IgE epitopes in the 11S globulin allergens of soya (glycinin) are located in the the acidic subunits of this protein. Beardslee et al. (2000) [19] identified epitope A (GGSI LSGFTLEFLEHAFSV, 217-235) and epitope B (GAIVTVKG GLSVI, 253-265) of P04776 as important. Xiang et al. (2002) [636] defined a shorter epitope from glycinin G2A (P04405) of 219-233 as dominant. They showed that residues E224, F225, L226, F230, G231 and V232 were most important for IgE binding. Only residue G231 is not conserved in Ara h 3 and this is adjacent to an epitope identified by Rabjohn et al. (1999) [166]. Another peptide, residues 247-261, shows weak IgE binding. There is also evidence that the G2 form of the glycinin basic subunit contains IgE epitopes.
Helm et al. (2000) [66], [67] in two articles first characterized the 21 kDa soya allergen as the basic subunit of glycinin G2 and then identified epitopes over the complete glycinin G2 gene sequence. 11 epitopes were found in 6 regions of 1-23, 57-111, 169-215, 249-271, 329-383 and 449-471. Region 1 was soya specific but the other regions bound IgE from peanut allergic sera. The 3-D structure of glycinin shows that these epitopes are generally more buried and hydrophobic than those of Xiang et al. (2002) [636] and imply that the IgE binds to a denatured protein.
Allergen stability: Process, chemical, enzymatic
Glycinin forms stable intermediates of Mr ~ 280,000 on trypsinolysis known as glycinin T. These retain the quaternary structure of glycinin and result from clipping of the acidic subunits to form a 13,000 and a 16,000 molecular weight fragments, the basic subunits remaining intact, similar intermediates being found following chymotrypsinolysis. It is thermostable, requiring temperatures in excess of 95°C for denaturation and has the propensity to form large aggregates on heating which still retain a large degree of the native secondary structure (Mills et al 2003 [580]). In vivo digestion has been investigated in calves (Lalles et al. 1999 [1088]) and in rats (Perez et al. 1999 [1089]). Glycinin (but not beta-conglycinin) immunoreactivity could be identified after 10 hours in calf ileum. In rat both glycinin derived peptides and the intact basic subunit could be identified after 3 hours.
Nature of main cross-reacting proteins:
Cross-reactions may be seen with 11S globulin allergens from other plant food species such as peanut and lupin. This results from homologous IgE epitopes although peanut allergic individuals can usually tolerate soya.
Allergen properties & biological function:
11S seed storage globulin
Allergen purification:
Defatted soy flour was extracted with water (1:5, w/v) by stirring for 1h at 20C. After centrifugation (10,000xg, 20 min, 20C) and the supernatant retained. After incubation at 2C overnight the cold-insoluble fraction (CIF) is collected by centrifugation at 10,000xg for 30 min, 20C. The CIF is dissolved in 0.1M potassium phosphate buffer (pH 7.6) and applied to a 5x20cm hydroxyapatite Ultorgel column. After washing away the unbound material, the bound fraction is eluted with a 0.1-0.75M potassium phosphate buffer (pH 7.6) gradient using a flow rate of 0.5 ml/min. Following dialysis against 50mM Tris-HCl, pH 7.8, containing 0.2mM NaCl the peak fractions are further purified by gel filtration on Sephacryl S300 (2.6x100cm column) using a flow rate of 0.4 ml/min, to remove aggregates. (after Eldridge and Wolf, 1967 [49])
Other biochemical information:
References (11)
Adachi M, Kanamori J, Masuda T, Yagasaki K, Kitamura K, Mikami B, Utsumi S.
Crystal structure of soybean 11S globulin: glycinin A3B4 homohexamer.
Proc Natl Acad Sci U S A. 100(12):7395-400. 2003
PUBMED ID:
12771376
Perez, M. D.; Mills, E. N. C.; Lambert, N.; Johnson, I. T.; Morgan, M. R. A.
The use of anti-soya globulin antisera in investigating soya digestion in vivo.
J. Sci. Food Agric. 80, 513-521. 2000
PUBMED ID:
unknown
[1089]
Rabjohn, P., Helm, E.M., Stanley, J.S., West, C.M., Sampson,H.A., Burks, A.W.,Bannon, G.A
Molecular cloning and epitope analysis of the peanut allergen Ara h 3.
J. Clin. Invest. 103:535-542. 1999
PUBMED ID:
10021462
Xiang P, Beardslee TA, Zeece MG, Markwell J, Sarath G.
Identification and analysis of a conserved immunoglobulin E-binding epitope in soybean G1a and G2a and peanut Ara h 3 glycinins.
Arch Biochem Biophys. 408(1):51-57 2002
PUBMED ID:
12485602
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