7.5 Properties of Caseins

7.5.1 Molecular Biological Properties
 

Caseins (CAS)	References
Allergen Nomenclature  Bos d 8	(1) Larsen & Lowenstein 1999
Isoallergens and Variants CAS alpha-S1 alpha-S2 beta  kappa Genetic variants A, B, C, D (1), F (5) A, D (3) A1, A2, A3, B, C, E F-5P (4) A, B, B2 (2)	(1) Mercier et a. 1971, 1973 (2) Grosclaude et al. 1972 (3) Grosclaude et al. 1979 (4) Visser et al. 1995 (5) Prinzenberg et al. 1998
Molecular Mass CAS alpha beta  gamma  kappa SDS-PAGE  24 kDa (2) 32.4 kDa (1) 26.6 kDa (1) 19 kDa (1)	(1) Docena et al. 1996 (2) del Val et al. 1999
Isoelectric Point   alpha-S1 alpha-S2 beta-CAS gamma-CAS kappa-CAS pI 4.9-5 pI 5.2-5.4 pI 5.1-5.4 pI 5.4-5.6	(1) Wal 1998
Amino Acid Sequence, mRNA, and cDNA Bos d 8 alpha-S1 alpha-S2 beta kappa SWISS-PROT: P02662 P02663 P02666 P02668 GenBank: S72388, M33123, X59856, M38641, X00564 M16644 S67277 (A3), M55158, X06359, M15132, M16645 X00565 (A), M36641 (B2), X14908 PIR: KABOSB KABOS2 KBBOA2 KKBOB Amino acids 199 (1) 207 (4) 209 (3) 169 (2) mRNA 1123 bp (5) 1172 bp (6) 1134 bp (7) 1024 bp (11) 1094 bp (8) 755 bp (9) 1126 bp (A3, 17) 850 bp (6) 838 bp (10) cDNA 1862 bp (15) 2510 bp (16)     Gene 17508 bp (14) 18483 bp (16) 10338 bp (13) 7595 bp (12)	(1) Mercier et al. 1971, 1973 (2) Grosclaude et al. 1972 (3) Ribadeau-Dumas et al. 1972 (4) Brignon et al. 1977 (5) Nagao et al. 1984 (6) Stewart et al. 1984 (7) Gorodetskii et al. 1986 (8) Baev et al. 1987 (9) Jimenez-Flores et al. 1987 (10) Gorodetskii & Kaledin 1987 (11) Stewart et al. 1987 (12) Alexander et al. 1988 (13) Bonsing et al. 1988 (14) Koczan et al. 1991 (15) Chen et al. 1992 (16) Groenen et al. 1993 (17) Simons et al. 1993
recombinant Protein expression in  alpha-S1 alpha-S2 beta gamma kappa Escherichia coli (2) (1) Transgenic mice (5) (3), (4)	(1) Kang & Richardson 1988 (2) Simons et al. 1993 (3) Hitchin et al. 1996 (4) Jeng et al. 1997 (5) Rijnkels et al. 1998
3D-Structure Micelle aggregation: CAS subunits associate in solution forming complexes and ordered aggregates of micelles in lactoserum by colloidal calcium phosphate and phosphoserine interactions: ratio alpha-S1 / beta / alpha-S2 / kappa-CAS is 37% / 37% / 13% / 13% (2) Polymerisation kappa-CAS: monomer or multimer linked by disulfide bonds (1)	(1) SWISS-PROT (2) Wal 1998
Posttranslational Modifications Numbers of alpha-S1 alpha-S2 beta kappa Disulfide bonds - 1 - 1 Glycosylation sites - - 7-8 - Phosphorylation 8-9 10 4-5 2 Glycosylation of kappa-CAS: O-glycosation sites: distribution of monosaccharide, disaccharide, trisaccharide (straight), trisaccharide (branched), and tetrasaccharide chains were 0.8, 6.3, 18.4, 18.5, and 56.0%, respectively (means of five kappa-CAS) (2)	(1) SWISS-PROT (2) Saito & Itoh 1992
Biological Function alpha-CAS: Calcium phosphate transport capacity of milk (1) kappa: Micelle formation stabilizing, preventing CAS precipitation in milk (1)	(1) SWISS-PROT
Location alpha-CAS, kappa-CAS: production in mammary gland, extracellular secretion (1)	(1) SWISS-PROT
Sequence Homology alpha-S1 and S2 CAS from cow's milk: aa identity 22.5% (2) alpha-S1 CAS from sheep's and goat's milk: aa identity  87-89% (2) alpha-S2 CAS from sheep's and goat's milk: aa identity  87-89% (2) alpha-S1 and S2 CAS from sheep's and goat's milk: aa identity 97-98% (2) beta-CAS from sheep's and goat's milk: aa identity 91% (1) kappa-CAS from sheep's and goat's milk: aa identity 84% (1)	(1) BLAST at PIR (2) Spuergin et al. 1997
Stability   alpha-CAS beta-CAS kappa-CAS Ca2+ sensitivity + + -	(1) Wal 1998

7.5.2 Allergenic Properties
 

Caseins (CAS)	References
Frequency of Sensitization IgE-binding to CAS in 65-100% of patients (1)	(1) see 7.1 Sensitization to Cow's Milk Allergens
Allergenicity of Subunits Major IgE- binding CAS subunits in 4 patients with CMA and atopic dermatitis: in 1 patient alpha- and kappa-CAS, in 2 patients alpha-CAS, and in 1 patient kappa-CAS (tested: alpha-, beta-, and kappa-CAS) (1) 85% of 58 children presented IgE against each CAS, only 1 child was monosensitized (to kappa-CAS), allergenic potencies according to statistical distribution of specific serum IgE levels: alpha S1-CAS > beta-CAS >> alpha S2-CAS = kappa-CAS (RAST) (2) IgE-binding to alpha-CAS in 100%, beta + gamma-CAS in 50%, and kappa-CAS in 33% of 6 children with CMA (3)	(1) Shimojo et al. 1997 (2) Bernard et al. 1998 (3) Restani et al. 1999
B-Cell Epitopes: alpha S1 CAS IgE binding sites located on alpha S1 CAS: Peptides Positivity in Patients inhibition of IgE binding to alpha S1 CAS [%] Ref. 1-54 (fragment) + (a)   (1) 1-10 (synthetic peptide)  67% (b) 5% (max. 14%) (2) 19-30 (synthetic peptide)  100% (c)   (2)* 20-31 (synthetic peptide)  58% (b) 7% (max. 40%) (2) 34-45 (synthetic peptide)  50% (b) 5% (max. 18%) (2) 58-73 (synthetic peptide)  42% (b) 3% (max. 15%) (2) 61-123 (fragment) + (a)   (1) 86-103 (synthetic peptide) 100% (b) 19% (max. 42%) (2) 93-98 (synthetic peptide)  100% (c)   (2)* 124-135 (fragment) + (a)   (1) 136-196 (CNBr fragment) + (a)   (1, 3) 141-150 (synthetic peptide) 92% (b) 8% (max. 20%) (2)* 165-199 (fragment) + (a)   (1) 181-199 (synthetic peptide) 100% (a) 92% and 30% (n=2) (b) (3) 188-199 (synthetic peptide) 42% (b) 7% (max. 28%) (2) (a) EAST / RAST (b) EAST / RAST-inhibition (c) Pin-ELISA (1) 2 patients with CMA (2) 12 patients with CMA, *similar IgG binding (3) 9 patients with CMA	(1) Otani et al. 1989 (2) Spuergin et al. 1996 (3) Nakajima-Adachi et al. 1998
B-Cell Epitopes: beta-CAS IgE binding sites located on beta-CAS: Peptides Positivity in Patients Ref. 1-139 (fragment) + (a) (1) 1-93 (fragment) + (a) (1) 1-60 (fragment) + (a) (1) 26-93 (fragment) + (a) (1) 106-209 (fragment) + (a) (1) 110-144 (fragment) + (a) (1) 132-144 (fragment) + (a) (1) 157-185 (fragment) + (a) (1) 186-209 (fragment) + (a) (1) (a) EAST / RAST (1) 2 patients with CMA	(1) Otani et al. 1989
T-Cell Epitopes: alpha S1 CAS alpha S1 CAS specific T-Cell Lines responsive to: 1-54 (CNBr fragment) (1) 31-50 (synthetic peptide) (1) 76-95 (synthetic peptide) (1) 91-110 (synthetic peptide) (1) 124-135 (CNBr fragment) (1) 136-155 (synthetic peptide) (1) (1) 7 TCL from 2 patients with CMA	(1) Nakajima-Adachi et al. 1998
PBMC Proliferation stimulation with CAS (1)	(1) see Diagnostic Features of CMA
PBMC Stimulation / Cytokines PBMC stimulation with CAS: Significantly higher PBMC proliferation in cow's milk allergic children with atopic dermatitis as compared to children with atopic dermatitis without cow's milk allergy (1) 16 of 28 CAS- or ovalbumin-specific TCC from cow's milk and egg allergic children were CD8+; 75% of CD4+ TCC and 44% of CD8+ TCC secreted IL-4; all TCC secreted INF-gamma (1) 27% of CD4+ CAS- specific TCCs from adolescent or adult patients with cow's milk- responsive atopic dermatitis, and the majority of house dust mite- specific TCCs, produced IL-4 on mitogen stimulation; INF-gamma was produced by the majority of TCCs with both specificities (3) PBMC stimulation with kappa- CAS: 25 of 31 TCC from patients with milk- responsive atopic dermatits responded to mixed CAS (alpha-, beta-, kappa-) and kappa- CAS (2)	(1) Reekers et al. 1996 (2) Werfel et al. 1996 (3) Werfel et al. 1997b
T-Cell Lines (TCL) / Cytokines PBMC responsiveness to alpha s1- CAS activation was rather weak in cow's milk allergic patients; 26 alpha s1- CAS- specific T-cell lines were established; higher frequency of CD8+ T cells which produced INF-gamma and IL-4 (1)	(1) Nakajima al. 1996
Alteration of Allergenicity Treatment alpha- beta kappa heat denaturation NS (1)     acidic treatment (HCl) NS (1)     alkaline treatment (NaOH) NS (1)     sodium dodecyl sulfate NS (1)     urea denaturation NS (1)     NS no significant difference in IgE-binding (1) patients with CMA	(1) Kohno et al. 1994 see also 10 Stability of Cow's Milk Allergens