Characterization of the single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei using synthetic peptides and lipid-linked oligosaccharide analogs

Article abstract of DOI:10.1093/glycob/cwx017

The initial transfer of a complex glycan in protein N-glycosylation is catalyzed by oligosaccharyltransferase (OST), which is generally a multisubunit membrane protein complex in the endoplasmic reticulum but a single-subunit enzyme (ssOST) in some protists. To investigate the reaction mechanism of ssOST, we recombinantly expressed, purified and characterized the STT3A protein from Trypanosoma brucei (TbSTT3A). We analyzed the in vitro activity of TbSTT3A by synthesizing fluorescently labeled acceptor peptides as well as lipid-linked oligosaccharide (LLO) analogs containing a chitobiose moiety coupled to oligoprenyl carriers of distinct lengths (C₁₀, C₁₅, C₂₀ and C₂₅) and with different double bond stereochemistry. We found that in addition to proline, charged residues at the +1 position of the sequon inhibited glycan transfer. An acidic residue at the -2 position significantly increased catalytic turnover but was not essential, in contrast to the bacterial OST. While all synthetic LLO analogs were processed by TbSTT3A, the length of the polyprenyl tail, but not the stereochemistry of the double bonds, determined their apparent affinity. We also synthesized phosphonate analogs of the LLOs, which were found to be competitive inhibitors of the reaction, although with lower apparent affinity to TbSTT3A than the active pyrophosphate analogs.

Full text of DOI:10.1093/glycob/cwx017

Glycobiology, 2017, vol. 27, no. 6, 525–535
doi: 10.1093/glycob/cwx017
Advance Access Publication Date: 16 March 2017
Original Article
Chemical Biology
Characterization of the single-subunit
oligosaccharyltransferase STT3A from
Trypanosoma brucei using synthetic peptides
and lipid-linked oligosaccharide analogs
Ana S Ramírez2,†, Jérémy Boilevin3,†, Rasomoy Biswas3,5, Bee Ha Gan3,
Daniel Janser2, Markus Aebi4, Tamis Darbre3, Jean-Louis Reymond3,
and Kaspar P Locher2,1
2Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule (ETH), CH-8093 Zürich,
Switzerland, 3Department of Chemistry and Biochemistry, University of Berne, CH-3012 Berne, Switzerland,
and 4Institute of Microbiology, Eidgenössische Technische Hochschule (ETH), CH-8093 Zürich, Switzerland
1To whom correspondence should be addressed: Tel: +41-44-633-3991; Fax: +41-44-633-1182;
e-mail: locher@mol.biol.ethz.ch.
5Present address: Aenova Holding GmbH, 82319 Starnberg, Germany.
†These authors contributed equally to this work.
Received 16 January 2017; Revised 9 February 2017; Editorial decision 9 February 2017; Accepted 10 February 2017
Abstract
The initial transfer of a complex glycan in protein N-glycosylation is catalyzed by oligosaccharyl-
transferase (OST), which is generally a multisubunit membrane protein complex in the endoplas-
mic reticulum but a single-subunit enzyme (ssOST) in some protists. To investigate the reaction
mechanism of ssOST, we recombinantly expressed, purified and characterized the STT3A protein
from Trypanosoma brucei (TbSTT3A). We analyzed the in vitro activity of TbSTT3A by synthesiz-
ing fluorescently labeled acceptor peptides as well as lipid-linked oligosaccharide (LLO) analogs
containing a chitobiose moiety coupled to oligoprenyl carriers of distinct lengths (C10, C15, C20 and
C25) and with different double bond stereochemistry. We found that in addition to proline, charged
residues at the +1 position of the sequon inhibited glycan transfer. An acidic residue at the −2 pos-
ition significantly increased catalytic turnover but was not essential, in contrast to the bacterial OST.
While all synthetic LLO analogs were processed by TbSTT3A, the length of the polyprenyl tail, but
not the stereochemistry of the double bonds, determined their apparent affinity. We also synthesized
phosphonate analogs of the LLOs, which were found to be competitive inhibitors of the reaction,
although with lower apparent affinity to TbSTT3A than the active pyrophosphate analogs.
Key words: enzyme kinetics, lipid-linked oligosaccharide, N-glycosylation, oligosaccharyltransferase
Introduction
pathways (Kelleher and Gilmore 2006; Breitling and Aebi 2013; Xu
Protein N-glycosylation confers a multitude of functions to the
acceptor macromolecules, facilitating diverse interactions and signaling
and Ng 2015; Cherepanova et al. 2016). The initial transfer of the gly-
can moiety from a lipid-linked oligosaccharide (LLO) donor to the
© The Author 2017. Published by Oxford University Press.
525
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unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

526
AS Ramírez et al.
asparagine of the acceptor protein is catalyzed by oligosaccharyltrans-
ferase (OST), an enzyme located in the membrane of the endoplasmic
reticulum in eukaryotic cells or in the plasma membrane of bacteria.
The mechanism of OST-catalyzed N-glycosylation has been investi-
gated using the bacterial PglB protein, a single subunit OST (ssOST)
from Campylobacter lari. Structural and in vitro functional studies of
PglB have provided insight into peptide recognition and have identified
catalytically important residues (Schwarz et al. 2010; Lizak et al. 2011,
2013, 2014; Gerber et al. 2013). In contrast to bacterial, eukaryotic
OSTs have different levels of complexity, varying between different
species (Cherepanova et al. 2016). In fungi and vertebrates, OST is a
membrane complex composed of up to eight subunits, whereas in
insects and plants it is predicted to be composed of seven subunits. The
OST enzymes of certain protists are predicted to have six subunits (i.e.,
Cryptosporidium parvum), four subunits (i.e., Plasmodium falciparum)
or even a single subunit as in kinetoplastids (i.e., Trypanosoma brucei
and Leishmania major) (Kelleher and Gilmore 2006; Nasab et al.
2008). Intriguingly, certain kinetoplastids contain several orthologues
of ssOSTs that originate from gene duplication. The existence of more
than one ssOST has been proposed to allow a wider range of acceptor
sequons to be glycosylated (Izquierdo et al. 2009; Schwarz and Aebi
2011). T. brucei contains three distinct orthologues of ssOST, denoted
TbSTT3A, TbSTT3B and TbSTT3C. Two of them, TbSTT3A and
TbSTT3B, are endogenously expressed at different stages of the life
cycle of the parasite, whereas expression of TbSTT3C was not detected
(Izquierdo et al. 2009). In vivo studies suggested that TbSTT3A and
TbSTT3B have different preferences for the LLO donor as well as for
acceptor sequon (Jones et al. 2005; Manthri et al. 2008). Whereas
TbSTT3B requires a c-branch in the LLO (present in Man9GlcNAc2-PP-
Dol) and glycosylates sequons surrounded by neutral or basic residues,
TbSTT3A preferentially transfer glycans from LLO donors lacking the
c-branch such as Man5GlcNAc2-PP-Dol to sequons surrounded by
acidic side chains (Izquierdo et al. 2009, 2012).
(LbSTT3A, LbSTT3B, LbSTT3C), Leishmania infantum (LiSTT3A,
LiSTT3B, LiSTT3C), L. major (LmSTT3A, LmSTT3B, LmSTT3C,
LmSTT3D) and T. brucei (TbSTT3A, TbSTT3B, TbSTT3C).
Expression constructs were generated by fusing either a His10-YFP
tag to the N-terminus or a YFP-His10 tag to the C-termini of the pro-
teins. All constructs were heterologously expressed in HEK293 cells.
Expression levels and protein monodispersity after extraction with
detergent were evaluated by fluorescent size exclusion chromatog-
raphy. TbSTT3A showed the highest expression level and was selected
for all subsequent biochemical analyses. Even though TbSTT3A shares
81% identity with TbSTT3B and TbSTT3C, the expression of these
last two was significantly lower. The main difference between
TbSTT3A and its orthologs is the length of its C-terminal domain,
which is 23 amino acids shorter in TbSTT3A compared to TbSTT3B
and TbSTT3C, this might influence the recombinant expression of the
proteins. According to topology predictions, TbSTT3A has the same
transmembrane topology as the bacterial OST, PglB (Figure 1A).
Although TbSTT3A and PglB share a low percentage of sequence
similarity (15.4% identity, 28.1% similarity), some of the catalytic
residues of PglB are conserved (Figure 1A and Supplementary Fig. 1).
Large-scale overexpression of TbSTT3A with His10-YFP fused to
its N-terminus was performed in Sf9 cells. Various parameters, in
particular the choice and concentration of the detergent used to
solubilize and purify the protein, were carefully screened and opti-
mized. Purified and deglycosylated TbSTT3A ran as a single band in
SDS-PAGE electrophoresis and showed a single peak in size exclu-
sion chromatography, with a small shoulder at higher mass, indicat-
ing potential aggregation (Figure 1B). The obtained yield and purity
allowed detailed functional investigation of TbSTT3A and will
enable us to pursue structural studies in the future.
The structural and mechanistic basis of substrate recognition and
preference in ssOST enzymes is unknown. Structural and functional stud-
ies are needed to rationalize the in vivo function of eukaryotic OST
enzymes. Mechanistic studies using purified eukaryotic OST enzymes are
scarce, which is in part due to the challenges associated with their expres-
sion and purification. Therefore, much of the structural and mechanistic
insight has been derived from the bacterial (PglB) and archaeal (AglB)
ssOST enzymes (Lizak et al. 2011, 2014; Gerber et al. 2013; Matsumoto
et al. 2013; Liu et al. 2014). Protist ssOST enzymes offer an opportunity
to study eukaryotic OST with the reduced complexity of a single mem-
brane protein. Here we describe the heterologous expression, purification
and functional analysis of the T. brucei ssOST enzyme TbSTT3A. We
generated fluorescently labeled acceptor peptides and varied the residues
surrounding the glycosylation sequon to increase the affinity and turn-
over of the reaction. We also synthesized various LLO analogs with
polyprenyl chains of controlled length and coupled to chitobiose. In add-
ition, we generated inhibitory LLO analogs where the pyrophosphate
group was replaced with an unreactive pyrophosphonate moiety. This
allowed us to perform detailed in vitro studies on TbSTT3A, providing
functional insight into the mechanism of eukaryotic ssOST.
Fig. 1. (A) Schematic representation of the predicted transmembrane top-
ology of TbSTT3A. The positions of the expected catalytic residues based on
sequence alignment with PglB are shown as red asterisks. Purple asterisks
depict the two glycosylation sites in the luminal domain of TbSTT3A (N619
and N765). (B) SEC and SDS-PAGE analysis of purified TbSTT3A. 85 µg of
purified and deglycosylated TbSTT3A were loaded on a Superdex S200 col-
umn at 0.5 mL/min. SDS-PAGE analysis of purified TbSTT3A is shown in the
inset. 1.7 µg of purified TbSTT3A was loaded on a 10% polyacrylamide gel. A
single band is observed for the purified protein. This figure is available in
black and white in print and in color at Glycobiology online.
Results
Expression and purification of TbSTT3A
To identify the most suitable STT3 orthologue for functional and
structural studies, we screened 13 genes encoding ssOST enzymes
from protists (Parsaie Nasab et al. 2013): Leishmania braziliensis

Functional characterization of single-subunit oligosaccharyltransferase STT3A
527
glycine led to no detectable glycosylation under the conditions tested.
The highest turnover was observed for a tyrosine residue at this pos-
ition (Figure 2B). At the +3 position of the sequon, an additional
tyrosine residue resulted in the highest turnover compared to any
other amino acids inserted at this position. Lysine or arginine were
also tolerated at the +3 position, but the turnover rate was 4.9- and
2.7-fold lower when compared to that of tyrosine-containing sequon.
Among the peptides screened in this study, P11 showed the high-
est turnover rate (Figure 2B). Unfortunately, kinetic analysis of P11
did not allow accurate determination of KM and kcat values because
saturation was not reached, even at the highest peptide concentra-
tions experimentally possible (Figure 2C). To test whether an
increase in peptide length might affect the affinity and in vitro glyco-
sylation efficiency of P11, we synthesized peptides with two add-
itional residues, either at the N-terminus (P13) or at the C-terminus
(P14) (Table I). Elongation of the C-terminus led to an increase in
peptide affinity, and a KM value of 28 3 µM could be determined
(Figure 2C). The opposite was observed when the peptide was elon-
gated at the N-terminus: In this case, it was not possible to deter-
mine KM and kcat values, because there was an almost linear
response of the observed activity with increasing peptide concentra-
tion (Figure 2C, Table I). The combined changes in the sequence,
from the initial DANYTK to GSDANYTYTQ, led to an increase of
about 3-fold in glycosylation turnover and a significant increase of
the apparent affinity (KM: 28 3 µM). Therefore, peptide P14 was
used in all subsequent assays.
Synthesis and in vitro glycosylation of acceptor
peptides
Following previously reported assay designs used for in vitro studies
of bacterial PglB and archaeal AglB (Kohda et al. 2007; Gerber et al.
2013; Liu et al. 2014), we synthesized a range of peptides containing
a glycosylation sequon and a 5-carboxyfluorescein label that was
attached to the N-terminus for quantitation purposes. Starting from
the peptide sequence DANYTK, which was previously used to study
PglB and the cytoplasmic glycosyltransferase NGT (Schwarz et al.
2011; Liu et al. 2014), we explored how distinct side chains in and
around the sequon influenced the activity of TbSTT3A (Figure 2A
and B). In vitro glycosylation experiments were performed with puri-
fied TbSTT3A and farnesyl-PP-chitobiose 1b (see below) as a donor
substrate (Figure 2A and B).
Analysis of different residues at the −2 position revealed that high-
est turnover rates were obtained with negatively charged side chains
(5.2 0.3 for aspartate and 3.6 0.3 pep/min for glutamate respect-
ively). In contrast, the positively charged side chains arginine or lysine
caused a decrease in the turnover rate of ~28- and ~12-fold, respect-
ively (Figure 2B). The small, polar residue serine at the −2 position
caused a 3.7-fold decrease in turnover. Analysis of different residues
at the +1 position of the sequon showed that besides proline, other
amino acids strongly inhibited glycosylation by TbSTT3A. Charged
residues such as lysine or aspartate were not tolerated. Nonpolar resi-
dues such as alanine and leucine led to a pronounced decrease in
turnover (5.8- and 20-fold, respectively, compared to tyrosine) and
Fig. 2. Optimization of the acceptor peptide sequence. (A) Synthetic acceptor peptides are shown in single letter code. The acceptor asparagine (zero position of
the sequon) is indicated in red. Glycosylation experiments were performed and the fluorescently labeled substrate (G0) and product (G1) were quantified follow-
ing Tricine SDS-PAGE analysis. (B) Turnover rates after fitting the time points by linear regression using PRISM. Error bars indicate standard error of the fitting.
(C) Kinetic analysis of substrate peptides: glycosylation experiments were performed with 20 nM purified TbSTT3A, 50 µM farnesyl-PP-chitobiose, 10 mM MnCl2,
150 mM NaCl, 20 mM Hepes pH 7.5, 0.035% DDM, 0.007% CHS and different concentrations of peptides varying from 2 to 60 µM. Data points reflect the mean of
three separate measurements. Error bars indicate standard deviations. Data were fitted by nonlinear regression according to the Michaelis–Menten formula
using PRISM. This figure is available in black and white in print and in color at Glycobiology online.

528
AS Ramírez et al.
We synthesized four distinct analogs of the eukaryotic LLO and sys-
tematically varied the length of the lipid moiety (C10, C15, C20 and
Synthesis and kinetic analysis of LLO analogs
The native LLO in trypanosome is Dol-PP-GlcNAc2-Man9, but
in vivo studies have shown that TbSTT3A preferentially uses Dol-
PP-GlcNAc2-Man5 as a substrate (Izquierdo et al. 2009). Native
LLOs are difficult to extract in sufficient quantities and their solubi-
lities are low because of the long polyprenyl chain of dolichol. In
contrast to the bacterial PglB enzyme, for which milligram amounts
of pure LLO can be isolated by introducing the C. jejuni pgl operon
into Escherichia coli SCM6 cells (Kowarik et al. 2006), such an
approach is not available for the production of eukaryotic LLO. We
therefore synthesized LLO analogs as donor substrates, starting
C25) as well as the double bond stereochemistry (Figure 3A). The lipid
carrier in compound 1b (E,E-farnesol), has a double bond in the first
isoprenyl unit, whereas the lipid carriers in compounds 1a, 1c and 1d
are structurally similar to the native dolichol. These four polyprenyl
chains were coupled to a chitobiose moiety, which has been reported
to be recognized and transferred by the OST complex from yeast
microsomes (Fang et al. 1995).
The synthesis of LLO analogs included three distinct steps: The
synthesis of the chitobiose phosphate and of the lipid phosphate pre-
cursors, were performed in parallel and were followed by a coupling
step and deacetylation of monophosphates to the final pyrophosphate
(Figure 4). The peracetylated N,N′-diacetylchitobiose phosphate
5 was obtained from the commercially available N,N′-diacetylchito-
biose hexaacetate 2 as described earlier (Lee and Coward 1992)
(Figure 4A). Lipids 15c and 15d were synthesized in nine steps from
(S)-citronellol 6. The key intermediate 10 was prepared in 23% over-
all yield from silyl protected (S)-citronellol 7 by reductive ozonolysis
̌
from procedures reported earlier (Fang et al. 1995; Kocovský et al.
1999; Liu et al. 2014) but improving reaction conditions (see below).
Table I. Kinetic parameters for the synthetic substrate peptides
Peptide
KM (µM)
kcat (pep/min)
P11: 5CF-GSDANYTY
P13: 5CF-GSGSDANYTY
P14: 5CF-GSDANYTYTQ
50 10a
ND
37 5a
ND
̌
to aldehyde 8, stereoselective olefination (Kocovský et al. 1999) to
28
3
28
1
form alcohol 9, and chlorination. Intermediate 10 was then coupled
with either the phenylsulfonyl nerol 13c or the phenylsulfonyl E,E-
farnesol 13d, followed by desilylation and reductive removal to afford
the desired lipids 15c, d. Phosphorylation of the four lipids 15a–d
gave precursors 16a–d (Figure 4B). The lipid phosphates 16a–d were
then activated with 1,1′-carbonyldiimidazole and coupled to 5 to
aFor peptide P11, accurate determination of KM and kcat values is not pos-
sible because higher peptide concentrations could not be reached due to solu-
bility limitations.
ND: not determined. For this peptide, the apparent KM value estimated is
higher than the measured concentrations of peptide.
Fig. 3. (A) Structure of the LLO analogs synthesized (1a) (S)-Citronellyl-PP-Chitobiose, C10; (1b) farnesyl-PP-chitobiose, C15; (1c) (S)-NerylCitronellyl-PP-chito-
biose, C20; (1d) (S)-farnesylCitronellyl-PP-chitobiose, C25. (B) Kinetic analysis of synthetic LLO analogs. Glycosylation experiments were performed with 20 nM
purified TbSTT3A, 10 µM peptide P14, 10 mM MnCl2, 150 mM NaCl, 20 mM Hepes pH 7.5, 0.035% DDM, 0.007% CHS and different concentrations of synthetic
LLO analogs. Data points reflect the mean of 3 separate measurements. Error bars indicate standard deviations. Data were fitted by nonlinear regression accord-
ing to the Michaelis–Menten formula using PRISM software. This figure is available in black and white in print and in color at Glycobiology online.

Functional characterization of single-subunit oligosaccharyltransferase STT3A
529
Fig. 4. Synthesis of the LLO analogs used in this study. (A) Synthesis of Chitobiose phosphate precursor (5). (B) Synthesis of lipid phosphate precursors (16a–d).
(C) Coupling reaction and deacetylation of monophosphates 16a–d and 5 to final pyrophosphates 1a–d. Synthetic pathway is shown for 1c, yields and precursor
names for the synthesis of 1a, 1b and 1d are shown in parentheses. This figure is available in black and white in print and in color at Glycobiology online.
Table II. Kinetic parameters for the different synthetic LLO analogs
and inhibitors
yield after purification by chromatography and deacetylation pure
LLOs 1a–d (Figure 4C).
All of our synthetic LLO analogs were recognized as substrates
by TbSTT3A. As illustrated in Figure 3B, the length of the polypre-
nyl chain had a significant effect on the apparent affinity (KM value,
Table II). The highest KM value was observed for the LLO analog
LLO analog
KM (µM)
kcat (pep/min)
(1a) Citronellyl-PP-chitobiose
(1b) Farnesyl-PP-chitobiose
(1c) NerylCitronellyl-PP-chitobiose
(1d) FarnesylCitronellyl-PP-chitobiose
95 18
5.6 0.8
5.3 0.9
2.5 0.3
9.1 0.5
12
23
16
1
1
1
containing only two isoprenyl units (1a, KM of 95
18 µM),
whereas LLO analogs with 3, 4 or 5 isoprenyl units (1b, 1c, 1d)
resulted in lower KM values (5.6 0.8 µM, 5.3 0.9 µM and 2.5
0.3 µM, respectively). The glycosylation rate also increased with the
length of the polyprenyl chain, reaching a maximum when the LLO
Inhibitory LLO
(34b) Farnesyl-PP-C-chitobiose
(34d) FarnesylCitronellyl-PP-C-chitobiose
IC50 (µM)
167 14
26
3

530
AS Ramírez et al.
analog contained four isoprenyl units (C20). Although the length of
the lipid tail had an impact on the LLO affinities by TbSTT3A, the
difference between the kcat values calculated for each LLO analog
was maximum 2-fold (1a, kcat: 9.1 0.5 pep/min and 1c, kcat: 23
1 pep/min), indicating that the efficiency of glycosylation was not
drastically affected by the length of the lipid tail (Table II).
We also tested whether a synthetic LLO analog with a single gly-
can moiety, farnesyl-PP-GlcNAc, could be a substrate for TbSTT3A.
Previous studies showed that farnesyl-PP-GlcNAc stimulated
ATPase activity of the bacterial flippase PglK in detergent (Perez
et al. 2015), and the compound is also accepted as an in vitro sub-
strate of PglB (data not shown). In our studies, no glycopeptide
product was observed when farnesyl-PP-GlcNAc was used, even
after overnight incubation at vast excess substrate concentrations
(Supplementary Fig. 2). This indicates that two GlcNAc units is
the smallest glycan moiety required by TbSTT3A to perform
N-glycosylation.
Synthesis and kinetic analysis of inhibitory
LLO analogs
Fig. 5. (A) Structure of the LLO inhibitors synthesized (34b) farnesyl-PP-C-
chitobiose, C15; (34d) (S)-farnesylCitronellyl-PP-C-chitobiose, C25. (B) Kinetic
analysis of synthetic LLO inhibitors. Glycosylation experiments were per-
formed with 20 nM TbSTT3A protein, 10 µM peptide P11, 10 mM MnCl2,
150 mM NaCl, 20 mM Hepes pH 7.5, 0.035% DDM and 0.007% CHS. Samples
were incubated with LLO competitive inhibitors (1–2500 µM) for 5 min before
adding 15 µM of farnesyl-PP-chitobiose, 1b. Data points reflect the mean of
three separate measurements. Error bars indicate standard deviations. Data
were fitted by nonlinear regression using PRISM software. This figure is
available in black and white in print and in color at Glycobiology online.
Inhibitors based on nonhydrolyzable substrates analogs have been
extensively studied for glycosyltransferases that use nucleotide acti-
vated sugars (Compain and Martin 2001). Among those, substrate
analogs bearing a phosphonate group at the anomeric carbon of the
reducing-end or first sugar moiety (Gordon et al. 2006) or an elon-
gated sugar–phosphate bond (Luengo and Gleason 1992; Schäfer
and Thiem 2000) have been described in the literature. In the case of
transglycolases, which use lipid-linked sugars as substrate donors,
similar inhibitors have been synthesized coupling phosphonate or
elongated phosphate sugars to short lipid carriers (Qiao and
Vederas 1993; Garneau et al. 2004; Lin et al. 2015). In analogy to
these approaches, we designed and synthesized potentially inhibitory
LLO analogs containing a pyrophosphonate moiety, but otherwise
structurally identical to the substrate LLO analogs described above.
We chose the lipid (S),Z,E,E-farnesylcitronellol from the LLO ana-
log 1d, which showed the highest apparent affinity (lowers Km
value) in TbSTT3A assays , as well as the shorter lipid E,E-farnesol
from 1b, which has a higher solubility in water and showed an
apparent affinity only 2-fold lower compared to 1d (Table II). The
resulting compounds 34b and 34d (Figure 5A) contain an unreactive
pyrophosphonate group instead of the pyrophosphate moiety.
The synthesis of the pyrophosphonate analogs required 22 steps for
compound 34b and 31 steps for compound 34d from commercially
available starting materials. The glycosyl acceptor 24 was obtained in
seven steps from N-acetyl-D-glucosamine by acetylation/chlorination,
radical allylation, deacetylation, orthogonal protection of position 4
and 6 with benzaldehyde, benzyl protection of position 3, allyl double
bond isomerization and selective deprotection of position 4 (Bouvet
and Ben 2006) (Figure 6A). The glycosyl donor 28 was obtained in 4
steps from glucosamine hydrochloride as described earlier (Dullenkopf
et al. 1996) (Figure 6B). The coupling reaction between glycosyl
acceptor 24 and donor 28 formed the disaccharide 29, which was
converted into chitobiose phosphonate 32 and finally chitobiose
phosphonic acid 33 (Lin et al. 2015) (Figure 6C). Lipid phosphates
16b and 16d were then activated with 1,1′-carbonyldiimidazole
and coupled with 33 to yield pure LLOs, 34b and 34d, after deace-
tylation and purification by chromatography in moderate yields
(Figure 6D).
to the observations with reactive LLO analogs discussed above, the
length of the polyprenyl chain affected the apparent affinity of the
inhibitors (Figure 5B, Table II). The IC50 value obtained with com-
pound 34b was 167
was 26 3 µM. However, both inhibitors show a significantly lower
apparent affinity (IC50 vs. KM compared to their reactive,
14 µM, whereas that of compound 34d
)
pyrophosphate-containing counterparts.
Discussion
Single subunit OSTs have been mostly characterized in vivo and by
complementation studies in yeast in which the endogenous STT3
gene or other essential components of the multimeric OST complex
were deleted. These studies have shown that some ssOSTs could
replace the catalytic subunit STT3 (Castro et al. 2006; Izquierdo
et al. 2009) or even the whole octameric OST complex from yeast
(Nasab et al. 2008; Hese et al. 2009). Intriguingly, ssOSTs from
T. brucei have been shown to exhibit different preferences towards
LLO donor and sequon acceptors in vivo despite sharing high
sequence identity (Izquierdo et al. 2009, 2012). In vitro studies kine-
tically characterizing ssOSTs have been scarce because of the chal-
lenges associated with their purification in functional form from the
membrane as well as the low availability of substrates. Our work
describes the first large-scale recombinant expression, purification
and characterization of a eukaryotic ssOST.
Optimization of a substrate peptide for our in vitro studies was
achieved by analyzing the effect of variations in the amino acids at
position −2, +1 and +3 of the sequon on glycosylation turnover and
the KM values. We found that the highest rates were obtained when
an acid residue was present at position −2. The crystal structure of
Both compounds 34b and 34d inhibited TbSTT3A-catalyzed gly-
cosylation of acceptor peptides under the conditions tested. Similar

Functional characterization of single-subunit oligosaccharyltransferase STT3A
531
Fig. 6. Synthesis of LLO inhibitors 34b and 34d. (A) Preparation of glycosyl acceptor precursor 24. (B) Synthesis of glycosyl donor precursor 28. (C) Preparation
of chitobiose phosphonate precursor 33. (D) Coupling reaction and deacetylation of monophosphates 16b and 16d with 33 to final pyrophosphates 34b and 34d.
Synthetic pathway is shown for 34d, yields and precursor names for the synthesis of 34b are shown in parentheses. This figure is available in black and white in
print and in color at Glycobiology online.
PglB with bound substrate peptide revealed that the strict require-
ment of an acidic residue at the −2 position of the sequon was due
to the formation of a stabilizing salt bridge with a positively charged
R331 conserve in bacterial PglB homologs (Lizak et al. 2011). A
residue equivalent to R331 from PglB has not been found in eukary-
otic orthologues of STT3 (including TbSTT3A) which may explain
the more relaxed specificity of the protein towards amino acids at
the −2 position. We found that the activity of TbSTT3A is strongly
inhibited by presence of charged residues or, surprisingly, a glycine
at the +1 position of the sequon. This contrasts with findings
described for PglB and for the eukaryotic octameric OST complex,
where the only amino acid not tolerated at the +1 position was pro-
line. Our finding suggests that TbSTT3A has additional structural
requirements for the acceptor peptide, at least in vitro.
Generating sufficiently large amounts of functional LLO analogs
was one of the challenges for the development of in vitro studies

532
AS Ramírez et al.
with eukaryotic OSTs. Our approach of chemically synthesizing
functional LLO analogs was of paramount importance for studying
the interactions of ssOST with the LLO substrate. Our results
showed that several synthetic LLO analogs can serve as glycan
donor substrates of TbSTT3A in vitro, which is in line with findings
reported for the yeast OST complex (Fang et al. 1995). The length
of the isoprenyl tail seems to be an important determinant of the
apparent affinity, but does not influence the glycosylation efficiency
(turnover) of TbSTT3A (Table II), suggesting that it is not involved
in catalytic turnover, as it was previously reported for PglB (Liu
et al. 2014) and the O-oligosaccharyltransferase PglL (Musumeci
et al. 2013). Intriguingly, we did not observe a significant effect of
the presence or absence of a double bond in the first isoprenyl unit
of LLO on turnover, nor of the stereochemistry of the polyprenyl
tail in the affinity of TbSTT3A for the LLO analogs, suggesting that
there is little specificity in the interaction of TbSTT3A and the poly-
prenyl tail of bound LLO.
screened by transient transfection in human embryonic kidney
(HEK293) cells, followed by harvesting, resuspension in lysis buffer
(25 mM Tris pH 8.0, 250 mM NaCl, 10% glycerol) and solubiliza-
tion with a mixture of 1% w/v N-dodecyl-β-D-Maltopyranoside
(DDM), 0.2% w/v cholesteryl hemisuccinate Tris Salt (CHS,
Anatrace). Solubilized samples were analyzed by fluorescence size
exclusion chromatography using a TSKgel G3000SWxl Column
(TOSHO)(Kawate and Gouaux 2006).
Expression and purification of TbSTT3A
A synthetic gene encoding TbSTT3A, optimized for expression in
insect cells was purchased from Thermo Fischer Scientific. It was
fused to a N-terminal His10-YFP tag, and cloned into pOET1 vector
(Oxford Expression Technologies). Baculovirus production was per-
formed using flashBAC DNA (Oxford Expression Technologies) in
Spodoptera frugiperda (Sf9) insect cells following the manufacturer’s
instructions. Infected Sf9 cells were cultured in serum-free SF4
medium (Amimed) at 27 °C for 60 h. Cells were collected by centri-
fugation at 6500 × g and washed with phosphate-buffered saline.
Cell pellets were frozen in liquid nitrogen and stored at −80°C until
the time of use. For purification, cell pellets were thawed and resus-
pended in lysis buffer (25 mM K2HPO4/NaH2PO4, pH 7.0;
250 mM NaCl; 10% w/v Glycerol) supplemented with cOmplete™,
EDTA-free Protease Inhibitor Cocktail (Roche). Lysis was per-
formed by dounce homogenization on ice and the cell lysate was
incubated with 1% (w/v) DDM, 0.2% (w/v) CHS for two hours at
4°C, then submitted to high-speed centrifugation (35,000 rpm, Ti45i
rotor, 30 min). All subsequent buffers contained 0.035% (w/v) DDM,
0.007% (w/v) CHS. The supernatant was loaded onto a Ni/NTA
super flow affinity column (Qiagen), washed with the same lysis buf-
fer but containing 50 mM imidazole and eluted with the same buffer
but containing 200 mM imidazole. The protein was desalted into
20 mM phosphate, pH 7.0; 150 mM NaCl; 5% glycerol (v/v) using
a HiPrep 26/10 column (GE Healthcare) and incubated with home-
produced 3C protease and EndoF1 endoglycosidase overnight at 4°C
(Walker et al. 1994). His10-YFP, 3C and EndoF1 were removed by
incubation with Ni/NTA super flow. TbSTT3A was further purified
by size exclusion chromatography (Superdex 200 10/300 GL, GE
Healthcare) in desalting buffer and peak fractions were pooled and
concentrated to 2 µM for subsequent functional studies. The removal
of the glycans (leaving a single GlcNAc moiety in each glycosylated
residue) was confirmed by mass spectrometry (Chen et al. 2013).
In contrast to farnesyl-PP-chitobiose, which is recognized as a
substrate by TbSTT3A with a remarkably high apparent affinity
(KM: 5.6
0.8 µM), farnesyl-PP-GlcNac, which contained only a
single sugar moiety, was not recognized as a substrate. This suggests
that two GlcNAc units are the minimal glycan structure that can be
processed by TbSTT3A in vitro, which contrasts with earlier studies
performed with partially purified octa-subunit OST complex from
pig liver (Bause et al. 1995) as well as from yeast (Tai and Imperiali
2001). In both these studies, it was concluded that octameric OST
could recognize Dol-PP-GlcNAc as a substrate in vitro, albeit as a
poor donor.
The design and characterization of phosphonate inhibitors for
TbSTT3A was performed in analogy to previous approaches
reported for transglycosylases (Qiao and Vederas 1993; Garneau
et al. 2004; Lin et al. 2015). We synthesized two phosphonate LLO
analogs by varying the length of the polyprenyl tail. Both inhibitory
LLOs carried a phosphonate group coupled to chitobiose. Both pyr-
ophosphonate LLO analogs inhibited TbSTT3A in vitro, although
the affinities of the inhibitors were lower compared to their substrate
counterparts, which might indicate that the presence of the methy-
lene group instead of the oxygen in the natural pyrophosphate bond
has an influence in the binding by TbSTT3A. Intriguingly, for the
bacterial transglycosylase (TGase), lipid II analogs carrying
a
phosphonate group did not inhibit the protein in vitro, but the pres-
ence of an elongated sugar phosphate bond instead led to inhibition
(IC50: 25 μM) (Lin et al. 2015).
In summary, our results showed the in vitro substrate require-
ments of a eukaryotic ssOST enzyme. Purified TbSTT3A is active
and our synthetic compounds faithfully represent native LLOs in
our functional studies. Finally, these LLO analogs together with the
characterized inhibitors will facilitate future structural studies aimed
at visualizing LLO-bound states of OST at high resolution.
Synthesis of acceptor peptides labeled
with 5-carboxyfluorescein
Peptide synthesis was carried out either manually or with the CEM
Liberty Microwave automated peptide synthesizer. Manually, the
synthesis was initiated by loading TentaGel S RAM resin (300 or
500 mg, loading: 0.25 or 0.26 mmol/g) in a 10-mL polypropylene
syringe fitted with a polypropylene frit, a teflon stopcock and a stop-
per. The resin was swollen in DCM (5 mL, 20 min). After removal
of the DCM, the Fmoc-protecting group of the resin was removed
by using a solution of 20% piperidine in NMP. Stirring of the reac-
tion mixture at any given step was performed by attaching the
closed syringes to a rotating axis. The completion of the reaction
was checked using the TNBS test. Removal of the Fmoc-protecting
group of the attached amino acid was performed by using a solution
of 20% piperidine in NMP (5 mL, 2 × 10 min). After filtration, the
resin was washed with NMP (3 × 4 mL2), MeOH (3 × 4 mL2) and
DCM (3 × 4 mL2). Coupling of amino acids was performed by using
Materials and methods
Screening of different STT3 orthologues
The genes encoding the STT3 orthologues from L. braziliensis
(STT3A, STT3B, STT3C), L. infantum (STT3A, STT3B, STT3C),
L. major (STT3A, STT3B, STT3C, STT3D) and T. brucei (STT3A,
STT3B, STT3C) were amplified from previously reported constructs
(Parsaie Nasab et al. 2013) and cloned into a modified pUC57 vec-
tor carrying either a His10-YFP tag at the N-terminus or a YFP-
His10 tag at the C-terminus. Expression of genes fused to YFP was

Functional characterization of single-subunit oligosaccharyltransferase STT3A
533
Fmoc-protected amino acids (3 eq), PyBOP (3 eq) and DIPEA (6 eq)
in NMP (5 mL). The resin was stirred for 2 h before it was washed
three times with 4 mL NMP, MeOH and DCM. Acetylation of the
resin was performed after each amino-acid coupling by using a solu-
tion of acetic anhydride/DCM1:1. The synthesis performed on CEM
Liberty Blue was based on Fmoc solid phase peptide chemistry. The
synthesis was carried out by using Rink Amide MBHA resin
(100–200 mesh), unloaded (0.78 mmol/g), 0.25 mM scale (150 mg
of resin). The coupling reaction was performed in microwave at
75°C (40 W microwave power, 1 × 360 s) with the following para-
meters: 5 eq (relative to resin loading) of Fmoc amino acid and
Oxyma in DMF and 5 eq DIC. Fmoc deprotection was performed
using the default instrument protocol (20% piperidine in DMF, 1 ×
180 s) at 75°C (100 W microwave power). The resin was then
removed from the instrument and transferred to a synthesis syringe
for manual coupling of the 5-carboxyfluorescein. Coupling of 5-CF
was performed by using 5-CF (2 eq), HOBt (5 eq) and DIC (5 eq) in
NMP (5 mL). The resin was stirred overnight and protected from
light using aluminum foil, washed with NMP, MeOH and DCM
(3 × 4 mL2 each). A solution of 20% piperidine in NMP (8 × 4
mL2, until the supernatant was colorless) was added to the resin to
remove the excess of free 5-CF before the resin was finally washed
with DCM (5 × 4 mL2). After coupling of the fluorophore, the com-
pounds were protected from light with aluminum foil at all time.
Trifluoroacetic acid (TFA) cleavage was performed by adding a solu-
tion of TFA/TIS/H2O (94:5:1, v/v/v) to the resin for 1–2 h. The pep-
tide was precipitated with t-BuOMe and dissolved in H2O/MeCN
with 0.1% TFA mixture. Labeled acceptor peptides were purified by
preparative RP-HPLC. Analytical RP-UPLC was performed in Dionex
ULTIMATE 3000 Rapid Separation LC System (ULTIMATE-3000RS
diode array detector) using Dionex Acclaim® RSLC 120 C18 column
(2.2 μm, 120 Å, 3.0 × 50 mm2, flow 1.2 mL/min). Compounds were
detected by UV absorption at 214 nm. Data recording and processing
was performed with Dionex Chromeleon Management System Version
6.80 (analytical RP-HPLC). Preparative RP-HPLC was performed
either with a Waters PrepLC4000 chromatography or Waters Prep
150 LC system using a Dr. Maish GmbH Reprospher Column (C18-
DE, 5 μm, 100 × 30 mm2, pore size 100 Å, flow rate of 60 mL/min).
Compounds were detected by UV absorption at 214 nm using a
Waters 486 Tunable Absorbance detector. All RP-HPLC were using
HPLC-grade acetonitrile and Milli-Q deionized water. The elution
solutions were: A: H2O with 0.1% TFA; B: H2O/MeCN (50:50); C:
H2O/MeCN (10:90) with 0.1% TFA; D: H2O/MeCN (40:60) with
0.1% TFA. MS spectra, recorded on a Thermo Scientific LTQ
OrbitrapXL were provided by the Mass Spectrometry service of the
Department of Chemistry and Biochemistry at the University of Berne.
Characterization of each individual peptide by mass spectrometry can
be found in Supporting information.
diluted 200-fold prior to analysis by Tricine SDS-PAGE consisting
of a 16% resolving gel with 6 M urea, a 10% spacer gel and a 4%
stacking gel (Schägger 2006). Fluorescent bands for peptide and
glycopeptide were visualized by using a Typhoon Trio Plus imager
(GE Healthcare) with excitation set at 488 nm and using a 526 nm
SP emission filter. The amount of formed glycopeptide was deter-
mined from band intensities of fluorescence gel scans (ImageJ)
(Gerber et al. 2013). Turnover rate determination was performed
by fitting of the data to linear regression using PRISM software.
For Michaelis–Menten kinetics of the peptides, reaction mix-
tures containing 20 nM TbSTT3A protein, 50 µM farnesyl-PP-
chitobiose, 10 mM MnCl2, 150 mM NaCl, 20 mM Hepes pH 7.5,
0.035% DDM and 0.007% CHS were incubated with varying con-
centrations (2–60 µM) of peptides P11, P13 and P14. Samples were
taken at different time points and the turnover rate of the reactions
were determined as before. Data were fitted by nonlinear regres-
sion according to the Michaelis–Menten formula using PRISM
software.
For Michaelis–Menten kinetics of the LLO analogs, reaction
mixtures containing 20 nM TbSTT3A protein, 10 µM peptide P11,
10 mM MnCl2, 150 mM NaCl, 20 mM Hepes pH 7.5, 0.035%
DDM and 0.007% CHS were incubated with different concentra-
tions of LLO analogs (10–900 µM, for 1a; 2–60 µM, for 1b;
1–50 µM, for 1c and 1–20 µM, for 1d). Samples were taken at dif-
ferent time points and turnover rates of the reactions were deter-
mined as before. Data were fitted by nonlinear regression according
to the Michaelis–Menten formula using PRISM software.
For inhibition analysis, reaction mixtures containing 20 nM
TbSTT3A protein, 10 µM peptide P11, 10 mM MnCl2, 150 mM
NaCl, 20 mM Hepes pH 7.5, 0.035% DDM and 0.007% CHS
were incubated with different concentrations of LLO competitive
inhibitors (1–2500 µM) for 5 min at 30°C. Reactions were started
with addition of 15 µM of farnesyl-PP-chitobiose, 1b. Samples were
taken at different time points and turnover rates of the reactions
were determined as before. Data were fitted to nonlinear regression
and IC50 was determined using PRISM software.
Supplementary data
Supplementary data are available at Glycobiology online.
Funding
Swiss National Science Foundation (Transglyco Sinergia program to M.A.,
J.-L.R. and K.P.L.).
Acknowledgments
Chemical synthesis of LLO analogs and inhibitors
A detailed description of the synthesis of the LLO analogs can be
found in Supporting information.
We thank Prof. R. Zenobi and M. Köhler for MALDI-MS analysis of TbSTT3A
after deglycosylation (Department of Chemistry and Applied Sciences, ETH
Zurich). We thank the Mass Spectrometry Service (Department of Chemistry
and Biochemistry, University of Berne) for recording MS spectra of synthetic
substrates.
In vitro glycosylation assay
Reaction mixtures contained 20 nM purified TbSTT3A protein,
50µM farnesyl-PP-GlcNAc or farnesyl-PP-chitobiose, 10 mM MnCl2,
150 mM NaCl, 20 mM Hepes pH 7.5, 0.035% DDM, 0.007%
CHS and 20 µM of the peptide. Reactions were incubated at 30°C.
Samples were taken at different time points and reactions were
stopped by the addition of 4X SDS Laemmli buffer. Samples were
Conflict of interest
The authors declare that they have no conflicts of interest with the contents of
this article.

534
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