A glyco-gold nanoparticle based assay for α-2,8-polysialyltransferase from Neisseria meningitidis

Article abstract of DOI:10.1039/c3cc45147j

We designed a novel strategy for sensitively detecting the activity of α-2,8-polysialyltransferase (PST) by a combination of ganglioside GD3 functionalized gold nanoparticles and inactive endosialidase. We anticipate that this new method will facilitate the search for PST inhibitors as well as for improved mutant forms of PST in directed evolution experiments. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc45147j

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A glyco-gold nanoparticle based assay for
a-2,8-polysialyltransferase from Neisseria meningitidis†
Ching-Ching Yu,a Li-De Huang,a David H. Kwan,b Warren W. Wakarchuk,c
Stephen G. Withers*b and Chun-Cheng Lin*a
Cite this: Chem. Commun., 2013,
49, 10166
Received 9th July 2013,
Accepted 3rd September 2013
DOI: 10.1039/c3cc45147j
www.rsc.org/chemcomm
We designed a novel strategy for sensitively detecting the activity impede the development of enzymatic PSA synthesis. A key factor
of a-2,8-polysialyltransferase (PST) by a combination of ganglioside limiting the engineering of PSTs is the lack of activity-based, high-
GD3 functionalized gold nanoparticles and inactive endosialidase. throughput screening methodologies for PST activity.
We anticipate that this new method will facilitate the search for PST
The requirement for an oligosialyl acceptor (primer) and the
inhibitors as well as for improved mutant forms of PST in directed occurrence of multiple additions render high-throughput assay
evolution experiments.
development for PSTs considerably more challenging than is the
case for mono-sialyltransferases (STs).5 The most widely used PST
Polysialic acid (PSA) is a highly hydrophilic and negatively charged assays are the radioactivity-based assay using isotopically-labeled
biopolymer that is found in bacteria and mammals.1 In humans, CMP-sialic acid as a donor and the CE or HPLC-based assay with
PSA is found predominantly on neural cell adhesion molecules fluorescence detection in which a synthetic fluorescent di-sialyl-
(NCAMs) where it plays a role in modulating cellular interactions lactose (GD3 analogue) or directly derivatized tri-sialic acid is used
during migration and in promoting the plasticity of neuronal cells as an acceptor.6 While the latter method has provided sensitive
in early brain development, as well as being involved in cancer and accurate quantification of PST activity, as well as mechanistic
metastasis.2 In bacteria, PSA is a capsular virulence factor and plays insights into PST-catalyzed polymerization, it is instrument-
a role in the evasion of phagocytosis and in immune clearance by intensive, requires tedious sample pre-treatment, and is not a real-
the human host. In addition, since bacterial and human PSAs are time methodology, thus is not readily adapted to high-throughput
identical, PSA has very low immunogenicity and thus has great screening. Due to the increasing demand for high throughput
potential for use in a range of therapeutic applications such as the screens, a simple, sensitive, and homogeneous method for detec-
elongation of circulatory lifetimes of therapeutic proteins.3
The enzymes responsible for biosynthesis of PSA are known as washing and pretreatment steps is urgently needed.
polysialyltransferases (PSTs). Both vertebrate and bacterial PSTs4
Recently, nanoparticles (NPs) have emerged as promising new
tion of the presence of PSA activity without a need for elaborate
are template-independent polymerases, which catalyze the succes- reagents for biological applications.7 The unique quantum proper-
sive addition of sialyl moieties from CMP-sialic acid onto the non- ties of these nanoscale materials offer excellent prospects for the
reducing end of a growing PSA chain. While a number of biological design of highly sensitive and innovative biosensing devices and
studies have been reported for PSTs, the catalytic mechanisms of machines.8 Amongst these, gold nanoparticles (AuNPs) have already
PSA products are poorly understood, and as yet no 3-D structure of found use as biosensors,9 and in nanomedicines,10 bioseparations,11
a PST has been determined, in large part due to the difficulties in and cell imaging.12 When used in conjunction with a microtiter
producing stable and soluble enzymes. The same problems further plate format, colorimetric assays based on the aggregation of AuNPs
represent simple but sensitive methods for the detection of bio-
molecules and for the evaluation of enzymatic activity.13 To the best
a Department of Chemistry, National Tsing Hua University, 101, Sec. 2,
of our knowledge, the use of AuNP-based aggregation to detect
Kuang Fu Rd., Hsinchu, 30013, Taiwan. E-mail: cclin66@mx.nthu.edu.tw;
enzymatic activity has focused mostly on hydrolytic activity.13c,d
Fax: +886 3 5711082; Tel: +886 3 5753147
b Centre for High-throughput Biology (CHiBi) and Department of Chemistry,
University of British Columbia, 2036 Main Mall, Vancouver,
British Columbia V6T 1Z1, Canada. E-mail: withers@chem.ubc.ca;
Fax: +1 604 822 8869; Tel: +1 604 822 3402
Herein, we report a novel biosensing strategy to detect bond
formation catalyzed by glycosyltransferases, and we exemplify
this in an assay for a-2,8-PST activity.
The flow chart of the developed assay is illustrated in Fig. 1.
AuNPs decorated with a ganglioside GD3 analog (1) were used as
a primer for PST-catalyzed polymerization, generating polysialyl
structures on the NP surface. Detection is based upon the use of
c Department of Chemistry and Biology, Ryerson University, 350 Victoria St.,
Toronto, Ontario M5B 2K3, Canada
† Electronic supplementary information (ESI) available: Experimental details and
compound characterization. See DOI: 10.1039/c3cc45147j
c
10166 Chem. Commun., 2013, 49, 10166--10168
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Fig. 3 (A) UV-visible spectra of GD3@AuNP solutions (a) before and (b) after
incubation with PST and CMP-sialic acid and (c), following addition of EndoNF-DM. (B)
Photographs of the visual detection for the EndoNF-DM-mediated assembly AuNP
for PST assay using lactose-, GM3- and GD3-AuNPs as substrates. aPST 30 mg mLÀ1
,
CMP-sialic acid 1 mM in HEPES 50 mM, pH 7.5, 20 mM MgCl2, 37 1C, 1 h; then
EndoNF-DM 3 mM, r.t, 30 min. (C) The absorption spectra of GD3@AuNP solutions
incubated with PST at different concentrations in E. coli cell lysate and CMP-sialic acid,
followed by addition of a fixed concentration of EndoNF-DM; the inset shows the
linear plot of the A525nm versus the PST concentration.
Fig. 1 Schematic representation of the biosensing strategy for a-2,8-PST activity
based on EndoNF-DM-mediated assembly of gold nanoparticles decorated with
acceptor oligosaccharides that were subjected to enzymatic polysialylation.
an inactive mutant of the polysialic acid specific trimeric phage
tailspike endosialidase from the E. coli phage K1F, EndoNF-DM,
which serves as a cross-linker.14 Upon addition of EndoNF-DM, the
AuNPs with oligo/polymer PSA on the surface undergo aggregation.
This triggers a network-like assembly of the PSA-functionalized
AuNPs, resulting in aggregation of PSA@AuNPs and a significant
decrease of intensity in the plasmon resonance absorption peak of
the AuNP solution. The simplicity of application of this method
offers great potential as a generic approach for activity screening
of a-2,8-PSTs and, when other ‘‘cross-linkers’’ are used, of
glycosynthases and other glycosyltransferases.
To prepare a suitable oligosaccharide ligand to serve as the
primer for a PST catalyzed reaction, a long alkane-thiol chain with a
hydrophilic oligomeric ethylene glycol linker was chosen as the
aglycone not only to facilitate the purification by reverse-phase
chromatography but also to establish sufficient steric hindrance to
prevent the aggregation of the resulting glyco-AuNPs. LacPEG6C10SH
2 was synthesized and then sialylated using a-2,3-ST Cst-I15 and
bifunctional a-2,3/2,8-ST Cst-II,16 respectively, to afford ganglioside
analogs GM3PEG6C10SH 3 and GD3PEG6C10SH 1, as shown in
Fig. 2 (for detailed procedures, see ESI†).
(Fig. 3A, line a), with similar absorption bands also observed in
the spectra of Lac@AuNP and GM3@AuNP (Fig. S1, ESI†). The
lack of an obvious red-shift (from 519 nm to 525 nm) of the
maximum absorption band after oligosaccharide modification
also indicated that these glyco-AuNPs do not self-aggregate in
aqueous media. The GD3@AuNPs are well-dispersed in aqueous
solution and are stable for at least one week, even in the high
ionic strength of 0.1 M sodium chloride solution (Fig. S1, ESI†).
The size of the metal core of these glyco-AuNPs is 13 Æ 1.2 nm.
The feasibility of such a AuNP-based assay for directly monitoring
PSA formation catalyzed by PST was tested with the Neisseria
meningitidis a-2,8-PST.3 Incubation of GD3@AuNPs (final
concentration of 5 nM based on AuNPs)18 with 300 nM PST
and 1 mM CMP-sialic acid in 50 mM HEPES buffer at pH 7.5,
containing 20 mM MgCl2, resulted in polysialylation of the NPs, but
no visible change in the solution colour or shift in the absorption
peak of AuNP, indicating minimal effects of the enzymatic poly-
sialylation reaction on the stability of the GD3@AuNPs (Fig. 3A,
line b). Addition of the ‘‘cross-linker’’ EndoNF-DM14,19 at a final
concentration of 3 mM resulted in the precipitation of PSA@AuNP
aggregates from the solution and consequently in visible fading of
the solution colour over 30 min, quantified as a decrease in the
absorbance. This was observed consistently upon aggregation of
AuNPs (Fig. 3A, line c). By contrast, no aggregation was observed
when GD3@AuNPs that had not been polysialylated were incubated
with EndoNF-DM. This lack of binding between di-sialic acid and
EndoNF-DM is consistent with reports that the EndoNF-DM binding
site accommodates at least five residues.19,20
The GD3-functionalized AuNPs (GD3@AuNPs) were synthesized
according to a previously reported procedure17 by using GD3 ligand
1. Similarly, Lac@AuNP and GM3@AuNP were prepared using
ligands 2 and 3, respectively. The UV-visible spectrum of GD3@AuNP
clearly shows the characteristic plasmon absorption band at 525 nm
Further control experiments were performed to verify that the
EndoNF-DM-triggered assembly of the GD3@AuNPs was mediated
by active enzymatic polysialylation of PST. In these control experi-
ments, either PST or CMP-sialic acid was omitted from the reaction
mixture, and indeed no aggregation or precipitation of nano-
particles causing a loss of the intense color in solution was
observed after addition of EndoNF-DM (Fig. S2, ESI†). In a further
Fig. 2 Oligosaccharide ligands for glyco-AuNPs assembly.
c
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control experiment, the ineffective ‘‘acceptors’’, Lac@AuNP and
We thank the National Tsing Hua University, Academia
GM3@AuNP, were employed in parallel reactions. As expected, no Sinica, the National Science Council, Taiwan, and the Canadian
significant loss of colour in solution due to aggregate precipitation Institutes for Health Research (CIHR) for support of this work.
was seen after addition of EndoNF-DM, indicating that, as D.H.K. thanks the CIHR, the Canadian Blood Services and the
expected, GD3 is the minimal acceptor required for polysialylation Michael Smith Foundation for Health Research for fellowship
by a-2,8-PST (Fig. 3B).
support.
It is interesting that the more typical colour change of
AuNPs from the initial pinkish-red colour of well-dispersed
AuNPs to the purple colour that is normally seen with aggregated
AuNPs was not observed. Such a colour change is attributed to
the change of the inter-particle distance induced by binding and
the change of the dielectric constant around the surrounding
medium or the shell of the AuNPs. Since PST catalyzes formation
of PSA with 4 to 70 sialic acid residues and the EndoNF-DM
binds a minimum of 5 to 8 sialic acid residues, it seems probable
that the inter-particle distance of the cross-linked AuNPs is not
short enough to induce strong coupling between the adjacent
nanoparticles. Instead, a decrease in absorbance of the solution
at 525 nm resulted from the precipitation of PSA@AuNP aggre-
gates, which was facilitated by centrifugation.
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PSA recognition by EndoNF-DM was examined, as shown in Fig. S3
(ESI†). A linear correlation between PSA@AuNP absorbance and
EndoNF-DM concentration was found. Optimum conditions for
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near-linear correlation between the enzyme concentration and the
absorbance (Fig. 3C, inset), up to a PST concentration of 1.5 mM.
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be achieved using this assay. Moreover, since GD3@AuNPs are
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in the presence of cell lysate without the concern of self-aggregation,
making directed evolution screening a reality.
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