A sequential bioorthogonal dual strategy: ManNAl and SiaNAl as distinct tools to unravel sialic acid metabolic pathways

Article abstract of DOI:10.1039/c5cc08838k

Recent methodological developments in metabolic oligosaccharide engineering (MOE) pave the way for tremendous advances in glycobiology. Herein, we propose a Sequential Bioorthogonal Dual Strategy (SBDS) combining the use of two unprotected alkyne-tagged monosaccharide reporters (ManNAl and SiaNAl) with the bioligation of fluorescent probes by copper-catalysed azide-alkyne cycloaddition (CuAAC). With SBDS, we are able to shed light on trafficking and cellular uptake mechanisms of sialic acid. Using their corresponding analogues, we visualized that SiaNAl enters via endocytosis, whereas its biosynthetic intermediate ManNAl uptake is mediated by a yet unknown but specific plasma membrane transporter. Sialin, a lysosomal protein, is shown to be crucial for the export of exogenous sialic acid from lysosomes to the cytosol. Metabolic labeling with alkyne-tagged derivatives of N-acetylneuraminic acid (Neu5Ac) or N-acetylmannosamine (ManNAc) could thus be used to follow endocytosis in physiological vs. pathological conditions.

Full text of DOI:10.1039/c5cc08838k

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Sequential Bioorthogonal Dual Strategy: ManNAl and SiaNAl as
distinct tools to unravel sialic acids metabolic pathways††
Received 00th January 20xx,
Accepted 00th January 20xx
a, b
a, b
a, b
c
a, b
a, b
a, b
P.A. Gilormini, C. Lion, D. Vicogne, T. Levade, S. Potelle, C. Mariller, Y. Guérardel,
a, b
a, b
C. Biot, *† and F. Foulquier *†
DOI: 10.1039/x0xx00000x
www.rsc.org/
Recent
oligosaccharide engineering (MOE) pave the way for tremendous glycoconjugate families.
advances in glycobiology. Herein, we propose a Sequential specifically been developed for the study of sialylated
methodological
developments
in
metabolic very elegant strategy to visualize and quantify various
2
-6
Several methodologies have
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Bioorthogonal Dual Strategy (SBDS) combining the use of two glycoconjugates.
Sialic acids are a diverse family of
unprotected alkyne-tagged monosaccharide reporters (ManNAl monosaccharides that consistently occupy the non-reducing
1
and SiaNAl) with the bioligation of fluorescent probes by copper- termini of glycan chains in glycoproteins and glycolipids.
3-14
catalysed azide-alkyne cycloaddition (CuAAC). With SBDS, we are The sialic acid metabolism occurs in different compartments of
able to shed light on trafficking and cellular uptake mechanisms of the cell. Its synthesis starts in the cytosol from UDP-GlcNAc,
sialic acid. Using their corresponding analogues, we visualized that which is epimerized and phosphorylated into ManNAc-6P by
SiaNAl enters via endocytosis, whereas its biosynthetic UDP-GlcNAc 2-epimerase (GNE/MNK). ManNAc-6P is then
intermediate ManNAl uptake is mediated by a yet unknown but condensed with PEP by the Neu5Ac-9-Phosphate synthase
specific plasma membrane transporter. Sialin, a lysosomal protein, (NANS) to give rise to Neu5Ac-9P. After dephosphorylation by
is shown to be crucial for the export of exogenous sialic acid from N-acetylneuraminic acid phosphatase (NANP), the synthesis
lysosomes to the cytosol. Metabolic labeling with alkyne-tagged then continues in the nucleus where the Neu5Ac is activated
derivatives of N-acetylneuraminic acid (Neu5Ac) or N- into CMP-Neu5Ac by the CMP-Neu5Ac synthase (CSS). Once
acetylmannosamine (ManNAc) could thus be used to follow activated, the CMP-Neu5Ac is exported from the nucleus into
endocytosis in physiological vs pathological conditions.
the cytosol then to the Golgi apparatus via the SLC35A1
transporter, before its incorporation into glycoconjugates via
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With the advent of bioorthogonal click chemistry, the
emergence of metabolic oligosaccharide engineering (MOE)
has opened a completely new field of investigation in the past
sialyltransferases.
However, biological insights into the metabolism of
glycoconjugates have yet to emerge from such chemical
reporter strategies. To our knowledge, all reported studies but
1
few years. Indeed, a traditional limitation in the field of
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6-17
glycosciences used to be the molecular imaging of mono-,
oligo- or polysaccharides in terms of subcellular localization
and quantification. Metabolic inclusion of a synthetically
modified monosaccharide reporter in living cells or organisms,
followed by bioorthogonal ligation of a probe, now provides a
two
have so far taken an interest in observing
glycoconjugates incorporated at the cellular membrane
exclusively.
The available chemical toolbox for such investigations mainly
consists of peracetylated saccharide derivatives bearing a
reporter group, typically an azide or a terminal alkyne. As
those unnatural monosaccharides are presumed not to be
membrane permeable when unprotected, they have so far
been systematically peracetylated in order to increase their
hydrophobicity and thus facilitate their cellular entry via
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passive diffusion. While powerful, these tools do not allow
the fine study of cellular uptake and metabolic
compartmentation.
In order to highlight such processes, we have successfully
developed a Sequential Bioorthogonal Dual Strategy (SBDS)
based on the distinct metabolic incorporation of two
unprotected monosaccharide reporters, namely N-(4-
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pentynoyl)neuraminic acid (SiaNAl) and N-(4-pentynoyl) Depending on the used chemical tool (ManNAl or SiaNAl), the
mannosamine (ManNAl) (Fig. 1). The visualization of tagged dynamics of incorporation into glycoconjugates are strikingly
glycoconjugates was then obtained by chemical ligation of an different in accordance with the known sialic acids metabolic
azido probe via copper-catalyzed azide-alkyne cycloaddition pathway.
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(
CuAAC), and imaging by confocal fluorescence microscopy.
While sialylated glycoconjugates can be observed in the Golgi
In order to study the dynamics of incorporation of our region after only 3h of cellular SiaNAl metabolic labeling, the
chemical reporters into glycoconjugates, we have successfully same Golgi pattern is only observed with ManNAl after 7h of
used SBDS with the two key players ManNAl and SiaNAl after labeling.
optimization of their synthesis and of our previously described
2
staining methodology.
0
In order to confirm the sialin-mediated uptake pathway proposed
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1
by Bardor et al., we applied our SBDS in sialin deficient patients’
cells. Indeed, sialin (Q9NRA2) is an anion transporter known to be a
membrane protein that exports sialic acids out of lysosomes into
the cytosol. To highlight the potential role of sialin in cellular
SiaNAl/ManNAl incorporation, control and sialin-deficient patients’
cells were metabolically labeled for 8h with either SiaNAl or
ManNAl. After incorporation of ManNAl, labeled sialylated
glycoconjugates were mainly observed in the perinuclear Golgi like
region of both control and sialin deficient patients’ cells (Fig. 2). In
Human fibroblast cells from healthy individuals were
metabolically labeled at different time points (2, 5 and 7h,
respectively) with SiaNAl or ManNAl. Figure 1 shows the
distinctive staining patterns observed as a function of labeling
time and alkyne-tagged monosaccharide. When comparing the
incorporation of SiaNAl with that of ManNAl, two pools of
labeled sialylated glycoconjugates can easily be differentiated:
one is perfectly localized in the Golgi compartment while the
22
other is localized in early endosomes (respectively shown by contrast, the sialin deficient cells metabolically labeled with SiaNAl
co-localizations with the Golgi marker TGN46 and the early displayed no staining. The sialin deficient patients’ cells have the
endosomal marker Eea1, Fig. S1).
capacity to transform ManNAl into CMP-SiaNAl, which can then be
incorporated into the newly synthesized glycoconjugates. These
result show the inability of SiaNAl to reach the cytosol and be
converted to CMP-SiaNAl in sialin deficient cells. In support of the
21
pathway suggested previously for Neu5Gc, the present study not
only confirms the crucial role of sialin in SiaNAl metabolism but also
the potential of this metabolic labeling methodology to decipher
deficiencies in sialic acid pathways.
Figure 1. A. Structure of the two chemical reporters, SiaNAl and ManNAl. B. Fibroblasts
from healthy individuals were metabolically labelled with 500 µM of SiaNAl or ManNAl
for 2, 5, and 7 h respectively. They were then stained with AzidoFluor545 fluorescent
probe (alkyne tagged sialic acid linked to glycoconjugates in red). Staining was
visualized using confocal microscopy. Scale bar: 50 µm
Figure 2. Fibroblasts from healthy individuals and Sialin deficient patients were either
metabolically labelled with 500 %M of ManNAl or SiaNAl for 8 h and stained with
AzidoFluor 545 fluorescent probe. Cells were then examined using confocal microscopy.
Scale bar: 50 µm.
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In summary, the present report based on the use of
unprotected monosaccharides demonstrates the power of
these two different metabolic strategies. SiaNAl and ManNAl
are shown to be suitable tools to track sialylated
glycoconjugate metabolism and particularly to dissect the sialic
acids metabolic routes. Indeed, our data bring evidences that
both synthetic monosaccharides are up-taken by differential
cellular mechanisms.
Then, we demonstrated that this Sequential Bioorthogonal
As no staining was visible in sialin deficient cells after SiaNAl Dual Strategy is suitable to elucidate fundamental intracellular
labeling, we wanted to discriminate between an endocytosis glycobiological questions. In this instance, we confirmed that
sialin is crucial to the export of exogenous sialic acids from
Figure 3. Quantification by HPLC after DMB derivatization (A) SiaNAl, both free and
lysosomes to cytosol. SiaNAl could be a major tool to identify
linked to glycoconjugates into Sialin deficient patients cells versus control fibroblast
lysosomal diseases or endocytosis deficiencies. Such labeling
cells. (B) Free and linked Neu5Ac into ΔSialin cells versus control fibroblast cells.
strategies could also prove valuable in the search of
Bars represent arithmetic means +/- SEM of two independent DMB experiments.
therapeutic molecules to restore sialin functions.
or a lysosomal export defect of sialic acids mediated by sialin. Complementary to SiaNAl metabolic labeling, ManNAl is an
Control and sialin deficient cells were labeled with SiaNAl for alternative tool to visualize the ManNAc downstream pathway.
2
4
8
h and the amount of free and linked SiaNAl was quantified by For example, in case of GNE deficiency, this methodology
2
5
reverse phase fluorescence HPLC after DMB (1,2-diamino-4,5- could simply be used to assess treatment efficacy.
methylenedioxybenzene dihydrochloride) derivatization (see
SI). As shown in Fig. 3, a similar amount of total SiaNAl is
observed in sialin deficient cells and control cells. However,
while 15% of the observed SiaNAl is incorporated in References
glycoconjugates in control cells, SiaNAl is exclusively observed
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Figure 4. A working hypothesis of cellular uptake and metabolization of ManNAl and SiaNAl. SiaNAl enters via endocytosis and is recycled from lysosomes via Sialin. Once inside the
cytosol, SiaNAl is converted into CMP-SiaNAl in the nucleus lumen and used at the Golgi level by the sialyltransferases. The newly sialylated glycoconjugates are then exported
either to the plasma membrane or at the early endosomes. We hypothesize that ManNAl enters via a facilitate transporter uncharacterized yet. The cytosolic ManNAl is converted
into SiaNAl by the different enzymes (GNE, NANS, NANP). In sialin deficient patients and after ManNAl labelling. In conclusion, SiaNAl labelling can be extremely to identify
endosomal/lysosomal dysfunctions while ManNAl labelling allow the sialylated glycoconjugates monitoring.
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