Biochemical characterization of Helicobacter pylori α1–3-fucosyltransferase and its application in the synthesis of fucosylated human milk oligosaccharides

Article abstract of DOI:10.1016/j.carres.2019.05.007

Fucosylated human milk oligosaccharides (HMOs) have important biological functions. Enzymatic synthesis of such compounds requires robust fucosyltransferases. A C-terminal 66-amino acid truncated version of Helicobacter pylori α1–3-fucosyltransferase (Hp3FT) is a good candidate. Hp3FT was biochemically characterized to identify optimal conditions for enzymatic synthesis of fucosides. While N-acetyllactosamine (LacNAc) and lactose were both suitable acceptors, the former is preferred. At a low guanosine 5′-diphospho-β-L-fucose (GDP-Fuc) to acceptor ratio, Hp3FT selectively fucosylated LacNAc. Based on these enzymatic characteristics, diverse fucosylated HMOs, including 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, lacto-N-neofucopentaose (LNnFP) V, lacto-N-neodifucohexaose (LNnDFH) II, difuco- and trifuco-para-lacto-N-neohexaose (DF-paraLNnH and TF-para-LNnH), were synthesized enzymatically by varying the ratio of the donor and acceptor as well as controlling the order of multiple glycosyltransferase-catalyzed reactions.

Full text of DOI:10.1016/j.carres.2019.05.007

Accepted Manuscript
Biochemical characterization of Helicobacter pylori α1–3-fucosyltransferase and its
application in the synthesis of fucosylated human milk oligosaccharides
Jing Bai, Zhigang Wu, Go Sugiarto, Madhusudhan Reddy Gadi, Hai Yu, Yanhong Li,
Cong Xiao, Alice Ngo, Baohua Zhao, Xi Chen, Wanyi Guan
PII:
S0008-6215(19)30195-8
https://doi.org/10.1016/j.carres.2019.05.007
CAR 7714
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 2 April 2019
Revised Date: 6 May 2019
Accepted Date: 15 May 2019
Please cite this article as: J. Bai, Z. Wu, G. Sugiarto, M.R. Gadi, H. Yu, Y. Li, C. Xiao, A. Ngo, B. Zhao,
X. Chen, W. Guan, Biochemical characterization of Helicobacter pylori α1–3-fucosyltransferase and its
application in the synthesis of fucosylated human milk oligosaccharides, Carbohydrate Research (2019),
doi: https://doi.org/10.1016/j.carres.2019.05.007.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Biochemical characterization of Helicobacter pylori α1–3-fucosyltransferase and its
application in the synthesis of fucosylated human milk oligosaccharides
Jing Baia#, Zhigang Wub#, Go Sugiartoc#, Madhusudhan Reddy Gadid, Hai Yuc, Yanhong Lic, Cong
Xiaod, Alice Ngoc, Baohua Zhaoa*, Xi Chenc*, Wanyi Guana*
aCollege of Life Science, Hebei Normal University, Shijiazhuang, Hebei 050024, China.
bCollege of Bioscience and Bioengineering, Hebei University of Science and Technology,
Shijiazhuang, Hebei 050018, China.
cDepartment of Chemistry, University of California, One Shields Avenue, Davis, CA, 95616,
USA.
dDepartment of Chemistry and Center of Diagnostics & Therapeutics, Georgia State University,
Atlanta, GA 30303, USA.
# These authors contributed equally to this work.
* Corresponding author.
E-mail addresses: guanwanyi@hebtu.edu.cn (W. Guan); xiichen@ucdavis.edu (X. Chen);
zhaobaohua@hebtu.edu.cn (B. Zhao)
Abstract
Fucosylated human milk oligosaccharides (HMOs) have important biological functions. Enzymatic
synthesis of such compounds requires robust fucosyltransferases. A C-terminal 66-amino acid
truncated version of Helicobacter pylori α1–3-fucosyltransferase (Hp3FT) is a good candidate.
Hp3FT was biochemically characterized to identify optimal conditions for enzymatic synthesis of
fucosides. The acceptor substrate specificity is N-acetyllactosamine (LacNAc) and lactose, with
LacNAc as its preferred acceptor. At a low guanosine 5’-diphospho-β-L-fucose (GDP-Fuc) to
acceptor ratio, Hp3FT selectively fucosylated LacNAc. Based on these enzymatic characteristics,
diverse fucosylated HMOs, including 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III,
lacto-N-neofucopentaose (LNnFP) V, lacto-N-neodifucohexaose (LNnDFH) II, difuco- and
trifuco-para-lacto-N-neohexaose (DF-paraLNnH and TF-para-LNnH), were synthesized
enzymatically by varying the ratio of the donor and acceptor as well as controlling the order of
multiple glycosyltransferase-catalyzed reactions.
Keywords
fucosyltransferase; substrate specificity; human milk oligosaccharide; enzymatic synthesis
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1. Introduction
Lactose (Lac), lipid, and human milk oligosaccharides (HMOs) are the three major components
of human milk [1, 2]. HMOs are a group of oligosaccharides that can provide beneficial effects to
the health of breast-fed infants [3, 4]. In a typical human milk sample, around 70 percent of HMOs
are fucosylated [5], and they have crucial functions. For example, 3-fucosyllactose [3-FL, Galβ1–
4(Fucα1–3)Glc] and lacto-N-fucopentaose III (LNFP III, Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–
4Glc) can provide protection to infants as prebiotics to nourish specific gut bifidobacterial strains
[6]. Fucosylated HMOs resemble intestinal cell surface glycans and act as soluble receptors of
intestinal pathogens to prevent infection caused by their adhesion to intestinal epithelial surfaces [7].
Moreover, some fucosylated HMOs have potential pharmaceutical applications. LNFP III can
improve glucose tolerance and insulin sensitivity [8] and reduce experimental allergic
encephalomyelitis (EAE) severity and central nervous system (CNS) inflammation [9]. Therefore,
synthesis of fucosylted HMOs is important for related functional studies. Compared with chemical
synthesis, enzymatic fucosylation is an efficient approach to synthesize fucosylated HMOs since it
does not require harsh chemicals.
A truncated version of Helicobacter pylori α1–3-fucosyltransferase (Hp3FT) lacking the
C-terminal 66 amino acid residues was successfully cloned and expressed in E. coli and
characterized [10, 11]. Its crystal structure was solved [12]. It can catalyze the transfer of L-fucose
(Fuc) from sugar nucleotide donor guanosine 5’-diphospho-β-L-fucose (GDP-Fuc) to acceptors
such as N-acetyllactosamine (LacNAc, Galβ1–4GlcNAc) to form Lewis X (LeX) antigen. The
enzyme was first reported by the Lin group [10, 12] and has been applied for oligosaccharide
synthesis by our labs [13-17] and others including the groups of Wu [18], Boons [19], Cao [20],
Wang and Li [21, 22]. However, discrepancies on whether Lac moiety can be recognized as an
acceptor by the enzyme were unclear. The Lin group reported that the enzyme was not able to
recognize Lac as an acceptor [10]. In addition, the Cao group [20] and one of our groups [16]
independently used it for the synthesis of LNFP III, a monofucosylated oligosaccharide, from
Galβ1–4GlcNAcβ1–3Galβ1–4Glc successfully, without fucosylation of the Lac moiety in the
acceptor. On the contrary, the Boons group reported that it catalyzed the transfer of two fucose
residues to Siaα2–3Galβ1–4GlcNAcβ1–3Galβ1–4Glc to form Siaα2–3Galβ1–4(Fucα1–
3)GlcNAcβ1–3Galβ1–4(Fucα1–3)Glc [19], indicating that Lac component was also suitable for
fucosylation by the enzyme. Only the clarification of these conflicting results will enable a clear
future application of Hp3FT in fucosides preparation. Therefore, in this study, its acceptor substrate
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specificity to Lac and LacNAc was elucidated by kinetic study. An HPLC-based assay that detects
non-derivatized acceptor and product glycans for kinetic study was developed for the first time by
combining hydrophilic interaction liquid chromatography (HILIC) separation and evaporative light
scattering detector (ELSD). Meanwhile, systematic biochemical characterization of Hp3FT was
carried out to obtain its optimal reaction condition. The information learned was applied in efficient
synthesis of a series of fucosylated HMOs, including 3-FL, LNFP III, lacto-N-neofucopentaose
(LNnFP)
V
[23],
lacto-N-neodifucohexaose (LNnDFH)
II [24],
difuco- and
trifuco-para-lacto-N-neohexaose (DF-paraLNnH and TF-para-LNnH) [25].
2. Results and discussion
2.1 Cloning, expression and purification of Hp3FT
A C-terminal 66 amino acids truncated Hp3FT was cloned from a codon-optimized synthetic
gene of H. pylori α1–3-fucosyltransferase into pET15b vector and expressed in Escherichia coli
BL21(DE3) as an N-terminal His6-tagged fusion protein. One-step affinity column purification
using a nickel-nitrilotriacetic acid (Ni2+-NTA) agarose column was able to provide a purified
sample containing more than 95% of the purified Hp3FT with molecular weight close to its
theoretical value (44.2 kDa) (Fig. 1). An expression level of about 27 mg of purified protein per
liter culture was typically achieved when Terrific Broth (TB) was used.
2.2 pH profile of Hp3FT and metal ion effects on Hp3FT
The pH profile study of Hp3FT showed that it was active in a broad pH range between pH 5.0
and pH 9.5 with an optimal activity at pH 7.5 in Tris-HCl buffer (Fig. 2).
Similar to E. coli α1–2-fucosyltransfearse EcWbgL [26] and H. pylori α1–
3/4-fucosyltransferase (Hp3/4FT) [27], a metal cation is not required for the fucosyltransfer activity
of Hp3FT, as shown in the metal effect studies using Mg2+, Mn2+, or ethylenediaminetetraacetic
acid (EDTA) in Tris-HCl buffer at pH 7.5 (Fig. 3), despite the fact that it uses a nucleotide
diphosphate sugar as the donor substrate. This can be explained by its GT-B structure fold [12] and
the absence of a DXD domain in its sequence [28].
2.3 Kinetic study of Hp3FT
An HPLC-based assay using HILIC separation and ELSD was developed for kinetics studies of
Hp3FT using non-derivatized LacNAc and Lac as acceptors. The obtained apparent kinetic
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parameters showed that, compared to Lac, LacNAc has a lower Km value and a higher kcat value,
leading to a catalytic efficiency 161-fold higher than that of Lac. The results clearly indicate that
LacNAc is a preferred acceptor for Hp3FT. A possible explanation for the preferential binding of
Hp3FT to LacNAc is that, the hydrophobic interaction formed between the methyl group in the
N-acetyl group on C2 of GlcNAc in LacNAc and the side chain of Leu-124 of Hp3FT [12]
contributes to the better binding of LacNAc to the active site cleft of Hp3FT, whereas no such
interaction is formed between the hydroxyl group on C2 of glucose residue in Lac and Leu-124.
2.4 Synthesis of fucosylated HMOs
With the kinetics toward the two acceptor substrates characterized, Hp3FT was subsequently
employed to synthesize a series of fucosylated HMOs (Scheme 1). In our synthesis, oligosaccharides
with only Lac moiety or both LacNAc and Lac moieties were used as acceptors, and effective
fucosylation was achieved by the catalysis of Hp3FT. From Lac, 3-FL (1) can be efficiently
synthesized by the enzyme. However, when synthesizing the monofucosylated LNFP III (4) from
lacto-N-neotetraose (LNnT, 3), a tetrasaccharide containing two potential fucosylation sites (one Lac
moiety and one LacNAc moiety), Fuc was preferentially transferred onto LacNAc rather than Lac at
lower ratio of GDP-Fuc to acceptor (1.2:1), consistent with the kinetic study. The structure of the
product was identified by tandem MS (Fig. 4a) and 2D NMR (the data is in match with the data in ref
[27]). If the ratio of GDP-Fuc to LNnT was over 2:1 (in this case, 2.6:1 was used), two fucoses can be
added and difucosylated LNnDFH II (5) was acquired and identified by tandem MS (Fig. 4b).
According to tandem MS results, specific fragment of Galβ1–4(Fucα1–3)GlcNAc was found in
LNFP III (4), whereas Galβ1–4(Fucα1–3)Glc was not (Fig. 4a). However, specific fragments of both
Galβ1–4(Fucα1–3)GlcNAc and Galβ1–4(Fucα1–3)Glc can be found in LNnDFH II (5). These
results are consistent with previously reported synthesis of fucosides, in which LNFP III was
generated when GDP-Fuc to acceptor ratio was at most 1.5:1 [20], whereas LNnDFH II was formed
with a GDP-Fuc to acceptor ratio at ~ 4:1 [19].
For para-lacto-N-neohexaose (para-LNnH, 8) [25] with single Lac and double LacNAc moieties,
the preferences of Hp3FT were similar to those for LNnT, with the first two Fuc residues transferred
to the two LacNAc moieties to give DF-para-LNnH (9) at lower ratio (2.4:1) of GDP-Fuc to acceptor,
and the third Fuc attached onto Lac moiety to generate TF-para-LNnH (10) only when the ratio of
GDP-Fuc over acceptor was over 3:1 (in this case, 4:1 was used). The structures of both glycans (9
and 10) were identified by tandem MS (Fig. 4d and 4e).
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Given its broad acceptor specificity and preferential fucosylation activity on LacNAc over Lac,
an approach with altered sequence of glycosyltransferases was adopted to achieve site-specific
fucosylation of Lac moiety at the reducing end with the existence of LacNAc moiety in the acceptor,
similar to the strategy used for selective sialylation of galactose close to the reducing end of the
acceptor catalyzed by a bacterial α2–6sialyltransferase with broad acceptor substrate specificity [29].
For example, by first using Hp3FT and then NmLgtB, LNnFP V (also named LNFP VI, 7), an isomer
of LNFP III (4), can be obtained from lacto-N-triose II (LNT II, 2) (Scheme 1), as identified by
tandem MS (Fig. 4c) and 2D NMR (Fig. S1). For the synthesis of all the 7 fucosylated compounds
(compounds 1, 4, 5, 6, 7, 9, 10) shown in Scheme 1, no remaining acceptors were detected by
MALDI-TOF MS, indicating the efficient fucosylation catalyzed by Hp3FT.
3. Conclusions
The commonly used fucosyltransferase Hp3FT has been previously reported to generate
discrepant fucoside products from the same acceptor glycan [16, 19, 20]. According to the acceptor
specificity study of recombinant Hp3FT performed in this study, the inconsistency can be explained
by its recognition of both Lac and LacNAc as acceptors, but with a higher catalytic efficiency
towards LacNAc. Therefore, lower donor (GDP-Fuc) to acceptor ratio used in synthetic reaction
mixture in the previous reports resulted in fucosylation at LacNAc only [16, 20], whereas higher
donor to acceptor ratio provided fully fucosylated glycans at both LacNAc and Lac sites [19]. In the
case that Lac is present with multiple LacNAc moieties within the same glycan, Lac will be
fucosylated only after LacNAc moieties are all fucosylated (as demonstrated by the synthesis of
compounds 9 and 10 herein). If site-specific fucosylation of the reducing terminal Lac is desired
with the coexistence of LacNAc in the target glycan (as illustrated by LNnFP V, compound 7 in this
study), synthetic routes with appropriate sequences of glycosylation need to be designed. Based on
this principle, fucosylated HMOs with fucosylation specifically on LacNAc, specifically on Lac, or
both on LacNAc and Lac can be prepared, as exemplified in this study (including 3-FL, LNFP III,
LNnFP V, LNnDFH II, DF-para-LNnH, and TF-para-LNnH). The products obtained are important
probes for further biological studies on their nutraceutical and pharmaceutical potentials.
4. Experimental
4.1 Bacterial strains, vector, materials and general methods
Full-length H. pylori α1–3-fucosyltransferase synthetic gene with codons optimized for
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Escherichia coli expression system was custom-synthesized by Geneart, Inc. (Burlingame, CA,
USA). E. coli DH5α electrocompetent cells and chemically competent BL21 (DE3) cells were from
Invitrogen (Carlsbad, CA, USA). Vector pET15b was purchased from Novagen (EMD Biosciences
Inc., Madison, WI, USA). Restriction enzymes NdeI and BamHI were purchased from New
England BioLabs (Beverly, MA, USA). Chemicals were purchased and used without further
purification. Fucose (Fuc), ethylenediaminetetraacetic acid (EDTA), adenosine 5'-triphosphate
(ATP), guanosine 5'-triphosphate (GTP), magnesium chloride, manganese chloride, and nickel
sulfate were purchased from Sigma Aldrich (Saint Louis, MO, USA). Gel filtration chromatography
was performed with a column (100 cm × 2.5 cm) packed with BioGel P-2 Fine resins (Bio-Rad).
BfFKP [30], PmPpA [31], NmLgtA [32], and NmLgtB [33] were overexpressed as reported.
GDP-Fuc was prepared by one-pot multi-enzyme synthesis followed by gel filtration
chromatography as previously reported [34].
4.2 Cloning of Hp3FT
N-His6-tagged and 66 amino acids at C-terminal truncated H. pylori α1–3-fucosyltransferase
(Hp3FT)
was
cloned
into
pET15b
vector.
Primers
used
were:
forward
5’-GATCCATATGTTTCAGCCGCTGCTG-3’ (NdeI restriction site is underlined) and reverse
5’-CGCGGATCCTTATTAGTAGTTAACACGCAGATC-3’ (BamHI restriction site is underlined).
Plasmids were purified and positive clones were verified by restriction mapping and DNA
sequencing performed by the Davis Sequencing Facility at the University of California-Davis.
4.3 Expression and purification of Hp3FT
The E. coli BL21 (DE3) cells containing recombinant plasmid with gene encoding Hp3FT were
cultured in Terrific Broth (TB) containing 100 µg mL−1 of ampicillin at 37 °C with orbital shaking
(250 rpm) until the OD600 nm reached 0.8–1.0. Overexpression of the target protein was achieved by
adding 0.1 mM of IPTG to the E. coli culture followed by further incubation at 20 °C for 20 h. Then,
the cells were harvested by centrifugation at 4,000 rpm at 4 °C for 30 min. The cell pellet was
resuspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing 0.1% Triton X-100) (20 mL per
liter cell culture) and the mixture went through two freeze-thaw cycles. Lysozyme (50 µg mL-1) and
DNase I (3 µg mL-1) were then added and the mixture was incubated at 37 °C for 1 h with gentle
shaking (125 rpm). Then, the supernatant was obtained by centrifugation at 11,000 rpm for 20 min
at 4 °C, and was loaded onto a Ni-NTA column pre-equilibrated with binding buffer (50 mM
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Tris-HCl, pH 7.5, 0.5 M NaCl, 5 mM imidazole). The column was washed with 8 column volumes
of binding buffer and 8 column volumes of washing buffer (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl,
30 mM imidazole), and then the protein of interest was eluted with an elution buffer (50 mM
Tris-HCl, pH 7.5, 0.5 M NaCl, 200 mM imidazole). The fractions containing purified recombinant
protein were pooled and dialyzed against Tris-HCl buffer (pH 8.0, 20 mM) containing 10% glycerol
and NaCl (100 mM). Dialyzed proteins were stored at 4 °C. The purified protein was analyzed by
SDS-PAGE and was quantified by a BCA protein assay kit (Pierce Biotechnology, Rockford, IL).
4.4 pH profiles of Hp3FT
All assays were carried out in duplicate. Each reaction in a total volume of 10 µL contains a
buffer (200 mM) with pH varying from 4.0 to 11.0, MnCl2 (20 mM), GDP-Fuc (1 mM),
LacNAcβPro2AA (1 mM) which is labeled with 2-anthranilic acid (2AA), and 1.2 µg Hp3FT. The
buffers used were sodium acetate, pH 4.0–4.5; MES, pH 5.0–6.5; HEPES, pH 7.0; Tris-HCl, pH
7.5–9.0; CAPSO, pH 9.5; and CAPS, pH 10.0–11.0. Reactions were carried out at 37 °C for 15 min
before being quenched by adding pre-chilled 20% acetonitrile (20 µL). The samples were kept on
ice until aliquots of 8 µL were injected and analyzed by a Shimadzu LC-2010A HPLC system
equipped with a membrane online degasser, a temperature control unit, and a fluorescence detector.
A reverse-phase Waters Spherisorb C18 column (250 × 4.6 mM i.d., 5 µm particle size) protected
with a C18 guard column cartridge was used. The mobile phase was 18% acetonitrile. Fluorescently
labeled compounds LacNAcβPro2ΑΑ and LeXβPro2ΑΑ were detected by excitation at 315 nm and
emission at 400 nm.
4.5 EDTA and metal effects on the α1–3-fucosyltransferase activity of Hp3FT
The reactions were carried out in duplicate in a total volume of 10 µL in Tris-HCl buffer (100
mM, pH 7.5) containing GDP-Fuc (1 mM), LacNAcβPro2AA (1 mM), and recombinant enzyme
(1.2 µg) in the presence of EDTA (5 mM) or a divalent metal salt (MgCl2 or MnCl2) (5 mM, 10
mM, or 20 mM). Reactions without EDTA or a metal ion were used as controls. Reactions were in
carried out in duplicate at 37 °C for 15 min, quenched and assayed using the same method as
described above for pH profile.
4.6 Kinetics study for the
α
1–3-fucosyltransferase activity of Hp3FT
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To obtain the apparent kinetic parameters of LacNAc and Lac, the kinetic assays were
performed in triplicates in reaction mixtures of 20 µL containing Tris-HCl buffer (50 mM, pH 7.5),
10 mM of MgCl2, a fixed concentration of GDP-Fuc (15 mM), different concentrations of LacNAc
(0.5, 0.8, 1, 1.5, 2, 3 and 5 mM) or Lac (1, 2, 3, 5, 6, 8 and 10 mM), and Hp3FT (1.6 µg for
LacNAc, 100 µg for Lac). To obtain the apparent kinetic parameters of GDP-Fuc, the kinetic assays
were performed in triplicates in reaction mixtures of 20 µL containing Tris-HCl buffer (50 mM, pH
7.5), a fixed concentration of LacNAc (3 mM) or Lac (10 mM), different concentrations of
GDP-Fuc (2, 5, 8, 12, 18 and 20 mM), and Hp3FT (1.6 µg for LacNAc, 100 µg for Lac). All
reactions were allowed to proceed at 37 °C for 20 min and the reactions were terminated by
incubating the tubes containing the reaction mixtures in a boiling water bath for 5 minutes. After
centrifugation at 13000 rpm in a table-top centrifuge for 20 min, the supernatant of each sample was
obtained and 10 µL of each was injected and analyzed by a Waters e2695 Alliance system equipped
with a 2424 evaporative light scattering detector (ELSD). An XBridge BEH Amide Column (130Å,
5 µm, 4.6 mm × 250 mm, Waters) was used. Mobile phase A: 100 mM ammonium formate, pH 3.4;
Mobile phase B: acetonitrile; B% Gradient: 90% to 80% over 7 min, 80% to 70% over 10 min, 70%
to 60% over 13 min, 10% for 10 min, 90% for 10 min; Flow rate: 1 mL min-1. LacNAc, Lac, and
their fucosylated product were detected by ELSD. Apparent kinetic parameters were obtained by
fitting the experimental data (the average values of triplicate assay results) into the Michaelis–
Menten equation using Graph Pad 5.0.
4.7 HMOs synthesis
Reaction mixtures contained Tris-HCl (100 mM, pH 8.0), an acceptor glycan (as a pure
compound or in the reaction mixture from the previous reaction, 10 mM), an appropriate sugar
nucleotide (1.2 equiv. for UDP-GlcNAc or UDP-Gal, 1.2–4.0 equiv. for GDP-Fuc, depending on
the number of fucose residues to be transferred), MnCl2 (10 mM), varying amounts of appropriate
glycosyltransferases (NmLgtA, NmLgtB, or Hp3FT), and FastAP (1 U/200 µL, to digest the
byproduct NDP and drive the reaction forward). Reactions were incubated at 37 °C for overnight
and monitored by MALDI-TOF MS. When all acceptors were converted, the reactions were
quenched by incubating the tubes containing the reaction mixtures in a boiling water bath for 5 min.
If the product of a glycosylation step was not the final product, no purification was performed, and
the reaction mixture was directly used for the next enzymatic extension. In the cases of the
compounds being final compounds, purification was performed by HPLC with a semi-preparative
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amide column (130 Å, 5 µm, 10 mm × 250 mm). Mobile phase A: 100 mM ammonium formate, pH
3.4; Mobile phase B: acetonitrile; B% Gradient: 80% to 60% over 35 min; Flow rate: 4 mL min-1.
HPLC eluent was monitored by absorption at 210 nm, and glycan-containing fractions were
1
13
analyzed by MALDI-TOF MS. H NMR and C NMR spectra of the purified compounds were
recorded on a Bruker Avance-III 600 MHz NMR spectrometer.
1
4.7.1. Gal
β
1–4(Fuc
α
1–3)Glc (1). 5 mg, 88%; H NMR (600 MHz, D2O) δ 5.34 (d, J = 4.2, 0.5H),
5.28 (d, J = 4.2 Hz, 0.4H), 5.08 (d, J = 3.6 Hz, 0.4H), 4.80-4.72 (m, 1H), 4.55 (d, J = 7.8 Hz, 0.6H),
4.33 (d, J = 7.8 Hz, 1H), 3.84-3.35 (m, 16 H), 1.09-1.07 (m, 3H); 13C NMR (150 MHz, D2O) δ
101.77, 101.74, 98.48, 98.34, 95.79, 92.05, 76.97, 75.50, 75.33, 74.91, 74.68, 72.64, 72.58, 72.38,
71.93, 71.11, 70.88, 69.25, 69.20, 68.29, 68.09, 68.01, 66.47, 66.43, 61.51, 61.48, 59.74, 59.66,
15.22. MALDI-TOF m/z calculated for C18H32NaO15 (M+Na): 511.16, found 511.36.
4.7.2. Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4Glc (4). 10 mg, 80%; NMR data see ref [27].
MALDI-TOF m/z calculated for C32H55NNaO25 (M+Na): 876.31, found 876.71.
4.7.3. Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4(Fucα
1–3)Glc (5). 8 mg, 68%; 1H NMR (600 MHz,
D2O) δ 5.28 (s, 0.7H, 5.22 (s, 0.7 H), 5.03-4.98 (m, 2.6H), 4.56-4.49 (m, 5.4 H), 4.31 (d, J = 7.2 Hz,
1.4H), 4.26 (d, J = 7.8 Hz, 1.3H), 3.94 (s, 1H), 3.80-3.33 (m, 30H), 1.86 (s, 3H), 1.02-1.01 (m, 6H);
13C NMR (150 MHz, D2O) δ 174.72, 102.48, 101.73, 98.56, 98.42, 95.82, 92.11, 81.55, 81.49,
76.98, 75.53, 75.35, 75.06, 74.90, 74.73, 74.67, 74.47, 73.02, 72.66, 72.46, 72.35, 72.26, 71.90,
71.03, 70.93, 70.67, 70.63, 69.26, 69.18, 68.33, 68.25, 68.22, 68.00, 67.68, 66.66, 66.46, 61.49,
59.78, 59.62, 55.93, 22.22, 15.28, 15.19. MALDI-TOF m/z calculated for C38H68NNaO29 (M+Na):
1022.36, found 1022.46.
4.7.4. GlcNAcβ1–3Galβ1–4(Fucα
1–3)Glc (6). 12 mg, 84%; 1H NMR (600 MHz, D2O) δ 5.33 (d, J
= 4.2 Hz, 0.6H), 5.27 (d, J = 4.2 Hz, 0.5H), 5.08 (d, J = 4.2 Hz, 0.5H), 4.74-4.73 (m, 1H), 4.58 (dd,
J = 2.4 and 8.4 Hz, 1H), 4.55 (d, J = 8.4 Hz, 0.5H), 4.32 (d, J = 7.8 Hz, 1H), 3.99 (d, J = 3.6 Hz,
13
1H), 3.87-3.33 (m, 22H), 1.93 (s, 3H), 1.07-1.06 (m, 3H); C NMR (150 MHz, D2O) δ 174.90,
102.76, 101.70, 98.51, 98.40, 95.81, 92.09, 81.44, 81.39, 76.97, 75.58, 75.32, 74.65, 74.46, 73.44,
72.65, 72.35, 72.27, 71.90, 70.90, 70.70, 70.65, 69.67, 69.23, 69.18, 68.23, 68.01, 67.98, 66.44,
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61.43, 60.42, 59.76, 59.69, 55.60, 22.14, 15.20. MALDI-TOF m/z calculated for C26H45NNaO20
(M+Na): 714.25, found 714.67.
1
4.7.5. Gal
β
1–4GlcNAc
β
1–3Gal
β
1–4(Fuc
α
1–3)Glc (7). 6 mg, 80%; H NMR (600 MHz, D2O)
δ 5.27 (d, J = 4.2 Hz, 0.5 H), 5.21 (d, J = 3.6 Hz, 0.5 H), 5.02 (d, J = 4.2 Hz, 0.5 H), 4.67-4.65 (m,
2H), 4.53 (d, J = 8.4 Hz, 1H), 4.49 (d, J = 7.8 Hz, 0.5 Hz), 4.32 (d, J = 7.8 Hz, 1H), 4.25 (d, J = 7.8
13
Hz, 1H), 3.94 (d, J = 3.0 Hz, 1H), 3.80-3.28 (m, 28H), 1.87 (s, 3H), 1.09-0.99 (m, 3H); C DEPT
135 NMR (150 MHz, D2O) δ 102.83, 102.67, 101.70, 98.52, 98.41, 95.81, 92.09, 81.54, 81.49,
78.12, 76.97, 75.52, 75.32, 74.66, 74.49, 74.46, 72.65, 72.47, 72.35, 72.26, 72.09, 71.90, 70.94,
70.64, 70.60, 69.24, 69.19, 68.22, 68.01, 67.98, 66.46, 66.41, 61.43, 59.81, 59.75, 59.68, 55.14,
22.14, 15.19. MALDI-TOF m/z calculated for C32H55NNaO25 (M+Na): 876.31, found 876.06.
4.7.6. Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4Glc (9). 4 mg, 80%;
1H NMR (600 MHz, D2O) δ 5.15 (d, J = 4.2 Hz, 0.4H), 5.06 (d, J = 3.6 Hz, 1H), 5.04 (d, J = 4.2 Hz,
1H), 4.65-4.63 (m, 2.6H), 4.59 (d, J = 8.4 Hz, 1H), 4.40-4.36 (m, 3H), 4.08 (d, J = 3.6 Hz, 1H),
4.02 (d, J = 3.0 Hz, 1H), 3.90-3.41 (m, 48H), 1.95 (s, 3H), 1.95 (s, 3H), 1.10 (d, J = 6.6 Hz, 3H),
13
1.07 (d, J = 6.6 Hz, 3H). C DEPT 135 NMR (150 MHz, D2O) δ 102.84, 102.47, 101.67, 98.65,
98.54, 95.66, 91.76, 81.99, 81.52, 78.09, 74.98, 74.84, 74.67, 74.37, 73.70, 72.90, 72.64, 72.37,
71.83, 70.96, 70.45, 69.89, 69.09, 68.27, 67.60, 66.62, 61.46, 60.90, 59.96, 59.52, 59.27, 55.87,
22.16, 15.24. MALDI-TOF m/z calculated for C52H88N2NaO39 (M+Na) 1387.50, found 1387.45.
4.7.7. Gal
β
1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4(Fucα1–3)Glc (10).
1
4.8 mg, 94%; H NMR (600 MHz, D2O) δ 5.35 (d, J = 4.2 Hz, 1H), 5.29 (d, J = 3.6 Hz, 1H), 5.10
(d, J = 4.2 Hz, 1H), 5.05 (dd, J = 3.6, 8.4 Hz, 3H), 4.63 (d, J = 8.4 Hz, 3H), 4.57 (d, J = 7.8 Hz, 1H),
4.39-4.36 (m, 2H), 4.33 (d, J = 7.8 Hz, 1H), 4.02 (bs, 2H), 3.90-4.40 (m, 60H), 1.94 (s, 6H),
13
1.10-1.07 (m, 9H). C DEPT 135 NMR (150 MHz, D2O) δ 102.47, 101.72, 98.56, 95.82, 92.18,
81.60, 75.53, 75.35, 75.05, 74.90, 74.74, 74.43, 73.01, 72.75, 72.46, 71.90, 71.04, 70.52, 69.17,
68.34, 68.22, 68.00, 67.69, 66.67, 66.47, 61.45, 59.62, 55.94, 22.23, 15.29, 15.26, 15.20.
MALDI-TOF m/z calculated for C58H98N2NaO43 (M+Na) 1533.55, found 1533.54.
4.8 MALDI-TOF/TOF MS analyses
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One microliter of sample solution with the same volume of DHB (15 mg mL-1 in 50:50 ACN–
water) was spotted on the MALDI plate (Bruker Daltonics, MTP 384 polished steel) for MS
analysis. The MS and MS/MS spectra were acquired on an ultrafleXtreme MALDI TOF-TOF
mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with 1 kHz Smartbeam-II
laser in reflectron positive mode. The operation parameters for MS spectrum acquisition were ion
source 1, 25.00 kV; ion source 2, 22.70 kV; lens, 8.30 kV; reflectron 1, 26.45 kV; and reflectron 2,
13.45 kV; detector gain, × 4.0. The operation parameters for MS/MS spectrum acquisition were
ion source 1, 7.50 kV; ion source 2, 6.80 kV; lens 3.50 kV; reflectron 1, 29.50 kV; reflectron 2,
13.80 kV; LIFT 1, 19.00 kV; LIFT 2, 2.9 kV; detector gain, ×10.0. A total of 2000 shots were
accumulated for each MS and MS/MS spectrum. The interpretation of the spectra was manually
analyzed with GlycoWorkbench software (Euro-CarbDB) [35].
Acknowledgments
This work was supported by Hebei Science and Technology Plan Project grant 17226701D (to B.Z.),
Hebei Provincial Department of Human Resources and Social Security grants C201812 and
E2018100010 (to Z.W.), as well as United States National Institutes of Health grant numbers
U01GM120419 and R01HD065122 (to X.C.).
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Table 1. Apparent kinetic parameters of recombinant Hp3FT.
Substrate
LacNAc
kcat (s-1)
Km (mM)
kcat/Km (s-1mM-1)
8.7 × 10-1
0.3677±0.0080
0.4225±0.0393
GDP-Fuca
Lac
9.6 × 10-2
5.4 × 10-3
7.9 × 10-3
0.6168±0.0417
0.0201±0.0006
0.0428±0.0007
6.4480±1.1360
3.7290±0.2601
5.3920±0.2868
GDP-Fucb
aAssayed with LacNAc as the acceptor for Hp3FT.
bAssayed with Lac as the acceptor for Hp3FT.
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Figure Captions
Fig. 1. SDS-PAGE analysis of Hp3FT. The apparent molecular weight of the purified enzyme
appears approximate 44 kDa (calculated molecular weight 44.2 kDa). Lane 1, whole cell extraction
before induction; Lane 2, whole cell extraction after induction; Lane 3, lysate after induction; Lane
4, Ni2+-NTA agarose column purified protein; Lane M, protein markers (Blue Plus IV Protein
Marker, 10–180 kDa, from TransGen Biotech, Beijing, China).
Fig. 2. pH profile of Hp3FT with LacNAcβPro2AA as the acceptor substrate. Buffers used were:
sodium acetate, pH 4.0–4.5; MES, pH 5.0–6.5; HEPES, pH 7.0; Tris-HCl, pH 7.5–9.0; CAPSO, pH
9.5; and CAPS, pH 10.0–11.0.
Fig. 3. Metal cation effects on Hp3FT assayed using a reaction mixture containing Mn2+ (Black
columns) or containing Mg2+ (grey columns), in the presence of EDTA (5 mM) (striped column) or
in the absence of a metal (dotted column).
Fig. 4. Acceptor substrate specificity of Hp3FT was identified by tandem MS during the synthesis of
(a) LNFP III (4), (b) LNnDFH II (5), (c) LNnFP V (7), (d) DF-para-LNnH (9), and (e)
TF-para-LNnH (10).
Scheme Caption
Scheme 1. Synthesis of fucosylated HMOs by Hp3FT according to its acceptor specificity. (a)
NmLgtA, UDP-GlcNAc, Mn2+; (b) NmLgtB, UDP-Gal, Mn2+; (c) Hp3FT, GDP-Fuc, Mn2+,
GDP-Fuc to acceptor ratio = 1.2:1; (d) Hp3FT, GDP-Fuc, Mn2+, GDP-Fuc to acceptor ratio = 2.6:1;
(e) Hp3FT, GDP-Fuc, Mn2+, GDP-Fuc to acceptor ratio = 2.4:1; (f) Hp3FT, GDP-Fuc, Mn2+,
GDP-Fuc to acceptor ratio = 4:1.
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ó
Helicobacter pylori α1–3-fucosyltransferase (Hp3FT) is biochemically
characterized
ó
ó
ó
Hp3FT prefers LacNAc over Lac as an acceptor substrate
Hp3FT selectively fucosylates LacNAc at a low GDP-Fuc to acceptor ratio
Selective fucosylation of Lac can be achieved by controlling glycosylation
sequence
ó
Diverse fucosylated HMOs have been synthesized using Hp3FT