Facile Formation of β-thioGlcNAc Linkages to Thiol-Containing Sugars, Peptides, and Proteins using a Mutant GH20 Hexosaminidase

Article abstract of DOI:10.1002/anie.201809928

Thioglycosides are hydrolase-resistant mimics of O-linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. The development of an efficient, enzyme-mediated synthesis of thioglycosides, including S-GlcNAcylated proteins, is reported, using a thioglycoligase derived from a GH20 hexosaminidase from Streptomyces plicatus in which the catalytic acid/base glutamate has been mutated to an alanine (SpHex E314A). This robust, easily-prepared, engineered enzyme uses GlcNAc and GalNAc donors and couples them to a remarkably diverse set of thiol acceptors. Thioglycoligation using 3-, 4-, and 6-thiosugar acceptors from a variety of sugar families produces S-linked disaccharides in nearly quantitative yields. The set of possible thiol acceptors also includes cysteine-containing peptides and proteins, rendering this mutant enzyme a promising catalyst for the production of thio analogues of biologically important GlcNAcylated peptides and proteins.

Full text of DOI:10.1002/anie.201809928

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Accepted Article
Title: Facile formation of β-thioGlcNAc linkages to thiol-containing
sugars, peptides, and proteins using a mutant GH20
hexosaminidase
Authors: Gregor Tegl, John Hanson, Hong-Ming Chen, David Kwan,
Andres Gonzalez Santana, and Stephen Withers
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201809928
Angew. Chem. 10.1002/ange.201809928
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Facile formation of β-thioGlcNAc linkages to thiol-containing sugars,
peptides, and proteins using a mutant GH20 hexosaminidase.
Gregor Tegl[a], John Hanson[b], Hong-Ming Chen[a], David H Kwan[a], Andrés G. Santana[a] and Stephen
G. Withers*[a]
Abstract: Thioglycosides are hydrolase-resistant mimics of O-linked
glycosides that can serve as valuable probes for studying the role of
glycosides in biological processes. Traditional chemical syntheses of
thioglycoside analogues of O-GlcNAc-modified peptides and proteins
require lengthy, multi-step approaches. We report the development of
efficient, enzyme-mediated syntheses of thioglycosides, including S-
GlcNAcylated proteins, using a thioglycoligase derived from a GH20
hexosaminidase from Streptomyces plicatus in which the catalytic
acid/base glutamate has been replaced with an alanine (SpHex
E314A). This robust, easily-prepared, engineered enzyme uses
readily available GlcNAc and GalNAc donors and couples them to a
remarkably diverse set of thiol acceptors. Thioglycoligation using 3-,
4-, and 6-thiosugar acceptors from a variety of sugar families
produces S-linked disaccharides in nearly quantitative yields. The set
of possible thiol acceptors also includes cysteine-containing peptides
and proteins, rendering this mutant enzyme a promising catalyst for
the production of thio analogs of biologically important GlcNAcylated
peptides and proteins.
replaced with sulfur) for use as GH inhibitors or as stable ligands
for structural and functional biology studies.[6,7] Despite advances
in the conventional chemical synthesis of thioglycosides, common
bottlenecks remain.[8] Enzyme-based methods have the potential
to streamline the preparation of thioglycosides. However, due to
the paucity of natural thioglycosides, there are relatively few
enzymes in nature that normally catalyze the synthesis of
thioglycosides.[9,33] One proven strategy for producing enzymes
that can form thioglycosidic linkages, is to use mutant GHs in
which the catalytic acid/base residue has been replaced by a non-
catalytically active residue (Scheme S1c);[6] this approach has
resulted in several α and β-thioglycoligases derived from retaining
Scheme 1. (a) Wild type neighbouring group-assisted hexosaminidase
mechanism, (b) oxazoline stabilizer mutant hexosaminidase acting as
a
glycosynthase using an oxazoline as the donor, (c) acid/base mutant
hexosaminidase acting as a thioligase using a donor with a good leaving group
(alternatively the oxazoline may be used as donor).
Advances in glycobiology are often dependent on the preparation
of specific oligosaccharides or complex glycoconjugates.
Conventional chemical synthesis of such compounds typically
requires time-consuming protecting group manipulations in order
to achieve the desired regiochemical and stereochemical control.
Thus, enzyme-based approaches have become particularly
valuable for
the synthesis
of
oligosaccharides
or
glycoconjugates.[1] While glycosyltransferases are the primary
synthetic enzymes in nature, a variety of glycoside hydrolases
(GHs) have been engineered to convert them into effective
catalysts for forming glycosidic bonds.[2] Most retaining GHs
contain two key active site residues, a catalytic nucleophile and a
catalytic acid/base, and catalyze reactions through a glycosyl-
enzyme intermediate (Scheme S1a).[3] One common strategy for
converting such a retaining GH into a glycosynthase, is to remove
the active site nucleophile; the resulting mutant enzyme, with
greatly diminished hydrolytic activity, can form glycosidic linkages
when used in combination with an activated donor that mimics the
glycosyl-enzyme intermediate (Scheme S1b).[4,32]
A desire for carbohydrate mimetics that are resistant to enzymatic
hydrolysis in vivo has elicited interest in the preparation of
thioglycosides (in which the typical glycosidic oxygen atom is
[a]
[b]
Dr. G.Tegl, Dr. Hong-Ming Chen, Prof. D. H. Kwan, Dr. A. G.
Santana, Prof. S.G. Withers
Department of Chemistry, University of British Columbia
GHs which proceed via a double displacement (Koshland)
mechanism.[10–14] The glycosyl-enzyme intermediate is formed by
using a donor possessing a good leaving group (LG), avoiding the
requirement of acid catalysis for the glycosylation step. De-
glycosylation would otherwise be slow due to the absence of a
general base to deprotonate the water during nucleophilic attack.
Vancouver, British Columbia, V6T 1Z1 (Canada)
E-mail: withers@chem.ubc.ca
Prof. J. Hanson
Department of Chemistry, University of Puget Sound
Tacoma, WA 98416 (USA)
Supporting information for this article is given via a link at the end of
the document
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However, when using a nucleophile with a low pKa, such as a
thiol, activation by the catalytic base is not required, thus allowing
the reaction to proceed at a reasonable rate.[6]
E314A. The mutant enzyme containing a polyhistidine tag was
readily produced in large quantities as a robust, soluble protein.
The enzyme retained high activity over several hours at 37 °C at
pH values ranging from pH 5 to 9 in solutions containing up to
10% DMSO. Use of this co-solvent was occasionally necessary
to help solubilize acceptor or donor saccharides.
Oligosaccharides and glycoconjugates containing beta-linked
GlcNAc or GalNAc residues have many important biological
roles.[15] Studies on their effects would benefit from the ability to
readily prepare hydrolytically stable thioglycoside analogs. For
example, O-GlcNAc modification of nuclear and cytosolic proteins
is a dynamic process influencing a wide range of cellular
processes.[16] Despite advances in analytical methods, the study
of the effects of O-GlcNAcylation is still a troublesome endeavor
due to omnipresent hexosaminidases capable of removing
GlcNAc from proteins and thus dramatically reducing their in vivo
half-life.[14] Cysteine-based S-GlcNAcylated proteins have been
prepared and are reported to mimic O-GlcNAc linkages without
altering their biological properties, but were very challenging to
synthesize.[17] An attractive alternative strategy to prepare S-
GlcNAcylated proteins would be to replace the amino acid that is
normally post-translationally modified (serine or threonine) with a
cysteine, and then use an appropriate thioglycoligase to attach
the GlcNAc to the cysteine. With these objectives in mind we set
out to modify a glycoside hydrolase that normally cleaves terminal
GlcNAc and GalNAc residues (a hexosaminidase) to produce a
thioglycoligase capable of forming S-linked β-GlcNAc (or β-
GalNAc) oligosaccharides and glycoconjugates when reacted
with the appropriate thiol acceptors.
Hexosaminidases from GH18, GH20, GH25, GH56, GH84, and
GH85 operate by a variant of the Koshland mechanism involving
neighboring group participation from the 2-acetamido group of the
respective sugar substrate, producing an oxazoline intermediate
rather than a glycosyl-enzyme intermediate (Scheme 1a).[18,19]
While several mutant hexosaminidases have been used as
glycosynthases[20], no thioglycoligases from these families have
been previously reported. Since the mechanism and charge
balance in the active site is substantially different between the
double displacement (Koshland) type enzymes and the
neighboring group participation type enzymes, it was not clear
whether the strategies used to convert the former into
thioglcoligases would work for the latter.
The hexosaminidase from Streptomyces plicatus (SpHex) is a
retaining GH20 exo-hexosaminidase that uses the neighboring
group mechanism to cleave terminal β-linked GlcNAc or GalNAc
residues from the non-reducing end of oligosaccharides (Scheme
1a).[21] A conserved aspartate (D313) in the active site assists by
helping to polarize and position the 2-acetamido group.[18]
Mutation of this conserved aspartate to alanine produces a mutant
enzyme SpHex D313A that catalyzes formation of glycosidic
bonds when synthetically prepared oxazoline is used as the donor
(Scheme 1b).[22] The active site of SpHex also contains a
conserved glutamate (E314) that acts as the catalytic acid/base,
promoting cleavage of the glycosidic linkage by general acid
catalysis and subsequent nucleophilic opening of the oxazoline
by general base catalysis.[23] In analogy with previous strategies
for producing thioglycoligases, we hoped that replacement of
glutamate 314 by an alanine would produce an enzyme capable
of catalyzing thioligation of activated GlcNAc donors to
appropriate thiol acceptors (Scheme 1c).
As expected, and as previously reported for the E314Q mutant,[22]
the removal of the catalytic acid/base results in a significant drop
in the hydrolytic efficiency of the enzyme; kcat/KM (and kcat
)
dropped to slightly less than 10% of the wild type values using
pNP-GlcNAc as substrate (Table 1) The pH profile was fairly flat
from pH 5 to 9, with kcat/KM values staying within a factor of 2 over
this range (Figure S1). pNP-GalNAc was used as substrate by the
mutant with rate constants approximately 3-fold lower. This
substrate and pH-tolerance will facilitate the use of this mutant in
synthesis.
To explore the ability of SpHex E314A to act as a thioglycoligase,
an excess of the donor GlcNAc oxazoline was incubated at 37 °C
and pH 7 with a variety of sugar thiol acceptors (5 mM) in the
presence of SpHex E314A (0.5 mg/mL) (Figure S2 + S3, Table
2); formation of the corresponding thioglycosides was monitored
by TLC and ESI MS analysis. Ligation to the thiol residue, rather
than to sugar hydroxyls, was demonstrated by TLC using a DTNB
stain which reacts with free thiols;[24] the products did not react
with this stain (Figure S4). Since wildtype SpHex cleaves β-linked
glycosides of GlcNAc, we initially tested the 4-thio analogue of
pNP-GlcNAc as an acceptor.[24] Using excess oxazoline donor, we
observed quantitative formation of the desired disaccharide
containing the thioglycosidic linkage. Encouraged by this result,
we also tested the 3-thio- and 6-thio- pNP-GlcNAc analogs as
acceptors and were pleasantly surprised to again observe rapid
conversion to the corresponding disaccharides (Figure S3). Given
the observed acceptor promiscuity, several other 3- and 4-thio
analogues were tested, comprising representatives of the D-gluco,
D-galacto and D-manno series (Table 2). Remarkably,
thioglycoside formation was observed for all candidates (Figure
S2 and S3). The donor was typically consumed within an hour; if
unreacted thiol acceptor was still present, additional oxazoline
donor was added as necessary, resulting in complete conversion
of the respective thio-acceptor and quantitative product yields.
Sub-stoichiometric product formation was observed only for 4-thio
ManNAc and 3-thio GalNAc acceptors. No detectable
disaccharide formation was observed in control reactions using
either wt or SpHex D313A.
Although the GlcNAc oxazoline donor could be synthesized from
GlcNAc,[26] we found that the commercially available pNP-GlcNAc
was an even more convenient glycosyl donor for these thioligation
reactions, especially on a preparative scale; pNP-GlcNAc is
presumably converted into the intermediate GlcNAc oxazoline by
SpHex E314A (Scheme 1c). We also showed that pNP-GalNAc
is an effective donor substrate, as demonstrated by synthesis of
β-GalNAc-1,4-S-GlcNAc-pNP in 98% isolated yield (SI 1.4 NMR
Data). Further confirmation of the identity of the products from
preparative scale (approximately 10 mg (20 µmol) of product)
reactions using pNP-GlcNAc and pNP-GalNAc as donors (Table
2, Entries 1-4) was obtained by NMR analysis of HPLC purified
products (SI 1.1-1.4 NMR Data). Interestingly self-condensation
of the thio-pNP-GlcNAc sugars listed in Table 2 was not observed,
Site-directed mutagenesis was used to convert the catalytic
acid/base glutamate of SpHex to alanine, producing SpHex
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in-silico models confirms that, as expected, mutation of glutamate
314 to alanine (in SpHex) results in a significant increase in space
within the +1 subsite (Figure 1). In the wt enzyme, the glutamate
314 sidechain interacts with the glycosidic oxygen (2.5 Å H-bond).
Replacing the glutamate by alanine widens the binding pocket by
3.5 Å, presumably making steric hindrance much less of an issue
and enabling nucleophilic attack by a wide array of thiol acceptors.
Thioglycoligases formed by mutation of the catalytic acid/base
residue of GHs do not normally act as O-glycoligases since there
is no base present to help deprotonate the otherwise poorly
nucleophilic sugar hydroxyl (pKa ~14-20) on the acceptor.[28]
However, thioglycoligases can catalyze the formation of O-linked
glycosides if acceptors with more acidic hydroxyl groups (e.g.,
phenols) are utilized, as previously reported for α-
thioglycoligases.[11] The ability of SpHex E314A to catalyze the
synthesis of O-aryl glycosides was investigated using a variety of
substituted phenols covering a pKa range of 4.1-10 (Scheme 3).
Reactions were performed at pH 8.0 using GlcNAc oxazoline as
donor; TLC analysis revealed O-aryl glycoside formation for all
acceptor candidates within short reaction times (< 1 h). The
absence of any significant difference in the synthesis efficiency
between the phenols suggested that neither the pKa value nor the
substitution patterns influenced the reaction significantly. By
contrast, steric hindrance was a previously reported issue in the
use of ortho- substituted phenols by an α-xylosidase
thioglycoligase in O-aryl glycoside synthesis[28]; our results
suggest that no such steric issues occur in the +1 subsite of
SpHex E314A.
Unsurprisingly, after further incubation overnight, the more
activated O-aryl glycosides formed (2,4-DNP and 4-NP), which do
not require acid catalysis for cleavage, were hydrolysed (Figure
S5). No such hydrolysis of the other glycosides (2-chlorophenol,
pKa 8.5; phenol, pKa 10.0) was seen, indicating that a phenol pKa
of less than approximately 8 is required for efficient hydrolysis by
SpHex E314A. The effect of the pH on the rate of the synthetic
reaction was measured using GlcNAc-oxa as donor and 4-NP as
acceptor. Only a narrow pH range (7-9) could be studied due to
the instability of GlcNAc-oxa at a pH < 7 and the previously
determined complete loss of enzyme activity at pH 10. The ligase
activity dropped only gradually from pH 7>8>9, presumably due
to a decreasing ability to activate the oxazoline by protonation.
Table 1. Kinetic parameters for SpHex and its mutants at pH5 and 7
(KM in µM, kcat in s-1 and kcat/KM in s-1*µM-1);
n.d.: not determined
pH 5
SpHex
wt
pH 5
SpHex
E314A
pH 7
SpHex
wt
pH 7
SpHex
E314A
pNP-GlcNAc
KM
36
20
13
16
kcat
kcat/KM
237
6.6
9.1
0.45
64
4.92
5.9
0.37
pNP-GalNAc
KM
kcat
kcat/KM
n.d.
n.d.
n.d.
34
2.7
0.08
n.d.
n.d.
n.d.
11
1.2
0.11
presumably because the bulkier thiosugar is not accepted in the
donor (-1) subsite.
Table 2. SpHex E314A catalyzed thioligation using thiosugar acceptors.
Reactions using pNP-GlcNAc or pNP-GalNAc as donor were performed on a
preparative scale (~20 µmol); products were purified by HPLC and
characterized by NMR spectroscopy. Yields were essentially stoichiometric with
respect to acceptor. Reactions using GlcNAc oxa as donor were performed on
an analytical scale; products were characterized by TLC and MS.
Having shown that SpHex E314A has relatively broad acceptor
specificity, we decided to try other thiols with biological
significance, such as cysteine; thioglycosides of cysteine would
be stable analogs of O-GlcNAc post-translationally modified
serines and threonines. When 4-NP-GlcNAc donor was mixed
with cysteine ethyl ester (Scheme 4) as acceptor in the presence
of SpHex E314A, thioligation proceeded in a manner similar to
that observed with the sugar acceptors and resulted in the
GlcNAc-cysteine conjugate whose structure was confirmed by
NMR (SI 1.5 NMR Data, Figure S6). Inspection of the in-silico
model structure of SpHex E314A suggested that the active site
Given the relatively narrow substrate specificities of most
enzymatic systems, the broad acceptor specificity observed in
this system is somewhat surprising. Similar acceptor promiscuity
has been reported for a thioglycoligase derived from the β-
glucosidase from Agrobacterium sp., Abg E171A.[6,27] Apparently
the high nucleophilicity of the thiolate, more so than specific
binding interactions of the acceptor, is the major determinant for
formation of the thioglycosides in these thioligations. Inspection of
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Figure 1. The (in-silico) mutation of glutamate 314 to alanine (right) in SpHex opens up space in the +1 subsite, compared to the wiltype (left)
pocket is oriented in a manner that would accommodate peptide
chains attached to the cysteine.
hydrolase. However enzymatic S-GlcNAcylation of peptides had
been previously demonstrated using native O-GlcNAc transferase
(OGT).[33]
Consequently, we tested the S-GlcNAcylation ability of SpHex
E314A on two model peptides derived from synuclein. The
aggregation of α-synuclein, a protein found in presynaptic
neurons, is associated with Parkinson’s disease; interestingly, O-
GlcNAcylation of α-synuclein has an inhibitory effect on this toxic
aggregation.[29]
Scheme 4. Investigated peptide thiol acceptors; cysteine ethyl ester as well as
synuclein model peptides containing a known GlcNAcylation site (72 and 87)
which was replaced by a cysteine.
The natural extension of this peptide work was to investigate
whether SpHex E314A could be used to modify a cysteine in a
folded protein. Tau is a microtubule-associated protein suggested
to be a key player in Alzheimer´s disease (AD) pathogenesis. O-
GlcNAcylation of Tau commonly prevents phosphorylation of the
glycosylation site that was found to be the key factor of Tau
pathogenesis.[30] Tau naturally contains two cysteine residues
(Cys301 and Cys322). In the version we employed, Tau(244-441),
those two Cys residues had been replaced by Ser. We further
replaced the critical O-GlcNAcylation site, Ser 400, with a cysteine
designating the triply modified protein as (Tau S400C). After 3 h
incubation of Tau S400C (mass = 23,377) with pNP-GlcNAc and
SpHex E314A, ESI-TOF MS analysis of the protein showed the
formation of a GlcNAcylated version (mass = 23,580), heavier by
the expected 203 mass units (Figure 2b). GlcNAcylation of the
thiol of Cys 400 was further confirmed by thiol titration using
Scheme 3. (a) Mechanism of aryl glycoside synthesis by SpHex E314A and (b)
Substituted phenols used for aryl glycoside synthesis by SpHex E314A.
Pentapeptides were synthesized corresponding to residues 70-74
and 85-89 of α-synuclein, in which threonine 72 and serine 87 had
been replaced by cysteine (Scheme 4). MALDI-TOF (Figure 2a)
and HPLC analysis (Figure S9) indicated GlcNAcylation of the
cysteine residue after short incubation times for both
pentapeptides; control reactions using the native pentapeptides
(Thr/Ser) in the presence of SpHex E314A did not result in
GlcNAcylated reaction products. Product yields of 44% for VVC(-
GlcNAc)GV and 30% for AGC(-GlcNAc)IA were obtained, as
determined by HPLC.
To our knowledge, this is the first report of the direct enzymatic S-
GlcNAcylation of a peptide using an engineered glycoside
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DTNB, revealing 44% modification of the thiol (Figure S8), in
agreement with the ESI-TOF data shown in Figure 2b.
Figure 2. (a) S-GlcNAcylation of α-synuclein model peptides representing cysteine mutations of the GlcNAcyation sites 72 (AGCIA) and 87 (VVCGV); (b) Whole
protein ESI-TOF spectrum of TauS400C that has been S-GlcNAcylated by SpHex E314A. Mass of TauS400C = 23377.10, GlcNAcylated TauS400C = 23580.20
and double GlcNAcylated TauS400C = 23783.
This yield can likely be improved by using more appropriate
hexosaminidases.
neighboring group participation in the initial cleavage step. The
mutant enzyme, SpHex E314A, catalyzes the attachment of
terminal GlcNAc or GalNAc residues onto a range of thiol
acceptors, including peptides and proteins, through β linked-S-
linkages. These thioglycosides are stable mimics of biologically
important O-GlcNAc and O-GalNAc oligosaccharides and
glycoconjugates. State of the art approaches such as the
semisynthesis of O/S-GlcNAcylated proteins have required
multiple preparation steps, including chemical synthesis.[17,32] In
contrast, the direct enzymatic GlcNAcylation of proteins in a single
step embodies a greatly simplified concept, providing a fast and
easy-to-handle tool for protein S-GlcNAcylation. It is noteworthy
that this thioglycoligase utilizes an inexpensive, commercially
available glycosyl donor (pNP-GlcNAc) which greatly reduces the
workload and increases the practicability of this S-GlcNAcylation
system. Whether this strategy can be applied to proteins
displaying more than one cysteine is the subject of ongoing
studies.
A small amount of a double GlcNAcylated product (+406) was
also detected which might be caused by non-enzymatic glycation,
a process previously observed by Parsons et al. in performing
transglycosylations of N-linked glycans using a large excess of
oxazoline donor substrates and a mutant Endo-S.[31] To test this
possibility, blank reactions were performed using different donor
substrates (GlcNAc, pNP-GlcNAc or GlcNAc oxazoline) in the
absence of the thioglycoligase. ESI-TOF analysis indicated traces
of glycation product when using GlcNAc oxazoline as donor, while
no reaction product was observed with just GlcNAc or pNP-
GlcNAc (Figure S7). The non-enzymatic formation of glycation
product in the presence of GlcNAc oxazoline concurs with the
observations of Parsons et al.;[31] our observation that pNP-
GlcNAc produces small amounts of similar non-enzymatic
glycation, but only in the presence of SpHex E314A, seems to
suggest that GlcNAc oxazoline may be liberated during incubation
of pNP-GlcNAc with SpHex E314A, although efforts at detecting
released GlcNAc oxazoline under these conditions were not
successful.
In conclusion, the strategy of producing thioglycoligases by
removing the catalytic acid/base sidechain in a glycoside
hydrolase has been extended to a new mechanistic class of
glycoside hydrolases (GH20 hexosaminidase) that utilizes
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Acknowledgements
We thank Luke Lairson and Kieran Hudson for preliminary work
on this project as well as Jacob Brockerman and Lawrence
McIntosh for a generous donation of the Cys mutant of Tau. We
acknowledge financial support from the Canadian Institutes for
Health Research (CIHR) and the Canadian Glycoscience
Network GlycoNet, as well as a Schroedinger fellowship to GT
from the FWF.
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Keywords: Glycoprotein synthesis • Glycosynthase •
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10.1002/anie.201809928
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Thioglycosides are stable carbohydrate mimetics frequently used for structural and functional biology studies. A thioglycoligase was
developed from a GH 20 hexosaminidase enabling direct S-GlcNAcylation of a diverse set of thio-acceptors. Thio-sugars of various
sugar families were GlcNAcylated in quantitative yields and the acceptor pool was successfully expanded to peptides and proteins.
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