Adachi, M., Kanamori, J., Masuda, T., Yagasaki, K., Kitamura, K., Mikami, B., & Utsumi,
S. (2003). Crystal structure of soybean 11S globulin: glycinin A3B4 homohexamer.
Proceedings of the National Academy of Sciences of the United States of America, 100
(12), 7395-7400. doi: 10.1073/pnas.0832158100.
Choi, S. M., Mine, Y., & Ma, C. Y. (2006). Characterization of heat-induced aggregates of
globulin from common buckwheat (Fagopyrum esculentum Moench). International
Journal of Biological Macromolecules, 39, 201–209. https://doi.org/10.1016/j.
ijbiomac.2006.03.025
Gim´enez-Bastida, J. A., & Zieli´
nski, H. (2015). Buckwheat as a functional food and its
effects on health. Journal of Agricultural Food Chemistry, 63, 7896–7913.
Heffler, E., Pizzimenti, S., Badiu, I., Guida, G., & Rolla, G. (2014). Buckwheat allergy: An
emerging clinical problem in Europe. Journal of Allergy & Therapy, 5, 2.
Huda, M. N., Lu, S., Jahan, T., Ding, M., Jha, R., Zhang, K., … Zhou, M. (2020). Treasure
from garden: Bioactive compounds of buckwheat. Food Chemistry, 335, Article
127653. https://doi.org/10.1016/j.foodchem.2020.127653
Jin, T., Guo, F., Chen, Y., Howard, A., & Zhang, Y. Z. (2009). Crystal structure of Ara h 3,
a major allergen in peanut. Molecular Immunology, 46, 1796–1804. https://doi.org/
10.1016/j.molimm.2009.01.023
Katayama, S., Yamaguchi, D., Suzuki, Y., Athamneh, A. M. A., Mitani, T., Satoh, R., …
Nakamura, S. (2018). Oral immunotherapy with a phosphorylated hypoallergenic
allergen ameliorates allergic responses more effectively than intact allergen in a
murine model of buckwheat allergy. Molecular Nutrition & Food Research, 62,
e1800303.
Katsube, T., Gidamis, A. B., Kanamori, J., Kang, I. J., Utsumi, S., & Kito, M. (1994).
Modification tolerability of the hypervariable region of soybean proglycinin. Journal
of Agricultural and Food Chemistry, 42, 2639–2645.
Katsube-Tanaka, T. (2016). Buckwheat production, consumption, and genetic resources
in Japan. In M. Zhou, I. Kreft, S. H. Woo, N. Chrungoo, & G. Wieslander (Eds.),
Molecular breeding and nutritional aspects of buckwheat (pp. 61–80). Amsterdam:
Elsevier.
Katsube-Tanaka, T., & Monshi, F. I. (2022). Characterization of 2S albumin allergenic
proteins for anaphylaxis in common buckwheat. Food Chemistry: Molecular Sciences,
100127, doi.org/10.1016/j.fochms.2022.100127.
Katsube-Tanaka, T., Nakagawa, M., Sano, M., & Yasui, Y. (2014). Development of novel
common buckwheat (Fagopyrum esculentum M.) plants with lowered contents of
tandem repeat-less 13S globulin-Discrimination methods of the tandem repeat-less
genes. Journal of Crop Research, 59, 31–35. In Japanese with English abstract https://
doi.org/10.18964/jcr.59.0_31.
Kelley, L., Mezulis, S., Yates, C., Wass, M., & Sternberg, M. (2015). The Phyre2 web
portal for protein modeling, prediction and analysis. Nature protocols, 10, 845–858.
https://doi.org/10.1038/nprot.2015.053
Khan, N., Katsube-Tanaka, T., Iida, S., Yamaguchi, T., Nakano, J., & Tsujimoto, H.
(2008a). Diversity of rice glutelin polypeptides in wild species assessed by the
higher-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
subunit-specific antibodies. Electrophoresis, 29, 1308–1316.
Khan, N., Katsube-Tanaka, T., Iida, S., Yamaguchi, T., Nakano, J., & Tsujimoto, H.
(2008b). Identification and variation of glutelin α polypeptides in the genus Oryza
assessed by two-dimensional electrophoresis and step-by-step immuno detection.
Journal of Agricultural and Food Chemistry, 56, 4955–4961.
Khan, N., Takahashi, Y., & Katsube-Tanaka, T. (2012). Tandem repeat inserts in 13S
globulin subunits, the major allergenic storage protein of common buckwheat
(Fagopyrum esculentum Moench) seeds. Food Chemistry, 133, 29–37.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature, 227(5259), 680–685.
Fig. 5. Comparison in trypsin digestibility between the α polypeptides of
native and 10aa zero-repeat subunits. Globulin fractions extracted from 10aa
heterozygotes (A) and a mixture of 10aa homozygotes (10aa) and native sub
units (Native) (B) were incubated with trypsin for 8 h. The digested samples
were analyzed by SDS-PAGE and western blotting, using anti-5rep and antiGlbNA antibodies. (A, B) Representative images of western blot data (α poly
peptide of 13S globulin). Estimated molecular size is 28.3, 29.4, 33.5, 35.9,
37.4, 39.4, and 41.3 kDa for Native, 10aa, and 1–5 rep, respectively. (C) Time
course of trypsin digestion in the mixture of 10aa and native subunits. Asterisks
indicate significant differences between 10aa and Native at each incubation
time (Student’s t-test: * P < 0.05, ** P < 0.01, *** P < 0.001). n=6; error bars
represent SE.
5. Conclusion
13S globulin of common buckwheat is an important allergen in
buckwheat-sensitized patients. There is large variation in the molecular
size of the protein caused by the insertion of tandem repeats of various
lengths. As the tandem repeat is hydrophilic and contains many arginine
residues, 1–6 repeat subunits containing the tandem repeat are more
digestible with trypsin than zero-repeat subunits containing no tandem
repeats. In this study, we found a new zero-repeat subunit, which con
tains additionally inserted ten amino acid residues (10aa) at the
equivalent position to the tandem repeat region by amplicon deep
sequencing. The 10aa subunit was predicted to possess no β-hairpin
structure, which was found in the zero-repeat (non-10aa, native) subunit
but not in the 1–6 repeat subunits. The 10aa subunit was more digestible
with trypsin than the non-10aa (native) subunit. However, the trypsin
digestibility of zero-repeat subunits, even the 10aa allele homozygote,
was still much lower than that of the 1–5 repeat subunits. Thus, another
new zero-repeat allele, for example, a non-functional allele, would be
required to develop hypoallergenic buckwheat.
T. Okada et al.
Food Chemistry: Molecular Sciences 6 (2023) 100159
Sano, M., Nakagawa, M., Oishi, A., Yasui, Y., & Katsube-Tanaka, T. (2014).
Diversification of 13S globulins, allergenic seed storage proteins, of common
buckwheat. Food Chemistry, 155, 192–198.
Utsumi, S., Katsube, T., Ishige, T., & Takaiwa, F. (1997). Molecular design of soybean
glycinins with enhanced food qualities and development of crops producing such
glycinins. In S. Damodaran, & A. Paraf (Eds.), Food proteins and lipids 1st edition (pp.
1–15). New York: Plenum Press.
Wieslander, G., & Norb¨
ack, D. (2001). Buckwheat allergy. Allergy, 56, 703–704.
Yasui, Y., Mori, M., Matsumoto, D., Ohnishi, O., Campbell, C. G., & Ota, T. (2008).
Construction of a BAC library for buckwheat genome research - an application to
positional cloning of agriculturally valuable traits. Genes & Genetic Systems, 83,
393–401.
Zhang, Z., Zhou, M., Tang, Y., Li, F., Tang, Y., Shao, J., … Wu, Y. (2012). Bioactive
compounds in functional buckwheat food. Food Research International, 49(1),
389–395.
Monshi, F. I., & Katsube-Tanaka, T. (2022). 2S albumin g13 polypeptide, less related to
Fag e 2, can be eliminated in common buckwheat (Fagopyrum esculentum Moench)
seeds. Food Chemistry: Molecular Sciences, 5, Article 100138. https://doi.org/
10.1016/j.fochms.2022.100138
Monshi, F. I., Khan, N., Kimura, K., Seita, S., Yamamoto, Y., & Katsube-Tanaka, T. (2020).
Structure and diversity of 13S globulin zero-repeat subunit, the trypsin-resistant
storage protein of common buckwheat (Fagopyrum esculentum M.) seeds. Breeding
Science, 70(1), 118–127.
Piersma, S. R., Gaspari, M., Hefle, S. L., & Koppelman, S. J. (2005). Proteolytic processing
of the peanut allergen Ara h 3. Molecular nutrition & food research, 49(8), 744–755.
https://doi.org/10.1002/mnfr.200500020
Radovi´c, S. R., Maksimovi´c, V. R., & Varkonji-Gaˇsi´c, E. I. (1996). Characterization of
buckwheat seed storage proteins. Journal of Agricultural and Food Chemistry, 44,
972–974.
...