Cloning and characterization of a viral α2-3-sialyltransferase (vST3Gal-I) for the synthesis of sialyl Lewisx

Article abstract of DOI:10.1093/glycob/cwq172

Sialyl Lewis^x (SLe^x, Siaα2-3Galβ1- 4(Fucα1-3)GlcNAcβOR) is an important sialic acid-containing carbohydrate epitope involved in many biological processes such as inflammation and cancer metastasis. In the biosynthetic process of SLe^x, α2-3-sialyltransferase-catalyzed sialylation generally proceeds prior to β1-3-fucosyltransferase-catalyzed fucosylation. For the chemoenzymatic synthesis of SLex containing different sialic acid forms, however, it would be more effi- cient if diverse sialic acid forms are transferred in the last step to the fucosylated substrate Lewis^x (Lex). An α2-3- sialyltransferase obtained from myxoma virus-infected European rabbit kidney RK13 cells (viral α2-3-sialyltransferase (vST3Gal-I)) was reported to be able to tolerate fucosylated substrate Lex. Nevertheless, the substrate specificity of the enzyme was only determined using partially purified protein from extracts of cells infected with myxoma virus. Herein we demonstrate that a previously reported multifunctional bacterial enzyme Pasteurella multocida sialyltransferase 1 (PmST1) can also use Le^x as an acceptor substrate, although at a much lower efficiency compared to nonfucosylated acceptor. In addition, N-terminal 30-aminoacid truncated vST3Gal-I has been successfully cloned and expressed in Escherichia coli Origami B(DE3) cells as a fusion protein with an N-terminal maltose binding protein (MBP) and a C-terminal His6-tag (MBP-δ30vST3Gal-IHis₆). The viral protein has been purified to homogeneity and characterized biochemically. The enzyme is active in a broad pH range varying from 5.0 to 9.0. It does not require a divalent metal for its α2-3-sialyltransferase activity. It has been used in one-pot multienzyme sialylation of Lex for the synthesis of SLex containing different sialic acid forms with good yields.

Full text of DOI:10.1093/glycob/cwq172

Glycobiology vol. 21 no. 3 pp. 387–396, 2011
doi:10.1093/glycob/cwq172
Advance Access publication on October 26, 2010
Cloning and characterization of a viral
α2–3-sialyltransferase (vST3Gal-I) for the synthesis
of sialyl Lewisx
Go Sugiarto, Kam Lau, Hai Yu, Stephanie Vuong,
Vireak Thon, Yanhong Li, Shengshu Huang,
and Xi Chen1
Keywords: cloning / sialic acid / Lewisx / sialyltransferase /
sialyl Lewisx
Department of Chemistry, University of California, One Shields Avenue,
Davis, CA 95616, USA
Introduction
Received on July 14, 2010; revised on September 23, 2010; accepted on
October 22, 2010
Sialic acids belong to an important family of negatively
charged monosaccharides. They have a common nine-carbon
backbone of a polyhydroxy α-keto acid nature and are usually
presented as the terminal residues on cell surface glycoconju-
gates of higher eukaryotes (Schauer 2000; Angata and Varki
2002; Chen and Varki 2010). The carboxyl group of sialic acids
is normally deprotonated under physiological pH (Vimr et al.
2004), which makes them bear a net negative charge that affects
their properties. Aside from their common features, sialic acids
are structurally diverse in nature with more than 50 different
sialic acid forms that have been identified. The diversity
includes modifications at the carbon 5 which can link to an
acetamido group providing N-acetylneuraminic acid (Neu5Ac),
the most abundant sialic acid broadly presented in humans,
animals, bacteria and protozoa. A hydroxyacetamido group on
carbon 5 leads to N-glycolylneuraminic acid (Neu5Gc), a
nonhuman sialic acid form, and a hydroxyl group at carbon 5
gives 2-keto-3-deoxy-nonulosonic acid (Kdn). Furthermore,
additional modifications include single or multiple acetylation
at hydroxyl groups at C4, C5, C7, C8 and/or C9, sulfation at
C8-OH, phosphorylation or lactylation at C9-OH and methyl-
ation at C8-OH, C5-OH (for Kdn), or N-glycolyl hydroxyl
group (for Neu5Gc) (Angata and Varki 2002; Schauer 2004;
Chen and Varki 2010). Owing to their negative charge, exposed
position and diversity, sialic acids play important roles in either
masking recognition sites or facilitating cell recognition and
adhesion. These biological processes are normally carried out
through carbohydrate and protein interactions. Both sialic acids
and underlying sugars are involved in determining the binding
characteristics of the interactions. Among sialic acid-containing
carbohydrates, sialyl Lewisx (SLex) Neu5Acα2–3Galβ1–4
(Fucα1–3)GlcNAcβOR is one of the most well-studied struc-
tures. SLex is involved in inflammation (Rosen 2004). Its inter-
action with E-, P-, and L-selectins (Lowe et al. 1990; Phillips
et al. 1990; Polley et al. 1991; Foxall et al. 1992; Lasky 1992)
facilitates the recruitment of leukocytes to inflammation sites.
SLex is also a well-known tumor-associated carbohydrate
antigen that is involved in adhesion and metastasis of cancer
cells (Takada et al. 1993; Ugorski and Laskowska 2002).
Sialyl Lewisx (SLex, Siaα2–3Galβ1–4(Fucα1–3)GlcNAcβOR)
is an important sialic acid-containing carbohydrate epitope
involved in many biological processes such as inflammation
and cancer metastasis. In the biosynthetic process of SLex,
α2–3-sialyltransferase-catalyzed sialylation generally pro-
ceeds prior to α1–3-fucosyltransferase-catalyzed fucosyla-
tion. For the chemoenzymatic synthesis of SLex containing
different sialic acid forms, however, it would be more effi-
cient if diverse sialic acid forms are transferred in the last
step to the fucosylated substrate Lewisx (Lex). An α2–3-sia-
lyltransferase obtained from myxoma virus-infected
European rabbit kidney RK13 cells (viral α2–3-sialyltrans-
ferase (vST3Gal-I)) was reported to be able to tolerate fuco-
sylated substrate Lex. Nevertheless, the substrate specificity
of the enzyme was only determined using partially purified
protein from extracts of cells infected with myxoma virus.
Herein we demonstrate that a previously reported multi-
functional bacterial enzyme Pasteurella multocida sialyl-
transferase 1 (PmST1) can also use Lex as an acceptor
substrate, although at a much lower efficiency compared to
nonfucosylated acceptor. In addition, N-terminal 30-amino-
acid truncated vST3Gal-I has been successfully cloned and
expressed in Escherichia coli Origami™ B(DE3) cells as a
fusion protein with an N-terminal maltose binding protein
(MBP) and a C-terminal His6-tag (MBP-Δ30vST3Gal-I-
His6). The viral protein has been purified to homogeneity
and characterized biochemically. The enzyme is active in a
broad pH range varying from 5.0 to 9.0. It does not require
a divalent metal for its α2–3-sialyltransferase activity. It has
been used in one-pot multienzyme sialylation of Lex for the
synthesis of SLex containing different sialic acid forms with
good yields.
1To whom correspondence should be addressed: Tel: +1-530-754-6037;
Fax: +1-530-752-8995; e-mail: chen@chem.ucdavis.edu
© The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
387

G Sugiarto et al.
Both chemical and enzymatic methods have been developed
to study the biological functions of SLex and its potential thera-
peutic applications such as cancer vaccine candidates.
Chemical sialylation in the synthesis of SLex is usually low
yielding and complicated by tedious protection and deprotec-
tion processes, steric hindrance of sialic acid for sialylation as
well as difficulties in choosing suitable protecting groups and
controlling stereospecificity (Boons and Demchenko 2000;
Halcomb and Chappell 2002; Muthana et al. 2009).
Chemically or enzymatically synthesized sialyloligosacchar-
ides have been used as building blocks to improve the synthetic
yields of SLex (Hayashi et al. 1996; Hanashima et al. 2007;
Cao et al. 2008). Glycosyltransferase-catalyzed enzymatic
methods have been developed with or without the regeneration
of sugar nucleotides (Ichikawa et al. 1992; DeFrees et al. 1993,
1995; Lin et al. 1995; Duffels et al. 2000; Koeller and Wong
2000; Huang et al. 2006; Soriano del Amo et al. 2010; Zhang
et al. 2010). Nevertheless, only Neu5Ac, the most abundant
sialic acid form, was the sialic acid form in previous SLex syn-
thetic targets.
In order to study the importance of naturally occurring sialic
acid forms in SLex, instead of following its natural biosynthetic
pathway in which sialylation takes place before fucosylation, a
more efficient enzymatic synthetic approach would be to carry
out the fucosylation before the final sialylation process to
introduce diverse sialic acid forms. Although most bacterial
α1–3-fucosyltransferases can tolerate both sialylated and non-
sialylated N-acetyllactosamine (LacNAc) as acceptors (Lin
et al. 2006; Soriano del Amo et al. 2010; Zhang et al. 2010),
α2–3-sialyltransferases are usually restricted to nonfucosylated
galactoside acceptors. Therefore, the key issue for efficient
chemoenzymatic synthesis of SLex-containing diverse sialic
acid forms is to find an α2–3-sialyltransferase that can tolerate
fucosylated galactoside Lex as acceptor substrate.
sialyltransferase 1 (PmST1 or tPm0188Ph; Yu et al. 2005; Ni
et al. 2006, 2007) was demonstrated and compared to the recom-
binant vST3Gal-I. Partially purified recombinant vST3Gal-I was
used in one-pot multienzyme sialylation of Lex for the synthesis
of SLex containing different sialic acid forms with good yields.
Results
Sequence comparison of vST3Gal-I and mammalian
ST3Gal-IVs
The amino-acid sequence of vST3Gal-I shares high identity
to mouse ST3Gal-IV (37%) (Sujino et al. 2000), human
ST3Gal-IV (38%) (Sasaki et al. 1993; Kitagawa and Paulson
1994), human ST3Gal-III (36%) (Kitagawa and Paulson
1993) and porcine ST3Gal-I (26%) whose X-ray crystal struc-
ture was recently reported (Rao et al. 2009). As shown in
Figure 1, vST3Gal-I has all four conserved sialyl motifs
including large (L), small (S), motif 3 and very small (VS)
motifs identified previously (Datta and Paulson 1995;
Geremia et al. 1997; Datta et al. 1998, 2001; Jeanneau et al.
2004). In addition, it has conserved amino-acid residues
(shown by asterisks in Figure 1) including a conserved cataly-
tic base H268 in sialyl motif VS identified in the porcine
ST3Gal-I X-ray crystal structures (Rao et al. 2009).
Cloning, expression and purification of recombinant protein
To obtain a soluble and active recombinant vST3Gal-I
in E. coli expression system, a truncated protein with the
deletion of an N-terminal cytoplasmic domain (1–6 aa) and
a noncleavable signal-transmembrane sequence (7–30 aa)
(Jackson et al. 1999) was cloned from a synthetic gene with
codons optimized for E. coli expression. As shown in
Figure 2, the codon-optimized Δ30vST3Gal-I gene contained
24% adenine, 27% cytosine, 25% guanine and 24% thymine
as compared to the original sequence containing 28%
adenine, 25% cytosine, 23% guanine and 24% thymine
(GenBank accession number U46578.1). The recombinant
protein was obtained as a fusion protein with an N-terminal
MBP and a C-His6-tag. The MBP tag was introduced by
using pMAL-c4X vector, while the C-His6-tag was introduced
by including the His6-tag codons in the 3′-primer used for
cloning. Optimal expression was achieved by incubating E.
coli Origami™ B(DE3) cells at 20°C for 24 h with vigorous
shaking (250 rpm) after the addition of isopropyl-1-thio-
β-D-galactopyranoside (IPTG) (0.5 mM) for induction.
Sodium dodecylsulfate–polyacrylamide gel electrophoresis
(SDS–PAGE) analysis (Figure 3) indicated that the recombi-
nant protein was overexpressed which constituted about 70%
of the total protein in the whole-cell extraction. Nevertheless,
only a small portion of the recombinant protein was seen in
the cell lysate, the soluble portion of the cell extraction.
Ni2+-NTA column purification of the fusion protein using
an ÄKTATM fast protein liquid chromatography (FPLC)
system yielded the purified protein, showing a molecular
weight of around 72 kDa in the SDS–PAGE. The molecular
weight was similar to that calculated (72.5 kDa) for the
MBP-Δ30vST3Gal-I-His6.
Among reported α2–3-sialyltransferases,
a viral α2–
3-sialyltransferase (vST3Gal-I) was shown to be able to toler-
ate fucosylated acceptors (Jackson et al. 1999; Sujino et al.
2000). The sialyltransferase was classified together with mam-
malian sialyltransferases in Carbohydrate-Active enZymes
(CAZy, http://www.cazy.org/) glycosyltransfersae family 29
(GT 29; Coutinho et al. 2003; Cantarel et al. 2009). This
vST3Gal-I, encoded by myxoma virus gene MST3N, was
obtained from RK13 cells (European rabbit kidney cells)
infected with myxoma virus (Jackson et al. 1999; Sujino et al.
2000). Partially purified extracts of the cells were used to test
the acceptor substrate specificity of vST3Gal-I. It was shown
that the enzyme sialylated both Lewisx and Lewisa with rela-
tive Vmax of 30 and 40%, respectively, compared to that of
LacNAc (Sujino et al. 2000). Nevertheless, the vST3Gal-I has
not been purified to homogeneity and hence the detailed kin-
etics data were not available.
Herein we report the cloning, expression (in Escherichia coli),
purification and characterization of an N-terminal truncated
vST3Gal-I as a fusion protein with an N-terminal maltose
binding protein (MBP) and a C-terminal His6-tag (MBP-
Δ30vST3Gal-I-His6). Its activities toward fucosylated LacNAc
(Lewisx) and nonfucosylated lactoside were compared. In
addition, the tolerance of fucosylated LacNAc as an acceptor by
a previously reported multifunctional Pasteurella multocida
388

Viral α2–3-sialyltransferase vST3Gal-I
Fig. 1. Alignment of vST3Gal-I, hST3Gal-IV (GenBank accession number NP_006269) and pST3Gal-I (GenBank accession number AAA31125.1).
pH profile of MBP-Δ30vST3Gal-I-His6
efficiency of PmST1 (kcat/Km = 23 mM−1 min−1) was actually 15
times higher than that of MBP-Δ30vST3Gal-I-His6 (kcat/Km =
1.5 mM−1 min−1). Nonetheless, this PmST1 activity with
LexβMU acceptor was 87-fold less efficient compared to the
PmST1 activity with LacβMU acceptor due to a much (about
13-fold) higher Km value and a much (7-fold) lower kcat value
for LexβMU.
High-performance liquid chromatography (HPLC)-based pH
profile study using a 4-methylumbelliferyl (MU)-labeled
lactose (LacβMU or Galβ1–4GlcβMU) as an acceptor sub-
strate demonstrated that MBP-Δ30vST3Gal-I-His6 was active
in a wide pH range varying from 5.5 to 8.5 with minimum
variation of activity (Figure 4). About 80% of the optimum
activity was observed at pH 5.0 and 9.0. The activity
decreased drastically when the pH was below 5.0 or above
9.0. Only about 20% of the optimum activity was observed at
pH 4.5 and 11.0.
Confirmation of the α2–3-sialyltransferase activity of
MBP-Δ30vST3Gal-I-His6 in using Lex acceptor by one-pot
two-enzyme synthesis of SLex Neu5Acα2–3Galβ1–4(Fucα1–
3)GlcNAcβProN3
In order to confirm that Lex was indeed a tolerable acceptor for
the α2–3-sialyltransferase activity of MBP-Δ30vST3Gal-I-His6,
a preparative-scale synthesis of SLex tetrasaccharide Neu5Acα2–
3Galβ1–4(Fucα1–3)GlcNAcβProN3 was carried out from Lex
trisaccharide Galβ1–4(Fucα1–3)GlcNAcβProN3, Neu5Ac and
cytidine 5′-triphosphate (CTP) using a one-pot two-enzyme
reaction (Figure 5) containing partially purified recombinant
sialyltransferase MBP-Δ30vST3Gal-I-His6 and a recombinant
Neisseria meningitidis CMP-sialic acid synthetase (NmCSS; Yu
et al. 2004). The presence of NmCSS allowed in situ synthesis
of CMP-Neu5Ac, the sugar nucleotide donor for MBP-
Δ30vST3Gal-I-His6, from inexpensive, commercially readily
available Neu5Ac and CTP. SLex Neu5Acα2–3Galβ1–4(Fucα1–
3)GlcNAcβProN3 was obtained in a yield of 61%. The product
was confirmed by nuclear magnetic resonance (NMR) and high-
resolution mass spectrometry (HRMS) studies. As shown in
Table II, comparing the 13C NMR chemical shifts of the purified
Kinetics
Both fluorophore-tagged lactoside (LacβMU) and Lewisx
(LexβMU) were tolerable acceptor substrates by the
MBP-Δ30vST3Gal-I-His6. As shown in Table I, LacβMU was a
preferred acceptor for MBP-Δ30vST3Gal-I-His6 compared to
LexβMU. Compared to LexβMU as an acceptor (kcat/Km = 1.5
mM−1 min−1), the catalytic efficiency of the recombinant viral
enzyme was 19 times greater when LacβMU was used as an
acceptor (kcat/Km = 28 mM−1 min−1). The difference was contrib-
uted by both a lower Km and a higher kcat value for LacβMU.
Nevertheless, when LacβMU was used as an acceptor, the α2–
3-sialyltransferase activity of MBP-Δ30vST3Gal-I-His6 was
much weaker (about 70-fold difference) than a previously
reported multifunctional P. multocida α2–3-sialyltransferase
(PmST1; Yu et al. 2005; Ni et al. 2006, 2007). Quite interest-
ingly, LexβMU was also a tolerable acceptor substrate for
PmST1. When LexβMU was used as an acceptor, the catalytic
389

G Sugiarto et al.
Fig. 2. Gene and protein sequences of the codon-optimized MBP-Δ30vST3Gal-I-His6. Only the C-terminal sequence of MBP is shown (in italics). The multiple
cloning sites of pMAL-c4X vector are underlined. The six histidine residues introduced at the C-terminus during cloning are in bold.
product and the starting trisaccharide Lex indicated a significant
downfield shift (72.58 ppm to 75.82 ppm) of the C-3 signal of
the galactose (Gal) residue in the product and a moderate upfield
shift (71.16 ppm to 69.43 ppm) of the neighboring C-4 signal in
the Gal. This confirmed that the sialylation took place at the C-3
of the Gal in Lex. High resolution mass spectrometry electro-
spray ionization (HRMS ESI) spectrum obtained showed the
desired m/z of 926.3309 for C34H57N5O23Na (M+Na) (calcu-
lated: 926.3342).
One-pot three-enzyme synthesis of SLex analogs containing
different sialic acid forms
To demonstrate the application of MBP-Δ30vST3Gal-I-His6 in
enzymatic synthesis of SLex containing different sialic acid
forms from Lex, partially purified MBP-Δ30vST3Gal-I-His6
was used with NmCSS (Yu et al. 2004) and a recombinant P.
multocida sialic acid aldolase (Li et al. 2008) in a one-pot three-
enzyme system (Yu et al. 2005, 2006) for the preparative-scale
synthesis of SLex tetrasaccharides Kdnα2–3Galβ1–4(Fucα1–3)
390

Viral α2–3-sialyltransferase vST3Gal-I
Table I. Apparent kinetic parameters of recombinant MBP-Δ30vST3Gal-I-His6
and PmST1
Enzyme
Substrate
Km
(mM)
kcat (min−1
)
kcat/Km
(mM−1
min−1
)
MBP-Δ30vST3Gal-I-His6 CMP-Neu5Ac 1.4
0.2
1.2
0.1
3.3
0.5
32
33
2
1
23
LacβMU
LexβMU
28
4.7 0.2
1.5
PmST1
CMP-Neu5Aca 0.44 1.9 × 103
4.4 × 103
2.0 × 103
LacβMUa
LexβMU
1.4
2.8 × 103
Fig. 3. SDS–PAGE analysis for MBP-Δ30vST3Gal-I expression and
purification. Lanes: 1, protein standard; 2, whole-cell extraction before
induction; 3, whole-cell extraction after induction; 4, cell lysates after
induction; 5, Ni-column purified fraction.
18 2 (4.0 0.2) × 102 23
aYu et al. (2005).
C32H54N4O23Na (M+Na) (calculated: 885.3077) and 967.396
for Neu5AcN3-containing SLex C34H56N8O23Na (calculated:
967.3356).
Discussion
Synthesizing SLex with different sialic acid forms is crucial to
understand the importance of structural diversity of naturally
occurring sialic acids and to generate nonnatural carbohydrate
probes. Variation of sialic acid structures, especially at the C-5
position, has been demonstrated to enhance the immunogeni-
city of some carbohydrate vaccines (Livingston 1995; Liu
et al. 2000; Lemieux and Bertozzi 2001; Krug et al. 2004;
Zou et al. 2004; Pan et al. 2005). We demonstrate here that
SLex oligosaccharides containing natural and nonnatural sialic
acid forms can be directly synthesized by enzyme-catalyzed
sialylation of fucose-containing acceptor Lex. Although viral
vST3Gal-I and bacterial PmST1 do not share significant
protein sequence homology and belong to different CAZy
glycosyltransferase families (GT 29 and GT 80, respectively),
both have α2–3-sialyltransferase activity and can tolerate non-
fucosylated and fucosylated galactosides as acceptors.
Nonetheless, their activities toward fucosylated acceptors are
much lower than the nonfucosylated acceptor substrates. We
have successfully demonstrated here that the flexible acceptor
substrate specificity of vST3Gal-I can be applied in a pre-
viously described one-pot three-enzyme sialylation system
containing a sialic acid aldolase, a CMP-sialic acid synthetase
and a suitable sialyltransferase (Yu et al. 2005, 2006) to intro-
duce different sialic acid structures onto Lex to generate
structurally diverse SLex and analogs. In comparison, the
application of PmST1 in sialylation of LexβMU for the for-
mation of Neu5Acα2–3LexβMU was unsuccessful as less
than 5% yields were observed by HPLC assays due to
its strong donor hydrolysis activity (data not shown).
Nevertheless, the relatively low expression level of soluble
and active vST3Gal-I hampers its application in large-scale
synthesis of SLex. Due to the high expression level and high
activity of PmST1, generating PmST1 mutants with increased
activity toward fucosylated acceptors may be a preferred
approach to achieve efficient synthesis of SLex with diverse
sialic acid forms.
Fig. 4. The pH profile of MBP-D30vST3Gal-I-His6. Activity was measured at
indicated pH at 37°C for 30 min. Buffers (200 mM) used: sodium acetate (pH
4.5), MES (pH 5.0–6.5), HEPES (pH 7.0–7.5), Tris–HCl (pH 8.0–9.0), 3-
(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (pH 9.5–11.0).
GlcNAcβProN3 and Neu5AcN3α2–3Galβ1–4(Fucα1–3)GlcNAc
βProN3 from mannose and N-azidoacetylmannosamine
(ManNAcN3; Yu et al. 2005), respectively, in the presence of
Lex, sodium pyruvate, CTP and MgCl2. In this system, aldolase
catalyzed the formation of Kdn and N-azidoacetylneuraminic
acid (Neu5AcN3) from mannose and ManNAcN3 in the presence
of an excess amount of pyruvate. These sialic acids were con-
verted into their activated CMP-sialic acid by NmCSS which
were used by the viral sialyltransferase to transfer sialic acids
to Lex for the formation of SLex containing different sialic
acid forms. SLex tetrasaccharides Kdnα2–3Galβ1–4(Fucα1–3)
GlcNAcβProN3 and Neu5AcN3α2–3Galβ1–4(Fucα1–3)GlcNAc
βProN3 were formed with 64% and 58% yields, respectively.
The products were confirmed by NMR as shown in Table II and
HRMS studies. Similar to that observed for Neu5Acα2–
3Galβ1–4(Fucα1–3)GlcNAcβProN3, a significant downfield
shift of the C-3 signal of the Gal residue in the product and a
moderate upfield shift of the neighboring C-4 signal in the Gal
were observed in the 13C NMR spectra of both products when
compared to that of Lex starting material, indicating the for-
mation of a 2–3-sialyl linkage. HRMS (ESI) spectrum obtained
showed the desired m/z of 885.3103 for Kdn-containing SLex
391

G Sugiarto et al.
Fig. 5. Schematic illustration for the one-pot two-enzyme preparative scale synthesis of sialyl LexβProN3 Neu5Acα2–3Galβ1–4(Fucα1–3)GlcNAcβProN3 from
Neu5Ac, CTP and LexβProN3 Galβ1–4(Fucα1–3)GlcNAcβProN3. Enzymes used: MBP-Δ30vST3Gal-I-His6 and a recombinant NmCSS.
Compared to the common biosynthetic route for the for-
mation of SLex structure with α2–3-sialylation followed by
α1–3-fucosylation, enzymatic synthesis with an alternative
glycosylation sequence of α1–3-fucosylation followed by α2–
3-sialylation has a great advantage for introducing different
sialic acid forms in the last step. Obtaining a sialyltransferase
that can tolerate fucosylated acceptor substrate (e.g. Lex),
such as the recombinant vST3Gal-I described here,
provides an efficient catalyst to do so. With the availability of
MBP-Δ30vST3Gal-I-His6 by expression in E. coli system,
SLex with diverse naturally occurring and nonnatural sialic
acid forms can be obtained from a common acceptor substrate
Lex and a sialic acid precursor such as mannose, ManNAc or
their derivatives, using an efficient one-pot three-enzyme
system containing the sialyltransferase, a sialic acid aldolase
and a CMP-sialic acid synthetase.
(Valencia, CA). Herculase-enhanced DNA polymerase was
from Stratagene (La Jolla, CA). T4 DNA ligase and 1 kb DNA
ladder were from Promega (Madison, WI). Bicinchoninic acid
(BCA) Protein Assay Kit was from Pierce Biotechnology, Inc.
(Rockford, IL). CTP, D-N-acetylmannosamine (ManNAc) and
sodium pyruvate were purchased from Sigma (St. Louis, MO).
Full-length vST3Gal-I synthetic gene with the codons opti-
mized for E. coli expression system was custom-synthesized
by Codon Devices, Inc. (Cambridge, MA).
Synthesis of CMP-Neu5Ac
CMP-Neu5Ac was synthesized enzymatically from ManNAc,
sodium pyruvate and CTP by a one-pot two-enzyme system
using a recombinant sialic acid aldolase cloned from E. coli
K12 and a recombinant NmCSS as described previously (Yu
et al. 2004).
vST3Gal-I has high sequence homology with mouse
ST3Gal-IV (Kono et al. 1997) and human ST3Gal-IV
(Sasaki et al. 1993; Kitagawa and Paulson 1994). Similar
to human placenta hST3Gal-IV expressed in COS-1 cells
which showed marginal activity toward Lex with the rela-
tive rate of the reaction 33-fold less than that of type-II
acceptor (Kitagawa and Paulson 1994), the recombinant
MBP-Δ30vST3Gal-I-His6 shows a 19-fold less efficiency in
its sialyltransferase activity toward Lex acceptor (LexβMU)
compared to an acceptor (LacβMU) closely resembling
type-II glycan.
As shown in Figures 1 and 2, vST3Gal-I has several poten-
tial N-linked glycosylation sites at 34, 45, 95, 147 and 283
(Jackson et al. 1999) with conserved NxS/T where x is any
amino acid other than a proline. We demonstrate in this report
that similar to pST3Gal-I (Rao et al. 2009), glycosylation of
vST3Gal-I is not essential for its activity in vitro.
Synthesis of 4-methylumbelliferyl β-D-lactoside (LacβMU)
and Galβ1–4GlcNAcβProN3 (LacNAcβProN3)
LacβMU was synthesized from lactose through
hepta-O-acetyllactosyl trichloroacetimidate intermediate as
reported by Yu et al. (2005). The synthesis of LacNAcβProN3
was carried out as described previously (Chokhawala et al.
2008).
a
Synthesis of LexβProN3 and LexβMU as the acceptors for
MBP-Δ30vST3Gal-I-His6
Both LexβProN3 and LexβMU were synthesized from the
corresponding LacNAc derivatives LacNAcβProN3 and
LacNAcβMU using a one-pot two-enzyme system containing
a
recombinant N-His6-tagged C-terminal truncated
Helicobacter pylori α1–3-fucosyltransferase (Hp1-3FTΔ66;
Sun et al. 2007) and a recombinant bifunctional L-fucokinase/
GDP-fucose pyrophosphorylase (FKP) from Bacteroides
fragilis (Yi et al. 2009). To synthesize LewisxβProN3,
LacNAcβProN3 (47 mg, 0.10 mmol), L-fucose (25 mg, 0.15
mmol), adenosine 5'-triphosphate (ATP) (84 mg, 0.15 mmol),
guanosine 5'-triphosphate (GTP) (79 mg, 0.15 mmol) and
MgCl2 (41 mg, 0.20 mmol) were dissolved in H2O (5 mL). A
stock solution of Tris–HCl buffer (1 M, pH 7.5, 1 mL) was
added. After the addition of a recombinant FKP (3.4 mg)
which catalyzes the synthesis of GDP–fucose from L-fucose,
ATP and GTP via a fucose-1-phosphate intermediate and
Materials and methods
Bacterial strains, plasmids and materials
E. coli electrocompetent cells DH5α were from Invitrogen
(Carlsbad, CA) and chemically competent Origami™ B(DE3)
from Novagen (Madison, WI). EcoRI and SalI restriction
enzymes as well as vector plasmid pMAL-c4X were purchased
from New England BioLabs (Beverly, MA). Ni2+-NTA agarose
(nickel-nitrilotriacetic acid agarose), QIAprep Spin Miniprep
Kit and QIAquick Gel Extraction Kit were from Qiagen
392

Viral α2–3-sialyltransferase vST3Gal-I
Table II. 13C NMR chemical shift assignments of LexβProN3 Galβ1–4(Fucα1–3)GlcNAcβProN3 and SLexβProN3 including Neu5Acα2–3Galβ1–4(Fucα1–3)
GlcNAcβProN3, Kdnα2–3Galβ1–4(Fucα1–3)GlcNAcβProN3 and Neu5AcN3α2–3Galβ1–4(Fucα1–3)GlcNAcβProN3
Residue
Carbon atom
Chemical shift (ppm)
βDGlcNAc
C
1
2
3
4
5
6
C=O
CH3
1
2
3
4
5
6
1
2
3
4
5
CH3
1
2
3
4
5
6
7
8
9
C=O
CH3
LexβProN3
101.09
55.94
75.05
75.47
73.49
59.88
174.40
22.36
101.96
71.16
72.58
68.47
75.10
61.63
98.78
67.82
69.33
72.03
66.85
15.43
Neu5Acα2–3LexβProN3
Kdnα2–3LexβProN3
101.09
55.91
75.02
75.35
73.43
59.73
174.17
22.31
101.72
69.34
75.70
69.26
74.93
61.60
98.74
67.79
69.84
71.99
Neu5AcN3α2–3LexβProN3
101.15
55.99
75.07
75.43
73.52
59.83
174.41
22.40
101.79
69.43
75.82
68.48
74.99
61.65
98.77
67.88
69.36
72.03
66.85
15.44
174.05
99.84
39.95
67.48
51.87
73.08
68.29
72.07
62.78
175.20
22.21
101.17
55.98
75.08
75.40
73.47
59.79
174.43
22.38
101.75
69.43
75.79
68.35
74.99
61.67
98.81
67.86
69.33
72.06
66.86
15.44
βDGal(1–4)
αLFuc(1–3)
66.79
15.37
αDNeu5Ac(2–3)
αDKdn(2–3)
1
2
3
4
5
6
7
8
174.36
99.76
39.51
67.28
70.32
74.04
67.28
72.26
62.73
9
αDNeu5AcN3(2–3)
1
2
3
4
5
6
7
8
174.09
99.82
39.97
67.44
51.92
72.74
68.19
72.10
62.72
171.36
52.06
67.35
28.28
47.91
9
C=O
NHCOCH2N3
OCH2CH2CH2N3
OCH2CH2CH2N3
OCH2CH2CH2N3
ProN3
67.32
28.24
47.88
67.37
28.28
47.94
67.36
28.21
47.84
Hp1–3FTΔ66 (2.2 mg), water was added to bring the volume
of the reaction mixture to 10 mL. The reaction mixture was
then incubated in an incubator shaker at 37°C for 48 h. The
reaction was stopped by adding cold EtOH (10 mL). The
mixture was incubated on ice for 30 min and was centrifuged
to remove precipitates. The supernatant was concentrated by
rotavaporation. A Bio-Gel P-2 filtration column and a silica
gel column (eluted with EtOAc:MeOH:H2O = 7:2:1, v/v/v)
were used to purify the product LexβProN3 Galβ1–4(Fuc1–3)
GlcNAcβProN3 (40 mg, 65% yield). The LexβMU Galβ1–4
(Fucα1–3)GlcNAcβMU was synthesized similarly with a 59%
yield.
Cloning
To clone MBP-Δ30vST3Gal-I-His6 in pMAL-c4X vector, the
forward primer used was 5′-CCGGAATTCGGTACGAACAA
CGTTTCAG-3′ (EcoRI restriction site is underlined) and the
393

G Sugiarto et al.
reverse primer used was 5′-ACGCGTCGACTTAGTGGTGG
TGGTGGTGGTGGCGGATACACAGAATG-3′ (SalI restriction
site is underlined and the sequence encoding the hexahistidine
tag is italicized). Polymerase chain reactions (PCRs) for ampli-
fying the target gene were performed in a 50 μL of reaction
mixture containing plasmid DNA (10 ng), forward and reverse
primers (0.2 μM each), 10× Herculase buffer (5 μL), dNTP
mixture (0.2 mM) and 5 U (1 μL) of Herculase-enhanced DNA
polymerase. The reaction mixture was subjected to 30 cycles
of amplification at an annealing temperature of 53°C. The
resulted PCR product was purified and double-digested with
EcoRI and SalI restriction enzymes. The digested PCR product
was purified, ligated with the predigested pMAL-c4X vector
and transformed into electrocompetent E. coli DH5α cells.
Selected clones were grown for minipreps and characterization
by restriction mapping. DNA sequencing was performed by
the Davis Sequencing Facility in the University of
California-Davis.
protein standard. The absorbance of each sample was
measured at 562 nm by a BioTek Synergy™ HT Multi-Mode
Microplate Reader.
Sodium dodecylsulfate–polyacrylamide gel electrophoresis
SDS–PAGE was performed in a 12% Tris–glycine gel using a
Bio-Rad Mini-protein III Cell Gel Electrophoresis Unit
(Bio-Rad) at a DC voltage of 120 V. Bio-Rad Precision Plus
Protein Standards (10–250 kDa) were used as molecular
weight standards. Gels were stained with Coomassie Blue.
pH profile by HPLC
Assays were performed in a total volume of 10 μL in a buffer
(200 mM) with pH varying from 4.5 to 11.0 containing
CMP-Neu5Ac
(1 mM),
LacβMU
(1 mM)
and
MBP-Δ30vST3Gal-I-His6 (0.3 μg). The buffers used were:
sodium acetate, pH 4.5; MES, pH 5.0–6.5; HEPES, pH 7.0–
7.5; Tris–HCl, pH 8.0–9.0; 3-(cyclohexylamino)-2-hydroxy-1-
propanesulfonic acid, pH 9.5–11.0. Reactions were allowed to
proceed at 37°C for 30 min before being quenched by adding
10 μL of ethanol. An aliquot (2 μL) of the mixture was then
added to 138 μL of 25% acetonitrile to make 70-fold
dilutions. The samples were then kept on ice until aliquots of
8 μL were injected and analyzed by a Shimadzu LC-6AD
system equipped with a membrane on-line degasser, a temp-
erature control unit and a fluorescence detector (Shimadzu
RF-10AXL). A reversed-phase Premier C18 column (250 ×
4.6 mm i.d., 5 μm particle size, Shimadzu) protected with a
C18 guard column cartridge was used. The mobile phase was
25% acetonitrile. The fluorescent compounds LacβMU and
Neu5Acα2–3LacβMU were detected by excitation at 325 nm
and emission at 372 nm (Yu et al. 2005). All assays were
carried out in duplicates.
Expression
The plasmids of positive clones were selected and trans-
formed into E. coli Origami™ B(DE3) chemically competent
cells. The plasmid-bearing E. coli strain was cultured in an
LB-rich medium (10 g L−1 tryptone, 5 g L−1 yeast extract and
10 g L–1 NaCl) supplemented with ampicillin (100 μg mL–1).
Overexpression of the target protein was achieved by inducing
the E. coli culture with 0.5 mM IPTG when the OD600 nm of
the culture reached 1.0 followed by incubating the induced
culture at 20°C for 24 h with vigorous shaking at 250 rpm in
a C25KC incubator shaker (New Brunswick Scientific,
Edison, NJ).
Purification
His6-tagged target protein was purified from cell lysates. To
obtain the cell lysate, cell pellets harvested by centrifugation
at 3452 × g for 2 h were resuspended in lysis buffer (pH 8.0,
100 mM Tris–HCl containing 0.1% Triton X-100) (20 mL
for cells obtained from 1 L of cell culture). Lysozyme (50 μg
mL–1) and DNaseI (3 μg mL–1) were then added and the
mixture was incubated at 37°C for 60 min with vigorous
shaking. Cell lysates were obtained as the supernatant after
centrifugation at 11,000 rpm for 20 min. Purification of
His6-tagged proteins from the lysate was achieved using an
AKTA FPLC system (GE Healthcare) equipped with a
HisTrap™ FF 5 mL column. The column was pre-equilibrated
with 10 column volumes of binding buffer (5 mM imidazole,
0.5 M NaCl, 50 mM Tris–HCl, pH 7.5) before the lysate was
loaded. After washing with 10 column volumes of binding
buffer, stepwise gradient elution was carried out sequentially
with 10 column volumes of elute buffers containing 34.25,
102.5 and 200 mM imidazole, respectively, in Tris–HCl
buffer (50 mM, pH 7.5, 0.5 M NaCl). The fractions contain-
ing the purified enzyme were collected and stored at 4°C.
Expression and purification of the recombinant PmST1
The recombinant PmST1 was expressed as a His6-tag protein
and purified using an ÄKTATM FPLC system (GE Healthcare)
equipped with a HisTrap™ FF 5 mL column as previously
described (Yu et al. 2005).
Kinetics by HPLC assay
The enzymatic assays were carried out in a total volume of
10 μL in a Tris–HCl buffer (200 mM, pH 8.0) containing
CMP-Neu5Ac, acceptor substrates (LacβMU or LexβMU) and
the recombinant proteins (MBP-Δ30vST3Gal-I-His6 or
PmST1). All reactions for the MBP-Δ30vST3Gal-I-His6 were
allowed to proceed at 37°C for 60 min. The PmST1 reactions
with LexβMU acceptor were allowed to proceed for 7 min at
37°C. Apparent kinetic parameters of CMP-Neu5Ac for
MBP-Δ30vST3Gal-I-His6 (0.2 μg was used) were obtained by
varying the CMP-Neu5Ac concentrations (0.1, 0.2, 0.4, 1.0,
2.0 and 4.0 mM), while the concentration of LacβMU was
fixed at 1 mM. Kinetic parameters of LacβMU (concentrations
used were 0.1, 0.2, 0.4, 1.0, 2.0, 4.0, 6.0 and 8.0 mM) and
LexβMU (concentrations used were 0.1, 0.2, 0.4, 1.0, 2.0, 4.0,
8.0 and 14.0 mM) for MBP-Δ30vST3Gal-I-His6 (0.3 and
0.4 μg were used for determining the parameters for LacβMU
and LexβMU, respectively) were obtained with a fixed
Quantification of purified protein
The concentration of purified enzyme was obtained in a
96-well plate using a BCA Protein Assay Kit (Pierce
Biotechnology, Rockford, IL) with bovine serum albumin as a
394

Viral α2–3-sialyltransferase vST3Gal-I
concentration of CMP-Neu5Ac (1 mM). The same concen-
tration of CMP-Neu5Ac (1 mM) was used for the kinetic
study of PmST1 (40 ng was used) on LexβMU (concentrations
used were 1.0, 5.0, 10.0, 15.0, 25.0 and 35.0 mM). Apparent
kinetic parameters were obtained by fitting the experimental
data (the average values of duplicate assay results) into the
Michaelis–Menten equation using Grafit 5.0.
Funding
This work was supported by NIH grant R01GM076360. X.C.
is an Alfred P. Sloan Research Fellow, a Camille Dreyfus
Teacher-Scholar and a UC-Davis Chancellor’s Fellow.
Conflict of interest statement
One-pot two-enzyme synthesis of Neu5Acα2–3LexβProN3
from LexβProN3 using MBP-Δ30vST3Gal-I-His6
None declared.
For the synthesis of Neu5Acα2–3LexβProN3, LexβProN3 (21
mg, 0.03 mmol), Neu5Ac (16 mg, 0.05 mmol), CTP (29 mg,
0.05 mmol) and MgCl2 (22 mg, 0.11 mmol) were dissolved in
H2O (2.5 mL). A stock solution of Tris–HCl buffer (1 M, pH
8.5, 0.5 mL) was added. After the addition of a recombinant
N. meningitidis CMP–sialic acid synthetase (1.1 mg) (Yu
Abbreviations
BCA, bicinchoninic acid; CAZy, Carbohydrate-Active
enZymes; CMP, cytidine 5′-monophosphate; CTP, cytidine
5′-triphosphate; Hp1–3FTΔ66, Helicobacter pylori α1–3-fuco-
syltransferase; FKP, bifunctional L-fucokinase/GDP-fucose pyr-
ophosphorylase; FPLC, fast protein liquid chromatography;
Gal, galactose; HPLC, high-performance liquid chromato-
graphy; HRMS, high-resolution mass spectrometry; IPTG,
isopropyl-1-thio-β-D-galactopyranoside; Kdn, 2-keto-3-deoxy-
nonulosonic acid; LacβMU, 4-methylumbelliferyl-β-D-lactoside;
LexβMU, 4-methylumbelliferyl-β-D-Lewisx; ManNAc, D-N-
acetylmannosamine; ManNAcN3, N-azidoacetylmannosamine;
MBP, maltose binding protein; MES, 2-(N-morpholino)
ethanesulfonic acid; MU, 4-methylumbelliferyl; Neu5Ac,
N-acetylneuraminic acid; Neu5AcN3, N-azidoacetylneuraminic
acid; Neu5Gc, N-glycolylneuraminic acid; NmCSS, Neisseria
meningitidis CMP-sialic acid synthetase; NMR, nuclear
magnetic resonance; PCR, polymerase chain reactions; PmST1,
Pasteurella multocida sialyltransferase 1; SDS–PAGE, sodium
dodecylsulfate–polyacrylamide gel electrophoresis; SLex, sialyl
Lewisx; vST3Gal-I, viral α2–3-sialyltransferase.
et
al.
2004)
and
Ni2+-NTA
column
purified
MBP-Δ30vST3Gal-I-His6 (262 μg, estimated), water was
added to bring the volume of the reaction mixture to 5 mL.
The reaction was carried out by incubating the solution in an
incubator shaker at 37°C for overnight. The reaction was
stopped by adding cold EtOH (5 mL) followed by incubation
on ice for 30 min. The mixture was then centrifuged to
remove precipitates. The supernatant was concentrated by
rotavaporation. A BioGel P-2 filtration column and a silica
gel column (eluted with EtOAc:MeOH:H2O = 5:2:1, v/v/v)
were used to purify the product SLexβProN3Neu5Acα2–
3Galβ1–4(Fucα1–3)GlcNAcβProN3 (19 mg, 61% yield).
One-pot three-enzyme synthesis of Kdnα2–3LexβProN3 and
Neu5AcN3α2–3LexβProN3 from LexβProN3 using
MBP-Δ30vST3Gal-I-His6
For the synthesis of Kdnα2–3LexβProN3 and Neu5AcN3α2–
3LexβProN3, LexβProN3 (20 mg, 0.03 mmol), mannose (9
mg, 0.05 mmol) or ManNAcN3 (Yu et al. 2005) (12 mg, 0.05
mmol), sodium pyruvate (18 mg, 0.16 mmol), CTP (28 mg,
0.05 mmol) and MgCl2 (22 mg, 0.11 mmol) were dissolved in
H2O (2.5 mL). A stock solution of Tris–HCl buffer (1 M, pH
8.5, 0.5 mL) was added. After the addition of a recombinant
P. multocida sialic acid aldolase (Li et al. 2008) (1.7 mg), a
recombinant N. meningitidis CMP–sialic acid synthetase (Yu
et al. 2004) (1.1 mg), and Ni2+-NTA column purified
MBP-Δ30vST3Gal-I-His6 (262 μg, estimated), water was
added to bring the volume of the reaction mixture to 5 mL in
both reactions. The reactions were carried out by incubating
the solution in an incubator shaker at 37°C for overnight. The
reactions were stopped by adding cold EtOH (5 mL) followed
by incubation on ice for 30 min. The mixtures were then
centrifuged to remove precipitates. The supernatants were
concentrated by rotavaporation. A BioGel P-2 filtration
column and a silica gel column (eluted with EtOAc:MeOH:
H2O = 5:2:1, v/v/v) were used to purify the product
SLexβProN3Kdnα2–3LexβProN3 (18 mg, 64% yield) and
Neu5AcN3α2–3LexβProN3 (18 mg, 58% yield).
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