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식품면역연구회 2012.03.09
대두, 메밀 알레르겐의 특성분석
이화여자대학교
오상석
Food Allergy
 “An immunological (IgE mediated) adverse reaction to a food”
Stefano Luccioli, MD(Office of Food Additive Safety, FDA), 2007
2
Food Allergy Mechanism
step 1 (Sensitization) : (1) IgE production
step 3 (Elicitation) :
Mediator release
Food protein(1) / pollen (2)
B cell
T cell
IgE
Antibody
Specific to food or pollen
step 1 (Sensitization) :
(2) Priming
IgE
Antibody
FcєR1
(IgE receptor)
Mast cell/ Basophil
step 2 (Challenge) :
Receptor crosslinking
Food
Mediators: Histamine,
leukotrienes, prostaglandins,
cytokines, etc.
Effects: blood vessel leakage and
dilation, smooth muscle
contraction, nerve irritation, etc.
Symptoms
Stefano Luccioli, MD(Office of Food Additive Safety, FDA), 2007
3
각국의 알레르기 유발 표시 식품
한국
EU
미국
일본
난류(가금류)
곡류 (글루텐)
우유
우유
난류
우유
갑각류
난류
메밀
땅콩
메밀
난류
생선
대두
호두
땅콩
어류
갑각류
밀
전복
대두
유제품
견과류
오징어
연어
밀
대두
밀
연어알젓
게
고등어
너트류
땅콩
새우,
고등어
게
샐러리
대두
쇠고기
닭고기
돼지고기
겨자
아황산염
돼지고기
오렌지
복숭아
참깨
키위
송이
토마토
연체동물류
복숭아
버섯
새우
루핀
사과
참마
아황산염
4
Classification of storage protein
• Based on extraction and solubility
- albumin in water
- globulin in saline
- prolamine in alcohol/water
- glutelin in dilute acid or alkali
(Osborne, 1924)
2S albumin
• Defined as a group of the basis of their
sedimentation coefficients (S20) of ~2
(Youle and Huang, 1981)
All the 2S albumins are compact globular
proteins with conserved cysteine residues
(Shewry et.al. 1995)
7S globulin
7S: trimeric proteins ~140 – 170 kDa
difficult to purify and characterize
extensive glycosylation
11S globulin proteins
11S :
six subunit pairs
each subunit:
acidic subunit (~40 kDa) +
basic subunit (~20 kDa)
(linked by a disulfide bond)
대두 단백질의 조성
분획
2S
7S
함량(%)
10~20
30~35
단백질성분
Trypsin inhibitor (Bowman-Birk)
7,900
Cytochrome C
12,500
Trypsin inhibitor (Kunitz)
21,500
Enzymes
15,000~60,000
Enzymes
70,000~240,000
Lipoxygenase
100,000
7S Globulin(γ-conglycinin)
104,000
Agglutinins
110,000
7S Globulin(β-conglycinin)
11S
15S
30~50
5~10
분자량
140,000~170,000
11S Globulin(glycinin)
350,000
Urease
480,000
15S Globulin
600,000
(김우정, 2006)
9
대두 단백질 주요 알레르겐
Protein/Glycoproteins
Allergen
Nomenclature
% of the total
protein of soya
Molecular mass
(Da)
Soybean Hydrophobic Protein
Gly m 1.0101,
Gly m 1.0102
7500
Soybean Hull Protein
Gly m 2
8000
Soybean Profilin
Gly m 3
14000
Soybean Vacuolar Protein
Comments
~34000
Glycinin
31~38
320000~360000
11S
β-conglycinin
17~21
140000~180000
7S
2
21500
2S
Kunitz-Trypsin Inhibitor
(Cordle, C.T., 2004)
10
7S globulin과 가수분해물의 전기영동적 특성
 SDS-PAGE gel of 7S-globulin and peptic and
chymotryptic hydrolysates of 7S globulin
1. 7S globulin with 2-ME
2. 7S globulin without 2-ME
3. 11S globulin with 2-ME
4. 11S globulin without 2-ME
5. 7S hydrolysate by peptic hydrolysis
6. 7S hydrolysate by chymotryptic
hydrolysis
11
11S globulin과 가수분해물의 전기영동적 특성
 SDS-PAGE gel of 11S globulin and peptic and
chymotryptic hydrolysates of 11S globulin
MM : Molecular Marker
1 : 11S globulin with 2-mercaptoethanol
2 : 11S globulin without 2-mercaptoethanol
3 : 11S globulin hydrolysate by peptic hydrolysis
4 : 11S globulin hydrolysate by peptic and
chymotryptic hydrolysis
12
7S와 11S globulin의 allergenicity
 Allergenicity of soybean 7S globulin and 11S globulin
CAP IgE FEIA
Patient
Age
No.
(yr)
soybean IgE
4
(kUA/L)
1
0.63
2
2
1.01
3
3
101
4
8
2.06
5
3
17.6
6
3
0.52
7
2
22.2
8
2
16.5
9
3
101
10
4
7.84
3.5
Absorbance at 450nm
1
3
2.5
Blank
7S
11S
2
1.5
1
0.5
0
정상
혈청1 혈청2 혈청3 혈청4 혈청5 혈청6 혈청7 혈청8 혈청9 혈청10
13
7S와 11S globulin의 allergenicity
 Allergenicity of soybean 7S globulin and 11S globulin
4
Age
CAP IgE FEIA
No.
(y)
soybean IgE(KUA/L)
1
9
0.35
2
2
0.36
3
9
18.4
4
4
12
5
4
3.7
6
1
2.39
3.5
3
Absorbance at 450nm
Patient
2.5
11S
7S
2
1.5
1
0.5
0
정상
혈청1
혈청2
혈청3
혈청4
혈청5
혈청6
14
가수분해에 따른 7S globulin의 allergenicity변화
 Effects of peptic and sequential peptic and
chymotryptic hydrolysis of 7S globulin on allergenicity
4
Blank
Absorbance at 450nm
3.5
3
7S globulin
2.5
Peptic
hy droly s ate
2
Peptic &
Chy motry ptic
hy droly s ate
1.5
1
0.5
0
정상
혈청1
혈청2
혈청3
혈청4
혈청5
혈청6
혈청7
혈청8
혈청9 혈청10
15
가수분해에 따른 11S globulin의 allergenicity변화
 Effects of peptic and sequential peptic & chymotryptic
hydrolysis of 11S globulin on allergenicity
4
3.5
11S globulin
Absorbance at 450nm
3
2.5
Peptic
hydrolysate
2
1.5
Peptic &
Chymotryptic
hydrolysate
1
0.5
0
정상
혈청1
혈청2
혈청3
혈청4
혈청5
혈청6
16
7S globulin 가수분해물의 allergenic fraction 분리
 Fractionation on Sephacryl S-100 HR of 7S hydrolysates
by peptic & chymotryptic hydrolysis and SDS-PAGE of each fraction
17
7S globulin 가수분해물의 allergenic peptides 확인
 Allergenicity of fractions by sequential peptic &
chymotryptic hydrolysis of soybean 7S globulin
18
11S globulin 가수분해물의 allergenic fraction 분리
 Fractionation on Sephacryl S-100 HR of 11S hydrolysates
by peptic & chymotryptic hydrolysis and SDS-PAGE of each fraction
0.14
0.12
F
G
Absorbance at 280nm
0.1
0.08
0.06
0.04
B
0.02
A
A
9
17
C
D
H
E
I
0
1
25
33
41
49
57
65
73
81
89
97
105 113
-0.02
Fraction number
19
11S globulin 가수분해물의 allergenic peptides 확인
 Allergenicity of fractions by sequential peptic &
chymotryptic hydrolysis of soybean 11S globulin
3.5
Absorbance at 450nm
3
2.5
정상
혈청1
혈청2
혈청3
혈청4
혈청5
혈청6
2
1.5
1
0.5
0
A
B
C
D
E
F
G
H
I
-0.5
Fraction number
20
soybean Kunitz trypsin inhibitor
www.allergom.org
21
soybean Kunitz trypsin inhibitor
 The sequence of Kunitz trypsin inhibitor (Koide & Ikenaka, 1976)
Asp-Phe-Val-Leu-Asp-Asn-Glu-Gly-Asn-Pro-Leu-Glu-Asn-Gly-Gly-Thr-Tyr-Tyr-Ile-Leu-Ser-Asp-Ile-Thr-Ala-PheGly-Gly-Ile-Arg-Ala-Ala-Pro-Thr-Gly-Asn-Glu-Arg-Cys-Pro-Leu-Thr-Val-Val-Gln-Ser-Arg-Asn-Glu-Leu-Asp-LysGly-Ile-Gly-Thr-Ile-Ile-Ser-Pro-Ser-Tyr-Arg*-Ile*-Arg-Phe-Ile-Ala-Glu-Gly-His-Pro-Leu-Ser-Leu-Lys-Phe-Asp-Ser-Phe-Ala-Val-Ile-Met-Leu-Cys-Val-Gl
y-Ile-Pro-Thr-Glu-Trp-Ser-Val-Val-Glu-Asp-Leu-Pro-Glu-Gly-Pro-Ala-Val-LysIle-Gly-Glu-Asn-Lys-Asp-Ala-Met-Asp-Gly-Trp-Phe-Arg-Leu-Glu-Arg-Val-Ser-Asp-Asp-Glu-Phe-Asn-Asn-Tyr-LysLeu-Val-Phe-Cys-Pro-Gln-Gln-Ala-Glu-Asp-Asp-Lys-Cys-Gly-Asp-Ile-Gly-Ile-Ser-Ile-Asp-Asp-Asp-Gly-His-Thr-Arg-Arg-Leu-Val-Val-Ser-Lys-Asn-LysPro-Leu-Val-Val-Gln-Phe-Gln-Lys-Leu-Asp-Lys-Glu-Ser-Leu
* Arg63-Ile64 : active center
 Inhibit trypsin > chymotrypsin, plasmin
 181 amino acids
 Single polypetide
 Linked by 2 disulfide bonds
 Synonyms : Tia1, STI, SBTI, Gly m TI
 pI : 4.5
22
Stability of the allergenic soybean Kunitz trypsin inhibitor
 SGF proteolysis of native and reduced
SKTI for 5 s, 2, 20 and 30min as
analyzed by SDS-PAGE
1. Panel A : native SKTI
2. Panel B : SKTI with 30mM 2-mercaptoethanol and SGF
3. Panel C : SKTI with 30mM dithiothreitol and SGF
(Biochimica et Biophysica Acta 1699 (2004) 207-212)
 In vitro digestibility of the soybean extract
in SGF
* Lane U represents protein samples without treatment
with SGF.
(Int Arch Allergy Immunol 2006 ; 141 : 91-102)
23
Stability of the allergenic soybean Kunitz trypsin inhibitor
 2S globulin의 주요 알레르겐인 soybean Kunitz trypsin inhibitor(SKTI)는 가열,
pepsin, chymotrypsin에 비교적 안정
 11S, 7S, 2S globulin이 대두의 주된 알레르기 관련 단백질임
24
Enzymatic hydrolysis of 7S, 11S
 7S globulin (10개 혈청)
- pepsin 가수분해보다 pepsin 처리 후 chymotrypsin처리 한 경우 알레르기성이 일
부 증가
 11S globulin (6개 혈청)
- 환자 혈청 6개 모두에서 효소 가수분해물의 알레르기성이 유의적으로 감소
25
대두단백질 알레르겐 저감화
번호
가열
제목
1 열처리에 따른 콩 단백질 Allergenicity 변화 (2000)
2 Allergenicity of major component protein of soybean (1980)
3 Allergenicity of proteolytic hydrolysates of the soybean 11S globulin* (2007)
protease
4
Effect of Enzymatic Hydrolysis of 7S Globulin, a Soybean Protein, on Its Allergenicity and Identification of its Allergenic Hydrolyzed
Fragments Using SDS-PAGE* (2006)
5 Preparation of Hypoallergenic Soybean Protein with Processing Functionality by Selective Enzymatic Hydrolysis (1999)
6 Reduction of the allergenicity of soybean by treatment with preteases (1996)
7 발효를 이용한 allergenicity 감소 - 발효 과정에 따른 대두의 알레르기성 분석 (2008)
8 Immunoreactivity and Amino Acid Content of Fermented Soybean Products (2008)
발효
9
Immunoreactivity reduction of soybean meal by fermentation, effect on amino acid composition and antigenicity of commercial
soy products (2008)
10 Reduction of the Soybean Allergenicity by the Fermentation with Bacillusnatto (1995)
11 Soybean allergens and hypoallergenic soybean products (2000)
유전자 조작
12
Substantially Complete removal of Three Major Allergenic Soybean Proteins from Soy Protein by Using a mutant soybean,
Tohoku 124 (1997)
13 Genetic Modification Removes an Immunodominant Allergen from Soybean (2003)
비가열처리
비가열처리
+ 효소
glycosylation
salting out
+ centrifugation
14 Use of the pulsed ultraviolet light to reduce the allergenic potency of soybean extracts (2010)
15
16
Assessment of the residual immunoreactivity of soybean whey hydrolysates obtained by combined enzymatic proteolysis and
high pressure (2005)
Effect of polysaccharide conjugation or transglutaminase treatment of the allergenicity and functional properties of soy protein
(1998)
17 Simple and efficient procedure for removing the 34kDa allergenic soybean protein, Gly m I, from defatted soy milk (1994)
26
메밀의 구성
메밀 종실의 조단백질 함량과 아미노산 조성
단백질 및 아미노산
최고치(%)
최저치(%)
평균(%)
CV(%)
단백질
15.4
12.6
13.7
6.2
리신(Lys)
7.0
5.0
6.1
7.5
트레오닌(Thr)
4.1
3.6
3.9
3.5
메티오닌(Met)
3.0
1.8
2.5
13.5
Lys + Thr + Met
13.0
11.7
12.5
3.1
27
메밀 알레르기
 임상증세
 메밀 알레르기 임상 연구
표1. 502명의 알레르기 환자의 알레르기 식품 분석(손대열 et al., 2002).
•
두드러기
•
부풀어오름
•
Allergic rhinoconjunctivitis
•
호흡곤란
•
아낙필락시스
순위
식품
알레르기 증상 환자(%)
1
egg white
32.9
2
cow milk
26.0
3
egg yolk
9.3
4
soybean
7.4
5
buckwheat
3.8
6
wheat
1.2
7
beef
<1
8
crab
<1
9
pork
<1
10
chicken
<1
28
buckwheat allergens
분자량
특징
10kDa
•
•
identical to 8kDa buckwheat allergen
a member of the 2S albumin family
16kDa
•
•
pepsin-resistant
associated with immediate hypersensitivity reactions
19kDa
•
•
has homology with the rice α-globulin protein
specific for buckwheat allergy patients
•
100% binding activity to Ig E antibodies of the buckwheatallergic patient sera
the β subunit of a legumin-like storage molecule
24kDa
•
년도
Matsumoto et al.,
2004
Kazuko et al., 2001
J.W. Park et al.,
2000
Urisu A et al.,
1994
29
24-kDa
•
Using the immunoblotting method, radioallergosorbent test(RAST) and RAST inhibition assay,
BW24KD binding to IgE antibodies from 100% of the patients’ sera.(Urisu A et al., 1994)
•
A 24-kDa buckwheat protein(BW24KD) was the most frequently recognized allergenic
component, binding to IgE antibodies from 100% of the patients’ sera.(G. Wieslander et al.,
2001)
30
22-kDa
•
A 22-kDa protein has been reported as the major allergens, and its N-terminal sequence
differed only in three amino acids to that of BW24KD. (Nair, A et al., 1999)
•
22-kDa protein, which displayed binding activity with almost all sera from different patients is
considered a major allergenic protein. (Hiroyuki, Y et al., 2004)
31
19-kDa

J.W. Park et al., Identification and characterization of the major allergens of buckwheat.
Allergy 2000: 55:1035-1041
- The prevalence of IgE binding in BW-allergic sera
1. 19-kDa (73.7%)
2. 16-kDa (73.7%)
3. 24-kDa (68.4%)
4. 9-kDa (52.6%)
- In the asymptomatic sera
1. 24-kDa (80%)
2. 9-kDa (80%)
3. 16-kDa (66.7%)
4. 19-kDa(Only one subject)
19-kDa allergens
were relatively
specific in BWallergic patients.
32
16-kDa
•
The 16-kD buckwheat protein was resistant to pepsin digestion and appeared to be responsible
for IHR including anaphylaxis, while the pepsin-sensitive 24-kD protein was responsible for
CAP-FEIA but not IHR. (Kazuko et al., 2001)
•
Mutations of Cys65 and Cys66 in rBWp16 decreased the pepsin digestibility of the protein, and
an ELISA inhibition assay revealed a weaker inhibitory effect of rBWp16 C65S than that of
rBWp16 WT. (Rie Satoh et al., 2008)
33
메밀 알레르겐 요약
 24-kDa
•
•
이전 연구에 의해 24-kDa이 메밀의 주요 알레르겐이라고 알려져 있었음.
증상에 관계 없이 모든 환자 혈청과 반응함 (19/20) (Urisu A et al., 1994).
 16, 19-kDa
•
•
16-kDa과 19-kDa은 아낙필러시스 증상을 동반한 IHR(immediate hypersensitivuty reactions) 증
상을 보이는 환자(9/10)의 혈청과 반응 (J.W. Park et al, 2000).
펩신 처리 후, 19-와 24-kDa 단백질은 분해가 되나, 16-kDa 단백질은 어느정도
의 저항력을 보이는 것으로 보아 현재 메밀의 주요 알레르겐을 16-kDa으로 보는
추세임 (Tanaka et al., 2001).
34
Characteristics of 16-kDa

No homology with the 24-kDa protein by the N-terminal amino acid sequences.

100% identical with 18-kDa buckwheat protein.

Belongs to 2S albumin family.

While 19- and 24-kDa proteins are pepsin-sensitive, 16-kDa is pepsin-resistant.

Cys residues(having disulfide bonds) contained in 16-kDa are resistant to proteolysis.
 Schematic representation of BWp16. The predicted signal peptide is illustrated as the gray portion
of the bar. The white portion of the bar represents the mature BWp16. Ten Cys residues are shown
as filled circles, and the numbers of the residues are shown at the bottom. (Satoh., 2008)
35
16kDa
 Allergen Details:
Allergen name:
Fag e 2
Lineage:
Source: Plantae Magnoliopsida
Order: Caryophyllales
Species: Fagopyrum esculentum (Common buckwheat)
Obsolete name:
BWp16
Biochemical name:
2S albumin
MW(SDS-PAGE):
16 kDa
Food allergen:
Yes
Date Created:
2010-05-27 02:42:37
Last Updated:
2010-05-27 02:42:37
 16kDa sequence
출처:ALLERGEN NOMENCLATURE
IUIS Allergen Nomenclature SubCommittee
36
메밀 알레르겐 저감화 방법

Salting 방법 (류정우, 2000)

Polysaccharide와의 conjugation 방법 (Suzuki et al., 2009)

곰팡이 발효법 (Handoyo, 2006)

Amino sequence mutation 방법 (Satoh et al., 2008)

메밀의 배젖과 배아를 분리하여 메밀 알레르겐이 많은 배아를 제거하는 방법 (Licen, 2007).
37
Pepsin Digestion
메밀에서 단백질 분리
Pepsin digestion soln.에 분리한 메밀
단백질을 넣고 시간별로 digestion 진행
(0, 0.5, 1, 5, 10, 20, 30, 60 분)
메밀 알레르기 환자의 pooled serum을
사용하여 Western blot
•
Pepsin 효소를 넣지 않은 buffer에
메밀 단백질만 넣고 60분 진행,
Pepsin digestion soln.에 메밀 단백
질이 들어있지 않은 단백질 수용용
액을 넣고 60분 진행.
ELISA
Pepsin digestion soln.(pH 1.2) : pepsin 1.157mg/1ml, NaCl(2mg/ml)
pH에 따른 pepsin digestion degree 고찰
pH
결과
3
16kDa 이하의 proteins은 120min 까지 반응 시킨 결과 펩신에 의해 분해되지
않음. (Tanaka et al., 2002)
2
16kDa protein은 0min부터 서서히 반응하다가 10min이 되면 완전히 분해됨.
(Koyano et al., 2005)
1.2
16kDa protein은 0min부터 서서히 반응하다가 10min이 되면 완전히 분해됨.
(Satoh et al., 2008)
Pepsin Digestion
• SDS-PAGE
MW
(KDa)
21111979 –
53 –
M
1
2
3
4
5
6
7
8
9
10 11
12
13
36.8 –
28.6–
17.8–
6 –
•
M: Marker, 1: Pepsin only 0’, 2: Pepsin only 60’, 3: Digestion 0’, 4: Digestion 0.5’, 5: Digestion 1’,
6: Digestion 2’, 7: Digestion 5’, 8: Digestion 10’, 9: Digestion 20’, 10: Digestion 30’, 11:
Digestion 60’, 12: BW Protein only 0’, 13: BW Protein only 60’,
Pepsin Digestion
• Western Blot
M
1
2
3
4
5
6
7
8
9
10
11
MW
(KDa)
21111979 –
53 –
37 –
24 –
16 –
•
M: Marker, 1: Pepsin only 60’, 2: Digestion 0’, 3: Digestion 0.5’, 4: Digestion 1’, 5: Digestion 5’, 6:
Digestion 10’, 7: Digestion 20’, 8: Digestion 30’, 9: Digestion 60’, 10: BW Protein only 0’, 11:
BW Protein only 60’,
ELISA(Pepsin)
0.45
Pepsin 가수분해에 따른 buckwheat의 allergenicity 변화
0.4
Absorbance at 595 nm
0.35
3853
0.3
4192
Serum
나이
CAP
5747
3853
12
58.2
855
4192
1
33
4750
7
16.4
5747
16
41
855
3
13
5254
4
47.2
0.25
4750
0.2
0.15
5254
0.1
normal
control
0.05
0
1
2
3
4
5
6
7
8
Time of digestion
Protein states; 1: Digestion 0’, 2: Digestion 0.5’, 3: Digestion 1’, 4: Digestion 5’, 5: Digestion 10’, 6: Digestion 20’, 7: Digestion 30’,
8:Digestion 60’