KDEL tagging: A method for generating dominant-negative inhibitors of the secretion of TGF-β superfamily proteins

Shinya Matsukawa, Yuki Moriyama, Tadayoshi Hayata, Haruka Sasaki, Yuzuru Ito, Makoto Asashima, Hiroki Kuroda

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Most endoplasmic reticulum (ER)-retained proteins contain a carboxy-terminal signal sequence called the ER retention signal motif such as the Lys-Asp-Glu-Leu (KDEL) motif. Using this molecular mechanism, we developed a new dominant-negative assay, designated the KDEL-tag trap assay, to negatively regulate secretion of disulfide bond-dependent protein dimers, as typified by TGF-β superfamily proteins. First, we tested this method on the Nodal protein Xnr5, which is a well-studied mesoderm inducer in vertebrates. Tagging of Xnr5 protein with KDEL at the carboxy-terminus effectively blocked the secretion of Xnr5, resulting in complete inhibition of mesoderm induction in Xenopus embryogenesis. Second, we examined the usefulness of the KDEL-tag trap assay on BMPs, which are well-known negative regulators of neural induction and ventralizing factors during early development, and demonstrated that the functions of the BMP family proteins BMP4 and ADMP were blocked by the KDEL-tag trap assay. Moreover, the technical feasibility of the KDEL-tag trap assay was confirmed in a cell culture system using mouse osteoblasts. Taken together, these results suggest that the KDEL-tag trap assay can be adapted to inhibit a variety of plasma membrane or secreted proteins of a multimeric nature.

Original languageEnglish
Pages (from-to)351-356
Number of pages6
JournalInternational Journal of Developmental Biology
Volume56
Issue number5
DOIs
Publication statusPublished - 2012
Externally publishedYes

Fingerprint

Proteins
Mesoderm
Endoplasmic Reticulum
lysyl-aspartyl-glutamyl-leucine
Nodal Protein
Protein Sorting Signals
Xenopus
Osteoblasts
Disulfides
Embryonic Development
Vertebrates
Cell Culture Techniques
Cell Membrane
5-amino-5-deoxymannopyranoside

Keywords

  • BMP
  • ER retention signal
  • KDEL
  • Nodal
  • TGF-β superfamily

ASJC Scopus subject areas

  • Developmental Biology
  • Embryology

Cite this

KDEL tagging : A method for generating dominant-negative inhibitors of the secretion of TGF-β superfamily proteins. / Matsukawa, Shinya; Moriyama, Yuki; Hayata, Tadayoshi; Sasaki, Haruka; Ito, Yuzuru; Asashima, Makoto; Kuroda, Hiroki.

In: International Journal of Developmental Biology, Vol. 56, No. 5, 2012, p. 351-356.

Research output: Contribution to journalArticle

Matsukawa, Shinya ; Moriyama, Yuki ; Hayata, Tadayoshi ; Sasaki, Haruka ; Ito, Yuzuru ; Asashima, Makoto ; Kuroda, Hiroki. / KDEL tagging : A method for generating dominant-negative inhibitors of the secretion of TGF-β superfamily proteins. In: International Journal of Developmental Biology. 2012 ; Vol. 56, No. 5. pp. 351-356.
@article{fabbcaa087fb41c587341e4a870406d9,
title = "KDEL tagging: A method for generating dominant-negative inhibitors of the secretion of TGF-β superfamily proteins",
abstract = "Most endoplasmic reticulum (ER)-retained proteins contain a carboxy-terminal signal sequence called the ER retention signal motif such as the Lys-Asp-Glu-Leu (KDEL) motif. Using this molecular mechanism, we developed a new dominant-negative assay, designated the KDEL-tag trap assay, to negatively regulate secretion of disulfide bond-dependent protein dimers, as typified by TGF-β superfamily proteins. First, we tested this method on the Nodal protein Xnr5, which is a well-studied mesoderm inducer in vertebrates. Tagging of Xnr5 protein with KDEL at the carboxy-terminus effectively blocked the secretion of Xnr5, resulting in complete inhibition of mesoderm induction in Xenopus embryogenesis. Second, we examined the usefulness of the KDEL-tag trap assay on BMPs, which are well-known negative regulators of neural induction and ventralizing factors during early development, and demonstrated that the functions of the BMP family proteins BMP4 and ADMP were blocked by the KDEL-tag trap assay. Moreover, the technical feasibility of the KDEL-tag trap assay was confirmed in a cell culture system using mouse osteoblasts. Taken together, these results suggest that the KDEL-tag trap assay can be adapted to inhibit a variety of plasma membrane or secreted proteins of a multimeric nature.",
keywords = "BMP, ER retention signal, KDEL, Nodal, TGF-β superfamily",
author = "Shinya Matsukawa and Yuki Moriyama and Tadayoshi Hayata and Haruka Sasaki and Yuzuru Ito and Makoto Asashima and Hiroki Kuroda",
year = "2012",
doi = "10.1387/ijdb.123514sm",
language = "English",
volume = "56",
pages = "351--356",
journal = "International Journal of Developmental Biology",
issn = "0214-6282",
publisher = "University of the Basque Country Press",
number = "5",

}

TY - JOUR

T1 - KDEL tagging

T2 - A method for generating dominant-negative inhibitors of the secretion of TGF-β superfamily proteins

AU - Matsukawa, Shinya

AU - Moriyama, Yuki

AU - Hayata, Tadayoshi

AU - Sasaki, Haruka

AU - Ito, Yuzuru

AU - Asashima, Makoto

AU - Kuroda, Hiroki

PY - 2012

Y1 - 2012

N2 - Most endoplasmic reticulum (ER)-retained proteins contain a carboxy-terminal signal sequence called the ER retention signal motif such as the Lys-Asp-Glu-Leu (KDEL) motif. Using this molecular mechanism, we developed a new dominant-negative assay, designated the KDEL-tag trap assay, to negatively regulate secretion of disulfide bond-dependent protein dimers, as typified by TGF-β superfamily proteins. First, we tested this method on the Nodal protein Xnr5, which is a well-studied mesoderm inducer in vertebrates. Tagging of Xnr5 protein with KDEL at the carboxy-terminus effectively blocked the secretion of Xnr5, resulting in complete inhibition of mesoderm induction in Xenopus embryogenesis. Second, we examined the usefulness of the KDEL-tag trap assay on BMPs, which are well-known negative regulators of neural induction and ventralizing factors during early development, and demonstrated that the functions of the BMP family proteins BMP4 and ADMP were blocked by the KDEL-tag trap assay. Moreover, the technical feasibility of the KDEL-tag trap assay was confirmed in a cell culture system using mouse osteoblasts. Taken together, these results suggest that the KDEL-tag trap assay can be adapted to inhibit a variety of plasma membrane or secreted proteins of a multimeric nature.

AB - Most endoplasmic reticulum (ER)-retained proteins contain a carboxy-terminal signal sequence called the ER retention signal motif such as the Lys-Asp-Glu-Leu (KDEL) motif. Using this molecular mechanism, we developed a new dominant-negative assay, designated the KDEL-tag trap assay, to negatively regulate secretion of disulfide bond-dependent protein dimers, as typified by TGF-β superfamily proteins. First, we tested this method on the Nodal protein Xnr5, which is a well-studied mesoderm inducer in vertebrates. Tagging of Xnr5 protein with KDEL at the carboxy-terminus effectively blocked the secretion of Xnr5, resulting in complete inhibition of mesoderm induction in Xenopus embryogenesis. Second, we examined the usefulness of the KDEL-tag trap assay on BMPs, which are well-known negative regulators of neural induction and ventralizing factors during early development, and demonstrated that the functions of the BMP family proteins BMP4 and ADMP were blocked by the KDEL-tag trap assay. Moreover, the technical feasibility of the KDEL-tag trap assay was confirmed in a cell culture system using mouse osteoblasts. Taken together, these results suggest that the KDEL-tag trap assay can be adapted to inhibit a variety of plasma membrane or secreted proteins of a multimeric nature.

KW - BMP

KW - ER retention signal

KW - KDEL

KW - Nodal

KW - TGF-β superfamily

UR - http://www.scopus.com/inward/record.url?scp=84863918591&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84863918591&partnerID=8YFLogxK

U2 - 10.1387/ijdb.123514sm

DO - 10.1387/ijdb.123514sm

M3 - Article

C2 - 22811269

AN - SCOPUS:84863918591

VL - 56

SP - 351

EP - 356

JO - International Journal of Developmental Biology

JF - International Journal of Developmental Biology

SN - 0214-6282

IS - 5

ER -