ovalbumin (Gal d 2)

4.3 Properties of Ovalbumin

4.3.1 Molecularbiological Properties

Ovalbumin (OA) References

Allergen Nomenclature Gal d 2 (1) King et al. 1994

Molecular Mass   Mr 44.0-45.0 kDa
6 and 4 isoforms by mass spectrometry, respectively (1, 2) (1) Kelly et al. 1996
(2) Chakel et a. 1997

Isoelectric Point pI 4.6
(1) Holen & Elsayed 1990

Amino Acid Sequence
SWISS-PROT: P01012
385 residues (1) (1) Nisbet et al. 1981

cDNA Sequence
EMBL: J00895
7.564 kb (4) Sequence (1, 2, 3) (1) Dugaiczyk et al. 1979
(2) Gannon et al. 1979
(3) Caterall et al. 1980
(4) Woo et al.1981

mRNA Sequence
1859 nucleotides (1), 1872 nucleotides (2, 4)
Sequence (2, 3) (1) McReynolds et al. 1978
(2) O'Hare et al. 1979
(3) Caterall et al. 1978
(4) Woo et al.1981

recombinant OA
expression in Escherichia coli (1)
Japanese quail OA expression in Saccharomyces cerevisiae (7)
expression in mouse cells (2)
expression in Xenopus laevis oocytes (3)
OA and OA mutant expression in mouse L cells (5, 6)
expression in human breast carcinoma cell line (4) (1) Fraser & Bruce 1978
(2) Lai et al. 1980
(3) Colman et al. 1981
(4) Lai et al. 1983
(5) Sheares & Robbins 1986
(6) Sheares 1988
(7) Krizkova et al. 1992

3D-Structure
X-ray studies of OA (1, 3)
X-ray of plakalbumin (2)
OA 257-264 in complex with the murine MHC class I H-2Kb molecule (4) (1) Stein et al. 1990
(2) Wright et al. 1990
(3) Stein et al. 1991
(4) Fremont et al. 1995

Posttranslational Modifications
Acetylation:
N-terminal acetylation (3)
Disulfide Bridges:
1 disulfide bond: 73-120 (3)
Glycosylation of OA:
carbohydrate content: 3.2% of whole Mr (1)
carbohydrate composition: 1.2% GlcNAc, 1.7-2.0% Man (1)
1 N-glycosylation site: Asn-292 (3)
hydrazinolysis of sialyl-oligosaccharides and sialydase digestion (4)
isolation and mass spectrometry of OA-glycans (5)
purified glycopeptides characterized by sequential exoglycosidase digestion (9)
sequential exoglycosidase digestion and mass spectrometry of glycans (11)
transiently diglycosylated ovalbumin (Asn-292, Asn-311) in hen oviduct (12)
glycosylation of recombinant OA (6, 7)
Phosphorylation of OA:
2 phosphorylation sites: Ser-68, Ser-344 (2, 3)
posphate content: 1.73 mol/mol OA (10)
variants: A1, A2, or A3 depending on 2, 1, or no phosphorylation, ratios A1:A2:A3 = 12:7:1 (8)
separation by capillary isoelectric focussing and mass spectrometry of mono- and diphospho-OA (13)
(1) Robinson 1972
(2) Henderson et al. 1981
(3) Nisbet et al. 1981
(4) Yamashita et al. 1984
(5) Chen et al. 1988
(6) Sheares & Robbins 1986
(7) Sheares 1988
(8) Burley & Vehedra 1989
(9) Rago et al. 1992
(10) Ekman & Jäger 1993
(11) Kuster et al. 1997
(12) Suzuki et al. 1997
(13) Wei et al. 1998

Genetic Variants
Asn / Asp replacement at residue 311 (1) (1) Nisbet et al. 1981

Biological Function
function unknown, OA belongs to serpin family of protease inhibitors (1)
active site: 352-353 (1) Burley & Vehedra 1989

Stability
conversion to S-Ovalbumin by heat or storage (1, 2)
5% S-form in fresh eggs, 81% in eggs stored for 6 months (1)
S-form more resistant to denaturation by heat, urea, or guanidine (2) (1) Smith 1964
(2) Smith & Back 1965

Ovalbumin (OA)	References
*Allergen Nomenclature* Gal d 2	(1) King et al. 1994
*Molecular Mass* Mr 44.0-45.0 kDa 6 and 4 isoforms by mass spectrometry, respectively (1, 2)	(1) Kelly et al. 1996 (2) Chakel et a. 1997
*Isoelectric Point* pI 4.6	(1) Holen & Elsayed 1990
*Amino Acid Sequence* SWISS-PROT: P01012 385 residues (1)	(1) Nisbet et al. 1981
*cDNA Sequence* EMBL: J00895 7.564 kb (4) Sequence (1, 2, 3)	(1) Dugaiczyk et al. 1979 (2) Gannon et al. 1979 (3) Caterall et al. 1980 (4) Woo et al.1981
*mRNA Sequence* 1859 nucleotides (1), 1872 nucleotides (2, 4) Sequence (2, 3)	(1) McReynolds et al. 1978 (2) O'Hare et al. 1979 (3) Caterall et al. 1978 (4) Woo et al.1981
*recombinant OA* expression in Escherichia coli (1) Japanese quail OA expression in Saccharomyces cerevisiae (7) expression in mouse cells (2) expression in Xenopus laevis oocytes (3) OA and OA mutant expression in mouse L cells (5, 6) expression in human breast carcinoma cell line (4)	(1) Fraser & Bruce 1978 (2) Lai et al. 1980 (3) Colman et al. 1981 (4) Lai et al. 1983 (5) Sheares & Robbins 1986 (6) Sheares 1988 (7) Krizkova et al. 1992
*3D-Structure* X-ray studies of OA (1, 3) X-ray of plakalbumin (2) OA 257-264 in complex with the murine MHC class I H-2Kb molecule (4)	(1) Stein et al. 1990 (2) Wright et al. 1990 (3) Stein et al. 1991 (4) Fremont et al. 1995
*Posttranslational Modifications* Acetylation: N-terminal acetylation (3) Disulfide Bridges: 1 disulfide bond: 73-120 (3) Glycosylation of OA: carbohydrate content: 3.2% of whole Mr (1) carbohydrate composition: 1.2% GlcNAc, 1.7-2.0% Man (1) 1 N-glycosylation site: Asn-292 (3) hydrazinolysis of sialyl-oligosaccharides and sialydase digestion (4) isolation and mass spectrometry of OA-glycans (5) purified glycopeptides characterized by sequential exoglycosidase digestion (9) sequential exoglycosidase digestion and mass spectrometry of glycans (11) transiently diglycosylated ovalbumin (Asn-292, Asn-311) in hen oviduct (12) glycosylation of recombinant OA (6, 7) Phosphorylation of OA: 2 phosphorylation sites: Ser-68, Ser-344 (2, 3) posphate content: 1.73 mol/mol OA (10) variants: A1, A2, or A3 depending on 2, 1, or no phosphorylation, ratios A1:A2:A3 = 12:7:1 (8) separation by capillary isoelectric focussing and mass spectrometry of mono- and diphospho-OA (13)	(1) Robinson 1972 (2) Henderson et al. 1981 (3) Nisbet et al. 1981 (4) Yamashita et al. 1984 (5) Chen et al. 1988 (6) Sheares & Robbins 1986 (7) Sheares 1988 (8) Burley & Vehedra 1989 (9) Rago et al. 1992 (10) Ekman & Jäger 1993 (11) Kuster et al. 1997 (12) Suzuki et al. 1997 (13) Wei et al. 1998
*Genetic Variants* Asn / Asp replacement at residue 311 (1)	(1) Nisbet et al. 1981
*Biological Function* function unknown, OA belongs to serpin family of protease inhibitors (1) active site: 352-353	(1) Burley & Vehedra 1989
*Stability* conversion to S-Ovalbumin by heat or storage (1, 2) 5% S-form in fresh eggs, 81% in eggs stored for 6 months (1) S-form more resistant to denaturation by heat, urea, or guanidine (2)	(1) Smith 1964 (2) Smith & Back 1965

4.3.2 Allergenic Properties

Ovalbumin (OA) References

Immunoglobulines
IgE-binding studies of OM (1)
IgM, IgG, IgE production by OA-specific B-Cells (2)
lower OA-specific IgA production in egg allergic children (3) (1) see 4.1 Sensitization to Egg White Allergens
(2) Ohshiba & Yata 1991
(3) Noma et al. 1996a

B-Cell Epitopes
IgE binding sites located on:
OA 1-10 (synthetic peptide) (c) (1)
OA 11-19 (synthetic peptide) (c) (4)
OA 41-171 (CNBr-fragement) (a) (3)
OA 56-70 (synthetic peptide) (c) (4)
OA 301-385 (CNBr-fragment) (a) (3)
OA 323-339 (synthetic peptide) (b, c) (2)
OA 347-385 (V8 protease digest) (a) (5)
OA 347-366 (synthetic peptide) (d) (5)
OA 357-366 (synthetic peptide) (e) (5)
OA 357-376 (synthetic peptide) (d) (5)
OA 367-385 (synthetic peptide) (d) (5)
(a) SDS-PAGE / immunoblot
(b) CRIE
(c) EAST / RAST-inhibition
(d) Histamine Release
(e) Histamine Release inhibition
(1) Elsayed et al. 1988
(2) Johnsen & Elsayed 1990
(3) Kahlert et al. 1992
(4) Elsyed & Stavseng 1994
(5) Honma et al. 1996

T-Cell Epitopes
Specific T-Cell Proliferation with:
OA 1-33 (synthetic peptide) (4)
OA 105-122 (synthetic peptide) (3)
OA 198-231 (synthetic peptide) (4)
OA 201-213 (synthetic peptide) (4)
OA 261-277 (synthetic peptide) (4)
OA 323-339 (synthetic peptide) (1, 3)
with whole OA (1, 2, 3)
(1) Shimojo et al. 1994
(2) Eigenmann et al. 1996
(3) Holen & Elsayed 1996
(4) Katsuki et al. 1996

PBMC Proliferation
stimulation with OA
decreased proliferation during elimination diets in egg allergic patients with atopic dermatitis (2)
higher proliferation in egg allergic patients with atopic dermatitis showing non-immediate symptoms to oral challenge than in patients showing immediate type symptoms (1, 3)
increased proliferation in allergy developing infants (4)
higher proliferation in egg allergic children with persisting symptoms of atopic dermatitis (5) (1) Kondo et al. 1990
(2) Agata et al. 1993
(3) Fukutomi et al. 1994
(4) Miles et al. 1996
(5) Shinoda et al. 1997

PBMC Stimulation / Cytokines
PBMC stimulation with OA:
increase of PBMC proliferation and decrease in IFN-gamma in egg allergic infants with atopic dermatitis (1)
increase of IL4 and decrease in IFN-gamma in egg allergic children with atopic dermatitis (not in outgrown and healthy children) (2)
increase of IFN-gamma (exclusively by simultaneous IL-2 stimulation) in egg allergic patients (3)
increase of IL-5 mRNA in egg allergic children (not in outgrown and healthy children) (4) (1) Warner et al. 1994
(2) Noma et al. 1996b
(3) Shinbara et al. 1996
(4) Tomiita et al. 1998

T-Cell Lines (TCL) / Cytokines
OA-stimulated lymphocytes from egg allergic patients induced IL-2-responsiveness of T-Cells (CD4+), induction of T-Cell responsiveness inhibited by anti-HLA-DP and anti-HLA-DQ monoclonal antibodies (1, 3)
antibody to CD45RA+ (CD4+ T-Cells) induced IL-2 responsiveness in lymphocytes of non-allergic patients, but did not increase responsiveness in egg allergic patients (2)
anti-HLA-DP monoclonal antibodies inhibited T-Cell proliferation in 5 egg allergic patients, anti-HLA-DQ monoclonal antibodies restored T-Cell proliferation in 2 egg allergic patients with atopic dermatitis (5)
OA-specific TCL ( CD4+, alpha beta T-Cell receptors) recognized OA presented by HLA-DR10, production of IL-5 on stimulation with OA or OA 323-339 observed (1 egg allergic patient) (4)
30 OA-specific TCL mainly CD4+ T-Cells significant production of IL-4 and IL-5, low production of IFN-gamma (6 egg allergic patients with atopic dermatitis) (6)
28 OA- or casein-specific T-Cell clones (TCC) (16 CD8+) from egg and cow's milk allergic patient, 75% of CD4+ TCC and 44% of CD8+ TCC secreted IL-4 (7) (1) Noma et al. 1990
(2) Kawano et al. 1992
(3) Noma et al. 1994
(4) Shimojo et al. 1994
(5) Shinbara et al. 1995
(6) Katsuki et al. 1996
(7) Reekers et al. 1996

Alteration of Allergenicity
acidic treatment:
HCl, pH 3.0: increased IgE-binding in EAST (157% of binding to native OA, for IgG 122%, IgA 104%) (2)
alkaline treatment:
NaOH, pH 11 overnight: decreased IgE-binding in EAST (0.4% of binding to native OA, for IgG 5.2%, IgA 27%) (2)
cyanogen bromide cleavage:
decreased 50%-inhibition of IgE-binding in RAST (1)
pepsin hydrolysis:
limited hydrolysis decreased IgE-binding (max. inhibition: 50%) in RAST (1)
reduction and alkylation:
decreased IgE-binding (max. inhibition: 20%) in RAST (1)
no change in in IgE-binding in EAST (89% of binding to native OA, for IgG 100%, IgA 111%) (2)
heat denaturation:
80°C, 10 min and treatment with urea or guanidine HCl resulted in no change in RAST inhibition (1)
100°C, 3 min: decreased IgE-binding in EAST (9.4% of binding to native OA, for IgG 39%, IgA 76%) (2)
trypsin hydrolysis:
decreased 50%- inhibition of IgE-binding in RAST (1)
urea denaturation:
increased IgE-binding in EAST (136% of binding to native OA, for IgG 120%, IgA 133%) (2)
(1) Elsayed et al. 1986
(2) Honma et al. 1994

Ovalbumin (OA)	References
*Immunoglobulines* IgE-binding studies of OM (1) IgM, IgG, IgE production by OA-specific B-Cells (2) lower OA-specific IgA production in egg allergic children (3)	(1) see 4.1 Sensitization to Egg White Allergens (2) Ohshiba & Yata 1991 (3) Noma et al. 1996a
*B-Cell Epitopes* IgE binding sites located on: OA 1-10 (synthetic peptide) (c) (1) OA 11-19 (synthetic peptide) (c) (4) OA 41-171 (CNBr-fragement) (a) (3) OA 56-70 (synthetic peptide) (c) (4) OA 301-385 (CNBr-fragment) (a) (3) OA 323-339 (synthetic peptide) (b, c) (2) OA 347-385 (V8 protease digest) (a) (5) OA 347-366 (synthetic peptide) (d) (5) OA 357-366 (synthetic peptide) (e) (5) OA 357-376 (synthetic peptide) (d) (5) OA 367-385 (synthetic peptide) (d) (5) (a) SDS-PAGE / immunoblot (b) CRIE (c) EAST / RAST-inhibition (d) Histamine Release (e) Histamine Release inhibition	(1) Elsayed et al. 1988 (2) Johnsen & Elsayed 1990 (3) Kahlert et al. 1992 (4) Elsyed & Stavseng 1994 (5) Honma et al. 1996
*T-Cell Epitopes* Specific T-Cell Proliferation with: OA 1-33 (synthetic peptide) (4) OA 105-122 (synthetic peptide) (3) OA 198-231 (synthetic peptide) (4) OA 201-213 (synthetic peptide) (4) OA 261-277 (synthetic peptide) (4) OA 323-339 (synthetic peptide) (1, 3) with whole OA (1, 2, 3)	(1) Shimojo et al. 1994 (2) Eigenmann et al. 1996 (3) Holen & Elsayed 1996 (4) Katsuki et al. 1996
*PBMC Proliferation* stimulation with OA decreased proliferation during elimination diets in egg allergic patients with atopic dermatitis (2) higher proliferation in egg allergic patients with atopic dermatitis showing non-immediate symptoms to oral challenge than in patients showing immediate type symptoms (1, 3) increased proliferation in allergy developing infants (4) higher proliferation in egg allergic children with persisting symptoms of atopic dermatitis (5)	(1) Kondo et al. 1990 (2) Agata et al. 1993 (3) Fukutomi et al. 1994 (4) Miles et al. 1996 (5) Shinoda et al. 1997
*PBMC Stimulation / Cytokines* PBMC stimulation with OA: increase of PBMC proliferation and decrease in IFN-gamma in egg allergic infants with atopic dermatitis (1) increase of IL4 and decrease in IFN-gamma in egg allergic children with atopic dermatitis (not in outgrown and healthy children) (2) increase of IFN-gamma (exclusively by simultaneous IL-2 stimulation) in egg allergic patients (3) increase of IL-5 mRNA in egg allergic children (not in outgrown and healthy children) (4)	(1) Warner et al. 1994 (2) Noma et al. 1996b (3) Shinbara et al. 1996 (4) Tomiita et al. 1998
*T-Cell Lines (TCL) / Cytokines* OA-stimulated lymphocytes from egg allergic patients induced IL-2-responsiveness of T-Cells (CD4+), induction of T-Cell responsiveness inhibited by anti-HLA-DP and anti-HLA-DQ monoclonal antibodies (1, 3) antibody to CD45RA+ (CD4+ T-Cells) induced IL-2 responsiveness in lymphocytes of non-allergic patients, but did not increase responsiveness in egg allergic patients (2) anti-HLA-DP monoclonal antibodies inhibited T-Cell proliferation in 5 egg allergic patients, anti-HLA-DQ monoclonal antibodies restored T-Cell proliferation in 2 egg allergic patients with atopic dermatitis (5) OA-specific TCL ( CD4+, alpha beta T-Cell receptors) recognized OA presented by HLA-DR10, production of IL-5 on stimulation with OA or OA 323-339 observed (1 egg allergic patient) (4) 30 OA-specific TCL mainly CD4+ T-Cells significant production of IL-4 and IL-5, low production of IFN-gamma (6 egg allergic patients with atopic dermatitis) (6) 28 OA- or casein-specific T-Cell clones (TCC) (16 CD8+) from egg and cow's milk allergic patient, 75% of CD4+ TCC and 44% of CD8+ TCC secreted IL-4 (7)	(1) Noma et al. 1990 (2) Kawano et al. 1992 (3) Noma et al. 1994 (4) Shimojo et al. 1994 (5) Shinbara et al. 1995 (6) Katsuki et al. 1996 (7) Reekers et al. 1996
*Alteration of Allergenicity* acidic treatment: HCl, pH 3.0: increased IgE-binding in EAST (157% of binding to native OA, for IgG 122%, IgA 104%) (2) alkaline treatment: NaOH, pH 11 overnight: decreased IgE-binding in EAST (0.4% of binding to native OA, for IgG 5.2%, IgA 27%) (2) cyanogen bromide cleavage: decreased 50%-inhibition of IgE-binding in RAST (1) pepsin hydrolysis: limited hydrolysis decreased IgE-binding (max. inhibition: 50%) in RAST (1) reduction and alkylation: decreased IgE-binding (max. inhibition: 20%) in RAST (1) no change in in IgE-binding in EAST (89% of binding to native OA, for IgG 100%, IgA 111%) (2) heat denaturation: 80°C, 10 min and treatment with urea or guanidine HCl resulted in no change in RAST inhibition (1) 100°C, 3 min: decreased IgE-binding in EAST (9.4% of binding to native OA, for IgG 39%, IgA 76%) (2) trypsin hydrolysis: decreased 50%- inhibition of IgE-binding in RAST (1) urea denaturation: increased IgE-binding in EAST (136% of binding to native OA, for IgG 120%, IgA 133%) (2)	(1) Elsayed et al. 1986 (2) Honma et al. 1994