MicroPlate Sialyltransferase Assay: A Rapid and Sensitive Assay Based on an Unnatural Sialic Acid Donor and Bioorthogonal Chemistry

Article abstract of DOI:10.1021/acs.bioconjchem.8b00529

Mammalian sialyltransferases transfer sialic acids onto glycoproteins and glycolipids within the Golgi apparatus. Despite their key role in glycosylation, the study of their enzymatic activities is limited by the lack of appropriate tools. Herein, we developed a quick and sensitive sialyltransferase microplate assay based on the use of the unnatural CMP-SiaNAl donor substrate. In this assay, an appropriate acceptor glycoprotein is coated on the bottom of 96-well plate and the sialyltransferase activity is assessed using CMP-SiaNAl. The alkyne tag of SiaNAl enables subsequent covalent ligation of an azido-biotin probe via CuAAC and an antibiotin-HRP conjugated antibody is then used to quantify the amount of transferred SiaNAl by a colorimetric titration. With this test, we evaluated the kinetic characteristics and substrate preferences of two human sialyltransferases, ST6Gal I and ST3Gal I toward a panel of asialoglycoprotein acceptors, and identified cations that display a sialyltransferase inhibitory effect.

Full text of DOI:10.1021/acs.bioconjchem.8b00529

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Article
MicroPlate Sialyltransferase Assay (MPSA): a rapid and sensitive assay
based on an unnatural sialic acid donor and bioorthogonal chemistry
Maxence Noel, Pierre-Andre Gilormini, Virginie Cogez, Cédric
Lion, Christophe Biot, Anne Harduin-Lepers, and Yann Guerardel
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00529 • Publication Date (Web): 07 Sep 2018
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MicroPlate Sialyltransferase Assay (MPSA): a rapid and
sensitive assay based on an unnatural sialic acid donor and
bioorthogonal chemistry
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Maxence Noel†, PierreꢀAndré Gilormini†, Virginie Cogez†, Cédric Lion†, Christophe Biot†,
Anne HarduinꢀLepers†, Yann Guérardel†*
†Univ. Lille, CNRS, UMR 8576 ꢀ UGSF ꢀ Unité de Glycobiologie Structurale et
Fonctionnelle, F 59000 Lille, France
*Corresponding author: Yann Guérardel, Unité de Glycobiologie Structurale et Fonctionnelle,
UMR CNRS 8576, Université Lille Nord de France, Lille1, 59655 Villeneuve d'Ascq, France.
Phone: +33 320 33 62 46; Fax: +33 320 43 65 55; Eꢀmail: yann.guerardel@univꢀlille.fr
Key words: Sialyltransferases, biorthogonal chemistry, CMPꢀSiaNAl, microplate
sialyltransferase assay
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ABSTRACT
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Mammalian sialyltransferases transfer sialic acids onto glycoproteins and glycolipids within the Golgi
apparatus. Despite their key role in glycosylation, the study of their enzymatic activities is limited by
the lack of appropriate tools. Herein, we developed a quick and sensitive sialyltransferase microplate
assay based on the use of the unnatural CMPꢀSiaNAl donor substrate. In this assay an appropriate
acceptor glycoprotein is coated on the bottom of 96ꢀwell plate and the sialyltransferase activity is
assessed using CMPꢀSiaNAl. The alkyne tag of SiaNAl enables subsequent covalent ligation of an
azidoꢀbiotin probe via CuAAC and an antiꢀbiotinꢀHRP conjugated antibody is then used to quantify
the amount of transferred SiaNAl by a colorimetric titration. With this test, we evaluated the kinetic
characteristics and substrate preferences of two human sialyltransferases, ST6Gal I and ST3Gal I
toward a panel of asialoglycoprotein acceptors, and identified cations that display a sialyltransferase
inhibitory effect.
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INTRODUCTION
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In the world of glycobiology, the sialic acids family occupies a quite unique position. Comprising
more than 50 natural members, this family of nine carbon carboxylated monosaccharides is often
found at the nonꢀreducing extremity of glycans.1 Due to their terminal position and negatively charged
nature, sialic acids are involved in many biological phenomena such as cellꢀcell adhesion, hostꢀ
pathogen interactions, or regulation of pathophysiological events.2 Sialyltransferases (STs) of the
CAZy GTꢀ29 family are found in all eukaryotic branches and in alphaꢀproteobacteria and represent a
subset of glycosyltransferases responsible for the introduction of sialic acid residues onto
glycoconjugates.3 In the past two decades, a growing interest for STs has been observed in various
fields such as clinical biomarkers, biocatalysis, natural products synthesis or biotherapeutics.4–6
Twenty different STs have been identified in mammalian genomes.7 Mainly human and mouse STs
have been biochemically characterized up to now and were shown to catalyze the transfer of a sialic
acid residue from its activated form, cytidyl monoꢀphosphate sialic acid (CMPꢀSia), onto galactose
(Gal), Nꢀacetylgalactosamine (GalNAc) or another sialic acid (Sia) residue of glycoproteins or
glycolipids defining four families of STs (ST3Gal, ST6Gal, ST6GalNAc and ST8Sia). All
characterized STs use CMPꢀSia as a donor substrate, each enzyme presents its own specificity with
regards to acceptors and type of glycosidic linkage formed.8 Even though sialylation diversity
encountered in Nature mainly relies on the activity, specificity and regulation of STs, these enzymatic
properties are not completely understood yet. Methods and specific tools are therefore needed for the
study of sialylation reactions and a complete understanding of the associated biochemical processes.
Commercial radiolabeled CMPꢀ[14C]Neu5Ac or synthetized CMPꢀ[14C]Neu5Gc have been widely
used for the characterization of ST activity since the work of Weinstein and Paulson.9 Although very
sensitive, these approaches are expensive and the use of radiolabeled substrates necessitates access to
highly controlled and specific facilities. As an alternative, the structural and quantitative analysis of
the acceptor glycan before and after sialylation provides valuable information about STs enzymatic
characteristics. Mass spectrometry combined to liquid chromatography (LCꢀMS) has long been used
for the analysis and characterization of sialylated glycans10 and we recently described the use of
microꢀLC/ESIꢀMRMꢀMS3 analysis to quantify transfer rates of STs, using various CMPꢀSia donors
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and various acceptor substrates.11 Despite being precise, sensitive, and quantitative, such analyses are
time consuming and require specific equipment and knowꢀhow. Some efforts have been made in the
development of simple and accessible methods to open sialylation assessment to nonꢀspecialists. For
instance, a method relying on indirect colorimetric detection of inorganic pyrophosphate has been
developed with Malachite green reagent.13 First developed for soil and water analyses, this
colorimetric assay has been used to probe ST6Gal I and ST6GalNAc V activities.14,15 However, this
assay presents several limitations such as high background when nonꢀpurified enzymes are used,
impossibility to use phosphate buffers or interferences with detergents. Besides, the Malachite green
reagent assay has been shown to interact with arsenate, a major component of the cacodylate buffer,
the gold standard buffer for in vitro assays with STs.16
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Therefore, alternative methods are needed to probe ST activity; one of them is to monitor the reaction
through quantification of the transferred sialic acid. Toward this aim, the activated sialic acid (CMPꢀ
Sia) has to be conjugated with a molecule allowing its detection. In the ‘80s, Brossmer and
collaborators synthesized unnatural CMPꢀSias donors that proved to be suitable for enzymatic
sialylation using purified STs,17,18 and more recently fluorogenic or chromogenic substrates were
designed to monitor sialylation reactions by FRET or HPLC.19,12 Synthesis of activated CMPꢀSias
conjugated with a fluorescent probe is however hard to achieve. The chemical reporter strategy,
pioneered in the ‘90s, successively by the groups of Reutter and Bertozzi,20,21 has opened the way for
new tools to study and visualize sialoglycoconjugates. In this strategy, a small bioorthogonal moiety is
chemically introduced onto a monosaccharide which can subsequently enter the cell and hijack its
metabolism. Once introduced into glycoconjugates, the chemical modification is ligated to a specific
complementary probe through bioorthogonal ligation, allowing the detection of the target molecule. In
a seminal study, Mbua et al. combined chemical reporter strategy and activated sialic acids with the
use of recombinant ST6Gal I to transfer an azide analog of sialic acid onto living cells.22 Subsequent
strainꢀpromoted azideꢀalkyne cycloaddition (SPAAC) with a cyclooctyneꢀfunctionalized fluorophore
allowed them to specifically detect cell surface α2ꢀ6ꢀsialylated glycoproteins. Such chemoenzymatic
glycan labeling has been increasingly applied to detect glycoproteins over the past few years through
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the use of unnatural CMPꢀactivated sialic acids23–27 and other nucleotide monosaccharide donors.28
Based on this method, we developed sensitive assays that can be used to study the biochemical
properties of STs, to screen potential ST inhibitors, or to characterize new enzymatic activities.
Recently, we reported a simple chemoenzymatic procedure that provides synthetically modified CMPꢀ
Sias without any purification steps29 and the joint use of LCꢀMS, radioactive assays, and enzymatic
labelling with nonꢀnatural sialic acids analogs to probe the activity and specificity of two human STs
(ST3Gal I and ST6Gal I).11 In this study, we propose a rapid and sensitive microplate sialylation assay
(MPSA) that allows easy monitoring of ST activity in various conditions using a alkynyl analog of
CMPꢀNeu5Ac (i.e., CMPꢀSiaNAl).
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RESULTS AND DISCUSSION
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Scheme 1: Sialylation reaction and enzymes preferential acceptor substrates with ST6Gal I on Nꢀglycans and
ST3Gal I on Oꢀglycans. Sialic acid is represented in purple.
Two human recombinant STs were chosen for the development of our sialylation assay
(Scheme 1): whereas ST6Gal I exclusively transfers sialic acid with an α2ꢀ6 glycosidic linkage on the
galactose residue of Galβ1ꢀ4GlcNAc of Nꢀglycans, ST3Gal I specifically targets Oꢀglycans for the α2ꢀ
3 transfer of sialic acid on Galβ1ꢀ3GalNAc. ST3Gal I and ST6Gal I are the two most widely known
and studied sialyltransferases. In addition to their respective specificity toward either Nꢀ or O-glycans,
this made them relevant models to set up and illustrate the versatility of MPSA. Recombinant human
ST6Gal I and ST3Gal I deleted of their first 56 amino acids (i.e. cytoplasmic tail, transmembrane and
stem domains) and bearing a 3XFlag tag at their Nꢀterminus were therefore produced into transfected
HEK293 (Human Embryonic Kidney) cell culture medium. We previously reported the tolerance of
both ST6Gal I and ST3Gal I toward CMPꢀSiaNAl, the alkynyl analog of CMPꢀNeu5Ac, and showed
that they exhibit similar kinetic parameters with natural and unnatural donor substrates.11 A simple
four step procedure was setꢀup: (1) the acceptor glycoconjugate is coated overnight into a 96ꢀwell
plate. (2) After saturation of wells with oxidized Bovine Serum Albumine (BSA), crude enzymatic
solution of either ST6Gal I or ST3Gal I and freshly prepared CMPꢀSiaNAl (Figure 1A) are incubated
in the wells at 37 °C. (3) AzidoꢀPEGꢀbiotin is covalently attached to the alkyne of each transferred
sialic acid through copper catalyzed alkyneꢀazide cycloaddition (CuAAC). (4) Biotin is then
specifically detected by an antiꢀbiotin antibody coupled with Horse Radish Peroxydase (HRP). The
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readout can be readily achieved with a simple measure of each well absorbance with TMB (3,3′,5,5′ꢀ
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Tetramethylbenzidine) substrate and a microplate spectrophotometer at 620 nm (Figure 1B).
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To assess the specificity of MPSA for sialylation reaction we set up the standard procedure
described above using fetuin, a model glycoprotein containing 3 sialylated Nꢀglycans and 3 sialylated
Oꢀglycans.30,31 Before the coating, fetuin was chemically desialylated (yielding asialofetuin) with
trifluoroacetic acid (TFA). Culture medium containing crude ST6Gal I expressed and produced in
transfected HEK cells or an STꢀfree cell culture medium (mock medium) and 0ꢀ100 µM CMPꢀSiaNAl
were incubated with the coated asialoꢀglycoprotein. Subsequently, reꢀsialylated glycoproteins were
reacted with azidoꢀPEGꢀbiotin in presence of CuSO4 and sodium ascorbate to perform a CuAAC
ligation. After washing steps, the covalently linked biotin was finally detected with antiꢀbiotinꢀHRP
antibody and the absorbance measured at 620 nm. Figure 1B illustrates the significant increase of the
absorbance with the concentration of donorꢀsubstrate, whereas only a slight increase of the absorbance
was detected in mock medium. Therefore, in the subsequent experiments, signal obtained with the
mock medium were systematically subtracted from the one obtained with sialyltransferases in order to
only consider specific signal.
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Figure 1: (A) Chemical structure of the alkynyl analog of CMPꢀNeu5Ac: CMPꢀSiaNAl. (B) MPSA experiment
after incubation with either ST6Gal I or mock medium and various concentrations of CMPꢀSiaNAl (0ꢀ100 µM).
All the wells have been treated with azidoꢀPEGꢀbiotin with CuAAC conditions and finally revealed with an antiꢀ
biotin antibody HRP. Absorbance measured at 620 nm for all CMPꢀSiaNAl concentrations. Error bars represent
SEM (n = 2).
To assess the robustness of our test and to set up the essential conditions of an enzymatic
assay as simple and fast as possible, we use directly the transfected cell culture medium as an enzyme
source for both human ST6Gal I and ST3Gal I. As previously reported, low levels of Δ56ST6Gal I
were consistently obtained in the cell culture medium of transfected cells, whereas Δ56ST3Gal I was
expressed at much higher level.11 Consistent with this data, we observed a dependence of the reaction
velocity on the enzyme amount used in the MPSA (Figure 2A). To keep the amount of enzyme as low
as possible and a reaction velocity in the same range for the two recombinant enzymes, the
Δ56ST3Gal I enzymatic source was diluted 5 times while ST6Gal I was used undiluted in the
subsequent assays. Since the enzyme amount is directly related with the reaction time, the best time
for sialylation reaction within the initial velocity was then determined (Figure 2 B). For both enzymes,
absorbance value reaches a plateau after 1h30 of reaction and to perform all reactions in conditions of
initial velocity, the sialylation reaction was therefore fixed to 1 h. The concentration of both donor and
acceptor substrates involved in the sialylation reaction should be saturating so that no substrates will
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be rate limiting. We thus evaluated the kinetics parameters of each enzyme preparation toward donor
substrate (CMPꢀSiaNAl) and acceptor substrate (asialofetuin). To that aim, either ST6Gal I or ST3Gal
I were incubated with 400 ng of coated asialofetuin in presence of various concentrations of CMPꢀ
SiaNAl (Figure 2, C). Values of 28.2 ± 7.8 µM and 14.8 ± 2.9 µM were calculated for Km toward
CMPꢀSiaNAl for ST6Gal I and ST3Gal I respectively. We fixed the donor substrate concentration to
100 µM in order to obtain nonꢀlimiting substrate conditions. Similar experiments were performed to
determine the kinetics of enzymes toward asialofetuin. Sialylation reactions were performed with
variable quantities of coated asialofetuin, in presence of 100 µM CMPꢀSiaNAl (Figure 2, D). We
determined that ST6Gal I had an arbitrary Km toward asialofetuin of 319 ± 151 ng whereas an
arbitrary Km of 88.1 ± 41.1 ng was found for ST3Gal I, indicating a higher affinity of this latter
enzyme toward asialofetuin. The plateau reached with 400 ng of coated asialofetuin shows that this
quantity of acceptor substrate provides the maximal absorbance for both ST6Gal I and ST3Gal I.
These results are in accordance with others found in the literature with more traditional
methodologies.11,32
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Figure 2. Determination of kinetic parameters with MPSA. Otherwise specified, 100 µM of CMPꢀSiaNAl and
400 ng of asialofetuin were incubated for 1 h with either ꢁ56ST6Gal I and ꢁ56ST3Gal I. (A) Activity in
function of the dilution factor of the enzymatic source (B) Reaction time. MPSA absorbance readout for both
ST6Gal I and ST3Gal I in function of time. (C) Km determination of both ꢁ56ST6Gal I and ꢁ56ST3Gal I
toward CMPꢀSiaNAl (0 ꢀ 100 µM) (D) and coated asialofetuin (0 – 400 ng). Error bars represent SEM, (n = 2).
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The specificity of STs toward acceptor substrates is rather difficult to assess as it usually
requires the ability to detect the acceptor glycan. On one hand, biologically relevant synthetic acceptor
are hardly available and on the other hand, available short diꢀ or triꢀsaccharides do not fully reflect the
structural complexity of STs’ substrate and are therefore not representative of the specificity of these
enzymes. To assess the ability of MPSA to reflect the preferences of both enzymes toward Nꢀ and Oꢀ
glycans, several glycoproteins were used as specific acceptors: Bovine Serum Albumin (BSA) is not
glycosylated and was used as a negative control; Porcine gastric mucin (PGM) bears multiple core 1
and 2 Oꢀglycans;33 orosomucoïd, also known as alphaꢀ1ꢀacid glycoprotein, bears five triꢀ and tetraꢀ
antennary Nꢀglycans chains;34 and, finally, fetuin exhibits both Nꢀ and Oꢀ glycans. Acceptor
glycoproteins were compared either under their native form or after removal of sialic acids in presence
of trifluoroacetic acid (TFA). Each acceptor was coated in the microplate overnight, incubated for 1 h
with either ST3Gal I, ST6Gal I or mock medium in presence of 100 µM of CMPꢀSiaNAl. As
expected, none of the native glycoproteins was efficiently used by recombinant sialyltransferases
because of the preexisting sialic acid residues that occupy the sialylation sites (Figure 3). No
significant difference between mock or any ST was observed for BSA, even after TFA treatment.
ST3Gal I was found more active toward desialylated PGM whereas ST6Gal I showed more activity
onto desialylated orosomucosoid and as already observed, asialofetuin was sialylated by the two
recombinant enzymes. These results highlight the versatility of MPSA for acceptor preferences
determination. Indeed, in only a few hours, the specificity of any ST can potentially be assessed
toward any glycoproteins including bearing several glycosylation motifs. In the last decade, in vitro
promiscuity toward substrate modifications of Nꢀacyl chain has been reported for ST6Gal, ST3Gal,
ST6GalNAc, and ST8Sia families, with both mammalian and bacterial systems.27,35–38 This
promiscuity should allow to extend MPSA to other STs in the future.
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Figure 3. Preference of Δ56ST6Gal I and Δ56ST3Gal I toward different acceptor glycoproteins. Either mock,
Δ56ST6Gal I or Δ56ST3Gal I were incubated with 100 µM of CMPꢀSiaNAl for 1 hour with 400 ng of the
indicated coated protein. Error bars represent SEM, (n = 4).
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To assess the relevance of MPSA as a screening assay for potential inhibitors of STs activities,
we measured the relative activity of Δ56ST6Gal I and Δ56ST3Gal I in presence of 10 mM of various
compounds. Activity of STs is usually assessed in Cacodylate buffer containing 10 mM of MnCl2. In
these experiments, we used a Cacodylate buffer with no MnCl2 as a control and STs activities were
subsequently expressed as percentage of this control condition (Figure 4A). Cytidylꢀmonoꢀphosphate
(CMP, 10 mM) was used as a positive control of inhibition as it has been shown to be a competitive
inhibitor of STs’ activity.39,40 As expected, 10 mM of CMP strongly inhibited the activity of both
enzymes. It has long been known that most glycosyltransferases require the presence of a divalent
cation to be active. MPSA was therefore conducted in presence of 10 mM of EDTA, a strong divalent
cations chelating reagent leading to reduced activities of ST6Gal I and ST3Gal I. A first hypothesis
would be that EDTA chelates divalent cations from the buffer and from the culture medium containing
the enzyme. Therefore, the resulting lower STs activity suggests that divalent cations play an
important role in sialyltransferase activity, in contradiction with works of Shah et al.41 Another
hypothesis is that cations’ role is to stabilize the CMPꢀSia as previously proposed by Kolter and
Sandhoff.42 We then investigated whether commonly used cations could exhibit any increasing or
decreasing effect on STs’ activity. MPSA was realized with 10 mM of MgCl2, CaCl2, MnCl2, CoCl2
and ZnCl2 (Figure 4A). Magnesium and calcium appear to have no major effect on ST6Gal I nor
ST3Gal I. Although not statistically significant, manganese appeared to inhibit ST6Gal I activity as
previously reported for serum sialyltransferase41,43 and to slightly stimulate ST3Gal I activity. At the
opposite, we observed that 10 mM of cobalt or zinc strongly inhibited the two sialyltransferase
activities. Although inhibition effects have been reported with polyoxometalate complexes,44 this is
the first time that sialyltransferases’ inhibition is reported with these cations. We therefore studied the
doseꢀresponse inhibition for cobalt and zinc for ST6Gal I and ST3Gal I (Figure 4B). We found that
ST6Gal I is more sensitive to ZnCl2 and CoCl2 inhibition than ST3Gal I. Indeed, in presence of 100
µM of ZnCl2 or CoCl2, ST6Gal I activity strongly decreases (approximatively 25 %) whereas ST3Gal
I activity remains high (80 %). Although these first findings would deserve further investigations, they
highlight the potential of MPSA for interesting advances in the study of STs inhibition.
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Figure 4. Inhibition effects on Δ56ST6Gal I and Δ56ST3Gal I activities. (A) Relative activity of Δ56ST6Gal I
(left) and Δ56ST3Gal I (right) in presence of 10 mM of CMP, EDTA, MgCl2, CaCl2, MnCl2, CoCl2, ZnCl2.
Cacodylate buffer (10 mM) was used as control and set at 100 %. Enzymes were incubated for 1 h with CMPꢀ
SiaNAl (100 µM) and asialofetuin (400 ng). Error bars represent SEM, (n = 3), pꢀvalue = 0.0001. (B) Doseꢀ
effect relationship of CoCl2 (left) and ZnCl2 (right) on Δ56ST6Gal I and Δ56ST3Gal I activity. Enzymes were
incubated for 1 h with CMPꢀSiaNAl (100 µM) and asialofetuin (400 ng) in presence of either CoCl2 or ZnCl2 (1
µM – 10 mM). Error bars represent SEM, (n = 6).
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CONCLUSION
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We presented here a new fast, sensitive, versatile and easy to set up MPSA that can be applied to
probe ST activities without any dedicated facilities or equipment. With the use of two recombinant ST
preparations (the human Δ56ST6Gal I and Δ56ST3Gal I), we showed that MPSA can be applied to
different investigations such as kinetic parameters determination, substrate specificity assessment and
inhibitors screening. MPSA using CMPꢀSiaNAl should be applicable to other sialyltransferases and
will provide a powerful and simple method for future advances in sialylation research.
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EXPERIMENTAL PROCEDURES
Material. Fetuin, orosomucoid, mucin from porcine stomach Type III, TMB and azidoꢀPEG3ꢀbiotin
were purchased from Sigma. Antiꢀbiotin HRP conjugated was purchased from Jackson
Immunoresearch. CMP was from TCI chemicals. BTTAA was synthesized in our laboratory.28 Sodium
cacodylate was from Prolabo, EDTA, magnesium acetate, potassium chloride and calcium chloride
were from Acros organics, sodium chloride and magnesium chloride were from euromedex, lithium
chloride is from Bio Basic Canada Inc., manganese chloride from RiedelꢀdeHaen and cobalt chloride
from Merck and zinc chloride from Fluka Chemika.
Production of recombinant enzymes. Preparation of expression plasmids for human ꢁ56ST6Gal I
and ꢁ56ST3Gal I was reported recently.11 For production of sialyltransferases in cell culture medium,
seventy percent confluent HEK293 cells were transiently transfected using lipofectAMINE reagent
(InVitrogen) and ultraMEM (LONZA), following the instruction of the manufacturer. Thirtyꢀsix hours
postꢀtransfection, cell culture medium was collected, centrifuged to eliminate cell debris and used as
crude enzyme fraction. The cell culture medium containing ꢁ56ST6Gal I was used undiluted and
ꢁ56ST3Gal I was diluted by 5.
Synthesis of CMP-SiaNAl. Synthesis of CMPꢀSiaNAl was carried out from Dꢀmannosamine as
previously described.29 Dꢀmannosamine hydrochloride (1 eq.) and pentynoic acid NHS ester (1 eq.)
were dissolved into DMF under nitrogen atmosphere. Triethylamine (3 eq.) was added and reaction
was stirred overnight. Solvent was removed under reduced pressure and the crude product was
purified by flash chromatography on silica column (CH2Cl2/EtOAc/MeOH; 45 : 45 : 10). After
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removing of the solvents, ManNAl was obtained as a white crystalline solid (95 %). ManNAl (1 eq.),
sodium pyruvate (2 eq.) and Neu5Ac aldolase (Sigma Aldrich, EC 4.1.3.3) from E. Coli K12 (5 U)
were dissolved into 1.5 mL of phosphate sodium buffer containing 20 mM of MgCl2. The reaction was
stirred overnight at 37 °C. The reaction was diluted with water and loaded onto anionꢀexchange resin
column (Dowex 1X8). Alkynyl acid was eluted with 0.2 M of NH4HCO3. Salts were subsequently
removed through a gel filtration resin (P2). SiaNAl (0.028 mmol, 1 eq.) and cytidine 5’ꢀtriphosphate
disodium salt (15 mg, 0.028mmol, 1 eq.) were dissolved into TrisꢀHCl buffer (100 mM, pH 8.8)
containing 20 mM MgCl2. Then, CMPꢀSialic acid synthetase from Neisseria Meningitidis group B
(Sigma Aldrich, EC 2.7.7.43), 1U, and inorganic pyrophospatase from baker’s yeast (S. cerevisiae),
(Sigma Aldrich, EC 3.6.1.1), 1U were added to the mixture. Reaction was incubated for 1 h at 37 °C.
The solution was then cooled down to 4 °C and directly diluted at the right concentration for
subsequent sialylation assays with no further purification in order to avoid the hydrolysis of the
product which is quite unstable in solution. CMPꢀSiaNAl was characterized by 1H, 13C and 31P NMR.
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NMR H (400 MHz, D2O): δ = 7.51 (d, J = 7.6, 1H, H20), 5.72 (d, J = 7.6, 1H, H21), 5.58
(d, J = 3.9, 1H, H19), 4.00 – 3.87 (m, 4H, ribose), 3.85 (d, J = 5.5, 1H, H15), 3.76 (d, J = 10.4, 1H),
3.74 – 3.66 (m, 1H), 3.61 (d, J = 10.3, 1H, H5), 3.56 (dd, J = 10.2, 3.0, 1H), 3.51 (d, J = 12.2, 1H,
H9 a), 3.26 (dd, J = 14.2, 7.1, 1H), 3.20 (dd, J = 10.8, 6.0, 1H, H9 b), 2.22 – 2.03 (m, 5H, H11, H12,
H3 eq), 1.99 (s, 1H, H14), 1.26 (ddd, J = 13.0, 11.7, 5.8, 1H, H3 ax). 13C (101 MHz, D2O): δ = 175.24
(C1), 174.38 (C10), 165.92 (C22), 157.54 (C23), 141.42 (C20), 99.90 (C2), 96.53 (C21), 88.98 (C19),
83.22 (C14), 82.31, 73.94, 71.65, 70.31 (C13), 69.18, 69.01, 68.78, 66.57, 64.75 (C15), 62.90 (C9),
51.63 (C5), 40.98 (C3), 34.65 (C11), 14.56 (C12). 31P (162 MHz, D2O): δ = ꢀ4.63
Preparation of cations solutions. Each salt was dissolved in 100 mM of cacodylate buffer at
concentration of 25 mM before sialylation. For the sialylation reaction, cacodylate was at 40 mM and
salts at 10 mM once diluted with CMPꢀSiaNAl and sialyltransferaseꢀcontaining medium.
Desialylation of glycoproteins. Glycoproteins were desialylated using 0.1 M of trifluoroacetic acid 2
hours at 80 °C, and free sialic acid was eliminated using dialysis membrane MWCO 3500 spectra/por3
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(18 mm flat width; Spectrum Laboratories) during 24 hours. Content was then transferred in glassꢀtube
and lyophilized.
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Oxidation of BSA. Four grams of BSA were dissolved in 100 mL of sodium acetate 0.1 M pH 4,5 and
10 mM of sodium metaperiodate. After 6 hours incubation at room temperature, periodate was
neutralized with 10 mM of glycerol and solution was dialyzed in membrane membraꢀcel
MC30x500CLR against distilled water for 24 h. Then, the solution was lyophilized.
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MPSA general procedure. 400 ng of asialofetuin in 100 µL of sodium bicarbonate buffer (20 mM pH
9,6) were coated in 96ꢀwell plate (F8 MaxiSorp Loose NuncꢀImmuno Module ThermoScientific)
overnight at 4 °C. After 3 washes with 150 µL of PBSTꢀ 0,05 % (Phosphate Buffer SalineꢀTween)
saturation was made 1 hour at room temperature using 100 µL of oxidized BSA at 0.05% dissolved in
sodium bicarbonate buffer. After 3 washes with 150 µL of PBSTꢀ0.05%, sialylation step was realized
in 100 µL containing 40 µL of 40 mM cacodylate buffer (pH 6.4), 55 µL of medium containing
sialyltransferase and 5 µL of freshly prepared CMPꢀSiaNAl (to limit any degradation of the donor
substrate) at 2 mM. After 1 hour incubation at 37 °C, wells were washed with PBSTꢀ0,05 % and 100
µL of mix for CuAAC was added (300 µM of CuSO4, 600 µM of BTTAA, 2.5 mM of sodium
ascorbate (Sigma), 250 µM of azidoꢀbiotin completed with PBS). After 1 hour incubation at 37 °C,
washes were made with PBSTꢀ0.05 % and wells were incubated with 100 µL of antiꢀbiotin antibody
HRPꢀconjugated diluted to 1/25000 in PBSTꢀ0.05% for 1 hour at 37 °C. After washes, 100 µL of
TMB (3,3’,5,5’ꢀtetramethylbenzidine) was added and incubated 20 min at room temperature in dark.
Finally, absorbance was quantified at 620 nm using spectrophotometer (SpectroStar Nano; BMG
Labtech). Data were then analyzed in GraphPad Prism 5 using KruskalꢀWallis test statistic between
samples.
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AUTHOR INFORMATION
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Corresponding Author
*Email: yann.guerardel@univꢀlille.fr
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ORCID
Yann Guérardel: 0000ꢀ0003ꢀ4967ꢀ9512
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Anne HarduinꢀLepers: 0000ꢀ0002ꢀ1233ꢀ3799
Notes
The authors declare no conflicts of interest.
ACKNOWLEDGMENTS
We thank M. Decloquement for his contribution in the MPSA experiments. We are indebted to the
Research Federation FRABio (Univ. Lille, CNRS, FR 3688, FRABio, Biochimie Structurale et
Fonctionnelle des Assemblages Biomoléculaires) for providing the technical environment conducive
to achieve this works. These experiments were supported by grants from the Ministère de l’Éducation
Nationale et de l’Enseignement Supérieur to M.N. and P.A.G.
ABBREVIATIONS
BSA, Bovine Serum Albumin; CMP, Cytidine monoꢀphosphate; CMPꢀSia, Cytidineꢀ5’ꢀ
monophosphateꢀN-acetylneuraminic acid; CuAAC, Copper Catalyzed Alkyne Azide Cycloaddition:
EDTA, ethylenediaminetetraacetic acid; ESI, electrospray ionization; FRET, Forster resonance energy
transfer; Gal, Galactose; GalNAc, Nꢀacetyl galactosamine; HEK, Human embryonic kidney; HPLC,
High performance liquid chromatography; HRP, Horse radish peroxidase; LCꢀMS, Liquid
chromatography combined to massꢀspectroscopy; MPSA, Microplate sialylation assay; MRM:
Multiple reaction monitoring; PGM, Porcine gastric mucin; Sia, sialic acid; SPAAC, Strain promoted
azide alkyne cycloaddition; ST, sialyltransferase; TFA, trifluoroacetic acid; TMB, 3,3′,5,5′ꢀ
Tetramethylbenzidine.
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Figure 1: (A) Chemical structure of the alkynyl analog of CMPꢀNeu5Ac: CMPꢀSiaNAl. (B) MPSA experiment
after incubation with either ST6Gal I or mock medium and various concentrations of CMPꢀSiaNAl (0ꢀ100
µM). All the wells have been treated with azidoꢀPEGꢀbiotin with CuAAC conditions and finally revealed with
an antiꢀbiotin antibody HRP. Absorbance measured at 620 nm for all CMPꢀSiaNAl concentrations. Error bars
represent SEM (n = 2).
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Figure 2. Determination of kinetic parameters with MPSA. Otherwise specified, 100 µM of CMPꢀSiaNAl and
400 ng of asialofetuin were incubated for 1 h with either ∆56ST6Gal I and ∆56ST3Gal I. (A) Activity in
function of the dilution factor of the enzymatic source (B) Reaction time. MPSA absorbance readout for both
ST6Gal I and ST3Gal I in function of time. (C) Km determination of both ∆56ST6Gal I and ∆56ST3Gal I
toward CMPꢀSiaNAl (0 ꢀ 100 µM) (D) and coated asialofetuin (0 – 400 ng). Error bars represent SEM, (n =
2).
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Figure 3. Preference of ∆56ST6Gal I and ∆56ST3Gal I toward different acceptor glycoproteins. Either mock,
∆56ST6Gal I or ∆56ST3Gal I were incubated with 100 µM of CMPꢀSiaNAl for 1 hour with 400 ng of the
indicated coated protein. Error bars represent SEM, (n = 4).
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Figure 4. Inhibition effects on ∆56ST6Gal I and ∆56ST3Gal I activities. (A) Relative activity of ∆56ST6Gal I
(left) and ∆56ST3Gal I (right) in presence of 10 mM of CMP, EDTA, MgCl2, CaCl2, MnCl2, CoCl2, ZnCl2.
Cacodylate buffer (10 mM) was used as control and set at 100 %. Enzymes were incubated for 1 h with
CMPꢀSiaNAl (100 µM) and asialofetuin (400 ng). Error bars represent SEM, (n = 3), pꢀvalue = 0.0001. (B)
Doseꢀeffect relationship of CoCl2 (left) and ZnCl2 (right) on ∆56ST6Gal I and ∆56ST3Gal I activity. Enzymes
were incubated for 1 h with CMPꢀSiaNAl (100 µM) and asialofetuin (400 ng) in presence of either CoCl2 or
ZnCl2 (1 µM – 10 mM). Error bars represent SEM, (n = 6).
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