7.2.1 Molecular Biological Properties
 

Vicilin	References
Allergen Nomenclature  Ara h 1	(1) Larsen & Lowenstein 1999
Isoallergens and Variants 2 isoallergens in SDS-PAGE (1)	(1) Burks et al. 1991
Molecular Mass SDS-PAGE: 63.5 kDa  (1), rAra h 1: 68 kDa (2)	(1) Burks et al. 1991 (2) Burks et al. 1995c
Isoelectric Point  pI 4.55 (1)	(1) Burks et al. 1991
Amino Acid Sequence, mRNA, and cDNA Ara h 1 (precursor)* Clone P17 Clone P41B SWISS-PROT: P43237 P43238 GenBank: L38853 L34402 Amino acids 614 residues (1) 626 residues (1) mRNA 1949 bp,  2.3 kb (1) 2032 bp cDNA     Gene     * N-terminus of native protein (deduced from 2): aa 79 (P17) and  aa 85 (P41B)	(1) Burks et al. 1995b, 1995c (2) de Jong et al. 1998
Recombinant Protein cDNA expression library: Isolation of Ara h 1 mRNA by PCR  (1, 2) expression in Escherichia coli: Expression of recombinant Ara h 1 selected from cDNA library (1) Subcloning into pDS56/RBSII vector and expression of recombinant Ara h 1 in E.coli M15 cells (2)	(1) Burks et al. 1995b, 1995c (2) Kleber-Janke et al. 1999
Formation of Oligomers Formation of stable trimeric structures of Ara h 1 measured by fluorescence polarization followed by SDS-PAGE of cross- linked Ara h 1 (1)	(1) Shin et al. 1998
Posttranslational Modifications Glycosylation: Carbohydrate content: 2.4% (1)* Carbohydrate composition: Xyl, Man, Glu (2:1:1), and GlcNAc (1)* Concanavalin A reactive (1)* Periodic acid-Schiff staining positive (SDS-PAGE), ConA binding negativ  (2)	(1) Barnett & Howden 1986 (2) Burks et al. 1991
Biological Function Seed storage protein (1)	(1) Burks et al. 1998
Sequence Homology ConA-binding peanut allergen (65 kDa, pI 4.6, N-terminal aa: GSAPGERQRGCYPGN) (1) may be identical to Ara h 1: aa composition score 6 and 8, and 66.7% and 64.3% aa identity (of 15 N-terminal aa)  to Ara h 1 clones (2) Vicilins from broad beans and peas: 60-65% DNA homology to Ara h 1 (3)	(1) Barnett & Howden 1986 (2) Amino Acid Composition Search and Similarity Search (SIM) at ExPASy (3) Burks et al. 1995c

* Results obtained for 65 kDa allergen with similarity to Ara h 1 (see Sequence Homology)
 

7.2.2 Allergenic Properties
 

Vicilin	References
Frequency of Sensitization IgE-binding to Ara h 1 in 65-100% of patients (1)	(1) see 7.1 Sensitization to Peanut Allergens
Allergenic Potencies 3-48% inhibition of IgE binding to raw peanut extract (8 peanut allergic patients, RAST inhibition) (1)*	(1) Barnett & Howden 1986
Allergenicity of recombinant Ara h 1 95% of peanut allergic patients reacted to recombinant Ara h 1 (18 peanut- allergic patients with positive IgE-binding to native Ara h 1, SDS-PAGE immunoblot) (1)	(1) Burks et al. 1995c
IgE binding 3 different IgE binding sites indicated by RAST inhibition with mAb vs patients' IgE (1)	(1) Burks et al. 1994b
B-Cell Epitopes IgE binding sites located on Ara h 1 (precursor sequence): Peptides Positivity in Patients aa 25-34 (synthetic peptide) 80% (1)* aa 65-74 (synthetic peptide) 80% (1)* aa 89-98 (synthetic peptide) 80% (1)* aa 344-353 (synthetic peptide) 80% (1) aa 498-507 (synthetic peptide) 80% (1)* aa 578-587 (synthetic peptide) 80% (1) dot / immunoblot (SPOTs membrane technique) * bound more IgE from individual sera than any other epitopes (1) In total 23 IgE-binding epitopes were identified (10 patients with peanut allergy )	(1) Burks et al. 1997
Mutational Analysis of B-Cell Epitopes Critical aa residues for IgE binding identified by single site aa substitution: Peptides aa Substitution* IgE binding aa 25-34 (synthetic peptide) a) S28 b) P29 c) Y30 d) K32 a) - (1, 2) b) - (1, 2) c) - (1, 2) d) - (1, 2) aa 65-74 (synthetic peptide) a) Y67 b) D68 c) P69 d) V72 e) Y73 a) - (1, 2) b) - (1, 2) c) - (1, 2) d) - (1, 2) e) - (1, 2) aa 89-98 (synthetic peptide) a) R91 b) R93 c) G94 d) R95 e) Q96 a) - (1, 2) b) - (1) c) - (1, 2) d) - (1, 2) e) - (1, 2) aa 344-353 (synthetic peptide) a) G349 a) - (2) aa 498-507 (synthetic peptide) a) R498 b) R499 c) Y500 a) - (1, 2) b) - (1, 2) c) - (1, 2) aa 578-587 (synthetic peptide) a) S585 a) - (2) dot / immunoblot (SPOTs membrane technique) * aa substitution with A (1) and A or M (2), respectively (1, 2) Pooled serum from 15 patients with peanut allergy (2) In total 21 epitopes analyzed with similar results	(1) Burks et al. 1997 (2) Shin et al. 1998
Sequence Homology of B-Cell Epitopes Epitope 498-507 shares significant sequence homology to other vicilins from legumes, epitopes 25-34, 65-74, and 89-98 do not share significant homologies to other vicilins (1)	(1) Burks et al. 1997
Types of Critical Amino Acids In 21 epitopes studied by single mutational aa substitution contained 45% charged aa, 36% hydrohopic aa, and 18% polar aa; 35% of mutated hydrophobic residues, 25% of polar, and 17% of charged aa resulted in loss of IgE binding (1)	(1) Shin et al. 1998
Location of B-Cell Epitopes on 3D-Structure IgE-binding sites clustered into 2 main regions, despite their even distribution throughout the primary sequence; 10 IgE-binding residues buried beneath the surface and 25 residues exposed on the surface of Ara h 1 (homology based 3D-model of Ara h 1: alignment to x-ray crystal structure of phaseolin) (1) IgE-binding epitopes 311-320 and 325-334 as well as epitopes 551-560 and 559-568 of one monomer contact one another in trimeric formation of Ara h 1 (2)	(1) Shin et al. 1998 (2) Maleki et al. 2000
Alteration of Allergenicity Deglycosylation: IgE binding potency reduced about 25-50% after chemical deglycosylation (pooled sera from 5 peanut allergic patients, RAST inhibition) (1)*	(1) Barnett & Howden 1986 see also 10 Stability of Peanut Allergens

* Results obtained for 65 kDa allergen with similarity to Ara h 1 (see Sequence Homology)