Generation of Recombinant Mammalian Selenoproteins through Genetic Code Expansion with Photocaged Selenocysteine

Article abstract of DOI:10.1021/acschembio.0c00147

Selenoproteins contain the amino acid selenocysteine (Sec) and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a noncanonical translation mechanism requiring suppression of the UGA stop codon and a Sec insertion sequence (SECIS) element in the 3′ untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged Sec (DMNB-Sec) at the UAG amber stop codon. DMNB-Sec is successfully incorporated into GFP and uncaged by irradiation of living cells. Furthermore, DMNB-Sec is used to generate the native selenoprotein methionine-R-sulfoxide reductase B1 (MsrB1). Importantly, MsrB1 is shown to be catalytically active after uncaging, constituting the first use of genetic code expansion to generate a functional selenoprotein in mammalian systems. The ability to site-specifically introduce Sec directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.

Full text of DOI:10.1021/acschembio.0c00147

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Article
Generation of Recombinant Mammalian Selenoproteins through
Genetic Code Expansion with Photocaged Selenocysteine
Jennifer C Peeler, Julia A Falco, Rachel E Kelemen, Masahiro Abo, Benjamin V
Chartier, Laura C Edinger, Jingjia Chen, Abhishek Chatterjee, and Eranthie Weerapana
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.0c00147 • Publication Date (Web): 24 Apr 2020
Downloaded from pubs.acs.org on April 25, 2020
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Generation of Recombinant Mammalian Selenoproteins through Genetic
Code Expansion with Photocaged Selenocysteine
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Jennifer C. Peeler, Julia A. Falco, Rachel E. Kelemen, Masahiro Abo, Benjamin V.
Chartier, Laura C. Edinger, Jingjia Chen, Abhishek Chatterjee*, Eranthie Weerapana* 1
1Department of Chemistry, Boston College, Chestnut Hill, MA 02467
*
Corresponding authors: abhishek.chatterjee@bc.edu; eranthie@bc.edu
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ABSTRACT:
Selenoproteins contain the amino acid selenocysteine and are found in all domains of
life. The functions of many selenoproteins are poorly understood, partly due to difficulties
in producing recombinant selenoproteins for cell-biological evaluation. Endogenous
mammalian selenoproteins are produced through a noncanonical translation mechanism
requiring suppression of the UGA stop codon, and a selenocysteine insertion sequence
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(SECIS) element in the 3’ untranslated region of the mRNA. Here, recombinant
selenoproteins are generated in mammalian cells through genetic code expansion,
circumventing the requirement for the SECIS element, and selenium availability. An
engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a
photocaged selenocysteine (DMNB-Sec) at the UAG amber stop codon. DMNB-Sec is
successfully incorporated into GFP and uncaged by irradiation of living cells. Furthermore,
DMNB-Sec is used to generate the native selenoprotein methionine-R-sulfoxide reductase
1
(MsrB1). Importantly, MsrB1 is shown to be catalytically active after uncaging,
constituting the first use of genetic code expansion to generate a functional selenoprotein
in mammalian systems. The ability to site-specifically introduce selenocysteine directly in
mammalian cells, and temporally modulate selenoprotein activity, will aid in the
characterization of mammalian selenoprotein function.
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Selenoproteins contain the amino acid selenocysteine (Sec), which is a cysteine
Cys) cognate with selenium (Se) in place of sulfur (S). In the majority of characterized
(
selenoproteins, such as glutathione peroxidase and thioredoxin reductases, Sec performs
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redox-catalytic functions. Selenoproteins are found throughout all three domains of life,
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with 25 selenoproteins expressed in humans. However, in some species, Cys-containing
homologs nominally perform the same function as selenoproteins. The stringent
requirement for Sec in certain organisms has been partially attributed to the reversible
nature of Sec oxidation, compared to Cys, thereby conferring resistance to irreversible
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inactivation under oxidative stress. For example, replacement of Sec with Cys in
glutathione peroxidase 4 (GPX4) renders neurons susceptible to ferroptotic cell death due
to GPX4 inactivation through irreversible Cys oxidation.4
Sec incorporation deviates from canonical protein translation, requiring
suppression of the UGA stop codon. In eukaryotes, Sec biosynthesis occurs directly on the
suppressor tRNA (tRNA[Ser]Sec). Specifically, tRNA[Ser]Sec is aminoacylated with serine by
seryl-tRNA synthetase (SerS), followed by phosphorylation by phosphoseryl-tRNA kinase
(PSTK), and subsequent Se incorporation by Sec synthase (SecS), to generate Sec-
tRNA[Ser]Sec (Figure 1a).5, 6 Suppression of UGA by Sec-tRNA[Ser]Sec requires a Sec
insertion sequence (SECIS) element in the selenoprotein mRNA. The SECIS element
appears in the 3’ UTR of the selenoprotein mRNA, and binding of the SECIS binding
protein 2 (SBP2) and Sec-specific translation elongation factor (EFSec) are required for
successful Sec insertion (Figure 1a).7-9
Due to the complexity of endogenous selenoprotein production in eukaryotes,
recombinant expression has proven challenging. Several methods have therefore been
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developed to obtain recombinant selenoproteins. In vitro methods include the use of solid-
phase selenopeptide synthesis, coupled with native and expressed protein ligation, to
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produce full-length selenoproteins. The recombinant expression of selenoproteins in E.
coli has been enhanced by a variety of methods, including the use of reassigned sense
codons, engineered tRNAs, and recoded release factor 1 (RF-1)-deficient E. coli.11-15 These
prokaryotic expression systems have significantly expanded the ability to produce
recombinant mammalian selenoproteins for biochemical characterization. Similar
approaches have been taken to increase selenoprotein production yields in eukaryotic cells,
including the use of unique SECIS elements from Toxoplasma.16
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Genetic code expansion (GCE) technology allows site-specific incorporation of
noncanonical amino acids using engineered aminoacyl-tRNA synthetase (RS)/tRNA
pairs.17-19 GCE offers an attractive alternative for selenoprotein production that overrides
many limitations of the endogenous pathway, including the requirement of the SECIS
element and selenium availability. Additionally, GCE enables incorporation of protected
analogs of Sec, which can be deprotected and activated on demand. An allyl-protected Sec
(ASec) and a photocaged (methyl-(6-nitropiperonyloxymethyl), MeNPOM) Sec (PSc)
have been genetically encoded in E. coli using the pyrrolysyl-tRNA synthetase/tRNA pair,
and can be uncaged to Sec using palladium (Pd)-catalysis and light, respectively.20, 21 In
yeast, a photocaged (4,5-dimethoxy-2-nitrobenzyl, DMNB) Sec (DMNB-Sec) (Figure 1b)
was incorporated using the E. coli leucyl-tRNA synthetase/tRNA pair.22 To date, only
ASec has been successfully incorporated and uncaged in mammalian cells, therefore
additional technologies for mammalian expression of selenoproteins are needed.
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Here, we report an alternative platform to generate selenoproteins directly in
mammalian cells through incorporation of photocaged DMNB-Sec (Figure 1b). The
noncanonical amino acid DMNB-Sec is minimally toxic to mammalian cells at the
concentrations required for incorporation. DMNB-Sec was incorporated into the model
protein GFP, and the native human selenoprotein MsrB1, in HEK293T cells. Uncaging of
the resulting selenoproteins was achieved by minimally invasive 365 nm irradiation of
purified protein or living cells expressing the protein.23 Importantly, DMNB-Sec
incorporation in MsrB1 was shown to be non-disruptive to protein function, demonstrating
the utility of this genetic code expansion platform for studying native selenoprotein
function in mammalian cells.
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RESULTS AND DISCUSSION
Incorporation of DMNB-Sec into GFP in HEK293T cells. The feasibility of
incorporating a photocaged selenocysteine amino acid into proteins in mammalian cells
was initially evaluated using GFP as the target protein in HEK293T cells. Photocaged
DMNB-Sec (Figure 1b) was synthesized as previously reported.22 Incorporation of
DMNB-Sec into GFP in mammalian cells was achieved using the E. coli LeuRS BH5
T252A/tRNA pair, which was originally evolved for photocaged serine (DMNB-Ser)
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incorporation, and subsequently used for DMNB-Sec in yeast. LeuRS and 8 copies of
the Leu tRNA were cloned into the pAcBac2 vector containing an eGFP-39-TAG reporter
(
Supplemental Figure 1).25-27 Transient transfection of the pAcBac2 vector into
HEK293T cells was followed by growth in the presence or absence of 100 µM DMNB-
Sec. Fluorescence of GFP was measured to be 5-fold higher for lysates from cells exposed
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to DMNB-Sec (Figure 2a), consistent with previously reported data for the yeast LeuRS
system with DMNB-Sec and DMNB-Cys.22
The toxicity of DMNB-Sec was examined using an MTT assay, and DMNB-Sec
was found to be minimally toxic to HEK293T at low micromolar concentrations (EC of
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07 µM; Supplemental Figure 2). DMNB-Sec was used at concentrations in the range of
2.5-100 µM for all reported experiments. To purify GFP for mass-spectrometry
characterization, HEK293T cells were transfected with eGFP-WT or eGFP-39-TAG and
grown in the presence or absence of DMNB-Sec. The resulting proteins were purified via
a C-terminal 6XHis tag and analyzed by SDS-PAGE (Figure 2b). A measure of purified
protein concentration revealed that eGFP-DMNB-Sec39 was expressed and purified with
a yield of 45% compared to eGFP-WT. Intact-protein electrospray-ionization mass
spectrometry (ESI-MS) confirmed the presence of the expected DMNB-Sec-containing
eGFP species (Figures 2c, Supplemental Figure 3). Analysis of eGFP from cells grown
in the absence of DMNB-Sec identified a mass consistent with the incorporation of leucine
(Leu) or isoleucine (Ile) (Supplemental Figure 4). When eGFP was expressed in the
presence of DMNB-Sec, eGFP-Leu/Ile39 was not detectable by ESI-MS (Supplemental
Figure 3), suggesting that incorporation of these natural amino acids is suppressed by the
presence of DMNB-Sec.
To monitor uncaging of the DMNB protecting group, cells expressing eGFP-
DMNB-Sec39 were irradiated (365 nm, 10 mins), followed by purification and ESI-MS
analysis of the resulting protein. The presence of uncaged eGFP-Sec39 was confirmed by
ESI-MS (Figure 2c, Supplemental Figure 5), with no detectable ESI-MS peak observed
for the caged eGFP-DMNB-Sec39. In the previously published study that incorporated
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DMNB-Sec into proteins in yeast, irradiation of yeast lysates resulted in the formation of
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diselenide dimers as well as dehydroalanine species. However, here we report that only
the expected monomer was observed in ESI-MS analysis of mammalian-expressed eGFP-
Sec39. To further explore the kinetics of uncaging, purified eGFP-DMNB-Sec39 was
subjected to irradiation for various time periods (0-20 mins), followed by ESI-MS analysis.
Detectable levels of the uncaged eGFP-Sec39 species appears after ~0.5 mins of irradiation
at 365 nm, and complete uncaging is observed after ~5 mins of irradiation (Supplemental
Figure 6).
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Incorporation of DMNB-Sec into human MsrB1 in HEK293T cells. Upon
demonstrating the successful incorporation of DMNB-Sec into GFP, a photocaged version
of the native mammalian selenoprotein, human methionine-R-sulfoxide reductase 1
(MsrB1), was generated. MsrB1 is localized to the cytoplasm and nucleus, and utilizes an
active-site Sec to stereo-selectively reduce methionine-R-sulfoxide.28 Intriguingly, the
MsrB1 homologs in humans, MsrA, MsrB2 and MsrB3, utilize a Cys, instead of Sec, to
perform similar chemistry. Studies comparing Cys- versus Sec-containing Msr proteins
have shown that Sec is advantageous for reductase activity.1, 29, 30 MsrB1 protects proteins
from oxidative damage, and enables signaling via dynamic, enzyme-dependent methionine
oxidation and reduction.31, 32 The role of MsrB1 has been well characterized for the
substrate actin, but regulation of other potential substrates is poorly understood, due in part
to the difficulties with overexpression and temporally controlled activation of MsrB1 in
mammalian cells. Expression of MsrB1 with a caged active-site Sec residue would
facilitate further characterization of the cellular functions of MsrB1.
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To recombinantly express MsrB1, a pAcBac2 plasmid containing the following
elements was generated: (1) LeuRS BH5 T252A; (2) 8 copies of the E. coli Leu tRNA;
and, (3) the MsrB1 gene with the amber stop codon (MsrB1-TAG95) or a Cys codon
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(MsrB1-Cys95) at residue 95 (the site of the endogenous Sec residue), and a C-terminal
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XHis tag. To test for successful incorporation of DMNB-Sec, MsrB1 expression was
monitored via immunoblot using antibodies against MsrB1 and the C-terminal 6XHis tag.
Importantly, the 6XHis antibody will only detect full-length protein generated via
successful amber suppression. Full-length MsrB1 protein was observed for MsrB1-Cys95,
and DMNB-Sec treated MsrB1-TAG95 cells (Figure 3a). As expected, no protein was
detected in the MsrB1-TAG95 cells in the absence of DMNB-Sec. Comparison of protein
yields of MsrB1-Cys95 and MsrB1-DMNB-Sec95 by western blot indicated that
expression of the DMNB-Sec-containing MsrB1 was ~10% that of the Cys-containing
version.
Purification and analysis by ESI-MS confirmed the presence of MsrB1-Cys95 and
MsrB1-DMNB-Sec95 (Figures 3b, Supplemental Figure 7 and 8). Successful uncaging
was confirmed by subjecting purified MsrB1-DMNB-Sec95 to UV irradiation (365 nm, 10
mins), and subsequent ESI-MS analysis (Figure 3b, Supplemental Figure 9). As with
eGFP-Sec39, dimer and dehydroalanine formation was not observed by ESI-MS.
Additionally, MsrB1-Ile/Leu95 was not detected, confirming that misincorporation of
Ile/Leu is negligible in the presence of DMNB-Sec.
To demonstrate uncaging of MsrB1 directly in cells, we utilized an isoTOP-ABPP
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method that was modified for selenoprotein detection. Typical isoTOP-ABPP monitors
Cys reactivity in complex proteomes via the following steps: (1) cell-lysate labeling with
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an iodoacetamide (IA)-alkyne probe; (2) conjugation of IA-alkyne modified proteins to a
cleavable-biotin tag using copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC); (3)
enrichment of IA-alkyne modified proteins on streptavidin beads; (4) on-bead trypsin
digestion and cleavage of the linker to release IA-alkyne modified peptides for analysis by
LC/LC-MS/MS. Incorporation of isotopically light and heavy IA-alkyne probes, or
cleavable linkers, enable quantitative comparisons of cysteine reactivity across different
biological samples.34-36 Performing the initial IA-alkyne labeling step at low pH (~pH 5.75)
was shown to suppress Cys labeling, and thereby enhance the detection of Sec-containing
peptides for the analysis of Sec-reactivity changes.33
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Low-pH isoTOP-ABPP analysis was used to compare lysates from irradiated (IA-
light labeled) and non-irradiated (IA-heavy labeled) HEK cells expressing MsrB1-DMNB-
Sec95 (Supplemental Figure 10). The IA-labeled MsrB1 Sec-containing peptide
(R.FU*IFSSSLK.F) was identified via LC/LC-MS/MS, and shown to display a light:heavy
ratio (R) of 2.66, indicative of ~3-fold increased Sec reactivity in the irradiated sample
(
Figure 3c). In contrast, identified Cys-containing peptides showed R values of ~1 (Figure
c), indicating no reactivity change upon irradiation. Importantly, the identified MsrB1
peptide displayed an isotopic envelope characteristic of Se-containing peptide species,
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confirming Sec incorporation. Together, the data from the low pH isoTOP-ABPP analysis
confirms the successful incorporation and uncaging of DMNB-Sec in MsrB1 directly in
mammalian cells.
28
Endogenous MsrB1 is known to localize to the cytosol and nucleus of cells. To
confirm that MsrB1-DMNB-Sec95 shows similar localization patterns, we utilized
confocal microscopy to determine the cellular localization of MsrB1-DMNB-Sec95 in
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irradiated and non-irradiated cells. Regardless of irradiation, the recombinant MsrB1 was
shown to display both cytosolic and nuclear localization, consistent with the endogenous
protein (Figure 3d). As expected, cells cultured in the absence of DMNB-Sec did not show
a significant signal for 6XHis-MsrB1. These imaging studies confirm that photocaged
versions of endogenous selenoproteins can be produced and activated in mammalian cells
with the expected cellular localization for subsequent biological interrogation.
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To confirm that the MsrB1-DMNB-Sec95 expressed in HEK293T cells displays
methionine sulfoxide reductase activity, we monitored the ability of the purified uncaged
MsrB1 protein to reduce dabsyl-methionine sulfoxide (dabsyl-MetO). Briefly, dabsyl-
MetO was incubated with buffer (negative control), uncaged MsrB1-DMNB-Sec95
(MsrB1-Sec95), or human recombinant MsrB2 (positive control). The resulting reaction
mixtures were analyzed by LC/MS, whereby levels of oxidized and reduced dabsyl-Met
3
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were quantified using absorbance at 466 nm. MsrB1-Sec95 reduced dabsyl-MetO at
levels comparable to human recombinant MsrB2, producing ~3-fold greater dabsyl-Met
relative to the negative control (Figure 3e, Supplemental Figure 11). The observed
reductase activity of MsrB1-Sec95 on the dabsyl-MetO substrate confirms that
incorporation of DMNB-Sec into MsrB1 does not grossly disrupt protein structure, and
subsequent uncaging produces the catalytically active protein product.
In summary, studies of selenoprotein function have been hindered by the poor
efficiency of recombinant expression of Sec-containing proteins and related mutants,
stimulating the development of numerous technologies for the production of selenoproteins
in vitro, in E. coli, and in mammalian cells. Of these, GCE methods can introduce protected
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variants of selenocysteine for controlled activation, and circumvent the requirement for the
SECIS element and the dependence on selenium concentrations in the growth media. We
adapt the E. coli leucyl-tRNA synthetase/tRNA pair to incorporate the photocaged amino
acid DMNB-Sec into GFP and native selenoproteins directly in mammalian cells.
Currently, the only available technology for producing protected selenoproteins in
mammalian cells, is a GCE platform utilizing ASec, whereby ASec-containing GFP was
deprotected directly in mammalian cells using palladium-mediated strategies. ASec was
not incorporated into a native selenoprotein, so the effects of palladium-mediated
deprotection on selenoprotein function was not investigated. We show that DMNB-Sec
incorporation and uncaging by UV irradiation does not disrupt the catalytic activity of
MsrB1. Additionally, DMNB-Sec displays low toxicity at the concentrations required for
selenoprotein production (12.5-100 µM). In contrast, the required concentrations of ASec
(0.2 mM) are toxic to mammalian cells, necessitating supplementation with high
concentration of cysteine (3.2 mM) to mitigate toxicity.20 Addition of high cysteine
concentrations will perturb cellular redox status, and limit application of ASec for
investigating the redox functions of the majority of selenoproteins. Therefore, DMNB-Sec
incorporation provides a complementary GCE platform that can be utilized for the study
of redox-active selenoproteins in mammalian cells.
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The yield of MsrB1-DMNB-Sec95 is significantly lower than that of the Cys-
containing MsrB1, necessitating further optimization of the GCE platform. Efforts are
underway to improve the efficiency of DMNB-Sec incorporation through optimization of
the LeuRS/tRNA pair. Upon optimization, the technology described herein could enable
temporally controlled selenoprotein production in mammalian cells for further advancing
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our understanding of selenoprotein biology. Ultimately, the all-in-one pAcBac2 vector
system used to deliver the DMNB-Sec incorporation machinery can be readily packaged
into an engineered baculovirus vector that can facilitate highly efficient unnatural amino
acid incorporation into a wide variety of mammalian cells and tissues, including
challenging cells such as neurons and stem cells.25, 27
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METHODS
See the Supporting Information for details.
ASSOCIATED CONTENT
Supporting Information
Detailed experimental procedures and supplementary figures and tables. The Supporting
Information is available free of charge on the ACS Publications website at DOI:
AUTHOR INFORMATION
Corresponding Author
*
E-mail: eranthie.weerapana@bc.edu
E-mail: abhishek.chatterjee@bc.edu
*
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
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This work was supported in part by NIH grant R01GM117004 and R01GM118431-01A1
to E.W., grants R01GM124319 and R01GM126220 to A.C, and fellowship
F32GM131615-01 to J.C.P.
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FIGURE LEGENDS
Figure 1. Selenocysteine incorporation and DMNB-Sec structure. (a) Endogenous
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eukaryotic Sec-incorporation mechanism. (b) Structures of DMNB-Sec and Sec.
Figure 2. Generation of eGFP-DMNB-Sec39. (a) Fluorescence microscopy and
quantification of HEK293T cells transiently transfected with a pAcBac2 plasmid encoding
LeuRS BH5 T252A /Leu tRNA/eGFP-39-TAG, in the presence and absence of DMNB-
Sec. (b) SDS-PAGE of eGFP-WT and LeuRS BH5 T252A /Leu tRNA/eGFP-39-TAG
expressed in the presence and absence of DMNB-Sec (c) ESI-MS analysis of eGFP-
DMNB-Sec39 (expected mass: 29865.33) and irradiated eGFP-Sec39 (expected mass:
2
9669.24)
Figure 3. Generation of MsrB1-DMNB-Sec95. (a) Immunoblot of MsrB1 expression with
a Cys or TAG amber codon at residue 95 in the presence and absence of DMNB-Sec. (b)
ESI-MS analysis of MsrB1-Cys95 (expected mass: 13309.97), MsrB1-DMNB-Sec95
(expected mass: 13552.04), and uncaged MsrB1-Sec95 (expected mass: 13356.87). (c)
Low pH isoTOP-ABPP analysis showing extracted ion chromatograms for Cys- or Sec-
containing peptides from irradiated (red, IA-light) or non-irradiated (blue, IA-heavy) cells
expressing MsrB1-DMNB-Sec95. (d) Immunofluorescence of cells transfected with
MsrB1-TAG95 in the presence or absence of DMNB-Sec, before and after irradiation.
Scale bar represents 10 µm. (e) Methionine sulfoxide reductase activity of buffer alone
(negative control), purified uncaged MsrB1-Sec95, and MsrB2 (positive control),
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measured as the percent reduced dabsyl-Met (dabsyl-MetR) present within the total amount
of dabsyl-Met (oxidized and reduced). Immunoblot shows that levels of purified MsrB1-
Sec95 is comparable to the amount of MsrB2 used in the activity assay.
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Page 23 of 25 ACS Chemical Biology
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ACS Chemical Biology
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