity. Furthermore, the IgE binding capacity is not severely affected, as long as the protein is able to refold. The implications of these findings are discussed in the context of the development of allergic symptoms upon exposure to these PR-10 proteins.

AB - The allergen Bet v 1 is known as the primary sensitizer for birch pollen-related food allergy and is responsible for IgE cross-reactivity to pathogenesis-related 10 (PR-10) proteins from, in particular, fruits from the Rosaceae and vegetables from the Apiaceae families. The allergenic potential of PR-10 proteins is mainly characterized for specific recombinantly produced isoforms, which are used for research and diagnostic purposes. However, in natural food sources these allergens are often present as isoform mixtures. The first aim of this research was to purify and characterize PR-10 allergens as natural isoform mixtures to determine whether differences could be observed between natural and recombinant allergens and between plant families. The second aim was to find a relationship between the physico-chemical stability of PR-10 proteins and structural characteristics to explain differences in IgE binding potential and cross-reactivity. The PR-10 allergens Bet v 1 from birch, Api g 1 from celery, and Dau c 1 from carrot were purified under mild conditions following a standardized protocol. Different allergen isoforms were determined and circular dichorism (CD) analyses of the allergen mixtures showed a similar secondary structure composition as observed for other PR-10 proteins. The allergen mixtures and recombinant allergens were characterized by stability studies to pH, temperature and denaturant where CD was used to detect structural changes. Minor differences were observed in stability between natural isoform mixtures and between the recombinant isoforms, although recombinant Dau c 1 was likely destabilized by its attached His-tag. A general trend was observed for allergen stability, structural differences and their relationship to the IgE binding capacity in aqueous solutions. The allergenic potential decreases in the following order: Bet v 1, the primary allergen of birch pollen-related allergies, Mal d 1, Api g 1 and Dau c 1, in accordance with their amino acid sequence identity. Bet v 1 cross-reactive IgE antibodies preferably bind to the charged and polar residues of Mal d 1 for which the positive charge can be increased by the physiological pH of fruit. Api g 1 appears to be more stable than Dau c 1 as the result of a tighter hydrophobic packing. However, the thermodynamic stability of Api g 1 is similar to that of Bet v 1, but the higher proportion of hydrophobic residues and the reduced proportion of charged residues are responsible for the lower IgE binding capacity. Furthermore, the IgE binding capacity is not severely affected, as long as the protein is able to refold. The implications of these findings are discussed in the context of the development of allergic symptoms upon exposure to these PR-10 proteins.

KW - allergenen

KW - voedselallergieën

KW - kruisreactie

KW - betula

KW - daucus carota

KW - penen

KW - selderij

KW - apium graveolens

KW - hooikoorts

KW - allergens

KW - food allergies

KW - cross reaction

KW - betula

KW - daucus carota

KW - carrots

KW - celery

KW - apium graveolens

KW - pollen allergy

M3 - internal PhD, WU

SN - 9789085853763

PB - S.n.

CY - [S.l.]

ER -