Collagen labelling with an azide-proline chemical reporter in live cells

Article abstract of DOI:10.1039/c4cc07974d

We have developed a strategy for selective imaging of collagen in live foetal ovine osteoblasts. Our approach involves the incorporation of an azide-tagged proline in the biosynthesis of collagen followed by labelling using a strain-promoted [3+2] azide-alkyne cycloaddition reaction. This journal is

Full text of DOI:10.1039/c4cc07974d

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Collagen labelling with an azide-proline chemical
reporter in live cells†
Cite this: Chem. Commun., 2015,
51, 5250
Beatrice Amgarten,a Rakesh Rajan,b Nuria Martınez-Saez,a Bruno L. Oliveira,c
´
´
Ines S. Albuquerque,d Roger A. Brooks,b David G. Reid,a Melinda J. Duera and
ˆ
Received 8th October 2014,
Accepted 17th November 2014
Gonçalo J. L. Bernardes*ad
DOI: 10.1039/c4cc07974d
www.rsc.org/chemcomm
We have developed a strategy for selective imaging of collagen in Using this approach, tagging and imaging of temporal and spatial
live foetal ovine osteoblasts. Our approach involves the incorpora- variations of the glycome,10 lipidome,11 as well as subsets of the
tion of an azide-tagged proline in the biosynthesis of collagen proteome12 have been successfully achieved. Proteome tagging can
followed by labelling using a strain-promoted [3+2] azide–alkyne be facilitated through the incorporation of non-canonical amino
cycloaddition reaction.
acids bearing reactive handles using the host cell’s machinery.
Subsequent labelling with a fluorescent dye or radionuclide
Collagen is the most abundant mammalian protein, accounting for by means of bioorthogonal reactions allows the visualisation
one third of all proteins and representing the major component of the of newly synthesised proteins. Reactive metabolic precursors
extracellular matrix (ECM).1 The basic structure of collagen consists of applied previously for this purpose include the methionine
three single strands containing the repeating units Gly–Xaa–Yaa analogues azidohomoalanine13 and homopropargylglycine14
which form a triple helix, where X and Y are predominantly proline as well as the phenylalanine analogue ethynylphenylalanine.15
and hydroxyproline, respectively (both constitute approximately 23%
Azido-proline (N3-Pro) has been used for residue-specific
of the total of amino acids in collagen).2,3 Due to its high abundance labelling of collagen model peptides by functionalization with
in collagen, proline has been widely exploited as a marker of collagen triazolyl moieties.16–18 Moreover, the incorporation fluoro-proline
biosynthesis by measuring incorporation of either radioactive or non- was shown to enhance thermal stability of synthetic collagen
radioactive isotopically-labelled proline (3H-Pro, 14C-Pro, 13C-Pro, structures.19 Importantly, the introduction of modified proline
15N-Pro, cis-18F-Pro) as well as incorporation of heavy atoms (13C analogues still allows a collagen triple helix to form. Together these
and 15N) directly in tissues.3–5 Collagen plays a key role in many examples highlight the potential of introducing proline-based
pathogenic processes, as for example fibrosis6 and tumour growth chemical reporters for the bioorthogonal imaging of collagen. Here,
and metastasis,7 to mention the most prominent. Thus, we under- we report the metabolic incorporation of N3-Pro as a chemical
took to exploit bioorthogonal strategies that would allow non- reporter for the imaging of collagen using dibenzooctyne (DIBO)20
invasive imaging of collagen structures and provide a powerful as a fluorescent probe and exploiting a strain promoted [3+2] azide–
imaging tool to monitor and study diseases where abnormal alkyne cycloaddition (SPAAC) bioorthogonal reaction (Fig. 1). The
collagen production is a main feature, including cirrhosis, metabolic incorporation of a reporter group by foetal ovine osteo-
diabetic nephropathy or idiopathic pulmonary fibrosis.
blasts was achieved by supplementation of the growth medium
Recent advances in bioorthogonal chemical reactions enables the with cis-4-azido-L-proline (ESI† for synthesis details).
design of probes tailored for imaging tagged macromolecules.8,9
We chose ovine osteoblasts as our model system to exploit
the bioorthogonal imaging of collagen in live cells because they have
been shown to behave similarly to human osteoblasts in terms of
viability, and osteocalcin and interleukin-6 production, compared to
other animal derived cells, making them a suitable model system for
our proposed investigations.21 Furthermore, since foetal ovine osteo-
blasts are not auxotrophic for proline – meaning that they are able to
synthesise proline themselves – the levels of incorporation in the
presence of the chemical reporter N3-Pro is expected to be lower than
in pulse-labelling. However, here we make use of a low, physiological
concentration for osteoblast culturing which makes it relevant
for applications in in vivo models.
a Department of Chemistry, University of Cambridge, Lensfield Road,
CB2 1EW Cambridge, UK. E-mail: gb453@cam.ac.uk
b Division of Trauma & Orthopaedic Surgery, University of Cambridge,
Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK
c Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology,
Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA
d Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de
Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal.
E-mail: gbernardes@medicina.ulisboa.pt; Web: http://gbernardes-lab.com
† Electronic supplementary information (ESI) available: Fig. S1–S7, detailed
experimental procedures, IR and mass spectrometry analysis, NMR spectra. See
DOI: 10.1039/c4cc07974d
5250 | Chem. Commun., 2015, 51, 5250--5252
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HPLC and infrared spectroscopy on harvested ECM grown in
the absence or presence of N3-Pro was performed. Amino acid
analysis provided evidence of N3-Pro incorporation in the ECM
(Fig. S2, ESI†). Infrared spectral analysis further supports
incorporation of N3-Pro into ECM as a band attributed to the
stretching of the N3 group could be detected in the labelled
ECM samples as opposed to the unlabelled (Fig. S3, ESI†).
Next, we used fluorescence microscopy to image N3-Pro tagged
collagen in foetal ovine osteoblasts after SPAAC reaction with the
fluorescent probe DIBO (Fig. 2B and C). Cells were grown in the
presence or absence of 36 mg LÀ1 of N3-Pro, fixed and stained
with 5 mM DIBO Alexa Fluor 555 for 60 min at room temperature
and counterstained with the nuclear dye Hoechst 33342 (Fig. 2C).
We found that foetal ovine osteoblasts cultured with N3-Pro and
bioorthogonally labelled with DIBO showed a nearly 2-fold
increase in fluorescence (Fig. 2B and C). Confocal microscopy
on stained foetal ovine osteoblast showed a dotted structure that
likely corresponds to non-secreted pro-collagen (Fig. S4, ESI†)
showing that the cells take up the azide-modified proline.
Fig. 1 Incorporation of N3-Pro chemical reporter into collagen and
labelling using a strain-promoted azide–alkyne cycloaddition reaction
with the fluorescent probe DIBO.
We started by analysing the effect on cell viability of culturing
foetal ovine osteoblasts in the presence of 18 mg LÀ1 or 36 mg LÀ1
of N3-Pro. The presence of N3-Pro had no effect on cell viability
at either concentration (Fig. 2A and Fig. S1, ESI†). To assess
incorporation of N3-Pro into collagen, amino acid analysis by
Fluorescent images showed, however, a relatively high back-
ground staining in the samples cultured in the absence of N3-Pro
(Fig. 2C, i–iii), indicating unspecific DIBO labelling. The DIBO
cyclooctyne fluorescent probe has been successfully applied in
bioorthogonal approaches based on SPAAC, although only in
imaging molecules densely presented on the cell surface.22 Given
the observed unspecific staining, we decided to analyse whether
DIBO was reacting with intracellular proteins, by in-gel fluorescence
(Fig. S5, ESI†) and mass spectrometry analysis (Fig. S6, ESI†) of the
stained whole cell lysate. Despite previously reported selectivity of
cyclooctynes including DIBO for azides over thiols,20 mass spectro-
metry analysis of some of the most prominent bands in the gel
revealed unspecific labelling of intracellular structures such as
actin (Fig. S6, ESI†), suggesting unspecific reaction of the strained
alkyne probe with sulfhydryl-containing biomolecules.
In addition, bovine serum albumin (BSA), which is used as
a media supplement and blocking agent for the staining protocol
(Fig. S6, ESI†) was also detected. Bertozzi and co-workers also reported
the same issue in the in vivo imaging of glycoconjugates using DIFO.23
In an attempt to reduce unspecific labelling, we pre-incubated fixed
samples with the thiol alkylating agent iodoacetamide (IAM) prior to
staining with DIBO.24 Indeed, in-gel fluorescence showed a reduction
of unspecific staining and an increase of background to signal ratio
(Fig. S5, ESI†), which was further confirmed by flow cytometry analysis
(Fig. 3 and Fig. S7, ESI†). Our observations indicate that the use of
the fluorescent probe DIBO in SPAAC bioorthogonal approaches
Fig. 2 (A) Measurement of metabolic activity of foetal ovine osteoblasts
upon incubation with N3-Pro. Data was collected after 1 and 3 days. Error
bars represent the standard error of independent measurements from for imaging of molecules that are not densely presented on the
3 different cell culture wells. (B) Mean of total fluorescent intensity Æ SEM
in arbitrary units (a.u.) of foetal ovine osteoblasts cultured in the absence or
presence of 36 mg mLÀ1 of N3-Pro upon staining with 5 mM DIBO-Alexa
Fluor 555. Statistically significant differences found after two-tailed Mann–
Whitney’s U test are marked as * (P o 0.05) and values are average Æ SEM.
(C) Fluorescence labelling of proteins with DIBO in foetal ovine osteoblasts
cultured in the absence (i–iii) or presence (iv–vi) of 36 mg LÀ1 N3-Pro:
(i) and (iv) staining with 5 mM DIBO Alexa Fluor 555 (red); (ii) and (v) counter-
staining with Hoechst 33342 (blue); (iii) and (vi) overlay. DIBO-Alexa Fluor 555
has a peak excitation at 555 nm and a peak emission at 565 nm. Hoechst 33342
has a peak excitation at 350 nm and a peak emission at 461 nm. Scale bar
represents 20 mm.
cell surface meets with some limitations, namely, its high
reactivity towards cellular and plasma sulfhydryl containing
biomolecules limiting its application to challenging structures
namely intracellular targets.
Finally, we wished to demonstrate that our approach did not
interfere with the normal growth of ECM in foetal ovine osteo-
blasts. Scanning electron microscopy images (SEM) of cells cultured
in the presence of N3-Pro confirmed adequate integrity (Fig. 4).
In summary, we report the metabolic incorporation of N3-Pro
into collagen and its use as a chemical reporter for bioorthogonal
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either in cells or animal models, are not available.6 The cellular
model described here could be used for example for high-
throughput screening of therapeutic agents by fluorescent
microplate-based analysis and help in the identification of lead
anti-fibrotic drugs from a series of candidates.
We thank the EU (Erasmus studentship to B.A.; Marie Curie CIG
to G.J.L.B.), FCT Portugal (FCT Investigator to G.J.L.B.), the EPSRC
and the National Institute of Health Research for funding. We also
thank Karin Mu¨ller for help with confocal microscopy and SEM
and Dr Filipa Cruz for critical reading of the manuscript. G.J.L.B. is
a Royal Society University Research Fellow.
Notes and references
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Fig. 3 Effect of pre-incubation with iodoacetamide (IAM) on fluores-
cence intensity of N3-Pro tagged foetal ovine osteoblasts. N3-Pro tagged
foetal ovine osteoblasts were treated with 5 mM IAM for 60 min prior to
staining with the DIBO probe. Results are normalized to the corresponding
negative controls (cells cultured in the absence of N3-Pro).
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5252 | Chem. Commun., 2015, 51, 5250--5252
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