A Single Atom Change Facilitates the Membrane Transport of Green Fluorescent Proteins in Mammalian Cells

Article abstract of DOI:10.1002/anie.201902347

Direct delivery of proteins into mammalian cells is a challenging problem in biological and biomedical applications. The most common strategies for the delivery of proteins into the cells include the use of cell-penetrating peptides or supercharged proteins. Herein, we show for the first time that a single atom change, hydrogen to halogen, at one of the tyrosine residues can increase the cellular entry of ～28 kDa green fluorescent protein (GFP) in mammalian cells. The protein uptake is facilitated by a receptor-mediated endocytosis and the cargo can be released effectively into cytosol by co-treatment with the endosomolytic peptide ppTG21.

Full text of DOI:10.1002/anie.201902347

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Accepted Article
Title: A Single Atom Change Facilitates the Membrane Transport of
Green Fluorescent Proteins in Mammalian Cells
Authors: Govindasamy Mugesh, Surendar R. Jakka, and Vijayakumar
Govindaraj
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A Single Atom Change Facilitates the Membrane Transport of
Green Fluorescent Proteins in Mammalian Cells
Surendar R. Jakka, Vijayakumar Govindaraj and Govindasamy Mugesh*
Abstract: Direct delivery of proteins into mammalian cells is a
challenging problem in biological and biomedical applications. The
most common strategies for the delivery of proteins into the cells
include the use of cell-penetrating peptides or supercharged proteins.
Herein, we show for the first time that a single atom change,
hydrogen to halogen, at one of the tyrosine residues can increase
the cellular entry of 28 kDa green fluorescent protein (GFP) in
mammalian cells. The protein uptake is facilitated by a receptor-
mediated endocytosis and the cargo can be released effectively into
cytosol by co-treatment with the endosomolytic peptide ppTG21.
The halo variants of GFP (EmGFP, Section 1.3, SI) were
synthesized biochemically by expanding the genetic code of E.
coli with 3-halo-L-tyrosine. Briefly, the Methanococcus jannaschii
tyrosyl-tRNA synthetase and tRNA pair evolved for 3-iodo-L-
tyrosine[10] was used to incorporate 3-halo-L-tyrosine to EmGFP
using an amber codon (UAG) as shown in Figure 1A. The
surface tyrosyl residue at 39-position of EmGFP was selected
for the single atom modification to facilitate a favourable
interaction between the halogen atom and the cell plasma
membrane. Also, the introduction of heavier halogen atoms on
the surface may not alter the secondary structure of the proteins.
For the expression of the wild-type GFP, a construct having no
amber codon at the 39-position was used. The recoded E. coli
strain C321A.exp was used for the protein expression. The
fluorescence microscopy study showed significant expression of
all four proteins in E. coli cells (Figure 1B). After lysing the cells,
The transport of macromolecules across the cell plasma
membrane is a major challenge in biomedical applications,
including the development of drug candidates,[1] and therefore,
the biological applications of many exogenous proteins are
restricted to extracellular targets.[2] The inability of almost all
macromolecules to spontaneously enter cells led to the
development of strategies for the delivery into mammalian cells.
These methods are generally based on cationic cell-penetrating
peptides (CPPs),[1a,3] antibodies,
[4]
nanoparticles,[5] receptor
ligands,[ and virus-like particles. Another approach involves
the use of supercharged proteins (SCPs) for the delivery of
functional macromolecules.[8] Unfortunately, in most of these
processes, large amounts of purified proteins are required for a
reasonable cellular delivery. Although the attachment of cell
permeable peptides enhances the cellular delivery of proteins,
such modifications alter the protein function inside the cells.
6]
[7]
Recently, we reported that the introduction of halogen atoms
facilitates the active transport of small molecules across the
plasma membrane in mammalian cells.[9] The cellular uptake of
some of the commonly used fluorescent probes containing
naphthalimide, coumarin, BODIPY and pyrene moieties has
been shown to be enhanced significantly upon introduction of
iodine atoms. Although this study provided a general strategy for
enhancing the cellular uptake of small organic molecules, it is
not known whether such strategy can be used for the delivery of
difficult cargo such as proteins. In this paper, we report that a
single atom change, hydrogen to halogen (Cl, Br or I), at one of
the tyrosyl residues facilitates the delivery of a green fluorescent
protein (GFP) having a molecular weight of 28 kDa into
mammalian cells and the highest cellular uptake is observed for
GFP having an iodine atom.
[*]
S. R. Jakka, Dr. V. Govindaraj and Prof. Dr. G. Mugesh
Department of Inorganic and Physical Chemistry
Indian Institute of Science
Figure 1. (A) The strategy for site-specific incorporation of 3-halo-L-tyrosine at
3
9-position of EmGFP utilizing amber stop codon suppression by an
engineered orthogonal aaRS/tRNA pair. The proteins are represented as
TAG-3ClY, 1TAG-3BrY or 1TAG-3IY, respectively. (B) Confocal images
Bangalore 560012, India
E-mail: mugesh@iisc.ac.in
1
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.2019xxxx.
confirming the expression of 1TAG-3ClY or -3BrY or -3IY EmGFP in E.coli. (C-
D) Confirmation of purified proteins by SDS-PAGE and mass spectrometry.
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the histidine-tagged proteins were purified by using an affinity
chromatography. The purity of the proteins was confirmed by the
SDS-PAGE (Figure 1C) and the successful introduction of the
halogen atoms into the proteins was confirmed by mass
spectrometry (Figure 1D).
scanning microscopy and fluorescence microplate reader
techniques. The HepG2 cells treated with 1 µM concentration of
EmGFP-WT for 90 min showed a very low fluorescence (Figure
2D-F and S11-12), indicating that the WT protein is not taken up
readily by the cells. The cellular uptake was marginally
increased for 1TAG-3ClY, suggesting that the replacement of a
hydrogen atom with a chlorine atom at 39-position increases the
cellular uptake. A further increase in the uptake was observed
for the bromo analogue (1TAG-3BrY) and the amount of protein
entered the cells was found to be almost 2 times higher than that
of the WT. Remarkably, a much higher uptake (almost 6-fold
increase with respect to WT) was observed for 1TAG-3IY,
indicating that the iodine atom facilitates the transport of the
protein as observed earlier for the iodinated small molecules.
The absorption and fluorescence spectra of all four proteins
indicated that there is no change in the spectral properties
(
Figure 2A). No quenching of the fluorescence was observed
upon introduction of heavier halogen atoms. This is probably
because the 3-halo-L-tyrosine residue is located on the surface
of the modified proteins away from the GFP fluorophore. The
circular dichroism (CD) studies indicated that the heavier
halogen atoms do not alter the secondary structures of the
proteins (Figure 2B). As the substitution of halogen atoms into
GFP may increase the toxicity, the cell viability was determined
by using HepG2 (human liver carcinoma) cells. The toxicity of all
three halogenated proteins was found to be almost identical to
that of the wild-type protein (Figure 2C), indicating that the
substitution of a hydrogen atom with a heavier halogen does not
lead to toxicity in mammalian cells. The interesting fluorescent
properties of the EmGFPs prompted us to investigate the
cellular uptake in mammalian cells. We studied the uptake of the
wild-type (WT) as well as the modified proteins by using laser
It is known that hydrophobic small molecules such as
benzene and gaseous molecules such as O and CO can cross
2 2
the cell membrane by simple diffusion. However, such simple
diffusion through the plasma membrane is extremely difficult for
macromolecules. When the cellular uptake studies were carried
out at 4 ºC, essentially no fluorescence was observed for all four
proteins (Figure 3B), indicating that the WT or the modified
proteins do not enter the cells by diffusion. In our earlier studies,
we showed that the iodine-containing small molecules such as
1
-5 (Figure 3A) enter the cells through the monocarboxylate
transporter 8 (MCT8),[ which is a very specific transporter for
the iodine-containing thyroid hormone, thyroxine (T4).[11] The 2-
iodoanisole moiety serves as an efficient receptor recognition
unit for compound 1-5 as the iodine atoms can form halogen
bonding with the receptor, facilitating their cellular entry.[9,12] As
9]
the GFPs, particularly the iodo-GFP having the ability to form
halogen bonding with the receptor,[
12]
may enter the cells
through MCT8, we studied the cellular uptake in the presence of
silychristin (SY), which is a potent and selective inhibitor of
MCT8-mediated uptake.[13] Interestingly, no inhibition of cellular
uptake was observed in the presence of SY (Figure 3A,B),
indicating that MCT8 is a specific small-molecule transporter and
it does not mediate the uptake of proteins such as GFP even
when they contain iodine atoms. Further, the cellular uptake was
not inhibited by 3-iodo-L-tyrosine (Figure S13).
To understand whether the cellular uptake is an energy-
dependent process, we depleted the adenosine triphosphate
(
ATP) level in the cells by using sodium azide and deoxyglucose
[
14]
in glucose-free medium.
The uptake is blocked under these
conditions (Figure 3B, S14), suggesting that the proteins are
taken up by the cells via an energy-dependent pathway,
involving either the clathrin-mediated or caveolar endocytosis.
As several proteins are transported via the clathrin-mediated
endocytosis (CME),[15,16] we studied the uptake in the presence
chlorpromazine (CPZ), a cationic amphipathic compound that
inhibits the CME by depleting clathrin from the plasma
membrane.[
16a]
As observed for SY, no inhibition of cellular
uptake was observed in the presence of CPZ (Figure 3A,B, S14),
suggesting that a clathrin-independent endocytosis (CIE) is
probably responsible for the cellular uptake. Further support for
CIE came from the cellular uptake experiments with methyl--
cyclodextrin (MCD), which is known to disrupt lipid rafts by
removing cholesterol from the plasma membrane.[ While the
uptake of WT protein is not affected, the MCD treatment
Figure 2. (A-B) Normalized fluorescence intensity and CD spectra of the WT
and modified proteins. (C) Cell viability of HepG2 cells incubated with halo-
EmGFP (1TAG) for 90 min. (D) The fluorescence measured by a plate reader,
(E) Mean fluorescence as determined by flow cytometry and (F) confocal
16]
images after 90 min treatment of HepG2 cells with WT and modified EmGFP.
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significantly inhibited the uptake of the halogenated GFPs
(
Figure 3B, S14), indicating that the proteins enter the cells via
the caveolar endocytosis. The caveolae-mediated internalization
was further confirmed by carrying out the uptake experiments in
the presence of genistein (GST), a tyrosine-kinase inhibitor
known not only to cause local disruption of the actin network at
the site of endocytosis, but also to inhibit the recruitment of
dynamin II.[16a] In the presence of GST, an almost complete
inhibition of the cellular uptake was observed (Figure 3B, S14)
confirming that all four proteins are taken up by the cells through
the ATP-dependent, caveolae-mediated endocytosis.
Figure 4. (A) Representation of EmGFP having 3-IY at positions 39 and 214.
(B) Confocal images showing the relative expression of 2TAG-3IY and 1TAG-
3
IY in E. coli cells. (C-D) Confirmation of purified 2TAG-3IY by SDS-PAGE
Figure 3. (A) Chemical structures of compounds 1-5, thyroxine and various
inhibitors used. (B) The fluorescence measured by a plate reader after 90 min
treatment of HepG2 cells with 1 μM of 1TAG-3ClY, 1TAG-3BrY and 1TAG-3IY
EmGFP at 37 °C, 4 °C, under ATP depletion or after pre-treatment with MCT8
inhibitor or endocytosis inhibitors. After treatment with the inhibitors, the cells
were washed thoroughly with PBS buffer before incubating with EmGFP.
and mass spectrometry. (H-I) Mean fluorescence intensity as determined by
flow cytometry and the corresponding confocal images after treating HepG2
cells with the proteins for 90 min. DAPI was used for staining the nucleus.
It is known that biomolecules, particularly proteins, that enter
the cells by endocytosis pathways get entrapped in endosomes,
transported to lysosomes and are subsequently degraded by
specific enzymes. The time-dependent cellular uptake
experiments indicate that both 1TAG-3IY and 2TAG-3IY rapidly
enter the cells through the receptor-mediated endocytosis, but a
significant decrease in the fluorescence intensity was observed
after 24 h (Figure 5D-E, S17), probably due to degradation of
the proteins in lysosomes (Figure 5F). To release the entrapped
proteins from the endosomes before their degradation, we co-
treated the cells with the histidine-rich 20-mer peptide, ppTG21
(pI 7.7, charge +1.3 @ pH 7.4), which has been reported as a
promising endosomolytic agent for plasmid-based gene
delivery.[17] Interestingly, a remarkable enhancement in the
fluorescence intensity was observed for both 1TAG-3IY and
2TAG-3IY in the presence of ppTG21 after 90 min, and there
was no decrease in the fluorescence was observed even after
24 h (Figure 5E, S17). We obtained similar results when the
cells were treated with the peptide after 60 min treatment with
1TAG-3IY (Figure S18), indicating the intracellular effect of
ppTG21 in the cellular uptake of 1TAG-3IY. These observations
confirm that the iodinated proteins are taken up by the cells
To understand whether the introduction of an additional 3-
iodo-L-tyrosine residue at the other end of the EmGFP can
increase the cellular uptake by pushing the tail-end through the
receptor by forming a halogen bonding, we generated 2TAG-3IY
having the iodinated tyrosine residues at 39- and 214-positions
by using two amber codons (Figure 4A). The protein was
expressed in the recoded E. coli strain C321A.exp and the
resulting His-Tag protein was purified by an affinity
chromatography. The mass spectrometry study confirmed the
incorporation of two iodotyrosyl residues (Figure 4D). The
protein expression levels and spectral features are quite like that
of 1TAG-3IY (Figure 4B-E). Although the secondary structure is
not affected by the substitution of two iodotyrosyl residues
(
Figure 4F), only a marginal increase in the cellular uptake was
observed in HepG2 cells (Figure 4G-I, S15), indicating that the
initial recognition of the 3-iodo-L-tyrosine (3IY) moiety by the
receptor is sufficient for the cellular uptake. The transport under
ATP depleted conditions or in the presence of various inhibitors
indicated that 2TAG-3IY also enters the cells through caveolae-
mediated endocytosis pathway (Figure 5A,B, S16).
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preferentially through a receptor-mediated endocytosis and the
proteins can be released effectively into cytosol by using
endosomolytic agents such as ppTG21.
acknowledge IISc, Bangalore, and SERB, respectively, for a
research fellowship. We thank Mary Nirmala Sarkar, M.
Kishorkumar Reddy, Sritama Bose and Aswin Govindan for their
help with some of the experiments. G. M. thanks the SERB for
the J. C. Bose fellowship (SB/S2/JCB-067/2015).
Keywords: cellular uptake • endocytosis • green fluorescent
protein • halogen • membrane transport • tRNA synthetase
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In summary, we showed for the first time that proteins can
be transported across the cell plasma membrane by a single
atom substitution. The introduction of iodine atom to green
fluorescent proteins remarkably enhances the cellular uptake
and halogen bonding may play key roles in the caveolae-
mediated endocytosis. The proteins can be released effectively
into cytosol by co-treatment with the histidine-rich 20-mer
peptide ppTG21, which can mediate the rupture of endosomal
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Acknowledgements
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This study was supported by the Science and Engineering
Research Board (SERB), New Delhi. S. R. J. and V. G.
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Entry for the Table of Contents
COMMUNICATION
Introduction of an iodine
atom to one of the tyrosine
residues facilitates the
cellular entry of green
Surendar R. Jakka,
Vijayakumar Govindaraj,
Govindasamy Mugesh*
fluorescent proteins,
providing a strategy for the
delivery of macromolecules
into mammalian cells.
Page No. – Page No.
A Single Atom Change
Facilitates the Membrane
Transport of Green
Fluorescent Protein in
Mammalian Cells
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