7.3.1 Molecular Biological Properties
 

Conglutin-like Protein	References
Allergen Nomenclature  Ara h 2	(1) Larsen & Lowenstein 1999
Isoallergens and Variants 4 isoallergens in IEF/SDS-PAGE (1)	(1) Burks et al. 1992a
Molecular Mass SDS-PAGE: 17 kDa  (1)	(1) Burks et al. 1992a
Isoelectric Point  pI 5.2 (1)	(1) Burks et al. 1992a
Amino Acid Sequence, mRNA, and cDNA Ara h 2 (precursor)   SWISS-PROT:   GenBank: L77197 PIR:   Amino acids 157 residues (1) mRNA 0.7 kb (1) cDNA  717 bp (1) Gene   * signal peptide 1-18, native protein 19-157 (1)	(1) Stanley et al. 1997
recombinant Protein cDNA expression library: Isolation of Ara h 2 mRNA by PCR  (1, 2) expression in Escherichia coli: Subcloning into pDS56/RBSII vector and expression of recombinant Ara h 2 in E.coli M15 cells (2) Expression in bacterial cells: Expression of recombinant Ara h 2 and mutated Ara h 2 by using procaryotic expression vector pET24 plasmid (2)	(1) Stanley et al. 1997 (2) Burks et al. 1998, 1999 (3) Kleber-Janke et al. 1999
Posttranslational Modifications Glycosylation: Carbohydrate content: 20% (1) Carbohydrate composition: Galacturonic Acid, Ara, Xyl, Glu, Gal, Rha, Man, Fuc (1)	(1) Burks et al. 1992a
Biological Function Seed storage protein (1)	(1) Stanley et al. 1997
Sequence Homology Conglutin-gamma from lupin: 39% aa similarity (1) Mabinlin I (chain B) from caper: 32-35% aa similarity (1) 2S-albumins from sunflower and castor bean: 34% and 30% aa similarity (1)	(1) Stanley et al. 1997

7.3.2 Allergenic Properties
 

Conglutin-like Protein	References
Frequency of Sensitization IgE-binding to Ara h 2 in 71-100% of patients (1)	(1) see 7.1 Sensitization to Peanut Allergens
IgE binding 2 different IgE binding sites indicated by RAST inhibition with mAb vs patients' IgE (1)	(1) Burks et al. 1995a
B-Cell Epitopes IgE binding sites located on Ara h 2 (precursor sequence): Peptides Positivity in Patients aa 15-24 (synthetic peptide) 50% (1) aa 27-36 (synthetic peptide) 100% (1)* aa 57-66 (synthetic peptide) 100% (1)* aa 65-74 (synthetic peptide) 100% (1)* dot / immunoblot (SPOTs membrane technique), * bound more IgE from individual sera than any other epitopes (densitometry) (1) In total 10 IgE-binding epitopes were identified (10 patients with peanut allergy)	(1) Stanley et al. 1997
Sequence Homology of B-Cell Epitopes Epitopes 57-66 and 65-74 both contain the sequence DPYSP (1)	(1) Stanley et al. 1997
Mutational Analysis of B-Cell Epitopes Critical aa residues for IgE binding identified by single site aa substitution: Peptides aa Substitution Positivity in Patients aa 15-24 (synthetic peptide) a) Q20A b) W22A a) - (1) b) - (1) aa 27-36 (synthetic peptide) a) R28A b) R29A c) Q31A d) E35A e) R36A a) - (1) b) - (1) c) - (1) d) - (1) e) - (1) aa 57-66 (synthetic peptide) a) D60A b) P61A c) Y62A a) - (1) b) - (1) c) - (1) aa 65-74 (synthetic peptide) a) D67A b) P68A c) Y69A a) - (1) b) - (1) c) - (1) dot / immunoblot (SPOTs membrane technique) (1) Pooled serum from 15 patients with peanut allergy, in total 10 epitopes analyzed with similar results	(1) Stanley et al. 1997
Allergenicity of mutated rAra h 2 Amino acids critical for IgE- binding of 4 epitopes (27-36, 39-48, 57-66, and 65-74) of Ara h 2 gene were mutated to encode alanine; recombinant mutated protein showed decreased IgE- binding from 12 of 16 sera from peanut allergic patients as compared to recombinant wild-type Ara h 2, while 3 showed similar and 1 increased IgE- binding. No difference in IgG binding to mutated and wild-type Ara h 2 (1)	(1) Burks et al. 1998b, Burks et al. 1999
T-Cell Proliferation No difference in proliferative response of Ara h 2- specific T-cell lines from peanut sensitive patients after stimulation with mutated and wild-type Ara h 2 (1)	(1) Burks et al. 1999
T-Cell Epitopes Specific T-cell proliferation after stimulation with (synthetic peptides):  aa 19-28, 45-53, 96-114, and 131-139  (1)	(1) Burks et al. 1998b