7.2 Properties of Ovomucoid

7.2.1 Molecular Biological Properties
 

Ovomucoid (OM)	References
Allergen Nomenclature  Gal d 1	(1) King et al. 1994
Isoallergens and Variants 2 genetic variants a) whole sequence of 186 aa residues and b) deletion of Val-134-Ser-135 (1)	(1) Kato et al. 1987
Molecular Mass  Mr 28.0 kDa (1, 3) 26.0-27.7 kDa in mass spectrometry (2) 25-31 kDa unresolved distribution in mass spectrometry (3)	(1) Holen & Elsayed 1990 (2) Haginaka et al. 1995 (3) Besler et al. 1997
Isoelectric Point  pI 4.4 - 4.6 2 isoforms in IEF/PAGE (1)	(1) Holen & Elsayed 1990
Amino Acid Sequence, mRNA, and cDNA Gal d 1   SWISS-PROT: P01005 GenBank: J00902 PIR: A01239 Amino acids 186 residues (3) mRNA 821bp (2) cDNA 5.6 kb (1)	(1) Lai et al. 1979 (2) Catterall et al. 1980 (3) Kato et al. 1987
recombinant Protein expression in Escherichia coli: domain I (OM 1-68) (Mr 7500) (3) domain III  (OM 131-186) (1, 2)	(1) Hinck et al. 1993 (2) Kojima et al. 1994 (3) DeKoster & Robertson 1997
3D-Structure X-ray studies of enzyme complex with domain III from turkey OM (1) NMR studies of domain III from turkey OM (3, 4) NMR studies of OM-glycopeptide (2) NMR studies of a penta-antennary N-glycan (5)	(1) Bode et al. 1986 (2) Davis et al. 1994 (3) Krezel et al. 1994 (4) Hoogstraten et al. 1995 (5) Rutherford et al. 1995
Posttranslational Modifications Disulfide bonds 9 disulfide bonds: 5-44, 22-41, 30-62, 70-109, 87-106, 95-127, 138-168, 146-165, 154-186 (2, 6) Glycosylation of OM: carbohydrate content: 22-29% of whole Mr (1) carbohydrate composition: 14-16% GlcNAc, 6.5-8.5% Man, 0.5-4.0% Gal and, 0.04-2.2% NeuNAc (1) 5 N-glycosylation sites: 10, 53, 69, 75 and partly 175 (3, 7) covalent multiantennary structures of glycans (sequential exoglucosidase digest, mass spectrometry) (4, 5, 8) hydrazinolysis of sialyl-oligosaccharides and sialydase digestion (6)	(1) Beeley 1971 (2) Beeley 1976a (3) Beeley 1976b (4) Yamashita et al. 1982 (5) Yamashita et al. 1983 (6) Yamashita et al. 1984 (7) Kato et al. 1987 (8) Yet et al. 1988
Biological Function serin protease inhibitor, Kazal family of protease inhibitors (1) 3 tandem domains (1): domain I (OM 1-68) domain II  (OM 65-130) domain III  (OM 131-186) active sites: 24-25, 89-90, 148-149 (1)	(1) Kato et al. 1987
Stability lower trypsin-inhibitory activity and heat denaturation stability of chemically deglycosylated OM (1) glycosylated first domain has increased thermal stability in comparison with recombinant domain I (2)	(1) Gu et al. 1989 (2) DeKoster & Robertson 1997

 7.2.2 Allergenic Properties
 

Ovomucoid (OM)	References
Frequency of Sensitization IgE-binding to OM in 34 to 97 % of patients (1)	(1) see 6.1 Sensitization to Egg White Allergens
IgE-binding of OM Domains Average percentage of specific serum IgE*: Domain/Ref. (1) (2) (3) I (OM 1-68)  - ** 33% 12% II  (OM 65-130)  - ** 48% 16% III  (OM 131-186) glycosylated non-glycosylated - 35% < 4% 28% - - - 27% 47% No. of Patients  2 (medium percentage) 45 children with atopic dermatitis (median percentage) 9  children (medium percentage) * total OM-specific serum IgE = 100%  (RAST / EAST) ** domains I+II (OM 1-130) = 78%	(1) Matsuda et al. 1986 (2) Cooke & Sampson 1997 (3) Zhang & Mine 1998
B-Cell Epitopes IgE binding sites located on: OM 1-20 (synthetic peptide) (b) (3) OM 40-50 (synthetic peptide) (b) (6) OM 49-56 (synthetic peptide) (b) (3) OM 56-66 (synthetic peptide) (b) (6) OM 71-75 (synthetic peptide) (b) (6) OM 81-91 (synthetic peptide) (b) (6) OM 85-96 (synthetic peptide) (b) (3) OM 90-121 (trypsin digest) (a) (5) OM 115-122 (synthetic peptide) (b) (3) OM 134-186 (pepsin digest) (a) (5) OM 161-174 (synthetic peptide) (b) (6) OM 175-186 (synthetic peptide) (b) (3) OM 179-186 (synthetic peptide) (b) (6) Carbohydrate epitopes: Specific serum IgE against N-glycosylated domain III (Asn-175) higher than against non-glycosylated domain III in egg allergic patients (c) (1) Specific serum IgE against N-glycosylated domain III (Asn-175) lower than against non-glycosylated domain III in egg allergic patients (c) (4) No difference in IgE-binding of OM and deglycosylated OM (a, c) (2)	(1) Matsuda et al. 1985 (2) Besler et al. 1997 (3) Cooke & Sampson 1997 (4) Zhang & Mine 1998 (5) Besler et al. 1999 (6) Mine & Zhang 1999   Applied methods: (a) SDS-PAGE / immunoblot (b) dot / immunoblot (c) EAST / RAST inhibition
T-Cell Epitopes T-Cell Proliferation with: domain I (OM 1-68) domain II  (OM 65-130) (predominant) domain III  (OM 131-186) (predominant) in 4 egg allergic patients (1) domain I (OM 1-68)  in 28% ' domain II  (OM 65-130)   in 55% domain III  (OM 131-186)  in 64% of 33 (' 29) egg allergic children with atopic dermatitis (3) whole OM (2)	(1) Eigenmann et al. 1996 (2) Holen & Elsayed 1996 (3) Cooke & Sampson 1997
T-Cells / Cytokines PBMC stimulation with OM: expression of IL-5 in 4 egg allergic patients, OM specific T-cell lines mainly CD3+ (of which 75-97% were CD4+ T-cells), CD8+ phenotypes < 25% (1)	(1) Eigenmann et al. 1996
Alteration of Allergenicity cyanogen bromide cleavage: no change in IgE- and IgG-binding in 6 OM allergic patients (direct ELISA) (1) 6 of 6 sera showed IgE binding OM fragments (SDS-PAGE immunoblot) (3) deglycosylation: see carbohydrate epitopes digestion 5 of 6 sera showed IgE binding to trypsin digested OM, 1of 6 to pepsin digest, and 2 of 6 to thermolysin digest (SDS-PAGE immunoblot) (3) pepsin, trypsin, and chymotrypsin preparations of OM showed high IgE binding activities in DBPCFC positive egg white allergic children (RAST inhibition) (4) performic acid oxidation: no change in IgE- (max. inhibition 93%) and decreased IgG-binding (max. inhibition 39%) in 7 egg allergic patients (EAST inhibition) (2) increased PBMC proliferation in 11 egg allergic patients (2) reduction and alkylation: IgE-binding lost in 4/6  and retained in 2/6 OM allergic patients, respectively (1) IgG-binding lost in 4/6  and retained in 2/6 OM allergic patients, respectively (1) decreased IgE-(max. inhibition 72%) and decreased IgG-binding (max. inhibition 82%) in 7 egg allergic patients (EAST inhibition) (2) increased PBMC proliferation in 11 egg allergic patients (2)	(1) Djurtoft et al. 1991 (2) Cooke & Sampson 1997 (3) Besler et al. 1999 (4) Urisu et al. 1999