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 language | English |
|---|---|
| Pages (from-to) | 351-356 |
| Number of pages | 6 |
| Journal | International Journal of Developmental Biology |
| Volume | 56 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - 2012 |
| Externally published | Yes |
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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 journal › Article
}
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 -