A glycal-based photoaffinity probe that enriches sialic acid binding proteins

Article abstract of DOI:10.1016/j.bmcl.2019.07.054

To identify sialic acid binding proteins from complex proteomes, three photocrosslinking affinity-based probes were constructed using Neu5Ac (5 and 6) and Neu5Ac2en (7) scaffolds. Kinetic inhibition assays and Western blotting revealed the Neu5Ac2en-based 7 to be an effective probe for the labeling of a purified gut microbial sialidase (BDI_2946) and a purified human sialic acid binding protein (hCD33). Additionally, LC–MS/MS affinity-based protein profiling verified the ability of 7 to enrich a low-abundance sialic acid binding protein (complement factor H) from human serum thus validating the utility of this probe in a complex context.

Full text of DOI:10.1016/j.bmcl.2019.07.054

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A glycal-based photoaffinity probe that enriches sialic acid binding proteins
Peter S. Thuy-Boun, Dennis W. Wolan
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https://doi.org/10.1016/j.bmcl.2019.07.054
BMCL 26595
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Bioorganic & Medicinal Chemistry Letters
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Please cite this article as: Thuy-Boun, P.S., Wolan, D.W., A glycal-based photoaffinity probe that enriches sialic
acid binding proteins, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.07.054
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A glycal-based photoaffinity probe that enriches sialic acid binding proteins
Peter S. Thuy-Boun and Dennis W. Wolan*
Departments of Molecular Medicine and Integrative Structural and Computational Biology, The Scripps
Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
*To whom correspondence should be addressed. Tel. 1.858.784.7936, email: wolan@scripps.edu

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ABSTRACT
To identify sialic acid binding proteins from complex proteomes, three photocrosslinking affinity-based
probes were constructed using Neu5Ac (5 and 6) and Neu5Ac2en (7) scaffolds. Kinetic inhibition
assays and Western blotting revealed the Neu5Ac2en-based 7 to be an effective probe for the labeling
of a purified gut microbial sialidase (BDI_2946) and a purified human sialic acid binding protein
(hCD33). Additionally, LC-MS/MS affinity-based protein profiling verified the ability of 7 to enrich a low-
abundance sialic acid binding protein (complement factor H) from human serum thus validating the
utility of this probe in a complex context.
Keywords: sialic acid, activity-based probe, sialidase, lectin, Neu5Ac
2

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Mucus layer integrity is a key determinant of intestinal homeostasis in the context of host-microbiome
interactions. During active ulcerative colitis (UC), microbes can be found in contact with, or translocated
through the host epithelium where they can trigger host inflammatory responses. This is in contrast to
healthy or remissive states when microbes are held at bay by a thick, impenetrable, and continuously
shed mucus layer.1-9 Though the aftermath of microbial infiltration is often apparent, the initiation and
progression of this process is poorly understood. The early stages of infiltration likely involve microbial
adherence to, and degradation of, the mucus layer and it is thought that specific microbes and microbial
products are culpable.10-14 The mucus layer is principally composed of Muc2 mucin, a heavily O-
glycosylated gel-forming protein secreted from intestinal goblet cells.15,16 The structures of mucin O-
glycans are diverse but many glycans terminate with sialic acid (N-acetyl neuraminic acid in humans)
making this residue one of the most exposed host components of the mucus layer, and very likely an
early target of microbial adhesion. To understand the microbiome component of UC development, we
aim to identify microbes and microbial proteins responsible for mucus layer degradation in UC patients.
Our overall approach relies on LC-MS/MS shotgun proteomics to identify proteins of interest; however,
the species- and protein-level complexity of the gut microbiome renders it highly impervious to deep
proteome profiling efforts without a pre-enrichment step.17-22 Toward the goal of identifying gut microbes
and microbial proteins that degrade the mucus layer in UC patients, we focus our efforts herein on the
design and evaluation of affinity-based protein profiling probes for the enrichment of sialic acid binding
proteins (sialidases and lectins) from complex mixtures.
Several tools currently enable the identification of proteins that bind carbohydrates. With respect to
sialic acid, the Wong group has reported a mechanism-based alkynyl difluoro-sialic acid probe that
effectively targets sialidases but not sialic acid binding lectins.23,24 Tools based on a metabolic labeling
strategy are powerful standouts but we sought a culture-independent strategy to preserve the unique in
vivo phylogenetic and transcriptional configuration of our clinical samples and because many gut
microbes are anaerobic and/or “unculturable” outside of their native environments.25-31 Moreover, the
abundance of O-glycolytic activity originating from microbiome samples limits our ability to use O-linked
carbohydrates as affinity groups.32 We were inspired by early works of the Bednarski group where non-
hydrolyzable sialic acid C-glycosides were used to visualize whole influenza virus attachment (via
hemagglutinin), and sought to adapt this concept for modern mass spectrometry-based proteomics.33
Thus, we designed a set of probes with three requirements: a non-hydrolyzable affinity tag, an
enrichment tag, and a covalent crosslinker.
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We first set out to identify affinity scaffolds applicable to the gut microbiome. Using the commercially
available turn-on fluorescent substrate 4-methylumbelliferyl-α-N-acetyl neuraminic acid (4-MUNANA),
we found an abundance of sialidase activity in several human patient microbiome samples and
determined that neither commercially available 1, Zanamivir (3),34 or Oseltamivir (4)35 were able to
inhibit this sialidase activity despite being potent inhibitors of influenza sialidases (Fig. 1). We did
however identify 2 as a potent inhibitor of microbiome sialidase activity (Table 1, entry 2).36
Interestingly, 2 and 3 only differ by their C4 substituents, yet 2 has >100-fold more inhibitory aptitude
than 3. This observation informed our decision to refrain from excessively modifying the sialic acid-
based affinity group at this time, except at the anomeric carbon for the sake of probe generality. Due to
the limited supply of clinical microbiome samples, we also identified BDI_2946, a sialidase from the
commensal gut microbe Parabacteroides distasonis, as a suitable protein surrogate with a similar
inhibition profile for further testing (Fig. 1B, Table 1).
Fig. 1. A. Sialic acid and sialidase inhibitors. B. Inhibition of sialidase activity from a healthy patient’s
microbiome and from a purified gut microbial sialidase.
We first constructed a modular crosslinking enrichment tag utilizing a UV-active diazirine moiety as a
crosslinker and a terminal-alkyne as a proxy for biotin via the copper catalyzed azide alkyne
cycloaddition (CuAAC click reaction) (Fig. 2A, “DA”).37-41 We constructed three probes (5, 6, 7) based
on 1 and 2 (Fig. 2A). 5 and 6 were elaborated from C-allylated intermediates 9a and 9b generated via
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tin-mediated allylation of 8 (Fig. 2B, a).42-45 C-allylation yielded a mixture of α/β glycosides which were
chromatographically separated prior to further functionalization and conjugation to the crosslinking
enrichment tag “DA.” Na2HPO4 mediated HCl elimination of 8 yielded 10 which was fully deprotected to
give 2.46,47 7 was prepared from 2 after reacetylation of free hydroxyl groups followed by linker
conjugation to the C1 carboxylic acid (see Supplementary data, Scheme 5).
Fig. 2. A. Probes for sialic acid binding proteins examined in this study. B. Synthetic routes to 5, 6, and
7; a) AllylSnBu3, Bu6Sn2, THF, hν, r.t., N2; b) Na2HPO4, MeCN, reflux.
Table 1. Inhibition of sialidase activity from (1) a healthy patient microbiome sample or (2) a purified
bacterial sialidase by various compounds. N.D.= not determined.
IC50
% Inhibition
(BDI_2946)
N/A
>99%
N/A
N/A
N/A
N/A
51%
Entry Compound
IC50(microbiome)
(BDI_2946)
> 1 mM
9 ± 1 μM
> 1 mM
> 1 mM
> 1 mM
> 1 mM
181 ± 2 μM
1
2
3
4
5
6
7
1
2
3
4
5
6
7
> 1 mM
11 ± 1 μM
> 1 mM
> 1 mM
N.D.
N.D.
N.D.
5

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Compounds 5-7 were evaluated as inhibitors of BDI_2946 and 7 was found to be the only candidate to
appreciably inhibit this sialidase at a concentration < 1mM (Table 1, entries 5-7). The importance of the
C1-carboxylate on 2 for binding affinity is underscored by the 16-fold reduction in inhibitory aptitude
observed with 7.
Fig. 3. A. Western blot of purified BDI_2946 sialidase (0.2 mg/mL) labeled by various photocrosslinking
probes (1 mM), 1 h UV irradiation; B. Western blot of purified BDI_2946 labeled with 7 (1 mM) and the
blocking of 7-labeling using 2 (100 μM), 30 min UV irradiation. C. Western blot of recombinant human-
CD33 labeled with probes 5-7 (1 mM), 1 h UV irradiation D. Western blot of CD33 labeled with 7 (1
mM) and the blocking of 7-labeling using 2 (200 μM). E. Western blot of human serum labeled with
probes 5-7 (100 μM and 10 μM), 30 min UV irradiation. F. Western blot of a cultured murine distal gut
microbiome sample labeled with probe 7 (1 mM), 30 min UV irradiation.
We next evaluated compounds 5-7 as photoaffinity probes for BDI_2946. BDI_2946 was treated with 1
mM each of 5-7 then irradiated 1h with a 365 nm UV black light bulb. After irradiation, the mixture was
treated with Biotin-Peg3-azide under CuAAC conditions (see Supporting Information).40,41 Streptavidin
bead enrichment and Western blotting revealed strong labeling with 7 but not 5 or 6 (Fig. 3A), an
observation that is consistent with our kinetic inhibition data. Because 7 produced an intense labeling
signal, we evaluated its selectivity further by labeling BDI_2946 in the presence of ovalbumin and 2 (a
more potent inhibitor of BDI_2946). Signal for BDI_2946 appears to diminish with the application of 2
suggesting a degree of active site specificity (Fig. 3B, 0.02 mg/mL lanes). 7 also appears to non-
specifically label ovalbumin alongside BDI_2946 at higher concentrations (Fig. 3B, 2 mg/mL and 0.2
mg/mL lanes), but 7 labels BDI_2946 exclusively at low concentrations (Fig. 3B, 0.02 mg/mL lanes).
6

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We initially tested 5-7 using BDI_2946 because we were able to use sialidase activity as a
straightforward readout for target-engagement. We wondered whether 5-7 would be effective probes for
the enrichment of lectins which could be challenging targets because their preferred ligands are often
multiple monosaccharides in length, and because binding affinities for their preferred ligands can be
high (millimolar range) in the absence of multivalency.48,49 Using relatively high concentrations of
human siglec 3 (hCD33), we observed consistently strong labeling by 7 relative to 5 and 6 (Fig. 3C).
This is surprising given that the preferred monovalent ligand of hCD33 Neu5Acα(2,3)Galβ(1-
4)GlcNacβOR is three residues long while 7 is comprised of only one residue.50
Table 2. LC-MS/MS analysis of complement factor H (CFH) and serum albumin (SA) enrichment from a
healthy patient’s serum treated with probes 5-7 relative to unenriched serum (background). “n.s.” = not
significant.
Protein
Probe
mean Ln(NSAF)
Δ mean Ln(NSAF)
p-value
fold change
5
6
7
5
6
7
-9.461
-9.607
-8.401
-4.453
-4.754
-3.743
0.095
-0.050
1.156
-1.970
-2.272
-1.261
0.803
0.913
1.1
0.9
3.2
0.1
0.1
0.3
CFH
(P08603)
0.026
<0.0001
<0.0001
<0.0001
SA
(P02768)
Finally, we wondered whether 7 could enrich for sialic acid binding proteins from a complex mixture.
We chose human serum as a test proteome with the intention of targeting complement factor H
(CFAH_HUMAN or CFH, P08603), an important attenuator for the alternative pathway of the
complement system. CFH is a 139 kDA protein that possesses multiple polyanion binding sites and is
known to prefer Neu5Acα(2,3)Gal-containing glycoconjugates.51-53 We chose serum because it is
protein-dense (>50 mg/mL), though only a small fraction of this mixture is comprised of our protein of
interest (CFH: 0.1-0.5 mg/mL).54,55 Approximately half of the serum proteome is serum albumin (SA,
P02768) by weight and it is expected that this protein would be a suitable specificity control.56,57
Because of the complexity of serum, LC-MS/MS was used to both identify and quantify protein
enrichment.
We treated undiluted human serum with 100 μM and 10 μM of probes 5-7 preconjugated to biotin to
simplify labeling. Labeled proteins were then enriched with streptavidin beads and partitioned. One half
7

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of the partitioned sample was used for Western blotting (Fig. 3D) and the other half for LC-MS/MS
analysis. Triplicates of the 100 μM probe-treated samples were analyzed and protein abundances were
quantified by normalized spectral abundance factor (NSAF).58 No significant enrichments of CFH were
observed in 5- and 6-treated serum compared to unenriched serum control (background). However,
treatment with probe 7 was found to significantly enrich CFH 3.2-fold over background with a p-value of
<0.05. In addition, all probes (5-7) were found to negatively enrich for SA 0.1-0.3-fold compared to
background with p-values <0.0001 (Table 2).
The finding that probe 7 outperformed probes 5 and 6 in the labeling of sialidase BDI_2946 is not
surprising given that 7 is based on transition-state like inhibitor 2. However, it is surprising that probe 7
outperformed probes 5 and 6 for the labeling of purified hCD33, and again for CFH from human serum
especially in light of the fact that both of these sialic acid binding proteins have preferred ligands
containing α-linked sialic acid >1 monosaccharide in length. It is important to note that while 7
performed best in our labeling studies, we can only infer the binding mode of 7 to BDI_2946 because of
the availability of an enzymatic readout (4-MUNANA assay). Whether 7 interacts with lectins hCD33 or
CFH in their native sialic acid binding pockets is an open question. Also noteworthy is the diazirine’s
impact on our labeling studies. We chose to utilize diazrine as a crosslinking moiety because of its
small structural footprint and well-documented protein labeling aptitude. The wide range of labeling
intensities observed for probes 5-7 (Fig 3A and 3C) suggest that if there is any significant binding bias
imparted by the diazirine moiety, this bias can be overridden by judicious choice of affinity handle.
Overall, the positive results observed with probe 7 are encouraging and provide a lead for further
structural optimization. In addition, multivalent display strategies are being pursued to enhance probe
affinity.
In summary, we synthesized and evaluated 5-7 as photocrosslinking probes for sialic acid binding
proteins. Of compounds 5-7, Neu5Ac2en-based 7 was identified as a competent probe for both a gut
microbial sialidase and a purified sialic acid binding protein. We also demonstrate using LC-MS/MS
protein profiling that 7 is capable of enriching for a naturally occurring sialic acid binding protein (CFH)
from human serum 3.2-fold over background. We anticipate that 7 will prove to be a useful tool to
further our efforts in the area of microbiome proteomics, as the molecule labels distinct bacterial
proteins from a cultured murine distal gut microbiome sample that has measurable sialidase activity
(Fig. 3F). Protein profiling efforts employing 7 to identify bacterial sialidases and lectins will greatly
assist in determining the role these proteins have in health and microbiome-related diseases.
8

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ACKNOWLEDGMENTS
We gratefully acknowledge the National Institute of Allergy and Infectious Diseases (1R21AI139744 to
D.W.W.) and the US EPA (STAR predoctoral fellowship for P.S.T.-B. Assistance Agreement no.
FP917296-01-0). We thank I. Wilson and the Joint Center for Structural Genomics for the BDI_2946
clone, J. Paulson and S. Duan for human CD33-expressing cells, J. Yates, J. Moresco, and J. Diedrich
for technical assistance with mass spectrometry instrumentation, H. Rosen for access to
instrumentation, and J. Oakdale, A. Silvestri, and V. Fokin for TTTA ligand.
Author Contributions
P.S.T.-B. and D.W.W. conceived of the project. P.S.T.-B. synthesized all probes, purified recombinant
proteins, performed enzyme assays, and collected and analyzed all LC-MS/MS data. Both authors
contributed to the preparation and editing of the manuscript and have given approval to the final version
of the manuscript. The authors declare no competing financial interests.
Supplementary data
Supplementary data associated with this article can be found in the online version at http://xxxxxxx.
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