Int. J. Dev. Biol. 56: 351-356 (2012)
doi: 10.1387/ijdb.123514sm
www.intjdevbiol.com

KDEL tagging: a method for generating dominant-negative
inhibitors of the secretion of TGF-b superfamily proteins
SHINYA MATSUKAWA1, YUKI MORIYAMA2, TADAYOSHI HAYATA3, HARUKA SASAKI1, YUZURU ITO4,
MAKOTO ASASHIMA4 and HIROKI KURODA1,2,*
1
Department 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 Scienceand 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 thismolecular 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-b superfamily proteins. First, we tested this method on the Nodal protein Xnr5, which is awell-studied mesoderm inducer in vertebrates. Tagging of Xnr5 protein with KDEL at the carboxyterminus 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 ofplasma membrane or secreted proteins of a multimeric nature.

KEY WORDS: KDEL, ER retention signal, Nodal, BMP, TGF-b superfamily

Introduction
In eukaryotic cells proteins destined for the secretory pathway
have a 5–15-amino acid-long hydrophobic fragment, also known asa signal sequence, at the amino-terminal end (Blobel, 1980). Thissignal sequence allows the proteins to be translocated into the ER
lumen, and the proteins exported from the ER then enter the firstcis-cisterna of the Golgi apparatus. In the Golgi apparatus, further
posttranslational modification occurs, and the trans-cisterna of theGolgi apparatus, which contains ready-to-use proteins, forms thetrans-Golgi network and secretory granules that eventually fusewith the plasma membrane (Fig. 1A). However, some proteins do
not have the ability to traffic to the extracellular space, although
they contain a signal sequence at the amino-terminal end. Many
ER-retained proteins such as chaperones and folding enzymes
contain a carboxy-terminal signal sequence designated the ER
retention signal motif Lys-Asp-Glu-Leu (KDEL) (Munro et al., 1987).
In eukaryotes, for example, calreticulin (Crt), protein disulfide

isomerase (PDI), and the 78-kDa and 94-kDa of glucose-regulated
proteins (GRP78 and GRP94) are retained in the ER by the KDEL
sequence. Yeasts and plants use His-Asp-Glu-Leu (HDEL) insteadof KDEL, and a few other related sequences, including KEEL in
ERp72, HNEL in the LDL-receptor associated protein, HSEL in
Canopy1 (Hirate et al., 2006), and HDEF in Calumenin have been
described (reviewed in Capitani et al., 2009). Interestingly, all of these
KDEL-like sequences seem to act in an almost identical manner,and thus, they might be interchangeable. More importantly, they
enable ER proteins to bind to a seven-transmembrane-spanning
protein that is considered as a KDEL receptor, and known to cycle
between the ER and Golgi apparatus (Fig. 1A, B).
The transforming growth factor-b (TGF-b) superfamily consistsof a large number of structurally related, dimeric proteins that are
Abbreviations used in this paper: BMP, bone morphogenetic protein; Crt, calreticulin;
ER, endoplasmic reticulum; KDEL, Lys-Asp-Glu-Leu motif; TGF, transforming
growth factor.

*Address correspondence to: Hiroki Kuroda. Department of Education (Sciences), Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
Tel: +81-54-238-4304. Fax: +81-54-287-6693. e-mail: ehkurod@ipc.shizuoka.ac.jp - web: http://www.ipc.shizuoka.ac.jp/~ehkurod
Accepted: 11 April 2012. Final, author-corrected PDF published online: 26 June 2012. Edited by: Edward De Robertis

ISSN: Online 1696-3547, Print 0214-6282© 2012 UBC Press
Printed in Spain

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secreted into the extracellular matrix. They act as either hormones,
growth factors, or paracrine mediators, to regulate various typesof biological events in animals, including cell proliferation and differentiation, developmental patterning and morphogenesis, anddisease pathogenesis (Massagué, 1998). Because of their biological and medical importance, developing a method capable ofeffectively inhibiting the activities of TGF-b superfamily molecules
has always been a goal of many cell biologists. Currently, several
assays are in use: a small molecule called dorsomorphin selectively
blocks bone morphogenetic protein (BMP) type I receptors (Yu etal., 2008); the skeletal integrity of mice is improved by adding thetype II activin receptor soluble extracellular domain (Pearsall et al.,2008), and the secretion of Xnr5 is blocked by a cleavage mutant
of Xnr5 (Onuma et al., 2005).
In this study, we introduce a novel and simple approach for
inhibiting TGF-b superfamily proteins, which are first synthesizedAB

as larger precursor proteins containing a propeptide region that isremoved prior to secretion, and a mature region that is dimerized
by disulfide bonds to attain biological activity when the protein issecreted (Kingsley, 1994). On the basis of this phenomenon, we
hypothesized that the artificially KDEL-conjugated TGF-b superfamily proteins may dimerize with their endogenous counterparts.
If this occurred, the target TGF-b superfamily molecules wouldbind to the KDEL receptor, which would confine them inside thecell, with the resultant protein complex shuttling between the ER
and Golgi apparatus. These trapped TGF-b superfamily proteinswould not be secreted, and would, therefore, be absent from theextracellular space (Fig. 1B). Thus, here, we show that this, indeed,is the case, and that KDEL-tagged subunits are potent dominantnegative reagents. Therefore, the KDEL-tag trap assay could be
widely applicable to inhibit the function of multimeric secreted orplasma membrane proteins.

Experimental Procedures
To conjugate the KDEL motif to the carboxy-terminal end of atarget protein, six nucleotides encoding EL were added to the 5’
end of the forward primer, followed by a stop codon sequence,
while KD was added to the reverse primer (Fig. 1C). Plasmids
pCS2-Xnr5 and both pCS2-BMP4 and pCS2-ADMP, used for creating KDEL-tag mutants, have been described in previous reports(Takahashi et al., 2000; Reversade et al., 2005). The primers 5’
GAACTCTAACTCGAGCCTCTAGAACT 3’ and 5’ GTCCTTATAACATCCACATTCCTCTAC 3’ were used for constructing Xnr5-KDEL.
The primers 5’ GAACTCTGAGTCTGAGATCCAAACAAAA 3’ and
5’ GTCCTTACGGCACCCACACCCA 3’ were used for constructing BMP4-KDEL. The primers 5’ GAACTCTAACTCGAGCCTCTAGAACT 3’ and 5’ GTCCTTGTGGCACCCGCAG 3’ were used
for constructing ADMP-KDEL. The underlined sequences in theprimers were used for adding KD or EL. We performed long-length
polymerase chain reaction involving these primers and high-fidelity
DNA polymerase (KOD-plus Neo, Toyobo, Japan). The 5’ ends ofthe DNA fragments were phosphorylated using T4 polynucleotide
kinase, followed by self-ligation with T4 DNA ligase (Fig. 1C). The
mRNAs used for microinjection experiments were transcribed fromlinearized plasmids by using an mMessage mMachine kit (Ambion).

Results & Discussion

C

Fig. 1. Schematic representation of the KDEL-tag trap assay. (A) Normal pathway of secreted homodimer proteins such as TGF-b superfamily
proteins. (B) Scheme of the KDEL-tag trap assay. An experimentally generated carboxy-terminal KDEL-conjugated subunit may bind subunits in thecomplementary endogenous heterodimeric protein via the formation ofa disulfide bond. The resultant protein complex should then be trappedby the KDEL receptor (KDEL-R), resulting in its continuous shuttling between the ER and Golgi apparatus. (C) A method to conjugate KDEL tothe carboxy-terminal end of target molecules. Only a single long-range
polymerase chain reaction is required.

The KDEL-tag trap assay blocks Nodal signaling
Only one polymerase chain reaction is required to conjugate thefour KDEL amino acids at the carboxy-terminal end of a target protein(Fig. 1C). To evaluate whether the mutated molecules constructedby this simple method actually function, we used our experimental model Xenopus Nodal-related 5 (Xnr5), a TGF-b superfamily
Nodal protein that functions during Xenopus embryogenesis. Nodal
proteins, which belong to the TGF-b superfamily, are known to be
essential for mesoderm inductions during vertebrates embryonicdevelopment (De Robertis et al., 2004; De Robertis, 2009). In the
Xenopus species, Xnr5 is expressed on the dorsal-vegetal sideof blastula embryos prior to any other players in mesoderm inductions (Takahashi et al., 2000). Therefore, the mesoderm would
fail to develop if the secretion step in Xnr5 synthesis were to be
inhibited using the KDEL-tag trap assay. To study the location of
Xnr5 protein during the early developmental stages in Xenopus

KDEL-tag trap assay

A

Fig. 2. KDEL-conjugated Nodal protein inhibits the secretion of epitopetagged 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 °C 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 concentrationof 1% (middle panel). (B) Translocation of myc-tagged Xnr5 protein fromthe 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).

Bembryos, we injected myc-tagged Xnr5 mRNA with orwithout KDEL-conjugated Xnr5 (Xnr5-KDEL) mRNA
into these embryos, cut presumptive ectodermal
regions generally called animal caps (AC), andcollected secreted proteins from the supernatant,cytoplasmic proteins from the harvested cell pellet,
or proteins solubilized using Triton X-100 detergent
in the cellular fraction (Fig. 2A). As expected, thesecretion of Xnr5 protein was inhibited by Xnr5-KDEL
in a dose-dependent manner (Fig. 2B, top). It wasalso confirmed that the nonsecreted Xnr5 protein
remained in the cell pellet (Fig. 2B, middle), while
the positive control cytoplasmic ERK1/2 was notaffected (Fig. 2B, bottom). In addition, visualization of the protein fractions secreted on the SDSPAGE gel by using Coomassie blue showed that
Xnr5-KDEL did not change the amounts of otherkinds of secreted proteins (data not shown). Thesebiochemical results indicated that the KDEL-tag
trap assay worked during the secretion process ina target-specific manner.
We next investigated whether Xnr5-mediatedmesoderm induction might be inhibited by this assay.
In amphibian embryos the pigmented blastopore on
the surface of the dorsal-vegetal side appears as
the first visible sign of mesoderm formation in theearly gastrula stage. The blastopore then extends
to the ventral side, and becomes a circle by the
mid-gastrula stage (Fig. 3A). The size of this circle,
whose inside is generally known as the yolk plug, isgradually reduced during gastrulation movements
by the process called epiboly, and is eventuallyclosed by the beginning of the neurula stage. As
expected, this blastopore formation was influenced
by the KDEL-tag trap assay for Xnr5. In the embryos
injected with a low amount of Xnr5-KDEL mRNA,353

the yolk plug region was expanded, and the blastopore appearedslightly irregular (Fig. 3B). In case of high amounts of Xnr5-KDEL,
the ventro-lateral blastopore was not formed, and only a short,pigmented line was observed on the dorsal side (Fig. 3C). In addition, the expression of Xbrachyury (Xbra) (Smith et al., 1991),
which is a pan-mesodermal marker expressed at the gastrula stage,
was completely suppressed by the KDEL-tag trap assay for Xnr5
(Fig. 3D, E). In later stages, mesodermal and axial defects were
induced by Xnr5-KDEL mRNA in a dose-dependent manner (Fig.
3 F–H). Those phenotypes induced by the KDEL-tag trap assay
were very similar to the effects caused by Cerberus-short (Cer-S)

A

F

B

C

D

E

G

H

I

J

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 byusing in situ hybridization analysis for the pan-mesodermal marker Xbra in the embryos atstage 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.

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S. Matsukawa et al.

(Piccolo et al., 1999), a truncated form of Cerberus that functions
as a specific Nodal antagonist (Fig. 3I, compare to 3H). Specificinhibition of Xnr5 can also be provided by a cleavage mutant of
Xnr5, which lacks the RXXR cleavage site for the convertase
proteases located between the propeptide and mature domains(Onuma et al., 2005)); despite this, the efficiency of the inhibitory
action of the KDEL-tag trap assay is almost 30 times higher than

A

B

C

D

E

F

G

H

I

J

K

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, suchas expansion of the cement gland (cg), while posteriorization was inducedby microinjections of the mRNA encoding wild-type BMP4 or ADMP (each20 pg). (G,H) The effects of KDEL-conjugated BMPs were rescued by theoverexpression 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.

that of Cer-S, and 60 times higher than a cleavage type of mutant.
To experimentally obtain complete inhibition of mesoderm formation
as shown in Fig. 3H, which was induced by 16 pg of Xnr5-KDEL
mRNA, more than 500 pg of Cer-S mRNA or 1 ng of mRNA encoding a cleavage mutant of Xnr5 are usually required (Piccolo
et al., 1999; Onuma et al., 2005). This suggests that the proteinsconjugated with KDEL at the the carboxy-terminally might bindwild-type molecules with very high affinity and interfere with thenormal pathway of secretion of protein dimers to the extracellularspace. Moreover, similar to the effects of Cer-S, we found thatthe expression of major Nodal target genes such as Mix1, Mixer,
Sox17, and Xbra was inhibited by the co-injection of Xnr5-KDEL
mRNA (Fig. 3J). These results demonstrate that the KDEL-tag trap
assay is very effective at inhibiting Nodal activity during Xenopus
embryogenesis. In these experiments it was unclear whether other
Nodal proteins such as Xnr1, Xnr2, Xnr4, and Xnr6 were also
targeted by Xnr5-KDEL; nevertheless, its strong inhibitory effects
suggest that this is highly possible.
An HDEL sequence is the other major version of ER retention
signals, and it is broadly used in plants and yeasts (Capitani etal., 2009). Phenotypical analysis in Xenopus demonstrated that
almost the same events reported above for Xnr5-tagged KDEL
occurred with Xnr5 that had been tagged with the HDEL motif(data not shown). In zebrafish, both Canopy1-HSEL, which is theendogenous signal in Canopy1-mediated regulation of FGF signaling, and the experimentally generated Canopy1-KDEL (Hirate
and Okamoto, 2006) have similar functions, as was the case in ourstudy. This suggests that KDEL variants might function identicallyin the vertebrate secretory pathway.
The KDEL-tag trap assay is applicable to BMPs
BMPs were originally discovered on the basis of their ability
to induce bone and cartilage formation, and are now known tomediate various biological events (Reddi, 1997). In total, 12 of
BMP-like molecules are members of the TGF-b superfamily andhave been well studied as central players in neural formation anddorsoventral patterning during early embryogenesis (De Robertis
et al., 2004; De Robertis, 2009). We, therefore, tried to identify
whether the KDEL-tag trap assay was adaptable to BMPs. In vertebrates, spatially opposed expression of the BMP family proteins
BMP4 and antidorsalizing morphogenetic protein (ADMP), which ismost closely related to human BMP3, can promote restoration of
dorsovent of ventral structures during Xenopus embryogenesis. As
shown in Fig. 4A, we designed BMP4-KDEL and ADMP-KDEL toblock ventral and dorsal BMP signaling, respectively. As previously
reported (Moos et al., 1995; Reversade et al., 2005), the microinjection of normal BMP4 or ADMP mRNA in our studies induced
ventralization in Xenopus embryos (Fig. 4 B-D). In contrast, both
BMP4-KDEL and ADMP-KDEL mRNA did not induce ventralization (Fig. 4 E,F). Instead, BMP4-KDEL mRNA caused strong dorsalization with big cement gland formation, which is a feature of
dorsalization (Fig. 4E). ADMP-KDEL mRNA also induced weakerdorsalization, albeit in a weaker manner BMP4-KDEL, with minor
expansion of the cement gland (Fig. 4F). Similar to Nodal proteins,cleavage mutants for BMP have also been reported (Hawley etal., 1995), but our method seems to be more effective. It is likelythat entire ligand family proteins such as BMP2 and BMP7 were
inhibited using our method. We also confirmed that the effects of
the KDEL-conjugated proteins were rescued by co-injecting theKDEL-tag trap assay

355

it may also be applicable in studies involving different moleculesin various organisms. In future, it will be particularly interesting todetermine whether our assay can block the trafficking of multimeric plasma membrane receptors to the cell surface. The usesof this KDEL-tag trap assay should, therefore, allow researchers
in molecular and cellular biology to readily generate new tools
for inhibiting the activities of target dimeric or polymeric secreted
proteins or membrane receptors.

Fig. 5. BMP4-KDEL inhibits BMP4-induced transcriptional activation ina 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 reducedby co-transfection with pCS2-BMP4-KDEL in a dose-dependent manner.

tagged protein with mRNA encoding wild-type protein (Fig. 4 G,H).
Moreover, we showed that co-injection with mRNA encoding a
constitutively-active form of human SMAD1 (cahSMAD1) overcame
all upstream effects of BMP4 and BMP4-KDEL, resulting in normal
ventralization with small head (Fig. 4I). The cahSMAD1 protein has
phosphorylation-resistant mutations in its SMAD1 linker region,and is not affected by ERK/MAPK and GSK3, or phospho-mimetic
mutations of SMAD1, in which C-terminal SVS phosphorylationsites are mutated into EVE (Fuentealba et al., 2007). Moreover,the expression of BMP target genes such as Sizzled and Vent was
blocked by BMP4-KDEL and rescued by cahSMAD1 mRNA (Fig.
4K). As expected, co-injection of BMP4-KDEL and ADMP-KDEL
mRNAs into all four blastomeres at the four-cell stage induced
the formation of bigger cement gland and stronger dorsalization(Fig. 4J) compared to the induced phenotypes, following separate
injections of either BMP4-KDEL or ADMP-KDEL (Fig. 4 E,F).
Finally, we wished to determine whether this system also
worked in other cell systems, because the roles of BMP signaling
in cultured cells have been extensively studied in recent years byusing loss-of-function assays (Yu et al., 2008; Pearsall et al., 2008).
When mouse MC3T3-E1 osteoblast cells were transfected with aplasmid encoding the BMP-responsive element fused to the firefly
luciferase reporter (BRE-luc) gene (von Bubnoff et al., 2005), andstimulated by cotransfection with pCS2-BMP4, or a combinationof pCS2-BMP4 and pCS2-BMP4-KDEL, we observed significant
inhibition of the BMP signal with BMP4-KDEL (Fig. 5). These
results indicate that the KDEL-tag trap assay might be applicableto various cell biological systems.
In the present study, we have demonstrated a novel, yet simple
and very effective technique for generating dominant-negative
proteins, by conjugating the KDEL motif to the carboxy-terminalend of TGF-b superfamily proteins amino acid sequences. In thissystem, we used only one long-range polymerase chain reaction
requiring two primers, each having six additional nucleomers, foradding the K–D or E–L motifs (Fig. 1C). Since many studies havebeen focused on Nodal and BMP proteins in general, the reagentsdescribed in this study will be, at a minimum, useful to cell biologists who study these molecules. Furthermore, as we confirmed
our technique by using both microinjection and cell culture assays,

Acknowledgements
We thank Professor Shuji Takahashi, Yoshiki Sasai, Jim Smith, andCamille Bouissou for providing DNA constructs; Miduki Nishimura, Tsubasa
Mishiku, and Nana Saito for providing technical assistance, and Shoko
Mori and Yoshihisa Ohata for their valuable comments on the manuscript.
This research was supported by a grant-in-aid received from the Shizuoka
Research Institute.

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