Comparison of alternative nucleophiles for Sortase A-mediated bioconjugation and application in neuronal cell labelling

Article abstract of DOI:10.1039/c3ob42325e

The Sortase A (SrtA) enzyme from Staphylococcus aureus catalyses covalent attachment of protein substrates to pentaglycine cross-bridges in the Gram positive bacterial cell wall. In vitro SrtA-mediated protein ligation is now an important protein engineering tool for conjugation of substrates containing the LPXTGX peptide recognition sequence to oligo-glycine nucleophiles. In order to explore the use of alternative nucleophiles in this system, five different rhodamine-labelled compounds, with N-terminal nucleophilic amino acids, triglycine, glycine, and lysine, or N-terminal non-amino acid nucleophiles ethylenediamine and cadaverine, were synthesized. These compounds were tested for their relative abilities to function as nucleophiles in SrtA-mediated bioconjugation reactions. N-Terminal triglycine, glycine and ethylenediamine were all efficient in labelling a range of LPETGG containing recombinant antibody and scaffold proteins and peptides, while reduced activity was observed for the other nucleophiles across the range of proteins and peptides studied. Expansion of the range of available nucleophiles which can be utilised in SrtA-mediated bioconjugation expands the range of potential applications for this technology. As a demonstration of the utility of this system, SrtA coupling was used to conjugate the triglycine rhodamine-labelled nucleophile to the C-terminus of an Im7 scaffold protein displaying Aβ, a neurologically important peptide implicated in Alzheimer's disease. Purified, labelled protein showed Aβ-specific targeting to mammalian neuronal cells. Demonstration of targeting neuronal cells with a chimeric protein illustrates the power of this system, and suggests that SrtA-mediated direct cell-surface labelling and visualisation is an achievable goal. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3ob42325e

Organic &
Biomolecular Chemistry
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Comparison of alternative nucleophiles for Sortase
A-mediated bioconjugation and application in
neuronal cell labelling†
Cite this: Org. Biomol. Chem., 2014,
12, 2675
Samuel Baer,a Julie Nigro,a,b Mariusz P. Madej,a Rebecca M. Nisbet,a,b
Randy Suryadinata,a Gregory Coia,a Lisa P. T. Hong,a Timothy E. Adams,a
Charlotte C. Williams*‡a and Stewart D. Nuttall‡a,b
The Sortase A (SrtA) enzyme from Staphylococcus aureus catalyses covalent attachment of protein sub-
strates to pentaglycine cross-bridges in the Gram positive bacterial cell wall. In vitro SrtA-mediated
protein ligation is now an important protein engineering tool for conjugation of substrates containing the
LPXTGX peptide recognition sequence to oligo-glycine nucleophiles. In order to explore the use of
alternative nucleophiles in this system, five different rhodamine-labelled compounds, with N-terminal
nucleophilic amino acids, triglycine, glycine, and lysine, or N-terminal non-amino acid nucleophiles ethy-
lenediamine and cadaverine, were synthesized. These compounds were tested for their relative abilities to
function as nucleophiles in SrtA-mediated bioconjugation reactions. N-Terminal triglycine, glycine and
ethylenediamine were all efficient in labelling a range of LPETGG containing recombinant antibody and
scaffold proteins and peptides, while reduced activity was observed for the other nucleophiles across the
range of proteins and peptides studied. Expansion of the range of available nucleophiles which can be uti-
lised in SrtA-mediated bioconjugation expands the range of potential applications for this technology. As
a demonstration of the utility of this system, SrtA coupling was used to conjugate the triglycine rhoda-
mine-labelled nucleophile to the C-terminus of an Im7 scaffold protein displaying Aβ, a neurologically
important peptide implicated in Alzheimer’s disease. Purified, labelled protein showed Aβ-specific target-
ing to mammalian neuronal cells. Demonstration of targeting neuronal cells with a chimeric protein illus-
trates the power of this system, and suggests that SrtA-mediated direct cell-surface labelling and
visualisation is an achievable goal.
Received 21st November 2013,
Accepted 3rd March 2014
DOI: 10.1039/c3ob42325e
www.rsc.org/obc
pharmacokinetics,2 giving rise to heterogeneous mixtures as
conjugation generally occurs at a number of sites on the anti-
Introduction
Precise, reliable, and reproducible conjugation of chemical body.5 For example, the covalent attachment of the cytotoxic
substances to proteins is a long-held goal of the chemistry– maytansinoid, (DM1), to the anti-ErbB2 monoclonal antibody
biology research community.1,2 For example, chemical and bio- trastuzumab, an ADC referred to as T-DM1, which is approved
logical conjugation of small molecule cytotoxic drugs or for the treatment of metastatic breast cancer.5,6 DM1 is intro-
imaging agents to monoclonal antibodies and their recombi- duced to lysine-amine residues on trastuzumab through a
nant fragments, to form antibody-drug conjugates (ADCs), is linker, giving a heterogeneous mixture with an average of 3.5
an expanding area of study and commercial interest.3,4 A draw- DM1 molecules per trastuzumab molecule.5
back in many ADCs reported to date is the inability to
An alternative to chemical conjugation is to use biological
precisely conjugate agents to specific residues or domains means, for example, the Sortase family of enzymes.7,8 Sortases
on the antibody molecule without impacting activity or have evolved in Gram positive bacteria as a mechanism of
generic, yet precise and efficient, covalent attachment of pro-
teins (including virulence factors) to the outer cell wall.9
aCSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria
Sortase A (SrtA), the first member of the family to be
described, is an integral membrane protein attached to the
outer surface of the bacterial cell10 where it accepts proteins
exported through the secretory apparatus tagged with an
NLPETGXC (or equivalent) motif. The enzyme then covalently
couples the C-terminus of the target protein to the free
3052, Australia. E-mail: Charlotte.Williams@csiro.au
bPreventative Health Flagship, 343 Royal Parade, Parkville, Victoria 3052, Australia
†Electronic supplementary information (ESI) available: Further experimental
details for expression and purification of SrtA and conjugation optimisation and
time-course reactions. See DOI: 10.1039/c3ob42325e
‡These authors contributed equally to the manuscript.
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N-terminus of the abundant poly-glycine acceptor peptides least one N-terminal glycine, although two or more have been
located on and within the thick peptidoglycan matrix.11,12 reported as ‘optimal’.23 Substitution of alanine and valine fol-
Further variants of Sortase (B, C, D isoforms) are implicated in lowing the first glycine have been reported as not as efficient
specialized functions such as pilus growth and attachment, as glycine.24 Complicating factors include the possible reversi-
and sporulation.9,10,13 Unsurprisingly, this robust natural bility of the reaction with competition from the cleaved and
protein ligation system has been eagerly embraced by the bio- released glycine from LPETGX22 and the requirement for a
technology community and many applications have been minimal length of flexible peptide to ensure steric access to
described in the protein engineering and bioconjugation the enzyme active site and N-terminal amine accessibility.
fields14,15 including but ‘by no means limited to’ recent
advances in protein circularisation,16 antibody functionalisa- last nucleophilic attack, we describe a series of experiments
tion17 and half-life extension.18
aimed at exploring alternative nucleophiles for use in the SrtA
In order to further elucidate substrate requirements in this
When the target LPXTGX recognition sequence is within system. We successfully demonstrate that triglycine, glycine
the SrtA active site, it is subjected to nucleophilic attack by a and ethylenediamine are efficient nucleophiles with respect to
thiol, from cysteine184, a residue which is highly conserved SrtA-mediated labelling of a range of recombinant antibody
across the Sortase family.12 This attack at the carbonyl of the and scaffold proteins and peptides that contain the LPETGG
Thr-Gly bond, results in the formation of a short-lived tetra- recognition sequence, and further demonstrate the utility of
hedral oxyanion transition state that is stabilised by a hydro- this system in Aβ-mediated labelling of primary neuronal
gen bond with arginine197.15 Subsequent protonation of the cultures.
leaving group (Gly-X) leads to formation of an acyl-enzyme
intermediate. The acyl-enzyme intermediate then faces nucleo-
philic attack at the threonine carbonyl, forming a new amide
bond and releasing the Sortase-Cys. It is this last nucleophilic
attack that is the focus of this work. The N-terminal nucleo-
Results
Comparison of five SrtA nucleophiles
phile is an amino-group, usually from a terminal glycine A linear sequence consisting of 3–5 consecutive glycine resi-
residue. Interestingly, there have been divergent observations dues is well-established as required for efficient nucleophilic
reported regarding the rules for this amino-group to act as an reactivity for in vitro SrtA bioconjugation reactions. To
effective nucleophile and the potential for non-glycine peptide compare alternative nucleophiles, we synthesised five rhoda-
or non-peptide nucleophilic activity.19–21 For example, N-termi- mine (5/6-carboxytetramethylrhodamine, TAMRA) functiona-
nal glycine analogues such as alanine have appeared to be lised compounds: TAMRA-EDA-triglycine; TAMRA-EDA-glycine;
ineffective22 as are non-peptide, non-amine nucleophiles such TAMRA-EDA-lysine; TAMRA-EDA; and TAMRA-cadaverine
as dithiothreitol (DTT) or beta-mercaptoethanol (BME).19
A
(Fig. 1; EDA = ethylenediamine). The five variants were isolated
second amide bond after the C-terminal Gly-carbonyl seems to in high purity and characterised by ES-MS and analytical
be required19 and it is generally assumed that there must be at HPLC. The triglycine nucleophile (TAMRA-EDA-triglycine) was
Fig. 1 Synthesis of Rhodamine-labelled nucleophiles 6a–e. (A) Synthetic route to prepare amine functionalised, N-Boc-protected amino acids and
(B) Synthetic route to prepare rhodamine-labelled nucleophiles. Dichloromethane (DCM), ethanol (EtOH), N,N,N’,N’-tetramethyl-O-(N-succinimi-
dyl)-uronium tetrafluoroborate (TSTU), diisopropylethylamine (DIPEA), dimethylformamide (DMF), triethylamine (Et3N), trifluoroacetic acid (TFA).
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chosen as a positive control for SrtA-mediated conjugation and
the single terminal glycine nucleophile (TAMRA-EDA-glycine)
was investigated in order to clarify conflicting reports regard-
ing the ability of a single glycine to act as an effective nucleo-
phile. Understanding the efficiency of a single terminal glycine
to act as a nucleophile in SrtA-mediated conjugations is an
important factor considering the possible reversibility of the
reaction with competition from the cleaved and released
glycine from LPETGX.22 Two different length, non-amino acid
nucleophiles were chosen, ethylenediamine (1,2-diamino-
ethane) and cadaverine (1,5-diaminopentane), which contain
an N-terminal nucleophilic primary amine. Understanding the
efficiency of (and selectivity between) non-amino acid nucleo-
philes, of similar size and structure to the amino acid terminal
nucleophiles, will aid in understanding the mechanism of
SrtA-mediated ligation. All five compounds were tested for
nucleophilic efficiency in SrtA-mediated bioconjugation reac-
tions in our model system where an LPETGG acceptor
sequence is positioned between the N-terminal Im7 4-helical
scaffold protein25 and C-terminal dual FLAG affinity tags and
associated linker sequences. Successful addition of rhodamine
to the Im7 protein was measured both as a decrease in protein
molecular weight (due to loss of the FLAG epitopes; ∼2.4 kDa)
and rhodamine fluorescence measured at 605 nm. Both trigly-
cine (as expected) and glycine alone acted as nucleophiles, and
a significant signal was also observed for ethylenediamine
(Fig. 2). In these assays, a band representing the Im7-LPET/
SrtA reaction intermediate (SrtA + Im7) was clearly visible at
∼29 kDa (as well as dimers of SrtA) which dissociated upon
nucleophilic attack. Importantly, the Im7-rhodamine end pro-
ducts (Im7-LPET-TAMRA) for the TAMRA-EDA-triglycine and
TAMRA-EDA-glycine reactions were also characterised by Elec-
trospray ionization mass spectrometry, ESI-MS (Fig. 3D and E).
The Im7-LPET/SrtA reaction intermediate was also detected by
mass spectrometry for the TAMRA-EDA-lysine, TAMRA-ethyle-
nediamine and TAMRA-cadaverine reactions, (Fig. 3F–H). The
presence of greater amounts of this intermediate for the
TAMRA-EDA-lysine, TAMRA-ethylenediamine and TAMRA-
cadaverine reactions, while not quantitative, indicates that due
to poor nucleophilic attack these intermediates were not disso-
ciated, leading to lower amounts of product for this particular
protein. Prior to further experimentation, this system was opti-
mised with regard to nucleophile and SrtA concentrations
(Fig. S1†), consistent with our previously reported results,26
and confirmation was obtained that the SrtA-mediated label-
ling was specific for the LPETGG recognition sequence
(Fig. S2†).
Fig. 2 Five rhodamine-labelled compounds were tested for their ability
to act as nucleophiles in SrtA-mediated bioconjugation reactions utilis-
ing C-terminally LPETGG & dual-FLAG epitope-tagged Im7 protein. The
decrease in molecular weight associated with replacement of the FLAG
epitopes by rhodamine is clearly observed (top panel, Coomassie
stained non-reducing SDS-PAGE), which is reflected by the associated
gain in fluorescence (bottom panel, measured at 605 nm). Various inter-
mediates and SrtA dimeric species are visible at higher molecular
weights (SrtA + Im7: intermediate species prior to nucleophilic attack;
SrtA2: dimer of SrtA; SrtA2 + Im7 intermediate species consisting of SrtA
dimer
+ Im7 prior to nucleophilic attack). Approximate molecular
weights (kDa) are indicated to the left. Fluorescent bands at the bottom
of the gel (bottom panel, <3 kDa) are due to excess, unreacted rhoda-
mine-labelled compound.
clear activity was also observed for ethylenediamine. Interest-
ingly, for these additional examples, and in particular for the
two larger proteins (Fab and scFv), a modest but consistent
labelling was observed for both lysine and cadaverine,
suggesting at least a minimal ability of these two compounds
to act as nucleophiles in these systems (Fig. 4A and B). 1,5-Di-
aminopentane (cadaverine) has been reported to act as an
efficient nucleophilic acyl-acceptor in SrtA-mediated conju-
gation reactions, upon extended incubation either with an
LPXTG containing nonapeptide (9 h) or an LPETG containing
glycosyltransferase (overnight).20 This cadaverine nucleophile
is a biotin-labelled 1,5-diaminopentane and successful conju-
gations were determined by detection of product by
MALDI-MS for the short peptide, and by immunoblotting with
streptavidin–HRP for the glycosyltransferase conjugation.20 In
order to further investigate whether extended incubation of
To demonstrate that the results obtained were not confined
to the Im7 system, a series of experiments was performed uti-
lising a range of LPETGG-tagged acceptor proteins, including:
a recombinant antibody Fab fragment (52 kDa); a recombinant
scFv antibody (29 kDa),26 the Im7 protein displaying the
amyloid beta (Aβ1–42) peptide at the N-terminus (17 kDa); the
Im7 protein alone (12 kDa); and a 23 residue peptide consist-
ing of Aβ1–16 + LPETGG (2.6 kDa) (Fig. 4A and E). In all cases,
glycine alone was as efficient a nucleophile as triglycine and
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Fig. 3 Electrospray ionization (ESI+) Mass Spectra of reaction mixtures corresponding to lanes 1–8 from Fig. 2B. (A) No SrtA; (B) No TAMRA-
EDA-GGG; (C) no Im7; (D) TAMRA-EDA-GGG; (E) TAMRA-EDA-glycine; (F) TAMRA-EDA-lysine; (G) TAMRA-EDA; and (H) TAMRA-cadaverine-; (EDA =
ethylenediamine; TAMRA = 5/6-carboxytetramethylrhodamine). Arrows in panels D and E represent predicted molecular masses for rhodamine-
labelled Im7 conjugates using triglycine and glycine nucleophiles respectively. While a rhodamine-labelled product is faintly visible for TAMRA-EDA
by fluorescence (Fig. 1B, bottom panel), it is not detected by mass spectrometry. SrtA is present at 18.760 kDa in all spectra except for panel A,
which is a SrtA free control. The species at ∼29 kDa represents an intermediate complex of Im7-LPETGG and SrtA enzyme prior to nucleophilic
attack (Im7-LPET/SrtA).
our TAMRA-cadaverine nucleophile would promote more This is in contrast with the ability of the cadaverine-based acyl-
efficient labelling, we extended reaction periods to 5 and acceptor reported by Ito et al. to act as an effective nucleophile
24 hours, for both the Im7 system (which showed the poorest in SrtA-mediated conjugation reactions with the LPXTG-
reactivity for this nucleophile) and for the scFv antibody. For labelled peptide and protein reported by these workers.20
use in these extended reaction timed experiments, Im7- Thus, we conclude that reaction of the nucleophile appears
LPETGG was purified to high yields through a C-terminal hexa- to be greatly dependent upon the LPXTG-labelled peptide or
histidine tag (replacing the dual FLAG epitopes). Results for protein that is being used in the SrtA-mediated
the scFv conjugation reaction over extended reaction times, conjugation reactions. This is evident within our own work
confirmed the abilities of lysine and cadaverine to act as and in comparison with others, which suggests the mechan-
nucleophiles (Fig. S3A†). However, in the case of Im7-LPETGG ism of nucleophilic attack of the protein-LPXT-SrtA intermedi-
only the most barely detectable activity was observed (by fluo- ate is dependant not only on the nature of the nucleophile but
rescence overexposure) (Fig. S3B†). Incubation time on the nature of the protein containing the LPXTGX recog-
beyond 3 hours did not result in an increased yield of product. nition sequence.
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LPETG-rhodamine as expected (Fig. 5D–F). In contrast, Aβ1–42
-
Im7-LPETG-rhodamine bound strongly to the cell body
(including the cell membrane of this region) and nucleoli of
fixed neuronal cultures (Fig. 5G–I).
Discussion
We have demonstrated that N-terminal triglycine, glycine and
ethylenediamine labelled with the fluorescent dye rhodamine
are capable of acting as effective nucleophiles for bioconjuga-
tion to a range of recombinant antibody constructs and pep-
tides. In exploring the reactivity of these terminal amines we
have progressed our understanding of the requirements for
the enzyme-recognition sequences for SrtA-mediated ligations.
Specifically, our findings can be reconciled with previous work
as follows: (i) The Gram positive cell wall contains pentagly-
cine bridges as the naturally evolved nucleophile for SrtA con-
jugation and it is therefore unsurprising that the enzyme
demonstrates maximal activity for this configuration. (ii)
Evolution of different Sortase variants with altered recognition
sequences suggests a degree of flexibility in the system. For
example, in SrtD-mediated pilin assembly the ε-amino group
of lysine within the YPKN motif initiates nucleophilic attack,29
while SrtB from Bacillus anthracis accepts meso-diaminopimelic
acid as a nucleophile.30 (iii) There is also clearly further poten-
tial for nucleophile heterogeneity, with reports of transfer to
kanamycin and oligosaccharides.31 (iv) Our demonstration of
marginal activity for both lysine and cadaverine in our SrtA-
based systems suggests that any promiscuity in nucleophilic
utilisation is probably tightly controlled in biological systems,
in line with the distinction between household (SrtA) and
more tightly regulated (SrtB-D) isoforms and the nature of
these tightly controlled biological processes.
This study of the nucleophilic efficiency of different N-term-
inal amino-groups using different amino-acid, and different
length non-amino acid, nucleophiles in SrtA-mediated conju-
gations has led to some interesting outcomes. A single N-term-
inal glycine is not always reported as an effective
nucleophile;19,22 in our system it is equally as effective as trigly-
cine which is widely accepted as a good substrate. This result
is significant given the possible reversibility of the SrtA reac-
tion, because the terminal glycine residue is cleaved and
released from the LPETGX sequence; this is now able to
compete with the nucleophile for reaction at the threonine of
the LPET-SrtA intermediate. In our work, the non-glycine
peptide, that is N-terminal lysine, showed some, but limited
overall effective nucleophilic activity which may suggest
unfavourable steric bulk in the SrtA active site. The difference
in ability of the non-peptide terminal amines (ethylenedi-
amine and cadaverine) to work as nucleophiles in Sortase reac-
Fig. 4 Image of SDS-PAGE gel (measured at 605 nm, to detect fluor-
escence) of SrtA-mediated labelling reactions utilizing C-terminally
LPETGG-tagged proteins. (A) Antibody Fab fragment 39S-3. (B) Antibody
scFv fragment 38B-5. (C) The Im7 protein displaying Aβ1–42 peptide at
the N-terminus. (D) The Im7 protein. (E) Aβ1–16-LPETGG peptide.
SrtA-mediated labelling of Aβ peptide and neuronal cell
imaging
The Aβ1–42 peptide is recognised as a key molecule in the
aetiology of Alzheimer’s disease, through its ability to interfere
with neuronal function, as well as induce an inflammatory
response after aggregation into amyloid plaques.27,28 To
demonstrate an application of the rhodamine-based SrtA-
mediated bioconjugation system, the Im7 scaffold protein was
protein-engineered to display Aβ1–42 at the N-terminus
(Fig. 5A). Aβ-Im7-LPETGG, Im7-LPETGG and Im7 (no LPETGG
sequence) proteins were labelled with rhodamine via SrtA-
mediated conjugation reactions with TAMRA-EDA-GGG, and
were assessed for fluorescence. As expected, native Im7 with
no SrtA recognition sequence was not labelled (indicating the
absence of non-specific interactions), while both Im7-LPETGG
and Aβ1–42-LPETGG were efficiently labelled, with only small
amount of unreacted protein and Im7-LPET/SrtA intermediate
apparent in the reaction mixture (Fig. 5B). Prior to cell studies,
non-reacting components were removed by immunoprecipita-
tion using Ni2+-Sepharose (to remove SrtA) and anti-FLAG-
Sepharose beads (to remove Im7 and Aβ1–42-Im7), taking
advantage of the loss of the FLAG epitopes from the protein
scaffold upon rhodamine addition in this system. Upon
addition of labelled proteins to fixed murine primary neuronal
cultures, only very weak binding was observed for the Im7-
tions alludes to
Terminal ethylenediamine (1,2-diaminoethane) worked well as
nucleophile whereas cadaverine (1,5-diaminopentane),
a size-specific nucleophilic active site.
a
which is only three carbon atoms longer, generally led to
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Fig. 5 Neuronal cell imaging by rhodamine-labelled proteins. (A) The Im7 protein encodes C-terminal FLAG affinity and SrtA tags for affinity purifi-
cation and labelling respectively. The Aβ1–42 peptide is displayed at the scaffold’s N-terminus, where it is precisely N-terminally processed (arrow)
during protein production. (B) Coomassie stained non-reducing SDS-PAGE gel. (C) Image of the same gel in B measured at 605 nm, to detect fluor-
escence. (B–C) Im7-LPETGG and Aβ1–42-LPETGG are labelled with rhodamine using TAMRA-EDA-GGG (arrowed), but wild type Im7 which lacks an
LPETGG sequence is not. In this reaction (prior to affinity purification) the bands at ∼26- and ∼47-kDa represent SrtA and SrtA dimer respectively;
intermediate products (Im7-LPET/SrtA) are also present for Im7-LPETG and Aβ1–42-LPETG as described for Fig. 2. Only the final Im7-LPET-rhodamine
and Aβ1–42-Im7-LPET-rhodamine products are visible by fluorescence. (D–F) Im7-LPET-rhodamine binding to fixed murine primary neuronal cul-
tures. (G–I) Aβ1–42-Im7-LPET-rhodamine binding to fixed murine primary neuronal cultures. D and G show DAPI staining only; E and H show rhoda-
mine staining only. F is the merged micrograph of D and E. I is the merged micrograph of G and H. Micrographs are 40× magnification.
lesser amounts of conjugate. Nucleophilic efficiency of ethy-
The results we have presented are qualitative rather than
lenediamine in SrtA-mediated conjugations suggests promis- strictly quantitative, due to the difficulty of accurately quanti-
cuity in the SrtA active site, thus allowing for nucleophilic attack tating precise yield figures within acceptable levels of error in
without site specific recognition of an amino acid sequence. The gel fluorescence and by mass spectrometry. This is com-
ability of ethylenediamine to function as a nucleophile in our pounded by the downstream processes required for protein
SrtA-mediated conjugations also contradicts the traditional rules purification including immune-pull-down and gel filtration
for SrtA nucleophiles that allude to the requirement for an N- which result in progressive losses. However, as an example at
terminal glycine. The mechanism of SrtA-mediated conju- the conclusion of these procedures a recovery yield of 15–20%
gations, and in particular of nucleophilic attack is clearly a purified labelled protein is not uncommon in our hands.
complex one that potentially involves a size/shape-specific Additionally, by presenting relative labelling levels for five
nucleophilic active site as well as other potential considerations different proteins/peptides we provide a basis for direct com-
such as pKa of the nucleophiles, which has not been addressed parison between all five nucleophiles across a range of
here but is a potential contributor to this mechanism.
LPETGG substrates.
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This work has also initiated further systematic and compre- technology, for example the ability to reduce the nucleophile
hensive studies involving the use of a range of amino acid and to a single N-terminal glycine (or ethylenediamine) is of value
non-amino acid based nucleophiles for SrtA-mediated conju- in situations where the introduced sequence, post-conjugation,
gation, which will be reported in due course. These further is to be minimised. This study has progressed the understand-
studies will include considerations for the role that potential ing regarding determination of the nucleophilic specificity, for
differences in pKa of each of the terminal nucleophilic amino both amino acid and non-amino acid nucleophilic com-
groups for each of the nucleophiles may play in the efficiency pounds, in SrtA-mediated conjugations, which ultimately will
of conjugation reactions.
lead to a better understanding of the nucleophilic binding site
Beyond dissection of the workings of in situ bacterial struc- in the enzyme. Not only does the nature of the nucleophile
ture-function, our results are useful in application of SrtA lab- determine efficiency of SrtA-mediated conjugation, but the
elling technology for in vitro applications. Our demonstration nature and size of the target LPXTG-labelled protein also plays
that lysine and cadaverine activity appeared dependent upon a key role in reaction efficiency. The demonstration of target-
the target protein scaffold, with more significant conjugation ing of neuronal cells with a Sortase-labelled medically impor-
observed for the two larger test proteins (Fab and scFv anti- tant peptide–protein–rhodamine conjugate illustrates the
body fragments) than for the helical Im7 scaffold, is a signifi- power of this system and further suggests that direct SrtA-
cant consideration for planning of bioconjugation mediated cell-surface labelling and visualisation may be an
experiments; and suggests that variations may occur, depen- achievable goal.
dent not only upon the specific Sortase recognition signal and
nucleophilic composition, but also upon the nature and size
of the target protein. Obviously, steric considerations for
access to the enzyme active site are of concern and the differ-
ences observed for our constructs may not necessarily be
Experimental
Cloning, expression, and purification of SrtA
A DNA cassette encoding residues 60–208 of the S. aureus SrtA
was synthesised at Geneart AG (Regensburg, Germany, http://
www.geneart.com) in the expression vector pET28a (Novagen).
The sequence included 5′ NdeI and 3′ BamH1 sites and initiat-
ing methionine and N-terminal hexahistidine tag sequences.26
Details of pET28a–SrtA protein expression are described in
the ESI.†
apparent for more extended i.e., pentaglycine, nucleophiles.
Our previous work demonstrated site-specific ligation of small
molecule GGG constructs to the therapeutically relevant sc528
scFv without compromising the ability to bind antigen with
high affinity.26 The present study utilised the more chemically
and biologically tractable fluorophore rhodamine, which dis-
plays photostability across a pH range, and good fluorescence
intensity with a narrow red emission spectrum.
Synthesis of nucleophiles
As an example of this technology, we demonstrated success-
ful labelling of a protein scaffold in the presence and absence
of Aβ peptide and measured binding to fixed primary neuronal
cells cultured from mouse brain. The binding of Aβ to the cell
membrane of live cells has been reported previously,32,33
however, we utilised acetone-fixed cells which not only cross-
links the cellular structures but also permeabilises the cell
membrane. Hence, in our study the Aβ1–42-Im7-LPET-rhoda-
mine bound to the cell membrane and the cell body,
suggesting widespread localisation of the Aβ peptide, consist-
ent with previous reports.34,35 The binding of Aβ to nucleoli
has not been reported and our data may reflect the interaction
of Aβ with ribosomal proteins in this region.36 These results
demonstrate not only the utility of our SrtA-mediated rhoda-
mine labelling, but also represent a promising system for
investigation of compounds which interfere with Aβ-mediated
cellular binding and possibly neuronal toxicity.
General. All chemicals were used as received (Aldrich), NHS-
rhodamine (5/6-carboxytetramethylrhodamine succinimidyl
ester, 5/6-TAMRA SE) was purchased from Thermo Scientific.
NMR spectra were recorded with a Bruker ARX-400 spectro-
meter at ambient temperature and were referenced with
respect to residual solvent peaks in deuterated solvents. Elec-
trospray ionization (ESI) mass spectra were recorded on an
Applied Biosystems API 150EX mass spectrometer. N-Boc
glycine was purchased from Aldrich, N-Boc-GGG37 and diBoc-
lysine38 were prepared by modifications of published
procedures.
Benzylphenylcarbonate (1). To a stirred solution of benzyl-
alcohol (500 μL, 4.83 mmol) and pyridine (470 μL, 5.82 mmol)
in dichloromethane (DCM, 30 mL) was added drop wise, phe-
nylchloroformate (730 μL, 5.82 mmol). The solution was
stirred at room temperature under nitrogen for 16 h. The reac-
tion mixture was washed with H2O, then 2 M H2SO4, and the
organic layer was dried (MgSO4), filtered and concentrated
under reduced pressure to give crude benzylphenylcarbonate
(1) as a pale tan colored oil (1.10 g, quantitative yield). δH
(400 MHz, CD3OD) 7.40–7.15 (m, 10H, ArH × 10), 5.25 (s, 2H,
ArCH2).
Conclusion
SrtA-mediated protein ligation represents a precise, reliable
and reproducible tool for functionalisation of biologically
important molecules. Expansion of the range of available
Mono-Cbz-ethylenediamine (2). A solution of benzylphenyl-
nucleophiles which can be utilised in such bioconjugation carbonate (1.10 g, 4.83 mmol) and ethylenediamine (290 mg,
systems extends the range of potential applications for this 4.83 mmol) in ethanol (EtOH, 30 mL) was stirred at room
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temperature under nitrogen for 16 h. The reaction mixture was
NH2-(CH2)2-NH-GGG-NH-Boc (4a). Colourless oil (60.9 mg,
concentrated under reduced pressure then dissolved in H2O quantitative yield), δH (400 MHz, CD3OD) 3.88 (s, 2H, G-CH2),
(25 mL). The solution was acidified to pH 3 using 5% HCl 3.83 (s, 2H, G-CH2), 3.73 (s, 2H, G-CH2), 3.27 (t, J 6.0 Hz, 2H,
(2 mL) and extracted into DCM (2 × 50 mL). The aqueous layer NHCH2), 3.25 (t, J 6.0 Hz, 2H, CH2NH), 1.44 (s, 9H, Boc).
NH2-(CH2)2-NH-glycine-NH-Boc (4b). Colourless oil (20.0 mg,
85%). δH (400 MHz, CD3OD) 3.65 (s, 2H, G-CH2), 3.31 (t,
J 5.8 Hz, 2H, NHCH2), 3.23 (t, J 5.8 Hz, 2H, CH2NH), 1.42 (s,
9H, Boc).
NH2-(CH2)2-NH-lysine-NH-Boc (4c). Colourless oil (50.0 mg,
84%). δH (400 MHz, CD3OD) 3.89 (t, J 5.6 Hz, 1H, CH), 3.30 (t,
J 6.2 Hz, 2H, NHCH2), 3.22 (t, J 6.2 Hz, 2H, CH2NH), 1.70 (m,
8H, CH2 × 4) 1.41 (s, 9H, Boc) 1.39 (s, 9H, Boc).
General procedure for rhodamine-CONH-R-NH-Boc (5). To a
stirred solution of NH2-R-NH-Boc (4) (1.2 eq.) and triethyl-
amine (Et3N, 5 eq.) in DMF (3 mL) was added a solution of
NHS-rhodamine (1 eq.) in DMF (1 mL). The bright pink solu-
tion was stirred at room temperature under nitrogen and pro-
tected from light, for 16 h. The reaction mixture was
concentrated under reduced pressure. The residue was taken
up into 1 : 1 MeOH–H2O [20 mg mL−1] and filtered through a
0.45 μm Acrodisc for purification by reverse phase HPLC.
HPLC conditions: 0.1% TFA, 20–80% MeCN from 2–26 min,
flow 10 mL min−1. HPLC revealed the presence of rhodamine-
CONH-R-NH-Boc (5) as two major products (two structural
isomers of 5/6-carboxytetramethylrhodamine succinimidyl
ester).
was made alkaline by 1 M NaOH (2 mL) and extracted into
DCM (3 × 60 mL), dried (MgSO4), filtered and concentrated
under reduced pressure to give mono-Cbz-ethylenediamine (2)
as a pale tan colored oil (332 mg, 28% yield calculated from 1).
δH (400 MHz, CDCl3) 7.38–7.27 (m, 5H, ArH × 5), 5.11 (s, 2H,
ArCH2), 3.24 (t, J 6.0 Hz, 2H, NHCH2), 2.83 (t, J 6.0 Hz, 2H,
CH2NH2).
General procedure for mono-Cbz-ethylenediamine-amino
acids (3). To a stirred solution of N-Boc protected amino acid
(1 eq.) in DMF (2.0 mL) was added diisopropylethylamine
(DIPEA, 5 eq.) followed by a solution of N,N,N′,N′-tetramethyl-
O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, 1.3 eq.)
in DMF (2.0 mL). The solution was stirred at room tempera-
ture, under nitrogen for 30 min. mono-Cbz-ethylenediamine
(2, 1.4 eq.) was added as a solution in DMF (2.0 mL) and the
solution stirred for a further 16 h. The reaction mixture
was concentrated under reduced pressure to yield crude
mono-Cbz-ethylenediamine-amino acid (3). The residue was
taken up into 1 : 1 MeOH–H2O [20 mg mL−1] and filtered
through a 0.45 μm Acrodisc for purification by reverse phase
HPLC. HPLC conditions: 0.1% TFA, 20–80% MeCN from
2–26 min, flow 10 mL min−1
, λ = 214 nm, ambient
Rhodamine-CONH-(CH2)2-NH-GGG-NH-Boc (5a). HPLC purifi-
cation yielded two major peaks at Rt 15.0 min and Rt 16.0 min,
giving rhodamine-CONH-(CH2)2-NH-GGG-NH-Boc (5a) as a
red powder (10.7 mg, quantitative yield). m/z (ESI) found 744.4
(M+ + 1), calculated 743.81.
temperature.
Cbz-NH-(CH2)2-NH-GGG-NH-Boc (3a). HPLC purification
revealed a major peak at Rt 15.7 min, giving Cbz-NH-(CH2)2-
NH-GGG-NH-Boc (3a) as a colourless oil (80.0 mg, 50%). δH
(400 MHz, CD3OD) 7.31–7.28 (m, 5H, ArH × 5), 5.07 (s, 2H,
ArCH2), 3.88 (s, 2H, G-CH2), 3.83 (s, 2H, G-CH2), 3.73 (s, 2H,
G-CH2), 3.27 (t, J 6.0 Hz, 2H, NHCH2), 3.25 (t, J 6.0 Hz, 2H,
CH2NH), 1.44 (s, 9H, Boc). m/z (ESI) found 466.3 (M+ + 1),
calculated 465.51.
Cbz-NH-(CH2)2-NH-glycine-NH-Boc (3b). HPLC purification
revealed a major peak at Rt 18.1 min, yielding Cbz-NH-(CH2)2-
NH-glycine-NH-Boc (3b) as a colourless oil (38.0 mg, 95%). δH
(400 MHz, CD3OD) 7.34–7.28 (m, 5H, ArH × 5), 5.07 (s, 2H,
ArCH2), 3.66 (s, 2H, G-CH2), 3.31 (t, J 5.8 Hz, 2H, NHCH2), 3.23
(t, J 5.8 Hz, 2H, CH2NH), 1.44 (s, 9H, Boc). m/z (ESI) found
352.5 (M+ + 1), calculated 351.40.
Cbz-NH-(CH2)2-NH-lysine-NH-Boc (3c). HPLC purification
revealed a major peak at Rt 23.3 min, isolating Cbz-NH-(CH2)2-
NH-lysine-NH-Boc (3c) as a colourless oil (67.5 mg, 89%). δH
(400 MHz, CD3OD) 7.35–7.28 (m, 5H, ArH × 5), 5.07 (s, 2H,
ArCH2), 3.94 (t, J 5.6 Hz, 1H, CH), 3.30 (t, J 6.2 Hz, 2H,
NHCH2), 3.22 (t, J 6.2 Hz, 2H, CH2NH), 1.70 (m, 8H, CH2 × 4)
1.43 (s, 18H, Boc × 2). m/z (ESI) found 523.6 (M+ + 1), calcu-
lated 522.63.
General procedure for ethylenediamine-amino acids (4). A
solution of mono-Cbz-ethylenediamine-amino acid (3) in
methanol [0.1 M] was subjected to Cbz-deprotection using
H-cube flow instrument (1 mL min−1, 50 °C, H2 (1 bar), 10%
Pd/C) and concentrated under reduced pressure to yield ethy-
lenediamine-amino acid (4).
Rhodamine-CONH-(CH2)2-NH-Glycine-NH-Boc
(5b). HPLC
purification gave two major peaks at Rt 16.4 min and Rt
17.4 min, isolating rhodamine-CONH-(CH2)2-NH-glycine-
NH-Boc (5b) as a red powder (10.3 mg, quantitative yield). m/z
(ESI) found 630.6 (M+ + 1), calculated 629.70.
Rhodamine-CONH-(CH2)2-NH-Lysine-NH-Boc (5c). HPLC puri-
fication yielded two major peaks at Rt 20.5 min and Rt
21.2 min, giving rhodamine-CONH-(CH2)2-NH-lysine-NH-Boc
(5c) as a red powder (8.3 mg, 91%). m/z (ESI) found 801.5
(M+ + 1), calculated 800.94.
Rhodamine-CONH-(CH2)2-NH-Boc (5d). HPLC purification
gave two major peaks at Rt 18.3 min and Rt 19.2 min, yielding
rhodamine-CONH-(CH2)2-NH-Boc (5d) as
a
red powder
(7.3 mg, quantitative yield). m/z (ESI) found 573.5 (M+ + 1), cal-
culated 572.65.
Rhodamine-CONH-(CH2)5-NH-Boc (5e). HPLC purification
gave two major peaks at Rt 20.6 min and Rt 21.1 min, isolating
rhodamine-CONH-(CH2)5-NH-Boc (5e) as
a
red powder
(11.0 mg, quantitative yield). m/z (ESI) found 615.5 (M+ + 1),
calculated 614.73.
General procedure for rhodamine-CONH-R-NH2 (6). To a
stirred solution of rhodamine-CONH-R-NH-Boc (5) (1 eq.) in
DCM (2 mL) was added trifluoroacetic acid (TFA, 0.4 mL). The
resulting pink solution was stirred at room temperature, pro-
tected from light, for 2 h. The solution was concentrated
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Paper
under reduced pressure and the residue was taken up into 1 : 1 monoclonal antibody resin40). Proteins were stored in small
MeCN–H2O (3 × 1 mL) and the volatiles removed under aliquots at 4 °C prior to use.
reduced pressure to yield rhodamine-CONH-R-NH2 (6). The
product was analysed by analytical reverse phase HPLC. HPLC
Cell-based immunofluorescence assays
conditions: 0.1% TFA, 0–80% MeCN from 2–34 min, flow 1 mL
Murine primary neuronal cells were isolated as previously
min−1, λ = 214 nm. HPLC revealed the presence of rhodamine-
described41 from E14–16 embryos with approval from the
CONH-R-NH2 (6) as two major products (two structural
Animal Ethics Committee of the Florey Institute of Neuro-
science and Mental Health (Melbourne, Victoria, Australia).
isomers of 5/6-TAMRA SE).
Rhodamine-CONH-(CH2)2-NH-GGG-NH2
(TAMRA-EDA-GGG)
Neuronal cells were plated (6.5 × 104 cells cm−2) on coverslips
(ProSciTech, Kurwan, Queensland, Australia, coverglass no.1,
13 mm diameter) coated with poly-D-lysine (10 μg cm−2, Sigma-
Aldrich, St. Louis, MO) and incubated at 37 °C for 8–10 days
in vitro (DIV). Cultures were fixed with ice-cold acetone for
15 min, then rinsed in PBS (non-permeabilising fixative such
as paraformaldehyde was not used because the free aldehydes
bind non-specifically to the rhodamine leading to high fluo-
rescence background). Before labelling, the coverslips were
treated with blocking buffer (2% w/v bovine serum albumin in
1× PBS) for 1 h at room temperature. The blocking solution
was removed and replaced with PBS containing either Im7-
LPETG-rhodamine (15 μM) or Aβ1–42-Im7-LPETG-rhodamine
(7.5 μM). After an overnight incubation at room temperature,
the rhodamine proteins were removed and the coverslips were
washed in 1× PBS for 5 min for a total of three washes. The
nuclei were stained with 4′,6-diamidino-2-phenylindole (1 mg
mL−1, DAPI) in PBS for 10 min at room temperature. The
coverslips were washed in 1× PBS for 5 min for a total of three
washes. Coverslips were mounted onto slides with fluorescent
mounting medium (Dako, Carpenteria, CA). Micrographs were
captured using a fluorescence microscope (BX60, Olympus,
Tokyo, Japan) and a QICAM 12-bit mono cooled camera
(QImaging, Surrey, British Columbia, Canada).
(6a). Analytical HPLC gave two major peaks at Rt 13.3 min and
Rt 14.4 min corresponding to rhodamine-CONH-(CH2)2-
NH-GGG-NH2 (6a). m/z (ESI) found 644.6 (M+ + 1), calculated
643.69.
Rhodamine-CONH-(CH2)2-NH-glycine-NH2 (TAMRA-EDA-glycine)
(6b). Analytical HPLC gave two major peaks at Rt 13.2 min and
Rt 14.2 min corresponding to rhodamine-CONH-(CH2)2-
NH-Glycine-NH2 (6b). m/z (ESI) found 530.3 (M+ + 1), calcu-
lated 529.59.
Rhodamine-CONH-(CH2)2-NH-lysine-NH2 (TAMRA-EDA-lysine)
(6c). Analytical HPLC gave two major peaks at Rt 13.7 min and
Rt 14.7 min corresponding to rhodamine-CONH-(CH2)2-NH-
Lysine-NH2 (6c). m/z (ESI) found 601.4 (M+ + 1), calculated
600.71.
Rhodamine-CONH-(CH2)2-NH2 (TAMRA-EDA) (6d). Analytical
HPLC gave two major peaks at Rt 14.0 min and Rt 15.2 min
corresponding to rhodamine-CONH-(CH2)2-NH2 (6d). m/z (ESI)
found 473.3 (M+ + 1), calculated 472.54.
Rhodamine-CONH-(CH2)5-NH2
(TAMRA-cadaverine)
(6e).
Analytical HPLC gave two major peaks at Rt 14.5 min and Rt
15.6 min corresponding to rhodamine-CONH-(CH2)5-NH2 (6e).
m/z (ESI) found 515.7 (M+ + 1), calculated 514.62.
Expression and purification of recombinant antibodies.
Recombinant Fab and scFv antibodies and Im7 scaffold
protein were expressed as previously described.39,40 Proteins
were purified from the E. coli periplasmic fraction or cellular
fraction (for Im7-LPETGG used in Fig. 5, where a single hexa-
histidine tag replaced the dual FLAG tags) by affinity chrom-
atography and gel filtration, and validated by SDS-PAGE and
mass spectrometry. The Aβ-LPETGG peptide (NDAEFRHDS-
GYEVHHQKSLPETGGC) was synthesised by Keck Biotechno-
logy (New Haven, CT).
Abbreviations
SrtA
Aβ
PBS
Sortase A from Staphylococcus aureus
amyloid beta peptide residues 1–42
Phosphate-buffered saline
scFv
single chain variable fragment antibody
TAMRA 5/6-carboxytetramethylrhodamine
SrtA-mediated bioconjugation
Competing interests
Typical SrtA labelling reactions were performed in SrtA reac-
tion buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.5).
Reactions containing 20 µM recombinant protein, 2 mM
nucleophile-rhodamine and 60 µM SrtA were incubated for
3 hours at 37 °C and analysed by non-reducing SDS-polyacryl-
amide gel electrophoresis through 12% tris/glycine gels (Invi-
The authors declare no competing interests.
Authors contribution
trogen, Australia). Rhodamine immunofluorescence was SB, LH, & CW synthesised rhodamine compounds and per-
measured at 605 nm using a VersaDoc™ MP Imaging System. formed conjugation assays. JN & RN performed the neuronal
Where required, rhodamine-labelled proteins were purified by imaging studies. MM, RS, & SN cloned and produced recombi-
removal of unlabelled protein and SrtA from the reaction nant proteins and performed conjugation assays. GC & TA par-
mixture using small-scale His-tag and Flag-tag pull-down puri- ticipated in the design of study and helped to draft the
fication (Ni-NTA superflow, QIAGEN; in house anti-FLAG manuscript. CW & SN designed the experiments, conceived the
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research and wrote the manuscript. Final manuscript was read 18 M. W. Popp, S. K. Dougan, T. Y. Chuang, E. Spooner and
and approved by all the authors.
H. L. Ploegh, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 3169–
3174.
19 X. Huang, A. Aulabaugh, W. Ding, B. Kapoor, L. Alksne,
K. Tabei and G. Ellestad, Biochemistry, 2003, 42, 11307–
11315.
20 T. Ito, R. Sadamoto, K. Naruchi, H. Togame, H. Takemoto,
H. Kondo and S. I. Nishimura, Biochemistry, 2010, 49,
2604–2614.
Acknowledgements
We thank Mr Nick Bartone for performing the mass spec-
trometry and Dr Michael Bird for animal handling and obtain-
ing the murine embryos.
21 M. W. Popp and H. L. Ploegh, Angew. Chem., Int. Ed., 2011,
50, 5024–5032.
22 J. M. Antos, G. L. Chew, C. P. Guimaraes, N. C. Yoder,
G. M. Grotenbreg, M. W. Popp and H. L. Ploegh, J. Am.
Chem. Soc., 2009, 131, 10800–10801.
23 M. W. Popp, J. M. Antos and H. L. Ploegh, Curr. Protoc.
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