Glycan-specific metabolic oligosaccharide engineering of C7-substituted sialic acids

Article abstract of DOI:10.1002/anie.201108809

A new sialic acid in the house! A biosynthetic approach gives access to previously inaccessible C7 modifications of sialic acids in living cells. Metabolic incorporation of synthetically derived N-acetyl-4-azido-4- deoxymannosamine (see scheme) into glycans of mammalian cells and zebrafish larvae preserves the bioorthogonal functionality of its azido group for subsequent labeling with biophysical probes. Copyright

Full text of DOI:10.1002/anie.201108809

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DOI: 10.1002/anie.201108809
Sialic Acid Engineering
Glycan-Specific Metabolic Oligosaccharide Engineering of
C7-Substituted Sialic Acids**
Heinz Mçller, Verena Bçhrsch, Joachim Bentrop, Judith Bender, Stephan Hinderlich,* and
Christian P. R. Hackenberger*
Dedicated to Professor Werner Reutter on the occasion of his 75th birthday
Intact and integral glycosylation of membrane-associated as
well as secreted glycoproteins has been shown to be essential
for many aspects of the proper function of biological systems.
Recombinantly expressed glycoproteins, such as antibodies,
growth factors, hormones, vaccines, and contrast agents are
key elements in medical applications.[1] The quality of these
therapeutically administered glycoproteins can be efficiently
improved by the incorporation of chemically functionalized
monosaccharides into their glycan moieties,
a process
denoted as metabolic oligosaccharide engineering (MOE).[2]
In addition to these pharmaceutical applications, MOE has
greatly advanced diagnostics by localizing and visualizing
glycans even in living animals.[2]
Figure 1. Methods for the structural modification of glycan-bound
sialic acids by application of chemically modified ManNAc or direct
periodate oxidation of glycan-bound sialic acids (left). Specific modifi-
cation of the C7 position of sialic acids was achieved by C4-modified
ManNAc in this study (right; note that to date these methods were
carried individually, resulting in only one modification of a single sialic
acid molecule).
To date, a multitude of chemically modified monosac-
charides have been designed for MOE applications. Owing to
their terminal position at glycan structures of glycoproteins
and relevance for cellular recognition, sialic acids and their
metabolic precursor N-acetylmannosamine (ManNAc), are
the most prominent targets for MOE.[3] Several ManNAc
derivatives with N-acetyl side-chain modifications have been
synthesized and metabolically incorporated by the sialic acid
biosynthetic pathway into a corresponding sialic acid C5
analogue (Figure 1). This approach was beneficial to extend-
ing the understanding of the biological role of the N-acyl side
chain of sialic acids, for example, in virus infection[4] or
neuronal differentiation.[5] Alternatively, C9 modifications of
sialosides have also been achieved by directly administering
synthetic sialic acid analogues.[6] Additionally, selective cleav-
age of the glycol moiety led to a truncated sialic acid equipped
glycans with an aldehyde for labeling reactions (Figure 1).[7]
All of these modifications address sialylation of both, N- and
O-glycosylation of glycoproteins, to almost the same extent.
Herein we investigate whether the biosynthetic machinery
for sialic acids also tolerates other ManNAc derivatives as
substrates, which are modified directly at the six-membered
carbohydrate ring. The modification of the C4 position
appeared most attractive, because it is not enzymatically
modified during cellular glycoprotein production and would
deliver previously unknown C7-modified sialic acid contain-
ing glycoproteins (Figure 1). To probe the biosynthetic
promiscuity, we targeted a C4-modified ManNAc derivative,
N-acetyl-4-azido-4-deoxymannosamine (4-azido-ManNAc,
1), in our study to enable postglycosylational conjugation
and visualization by bioorthogonal reactions.[8]
[*] Dr. H. Mçller,[+] Prof. Dr. S. Hinderlich
Beuth Hochschule fꢀr Technik Berlin—University of Applied
Sciences, Department of Life Sciences and Technology
Seestrasse 64, 13347 Berlin (Germany)
E-mail: hinderlich@beuth-hochschule.de
Dr. V. Bçhrsch,[+] Prof. Dr. C. P. R. Hackenberger
Freie Universitꢁt Berlin, Institut fꢀr Chemie und Biochemie
Takustrasse 3, 14195 Berlin (Germany)
E-mail: hackenbe@chemie.fu-berlin.de
Dr. J. Bentrop, Dipl. Chem. J. Bender
Karlsruhe Institute of Technology (KIT), Zoologisches Institut,
Abteilung fꢀr Zell- und Neurobiologie
[+] These authors contributed equally to this work. V.B. is a member of
the joint medical faculty Charitꢂ of FU and HU Berlin.
[**] This work was supported by a grant to S.H. and C.P.R.H. of the
program “Arbeitsgruppenwettbewerb Glykobiotechnologie” of the
BMBF. Further financial support from the DFG (Emmy Noether
grant HA 4468/2-1 and SFB 765 to C.P.R.H., and grant BA 1034/15-
19 to Martin Bastmeyer and Jo.B.), the Boehringer-Ingelheim
Foundation (Plus 3-Programm to C.P.R.H.), the FCI (to C.P.R.H.),
the Europꢁischer Fonds fꢀr regionale Entwicklung (to S.H.) and the
Institut fꢀr Angewandte Forschung Berlin (to S.H.) is acknowl-
edged.
Supporting information for this article (detailed information about
the used materials, the chemical synthesis of compounds, and the
biochemical methods) is available on the WWW under http://dx.
doi.org/10.1002/anie.201108809.
N-acetyl-(1,3,6-O-acetyl)-4-azido-4-deoxy-mannosamine
(Ac3-4-azido-ManNAc) was generated by an optimized
literature method (Figure S1 in the Supporting Informa-
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tion),[9] which included the peracetylation to
ensure membrane permeability for the
metabolic uptake. Subsequently, we
explored whether this synthetic carbohy-
drate is a suitable substrate for MOE of cell
surface glycoproteins of mammalian cells.
We employed HEK293 cells lacking UDP-
N-acetylglucosamine 2-epimerase/ManNAc
kinase (GNE), the key enzyme of sialic acid
biosynthesis, to ensure increased incorpo-
ration rates of ManNAc analogues com-
pared to GNE expressing cells as demon-
strated for N-acylated ManNAc deriva-
tives.[10] Consequently, we incubated GNE-
deficient as well as GNE-expressing cells
with Ac3-4-azido-ManNAc, along with per-
acetylated
N-azidoacetylmannosamine
(Ac4ManNAz) and peracetylated ManNAc
(Ac4ManNAc, Figure 2a). Isolated mem-
brane fractions were treated with alkyny-
lated biotin and AlexaFluor 488 by copper-
catalyzed cycloaddition (CuAAC) to label
incorporated azido sugars.[11] After separa-
tion of the samples by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE), azido-
glycoproteins were detected by fluores-
cence and by western-blot analysis using
a specific anti-biotin antibody (Figure 2b).
In contrast to Ac4ManNAc- or azido-sugar-
treated GNE-expressing cells, concentra-
tion-dependent signals were detected in
membrane fractions from Ac3-4-azido-
ManNAc-treated GNE-deficient cells,
which were comparable to GNE-deficient
cells treated with Ac4ManNAz (Figure 2b).
These results gave the first clear indication
that Ac3-4-azido-ManNAc is metabolized
by cellular enzymes and is indeed incorpo-
rated into glycan structures of cell surface
glycoproteins while maintaining the acces-
sibility of its azido group for bioorthogonal
conjugation with labeling tags.
Figure 2. A) Biosynthetic incorporation of peracetylated (Ac3,Ac4-)ManNAc derivatives to the
corresponding glycoconjugate-bound sialic acids. B) Incorporation of Ac3-4-azido-ManNAc
and Ac4-ManNAz into cell surface glycoproteins of GNE-deficient HEK293 cells. Membrane
fractions of sugar treated cells (3 days) were labeled with alkynylated AlexaFluor 488 or
biotin by CuAAC enabling detection of incorporated azido sialic acids by fluorescence and
western-blot analysis. C) Western blot analysis of glycoconjugate bound sialic acid deriva-
tives by sialidase digestion of cells treated for three days with 500 mm solutions of ManNAc
analogues. For further details see the Supporting Information.
Membrane fractions of the GNE-defi-
cient cells revealed, after sialidase digestion
and incubation with an alkynylated biotin to
target remaining azido sialic acids, no
signals by western-blot analysis, which con-
firmed that Ac3-4-azido-ManNAc was con-
verted into the C7-modified 7-azido-7-deoxy-N-acetylneura-
minic acid (Sia7Az) in glycans of cell surface proteins
(Figure 2c). To determine the incorporation rate of Ac3-4-
azido-ManNAc we analyzed membrane fractions of sugar-
treated cell lines by flourescence-based HPLC. For this
purpose hydrolytically cleaved sialic acids were labeled with
1,2-diamino-4,5-methyleneoxybenzene (DMB) and identified
in comparison to chemo-enzymatically synthesized Sia7Az
(Figure 3).[9b,12] Sia7Az integrity was further verified by
combined liquid chromatography and mass spectrometry
(LC-MS; for details see the Supporting Information). We
found that incorporated Sia7Az accounts for up to 50% of
total sialic acids, which is in a similar range to the maximum
incorporation rate as Sia5Az derived from Ac4ManNAz
(60% of total sialic acids). The high incorporation rate of
modified sialic acids into glycoconjugates in this study is, at
least in parts, due to the use of GNE-deficient cells, which
promote efficient metabolization of ManNAc derivatives.[10]
On the other hand, several studies have shown that ManNAz
is also useful for the modification of sialic acids in “normal”
GNE-expressing cells. The incorporation rate for 4-azido-
ManNAc from this study therefore indicates, that the novel
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Figure 4. A) Concentration-dependent incorporation of Ac3-4-azido-
ManNAc into cell surface glycoproteins of Mucin-1-expressing MCF-7
cells. Cells were treated for 3 days either with increasing amounts of
Ac3-4-azido-ManNAc (100, 250, and 500 mm) or with Ac4-ManNAc, Ac4-
ManNAz, and Ac4-GalNAz (each 500 mm). Western blot analysis of
biotin-labeled cell surface glycoproteins indicated azido-modified sialic
acids or GalNAz in glycoproteins (top). Membrane associated Mucin-
1 expression was detected by immunoblotting (bottom). B) Incorpora-
tion of Ac3-4-azido-ManNAc into glycans of soluble O-glycosylated
Mucin-1. Soluble Mucin-1 from cell culture media of azido sugar-
treated MCF-7 cells (analogous to (A)) was immunoprecipitated,
treated with alkynylated biotin by CuAAC and analyzed by western
blotting. Ac4-ManNAz and Ac4-GalNAz treatment served as positive
controls (top). Amount of precipitated Muc-1 was detected by immu-
noblotting (bottom).
decrease of the azido-specific signals (Figure S3, left), which
indicates the predominant incorporation of the label into N-
linked chains. Incubation of Ac3-4-azido-ManNAc-treated
cells with PNGase F had virtually no effect on the signal
intensity of biotin labeled Sia7Az, as detected by western-blot
analysis (Figure S3, middle). In agreement with this data, cell
treatment with the N-glycosylation inhibitor tunicamycin
indicated no decrease in the incorporation of Ac3-4-azido-
ManNAc (Figure S3, right).
Figure 3. A) Chemoenzymatic synthesis of standards for HPLC assay.
B) Quantitative analysis of Sia5Az and Sia7Az incorporation into
glycans of GNE-deficient HEK293 cells. HEK293 cells were treated with
a 500 mm solution of Ac3-4-azido-ManNAc (top), Ac4-ManNAz (mid-
dle), or corresponding amounts of DMSO (bottom) for 3 days. FLU=
fluorescence light units. For further details see the Supporting Infor-
mation.
To gain direct evidence for the specific incorporation of
Ac3-4-azido-ManNAc into O-glycans, we investigated Mucin-
1, a heavily O-glycosylated protein highly expressed in MCF-
7 cells.[15] MCF-7 cells did not only reveal incorporation of
Ac3-4-azido-ManNAc into glycans of cell surface proteins
(Figure 4a) but also into glycans of secreted Mucin-1 (Fig-
ure 4b). For comparison, we also investigated the incorpo-
ration efficiency of Ac4ManNAz into sialic acids, and the O-
glycan-specific azido sugar Ac4GalNAz.[16] Both were incor-
porated into cell surface glycans of MCF-7 cells as well as into
glycans of soluble Mucin-1 efficiently and to a similar extent
as Ac3-4-azido-ManNAc (Figure 4a,b). The labeling of
Mucin-1 by all three compounds used clearly indicates
modification of O-glycans, whereby Ac3-4-azido-ManNAc is
the only molecule specifically targeting sialic acids in this kind
of oligosaccharide.
compound we introduce herein is as promising as ManNAz as
a general tool in glycoengineering.
After successful incorporation of Ac3-4-azido-ManNAc
into glycans of cell surface glycoproteins, we investigated the
applicability of this sugar for MOE of recombinantly
expressed glycoproteins. Ac3-4-azido-ManNAc was first
tested for glycan incorporation in the CEA-related cell
adhesion molecule 1 (CEACAM1) and in lactotransferrin
(LTF), which could be addressed successfully by
Ac4ManNAz.[10,13] Surprisingly, no incorporation of Ac3-4-
azido-ManNAc into the glycans of CEACAM1 or LTF could
be found (Supporting Information, Figure S2). Since both
proteins predominantly carry N-glycans,[14] we speculated that
Ac3-4-azido-ManNAc may be specifically incorporated into
O-glycans.
O-glycan specificity of Ac3-4-azido-ManNAc was probed
by treatment of cells with PNGase F, an enzyme that
specifically cleaves N-glycans. Cells treated with ManNAz,
then by PNGase F, showed a strong but not complete
Since azido sugars have been successfully applied to
render cellular membranes[17] we tested the potential of Ac3-
4-azido-ManNAc as a tool for glycan labeling in living
animals. Therefore, Ac3-4-azido-ManNAc was injected into
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of C8 and C9 upon periodate oxidation.[7] Our approach
emphasizes the application of C4-modified ManNAc deriva-
tives which are converted by cells, it thereby enables the
functional characterization of C7-modified sialic acid with an
otherwise maintained molecule composition. Interestingly,
Sia7Az was not incorporated into N-glycans of recombinantly
expressed proteins or of membrane proteins, but could be
detected in the heavily O-glycosylated protein Mucin-1. This
result makes the sugar a valuable tool for specific O-glycan
analysis, which was not possible with the rather unspecific
ManNAc derivatives used in previous MOE studies. In
addition, Ac3-4-azido-ManNAc was shown to be suitable for
labeling of membrane associated glycoproteins of cultured
mammalian cells and living animals, as demonstrated for
zebrafish larvae. All in all, this study confirms the relevance of
a new MOE tool in glycobiology research with distinct
specifications differing from synthetically accessible azido
sugars.
Received: December 14, 2011
Revised: February 22, 2012
Figure 5. In vivo labeling of zebrafish glycans applying Ac3-4-azido-
ManNAc and AlexaFluor 488-DIBO. At 24 hpf, zebrafish larvae were
intraventricularly injected with Ac3-4-azido-ManNAc (top rows) or with
DMSO (bottom rows). At 48 hpf, AlexaFluor 488-DIBO was injected as
a chemical reporter for incorporated Ac3-4-azido-ManNAc. At 72 hpf,
fluorescence labeling was analyzed. Long arrows indicate midbrain and
hindbrain regions, white arrows mark myosepts, magnified in insets.
DIC=differential interference contrast.
Keywords: in vivo labeling · mannosamine ·
.
metabolic oligosaccharide engineering · O-glycan · sialic acid
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