Photoactivatable glycopolymers for the proteome-wide identification of fucose-∝(1-2)-galactose binding proteins

Article abstract of DOI:10.1021/ja502482a

Although fucose-∝(1-2)-galactose (Fuc∝(1-2)Gal)-containing glycans have been implicated in cognitive processes such as learning and memory, their precise molecular mechanisms are poorly understood. Here we employed the use of multivalent glycopolymers to enable the first proteome-wide identification of weak affinity, low abundance Fuc∝(1-2)Gal glycan-binding proteins (GBPs). Biotin-terminated glycopolymers containing photoactivatable cross-linking groups were designed to capture and enrich GBPs from rat brain lysates. Candidate proteins were tested for their ability to bind Fuc∝(1-2)Gal, and the functional significance of the interaction was investigated for the synaptic vesicle protein SV2a using a knockout mouse system. The results suggest a role for SV2a-Fuc∝(1-2)Gal interactions in SV2a trafficking and synaptic vesicle recycling. More broadly, our studies outline a general chemical approach for the systems-level discovery of novel GBPs.

Full text of DOI:10.1021/ja502482a

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Photoactivatable Glycopolymers for the Proteome-Wide
Identification of Fucose-α(1-2)-Galactose Binding Proteins
Arif Wibowo,† Eric C. Peters,§ and Linda C. Hsieh-Wilson*,†
†Division of Chemistry and Chemical Engineering, California Institute of Technology and Howard Hughes Medical Institute, 1200
East California Boulevard, Pasadena, California 91125, United States
§Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, United States
S
* Supporting Information
many glycans and has been implicated in neuronal development,
learning, and memory.4 For example, treatment of animals with
ABSTRACT: Although fucose-α(1-2)-galactose (Fucα(1-
2)Gal)-containing glycans have been implicated in
cognitive processes such as learning and memory, their
precise molecular mechanisms are poorly understood.
Here we employed the use of multivalent glycopolymers to
enable the first proteome-wide identification of weak
affinity, low abundance Fucα(1-2)Gal glycan-binding
proteins (GBPs). Biotin-terminated glycopolymers con-
taining photoactivatable cross-linking groups were de-
signed to capture and enrich GBPs from rat brain lysates.
Candidate proteins were tested for their ability to bind
Fucα(1-2)Gal, and the functional significance of the
interaction was investigated for the synaptic vesicle protein
SV2a using a knockout mouse system. The results suggest
a role for SV2a-Fucα(1-2)Gal interactions in SV2a
trafficking and synaptic vesicle recycling. More broadly,
our studies outline a general chemical approach for the
systems-level discovery of novel GBPs.
2-deoxy-D-galactose (2-dGal), a compound that disrupts the
formation of Fucα(1-2)Gal linkages, caused reversible amnesia4a
and interfered with the maintenance of long-term potentiation,4b
an electrophysiological model of learning and memory. More-
over, previous work from our laboratory has suggested the
existence of both Fucα(1-2)Gal GBPs and glycoproteins as well
as their involvement in neurite outgrowth and synaptogenesis.4c,d
Understanding the molecular mechanisms underlying the
activity of Fucα(1-2)Gal sugars will require identification of
the key molecular components involved. However, no Fucα(1-
2)Gal GBPs have been identified from the mammalian brain.
Our early attempts to isolate Fucα(1-2)Gal GBPs employed
monovalent ligands conjugated to agarose beads with or without
photoactivatable cross-linking groups. Although a few candidate
GBPs were successfully detected, this approach failed to capture
sufficient quantities of protein for mass spectrometry (MS)
analysis. To overcome these challenges, we designed synthetic
glycopolymers that contain several key elements (Figure 1).
First, we exploited multivalent sugar epitopes to augment weak
glycan−GBP interactions.5 Second, we incorporated a photo-
reactive nitrophenylazide moiety into the glycopolymer to enable
covalent cross-linking of the associated proteins. Lastly, we end-
functionalized the glycopolymers with a biotin handle to facilitate
affinity enrichment and identification of the GBPs.
To synthesize the glycopolymers, we explored cyanoxyl
(OCN)-mediated free radical polymerization chemistry because
it affords water-soluble polymers of controlled length and narrow
polydispersity.6 The Fucα(1-2)Gal epitope or a control ligand
lacking the disaccharide was incorporated into acryloyl-function-
alized monomers 1 and 2, respectively (Figure 1). We chose a
nitrophenylazide cross-linking agent for monomer 3 because this
group has been successfully applied to proteins at membrane
interfaces.7 For end-labeling the polymers with a biotin moiety,
we employed the arylamine initiator 4 developed by Chaikof et
al. 6b
lycan-binding interactions play important roles in many
Gcomplex physiological processes, including the immune
response, tumor metastasis, and viral infection.1 The distinct
arrays of glycans presented on cell surfaces are recognized by
various glycan-binding proteins (GBPs) or lectins. The ability of
GBPs to bind and often cluster glycan-presenting receptors can
have important consequences for processes such as cell adhesion,
migration, and gene regulation.1,2
Despite their importance, mammalian GBPs that interact with
specific glycans of interest have been challenging to identify.3
The weak affinities of many glycan−protein interactions (Kassoc
=
103−106 M−1) have complicated efforts to capture and study
endogenous GBPs. Most of the well-characterized lectins are
derived from plants (e.g. concanavalin A) or were discovered
through classical biochemical purification or structural homology
to known lectins. Notably, general systems-level approaches for
the proteome-wide identification of GBPs have been lacking. The
development of such methods is critical for elucidating the
structure−function relationships of carbohydrates and under-
standing the diverse roles of GBPs. Here we address these
challenges through the synthesis and characterization of chemical
probes for the discovery of novel mammalian GBPs.
Fucα(1-2)Gal monomer 1 was prepared using several one-pot,
multistep reactions (Scheme S1). Briefly, disaccharide 58 was
treated with (phenylthio)trimethylsilane in the presence of zinc
iodide, deprotected with TBAF, and the resulting C-6 hydroxyl
group acetylated to afford thioglycoside 6. Although coupling of
3-azido-1-propanol to this thioglycoside using NIS/AgOTf in
We focused on targeting neuronal GBPs that interact with
glycans containing the fucose-α(1-2)-galactose (Fucα(1-2)Gal)
motif. Fucα(1-2)Gal is found on the nonreducing termini of
Received: March 11, 2014
Published: June 17, 2014
© 2014 American Chemical Society
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Table 1. Polymers Generated via OCN-Mediated Radical
Polymerization
a
b
b
b
c
c
pol
monomer [3]0/[1 or 2]0
x
y
z
Mn
PDI
9
1
−
1/3
19
30
−
−
9
−
−
42
26.3
23.9
23.0
1.17
1.22
1.17
10
11
1 and 3
2 and 3
1/6
7
a
b
Initial ratio of cross-linker [3]0 to ligand [1 or 2]0. Cross-linker and
ligand content in the resulting polymer were estimated by H NMR
(see SI for details). Mn in kDa and PDI values were determined by
1
c
SEC-MALS.
each disaccharide unit was replaced by 2 equiv of 3-
hydroxypropyl units (1:6 molar ratio). Characterization of the
polymers by size-exclusion chromatography-multiangle light
scattering (SEC-MALS) revealed narrow polydispersity index
(PDI) values (∼1.2) and number-average molecular weights
(Mn) of 23−26 kDa. We also estimated an average polymer
composition of up to 30 disaccharides per chain by comparing
the integrated signal from the phenyl protons to that of the
fucose methyl protons by 1H NMR (see SI for details).
Importantly, the relative ratios of ligand (1 or 2) to cross-linking
agent (3) could be tuned to reproducibly control the glycan/
cross-linker content of the polymer.
Figure 1. Strategy for the systems-level, proteome-wide identification of
We first validated our strategy using the well-established plant
lectin Ulex europaeus agglutinin I (UEAI), which binds with weak
affinity to the Fucα(1-2)Gal epitope (IC50 = 0.73 mM).10 UEAI
conjugated to fluorescein was incubated with polymer 10 or 11
for 3 h at 37 °C and subsequently exposed to 365 nm light for 15
min at 4 °C. The cross-linked proteins were isolated by
streptavidin affinity chromatography, resolved by SDS-PAGE,
and detected by in-gel fluorescence imaging. We found that
polymer 10, but not polymer 11, captured UEAI, indicating
selective recognition of the glycan moiety (Figure 2A).
GBPs.
CH2Cl2 yielded an anomeric α/β mixture, acetonitrile as the
solvent promoted formation of the β-linked glycoside 7 as the
major product in 64% yield, presumably due to nitrilium ion
coordination in the axial position.9 Catalytic hydrogenation of 7
over Pd/C gave the amine intermediate, which was directly
transformed without further purification into the corresponding
́
acrylamide. Finally, facile deacetylation under Zemplen con-
ditions afforded the desired disaccharide 1 in 90% yield.
Nitrophenylazide monomer 3 (Scheme S2) and arylamine
initiator 46b were readily synthesized using standard procedures.
With the monomers in hand, we generated glycopolymers 9−
11 using acrylamide as the comonomer for the polymer
backbone (Scheme 1 and Table 1). Treatment of 4 with HBF4
Scheme 1. Synthesis of Biotin-Functionalized,
Photoactivatable Glycopolymers
Figure 2. (A) Selective capture of UEAI by 10. (B) Representative
silver-stained gel of GBPs isolated from synaptosomal and membrane
fractions. (C) Relative binding profiles of candidate GBPs (n = 3).
and NaNO2 in degassed 1:1 H2O/THF gave the arenediazonium
cation, which upon reaction with NaOCN at 60 °C generated the
biotinyl aryl radical in situ to serve as the initiator. Addition of
disaccharide monomer 1 and acrylamide (1:4 molar ratio)
generated polymer 9 with desirable sugar density and water
solubility. Using the same sugar to acrylamide ratio, we also
copolymerized monomer 1 with nitrophenylazide monomer 3
(1:3 molar ratio) to give polymer 10. Furthermore, we
synthesized the control polymer 11 from 2 and 3, wherein
Moreover, photo-cross-linking of the polymer led to a 2-fold
increase in UEAI capture. These results validate the polymer
design and demonstrate that synthetic glycopolymers can be
used to capture lectins with weak glycan binding affinities.
Next, we investigated whether polymer 10 could be utilized to
identify novel, endogenous Fucα(1-2)Gal GBPs from mamma-
lian tissues. Various subcellular fractions from rat brain were
incubated with polymer 10 or 11, cross-linked by UV irradiation,
and isolated as described above. The captured proteins were
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resolved by SDS-PAGE, and the entire lanes of proteins captured
either by 10 or 11 were cut into 20 similarly sized gel pieces. All
gel pieces were individually subjected to in-gel proteolytic
digestion and liquid chromatography-tandem mass spectrometry
(LC-MS/MS) analysis. We observed enhanced, specific protein
capture by 10 compared to 11 in both synaptosomal and
membrane fractions (Figure 2B). Individual proteins were
identified using highly stringent criteria, and after filtering out
any proteins present in both sample sets, 44 candidate GBPs
were identified from six total experiments. Interestingly, the
GBPs represented a broad range of functions, including proteins
involved in signal transduction, protein trafficking, protein
scaffolding, and metabolism (Table S1).
natural N-azidoacetylgalactosamine (GalNAz) moiety onto the
C-3 position of galactose in Fucα(1-2)Gal glycans (Figure S1).
Subsequent bioorthogonal reaction with alkyne-functionalized
fluorophores or biotin enables rapid, sensitive detection of the
labeled Fucα(1-2)Gal glycans. We observed significantly reduced
expression of Fucα(1-2)Gal glycans on cortical glycoproteins
from FUT2 knockout mice (FUT2−/−) compared to WT
littermate controls (Figures 3A and S2). In contrast, FUT1
As Fucα(1-2)Gal carbohydrates have been implicated in
synaptic regulation and plasticity, we focused our attention on
GBPs associated with those functions. Four proteins were
selected for further investigation: double cortin-like kinase 1
(DCLK1), protein kinase C epsilon subunit (PKCε), guanine
nucleotide-binding protein G(o) alpha subunit (GαO), and
synaptic vesicle glycoprotein 2a (SV2a).11 Although DCLK1,
PKCε, and GαO are found in the cytoplasm, they also undergo
translocation to the plasma membrane.12 Activity-dependent
secretion of these proteins could provide a mechanism for
interaction with extracellular Fucα(1-2)Gal sugars. Indeed,
secretion of cytosolic galectin 1, a lectin that binds β-galactosides,
into the extracellular space has been reported to mediate T cell
clearance during tumor progression.13 SV2a could engage
Fucα(1-2)Gal sugars in synaptic vesicles or at the extracellular
surface after synaptic vesicle exocytosis. We assessed the binding
affinities of the candidate GBPs using enzyme-linked lectin
assays. Each protein was immobilized on a microtiter plate and
incubated with varying concentrations of a biotinylated Fucα(1-
2)Gal glycopolymer. Bound polymer was detected using
streptavidin conjugated to horseradish peroxidase. For compar-
ison, we also examined the binding of a corresponding
biotinylated glycopolymer bearing Galα(1-2)Gal epitopes. All
four proteins interacted with the Fucα(1-2)Gal glycopolymer in
a dose-dependent manner, albeit with different affinities (Figure
2C). Furthermore, SV2a, PKCε, and DCLK1 showed greater
(2.5−2.8-fold) binding to Fucα(1-2)Gal compared to Galα(1-
2)Gal glycopolymers, suggesting that the terminal fucose residue
was important for recognition. In contrast, GαO exhibited similar
binding affinity toward both Fucα(1-2)Gal and Galα(1-2)Gal
glycopolymers (KD,app = 2.2 and 2.1 μM, respectively). Together,
these results demonstrate that all four candidate GBPs recognize
Fucα(1-2)Gal sugars and that our glycopolymer-based approach
can successfully identify novel GBPs.
As the mere presence of a glycan-protein interaction is not
sufficient to establish biological function, elucidating the
physiological roles of GBPs remains a major challenge. To
assess whether the interaction of Fucα(1-2)Gal sugars with GBPs
might have important functional consequences, we studied SV2a.
SV2a is a 12-transmembrane glycoprotein that has no close
homology to any known animal lectins. The trafficking of SV2a
between secretory vesicles and synapses is critical for activity-
dependent neurotransmission.11c,d To investigate the role of
Fucα(1-2)Gal sugars in SV2a trafficking, we utilized genetically
altered mice lacking FUT1 and FUT2, the fucosyltransferase
genes responsible for Fucα(1-2)Gal biosynthesis.14 We exam-
ined whether expression of Fucα(1-2)Gal sugars was regulated
by FUT1 or FUT2 in the mouse cortex using our recently
reported chemoenzymatic strategy.15 This approach exploits an
exogenous bacterial glycosyltransferase BgtA to transfer a non-
Figure 3. (A) Fucα(1-2)Gal expression on glycoproteins in FUT1−/−
and FUT2−/− mice. (B) Enrichment of SV2a in the membrane fraction
of FUT2−/− mice (n = 3, *P < 0.02). (C) Increased colocalization
(yellow) of SV2a and syt1 puncta along neuronal processes in FUT2−/−
cortical neurons (n = 70−80, **P < 0.005). (D) Increased cell surface
biotinylation of SV2a in FUT2−/− cortical neurons (n = 4, *** P < 0.05).
See SI for experimental details.
knockout mice (FUT1−/−) showed a lower reduction in the
overall levels of Fucα(1-2)Gal protein fucosylation. These results
suggest that FUT2 is primarily responsible for Fucα(1-2)Gal
biosynthesis in the mouse cortex.
Trafficking of SV2a to the membrane influences synaptic
vesicle exocytosis, also known as neurotransmitter release.
Following neurotransmitter release, the endocytic reuptake of
SV2a from the membrane into synaptic vesicles is critical for the
proper regeneration of synaptic vesicles. To investigate whether
Fucα(1-2)Gal glycans influence SV2a trafficking, we examined
the distribution of SV2a at the membrane in cultured cortical
neurons from WT, FUT1−/−, or FUT2−/− mice. Notably, we
found that FUT2−/− cortical neurons exhibited a 2-fold increase
in membrane-associated SV2a compared to WT or FUT1−/−
cortical neurons, as determined by subcellular fractionation and
quantitative Western blot analysis (Figure 3B). In contrast, the
levels of synaptophysin (syp), another synaptic vesicle protein,
remained unchanged.
SV2a also regulates expression and endocytosis of synapto-
tagmin (syt1), a calcium sensor protein required for vesicle
fusion during neurotransmitter release.11d We next assessed the
effect of Fucα(1-2)Gal glycan loss on SV2a-mediated
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Notes
endocytosis of syt1 by measuring the colocalization of SV2a and
syt1 puncta at synapses in neurons from WT and FUT2−/− mice.
The density of colocalized SV2a and syt1 puncta increased by
1.6-fold in FUT2−/− neurons compared to WT neurons,
consistent with reduced endocytic reuptake (Figures 3C and S3).
Finally, analysis of SV2a trafficking requires measurement of
the SV2a subpopulation exposed to the cell surface during
synaptic vesicle cycling. We therefore conducted a cell-surface
biotinylation assay, in which living neurons were incubated with a
non-cell permeable, reactive biotin molecule to label surface
proteins. Following immunoprecipitation of SV2a, we analyzed
the immunoprecipitated protein by both streptavidin and
Western blotting to measure the amount of SV2a on the
neuronal surface compared to the total amount of SV2a
precipitated. We found that the level of SV2a on the cell surface
was 1.9-fold greater in FUT2−/− neurons compared to WT
neurons (Figure 3D). Thus, we show using three independent
methods that SV2a localization at the membrane is disrupted in
FUT2 knockout mice, strongly suggesting that Fucα(1-2)Gal
sugars influence SV2a function. Based on these results, we
propose that the interaction of SV2a with a Fucα(1-2)Gal
glycoprotein on the cell surface influences the endocytic reuptake
of SV2a into synaptic vesicles. As FUT2−/− mice have reduced
Fucα(1-2)Gal sugars on their glycoproteins, this interaction is
disrupted in FUT2−/− neurons, attenuating the reuptake of SV2a
into vesicles and increasing the localization of SV2a at the cell
surface. As such, Fucα(1-2)Gal glycans may serve as a sorting
signal for SV2a endocytosis during synaptic vesicle recycling.
Interestingly, while SV2a itself is a glycoprotein, we found that
SV2a is unlikely to be modified by Fucα(1-2)Gal glycans, as
assessed by blotting of rat brain SV2a with UEAI (Figure S4).
Together with the in vitro binding data, our results provide
support for an important functional interaction between SV2a
and Fucα(1-2)Gal sugars.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Drs. J. Lowe and S. Domino for the FUT KO mice, Dr.
S. Bajjalieh for the SV2a plasmid, Dr. J. Gleeson for the DCLK1
plasmid, Dr. T. Kosaza for GαO protein, Dr. M. Shahgoli in the
CCE Division Mass Spectrometry Facility, and Dr. C.
Krishnamurthy for BgtA enzyme. This work was supported by
the NIH (R01 GM084724).
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and data. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
lhw@caltech.edu
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