Soyygbean Aller gens Characterization and Remo … GleddieSaskatoon10.pdfSoyygbean Aller gens...
Transcript of Soyygbean Aller gens Characterization and Remo … GleddieSaskatoon10.pdfSoyygbean Aller gens...
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Soybean Allergensy gCharacterization and Remo alRemovalSteve Gleddie, Christine Gagnon, Elroy Cober Vaino Poysa (CElroy Cober, Vaino Poysa (C.
Zarkadas) AAFC
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OutlineOutline
Brief review of food allergiesBrief review of food allergies
Soybean proteomicsSoybean proteomics
Strategies to eliminate allergens from soy
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What is a Food Allergy?
An immunological reaction (IgE)An immunological reaction (IgE) resulting from the ingestion, inhalation, or atopic contact with a food or food ingredientg
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Hypersensitized Individual (follows 1st exposure)(follows 1 exposure)
Second Exposure to Food/Protein
Activated Mast CellsAdverse Reactionibl React to Food/Proteinpossible
Anaphylaxis
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IgE-Mediated Hypersensitivityg yp y
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Most common allergenic foods
•Eggs 25%
(1992 US study on children with food allergies)
•Eggs 25%•Peanuts 24%•Milk 23%•Tree nuts 10%•Soybeans 6%Fish 3%•Fish 3%
•Wheat 2.5%
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S b d t t
40% Protein
Soybean seed content
• 40% Protein
• 20% Oil20% Oil
• 12% Carbohydrates
• Other (tannins, phytates,
phytoestrogens, oligosaccharides
saponins)saponins)Photo: Kansas State University
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Soy in the food industryy y
S i• Soy presence in foods increasing every year
• Health benefits• Soy prevalence
increases chances of exposure (allergic reactions)( g )
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Soybean proteins
Globulins
Albumins
Globulins
G S b it3 G
Glycinin (11S)β-Conglycinin (7S)
Gene SubunitsG1 A1aB2G2 A2B1a
3 Genes(α, α’ and βsubunits) G2 A2B1a
G3 A1bB1bG4 A5A4B3
subunits)
G5 A3B4
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cCal
l
AC S
corp
io
icen
tenn
ial
G20
G30
G41
aple
Arro
w
C B
ravo
r
AC F
ront
ier
OA
C L
ibra
C H
arm
ony
S 30
11.2
4
aple
Am
ber
arly
Hak
ucho
10%
50ugMarch09-04
Mc
OA Bi KG KG KG Ma
AC OA O AC QS
Ma
Ea 10%
97
66
Lipoxygenaseα’α
45β
A3
31
A3
A1 A2 A4
Gly m Bd 30K (P34)l ti31
B3
lectins
21 B1 B2 B4Kunitz
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2D gel electrophoresis
β97
g p
A3A4 A2
βα1α66
45
A131
KTI
A1
B1,2,3,4
21
4 7HAROVINTON
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Identification of glycinin and β-conglycinin subunits
A1A2A4A5 NULL KTI NULLA4A5 NULLα'A3 NULL α'A2 NULL
A3A4 A2
βα
Ια
75
50
37
A1A2A3A4A5 NULL HAROVINTON
KTI
A125
20
P34
α' A1A2A3A4A5 NULL4 7HAROVINTON
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Identification of basic glycinin chainsIdentification of basic glycinin chains
6 11 6 11
976645
976645
B431
21
31
Harovinton
21
A3 B4 null
21
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Protein Quality of null SoybeansZarkadas et al 2007. Food Res Intl.
• Total protein 33 37%• Total protein 33 – 37%• EAA9 44.5 – 47.3%9
• Physiologically normal seeds, l tplants
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MethodologySample preparation
1D or 2D gel electrophoresis1D or 2D gel electrophoresis
Transfer to PVDF
Incubate with patient serum O/N
Incubate with anti-human IgE-HRP
Chemiluminescent detectionChemiluminescent detection
Phosphorimager scan
Spot pick for MS
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Characteristics of patients allergic to soybean
Code Sex Age RAST SymptomsLM F 23 68x1 laryngeal edema, bronchospasm, hives, itching, abdominal pain.
Also allergic to peanuts
CU F 42 66x hives, asthma, watery eyes, sneezing, rhinitis. Lost allergy to peanut.
CS F 25 5x respiratory difficulty bronchoconstriction wheezing AlsoCS F 25 5x respiratory difficulty, bronchoconstriction, wheezing. Also allergic to peanuts
RC M 54 3x hives, localized swelling, bronchoconstriction, metallic taste. Not allergic to peanuts
08 M 28 ND h l i h i li d it th t ti ht08 M 28 ND anaphylaxis, wheezing, generalized pruritus, throat tightness, redness, chest tightness. Also allergic to peanuts
DB M 42 34.8kUA/L anaphylaxis, blood pressure increase. Not allergic to peanuts
DP F 25 NA anaphylaxis, throat closure, total swelling of eyes, hives, 5 a ap y a s, oa c osu e, o a s e g o eyes, es,vomiting. Also allergic to peanuts
AG F 32 3.18kUA/L breathing trouble, nasal congestion. Not allergic to peanuts.
HS F 31 0.6kUA/L eczema. Not allergic to peanuts
CC F 35 11x hives, swelling, difficulty breathing, diarrhea, lip edema. Also allergic to peanuts
1 Internal RAST score. Indicates number of times great the counts were compared to negative serum
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Patient reactions to soy proteinsy p
37
7550
25
15
20
LM1/400
DB1/800
DP1/1200
AG1/1000
CC1/400
HS1/25
081/100
CS1/100
RC1/100
CU1/100
15%
Anti-human IgE-HRP 1/3000
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Screening of glycinin null lines ith LMwith LM serum
345-
3- 45-
345-
3- 45-
345-
345-
75
A3-
A3-
A45
-
A2- A12
3
A12
3
A12
4
A12
3
+ A3- A3-
A45
-
A2- A12
3
A12
4
A12
3
+A12
3
75
3750
25
A375
3750
2520
Coomassie stained PVDF membrane12.5%
Western blotLM 1/400
Anti-human IgE-HRP 1/2500g
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Patient reactions to soy proteinsy p
37
7550
Glycinin A3
25
15
20
LM1/400
DB1/800
DP1/1200
AG1/1000
CC1/400
HS1/25
081/100
CS1/100
RC1/100
CU1/100
15%
Anti-human IgE-HRP 1/3000
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Allergen identification by 2D gel
α subunit of β l i i
β subunits of β l i i
α subunit of β
β subunits of β l i i
g y g
75
β-conglycinin β-conglycinin
4 7
75
β-conglycinin β-conglycinin
4 7
755037
25
755037
25 252520
15
2-D gel of soybean12.5%
Western BlotCS 1/100
A ti h I E HRP 1/2000Anti-human IgE-HRP 1/2000
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β-conglycinin sequence homologyβ g y q gy
alpha 605 aa
alpha’
83%
621 aap
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Patient reactions to soy proteinsy p
β l i i ( )
37
7550
Glycinin A3
β-conglycinin(α)β-conglycinin(β)
25
15
20
LM1/400
DB1/800
DP1/1200
AG1/1000
CC1/400
HS1/25
081/100
CS1/100
RC1/100
CU1/100
15%
Anti-human IgE-HRP 1/3000
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Allergen identification by 2D gel6 11
37
25
7550
6 11
2D gel15
20
10
2D gel
6 11 6 117550
7550 50
2520
3750
2520
37
Western Blot DP 1/800Anti-human IgE 1/2500
Western Blot CU 1/100Anti-human IgE 1/2500
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Allergen identification by 2D gelAllergen identification by 2D gel
6 11 6 112520
2520
10
15
10
15
10 10
Western BlotRC 1/100
Anti-human IgE 1/2500
Coomassie stained PVDF membrane
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Tandem MS result for the spot reacting to RC DP CUspot reacting to RC DP CU
Protein candidate Mass (kDa) / pI
Peptide sequences Mascot Score
seed maturation protein PM30(4838147 –NCBI)
15.09 / 8.95 -GAAQGATEAVK-TNQTMGNIGEK-TNQTMGNIGEK + Ox. of M-DNTQGFLQQTGEK
314
-QTLGLGEHDQDNR-QTLGLGEHDQDNRR-ESAQSGKDNTQGFLQQTGEK
LEA (Late embryonic abundant) group III
MASHRQSYEAGQTKGRTEEKTNQTMGNIGEKAQAAKEKTQEMAQAAKEKTQQTAQAAKDKTCDTSQAAKEKTQQNTGAAQQKTSEMGQSTKESAQSGKDNTQGFLQQTGEKVKGAAQGATEAVKQTLGLGEHDQDNRRNY Q QQ Q Q Q
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Epitope Mapping of LEA protein
MASHRQSYEAGQTKGRTEEKTNQTMGNIGEKAQAAKEKTQEMAQAAKEKTQQTAQAAKDKTCDTSQAAKEKTQQNTGAAQQKTSEMGQAAKEKTQQTAQAAKDKTCDTSQAAKEKTQQNTGAAQQKTSEMGQSTKESAQSGKDNTQGFLQQTGEKVKGAAQGATEAVKQTLGLGEHDQDNRRNY
NCBI AF117884
ELISA results for PK-2 human serum
Peptide Sequence OD450p q
49 GKDNTQGFLQQT 1.99350 DNTQGFLQQTGE 0.71851 TQGFLQQTGEKV 0 70951 TQGFLQQTGEKV 0.709
Background 0.060
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LM DB DP AG HS CC 08 CS RC CU Gel
Canadian patient reactions against soybean proteinsLM DB DP AG HS CC 08 CS RC CU Gel
37kDa
75kDa50kDa 7
9
811
37kDa
25kDa
96
45
15kDa
20kDa
21
1/400 1/1200 1/25 1/50 1/1001/1000 1/1000 1/200 1/100 1/200
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100kDaMH SN CS-2 RB LN RM CC Gel
American patient reactions against soybean proteins
75kDa100kDa
118
50kDa7
910
8*11
37kDa
6
9
25kDa 134
5
20kDa 12
43
1/800 1/1600 1/8001/800 1/2000 1/400 1/200
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Frequency of occurrence, potential identity, sizeand isoelectric point (pI) of soy allergens.
6 117 7Heat Shock Protein (HSP)0 041
pISize (kDa)
IdentityFreq. in Patients
Sample
6.1
8.9
8.0
17.7
15.1
20.5
Heat Shock Protein (HSP)
Late Embryonic Abundant (LEA)
Glycinin G1 precursor (Bx)
0.04
0.15
0.42
1
2
3
5.9
5.6
4.9
24.5
30.9
31.6
Gly m Bd 28k (N-term)
Lectin
Glycinin G2 precursor (A2)
0.50
0.50
0.54
4
5
6
5.7
4.9
4.9
48.0
63.2
36.4
β-conglycinin (β)
β-conglycinin (α)
Glycinin G5 (A3)
0.50
0.31
0.80
7
8
9
8.7
6.3
8.0
46.4
56.0
20.3
Basic 7S globulin precursor (SBg7S)
Sucrose binding protein homolog (S-64)
Glycinin G2 precursor (B1a)
0.08
0.19
0.04
10
11
12
5.927.2Triosephosphate isomerase (TIM)0.0413
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Soy in the feed industryy y
• Piglets are allergic to• Piglets are allergic to soybean for up to one month after weaningg
• Costly and potentially dangerous animal-b d i i f dbased protein is fed
• Industry would benefit significantly fromsignificantly from cheaper feed costs and safer feed practices
Picture painted by Ron Kimball
p
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Piglet studySelect 18 pigs at weaning (18 days, 5.5kg)
Blood sample (negative)
fed 31% soybean meal fed regular post-weaning diet
Monitor pigs (symptoms, weekly body weight)
Blood samples on day 7 and 14 post-weaning
Screen 1D/2D soy containing gels with pig serum
HTS 15000 mutagenized soybean seedsg y
Pig feed trial with null lines identified
Create multiple “hypoallergenic” null lines by breedingCreate multiple hypoallergenic null lines by breeding
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MW Experimental group of Piglets (#) Negative Control group of Piglets (#)
Marker 2 11 1 3 4 6 5 7 8 9 10 12 17 13 14 15 16 18
50kDa
75kDa
Marker 2 11 1 3 4 6 5 7 8 9 10 12 17 13 14 15 16 18
1 1110
7
823 9
100kDa150kDa
1
37kDa
25kDa
20kDa
45
66
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Hypoallergenic SoybeanHypoallergenic Soybean
• Identify major allergenic proteins• Identify major allergenic proteins
• Raise antisera to each
• Protein arrays or HT mutant screens
Id tif li l ki ifi ll• Identify lines lacking specific allergens
• Cross lines to develop multiple knock-outsp p
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Potential OutcomesPotential Outcomes
• New Canadian cultivars with added-valueNew Canadian cultivars with added value• Reduced risk to consumers• Broader knowledge base of allergens• Broader knowledge base of allergens
(epitopes, expression, digestion )N l d t ti th d (MS b d)• Novel detection methods (MS-based)
• Regulatory data for novel foods• Safer feed supply for animals
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References:
Gagnon, Poysa, Cober, Gleddie 2009. Food Analytical Methods
Zarkadas,Gagnon,Gleddie,Khanizadeh,Cober,Guillemette 2007 Food Res Intluillemette 2007. Food Res Intl.
Zarkadas,Gagnon,Poysa,Khanizadeh,Cober,Zarkadas,Gagnon,Poysa,Khanizadeh,Cober, Chang, Gleddie 2007. Food Res Intl.
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Challenges & OpportunitiesChallenges & Opportunities
• Extend basic knowledge of food allergensExtend basic knowledge of food allergens• Improve analytical/detection methods• Faster more sensitive diagnostics• Faster, more sensitive diagnostics• Develop capacity to predict/regulate novel
f dfoods• Science capacity for food safety issues• Develop a “Branded” Canadian-made
hypoallergenic food product
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Acknowledgmentsg
•Drs. Sue Hefle, SteveTaylor (patient serum, U of N)•Drs. Vaino Poysa, Elroy Cober (glycinin/β-y , y (g y βconglycinin nulls, EMS mutants, breeding, AAFC)•Dr. Kees de Lange, Bruce Wilkie (pig study, U of G)g (p g y )