名古屋学芸大学健康 ･ 栄養研究所年報

創刊号

２００７年

《Review》

Rice Allergenic Proteins
Hidehiko Izumi＊, Chikako Yamada＊ and Takeshi Masaki＊

Food allergy is an adverse reaction to food defined as the reaction in which an immune response can bedemonstrated,1) and allergens can be defined as substances that provoke the allergic reactions. In IgE-mediatedfood allergy, the allergens are proteins found in the food. The allergenic foods tend to be commonly consumed
with comparatively high protein contents, especially food of animal or marine origin. However, not all proteins
contained in food can play a part as allergens. The major factors allotting the allergic sensitization to a particularfood protein are the characteristics of the protein itself.
Rice is a grain produced and consumed in large quantities around the world, especially in Asia, and isstaple food for Japanese. But there have been only a few reports on rice allergy. Some atopic patients show
positive RAST values for rice grain proteins.2) Several clinical studies have suggested that rice grains were
responsible for severe atopic dermatitis in some patients.3-5)
Rice grain contains proteins accounting for 8% of the dried endosperm, most of which are storage proteins
accumulated in protein bodies. Most plant storage proteins in seed tissues are used as nitrogen, sulfur and carbon
sources during the germination of development.
These proteins can be classified into four groups
based on their solubility. Depending on theanalytical method, genotype and environmentalcondition, rice seeds contain 4-10% salt-soluble
proteins (globulin and albumin), 5-10% alcohol
soluble proteins (prolamin) and 80-90% alkalisoluble proteins (glutelin).6) Fig.1 shows a sodium dodecyl sulfate-polyacrylamide gel electorophoresis (SDS-PAGE) of fractionated riceseed proteins. Comparison of the total proteins
with the proteins in each fraction demonstrates
that glutelin is the major constituent of rice-endosperm proteins. The electrophoretogram also
shows that the soluble proteins in the albumin
and globulin fractions contain more diverse protein components than the insoluble proteins inprolamin and glutelin fractions. Shibasaki et al.7)
first showed that a high degree of allergenicity
was found in a globulin fraction of rice seed

Fig. 1. Solubility-dependent fractionation of rice-seed proteins.
Rice-endosperm proteins were fractionation into water-soluble(albumin), salt-soluble (globulin), alcohol-soluble (prolamin), andalkali-soluble (glutelin) fractions. The component proteins in eachfraction, as well as seed total proteins, were analyzed by SDSPAGE (CBB-staining).

＊ School of Nutritional Sciences, Nagoya University of Arts and Sciences, Nissin, Aichi 470-0196, Japan
Key words: rice; allergen; a-amylase/trypsin inhibitor; food allergy59

proteins, but the exact properties of rice allergenic proteins had not been clalified.

1. Separation of the Rice Allergenic Protein
To isolate the allergenic protein in rice grain, polished rice grain was extracted with 1M NaCl solution.
The rice allergenic proteins were isolated from the extract by ion-exchange chromatography on DEAE cellulose
column and gel filtration chromatography on Sephadex G-100, based on the reactivity with specific IgE frompatients allergic to rice. The molecular weight of the purified allergenic protein was estimated to be about
16,000, named 16-kDa allergen.8)
The reactivity of the 16-kDa allergen was tested about 31 sera with positive RAST values for rice grain
extract.9) The sera examined all gave positive RAST values for the purified this 16-kDa allergen. Furthermore,
there was a close correlation between RAST values for rice grain extract and those for purified 16-kDa allergen. These indicated that the 16-kDa allergen is the major allergenic protein in rice grain with response toIgE binding activity. The same result was indicated by the close correlation in the histamine-release activities
between the 16-kDa allergen and rice grain extract.
The 16-kDa allergen was measured by using monoclonal antibody against that about various rice strains
mainly provided by IRRI (International Rice Research Institute in Philippine).10) All of the screened Japanesecultivars contain nearly the same amount of this protein.

2. Property of Rice Allergenic Protein
Since allergenic proteins obtained earlier often have high heat stability, experiments have been done about
the effects of heating against the antigenicity of the 16-kDa allergen.8) Antigenicity was measured using bothhuman sera of rice allergenic patients and rabbit antisera against the 16-kDa allergen. Almost the same results
were obtained from both experiments. No less than 60% of the allergenicity was still remained even after heating
at 100℃ for 60min. This high resistance for heat treatment may allow this protein to be a food allergen.

3. Primary Structure of Rice Allergenic Protein
A cDNA clone encoding the 16-kDa allergen was first isolated from cDNA libraries of maturing rice seeds
by screening with a 32P-labeled synthetic oligonucleotide mixture corresponding to N-terminal amino acid of
the 16-kDa allergen and its nucleotide sequence was analyzed.11) The deduced amino acid sequence of 16-kDa
allergen showed a similarity to the members of cereal a-amylase/trypsin inhibitor family. In Fig.2, the deduced
amino acid sequence of 16-kDa allergen is compared with two members of family. The most prominent featureof the amino acid sequence of this family is the ten conserved cysteine residues that are all considered to bepresent as cystine in 16-kDa allergen. Among them, both wheat and barely a-amylase/trypsin inhibitors wererecently identified major allergens associated with baker’s asthma.12,13) This seems to show that the proteins
belonging to a-amylase/trypsin inhibitor family may be potential allergens.

4. Structural Characterization of Rice Allergenic Protein
A 16-kDa allergen belonging to the a-amylase/trypsin inhibitor family was isolated from rice seed andstructurally characterized by identifying cystine-containing peptides and predicting secondary structure and60

Rice Allergenic ProteinsFig. 2. Alignment of the deduced amino acid sequence of Rice 16-kDa allergen with sequences of a-amylase/trypsin inhibitorfamily proteins, namely, wheat a-amylase inhibitor (AI 28), barley trypsin inhibitor (TI). The sequences aligned for maximum
homology, resulting in several gaps (shown as a dot) which may represent insertions/deletions. Conserved residues are shown inshaded region.

hydrophobic regions.14) Eight peptides, which constitute three sets of cystine-containing peptide, were purified
by HPLC from thermolytic digest of 16-kDa allergen and identified by amino acid-sequence and -composition analyses, indicating five intramolecular disulfide bridges; Cys34-Cys94, Cys26-(Cys50 or Cys51)-Cys110 andCys12-(Cys62or Cys64)-Cys122.
According to the results obtained in this study and to the previously reported disulfide cross-linking of
wheat a-amylase inhibitor,15) the polypeptide folding of the 16-kDa allergen was predicted and schematicallydrawn in Fig.3. The 16-kDa allergen appeared to be a globular and compactly folded molecule with fiveinatramolecular disulfide bridges. The N- and C-terminal regions might cover the hydrophobic core region,and contribute to antigenic and/or allergenic epitopes of the 16-kDa allergen. Such a prediction would agreewith the result that rabbit and mouse antibodies against the 16-kDa allergen recognized the N-terminal regionof recombinant the 16-kDa allergen molecules.16)
The 16-kDa allergen is the member of a planta-amylase/trypsin inhibitor family. Ten cysteine
residues are conserved well within this family.11)
The similarity of intramolecular disulfide bridges inaddition to the sequence homology suggests that theproteins in this inhibitor family have a common folding profile, and conformational and physio-chemicalproperties. The 16-kDa allergen has been identified
as an allergen in severe atopic dermatitis caused byrice ingestion,3,7) and a-amylase inhibitors of wheat
and barley seeds have also been identified as major
allergens associated with baker’s asthma caused by
the inhalation of cereal flour.12,13) Allergenic proteinsin food have been suggested to be stable against heatFig. 3. Speculated folding profile of rice 16 kDa allergen.
Cysteine residues are shown as open circles with residue
numbers, and disulfide bridges are represented by striped bar
between two circles.

and proteases.8) The five intramolecular disulfide
bridges of the 16-kDa allergen would stabilize thefolding of the ordered structures, resulting in heat61

stability and reversibility in heat-induced denaturation. The compactly folded structure of the 16-kDa allergenmight also contribute to resistance to proteolytic degradation in alimentary tract. This stability against heat
denaturation and proteolytic degradation would increase the allergenic potential of the 16-kDa allergen.

References
1)

Chandra, R. K., “Food Allergy”, ed. by Chandra, R. K. Nutrition Res. Education Foundation, St. John’s, Newfoundland,
1987, pp. 3-5.

2)

Hoffman, D. R., Immunochemistry, The specificity of human IgE antibodies combining with cereal grains., 1975, 12,535-538.

3)

Ikezawa, Z., Miyakawa, K., Komatsu, H. et al., A probable involvement of rice allergy in severe type of atopic dermatitis inJapan., Acta Derm. Venereol. Suppl., 1992, 176, 103-107.

4)

Miyakawa, K., Hirai, Y., Miyakawa, J. et al., Statistical analyses of the diagnostic criteria, clinical severity, IgE-RAST
score, and serum IgE value in patients with atopic dermatitis (AD)-probable involvement of food antigens, especially rice,in severe cases., Jap. J. Allergol., 1988, 37, 1101-1110.

5)

Lezaun, A., Igea, M. J., Quirce, S., et al., Asthma and contact urticaria caused by rice in a house wife., Allergy, 1994, 49,92-95.

6)Cagampand, G. B., Cruz, L. J., Espiritu, S. G. et al., Studies on the extraction and composition of rice proteins., CerealChem., 1966, 43, 145-155.

7)

Shibasaki, M., Suzuki, S., Nemoto, H. et al., Allergenicity and lymphocyte-stimulating property of rice protein., Allergy
Clin. Immunol., 1979, 64, 259-265.

8)

Matsuda, T, Sugiyama, M., Nakamura, R. et al., Purification and properties of an allergenic protein in rice grain., Agric.
Biol. Chem., 1988, 52, 1465-1470.

9)

Urisu, A., Wada, E., Kondo, Y. et al., Rice protein 16KD−a major allergen in rice grain extract., Jpn. J. Allergol., 1991, 40,1370-1376.

10) Adachi, T., Alvarez, A. M., Aoki, N. et al., Screening of rice strains deficient in 16-kDa allergenic protein., Biosci.
Biotechnol. Bioche., 1995, 59, 1377-1378.
11) Izumi, H., Adachi, T., Fujii, N. et al., Nucleotide sequence of cDNA clone encoding majior allergenic protein in rice seeds.,
FEBS Lett., 1992, 302, 213-216.
12) Barber, D., Sanchez-Monge, R., Gomez, L. et al., A barley flour inhibitor of insect alpha-amylase is a major allergenassociated with baker’s asthma disease., FEBS Lett., 1989, 248, 119-122.
13) Gomez, L., Martin, E., Hernandez, D. et al., Members of the alpha-amylase inhibitors family from wheat endosperm are
major allergens associated with baker’s asthma., FEBS Lett., 261, 85-89 (1990).
14) Izumi, H., Sugiyama, M., Matsuda, T. et al., Structural characterization of the 16-kDa allergen, RA17, in rice seeds.,

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Rice Allergenic Proteins

Biosci. Biotechnol. Biochem., 1999, 63, 2059-2063.
15) Nozal, M. J., Bernal, J. L., Toribio, L. et al., Determination of glutathione, cysteine and N-acetylcysteine in rabbit eye
tissues using high-performance liquid chromatography and post-column derivatization with 5,5’-dithiobis(2-nitrobenzoicacid)., J. Chromatogr. A., 1997, 778, 347-53.
16) Alvarez, A. M., Fukuhara, E., Nakase, M. et al., Four rice seed cDNA clones belonging to a-amylase/trypsin inhibitor genefamily encode potential rice allergens., Biosci. Biotech. Biochem., 1995, 59, 1304-1308.

和文抄録

米アレルゲンタンパク質
和泉＊秀彦

山田千佳子

＊

間崎＊

剛

近年、アレルギー患者が増加しており食物アレルギーも例外ではない。古くからよく知られる卵、牛乳に加えて最近では、植物性食品である大豆、米、小麦などに対するアレルギー症が増加している。
米アレルゲンについては、米タンパク質と米アレルギー患者血清中に存在する特異 IgE 抗体との反応性が調べられた。その結果、可溶性画分中の16-kDa のタンパク質と強く反応することが見出され、これを米主要アレルゲンとした。これまでに、この16-kDa アレルゲンについて構造解析を行った結果、16-kDa アレルゲンは、穀類や豆類の a-アミラーゼ / トリプシンインヒビターファ
ミリーに属し、分子内に存在する10残基のシステインはすべて S-S 結合を形成し、小さく折りたたまれた構造をとっていることが明らかとなった。その構造安定性が、アレルゲン特有の性質であるプロテアーゼ耐性や熱安定性に寄与していることが示唆された。

＊名古屋学芸大学

管理栄養学部管理栄養学科

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