Conditional glycosylate in eukaryotic cells using a biocompatible chemical inducer of dimerization

Article abstract of DOI:10.1021/ja8037728

Chemical inducers of dimerization (CIDs) are cell-permeable small molecules capable of dimerizing two protein targets. The most widely used CID, the natural product rapamycin and its relatives, is immunosuppressive due to interactions with endogenous targets and thus has limited utility in vivo. Here we report a new biocompatible CID, Tmp-SLF, which dimerizes E. coli DHFR and FKBP and has no endogenous mammalian targets that would lead to unwanted in vivo side effects. We employed Tmp-SLF to modulate gene expression in a yeast three-hybrid assay. Finally, we engineered the Golgi-resident glycosyltransferase FucT7 for tunable control by Tmp-SLF in mammalian cells. Copyright

Full text of DOI:10.1021/ja8037728

Published on Web 09/13/2008
Conditional Glycosylation in Eukaryotic Cells Using a Biocompatible
Chemical Inducer of Dimerization
Jennifer L. Czlapinski,† Michael W. Schelle,† Lawrence W. Miller,‡,§ Scott T. Laughlin,†
Jennifer J. Kohler,†,| Virginia W. Cornish,‡ and Carolyn R. Bertozzi*,†,#,¶
Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, UniVersity of
California, Berkeley, California 94720, and Department of Chemistry, Columbia UniVersity,
New York, New York 10027
Received May 20, 2008; E-mail: crb@berkeley.edu
The development of small molecules that modulate protein
function in a tunable fashion has been a major focus in chemical
biology. First described by Schreiber and co-workers,1 chemical
inducers of dimerization (CIDs) are cell-permeable, bidentate
molecules capable of dimerizing two substrates. The prototype is
the immunosuppressive natural product rapamycin, which binds
simultaneously2 to the FK506/rapamycin binding protein FKBP3
and a domain of the mTOR protein termed FRB.4 A variety of
biological processes have been probed by rapamycin-induced
dimerization of proteins fused to FKBP and FRB.1,5
We have recently employed the CID technique in studies of
glycobiology.6 Golgi-resident glycosyltransferases and sulfotrans-
ferases comprise discrete catalytic (Cat) and localization (Loc)
domains that are both required for cellular function.7 Taking
advantage of their modular nature, we separated the two domains
and fused them independently to FKBP and FRB. In the absence
of rapamycin, the Cat domain failed to localize to the Golgi
compartment and was therefore unable to access its normal
substrates (Figure 1A). The addition of rapamycin induced het-
erodimerization of the Loc and Cat domains, reconstituting the
enzyme and restoring cellular activity.
Figure 1. Conditional activation of a Golgi glycosyltransferase using a CID.
(A) The membrane-associated Loc and soluble Cat domains are separated and
fused to small molecule binding proteins. In the absence of the CID, the Cat
domain has no mechanism for Golgi retention and is secreted from the cell. In
the presence of the CID, the Cat domain associates with the Loc domain and
is therefore retained in the Golgi compartment where it can act on substrates.
In this depiction, the glycosyltransferase is fucosyltransferase 7, which adds
fucose to a glycan substrate forming sialyl Lewis x. Monosaccharide symbols:
([), sialic acid; (O), galactose; (9), GlcNAc; (2), fucose. (B) Tmp-SLF.
While useful for studies with cultured cells, rapamycin’s interac-
tion with endogenous mTOR leads to undesirable in ViVo side
effects.4 Thus, we sought to create a new CID that does not interfere
with critical endogenous processes. Here we report the development
of a trimethoprim (Tmp) conjugate (Tmp-SLF, Figure 1B) capable
of dimerizing a bacterial dihydrofolate reductase (DHFR) with
FKBP. The compound was employed to modulate transcription in
yeast and glycosylation in mammalian cells.
The new CID replaces the mTOR-binding component of rapa-
mycin-like analogues with a moiety, Tmp, that has no endogenous
protein targets. The compound was modeled after our previously
reported methotrexate (Mtx)-SLF conjugate,8,9 but unlike the
promiscuous DHFR inhibitor Mtx, Tmp exhibits a 12,000-fold
preference for E. coli versus human DHFR.10 Confined within the
Golgi compartment, E. coli DHFR should not perturb endogenous
folate metabolism. The other CID component, SLF, is a synthetic
analogue of the natural product FK50611,12 that lacks FK506’s
immunosuppressive activity,1,13 is cell-permeable, and binds FKBP
with nanomolar affinity.12 The synthesis of Tmp-SLF is described
in the Supporting Information (SI).
Figure 2. Yeast three-hybrid assay of Tmp-SLF activity. (A) Tmp-SLF (or
Mtx-SLF) heterodimerizes the DNA-binding LexA domain-DHFR fusion
protein and a B42-FKBP transcriptional activator fusion protein, effectively
reconstituting a transcriptional activator and activating transcription of the lacZ
reporter gene. (B) Yeast treated with 1 µM Tmp-SLF or Mtx-SLF grown on
X-gal plates. The extent of X-gal hydrolysis (indigo color) provides a measure
of the level of transcriptional activation.
As Mtx-SLF and Tmp-SLF bind the same protein pair (DHFR/
FKBP), we first tested the ability of Tmp-SLF to activate transcription
in a yeast three-hybrid assay (Figure 2A).8,9 Yeast were engineered
to express a B42 transcription activation domain-FKBP (B42-FKBP)
protein chimera and LexA DNA binding domain-DHFR fusion (LexA-
DHFR). A lacZ reporter gene under the control of four tandem LexA
operators was used. The cells were grown on X-gal plates under
standard conditions for 3 days in the presence of 1 µM Tmp-SLF or
Mtx-SLF (Figure 2B). Both Mtx-SLF and Tmp-SLF activated lacZ
transcription in yeast cells. Control strain investigations with previously
† Department of Chemistry, University of California, Berkeley.
‡ Department of Chemistry, Columbia University.
§ Present Address: Department of Chemistry, University of Illinois at Chicago,
Chicago, IL 60607.
| Present Address: Department of Internal Medicine, UT Southwestern Medical
Center, Dallas, TX 75390.
# Department of Molecular and Cell Biology, University of California, Berkeley.
¶ Howard Hughes Medical Institute.
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13186 J. AM. CHEM. SOC. 2008, 130, 13186–13187
10.1021/ja8037728 CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. Effect of Tmp-SLF on FucT7 activity in CHO cells. Cells were
transfected with plasmids encoding the indicated FucT7 constructs. Open bars:
vehicle-treated cells; filled bars: cells treated with 1 µM Tmp-SLF for 24 h.
Cells were then incubated with biotinylated Mab HECA-452 followed by
tricolor-streptavidin and analyzed by flow cytometry. MFI ) mean fluorescence
intensities of all live cells. Error bars ) SD of triplicates.
described FKBP mutants fused to B428 indicate that Tmp-SLF is
potentially more effective as a transcriptional activator than Mtx-SLF
(see SI). In contrast, the previously reported Tmp-Dex CID appeared
less active than Mtx-Dex in the same yeast three-hybrid system.14
Next, we evaluated the ability of Tmp-SLF to activate a Golgi-
resident glycosyltransferase in mammalian cells. Fucosyltransferase
VII (FucT7) adds an R1,3-linked fucose residue to sialyl N-acetyllac-
tosamine to generate the sialyl Lexis x (sLex) epitope.15 The enzyme
participates in the assembly of selectin ligands, thereby playing a critical
role in the immune system. Accordingly, immunosuppressive CIDs
based on rapamycin are not suitable for in ViVo studies of FucT7
function. A more biocompatible CID such as Tmp-SLF would
potentially enable conditional activation of FucT7 in ViVo.
Figure 4. Activation of FucT7 by Tmp-SLF is tunable and can be inhibited
with Tmp. (A) Cells transfected with either Loc-DHFR/3xFKBP-Cat (b) or
Loc-DHFR/3xDHFR-Cat (O) construct pairs were treated with Tmp-SLF at
various concentrations. sLex was probed as in Figure 3. (B) Free Tmp inhibits
the Tmp-SLF-dependent response. MFI ) mean fluorescence intensities of all
live cells. Error bars ) SD of three replicates.
Supporting Information Available: Synthetic procedures and spectral
data for Tmp-SLF, general biological procedures, yeast three-hybrid assays
with FKBP mutants, and plasmid maps for FucT7 fusions. This material is
available free of charge via the Internet at http://pubs.acs.org.
We fused DNA encoding the FucT7 Loc and Cat domains6a
separately to one, two, or three tandem copies of the FKBP or DHFR
gene. Matched pairs of the corresponding chimeric proteins were
transiently expressed in Chinese hamster ovary (CHO) cells, which
lack endogenous FucT7 activity, in the presence or absence of Tmp-
SLF. Cell-surface sLex was detected with a biotin-conjugated anti-
sLex antibody (Mab HECA-452) followed by tricolor-labeled strepta-
vidin and quantified by flow cytometry. Of the 18 protein pairs tested,
6 were active with the Loc-DHFR and 3xFKBP-Cat combination
giving the best response (see SI). Cells transfected with these two
constructs expressed sLex in the presence of Tmp-SLF, but not in its
absence (Figure 3). The CID had no effect on the activity of full-
length FucT7, and the 3xFKBP-Cat construct alone produced no
detectable sLex in the presence or absence of Tmp-SLF. Furthermore,
cells transfected with Loc-DHFR and 3xDHFR-Cat, a mismatched pair,
expressed no detectable sLex and were unresponsive to Tmp-SLF.
Importantly, modulation of sLex expression by Tmp-SLF was
tunable and inhibitable by free Tmp. Tmp-SLF-dependent FucT7
activity was dose-dependent with an EC50 value of 43 nM (Figure
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Acknowledgment. We thank Zeljka Cabrilo for E. coli DH5R
genomic DNA and Steven Rosen for the FucT7 cDNA. This work
was supported by grants from the National Institutes of Health to
C.R.B. (GM59907) and V.W.C. (GM071754). J.L.C. was supported
by an NIH postdoctoral fellowship (GM69156), and J.J.K. was
supported by an American Cancer Society postdoctoral fellowship (PF
TBE-101932).
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