Int J Dev Biol
January 1, 2012;
KDEL tagging: a method for generating dominant-negative inhibitors of the secretion of TGF-beta superfamily proteins.
Most endoplasmic reticulum
)-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
, 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
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.
Int J Dev Biol
Nodal 5.2 Ab1
[+] show captions
Fig. 2. KDEL-conjugated Nodal protein inhibits the secretion of epitope-tagged Nodal protein. (A) Diagram illustrating the preparation of protein solutions. Animal caps were cut from embryos injected with mRNAs, dissociated in calcium- and magnesium-free solution (CMF), cultured for 8 h at 14 in CMF, and centrifuged at 1,000 g for 1 min. Extracellular or cytoplasmic fractions were prepared from the supernatant or precipitate. The Triton X-100-treated cytoplasmic (TTC) fraction, comprising proteins inside the ER, was generated by adding Triton-X100 to a final concentration of 1% (middle panel). (B) Translocation of myc-tagged Xnr5 protein from the extracellular space (top fraction) to the ER space (middle fraction) following the addition of Xnr5-KDEL. ERK1/2 in the intracellular fraction was examined as a positive control (bottom).
Fig. 3. Mesoderm formation is inhibited by microinjection of Xnr5-KDEL mRNA. (A-E) Inhibition of mesoderm formation by using microinjection of Xnr5-KDEL mRNA (4 or 16 pg), as demonstrated by monitoring the presence of defects in the blastopore, as well as by using in situ hybridization analysis for the pan-mesodermal marker Xbra in the embryos at stage 11 of embryogenesis. (F-I) Resultant Cer-S mRNA-mediated phenotypic effects on the embryos that fail to develop mesoderm (shown in I) were also confirmed in embryos at later stages, as shown in (G,H). (J) The expression of all Nodal target markers such as Mix1, Mixer, Sox17, and Xbra was blocked following microinjection of Xnr5-KDEL mRNA. EF1a was used as a positive control. Total mRNA was prepared from the embryos at stage 9 of embryogenesis.
Fig. 4. Effect of the KDEL-tag trap assay on BMP signaling. (A) Schematic diagram showing ventrally expressed BMP protein known as BMP4, and dorsally expressed ADMP in the early gastrula embryonic stage. (B-F) Microinjections of the mRNA encoding KDEL-conjugated BMP4 (400 pg) or ADMP (400 pg) caused typical phenotypic effects of low-BMP activity, such as expansion of the cement gland (cg), while posteriorization was induced by microinjections of the mRNA encoding wild-type BMP4 or ADMP (each 20 pg). (G,H) The effects of KDEL-conjugated BMPs were rescued by the overexpression of wild-type mRNAs. (I) Microinjection of mRNA encoding a constitutively active form of human SMAD1 (cahSMAD1, 400 pg) overcame the rescue effect as shown in G, indicating that the effects of KDEL-conjugated molecules occur upstream of SMAD1. (J) The anti-BMP effects increased with the co-injection of both mRNAs than with separate injections of the individual KDEL-traps. (K) Expression of the ventral BMP target markers Sizzled and Vent was blocked by the microinjection of BMP4-KDEL mRNA, and rescued by the co-injection of mRNA encoding cahSMAD1. EF1a was used as a loading control.
Fig. 5. BMP4-KDEL inhibits BMP4-induced transcriptional activation in a concentration-dependent manner on mouse MC3T3-E1 osteoblast cells. In all cases, plasmids were transfected with 0.1 mg of BRE-luc (pOLuc-xId3-BRE) for the luciferase assay. pcDNA3 was used for adjusting the total amounts of transfected DNA to 0.4 mg. Luciferase activity increased following the transfection of pCS2-BMP4, but it was reduced by co-transfection with pCS2-BMP4-KDEL in a dose-dependent manner.