A chemical reporter strategy to probe glycoprotein fucosylation

Article abstract of DOI:10.1021/ja064619y

Fucosylated glycoproteins are involved in many cell-cell recognition events and are markers of embryonic and malignant tissue. Here we report a method for rapid profiling of fucosylated glycoproteins from human cells using 6-azido fucose as a metabolic la

Full text of DOI:10.1021/ja064619y

Published on Web 08/23/2006
A Chemical Reporter Strategy to Probe Glycoprotein Fucosylation
David Rabuka,† Sarah C. Hubbard,† Scott T. Laughlin,† Sulabha P. Argade,# and
Carolyn R. Bertozzi*,†,‡,§,¶
Departments of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, UniVersity of
California, Berkeley, California, Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California,
and Glycotechnology Core Resource, UniVersity of California, San Diego, La Jolla, California
Received June 29, 2006; E-mail: crb@berkeley.edu
It has been estimated that 50% of the proteins from eukaryotic
cells are post-translationally modified by glycosylation.1 However,
for most glycoproteins, the types of glycans attached to the
polypeptide have not been defined, nor are their functions under-
stood. Thus, the field of glycomics has focused considerable
attention on the development of methods for profiling protein
glycosylation at the systems level.1 Our contribution to this effort
is the chemical reporter strategy for marking protein-associated
glycans with bioorthogonal functional groups, such as the azide.2
Figure 1. Fucose salvage pathway.
Metabolic labeling of specific glycan subtypes with azido sugar
precursors enables their subsequent covalent reaction with fluo-
rescent probes for visualization, or with affinity tags for enrichment
and proteomic analysis. Several azide-specific chemistries can be
employed in these experiments, including the Staudinger ligation,
Cu(I)-catalyzed azide-alkyne condensation (i.e., “click chemistry”),
or strain-promoted [3 + 2] azide-alkyne cycloaddition.2 Mucin-
type O-linked glycoproteins,3 sialylated glycoproteins,4 and cytosolic
O-GlcNAc-modified proteins5 are examples of glycoprotein classes
that have been studied using the chemical reporter strategy. Here
we report the extension of the method to fucosylated glycoproteins.
Figure 2. Metabolic labeling of cellular glycoproteins with azido fucose
Among the nine monosaccharide building blocks employed in
analogues. Jurkat cells were cultured with azido fucose derivatives, 1, 2,
vertebrates, fucose is distinguished by its contribution to numerous
or 3, and their lysates were reacted with 4 in the presence of CuSO4, TCEP,
and triazolyl ligand.16 The labeled lysates were analyzed by Western blot
epitopes associated with cell-cell interactions and regulation of
protein function. For example, fucose is a component of the
tetrasaccharide sialyl Lewisx, a determinant of the selectin ligands
that mediate leukocyte-endothelial interactions at sites of inflam-
mation.6 Overexpression of sialyl Lewisx is characteristic of many
cancers,7 and glycoproteins displaying Lewisx epitopes promote
embryonic cell adhesion and have important roles in neurogenesis.8
Fucose is also a terminal sugar on a number of tumor-associated
antigens, including the tetrasaccharide Lewisy, a modification of
carcinoembryonic antigen (CEA) in malignant colon cancer.9 The
multitude of fucosylated antigens found upregulated in cancerous
tissue provides an opportunity to exploit these glycans as potential
cancer biomarkers.10 Fucose is also found directly attached to
proteins via serine or threonine residues in a rare form of protein
glycosylation known as O-linked fucosylation. This modification
is found on epidermal growth factor (EGF) repeats in the
developmental switch Notch.11 The ability to profile fucosylated
glycoproteins from complex cell or tissue samples might therefore
reveal new cancer biomarkers and provide a means to study changes
in fucosylation associated with development.
with an R-biotin antibody-HRP conjugate.
Scheme 1 a
a Reagents: (a) butane-2,3-dione, trimethylorthoformate, CSA, MeOH,
87%; (b) Tf2O, pyridine; (c) toluene, Bu4NOAc, 64% (2 steps); (d) MeOH,
NaOMe, 87%; (e) Tf2O, pyridine; (f) LiN3, DMF, 88% (2 steps); (g) TFA,
H2O, 98%; (h) NBS, acetone, H2O; (i) Ac2O, pyridine, 58% (2 steps).
ferases, 13 of which have been identified in humans, transfer the
fucose residue to glycans or proteins within the secretory pathway,
and the modified glycoproteins are delivered to the cell surface or
secreted. Although the earlier enzymes have not been studied in
detail with respect to unnatural substrate tolerance, at least two
fucosyltransferases have demonstrated in vitro activity on GDP-
fucose analogues bearing C6 modifications on the pyranose ring.12
We synthesized three azido fucose analogues (compounds 1-3,
Figure 2) and probed for their incorporation into glycoproteins in
the human T lymphoma cell line Jurkat. Compounds 1 and 3 were
prepared as previously reported in the literature,12,13 and compound
2 was synthesized as shown in Scheme 1. Briefly, thiophenyl
glycoside 514 was protected as the butane 2,3-diacetal (6), and the
4-OH was subjected to a double displacement process to introduce
Our approach exploits the fucose salvage pathway for the
incorporation of azido analogues into fucosylated glycans. This
pathway begins with the uptake of free fucose, followed by
phosphorylation by fucose kinase and conversion to GDP-fucose
by GDP-fucose pyrophosphorylase (Figure 1).8 Fucosyltrans-
† Department of Chemistry, University of California, Berkeley.
‡ Department of Molecular and Cell Biology, University of California, Berkeley.
§ Howard Hughes Medical Institute, University of California, Berkeley.
¶ Lawrence Berkeley National Laboratory.
# University of California, San Diego.
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10.1021/ja064619y CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. Western blot analysis of lysates from Jurkat cells treated with
1, 2, 3, or buffer (no azido sugar). Jurkat lysates were reacted with a biotin-
alkyne probe in the presence (+) or absence (-) of CuSO4. The blot was
probed with an R-biotin antibody conjugated to horseradish peroxidase
(HRP) (upper panel), then stripped and re-probed with an R-actin antibody
as a protein loading control (lower panel).
the azide (9). Global deprotection and acetylation produced 2. All
three compounds were prepared in peracetylated form in order to
facilitate passive diffusion across cell membranes.3-5
Figure 4. HPAEC-PAD analysis of hydrolysates from Jurkat cells
incubated with (A) compound 3 or (B) Ac4Fuc. The peaks labeled 1 and 2
correspond to native fucose and 6-azido fucose, respectively, as determined
by analysis of authentic standards. Details are in the Supporting Information.
Compounds 1-3 were initially screened for metabolic incorpora-
tion into Jurkat glycans by flow cytometry. Briefly, the cells were
treated with 1-3 at concentrations of 10-200 µM for a period of
up to 3 days, then reacted by Staudinger ligation with a phosphine
reagent bearing the FLAG peptide.3-5 The cells were then labeled
with a FITC-conjugated R-FLAG antibody and analyzed by flow
cytometry. Significant fluorescence was only observed after treat-
ment with 3 at concentrations above 100 µM (Supporting Informa-
tion); compounds 1 and 2 did not appear to label cell surface glycans
at any concentration tested. Interestingly, we determined that the
observed signal resulted from cell death upon treatment with 3 at
concentrations above 100 µM, as determined by propidium iodide
staining (Supporting Information). By contrast, compounds 1 and
2 did not produce any adverse cellular effects at a concentration of
200 µM, suggesting that the toxicity of 3 is related to its cellular
metabolism.
labeling, but a contribution from glycolipids is also possible and
should be explored. Finally, the toxicity of compound 3 is a matter
of interest and also concern. The correlation of metabolic conversion
with toxicity suggests a specific mechanism involving either enzyme
inhibition or altered protein function as a result of the azido sugar
modification. These areas warrant further study in order to define
the full potential of compound 3 for profiling protein fucosylation
in living systems.
Acknowledgment. We thank Dr. Cheryl McVaugh for a critical
reading of the manuscript, and Nick Agard for compound 4 and
the triazolyl ligand. This work was supported by a grant from the
NIH to C.R.B. (GM66047). S.C.H. is supported by an NSF
predoctoral fellowship.
Supporting Information Available: Synthetic procedures, spectral
data for all compounds, experimental procedures for Western blots,
flow cytometry, and HPAEC-PAD. This material is available free of
charge via the Internet at http://pubs.acs.org.
To identify the nature of the azide-labeled Jurkat glycoconjugates,
we analyzed cell lysates by Western blot. Jurkat cells were cultured
with 125 µM 1, 2, or 3 for 72 h and then lysed with NP-40, a
detergent known to be compatible with click chemistry.15 The
lysates were reacted with biotin-alkyne derivative 4 using the
conditions for click chemistry suggested by Cravatt15 (Figure 2),
and the Western blot was probed with an R-biotin antibody-
horseradish peroxidase (HRP) conjugate. Significant glycoprotein
labeling was only observed in lysates from cells treated with 6-azido
fucose analogue 3 (Figure 3).
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