The International Journal of Developmental Biology

Int. J. Dev. Biol. 56: 351 - 356 (2012)

https://doi.org/10.1387/ijdb.123514sm

Vol 56, Issue 5

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

Technical Article | Published: 26 June 2012

Shinya Matsukawa1, Yuki Moriyama2, Tadayoshi Hayata3, Haruka Sasaki1, Yuzuru Ito4, Makoto Asashima4 and Hiroki Kuroda*,1,2

1Department of Education (Sciences), Shizuoka University, 2Graduate School of Science and Technology, Shizuoka University, Shizuoka, 3Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo and 4Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan

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-beta 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

KDEL, ER retention signal, Nodal, BMP, TGF-beta superfamily

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