Soybean Allergensy gCharacterization and Remo alRemovalSteve Gleddie, Christine Gagnon, Elroy Cober Vaino Poysa (CElroy Cober, Vaino Poysa (C.

Zarkadas) AAFC

OutlineOutline

Brief review of food allergiesBrief review of food allergies

Soybean proteomicsSoybean proteomics

Strategies to eliminate allergens from soy

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

Hypersensitized Individual (follows 1st exposure)(follows 1 exposure)

Second Exposure to Food/Protein

Activated Mast CellsAdverse Reactionibl React to Food/Proteinpossible

Anaphylaxis

IgE-Mediated Hypersensitivityg yp y

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%

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

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 )

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

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

2D gel electrophoresis

β97

g p

A3A4 A2

βα1α66

45

A131

KTI

A1

B1,2,3,4

21

4 7HAROVINTON

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

Identification of basic glycinin chainsIdentification of basic glycinin chains

6 11 6 11

976645

976645

B431

21

31

Harovinton

21

A3 B4 null

21

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

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

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

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

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

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

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

β-conglycinin sequence homologyβ g y q gy

alpha 605 aa

alpha’

83%

621 aap

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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 )