Allergy information for: Soybean, Soya (Glycine max )

Clinical History

• Number of Studies:>20
• Number of Patients:>50
• Symptoms:

  There have been more than 20 studies describing allergy to soybean (Besler et al. 2000 [660]). In general individuals report cutaneous, gastrointestinal and respiratory symptoms and some studies report specifically oral allergy syndrome (Kleine-Tebbe et al. 2002 [658]). Symptoms can be severe, requiring emergency treatment (Kleine-Tebbe et al. 2002 [658]) and there is one report of exercise induced anaphylaxis to soy (Taramarcaz et al. 2001 [675]). The types of symptom reported by selected representative studies are summarised below.

  1. Sampson & Ho (1997) [652] reported cutaneous (68%), gastrointestinal (77%) and respiratory (23%) symptoms in 31 soybean-allergic children.

  2. Sampson (2001) [651] similarly reported cutaneous (67%), gastrointestinal (62%) and respiratory (24%) symptoms in 21 soybean-allergic children.

  3. Niggemann et al. (1999) [661] reported cutaneous (58%), gastrointestinal (14%) and both cutaneous and gastrointestinal symptoms (28%) in 7 soybean-allergic children.

  4. Kleine-Tebbe et al. (2002) [658] reported on symptoms from 20 patients eating a soy containing food supplement. 17/20 experienced facial swelling, 14/20 oral allergy syndrome, 11/20 throat tightness and/or swallowing discomfort, 6/20 dyspnea (chest tightness, wheezing), 6/20 hives, urticaria, 5/20 drowsiness and/or vascular dysregulation, 4/20 gastrointestinal discomfort and/or vomiting, 2/20 nasal secretion and/or nasal congestion. 13 patients required emergency treatment.

  5. Mittag et al. (2004) [857] described 22 patients with oral allergy symptoms. 12 had additional symptoms: 5 had urticaria, one angioedema, one blistering of the mouth, 3 tightness of throat or chest and one each with dysnpea or oedema of the larynx, 3 experienced nausea, 1 diarrhoea, and 2 showed a fall of blood pressure.

Skin Prick Test

• Number of Studies:11-20
• Food/Type of allergen:Commercial extract
• Protocol: (controls, definition of positive etc)

  The positive control was histamine (10mg/ml). Several criteria were compared with the positive is mean diameter greater than 3 mm rule. The definition of negative used was the mean diameter or the surface area was less than the upper 95% confidence interval of subjects with negative DBPCFC (Eigenmann and Sampson, 1998 [674]).

  Gu et al. (2001) [654] and Mittag et al. (2004) [857] defined positive as a wheal of at least 3 mm diameter.

• Number of Patients:

  Eigenmann and Sampson (1998) [674] tested 51 patients by SPT with soy extract (DBPCFC subsequently found 25 positive and 26 negative).

  Gu et al. (2001) [654] tested 6 soy-sensitive patients and 2 nonsensitive individuals.

  Mittag et al. (2004) [857] tested 17 soy allergic patients.

• Summary of Results:

  Eigenmann & Sampson (1998) [674] conclude that skin prick tests are a useful procedure for evaluating clinical reactivity to egg, milk, peanut and wheat, but not to soy. For soy, 13/26 negative DBPCFC were SPT positive and 9/25 positive DBPCFC were SPT negative.

  Gu et al. (2001) [654] report that all 6 soy sensitive patients gave positive SPT to soy extract and 2 nonsensitive indivuals were SPT negative.

  Mittag et al. (2004) [857] reported that 13/17 soy allergic patients gave clear positive SPTs and 2 gave borderline positive SPTs.

IgE assay (by RAST, CAP etc)

• Number of Studies:0
• Food/Type of allergen:Commercial extract (Sampson 2001 [651]).
  Mittag et al. (2004) [857] also used purified Gly m 4.
• IgE protocol:

  CAP FEIA (Sampson 2001 [651]; Mittag et al. (2004) [857]; Rihs et al. (1999) [170])

  Earlier articles generally used RAST.

• Number of Patients:

  Mittag et al. (2004) [857] used sera from 22 soy allergic patients and from 94 birch allergic patients (9 of whom reported symptoms with soy). 16 were DBPCFC positive, 1 had suffered anaphylaxis and 5 declined the challenge.

  Sampson (2001) [651] used sera from 100 patients of whom 25 were judged soy allergic, 21 by challenge and 4 by clinical history and IgE level.

  Gu et al. (2001) [654] used sera from 6 soy-sensitive individuals and 2 soy-insensitive individuals.

  Rihs et al. (1999) [170] used sera from 13 patients (10 female and 3 male patients; age range, 6-57 years) who were CAP positive to soy proteins. All were highly atopic and had atopic dermatitis. Food-associated symptoms included atopic eczema, anaphylaxis, and/or asthma

  Awazuhara et al. 1997 [1091] and Awazuhara et al. 1998 [1080] used sera from 30 soy-allergic patients of whom 7 were positive to oral challenge.

  Eigenmann et al. (1996) [48] used sera from 5 peanut allergic patients.

  Ogawa et al. (1991) [441], Ogawa et al. (1993) [131] and Ogawa et al. (1995) [653] used sera from 69 patients with atopic dermatitis.

  Herian et al. (1990) [1078] used sera from 7 patients allergic to soybean of whom 6 were allergic to other legumes (4 to peanut).

  Pedersen & Djurtoft (1989) [1093] used sera from 10 atopic patients with a history of reactions to soya (and some to other legumes) and a positive RAST to soy extract.

• Summary of Results:

  Mittag et al. (2004) [857] found that 10/22 soy allergic patients gave a positive CAP test with a commercial extract (0.35 - 5.05 kU/l) but 21/22 were positive with purified Gly m 4 (0.72->100 kU/l). All 22 showed positive reaction to Bet v 1 (only 4 to Bet v 2 and 1 to Bet v 6). 67/94 birch allergic patients showed specific IgE against Gly m 4.
  Birch pollen significantly inhibited IgE binding to soy protein isolate by EAST inhibition in all tested sera, with a minimum of 45% in one patient and maximum inhibitions of more than 80% in 9 of 11 patients. The maximum inhibition of IgE binding by rGly m 4 ranged between 36% and 100%, with 9 of 11 sera showing at least 60% inhibition.

  CAP IgE of 65 kU_A/L confidently predicted allergy to soy, and above 30 kU_A/L suggested soy allergy (Sampson 2001 [651]).

  Gu et al. (2001) [654] report that 6 sera were positive for soy specific IgE (0.79-54.1 kU/l) and the two sera from nonsensitive individuals were negative (>0.1 kU/l).

  Pedersen & Djurtoft (1989) [1093] reported that IgE from all the 10 sera bound subunits of glycinin and 9/10 bound native glycinin by ELISA. Most sera bound the subunits more strongly.

Immunoblotting

• Immunoblotting separation:

  Besler et al. 2000 [660]) review 22 EAST or immunoblotting studies and details of only some recent studies are described below.

  Mittag et al. (2004) [857] used 1D SDS-PAGE with 15% separation gels and 5% stacking gels. Samples were reduced by heating with 1,4-dithiothreitol and loaded onto the gel at a concentration of 1 µg/cm for rGly m 4 and rGly m 3 and 30 µg/cm for soy isolate extract, respectively.

  Gu et al. (2001) [654] used 1D SDS-PAGE with a 10%-20% linear gradient separating gel (BioRad) with samples that had been heated to 95° C for 3 minutes with 2% SDS and 100 mM 2-mercaptoethanol.

  Rihs et al. (1999) [170] used 1D SDS-PAGE of recombinant rGly m 3 (as a fusion protein and after cleavage) or of soybean extract on 10% NuPAGE Bis-TRIS gels (NOVEX) under reducing conditions.

• Immunoblotting detection method:

  Mittag et al. (2004) [857] transferred proteins onto 0.2-µm pore-sized nitrocellulose membranes by means of semidry blotting. Remaining binding sites were blocked twice by means of incubation with 0.3% (v/v) Tween 20 in Tris-buffered saline (50 mmol/L Tris/HCl and 150 mmol/L NaCl, pH 7.4). The nitrocellulose strips were incubated overnight with 130 µL of patient serum diluted to 1 mL with incubation buffer, Tris-buffered saline containing 0.05% (v/v) Tween 20 and 0.1% (w/v) BSA. Immunodetection followed standard procedures.

  Gu et al. (2001) [654] transferred proteins onto polyvinylidene difluoride membranes (Millipore) by using a semidry apparatus (BioRad) at temperature for 2 h. at 0.8 mA/cm². The membranes were blocked overnight with 10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.3% (v/v) Tween 20, 1% w/v BSA and 3 mM NaN₃, then incubated with human sera (diluted 1:20 (v/v) in blocking buffer). The membranes were washed with 10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.3% (v/v) Tween 20 and 3 mM NaN₃ then incubated with goat anti-human IgE (diluted 1:20,000 (v/v) in blocking buffer) for 12 h. at 4°C. After 3 further washes, Amersham Pharmacia's chemiluminescent substrate solution (0.1 ml/cm²) for 5 min. at room temperature revealed IgE binding.

  Rihs et al. (1999) [170] transferred proteins onto polyvinylidene difluoride membranes (Millipore) by using a semidry system (16 × 16 cm) of Roth (Karlsruhe, Germany), with a discontinuous buffer system at 0.8 mA/cm² membrane for 1 hour at room temperature. After blocking with a solution consisting of 1% (w/v) BSA and 1% (w/v) polyvinylpyrrolidone (Sigma) in Tris-buffered saline, TBS (50 mM Tris/HCl, pH 7.4, 150 mM NaCl containing 5 mM NaN₃and 1 mM EDTA) and washing with TBS, membranes were cut into individual 4-mm wide strips. The strips were incubated individually with 800-µL serum samples diluted 1:10 in TBST-BSA (2% BSA dissolved in TBS containing 0.05% (v/v) Tween 20) for 18 hours at 4° C. The binding of specific IgE antibodies to the proteins was detected with alkaline phosphatase–conjugated anti-human IgE antibodies (Sigma) diluted 1:2000 (v/v) in TBST-BSA for 2 hours at room temperature, and bands were visualized with a substrate solution containing 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate disodium salt and 0.37 mmol/L p-nitro blue tetrazolium chloride in 0.1 M NaHCO₃ buffer, containing 10 mM MgCl₂ (pH 9.6).

• Immunoblotting results:

  Mittag et al. (2004) [857] detected IgE binding to rGly m 4 in 19/22 patients. rGly m 3 bound in 6 patients, including 2 patients without IgE binding to rGly m 4 in immunoblots. Fourteen patients showed IgE binding to other soy proteins in the range of 8 to 10, 18, 20, 35, and 50 kDa.

  Gu et al. (2001) [654] detected IgE binding to proteins at 7, 12, 20, 39 and 57 kDa from soy lecithin. The bands at 12, 20 and 39 kDa were identified by N-terminal sequencing. The 39 Kda protein was only known from incomplete Soybean EST sequences such as gi|46492876, which are distantly related to proteins of unknown function from Arabidosis, including a glycosylphosphatidylinositol-anchored protein, P59833. The 20 kDa protein was the Kunitz trypsin inhibitor and the 12 kDa was a 2S albumin.

  Rihs et al. (1999) [170] report that after preincubation of sera with 50 µg of MBP-rGly m 3, a significant inhibition of IgE binding to a double band of the blotted soybean extract in the area of 14 kDa could be observed in all 4 serum samples tested, whereas the other IgE-binding bands were not affected.

  Awazuhara et al. 1998 [1080] reported that IgE antibodies in the sera from 15 out of the 30 patients reacted to the proteins in the soy lecithin by immunoblotting, whereas the IgE antibodies in only three out of 22 control sera reacted to them. 40% (12/30) of soy sensitive and 4.5% (1/22) of controls reacted to a 31 kDa protein.

  Müller et al. (1998) [1092] reported that IgE bound to the proteins in soy lecithin between 14 kDa and 94 kDa. The 39 kDa heat-stable allergen was identified using a monoclonal antibody as a subunit of glycinin in this study.

  Awazuhara et al. 1997 [1091] reported that IgE bound most frequently to 5 allergens. These were 20 and 58 kDa in the whey fraction (i.e. soluble after aqueous extract was adjusted to pH 4.5) and 26, 31 and 78 kDa in the globulin fraction. Other allergens were detected at 28, 41, 75 and 91 kDa in the whey fraction and 40, 47, 56, 64, 72 and 91 kDa in the globulin fraction. The 47 kDa allergen actually bound weakly to 50% of sera. The authors suggest that the 31 kDa allergen might be Gly m Bd30K and the 78 kDa allergen might be beta-conglycinin.

  Eigenmann et al. (1996) [48] reported IgE binding soy fractions at 46, 40, 33, 19, and 9 kDa in sera from patients allergic to peanut. Two patients allergic to peanut and soy reacted to protein fractions of 45 and 17 kDa while three soy tolerant patients bound fractions at 45 kDa, and to a lesser degree, at 21 kDa.

  Ogawa et al. (1991) [441] reported that at least 16 soybean proteins with Mr from approximately 70 kDa to 14 kDa were recognized by the sera of the patients: 10 major IgE-binding components were found in the 7S-globulin fraction, and the others mainly in the 2S-globulin and whey fractions. The IgE antibodies of the patients bound most strongly and frequently to a 30 kDa protein in the 7S-globulin fraction, which appeared to be the major allergen in soybeans and was named as Gly m Bd30K. The 11S fractions did not bind IgE strongly from the sera of these atopic dermatitis patients.

  Herian et al. (1990) [1078] reported that there were different patterns of IgE reactivity between sera from patients sensitised to soybeans alone which binds to protein(s) at 20 kDa and sera from those reactive to both soybeans and peanuts which binds to proteins of 50 to 60 kDa.

  The wide variation in the above results may be partly due to differences between US, often peanut allergic patients; Japanese, sensitised to soy; and Europeans sensitised via birch pollen.

Oral provocation

• Number of Studies:6-10
• Food used and oral provocation vehicle:

  Niggemann et al. (1999) [661], Sicherer et al. (2000) [673], Sampson & Ho (1997) [652] and Sampson (2001) [651] used similar methods. A maximum of 8 to 10 g dry weight of dehydrated food, or equivalent liquid form, was camouflaged in a food product and given over a 90-minute period. Six doses of food were given in progressively larger quantities such as 0.5 g, 1 g, 3 doses of 2 g, and a final dose of 2.5 g. Patients received 2 challenges per day, 1 placebo and 1 test food.

  Mittag et al. (2004) [857] gave two different drinks, identical in color, texture, and taste. The active drink contained 15 g of soy protein isolate (soy protein content, 90%; Protein technologies, Du Pont & Dow, Bad Homburg, Germany), 150 mL of milk, 50 mL of cream, 9 g of Sinlac (containing rice flour, and carob flour; Nestle, Vevey, Switzerland), 25 g of sugar, 7 g of cocoa, and 6 drops of banana flavor (Givaudan, Duebendorf, Switzerland). The soy protein isolate is a mildly heated soy product that was produced through extraction of defatted soybean flakes at pH 8 to 8.5, neutralization, and spray drying. The composition of the placebo drink was identical but did not contain soy isolate. Apart from soy, all ingredients were known to be tolerated by each patient.

• Blind:Niggemann et al. (1999) [661], Palm et al. (1999) [1081], Sicherer et al. (2000) [673], Sampson & Ho (1997) [652], Sampson (2001) [651] and Mittag et al. (2004) [857] used double blind challenge.
  Awazuhara et al. 1998 [1080] used open challenge.
• Number of Patients:

  16 patients (Mittag et al. 2004) [857].

  45 patients with 7 positive (Niggemann et al. 1999) [661].

  53 patients from 1980-1993 gave a positive challenge to soy (Sicherer et al., 2000 [673]).
  31 patients (Sampson & Ho 1997) [652] gave a positive challenge to soy.
  21 patients (Sampson (2001) [651] gave a positive challenge to soy.

  Palm et al. (1999) [1081] challenged a single 4 year old patient.

  Awazuhara et al. 1998 [1080] report that 7 patients were positive on challenge.

• Dose response:

  Sicherer et al. (2000) [673] report that 28% of soy challenge positive patients reacted to the first dose (0.4g) but 19% reacted only to the last dose or to open challenge. However, patients with severe symptoms were excluded from challenge. After 1992, strong IgE reaction also excluded some patients from challenge.

  Mittag et al. (2004) [857] used a 2-step spit (local mucosal challenge) and swallow procedure. For the local mucosal challenge, 4 doses containing 0.3, 0.5, 1, and 2 g of soy protein, respectively, were administered to the patients, whereas for the systemic challenge, 4 doses containing 0.5, 1, 2, and 4 g of soy protein, respectively, were used.

• Symptoms:

  Mittag et al. (2004) [857] report that all 16 patients showed oral allergy syndrome symptoms, sometimes with rhinitis or flush, and 4 of them also had more severe symptoms of tighness of the throat or chest.

  None of the patients described by Sicherer et al. (2000) [673] suffered severe symptoms from soy in contrast to patients challenged by egg, milk, fish and peanut.

  Niggemann et al. (1999) [661] reported early reactions in 4/7 (57%) and late reactions in 3/7 (43%) of 7 positive challenges to soybean in allergic children (i.e. 16% positive challenges)

IgE cross-reactivity and Polysensitisation

Other Clinical information

Reviews (1)

• Besler M., Helm R.M., Ogawa T.
  Allergen Data Collection - Update: Soybean (Glycine max)
  Internet Symposium on Food Allergens 2(Suppl.3): 1-35. 2000
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  Internet Symposium on Food Allergens 2(Suppl.3): 1-35. 2000
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Biochemical Information for 2S Albumin

• Allergen Name:2S Albumin
• Alternatve Allergen Names:Methionine rich protein, MRP, GM2S-1, AL3
• Allergen Designation:Minor
• Protein Family:

  2S albumin family, PFAM PF00234; Tryp_alpha_amyl

• Sequence Known?:Yes
• Allergen accession No.s:

  http://us.expasy.org/cgi-bin/niceprot.pl?P19594

• 3D Structure Accession No.:N/A
• Calculated Masses:8975.19 Da (large chain)
  5028.36 Da (small chain)
  18460 Da (precursor)
• Experimental Masses:

  12 kDa (Gu et al. 2000 [654])
  14 kDa (Ogawa et al. 1991 [441]; Herian et al. 1990 [1078])

  8975.14 and 5138.30 (Lin et al. 2004 [1077])

• Oligomeric Masses:Not known
• Allergen epitopes:Not known
• Allergen stability:
  Process, chemical, enzymatic:The disulphide cross-linked 2S albumins are normally stable to heat and proteolysis. Lin et al. (2004) [1077] show that recombinant proteins retain their secondary structure in 6M guanidinium hydrocloride and also when heated up to 97°C.
• Nature of main cross-reacting proteins:Not known
• Allergen properties & biological function:2S seed storage albumin.
• Allergen purification:

  (Lin et al. 2004 [1077]) reported the purification of both native soybean 2S albumin from soybean and recombinant soybean albumin, rAL, from Pichia pastoris.

  Native soybean 2S albumin:
  Soybean seeds were ground into a fine powder, defatted by extraction with hexane (1:4 w/v) and dried in vacuum. Soluble proteins were extracted from 100 g of defatted soybean powder with 400 ml albumin extraction buffer (10% v/v DMSO, 0.5% v/v butan-1-ol, 100 mM KCl, 83 mM sodium acetate, pH 5.2). Following centrifugation at 6000×g for 15 min, the supernatant was dialyzed against 0.5% v/v butan-1-ol, 100 mM KCl, 83 mM sodium acetate buffer, pH 5.2, lyophilised, and dissolved in distilled water (250 mg/ml). Insoluble proteins were removed by centrifugation at 6000×g for 15 min and the supernatant was loaded onto a Sephacryl S-100 column (2 cm×55 cm, Pharmacia) run (0.5 ml/min) with the albumin extraction buffer. Fractions containing the 2S albumins (~14 kDa) were dialyzed against distilled water, lyophilised, dissolved in 20 mM Tris/HCl, pH 8.5 (20 mg/ml) and further fractionated by ion-exchange chromatography using the BioCAD perfusion chromatography system equipped with a POROS 20 HQ (4.6 mm×10 cm) column, eluted with a gradient of NaCl (0\Z750 mM) in 20 mM Tris/HCl, pH 8.5 at a flow rate of 5 ml/min. The peak, which was eluted at 360 mM NaCl and contained the 2S albumin, was then further purified by reversed-phase HPLC on a C₁₈ column (25 cm×10 mm, Phenomenex), in 0.07% (v/v) trifluoroacetic acid (TFA) in water and eluted with a linear gradient of 0\Z50%, 100% acetonitrile in 0.05% (v/v) TFA at a flow rate of 2.5 ml/min for 40 min and the purified 2S albumin eluted after 25 min. The eluted peak was lyophilised and stored at −20 °C.

  Recombinant soybean albumin:
  The supernatant secreted by P. pastoris was filtered through a 0.45µm filter, then concentrated and salt eliminated by buffer exchange with 10 mM citric acid buffer (pH 4.3) using a Viva Flow 200 (5000 MWCO) (Vivascience, Lincoln, UK). The concentrate was loaded onto three in-series 5 ml Hi-Trap heparin columns (Amersham-Pharmacia, Uppsala, Sweden) previously equilibrated with 10 mM citric acid buffer (pH 4.3) and eluted using a step gradient of NaCl (0.2\Z1.0 M) at a flow rate of 3 ml/min. The column effluent was monitored at 280 nm. The collected fractions containing rAL were dialyzed against distilled water for 24 h at 4 °C. The rAL was then further purified by reversed-phase HPLC on a C₈ column (10 cm×10 mm, Varian, Walnut Creek, CA, USA), in 0.1% (v/v) formic acid in water and eluted with a linear gradient of 0\Z100% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 2.5 ml/min for 25 min. The eluted peak was lyophilised and stored at −20 °C.

• Other biochemical information:

  The sequences P19594 and AAD00178.1 are identical. A further sequence AAD09630 is related but distinct. Both albumins were purified by Lin et al. (2004) [1077].

  Gu et al. (2000) [654]) reported that the 2S albumin was strongly bound at 12 kDa by IgE binding from a single sera and weakly by IgE from another sera. The 14 kDa minor allergen reported by Ogawa et al. (1991) [441] and Herian et al. (1990) [1078] may also be the 2S albumin. The reported masses are high as the proteins were reduced before electrophoresis.

References (4)

• Ogawa T, Bando N, Tsuji H, Okajima H, Nishikawa K, Sasaoka K
  Investigation of the IgE-binding proteins in soybeans by immunoblotting with the sera of the soybean-sensitive patients with atopic dermatitis.
  J Nutr Sci Vitaminol (Tokyo) 37:555-65. 1991
  PUBMEDID: 1819644
• Lin J, Fido R, Shewry P, Archer DB, Alcocer MJ.
  The expression and processing of two recombinant 2S albumins from soybean (Glycine max) in the yeast Pichia pastoris.
  Biochim Biophys Acta 1698(2):203-212.. 2004
  PUBMEDID: 15134653
• Gu X, Beardslee T, Zeece M, Sarath G, Markwell J.
  Identification of IgE-binding proteins in soy lecithin.
  Int Arch Allergy Immunol. 126(3):218-25.. 2001
  PUBMEDID: 11752879
• Herian AM, Taylor SL, Bush RK.
  Identification of soybean allergens by immunoblotting with sera from soy-allergic adults.
  Int Arch Allergy Appl Immunol. 92(2):193-198.. 1990
  PUBMEDID: 2242931

Biochemical Information for Beta-Conglycinin

• Allergen Name:Beta-Conglycinin
• Alternatve Allergen Names:Conglycinin, 7S globulin
• Allergen Designation:None
• Protein Family:7S Cupin, Pfam PF00190
• Sequence Known?:Yes
• Allergen accession No.s:P13916 :Swissprot: http://ca.expasy.org/cgi-bin/niceprot.pl?P13916
  P25974:Swissprot: http://ca.expasy.org/cgi-bin/niceprot.pl?P25974
  P11827:Swissprot: http://ca.expasy.org/cgi-bin/niceprot.pl?P11827
  O22120:Swissprot: http://ca.expasy.org/cgi-bin/niceprot.pl?O22120
  O22121:Swissprot: http://ca.expasy.org/cgi-bin/niceprot.pl?O22121
  Q39853:Swissprot: http://ca.expasy.org/cgi-bin/niceprot.pl?Q39853
  Q9FZP9:Swissprot: http://ca.expasy.org/cgi-bin/niceprot.pl?Q9FZP9
  
• 3D Structure Accession No.:1IPK
  1IPJ
• Calculated Masses:63151.8 Da P13916 residues 63-605
• Experimental Masses:68 kDa
• Oligomeric Masses:180 kDa
• Allergen epitopes:On the basis of the IgE reactivity being confined to the alpha subunit and to an 18 kDa peptide starting YVVN, the IgE epitopes may be in the region 232-383 of the mature alpha chain and must differ from the non-allergenic alpha' subunit (Ogawa et al. 1995 [653]).
• Allergen stability:
  Process, chemical, enzymatic:The storage proteins are thermostable. Temperatures in excess of 75°C are required for denaturation of beta-conglycinin and it shows only partial loss of secondary and tertiary structure (Mills et al. 2001 [114]). The proteins' ability to aggregate and gel on heating has been widely exploited in food processing but its implications for allergenicity have not been studied. They also show resistance to proteolysis, being able to form large (>100,000 dalton) oligomeric partially cleaved intermediates.
• Nature of main cross-reacting proteins:

  Not known. IgE cross-reactivity occurs between legumes, such as peanut and soybean, but it is generally not clinically significant. The 7S globulins Ara h 1 and beta-conglycinin are about 43% identical and may contribute to peanut -soya IgE cross-reactivity although the 11S family is slightly more conserved and may be more important. Beta-conglycinin and Len c 1 are more similar at 55% identity.

• Allergen properties & biological function:The 7S globulin (beta-conglycinin) proteins are heterotrimeric, of Mr ~ 180,000, made up of N-glycosylated subunits. In soya these are the 50 kDa beta subunit and the 63 kDa alpha/alpha' subunits (after removal of propeptides). The IgE-reactivity appears to be confined to the alpha subunit N-terminal insert.
• 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, 20°C) and the supernatant retained. After incubation at 2°C overnight, the cold-insoluble fraction (CIF) was removed by centrifugation at 10,000xg for 30 min, 20°C. The pH of the supernatant was adjusted to pH 5.4 with 0.1M HCl and following centrifugation at 10,000xg for 20 min at 20°C, the 7S globulin precipitated by adjusting the pH to 4.8 with 1M HCl. Following dialysis against 50mM Tris-HCl, pH 7.8, containing 0.2M NaCl the crude 7S globulin was further purified by gel filtration on Sephacryl S300 (2.6x100cm column) using a flow rate of 0.4 ml/min, to remove aggregates (after Than and Shibasaki, 1976).
• Other biochemical information:

  A complex of the alpha' and alpha subunits of beta-conglycinin and the allergen Gly m Bd 30K was identified by Samoto et al. (1996) [1084]. A genetically mutated soybean cultivar lacking the alpha' and alpha subunits allowed easier removal of Gly m Bd 30K from defatted soy milk (Samoto et al. 1996) [1085].

References (8)

• Ogawa T, Bando N, Tsuji H, Nishikawa K, Kitamura K.
  Alpha-subunit of beta-conglycinin, an allergenic protein recognized by IgE antibodies of soybean-sensitive patients with atopic dermatitis.
  Biosci Biotechnol Biochem. 59(5):831-833.. 1995
  PUBMEDID: 7787297
• Mills, E.N.C., Huang, L., Noel, T.R., Gunning, A. P. Morris, V.J.
  Formation of thermally-induced aggregates of the soya globulin beta-conglycinin.
  Biochim Biophys Acta 1547(2):339-350.. 2001
  PUBMEDID: 11410290
• Than VH, Shibasaki K
  Major proteins of soybean seeds. A straightforward fractionation and their characterization.
  J Agric Food Chem 24:1111-1117.. 1976
  PUBMEDID: 1033950
• 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
  PUBMEDID: 10021462
• Beardslee, T.A., Zeece, M.G., Sarath, G., Markwell, J.P.
  Soybean glycinin G1 acidic chain shares IgE epitopes with peanut allergen Ara h 3
  Int. Arch. Allergy Immunol. 123:299-307.. 2000
  PUBMEDID: 11146387
• Maruyama N, Adachi M, Takahashi K, Yagasaki K, Kohno M, Takenaka Y, Okuda E, Nakagawa S, Mikami B, Utsumi S.
  Crystal structures of recombinant and native soybean beta-conglycinin beta homotrimers.
  Eur J Biochem. 268(12):3595-604.. 2001
  PUBMEDID: 11422391
• Samoto M, Miyazaki C, Akasaka T, Mori H, Kawamura Y.
  Specific binding of allergenic soybean protein Gly m Bd 30K with alpha'- and alpha-subunits of conglycinin in soy milk.
  Biosci Biotechnol Biochem. 60(6):1006-1010.. 1996
  PUBMEDID: 8695899
• Samoto M, Takahashi K, Fukuda Y, Nakamura S, Kawamura Y.
  Substantially complete removal of the 34kDa allergenic soybean protein, Gly m Bd 30 K, from soy milk of a mutant lacking the alpha- and alpha'-subunits of conglycinin.
  Biosci Biotechnol Biochem. 60(11): 1911-1913.. 1996
  PUBMEDID: 8987873

Biochemical Information for Gly m 3

• Allergen Name:Gly m 3
• Alternatve Allergen Names:Profilin, Gly m 3.0101 & Gly m 3.0102
• Allergen Designation:None
• Protein Family:Profilin, Pfam PF00235
• Sequence Known?:Yes
• Allergen accession No.s:O65809 http://ca.expasy.org/cgi-bin/niceprot.pl?O65809
  O65810 http://ca.expasy.org/cgi-bin/niceprot.pl?O65810
  
• 3D Structure Accession No.:Not determined
• Calculated Masses:14100 Da
• Experimental Masses:14 kDa
• Oligomeric Masses:
• Allergen epitopes:Epitope mapping with recombinant profilin indicated that only the full-length rGly m 3 was able to bind with IgE antibodies, whereas the 3 soybean profilin fragments did not show significant binding reactivity. It was concluded that IgE binding to rGly m 3 depends on the integrity of a conformational structure, which was not present in the overlapping profilin fragments.
• Allergen stability:
  Process, chemical, enzymatic:Not known
• Nature of main cross-reacting proteins:

  Recombinant Gly m 3 cross-reacted with IgE directed to birch pollen profilin (Bet v 2), and the IgE binding to Bet v 2 could be inhibited by rGly m 3. The sequence of peanut Ara h 5 is 83% identical.

• Allergen properties & biological function:Soybean profilin is thought to be an actin-binding protein.
• Allergen purification:Rihs et al. (1999) [170] expressed recombinant Gly m 3 as a maltose binding protein (MBP) fusion protein in E. coli and purified the fusion protein by affinity chromatography on amylose. The rGly m 3 was cleaved from MBP by incubation of affinity-purified fusion protein with proteinase Factor Xa (MobiTech, Goettingen, Germany).
• Other biochemical information:

References (1)

• Rihs HP, Chen Z, Rueff F, Petersen A, Rozynek P, Heimann H, Baur X
  IgE binding of the recombinant allergen soybean profilin (rGly m 3) is mediated by conformational epitopes.
  J. Allergy Clin Immunol 104:1293-1301.. 1999
  PUBMEDID: 10589015

Biochemical Information for Gly m 4

• Allergen Name:Gly m 4
• Alternatve Allergen Names:SAM22
• Allergen Designation:None
• Protein Family:Bet v 1 family, Pfam PF00407
• Sequence Known?:Yes
• Allergen accession No.s:http://ca.expasy.org/cgi-bin/niceprot.pl?P26987
• 3D Structure Accession No.:Not known
• Calculated Masses:16772 Da
• Experimental Masses:17 kDa
• Oligomeric Masses:Monomer
• Allergen epitopes:Not known
• Allergen stability:
  Process, chemical, enzymatic:Mittag et al. (2004) [857] reported that in highly fermented soy foods (soy sauce and miso) and in roasted soybeans, no Gly m 4 could be detected. The Gly m 4 content of soybeans was reduced after 30 minutes of cooking, and after 4 hours of cooking, no Gly m 4 was detectable.
• Nature of main cross-reacting proteins:Gly m 4 cross-reacts with Bet v 1 and the main route of sensitisation to Gly m 4 is believed to be birch pollen inhalation.
• Allergen properties & biological function:Gly m 4 (SAM22) is a stress-induced PR-10 protein (Crowell et al. 1992) [659] and is 47% identical in amino acid sequence with Bet v 1
• Allergen purification:Recombinant SAM22 was produced in E. coli and purified from the supernatant by means of chromatofocusing with an ÄKTA- FPLC system (Amersham Biosciences, Freiburg, Germany) by using a Mono P HR 5/5 column. The protein was judged better than 98% pure from silver-stained SDS-PAGE gels (Kleine-Tebbe et al. 2002) [658].
• Other biochemical information:Mittag et al. (2004) [857] reported that the mean content of extractable and immune-reactive Gly m 4 was 256 ± 30 ppm over 10 soybean varieties. Unripe green soybeans contained approximately 8 ppm, ripe fresh soybeans contained 96 ppm, and ripe soybeans after 3 years of storage contained 224 ppm. On the basis of total protein, there was no difference between freshly harvested or stored ripe soybeans, but their Gly m 4 content on the basis of total protein amount was 6 times higher than that in unripe soybeans.

References (3)

• Mittag D, Vieths S, Vogel L, Becker WM, Rihs HP, Helbling A, Wuthrich B, Ballmer-Weber BK.
  Soybean allergy in patients allergic to birch pollen: clinical investigation and molecular characterization of allergens.
  J Allergy Clin Immunol. 113(1):148-154.. 2004
  PUBMEDID: 14713921
• Crowell DN, John ME, Russell D, Amasino RM.
  Characterization of a stress-induced, developmentally regulated gene family from soybean.
  Plant Mol Biol 18:459-66.. 1992
  PUBMEDID: 1371403
• Kleine-Tebbe J, Vogel L, Crowell DN, Haustein UF, Vieths S.
  Severe oral allergy syndrome and anaphylactic reactions caused by a Bet v 1- related PR-10 protein in soybean, SAM22.
  J Allergy Clin Immunol. 110(5):797-804.. 2002
  PUBMEDID: 12417891

Biochemical Information for Gly m Bd 28k

• Allergen Name:Gly m Bd 28k
• Alternatve Allergen Names:
• Allergen Designation:Major
• Protein Family:Pfam PF00190; Cupin, 7S seed storage family
• Sequence Known?:Yes
• Allergen accession No.s:

  AB046874:EMBL
  http://ca.expasy.org/cgi-bin/niceprot.pl?Q9AVK8
  BAB21619.1 :EMBL:
  gi:12697781:Genbank:

• 3D Structure Accession No.:Not determined
• Calculated Masses:50413.23 Da (residues 22-471)
• Experimental Masses:26 kDa
• Oligomeric Masses:150 kDa
• Allergen epitopes:

  IgE antibodies in soya allergic individuals recognise N-linked glycans at Asn20 (Hiemori et al 2000 [70]).

  Xiang et al. (2004) [1074] report that a dominant linear C-terminal IgE binding epitope resides between residues S256 and A270. Alanine scanning of this dominant epitope indicated that five amino acids, Y260, D261, D262, K264 and D266, contribute most towards IgE-binding.

• Allergen stability:
  Process, chemical, enzymatic:Gly m Bd 28k is a glycoprotein with glycan moieties, which are supposed to be the Man(3)GlcNAc(2) backbone with the beta1-->2 xylose and alpha1-->3 fucose branches.
• Nature of main cross-reacting proteins:The sequence of Bd 28K is only very distantly related to other known vicilin allergens and cross-reactivity would not be expected. The closest relatives in food plants have been reported from pumpkin (56% identity) and carrot (45% identity).
• Allergen properties & biological function:Gly m Bd 28K is a vicilin and like others may act as a seed storage protein.
• Allergen purification:Gly m Bd 28K was purified from defatted soybean flakes by five purification steps, including immunoaffinity chromatography with the mAb as a ligand. The purified allergen was found to be a glycoprotein with a molecular mass of 26 kDa. During the purification process the allergen was converted to more acidic proteins with the same molecular mass, suggesting that the allergen is unstable (Tsuji et al., 1997 [211]).
• Other biochemical information:

References (7)

• Tsuji H; Hiemori M; Kimoto M; Yamashita H; Kobatake R; Adachi M; Fukuda T; Bando N; Okita M; Utsumi S
  Cloning of cDNA encoding a soybean allergen, Gly m Bd 28K.
  BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1518, Iss 1-2, pp 178-182. 2001
  PUBMEDID: 11267676
• Ogawa T; Bando N; Yamanishi R; Tsuji H; Hiemori M
  Characterization of Gly m Bd 28K, an allergenic glycoprotein from soybean.
  JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY 101, Iss 1, pp 380. 1998
  PUBMEDID:
• Bando N; Tsuji H; Hiemori M; Yoshizumi K; Yamanishi R; Kimoto M; Ogawa T
  Quantitative analysis of Gly m Bd 28K in soybean products by a sandwich enzyme-linked immunosorbent assay.
  JOURNAL OF NUTRITIONAL SCIENCE AND VITAMINOLOGY 44, Iss 5, 655-664. 1998
  PUBMEDID: 9919485
• Kanegae R; Obata A; Matsunaga R; Takashi M; Komatsu K
  Analysis of Gly m Bd 28 K, major soybean allergen, in several leading varieties in Japan.
  JOURNAL OF THE JAPANESE SOCIETY FOR FOOD SCIENCE AND TECHNOLOGY-NIPPON SHOKUHIN KAGAKU KOGAKU KAISHI 48, Iss 5, pp 344-348. 2001
  PUBMEDID:
• Hiemori M, Bando N, Ogawa T, Shimada H, Tsuji H, Yamanishi R, Terao J
  Occurrence of IgE antibody-recognizing N-linked glycan moiety of a soybean allergen, Gly m Bd 28K.
  Int Arch Allergy Immunol 122:238-45.. 2000
  PUBMEDID: 10971113
• Tsuji H; Bando N; Hiemori M; Yamanishi R; Kimoto M; Nishikawa K; Ogawa T
  Purification and characterization of soybean allergen Gly m Bd 28K.
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 61, Iss 6, pp 942-947. 1997
  PUBMEDID: 9214751
• Xiang P, Haas EJ, Zeece MG, Markwell J, Sarath G.
  C-Terminal 23 kDa polypeptide of soybean Gly m Bd 28 K is a potential allergen.
  Planta 220(1):56-63. 2004
  PUBMEDID: 15252733

Biochemical Information for Gly m Bd 30k

• Allergen Name:Gly m Bd 30k
• Alternatve Allergen Names:P34
• Allergen Designation:Major
• Protein Family:Cysteine protease C1, Pfam PF00112
  
• Sequence Known?:Yes
• Allergen accession No.s:

  Gi:3097321:Genbank http://ca.expasy.org/cgi-bin/niceprot.pl?O64458
  Gi:1199563:Genbank http://ca.expasy.org/cgi-bin/niceprot.pl?P22895
  Gi:1199562:Genbank http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?cmd=Retrieve&db=protein&list_uids=1199563&dopt=GenPept

• 3D Structure Accession No.:Not determined
• Calculated Masses:28642.64 Da (residues 123-379)
• Experimental Masses:30 kDa
• Oligomeric Masses:>300,000 kDa as complex with the alpha' and alpha subunits of beta-conglycinin (Samoto et al. 1996) [1084].
• Allergen epitopes:IgE reactive linear epitopes have been reported from soy Gly m Bd 30K (Helm et al., 1998 [69]) and the five immunodominant epitopes were identified as follows: 3-12 FLVLLLFSLL, 100-110 PQEFSKKTYQ, 229-238 RCKANKIQDK, 299-308 INHFVLLVGY, and 331-340 GYIWIQRNTG. These were analysed further by mutation (Helm et al. 2000 [68]), showing that IgE from individual sera reacted to different aminoacids. The epitopes 3-12 and 110-119 are not present in the mature protein.
• Allergen stability:
  Process, chemical, enzymatic:Gly m Bd 30k exists as an oligomer of molecular weight >300,000 with the alpha' and alpha subunits of beta-conglycinin (Samoto et al. 1996) [1084] and has a pI of 4.5. Two potential glycosylation sites are seen in the sequence of the precursor at residues 70 (prodomain) and 292 (170 in mature P34) and may account for higher molecular masses estimated from SDS-PAGE gels compared to predictions from the sequences. Bando et al. (1996) [655] have shown that residue 170 in P34 is glycosylated.
• Nature of main cross-reacting proteins:Distantly homologous to members of the cysteine proteinase family, actinidin from kiwi fruit, papain from papaya and bromelain from pineapple, but no IgE cross-reactivity has been reported.
• Allergen properties & biological function:Gly m Bd 30K is also known as the 34 kDa oil-body-associated protein or as P34. The P34 protein was initially falsely identified as oil body associated but was shown to be localized in protein storage vacoles (Kalinski et al., 1992 [80]). P34 was shown to accumulate during seed maturation although traces of the 46 kDa precursor were present even in 300 mg. mature seeds. Gly m Bd 30K is homologous to the thiol proteases of the papain family but is probably not a protease because the active site cysteine is mutated to glycine. P34 may function as part of the PR system of soy as it, but not its precursor, binds syringolide elicitors (Ji et al., 1998 [79]).
• Allergen purification:Crude soya 7S was precipitated with 40% ammonium sulphate and dissolved in 35mM potassium phosphate pH 7.6 containing 0.4M NaCl and chromatographed on Sepharose 6B, and the 30 kDa allergen fractions collected and pooled. These fractions were then applied to a Con A sepharose column and the allergen eluted with 20mM alpha-methylmannose. The protein was concentrated by precipitation with 40% ammonium sulphate, disolved in a minimum quantity of 0.1M sodium phosphate pH 7.6 and dialysed to give the oligomeric allergen. To make monomers, the protein was disolved in 4% (w/v) SDS and 10mM mercaptoethanol, heated to 98C for 10 min and applied to Sephacryl S200 equilibrated in 0.1M sodium phosphate pH7.6 containing 4% (w/v) SDS and 10mM mercaptoethanol (Ogawa et al 1993 [131]).
• Other biochemical information:

  Gly m Bd 30k has also been called Gly m I by Hessing et al. (1996) [656]. However, Gonzalez et al. (1992) [657] used Gly m 1A and Gly m 1B to refer to two different small aeroallergens from soybean.

  Herman et al. (2003) [1082] have engineered the removal of Gly m Bd 30k from soybeans.
  Usui et al. (2004) [1073] have made a protein-chitosan conjugate by the Maillard reaction which showed no allergenicity by immunoblotting.

References (12)

• Gonzalez R, Polo F, Zapatero L, Caravaca F, Carreira J.
  Purification and characterization of major inhalant allergens from soybean hulls.
  Clin Exp Allergy. 22(8):748-55.. 1992
  PUBMEDID: 1525693
• Hessing M; Bleeker H; Tsuji H; Ogawa T; Vlooswijk RAA
  Comparison of human IgE-binding soya bean allergenic protein Gly m I with the antigenicity profiles of calf anti-soya protein sera.
  FOOD AND AGRICULTURAL IMMUNOLOGY 8, Iss 1, 51-58. 1996
  PUBMEDID:
• Bando N, Tsuji H, Yamanishi R, Nio N, Ogawa T.
  Identification of the glycosylation site of a major soybean allergen, Gly m Bd 30K
  Biosci Biotechnol Biochem. 60(2):347-348.. 1996
  PUBMEDID: 9063988
• Kalinski A, Weisemann JM, Matthews BF, Herman EM.
  Molecular cloning of a protein associated with soybean seed oil bodies that is similar to thiol proteases of the papain family.
  J Biol Chem. 265(23):13843-8.. 1990
  PUBMEDID: 2380191
• Kalinski A, Melroy DL, Dwivedi RS, Herman EM.
  A soybean vacuolar protein (P34) related to thiol proteases is synthesized as a glycoprotein precursor during seed maturation.
  J Biol Chem. 267(17):12068-76.. 1992
  PUBMEDID: 1601876
• Ogawa T, Tsuji H, Bando N, Kitamura K, Zhu Y-L, Hirano H, Nishikawa K
  Identification of the soyabean allergenic proteins, Gly m Bd 30K , with the soybean seed 34kDa oil-body-associated protein.
  Biosci Biotech Biochem 57:1030-1033.. 1993
  PUBMEDID: 7763872
• Ji C; Boyd C; Slaymaker D; Okinaka Y; Takeuchi Y; Midland SL; Sims JJ; Herman E; Keen N
  Characterization of a 34-kDa soybean binding protein for the syringolide elicitors
  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 95, Iss 6, 3306-3311. 1998
  PUBMEDID: 9501258
• Helm R, Cockrell G, Herman E, Burks A, Sampson H, Bannon G
  Cellular and molecular characterization of a major soybean allergen.
  Int Arch Allergy Immunol 117:29-37.. 1998
  PUBMEDID: 9751845
• Helm RM, Cockrell G, Connaughton C, West CM, Herman E, Sampson HA, Bannon GA, Burks AW.
  Mutational analysis of the IgE-binding epitopes of P34/Gly m Bd 30K.
  J Allergy Clin Immunol. 105: 378-84. 2000
  PUBMEDID: 10669862
• Usui M, Tamura H, Nakamura K, Ogawa T, Muroshita M, Azakami H, Kanuma S, Kato A.
  Enhanced bactericidal action and masking of allergen structure of soy protein by attachment of chitosan through maillard-type protein-polysaccharide conjugation.
  Nahrung 48(1):69-72.. 2004
  PUBMEDID: 15053356
• Herman EM, Helm RM, Jung R, Kinney AJ.
  Genetic modification removes an immunodominant allergen from soybean.
  Plant Physiol. 132(1):36-43.. 2003
  PUBMEDID: 12746509
• Samoto M, Miyazaki C, Akasaka T, Mori H, Kawamura Y.
  Specific binding of allergenic soybean protein Gly m Bd 30K with alpha'- and alpha-subunits of conglycinin in soy milk.
  Biosci Biotechnol Biochem. 60(6):1006-1010.. 1996
  PUBMEDID: 8695899

Biochemical Information for Glycinin

• Allergen Name:Glycinin
• Alternatve Allergen Names:None
• Allergen Designation:Major
• Protein Family:Pfam PF00190; Cupin; 11S seed storage globulin
• Sequence Known?:Yes
• Allergen accession No.s:

  P04776 http://ca.expasy.org/cgi-bin/niceprot.pl?P04776
  P04405 http://ca.expasy.org/cgi-bin/niceprot.pl?P04405
  P11828 http://ca.expasy.org/cgi-bin/niceprot.pl?P11828
  P02858 http://ca.expasy.org/cgi-bin/niceprot.pl?P02858
  P40437 http://ca.expasy.org/cgi-bin/niceprot.pl?P40437

• 3D Structure Accession No.:1FXZ (proglycinin trimer)
  1OD5 (homohexamer)
• Calculated Masses:

  55706 Da
  53606 Da without signal

• Experimental Masses:20 kDa and 33 kDa by SDS-PAGE after reduction
• Oligomeric Masses:322 kDa
• Allergen epitopes:

  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)

• 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
  PUBMEDID: 12485602
• 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
  PUBMEDID: 12771376
• Mills EN, Marigheto NA, Wellner N, Fairhurst SA, Jenkins JA, Mann R, Belton PS
  Thermally induced structural changes in glycinin, the 11S globulin of soya bean (Glycine max)--an in situ spectroscopic study.
  Biochim Biophys Acta 1648(1-2):105-14. 2003
  PUBMEDID: 12758153
• Helm RM, Cockrell G, Connaughton C, Sampson HA, Bannon GA, Beilinson V, Nielsen NC, Burks AW.
  A soybean G2 glycinin allergen. 2. Epitope mapping and three-dimensional modeling.
  Int Arch Allergy Immunol 123:213-9.. 2000
  PUBMEDID: 11112857
• Helm RM, Cockrell G, Connaughton C, Sampson HA, Bannon GA, Beilinson V, Livingstone D, Nielsen NC, Burks AW
  A soybean G2 glycinin allergen. 1. Identification and characterization.
  Int Arch Allergy Immunol 123:205-12.. 2000
  PUBMEDID: 11112856
• Eldridge AC, Wolf WJ
  Purification of the 11S component of soybean protein.
  Cereal Chem 55:16-22.. 1967
  PUBMEDID:
• Beardslee, T.A., Zeece, M.G., Sarath, G., Markwell, J.P.
  Soybean glycinin G1 acidic chain shares IgE epitopes with peanut allergen Ara h 3
  Int. Arch. Allergy Immunol. 123:299-307.. 2000
  PUBMEDID: 11146387
• Adachi M, Takenaka Y, Gidamis AB, Mikami B, Utsumi S.
  Crystal structure of soybean proglycinin A1aB1b homotrimer.
  J Mol Biol 305:291-305.. 2001
  PUBMEDID: 11124907
• 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
  PUBMEDID: 10021462
• Lalles, J.P.; Tukur, H.M.; Salgado, P.; Mills, E.N.C.; Morgan, M.R.A.; Quillien, L.; Levieux, D.; Toullec, R.
  Immunochemical studies on gastric and intestinal digestion of soybean glycinin and beta-conglycinin in vivo.
  J. Agric. Food Chem. 47, 2797-2806.. 1999
  PUBMEDID: 10552568
• 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
  PUBMEDID:

Biochemical Information for Trypsin inhibitor

• Allergen Name:Trypsin inhibitor
• Alternatve Allergen Names:None
• Allergen Designation:Minor
• Protein Family:Pfam PF00197; Kunitz_legume.
• Sequence Known?:Yes
• Allergen accession No.s:P01070 http://ca.expasy.org/cgi-bin/niceprot.pl?P01070
• 3D Structure Accession No.:

  1BA7
  1AVU
  1AVW
  1AVX

• Calculated Masses:20094.68 Da (mature)
• Experimental Masses:20 kDa
• Oligomeric Masses:None
• Allergen epitopes:Not Known
• Allergen stability:
  Process, chemical, enzymatic:Trypsin inhibitors are usually destroyed by thermal treatments which form the basis for destroying the anti-nutritional effects of the protease inhibitors. They are resistant to digestion. There is little loss of protein with simulated gastric fluid for 60 minutes and the protein refolds after heating to 90°C (Roychaudhuri et al. 2004 [1072]). However, reduction leads to denaturation and rapid proteolysis.
• Nature of main cross-reacting proteins:None identified.
• Allergen properties & biological function:The allergen corresponds to the soya bean trypsin inhibitors A and C. These inhibitors are plant protective proteins and may be involved in inhibiting microbial and fungal proteases.
• Allergen purification:The inhibitors can be extracted in 1M NaCl containing 8M urea for 4h at 4°C. The extracts were loaded onto a Superdex 75 column to isolate the 20 kDalton protein (after Burks et al 1994 [28]).
• Other biochemical information:

  Trypsin inhibitor has been detected in soy lecithin (Gu et al. 2001 [654]).

References (3)

• Gu X, Beardslee T, Zeece M, Sarath G, Markwell J.
  Identification of IgE-binding proteins in soy lecithin.
  Int Arch Allergy Immunol. 126(3):218-25.. 2001
  PUBMEDID: 11752879
• Burks AW, Cockrell G, Connaughton C, Guin J, Allen W, Helm RM
  Identification of peanut agglutinin and soyabean trypsin inhibitor as minor legume allergens.
  Int. Arch. Allergy Immunol. 105:143-149.. 1994
  PUBMEDID: 7920014
• Roychaudhuri R, Sarath G, Zeece M, Markwell J.
  Stability of the allergenic soybean Kunitz trypsin inhibitor.
  Biochim Biophys Acta 1699(1-2):207-212.. 2004
  PUBMEDID: 15158729