FEBS Letters 580 (2006) 3315–3320

A transgenic rice seed accumulating an anti-hypertensive peptidereduces the blood pressure of spontaneously hypertensive ratsq

Lijun Yanga, Yoshifumi Tadaa, Masayuki P. Yamamotoa, Hui Zhaob,Masaaki Yoshikawab, Fumio Takaiwaa,*

a Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences (NIAS), 2-1-2 Kannondai,Tsukuba, Ibaraki 305-8602, Japan

b Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan

Received 31 March 2006; revised 28 April 2006; accepted 28 April 2006

Available online 8 May 2006

Edited by Ulf-Ingo Flugge

Abstract RPLKPW is a potent anti-hypertensive peptide de-signed according to the structure of ovokinin(2–7) (RADHPF).In this study, we generated transgenic rice plants that accumu-late the RPLKPW peptide as a fusion protein with the ricestorage protein glutelin. The engineered peptide is expressedunder the control of endosperm-specific glutelin promotersand specifically accumulates in seeds. Oral administration ofeither the RPLKPW-glutelin fraction or transgenic rice seedsto spontaneously hypertensive rats (SHRs) significantly reducedsystolic blood pressures. These results suggest the possibleapplication of transgenic rice seed as a nutraceutical deliverysystem and specifically for administration of active peptidesin hypertension.� 2006 Federation of European Biochemical Societies. Publishedby Elsevier B.V. All rights reserved.

Keywords: Endosperm-specific expression; Glutelin;Transgenic rice; Hypertension; Oryza sativa

1. Introduction

Hypertension is a disease that involves complex interactions

between genetic, dietary and environmental factors [1–3].

Approximately 1 billion people worldwide suffer with this dis-

ease. When hypertension is undiagnosed or uncontrolled, it

places patients at risk for other cardiovascular diseases, con-

tributing to an increase in mortality and morbidity. Hyperten-

sion is closely associated with diet, but a diet rich in foods with

anti-hypertensive activity can potentially prevent or reduce dis-

ease severity as a supplement to drug therapy.

RPLKPW [4] is a potent anti-hypertensive peptide that was

designed based on the structure of ovokinin(2–7) (RADHPF)

[5], a vasorelaxing peptide derived from ovalbumin. Oral

administration of RPLKPW at a dose of 0.1 mg/kg has been

shown to lower the systolic blood pressure of spontaneously

q The GenBank/EMBO/DDBJ accession numbers of the nucleic acidsequences used in this study are as follows: Glutelin A2: X05664;Glutelin B1: X54314; Glutelin C: AB016501; Glutelin B1 promoter:AY427569; Glutelin B2 promoter: AY427570; Glutelin B4 promoter:AY427571; Hygromycin phosphotransferase: AY373338.

*Corresponding author. Fax: +81 29 838 8397.E-mail address: takaiwa@nias.affrc.go.jp (F. Takaiwa).

0014-5793/$32.00 � 2006 Federation of European Biochemical Societies. Pu

doi:10.1016/j.febslet.2006.04.092

hypertensive rats (SHRs). When RPLKPW sequences were

introduced into three sites of the a0subunit of soybean b-con-

glycinin by base substitution mutation, the modified protein

lowered the systolic blood pressure of SHRs at a dose of

10 mg/kg [6]. Furthermore, for improvement of RPLKPW

peptide release from fusion protein, modification of the pep-

tide was attempted by addition of arginine (R) and glutamine

(Q) residues to the amino and carboxy termini respectively.

When these RRPLKPWQ sequences were introduced into

the corresponding regions of the subunit, the modified protein

reduced blood pressure at a dose of 2.5 mg/kg [7]. The result

demonstrates that the addition of the R–Q spacer facilitates

RPLKPW release in vivo by gastrointestinal proteases. While

the systolic blood pressure in normotensive rats was not af-

fected even by a high dose of RPLKPW peptide [5], which

indicates that it may be an ideal functional peptide with anti-

hypertensive activity, and is a candidate for further hyperten-

sive remediation study. Therefore, it is conceivable that a novel

oral deliverable food, or ‘nutraceutical’ can be developed that

could be used to control or prevent clinical hypertension.

Plants have many advantages as bioreactors for the produc-

tion of pharmaceuticals, vaccines and antibodies [8,9]. Com-

pared to current competing technologies such as microbial

and mammalian cell culture systems, plants provide a means

of low cost production with almost no risk of contamination

by animal pathogens. In addition, pharmaceutical products ex-

pressed in cereal crop seeds or tubers are highly stable and can

be stored for long periods at room temperature with no loss of

activity [10]. Functional peptides or vaccines accumulated in

seeds do not require any processing or purification, thus allow-

ing direct oral delivery [11]. Taken together, plants provide an

economic, safe and stable production system for recombinant

products.

In this study, we developed transgenic rice plants that accu-

mulate anti-hypertensive peptides (RRPLKPWQ) in seeds. To

increase accumulation, the peptide was expressed as fusion

proteins with the major rice glutelin storage proteins [12–14].

Under the control of strong rice endosperm-specific promoters

[15], the fusion proteins were accumulated to around 10% of

total seed protein. Oral administration of isolated fusion pro-

tein or transgenic rice seeds to SHRs significantly reduces sys-

tolic blood pressure. This is the first study that indicates the

potential of an active and effective role for rice based anti-

hypertensive bioactive peptides as a dietary supplement for

hypertension patients.

blished by Elsevier B.V. All rights reserved.

3316 L. Yang et al. / FEBS Letters 580 (2006) 3315–3320

2. Materials and methods

2.1. Plasmid construction and rice transformationFor the construction of pGluB1-HRP (glutelin B1 containing the

Hypertension Reducing Peptide), 15 and 13 amino acid residues of thevariable regions of the acidic subunit (between positions 210–224 and284–296 from the precursor initiation codon) of glutelin B1 (GluB1)[12] were substituted with a RRPLKPWQ dimer peptide (RRPLKPW-QRRPLKPWQ), and the C terminal variable region of the basic subunit(residues 492–499) was replaced with the RRPLKPWQ sequence(Fig. 1). Introduction of peptides into the variable regions was accom-plished using a method with two or three stages of PCR amplificationas previously described [16]. The modified GluB-1 cDNA, containing atotal of five copies of the RRPLKPWQ peptide, was linked to the2.3 kb glutelin GluB-1 promoter [15] and then inserted into the binaryvector pGTV-35S-HPT [17], resulting in pGluB1-HRP. The glutelinA2 and glutelin C HRP fusion (pGluA2-HRP/GluC-HRP) was con-structed by substituting the RRPLKPWQ dimer peptide for the 15and 14 amino acid residues in the variable regions of the acidic subunitbetween positions 211–225 and 270–283 of GluA2 [13], and for the 14and 15 amino acids between positions 214–227 and 303–317 of GluC[14]. GluA2-HRP and GluC-HRP thus each contained four copies ofthe RRPLKPWQ peptide, were fused to the 2.4 kb glutelin B-2 (GluB-2) and 1.4 kb glutelin B-4 (GluB-4) promoters, respectively [15], and wereinserted in tandem into the binary vector pGTV-35S-HPT (Fig. 1). Thefidelity of all DNA constructs was confirmed by sequencing.

Glu-HRP constructs were introduced into the rice genome (Oryzasativa cv. Kita-ake) by Agrobaterium tumefaciens-mediated transfor-mation and transgenic plants were selected by resistant to hygromycinas described by Goto et al. [18]. The transgenic plants were grown in acontrolled greenhouse (28 �C, 12 h light/dark cycle) until harvesting.

2.2. Southern blot analysisGenomic DNA was prepared from young leaves with a CTAB meth-

od [19]. Ten micrograms of DNA were digested with XbaI or SacI,fractionated by electrophoresis on 0.8% agarose gel and transferredonto a nylon membrane (HybondN+, Amersham Biosciences). South-ern blot analysis was carried out by using standard method [17].Hybridization was performed at 65 �C by using a full-length hpt probelabeled by random priming reaction (megaprime DNA labeling sys-tem, Amersham Biosciences) with 32P.

Fig. 1. Construction of expression plasmids for rice transformation. (A)hygromycin phosphotransferase gene; pAg7, 3 0 terminal region of agropine sycoding region; GluB-1, rice glutelin B1 coding region; GluC, rice glutelin C co1 P, GluB-2 P and GluB-4 P represent 2.3 kb glutelin B1, 2.4 kb B2 and 1.4 kB1 and 0.7 kb B4 3 0 terminal regions, respectively; blank triangle, RRPLKglutelin B1; solid triangles, dimer peptide RRPLKPWQRRPLKPWQ substitamino acid sequence at variable regions of glutelins. The amino acid residuesubunit are double underlined. Numbers indicate the position of the aminoprecursors; Asterisk stands for stop codon.

2.3. Antibody preparationThe NH2-GCRRPLKPWQRRPLKPW-OH peptide was synthe-

sized by BIO-Synthesis incorporated (USA) and used to raise anti-RPLKPW antibody in a rabbit (Qiagen, Tokyo).

2.4. Detection of GluB1-HRP and GluA2-HRP/GluC-HRP fusion

proteinsRice seeds were ground to a fine powder with a Multibeads shocker

(Yasui Kikai, Osaka, Japan), and total proteins were extracted with aUrea–SDS buffer (50 mM Tris–Cl (pH 6.8), 8 M urea, 4% SDS, 5%mercaptoethanol, 20% glycerol) as described previously [20]. After sep-aration by 12% SDS–PAGE and transfer to PVDF membranes (Milli-pore), fusion proteins were detected with anti-RPLKPW antibody(1:10000 dilution) followed by a goat anti-rabbit IgG secondary anti-body conjugated to horseradish peroxidase (1:5000 dilution) (Promega).Fusion protein accumulation levels were determined with quantita-tive immuno-dot-blots with an anti-RPLKPW antibody [20] andGlu-HRP fusion protein produced in an E. coli culture was used asstandard.

2.5. Extraction and purification of total glutelinsExtraction and purification of total glutelins were performed accord-

ing to Tada et al. [20]. Briefly, glutelins were extracted with 1% (v/v)lactic acid from 12 g seed powders after stepwise removal of albuminsand globulins with 12 ml saline buffer (10 mM Tris–HCl pH 7.5, 0.5 MNaCl) followed by removal of prolamins with 12 ml 60% (v/v) n-pro-panol solution. Each extraction step was accomplished by resuspend-ing seed powder in the solution and sonicating on ice for severalminutes. After centrifugation (10000 rpm, 10 min) the residues wereextracted twice more with the same solution. The concentration ofcombined glutelin fractions was determined by Bradford method witha Bio-Rad Protein Assay kit 1 (Catalog No. 500-0001, Bio-Rad).

2.6. AnimalsMale spontaneous hypertensive-Izumo rats (SHR) were purchased

from SLC, Shizuoka, Japan. Each group of six rats was fed with SPchow (Funakoshi Farm) and water ad libitum, and was housed in atemperature-controlled room on a 12 h light/dark cycle. This studywas performed in accordance with Kyoto University guidelines forthe care and use of laboratory animals.

Diagram of two expression plasmids used for transformation. hpt,nthase gene; RB, right border; LB, left border; GluA-2, rice glutelin A2ding region; Nos T, 3 0 terminal region of nopaline synthase gene; GluB-b B4 promoters, while GluB-1 T and GluB-4 T indicate 0.65 kb glutelinPWQ peptide substitution site at variable region of basic subunit ofution sites at variable regions of acidic subunits of glutelins. (B) Partials substituted in acidic subunits are underlined, while the ones in basicacid residues starting from the translation initiation codon of glutelin

L. Yang et al. / FEBS Letters 580 (2006) 3315–3320 3317

2.7. Measurement of blood pressureGlutelin fractions isolated from non-transgenic and transgenic rice

seeds were dissolved in 1% lactic acid and administrated orally toSHRs at 18–22 weeks after birth using a metal sonde in a volume of1.6–1.9 ml/rat. Pulverized seeds from either non-transgenic or trans-genic rice were homogenized in water with a Polytron homogenizerand orally administered in a volume of 3.0 ml/rat. Systolic blood pres-sured was measured by the indirect tail cuff method using an MK-2000sphygmomanometer (Muromachi Kikai). Blood pressure was mea-sured every 2 h following oral administration.

2.8. Data analysisStatistical significance was determined using Student’s t-test, with

P < 0.05 as the threshold for significance. Results of blood pressuremeasurements are expressed as means ± S.E.M.

3. Results

3.1. Characterization of transgenic rice plants

Thirty individual transgenic rice plants were generated for

each pGluB1-HRP and GluA2-HRP/GluC-HRP construct

and fusion protein expression levels were determined by quan-

titative immuno-dot blot using the anti-RPLKPW peptide

antibody (Fig. 2). The accumulation levels of GluB1-HRP ran-

ged from 0.2% to 9.6% (Fig. 2A), while GluA2-HRP/GluC-

HRP from 1.9% to 13.7% (Fig. 2B) of total seed protein.

The highest expression lines were selected from pGluB1-HRP

(No. 6) and pGluA2-HRP/GluC-HRP (No. 30) constructs.

Southern blot analysis was performed using genomic DNA di-

gested with SacI (line 6) and XbaI (line 30) to determine the

copy number of transgenes. As shown in Fig. 3A, at least three

copies of the transgene were estimated to be present in each

transgenic line.

Western blot analysis was used to examine the expression of

the glutelin-HRP fusion proteins in transgenic rice grain using

anti-RPLKPW antibody. Rice glutelin is synthesized as a pre-

cursor form and then post-translationally processed into ma-

ture acidic and basic subunits, thus it was necessary to

examine whether the glutelin-HRP fusion proteins were pro-

cessed after insertion of the exogenous peptide sequences. As

Fig. 2. Dot blot analysis of Glu-HRP fusion proteins in transgenic rice seedsGluA2-HRP/GluC-HRP (B) were spotted onto a nitrocellulose membrane. Tsequentially diluted standard Glu-HRP proteins (between 2.5 and 100 ng) pucorresponding constructs and NT stands for non-transgenic rice cv. Kita-ak

shown in Fig. 3B, there are three bands representing the pre-

cursor, acidic and basic subunits with molecular masses of

55, 33 and 22 kDa, respectively, in case of the pGluB1-HRP

construct (Fig. 3B, lanes 2 and 3). On the other hand, four

bands were detected for the pGluA2-HRP/GluC-HRP con-

struct, which represented the GluC-HRP (56 kDa) and

GluA2-HRP (55 kDa) precursors, GluC-HRP (34 kDa) and

GluA2-HRP (33 kDa) acidic subunits, respectively (Fig. 3B,

lanes 4 and 5). Since the molecular masses of the acidic sub-

units of GluC-HRP and GluA2-HRP were very close, it was

difficult to separate them by SDS–PAGE. After the same mem-

brane was re-blotted with anti-GluC antibody and the electro-

phoretic mobility was compared carefully, it was apparent that

the upper band was derived from GluC-HRP, and the lower

one was from GluA2-HRP (Data not shown). These results

indicate that all three of glutelin-HRP fusion proteins were

processed normally in rice endosperm tissues.

3.2. Anti-hypertensive activity of glutelin-HRP after oral

administration in SHRs

To eliminate the possibility of confounding variables due to

the presence of starch, albumin, globulin and prolamin in rice

seeds, and to evaluate the anti-hypertension activity of the mod-

ified glutelin-containing RRPLKPWQ peptides, we isolated a

crude glutelin fraction that contained both endogenous glutelin

and the GluB1-HRP glutelin fusion protein from mature seeds.

As shown in Fig. 4A, the glutelin fraction significantly lowered

systolic blood pressure at the maximum of �28 ± 7 mmHg in

SHRs at 4 h after oral administration at dose of 30 mg of total

glutelin protein/kg rat weight, which is equivalent to 2.2 mg of

GluB1-HRP fusion protein/kg or 140 lg of RPLKPW peptide/

kg. A half-dose (equivalent to 70 lg the peptide) gave a maxi-

mum decrease at 2 h after oral administration with a propor-

tional reduction (�12 ± 2.2 mmHg) (Fig. 4B). This result may

suggest a dose-dependent relationship to anti-hypertensive

activity. It is notable that the glutelin fraction isolated from

kita-ake seeds as a control showed no effect at any time or dose.

To further examine whether transgenic rice seeds possess

anti-hypertensive function without any purification steps, 1 g

. Extracts (1 lg of proteins) of mature seeds from GluB1-HRP (A) andhe fusion protein level in each dot was determined by comparing withrified from E. coli cells. The number below each dot indicates lines of

e.

Fig. 3. Analysis of transgenic rice expressing the RPLKPW peptide. (A) Southern blot analysis. Genomic DNA isolated from young leaves wasdigested with SacI (line 6 of GluB1-HRP) and XbaI (line 30 of GluA2-HRP/GluC-HRP) and hybridized with a 32P-labelled hpt probe. Non-transgenic rice cultivar Kita-ake served as control. (B) Western blot analysis. Total proteins extracted from two independent homozygous seeds ofGluB1-HRP (line 6) (lanes 2 and 3) and GluA2-HRP/GluC-HRP (line 30) (lanes 4 and 5), and from one seed of non-transgenic cv. Kita-ake (lane 1)were separated by 12% SDS–PAGE and immuno-detected with anti-RPLKPW antibody.

Fig. 4. Anti-hypertensive activities of GluB1-HRP fusion protein and transgenic rice seeds of GluA2-HRP/GluC-HRP. (A) Oral administration of30 mg/kg (equivalent to 140 lg of RPLKPW peptide/kg), and (B) 15 mg/kg (equivalent to 70 lg of the peptide/kg) of a crude glutelin fraction(containing endogeous glutelin and GluB1-HRP fusion protein); (C) oral administration of 1 g/kg (equivalent to 470 lg of peptide/kg) of unpolishedpulverized GluA2-HRP/GluC-HRP rice seeds. Solid square, GluB1-HRP glutelin fraction or GluA2-HRP/GluC-HRP seeds; Blank square, glutelinfraction or seeds from Kita-ake. *P < 0.05, **P < 0.01, ***P < 0.001.

3318 L. Yang et al. / FEBS Letters 580 (2006) 3315–3320

of pulverized GluB1-HRP seeds were directly administrated to

SHRs, their systolic blood pressure was somewhat reduced to

certain level but with no statistical significance (data not

shown). Therefore, pulverized GluA2-HRP/GluC-HRP seeds

with higher amounts of RRPLKPWQ peptide were used for test

instead. As shown in Fig. 4C, the systolic blood pressure of

SHRs was reduced an average of �15.6 ± 4.8 mmHg at 2 h

after oral administration at a dose of 1 g grain/kg, which corre-

sponds to 9.5 mg of the fusion protein/kg or 470 lg of

RPLKPW peptide/kg.

4. Discussion

Hypertension is a worldwide epidemic and is one of the most

important risk factors leading to other health problems. Apart

from medical treatment, lifestyle changes and dietary manage-

ment may be effective in reducing blood pressure in hyperten-

sion patients or prehypertension candidates. In this study, a

new approach for prevention of hypertension was attempted

by taking advantage of transgenic rice plants that accumulate

active anti-hypertensive peptides.

It has been reported that the production of small peptides of

less than 30 amino acids is difficult in plants, possibly due to

degradation by endogenous proteases or silencing by suppres-

sion or co-suppression mechanisms [17]. These problems have

been resolved by expressing the peptide as a part of common

seed storage proteins, inserted into the variable regions

[16,21,22]. In this study, we enhanced the accumulation levels

by introduction of optimized RRPLKPWQ sequence into the

variable regions of glutelins and expressed the fusion proteins

under the control of three strong rice storage protein glutelin

promoters [15]. The fusion proteins accounted for around

10% of the total seed protein (Fig. 2). It is notable that the fu-

sion proteins processed properly into a mature form with

acidic and basic subunits (Fig. 3B). These data suggest that

replacement of sequences in the variable region of glutelin does

not affect the sorting of glutelin-HRP fusion proteins into pro-

tein storage vacuoles (PBII), since only proteins that are cor-

rectly localized can be processed and folded to the stable and

L. Yang et al. / FEBS Letters 580 (2006) 3315–3320 3319

mature form. De Jaeger et al. [23] reported that use of common

bean seed storage protein arcelin 5-1 and b-phaseolin promot-

ers enhanced the accumulation level of functional murine scFv

to 12.5% and 36.5% of total soluble protein in transgenic dicot-

yledonous seeds, thus demonstrating that these seed storage

promoters provide a promising means for large-scale produc-

tion of economically useful recombinant products.

It has been reported that the effective dose required for

reduction of blood pressure of SHRs is 100 lg/kg [4]. In this

study, administration of 2.2 mg and 1.1 mg of GluB1-HRP fu-

sion protein (equivalent to 140 lg and 70 lg of the RPLKPW

peptide, respectively) to SHRs lowered the systolic blood pres-

sure significantly (Figs. 4A and B). The results indicated that

the RPLKPW sequence was released from fusion proteins

properly in small intestine by flanking the RPLKPW peptide

sequences with R and Q residues. When GluB1-HRP (contain-

ing five copies of RPLKPW sequences) and GluA2-HRP/

GluC-HRP (having eight copies of RPLKPW sequences) seeds

were administrated to SHRs at a dose of 1 g grain, only the lat-

ter reduced the systolic blood pressure significantly. The results

suggest that improvement of anti-hypertensive activity of

transgenic seeds can be achieved by increasing the accumula-

tion levels as well as copies of inserted RPLKPW peptide in fu-

sion proteins. The reason for much higher dose requirement

(equivalent to 470 lg/kg of the RPLKPW peptide) in case of

seeds is unclear (Fig. 4C), one possible explanation is that

seeds may not be easily digested and assimilated in an un-

cooked form in the digestive organs after oral administration.

The transgenic rice generated in this study is functional for

reducing blood pressure of SHRs after oral administration.

Our results showed that both the glutelin fraction containing

glutelin-HRP fusion proteins and the unpolished transgenic

rice seeds effectively reduced systolic blood pressure (Fig. 4).

At present, we are crossing high expression transgenic rice lines

to increase the accumulation of glutelin-HRP fusion proteins.

Primary results indicate that hybrid lines have about 50% high-

er levels than their parental lines. Alternatively, an attempt to

accumulate multiple RRPLKPWQ peptides directly in rice

seed is in progress. Our purpose in this study is to produce hy-

per-expression rice plants that can potentially serve as an effec-

tive food supplement for hypertension patients.

Plant-derived biopharmaceuticals should meet the same

standards of safety and performance as other production sys-

tems. However, one of the public concerns is the presence of

antibiotic resistance genes or their products in edible parts of

genetically modified crops. It can be overcome by using an

antibiotic-free selection system like a MAT-vector system

[24]. The other major concern is the possible contamination

of non-transgenic crop seeds by out-crossing with transgenic

pollens in the field or in the process of transportation. There-

fore, methods for transgene containment are required to be

developed. Although further investigation with regard to the

safety evaluation such as allergenicity and chronic toxicity re-

mained to be performed, incorporation of the bioactive pep-

tide into rice storage proteins could assume a greater role for

dietetics of hypertension in the near future.

Acknowledgments: We thank Ms. M. Utsuno, Ms. X. Wang, Ms. H.Ito and Ms. M. Yahara for technical assistance. This work was sup-ported by a grant of development and demonstration of crop genomicbreeding technology from the Ministry of Agriculture, Forestry andFishery of Japan to F. Takaiwa and M. Yoshikawa, and Bio-orientedTechnology Research Advancement Institution (BRAIN) to M.Y.

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