6.2 Properties of Bet v 1 - homologous Protein (Api g 1)

6.2.1 Molecular Biological Properties
 

Bet-v-1-homologous Protein	References
Allergen Nomenclature Api g 1	(1) Larsen & Lowenstein 2000
Isoallergens and Variants 2 isoallergens designated Api g 1.0101 and Api g 1.0201 sharing 72% aa similarity (1, 3) (BLAST at NBCI: 49% aa sequence identity and 65% similarity) 4 isoallergens in 2D-PAGE immunoblot (2)	(1) Breiteneder et al. 1995 (2) Schöning et al. 1995 (3) Hoffmann-Sommergruber et al. 2000
Molecular Mass    Api g 1.0101 Api g 1.0201 calculated:  16.2 kDa (1) 17 kDa (5) SDS-PAGE:  15 kDa (2), 16 kDa (4) 17 kDa (5) 2D-PAGE:  15.5 kDa (3)	(1) Breiteneder et al. 1995 (2) Ebner et al. 1995 (3) Schöning et al. 1995 (4) Jankiewicz et al. 1998 (5) Hoffmann-Sommergruber et al. 2000
Isoelectric Point  2D-PAGE: major protein pI 4.4-4.6 (1), minor proteins pI 4.9, 5.1, and 6.0 (1) calculated: pI 4.45 (Api g 1.0201) (2)	(1) Schöning et al. 1995 (2) Hoffmann-Sommergruber et al. 2000
Amino Acid Sequence, mRNA, and cDNA Api g 1 Api g 1.0101 Api g 1.0201 SWISS-PROT: P49372   GenBank: Z48967 Z75662 PIR: S63984   Amino Acids 154* 159 mRNA 643 bp 700 bp cDNA     * product 2-154	(1) Breiteneder et al. 1995 (2) Hoffmann-Sommergruber et al. 2000
recombinant Protein Expression in Escherichia coli: cDNA subcloned into expression plasmid pMW 175 and expression of rApi g 1 in E.coli BL21DE3 (1, 2) cDNA library: Api g 1.0201 cloned after selection of IgE-binding clone from celery cDNA expression library (3) Api g 1.0201- cDNA subcloned as a 6xHis-tagged protein into plasmid pDS56 and expression in E.coli M15[pREP4] (4)	(1) Breiteneder et al. 1995 (2) Hoffmann-Sommergruber et al. 1996, 1999a (3) Scheiner et al. 1997 (4) Hoffmann-Sommergruber et al. 2000
3D-Structure Model structure of Api g 1.0201 calculated by homology to Bet v 1 (3D-structure prediction using SWISS MODEL from ExPASy) (1)	(1) Hoffmann-Sommergruber et al. 2000
Biological Function Bet v 1 family of pathogenesis-related proteins (PR-family 10) (1)	(1) SWISS-PROT
Sequence Homology aa sequence identity Api g 1.0101 Api g 1.0201 Carrot allergen Dau c 1:  81% 47% 2 Parsley pathogenesis-related proteins: 61% 60% Korean ginseng ribonuclease 1: 56% 65% Dandelion pathogenesis-related protein: 44% 33% Birch pollen allergen Bet v 1: 42% 34-37% Cherry allergen Pru a 1: 42% 39% Apple allergen Mal d 1: 39% 36% Hazelnut allergen Cor a 1: 38% 37% Soybean stress-induced protein: 38% 37% 2 Potato pathogenesis-related proteins: 35-38% 32-33%	(1) Breiteneder et al. 1995 (2) Hoffmann-Sommergruber et al. 1998 (3) BLAST at NBCI

 6.2.2 Allergenic Properties
 

Bet v 1 - homologous Protein	References
Frequency of Sensitization IgE-binding to Api g 1 in 33-100% of patients (1)	(1) see 6.1 Sensitization to Celery Allergens
Allergenicity of recombinant Api g 1 (1) All 10 sera from celery allergic patients bound to natural and recombinant Api g 1 with comparable intensities (immunoblot); complete inhibition of IgE binding to natural Api g 1 by rApi g 1 and rBet v 1, only reduced inhibition by rApi g 1 to natural Bet v 1 (serum pool, immunoblot inhibition); (2) 24 celery allergic and Api g 1 sensitized patients: a) Equal diagnostic potency (100% positivity) of commercial crude celery extract, native celery tuber and rApi g 1 in skin testing (SPT, skin prick-to-prick test) b) 16 sera showed IgE binding to Api g 1 in SDS-PAGE immunoblot, 2 sera detected native Api g 1 without reactivity to rApi g 1 indicating the presence of additional isoforms in native Api g 1 (3) 22 celery allergic patients (DBPCFC positive): 13 sera bound to natural and rApi g 1 in SDS-PAGE immunoblot (3 sera weaker binding to natural Api g 1 than to rApi g 1) and to rApi g 1 in EAST; inhibition of IgE binding from 1 patient's serum to rApi g 1 by native celery extract and rApi g 1 and to native Api g 1 from celery extract by rApi g 1 (immunoblot)	(1) Breiteneder et al. 1995 (2) Hoffmann-Sommergruber et al. 1999a (3) Lüttkopf et al. 2000
Allergenicity of recombinant Api g 1 isoforms Purified recombinant isoform Api g 1.0201: recognized by 10 sera from celery allergic patients, yet with weaker  IgE-binding capacity as compared to rApi g 1 (Api g 1.0101) (SDS-PAGE immunoblot) (1) In immunoblot inhibition: IgE-binding to Api g 1.0201 not inhibited by rApi g 1 (Api g 1.0101), only weak inhibition by rBet v 1a, complete inhibition by natural Bet v 1 (1)	(1) Hoffmann-Sommergruber et al. 2000
Stability of Api g 1 Allergenic activity sensitive to heat treatment of celery tuber	(1) see 9 Stability of Celery Allergens