RATIONALE: An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized. METHODS: Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut-allergic donors. RESULTS: Following gel filtration, 75 +/- 7% of the applied protein and 76 +/- 16% (n=3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85 +/- 2%; n=3) of the recovered activity resided in a fraction with a theoretical average molecular weight of approximately 20 kDa and a range of 13-25 kDa. When all the individual fractions were recombined, the measured activity was similar to that of the original extract [140 +/- 43% when measured with a pool of serum (n=2) and 66 +/- 7% when measured with individual sera (n=4)]; when all individual fractions excluding the 20 kDa fraction were recombined, the measured activity was only 8 +/- 2% (n=2) of the original extract when assayed with the serum pool and 10 +/- 4% (n=3) when assayed with the individual sera. Two-dimensional gel electrophoresis of this biologically active fraction revealed >60 protein spots. Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that >97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins. CONCLUSIONS: Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.
RATIONALE: An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized. METHODS: Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut-allergic donors. RESULTS: Following gel filtration, 75 +/- 7% of the applied protein and 76 +/- 16% (n=3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85 +/- 2%; n=3) of the recovered activity resided in a fraction with a theoretical average molecular weight of approximately 20 kDa and a range of 13-25 kDa. When all the individual fractions were recombined, the measured activity was similar to that of the original extract [140 +/- 43% when measured with a pool of serum (n=2) and 66 +/- 7% when measured with individual sera (n=4)]; when all individual fractions excluding the 20 kDa fraction were recombined, the measured activity was only 8 +/- 2% (n=2) of the original extract when assayed with the serum pool and 10 +/- 4% (n=3) when assayed with the individual sera. Two-dimensional gel electrophoresis of this biologically active fraction revealed >60 protein spots. Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that >97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins. CONCLUSIONS:Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.
Authors: S J Maleki; R A Kopper; D S Shin; C W Park; C M Compadre; H Sampson; A W Burks; G A Bannon Journal: J Immunol Date: 2000-06-01 Impact factor: 5.422
Authors: Xiu-Min Li; Kamal Srivastava; James W Huleatt; Kim Bottomly; A Wesley Burks; Hugh A Sampson Journal: J Immunol Date: 2003-03-15 Impact factor: 5.422
Authors: Donald A Dibbern; G William Palmer; P Brock Williams; S Allan Bock; Stephen C Dreskin Journal: J Immunol Methods Date: 2003-03-01 Impact factor: 2.303
Authors: S J Koppelman; E F Knol; R A A Vlooswijk; M Wensing; A C Knulst; S L Hefle; H Gruppen; S Piersma Journal: Allergy Date: 2003-11 Impact factor: 13.146
Authors: M Kulis; X Chen; J Lew; Q Wang; O P Patel; Y Zhuang; K S Murray; M W Duncan; H S Porterfield; A W Burks; S C Dreskin Journal: Clin Exp Allergy Date: 2012-02 Impact factor: 5.018
Authors: H W Chu; C M Lloyd; W Karmaus; P Maestrelli; P Mason; G Salcedo; J Thaikoottathil; A J Wardlaw Journal: Clin Exp Allergy Date: 2010-11 Impact factor: 5.018
Authors: Jelena P Berglund; Nicole Szczepanski; Anusha Penumarti; Ayeshia Beavers; Janelle Kesselring; Kelly Orgel; Bruce Burnett; A Wesley Burks; Michael Kulis Journal: J Allergy Clin Immunol Pract Date: 2017-01-26
Authors: Audrey E Koid; Martin D Chapman; Robert G Hamilton; Ronald van Ree; Serge A Versteeg; Stephen C Dreskin; Stef J Koppelman; Sabina Wünschmann Journal: J Agric Food Chem Date: 2013-12-26 Impact factor: 5.279