title: Antihypertensive activity of transgenic rice seed containing an 18-repeat novokinin peptide localized in the nucleolus of endosperm cells.
abstract: Novokinin (Arg-Pro-Leu-Lys-Pro-Trp, RPLKPW) is a new potent antihypertensive peptide based on the sequence of ovokinin (2-7) derived from ovalbumin. We previously generated transgenic rice seeds in which eight novokinin were fused to storage protein glutelins (GluA2 and GluC) for expression. Oral administration of these seeds to spontaneously hypertensive rats (SHRs) reduced systolic blood pressures at a dose of 1 g seed/kg of SHR. Here, 10- or 18-tandem repeats of novokinin with an endoplasmic reticulum (ER) retention signal (Lys-Asp-Glu-Leu, KDEL) at the C terminus were directly expressed in rice under the control of the glutelin promoter containing its signal peptide. Only small amounts of the 18-repeat novokinin accumulated, and it was unexpectedly deposited in the nucleolus. This abnormal intracellular localization was explained by an endogenous signal for nuclear localization. The GFP reporter protein fused to this sequence targeted to nuclei by a transient assay using onion epidermal cells. Transgenic seed expressing the 18-repeat novokinin exhibited significantly higher antihypertensive activity after a single oral dose to SHR even at one-quarter the amount (0.25 g/kg) of the transgenic rice seed expressing the fusion construct; though, its novokinin content was much lower (1/5). Furthermore, in a long-term administration for 5 weeks, even a smaller dose (0.0625 g/kg) of transgenic seeds could confer antihypertensive activity. This high antihypertensive activity may be attributed to differences in digestibility of expressed products by gastrointestinal enzymes and the unique intracellular localization. These results indicate that accumulation of novokinin as a tandemly repeated structure in transgenic rice is more effective than as a fusion-type structure.
authors.affiliation: Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Kannondai 3-1-3, Tsukuba, Ibaraki, Japan.
authors.forename: Yuhya
authors.initials: Y
authors.lastname: Wakasa
authors.forename: Hui
authors.initials: H
authors.lastname: Zhao
authors.forename: Sakiko
authors.initials: S
authors.lastname: Hirose
authors.forename: Daiki
authors.initials: D
authors.lastname: Yamauchi
authors.forename: Yuko
authors.initials: Y
authors.lastname: Yamada
authors.forename: Lijun
authors.initials: L
authors.lastname: Yang
authors.forename: Kousaku
authors.initials: K
authors.lastname: Ohinata
authors.forename: Masaaki
authors.initials: M
authors.lastname: Yoshikawa
authors.forename: Fumio
authors.initials: F
authors.lastname: Takaiwa
mesh_terms: D000284:Administration, Oral; D000818:Animals; D000959:Antihypertensive Agents / Q000008:administration & dosage*; D001794:Blood Pressure / Q000187:drug effects; D002466:Cell Nucleolus / Q000737:chemistry; D056625:Endosperm / Q000737:chemistry; D005983:Glutens; D049452:Green Fluorescent Proteins; D006973:Hypertension / Q000188:drug therapy*; D008297:Male; D009842:Oligopeptides / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics; D012275:Oryza / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D010194:Pancreatin / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011918:Rats, Inbred SHR; D011993:Recombinant Fusion Proteins; D012639:Seeds / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D020080:Tandem Repeat Sequences; D013997:Time Factors
chemical_list: D000959:Antihypertensive Agents; C511871:Arg-Pro-Leu-Lys-Pro-Trp; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005983:Glutens; D010194:Pancreatin; D010434:Pepsin A