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Change in subcellular localization of overexpressed vaccine peptide in rice endosperm cell that is caused by suppression of endogenous seed storage proteins

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Abstract

Rice-based peptide vaccine based on T cell epitopes acts as an ideal oral tolerogen for the treatment of type 1 allergic diseases. To improve production yields of oral tolerogen against Japanese cedar pollen allergy, hybrid peptide comprising seven predominant human T cell epitopes (7Crp) derived from Japanese cedar pollen allergens, Cry j 1 and Cry j 2, was produced in transgenic rice seed by expression of its codon optimized gene under the control of the endosperm-specific 26 kD globulin (Glb-1) promoter containing its signal peptide and the simultaneous suppression of endogenous seed storage proteins (SSPs) by RNA interference. Accumulation level of 7Crp peptide produced as a secretory protein was remarkably enhanced by suppression of both the 13–14 kDa prolamins and GluA and GluB glutelins as compared to those under suppression of either of them or in wild type rice. When these SSPs were down-regulated, the 7Crp peptide was observed to be localized in ER lumen as well as ER derived PBs (PB-Is). Especially, accumulation as self-aggregates in ER lumen increased by reduction of the endogenous 13–14 kDa prolamins. It is interesting to note that the absence of C terminal KDEL ER retention signal from the 7Crp peptide resulted in higher level accumulation (116 µg/grain) than that containing the KDEL.

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Abbreviations

BiP:

Binding protein

CDS:

Coding sequence

CNX:

Calnexin

Cys:

Cysteine

ER:

Endoplasmic reticulum

GALT:

Gut-associated lymphoid tissue

Glb-1:

26 kDa Globulin

PB:

Protein body

PDIL:

Protein disulfide isomerase-like

RNAi:

RNA interference

2-MER:

2-Mercaptoethanol

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SSP:

Seed storage protein

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Acknowledgements

We thank Ms. K. Miyashita, Y. Ikemoto and Y. Yajima for technical assistance, Dr. S. Saito (Jikei University of School of Medicine) for providing anti-7Crp monoclonal antibody, and Dr. Kenjiro Ozawa for encourage of this research. M.E was supported by Ministry of Science, Research and Technology of Iran.

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Authors and Affiliations

  1. Division of Biotechnology, Plant Molecular Farming Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan

    Mehrnaz Entesari, Yuhya Wakasa & Fumio Takaiwa

  2. Department of Agronomy & Plant Breeding, Faculty of Agriculture, University of Zanjan, P.O. Box 45195-313, Zanjan, Iran

    Mehrnaz Entesari & Bahram Maleki Zanjani

Authors
  1. Mehrnaz Entesari
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  2. Yuhya Wakasa
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  3. Bahram Maleki Zanjani
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  4. Fumio Takaiwa
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Contributions

FT designed this research. ME, FT and YW conducted experiments. FT and ME wrote this manuscript. All authors read and approved the manuscript.

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Correspondence to Fumio Takaiwa.

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The authors declare that they have no conflict of interest.

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Communicated by Sergio Rosales-Mendoza.

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11240_2018_1380_MOESM1_ESM.pdf

Supplementary material 1. Fig. S1—Effect on accumulation levels of KDEL less 7Crp peptide (7CrpKDEL(−)) by suppression of endogenous seed storage proteins. a SDS-PAGE analysis of total proteins extracted from non-transgenic Kita-ake and transgenic rice seeds containing various expression constructs. For detection of 7Crp peptide in mature transgenic rice seeds, immunoblot analysis was carried out using anti-7Crp antibody. For each construct, mature seeds from four independent representative expression lines were used for this analysis. Arrow indicates the expressed 7Crp peptide and size marker is shown on the left of gel. b Expression levels of endogenous seed proteins in mature transgenic rice seeds containing various expression constructs. Glutelins levels were examined by immunoblotting using antibodies specific to GluA, GluB and GluC. c Prolamin levels were examined by immunoblotting using antibodies specific to 16 kDa, RM1 (Cys-rich 14 kDa prolamin), RM2 (Cys-poor 13 kDa prolamin), RM4 (Cys-poor 13 kDa prolamin), RM9 (Cys-rich 14 kDa prolamin) and 10 kDa prolamins. Fig. S2—Effect of accumulation levels of 7Crp peptide for individual expression constructs by the presence or absence of KDEL signal. a WT/7Crp, b Proless/7Crp, c Gluless/7Crp, d Proless/ Gluless/7Crp. Blue circle, − KDEL; red circle, + KDEL; p positive control (transgenic rice containing 7Crp peptide 20 µg/grain). Fig. S3—Comparison of accumulation levels of 7Crp peptide between Proless/Gluless/7Crp and Proless/Gluless/7CrpKDEL(−). a SDS-PAGE of total seed proteins. b 7Crp and BiP4/5 levels were examined by Immunoblotting using anti-7Crp and antiBiP4/5 antibodies. (PDF 497 KB)

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Entesari, M., Wakasa, Y., Zanjani, B.M. et al. Change in subcellular localization of overexpressed vaccine peptide in rice endosperm cell that is caused by suppression of endogenous seed storage proteins. Plant Cell Tiss Organ Cult 133, 275–287 (2018). https://doi.org/10.1007/s11240-018-1380-2

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