7.3 Properties of beta-Lactoglobulin

7.3.1 Molecular Biological Properties
 

beta-Lactoglobulin (beta-LG)	References
Allergen Nomenclature  Bos d 5	(1) Larsen & Lowenstein 1999
Isoallergens and Variants Main genetic variants A (Asp-64, Val-118) and B (Gly-64 and Ala-118) (1, 3) Minor genetic variants of subtype B with single substitution: variant C  (Gln-59 > His-59) (SWISS-PROT), variant D (Glu-45 > Gln-45) (2), variant W (Ile-56 > Leu-56) (4), variants I (Glu-108 > Gly-108) and J (Pro-126 > Leu-126) (5)	(1) Braunitzer et al. 1973 (2) Brignon & Ribadeau-Dumas 1973 (3) Ebeler et al. 1990 (4) Godovac-Zimmermann et al. 1990 (5) Godovac-Zimmermann et al. 1996
Molecular Mass   Mr in SDS-PAGE: 18 kDa (1, 2)	(1) Docena et al. 1996 (2) del Val et al. 1999
Isoelectric Point  pI 5.3 (2) variant A: 5.13 (1), variant B: 5.23 (1)	(1) Fredriksson 1972 (2) Wal 1998
Amino Acid Sequence, mRNA, and cDNA Bos d 5 beta-LG Variant B SWISS-PROT: P02754   GenBank: M19088, X14712, M27732, K01086, X52581 Z48305 PIR: LGBO   Amino acids 162 residues (1)   mRNA 790 bp (3)   cDNA 601 bp (2) 9432 bp (4)	(1) Braunitzer et al. 1973 (2) Jamieson et al. 1987 (3) Alexander et al. 1989 (4) Hyttinen et al. 1998
recombinant Protein expression in Escherichia coli: expression using a tac promoter vector, pTTQ18 (1) expression of 2 site-directed mutants with an additional disulfide bond, increased thermostability (3) expression in strain DH5alpha, positive IgE binding from 5 patients with CMA (4) expression in a denatured form in periplasm using the pET26 vector (8) expression in yeasts: expression of recombinant beta-LG (2) and a site directed mutant (6) in Saccharomyces cerevisiae, mutant inhibited the proliferation of CD4+ TCC from mice (6) expression in native conformation in Pichia pastoris (5) epression in mouse cells: expression in native conformation in COS-7 cells and in vivo in mouse tibialis muscle (8) expression in transgenic mice: Bovine beta-LG gene was expressed mammary gland- specifically in transgenic mice, expression levels of beta-LG in milk > 1 mg/mL (7)	(1) Batt et al. 1990 (2) Totsuka et al. 1990 (3) Cho et al. 1994 (4) Chatel et al. 1996 (5) Kim et al. 1997 (6) Totsuka et al. 1997 (7) Hyttinen et al. 1998 (8) Chatel et al. 1999
3D-Structure X-ray studies of beta-LG (1) NMR studies of recombinant beta-LG (2) 3D-models of native and oxidized beta-LG, and partly and fully reduced beta-LG mutants (3)	(1) Brownlow et al. 1997 (2) Kuwata et al. 1998 (3) del Val et al. 1999
Posttranslational Modifications Disulfide Bridges: beta-LG occurs naturally as a mixture of monomers and 36-kDa dimers (2) 2 disulfide bonds: 66-160, 106-(119 or 121) (1)	(1) Brownlow et al. 1997 (2) Wal 1998
Biological Function belongs to lipocalin family, binds retinol  (1)	(1) SWISS-PROT
Sequence Homology beta-LG from water buffalo and mouflon: aa sequence identies 98% and 95% (1) beta-LG from goat and sheep: aa sequence identies 94% and 93% (1) cockroach allergen Bla g 4: aa sequence homology about 20% (2)	(1) BLAST at PIR (2) Arruda et al. 1995

7.3.2 Allergenic Properties
 

beta-Lactoglobulin (beta-LG)	References
Frequency of Sensitization IgE-binding to in 13-76% of patients (1)	(1) see 7.1 Sensitization to  Cow's Milk Allergens
Allergenicity of Variants A and B No difference in IgE titers specific for genetic variants A and B of beta-LG (1)	(1) van Beresteijn et al. 1995
B-Cell Epitopes IgE binding sites located on beta-LG: Peptides Positivity in Patients inhibition of IgE binding to beta-LG [%] 3D-Location on beta-LG Ref. 1-8 (tryptic peptide) 58% (b)     (6) 8-24 (CNBr peptide) 53% (b) max. 34%   (4) 9-14 (tryptic peptide) 40% (b)     (6) 15-26 (synthetic peptide) 44% (c) max. 20%   (3) 25-107 (CNBr peptide) 79% (4), + (1) (b) max. 38% (4)   (1, 4) 25-61 (fragment) + (b)     (1) 25-40 (tryptic peptide) 72% (b)     (6) 35-46 (synthetic peptide) 25% (c) max. 40%   (3) 41-107 (fragment) + (b)     (1) 41-60 (tryptic peptide) 92% (b)   surface (6) 62-107 (fragment) + (b)     (1) 78-83 (tryptic peptide) 28% (b)     (6) 84-91 (tryptic peptide) 40% (b)   cryptic (6) 85-96 (synthetic peptide) 44% (c) max. 10%   (3) 92-100 (tryptic peptide) 52% (b)     (6) 95-113 (synthetic peptide) 100% (d) 14 - 38% (c, d)   (5) 97-108 (synthetic peptide) 100% (c) max. 70% (20%*)   (3) 102-124 (tryptic peptide) 97% (b)   surface (6) 108-145 (CNBr peptide) 68% (4), + (1) (b) max. 57% (4)   (1, 4) 117-128 (synthetic peptide) 13% (c) max. 30%   (3) 124-134 (synthetic peptide) + (b) 60%   (2) 125-145 (fragment) + (b)     (1) 125-135 (tryptic peptide) 28% (b)   surface (6) 146-162 (CNBr peptide) 42% (4), + (1) (b) max. 28% (4)   (1, 4) 149-162 (tryptic peptide) 89% (b)     (6) 151-162 (synthetic peptide) 31% (c) max. 20%   (3) (a) SDS-PAGE / immunoblot (b) direct ELISA, EAST / RAST (c) EAST / RAST-inhibition (d) Pin-ELISA (1) 2 patients with CMA (2) 2 patients with CMA (3) 16 patients with CMA, * pooled serum (4) 19 patients with CMA (5) 14 children with CMA (age 6 months to 9 years) (6) 46 patients with CMA (location of epitopes in 3D-model of beta-LG)	(1) Otani et al. 1989 (2) Adams et al. 1991 (3) Ball et al. 1994 (4) Selo et al. 1998 (5) Heinzmann et al. 1999 (6) Selo et al. 1999
Cross-Reactivity beta-LG peptide 124-134 and alpha-LA 5-18 (1)	(1) Adams et al. 1991
T-Cell Epitopes Specific T-Cell Proliferation with:  beta-LG 145-161 (peptide) (1)	(1) Piastra et al. 1994
PBMC Proliferation stimulation with beta-LG (1)	(1) see Diagnostic Features of CMA
PBMC Stimulation / Cytokines PBMC stimulation with beta-LG: decrease in IFN-gamma production in cow's milk allergic children with atopic dermatitis as compared to immediate- type allergic or tolerant children (1)	(1) Hill et al. 1993
Alteration of Allergenicity cyanogen bromide cleavage: no alteration of IgE-binding  in 50% of patients with CMA, in 10% increased IgE- binding to CNBr- cleaved beta-LG (EAST inhibition) (2) pepsin hydrolysis: IgE-binding in 40% of 10 patients with CMA to native beta-LG and in 100% to peptic and peptic- tryptic digested beta-LG (RAST) (1) trypsin hydrolysis: reduced IgE binding (about <50%) in 75% and increased IgE binding in 9% of sera from 46 patients with CMA (3) reduction of disulfide bonds: no alteration of IgE-binding (1) increased pepsin digestibility and IgE- binding capacity (in animal model) of b-LG after reduction of disulfide bonds with thioredoxin (4)	(1) Haddad et al. 1979 (2) Selo et al. 1998 (3) Selo et al. 1999 (4) del Val et al. 1999 see also  10 Stability of Cow's Milk Allergens