www.fems-microbiology.org
FEMS Yeast Research 5 (2005) 341–350
Co-expression of two mammalian glycosyltransferasesin the yeast cell wall allows synthesis of sLex
Hanna Salo a,*, Eeva Sievi a, Taina Suntio a, Maria Mecklin a, Pirkko Mattila c,Risto Renkonen c, Marja Makarow a,b
a Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finlandb Department of Applied Chemistry and Microbiology, University of Helsinki, Finland
c Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Finland
Received 8 July 2004; received in revised form 8 October 2004; accepted 24 November 2004
First published online 9 December 2004
Abstract
Interactions between selectins and their oligosaccharide-decorated counter-receptors play an important role in the initiation of
leukocyte extravasation in inflammation. LL-selectin ligands are O-glycosylated with sulphated sialyl Lewis X epitopes (sulpho-sLex).
Synthetic sLex oligosaccharides have been shown to inhibit adhesion of lymphocytes to endothelium at sites of inflammation. Thus,
they could be used to prevent undesirable inflammatory reactions such as rejection of organ transplants. In vitro synthesis of sLex
glycans is dependent on the availability of recombinant glycosyltransferases. Here we expressed the catalytic domain of human
a-1,3-fucosyltransferase VII in the yeasts Saccharomyces cerevisiae and Pichia pastoris. To promote proper folding and secretion
competence of this catalytic domain in yeast, it was fused to the Hsp150D carrier, which is an N-terminal fragment of a secretory
glycoprotein of S. cerevisiae. In both yeasts, the catalytic domain acquired an active conformation and the fusion protein was exter-
nalised, but remained mostly attached to the cell wall in a non-covalent fashion. Incubation of intact S. cerevisiae or P. pastoris cells
with GDP-[14C]fucose and sialyl-a-2,3-N-acetyllactosamine resulted in synthesis of radioactive sLex, which diffused to the medium.
Finally, we constructed an S. cerevisiae strain co-expressing the catalytic domains of a-2,3-sialyltransferase and a-1,3-fucosyltrans-ferase VII, which were targeted to the cell wall. When these cells were provided with N-acetyllactosamine, CMP-sialic acid and
GDP-[14C]fucose, radioactive sLex was produced to the medium. These data imply that yeast cells can provide a self-perpetuating
source of fucosyltransferase activity immobilized in the cell wall, useful for the in vitro synthesis of sLex.
� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Fucosyltransferase; sLex; Yeast; Secretion; Protein production
1. Introduction
Leukocyte tethering to and rolling on endothelial
cells are mediated by the interaction of selectins and
their oligosaccharide-bearing counter-receptors. LL-selec-
tin on leukocytes recognizes heavily O-glycosylated
mucins, GlyCAM-1, CD34 and MAdCAM-1, bearing
1567-1356/$22.00 � 2004 Federation of European Microbiological Societies
doi:10.1016/j.femsyr.2004.11.007
* Corresponding author. Tel.: +358 9 19159418;
fax: +358 9 19159570.
E-mail address: [email protected] (H. Salo).
sulphated sialyl Lewis X (sulpho-sLex) oligosaccharides
[1–6]. The primary role of LL-selectin is to guide lympho-
cytes to peripheral lymphoid tissues, where high endo-
thelial venules act as sites of extravasation. Sulphated
sLex epitopes are selectively expressed in peripheral
lymph node high endothelium. Other endothelial cells
lack sulpho-sLex epitopes, but they can be induced byinflammatory stimuli to express these oligosaccharides
[5,7,8]. Infiltration of lymphocytes is a hallmark of e.g.
acute rejections of solid organ transplants. Exogenous
sLex glycans have been shown both in vitro and in vivo
. Published by Elsevier B.V. All rights reserved.
342 H. Salo et al. / FEMS Yeast Research 5 (2005) 341–350
to inhibit selectin-dependent inflammations [9–14].
Oligosaccharides carrying multiple sLex epitopes were
found to be up to 100-fold more effective inhibitors of
LL-selectin-dependent inflammation as compared to
monovalent sLex [8,15,16].
Pure glycosyltransferases are needed for the synthesisof sLex. Attempts to purify enzymes for sLex synthesis
from human placenta and milk have been reported,
but the yields were insufficient, the enzymatic activities
varied from sample to sample, and contaminating en-
zyme activities were present [15,16]. Mammalian glyco-
syltransferases can be produced in yeast cells, which
are able to secrete proteins and can be grown to high cell
densities in inexpensive growth media. However, mam-malian proteins usually cannot exit the yeast endoplas-
mic reticulum (ER) due to misfolding [17–22]. This
secretion block can be overcome by fusing the heterolo-
gous protein to the Hsp150D carrier, which helps the
heterologous protein to acquire its proper conformation
[23–26]. The Hsp150D carrier consists of an N-terminal
fragment of Hsp150, a glycoprotein of S. cerevisiae,
which is secreted rapidly and efficiently to the yeast cul-ture medium [27,28]. We have earlier been able to ex-
press the catalytic domain of rat a-2,3-sialyltransferase(ST3Ne) as an Hsp150D fusion protein (Hsp150D–ST3Ne) in S. cerevisiae and P. pastoris [18,19,29]. The
glycosyltransferase portion was found to acquire a cata-
lytically active, disulfide-bonded and N-glycosylated
form in the yeast ER, and Hsp150D–ST3Ne was trans-
ported to the exterior of the cells. However, the fusionprotein was not secreted to the culture medium, but re-
mained attached to the porous cell wall [18,19,29]. Thus,
intact yeast cells could be used as an enzyme source for
the synthesis of sialyl-a-2,3-N-acetyllactosamine from
CMP-NeuNAc and N-acetyllactosamine [18,19,29].
Here we expressed the catalytic domain of human
a-1,3-fucosyltransferase VII (FucTe) [30,31] in S. cerevi-
siae and P. pastoris as the fusion protein Hsp150D–FucTe. The fusion protein was externalized, but
remained mostly bound to the cell wall of both yeasts
in a catalytically active form. Furthermore, a recombi-
nant S. cerevisiae strain co-expressing Hsp150D–FucTeand Hsp150D–ST3Ne was constructed. This yeast strain
harboring both transferase activities in the cell wall was
able to synthesize sLex from N-acetyllactosamine,
CMP-NeuNAc and GDP-[C14]Fuc.
2. Materials and methods
2.1. Strain construction and media
To create an S. cerevisiae strain expressing Hsp150D–FucTe (see Fig. 1(c)), a cDNA fragment encodingFucTe [30,31] was synthesized by PCR using primers
5226 (5 0-CAATGGTACCCCGGCACCCCAGCC-
CAC-3 0) and 5227 (5 0-ATGTAAGCTTCAGGCCT-
GAAACCAACCCTCAAGGTCCTC-3 0). The PCR
fragment was digested with KpnI/HindIII and ligated
into plasmid pKTH4636 [18] containing the cDNA cod-
ing for the Hsp150D carrier, creating plasmid
pKTH4641 containing the cDNA for the entire fusionprotein Hsp150D–FucTe under the control of the
HSP150 promoter. This plasmid was transformed into
S. cerevisiae strain H23 from which the HSP150 gene
had been deleted (Mata his 3-11 15 leu2-3 112 trp1-1
ade2-1 can1-100 hsp150::URA3) to create strain H649.
To create an S. cerevisiae strain expressing both
Hsp150D–ST3Ne (see Fig. 1(f)) and Hsp150D–FucTe,plasmid pKTH4641 (see above) was digested withNheI/XbaI, and the resulting fragment coding for
Hsp150D–FucTe, the HSP150 promoter and the
ADH1 terminator was ligated into plasmid pFL26, cre-
ating pKTH4748. pKTH4748 was used to transform S.
cerevisiae strain H626, which already contained the
cDNA encoding the fusion protein Hsp150D–ST3Ne
(Mata his3-11 15 leu2-3 112 ade2-1 can1-100
hsp150::URA3 TRP1::HSP150D�ST3Ne) [18]. Theresulting strain H970 thus expressed both Hsp150D–ST3Ne and Hsp150D–FucTe under the control of the
HSP150 promoter. S. cerevisiae strains were cultivated
at 24 �C in YPD medium overnight. For genotypes
and source of the yeast strains see Table 1, and for re-
combinant proteins see Fig. 1.
To construct a recombinant P. pastoris strain express-
ing Hsp150D–FucTe (Fig. 1(d)), the FucTe cDNA wassynthesized by PCR using primers F0674 (5 0-
GAATTCCCGGCACCCCAGCCCACGATC-3 0) and
F0675 (5 0-TCTAGATCAGGCCTGAAACCAACCCT-
CAAGGTCCTC-3 0) and plasmid pKTH4641 (see
above) as a template. The PCR fragment was inserted
into the pGEM�-T Easy vector (Promega) creating plas-
mid pKTH5124. This additional cloning step was
exploited to facilitate the following restriction reactions.pKTH5124 was EcoRI/XbaI-digested and the resulting
FucTe cDNA was inserted into pKTH4678 to create
plasmid pKTH5152 containing the cDNA elements for
the Hsp150D carrier, Kex2p cleavage site and the cata-
lytic domain of FucTVII. pKTH5152 was electropora-
ted into P. pastoris strain P714 (X-33 WT, Invitrogen)
to yield strain P1755 expressing Hsp150D–FucTe under
the control of the AOX1/2 promoter. PlasmidpKTH4678 (see above), which contained the cDNA
encoding the Hsp150D carrier followed by a Kex2p pro-
tease cleavage site, was constructed as follows. The
cDNA for the signal peptide of mating factor a was syn-
thesized by PCR using primers 7173 (5 0-CAT-
CAGATCACGTGAGCTAATGCGGAGGATGC-3 0)
and 7186 (5 0-ACTAGTTCGAAACGATGAGATTT-
CC-3 0) using plasmid pPICZaA (Invitrogen, Carlsbad,CA, USA) as a template. The PCR fragment was ligated
into the BstBI/PmlI site of plasmid pPICZaB (Invitro-
Fig. 1. Fusion proteins. (a) Hsp150 consists of a signal peptide (SP) and subunits (SU) I and II. SUII is composed of 11 repeats of homologous
peptides of mostly 19 amino acids, and a unique C-terminal fragment. (b) Human a-1,3-fucosyltransferase VII (FucT) consists of an N-terminal
cytoplasmic tail of 14 amino acids, a transmembrane domain (amino acids 15–36) and a catalytic domain (FucTe) of 305 amino acids. (c) Hsp150D–FucTe for expression in S. cerevisiae. FucTe was fused to the C-terminus of the Hsp150D carrier, which consists of the 321 first amino acids of
Hsp150. (d) Hsp150D–FucTe for expression in P. pastoris. FucTe was fused to the Hsp150D carrier, but the signal peptide is that of S. cerevisiae
mating factor a. The FucTe and Hsp150D portions are separated by a recognition site of the Kex2 protease, the amino acids of which are indicated.
(e) Rat liver a-2,3-sialyltransferase (ST3N) consists of an N-terminal cytoplasmic tail of eight amino acids, a transmembrane domain (amino acids 9–
28) and a catalytic domain (ST3Ne) of 346 amino acids. (f) Hsp150D–ST3Ne for expression in S. cerevisiae. ST3Ne was joined to the C-terminus of
the Hsp150D carrier. (g) Hsp150D–ST3Ne for expression in P. pastoris. ST3Ne is joined to the C-terminus of the Hsp150D carrier, but the signal
peptide (amino acids 1–19) is derived from S. cerevisiae mating factor a.
Table 1
Yeast strains
Relevant mutation Recombinant protein Source/reference
S. cerevisiae strain
H1 None R. Schekman
H23 Dhsp150 [28]
H626 Dhsp150 Hsp150D–ST3Ne [18]
H649 Dhsp150 Hsp150D–FucTe This study
H970 Dhsp150 Hsp150D–ST3Ne,
Hsp150D–FucTeThis study
P. pastoris strain
P714 None Invitrogen
P1402 None Hsp150D–ST3Ne [19]
P1755 None Hsp150D–FucTe This study
H. Salo et al. / FEMS Yeast Research 5 (2005) 341–350 343
gen) to create plasmid pKTH4676. The cDNA for theHsp150D carrier was synthesized by PCR using primers
7187 (5 0-CATCAGATGGTACCCAGAAGTCTTAC-
AGGA-3 0) and 7188 (5 0-ACTAGCACGTGGCCTA-
TGCTCCATCTGAGCC-3 0), using pKTH4570 [32] as
a template. The PCR fragment was PmlI/KpnI-digestedand ligated into the PmlI/KpnI site of plasmid
pKTH4676, creating plasmid pKTH4677. Bluescript
SK+ (Stratagene, La Jolla, CA, USA) was used as a
template to synthesize a cDNA fragment encoding b-lactamase by PCR using primers 7189 (5 0-CAT-
CAGTCTAGATTACCAATGCTTAATCAGTGAGG-30)
and 7190 (5 0-ACTAGGAATTCGCTCACCCAGAAA-
CGCTGG-3 0). The PCR fragment was ligated intoEcoRI/XbaI site of plasmid pPICZaA (Invitrogen), to
create plasmid pKTH4679. The cDNA for b-lactamase
flanked with Kex2 protease cleavage site was cut from
plasmid pKTH4679 with XhoI/XbaI digestion, and the
resulting fragment was ligated into XhoI/XbaI-digested
plasmid pKTH4677 creating plasmid pKTH4678. This
plasmid was used to provide us with cDNA elements
for the Hsp150D carrier followed by the Kex2p cleavagesite in order to create plasmid pKTH5152 with HSP150-
KEX2p-FUCTe.
344 H. Salo et al. / FEMS Yeast Research 5 (2005) 341–350
P. pastoris strains P1402 (Table 1) (X-33 WT,
HSP150D-ST3Ne zeocin�) [19] and P1755 were culti-
vated at 30 �C in buffered minimal glycerol yeast extract
(BMGY, 0.1% glycerol, v/v, Invitrogen) for 16–20 h,
and diluted in buffered minimal methanol yeast extract
(BMMY, 0.5% methanol, v/v, Invitrogen) to OD 1.0.Methanol was added every 24 h to induce expression
of the recombinant gene.
2.2. Pulse chase experiments and Western blotting
Labelling with [35S]methionine/cysteine (50 lCiml�1,
1000 Cimmol�1, Amersham Pharmacia Biotech, Little
Chalfont, UK), chase with cycloheximide (Sigma, St.Louis, MO, USA), tunicamycin (Riedel-de Haen, Seelze,
Germany) treatment, cell lysis and immunoprecipitation
with Hsp150 antiserum (1:400) were as described in [27].
The cell lysates were obtained by vortexing the cells in
the presence of glass beads at 4 �C after which the cells
remnants were pelleted by centrifugation. Western
immunoblot analysis was with Hsp150 antiserum
(1:10000), followed by HRP-conjugated anti-rabbit-IgG (1:10000, Promega) and ECL detection (Amersham
Pharmacia Biotech).
2.3. Cell wall isolation
[35S]Methionine/cysteine-labeled cells were lysed by
vortexing at 4 �C with glass beads in 10 mM Tris–
HCl, pH 7.0, containing 1 mM PMSF (Sigma) and cen-trifuged (3000g, 10 min). The pellet containing the cell
walls was washed three times with 1 M NaCl (Riedel-
de Haen) and 1 mM PMSF. The isolated cell walls were
boiled twice in 2% SDS (ICN Biomedicals, Irvine, CA,
USA) containing 100 mM EDTA (GIBCO BRL, Carls-
bad, CA, USA) and 40 mM b-mercaptoethanol (Sigma)
to release non-covalently bound proteins, and the ex-
tract diluted 1:10 for immunoprecipitation. For isola-tion of covalently bound proteins, the SDS-extracted
cell walls were pelleted and washed with 1 M NaCl con-
taining 1 mM PMSF, and digested overnight at 37 �Cwith b-1,3-glucanase (Quantazyme, Quantum Biotech-
nologies, Irvine, CA, USA) in 50 mM Tris–HCl, pH
7.5, containing 40 mM b-mercaptoethanol [33].
2.4. Transferase activity assays
To assay cell wall-bound FucT activity, the yeast cells
(5 · 107) were pelleted and resuspended in 50 ll of 20
mM MOPS buffer (Sigma), pH 7.0, containing 10 mM
NaN3 (Riedel-de Haen), 20 mM MnCl2 (J.T. Baker,
Phillipsburg, NJ, USA) and 10 mM ATP (Sigma), and
incubated with sialyl-a-2,3-N-acetyllactosamine (50 lg;210 nmol, Sigma) and GDP-[14C]fucose (200000 cpm;0.5 nmol, Amersham Pharmacia Biotech) for 18 h at
24 �C. The respective culture medium samples were con-
centrated with a Vivaspin 6 ml concentrator (Viva-
science, Hannover, Germany) to the final volume of 20
ll. Then, 100 ll of 20 mM MOPS buffer, pH 7.0, was
added and the concentration was repeated to the final
volume of 20 ll. Then 20 ll of MOPS was added, and
NaN3, MnCl2 and ATP to the final concentrations of10, 20 and 10 mM, respectively. Incubation with sialyl-
a-2,3-N-acetyllactosamine and GDP-[14C]fucose was as
above. To assay the intracellular activity, pelleted cells
(5 · 107) were resuspended in 50 ll of 20 mM MOPS
buffer, pH 7.0, containing 1% Triton X-100 (Merck), 1
mM PMSF (Sigma), 1 lg/ml leupeptin (Sigma), 6.8 lg/ml aprotinin (Boehringer, Mannheim, Germany), 10
mM NaN3, 20 mM MnCl2 and 10 mM ATP, and lyzedwith glass beads by vortexing for 6 min at +4 �C. Theglass beads and cell remnants were let to sediment,
and the supernatant was transferred to a new tube. Incu-
bation with sialyl-a-2,3-N-acetyllactosamine and GDP-
[14C]fucose was as above. To assay the combined
a-2,3-sialyltransferase and a-1,3-fucosyltransferase VII
activities, whole yeast cells (5 · 107 cells) were incubated
with N-acetyllactosamine (80 lg; 210 nmol, Sigma),CMP-NeuNAc (133 lg; 210 nmol, Sigma) and GDP-
[14C]fucose (200000 cpm; 0.5 nmol, Amersham Pharma-
cia Biotech) in 20 mM MOPS buffer, pH 7.0, containing
10 mM NaN3, 20 mM MnCl2 and 10 mM ATP, for 18 h
at 24 �C. The above reaction mixtures were desalted on
columns of Dowex AG 1 (Ac�, BioRad, Hercules, CA,
USA) and Dowex 50 (H+, Fluka, St. Louis, MO, USA)
eluted with 6 ml of deionised water followed by 20 ml of0.5 M acetic acid (Riedel-de Haen) [34]. Radioactivity of
the eluted fractions was counted in OptiPhase �HiSafe�3(Perkin–Elmer, Wellesley, MA, USA) using an LKB
1214 RACKBETA (Wallac, Turku, Finland) liquid scin-
tillation counter.
The Km value of Hsp150D–FucTe for the acceptor
glycan sialyl-a-2,3-N-acetyllactosamine was determined
according to Lineweaver–Burk essentially like describedbefore [29]. To this end, H649 cells (108) were incubated
with varying concentrations of sialyl-a-2,3-N-acetyllac-
tosamine (0.5–300 lg) in the presence of 5 mM GDP-
Fuc (200000 cpm GDP-[14C]Fuc) for 18 h at 24 �C.The supernatants were subjected to ion exchange chro-
matography over Dowex columns, and the radioactivity
of the eluted fractions was counted as described above.
3. Results
3.1. Expression of Hsp150D–FucTe in S. cerevisiae
The cDNA fragment encoding the Hsp150D carrier
(Fig. 1(a), amino acids 1–321) was fused to that encod-
ing the catalytic domain of human a-1,3-fucosyltransfer-ase VII (FucTe) (Fig. 1(b), amino acids 37–341). The
chimeric cDNA encoding Hsp150D–FucTe (Fig. 1(c))
H. Salo et al. / FEMS Yeast Research 5 (2005) 341–350 345
was integrated into the genome of S. cerevisiae strain
H23, from which the HSP150 gene had been deleted,
creating strain H649 (Table 1). The Hsp150D portion
is heavily O-glycosylated with di-, tri-, tetra- and pen-
tamannosides [28], and the FucTe portion has two po-
tential N-glycosylation sites (arrows in Fig. 1(b)). Tostudy whether the fusion protein was expressed, the cells
were pulse-labeled with 35S-methionine/cysteine, and
chased with cycloheximide. Tunicamycin was present
to inhibit N-glycosylation of the FucTe portion, because
in S. cerevisiae the N-glycans may be extended with
more than 200 mannose residues resulting in poorly
detectable smears in SDS–PAGE [35]. It should be
noted that in yeast prevention of N-glycosylation usu-ally does not inhibit ER exit, like in mammalian cells.
The chimeric genes were placed under the control of
the HSP150 promoter, which confers basal expression
at the physiological temperature of 24 �C and is up-reg-
ulated when the cells are shifted to 37 �C [19,36]. Thus,
the incubations were mostly at 37 �C to up-regulate
expression of the recombinant gene. After the pulse, a
protein of 102 kDa predominated in the immune precip-itate obtained with Hsp150 antiserum from the cell ly-
sates (Fig. 2, lane 2). After 30 min of chase, the 102
kDa species had disappeared. Instead, a broad band of
about 150 kDa was detected, plus a fainter band of
120 kDa (lane 4). No protein could be detected in the
medium after pulse (lane 1) or chase (lane 3). A 60-
min chase gave similar results (not shown). No protein
could be immunoprecipitated with Hsp150 antiserumfrom lysates of the parental cells (lane 5). We suggest
that the 150-kDa species represents the mature, exten-
sively O-glycosylated fusion protein, which was not se-
creted to the medium, but remained cell-associated,
Fig. 2. Metabolic labeling of Hsp150D–FucTe. S. cerevisiae strain
H649 expressing Hsp150D–FucTe was preincubated for 15 min with
tunicamycin at 37 �C, labeled with [35S]methionine/cysteine for 5 min
(lanes 1 and 2), and chased for 30 min (lanes 3 and 4). Strain H23
lacking theHSP150D–FUCTe gene was pulse-chased similarly (lane 5).
Medium (m) and cell lysate (c) samples were immunoprecipitated with
Hsp150 antibody before SDS–PAGE analysis. Molecular weight
markers (kDa) are indicated on the left, and different Hsp150–FucTe
species (kDa) on the right (for details of the experimental setup, see
Section 2).
and was thus intracellular or cell wall-bound. The 102-
kDa species (lane 2) was probably the primary O-glycos-
ylated form located in the ER. Its single O-linked
mannose residues were apparently extended during the
chase, resulting in the 120-kDa species. This was proba-
bly brought about by Golgi glycosyltransferases, whichhave been shown to recycle between the Golgi and the
ER, and to glycosylate in the ER target glycoproteins,
which remain there for a sufficiently long time [37].
Retention of part of the Hsp150D–FucTe molecules in
the ER was likely due to problems in protein folding.
3.2. Binding of Hsp150D–FucTe to the S. cerevisiae cell
wall
Next we studied whether Hsp150D–FucTe remained
intracellular or was transported to the yeast cell wall.
To this end, S. cerevisiae strain H649 was pulse-labeled
with [35S]-methionine/cysteine and chased for an hour.
After the chase, one cell lysate sample was immunopre-
cipitated with Hsp150 antiserum directly, and a parallel
sample after proteinase K digestion of the intact cells inthe presence of DTT. Both samples were subjected to
SDS–PAGE analysis. Mature Hsp150D–FucTe of 150
kDa was detected in the absence of proteinase K diges-
tion, whereas it had disappeared after the digestion.
Thus, Hsp150D–FucTe most probably was located in
the cell wall and not inside the cells. To confirm that
proteinase K had had access to the cell wall but not to
intracellular proteins, parallel samples of labeled intactcells before and after proteinase K treatment were lysed,
and subjected to SDS–PAGE and Western blotting with
antiserum against cytosolic GAPDH. Both samples gave
similar signals suggesting that proteinase K had not at-
tacked cytosolic components. When the experiment was
repeated using antiserum against the cell wall protein
Bgl2p, only the untreated cell sample gave a signal,
implying that proteinase K had destroyed cell wall-bound Bgl2p (not shown).
Many secretory glycoproteins of S. cerevisiae remain
bound to the cell wall, either non-covalently, or cova-
lently via b-1,3-linkages to cell wall glucan [38–40]. Next
we studied whether Hsp150D–FucTe was bound cova-
lently to the cell wall b-glucan. S. cerevisiae cells were
metabolically labeled in the presence of tunicamycin,
and the cell lysates were subjected to immunoprecipita-tion with Hsp150 antiserum and SDS–PAGE analysis
(Fig. 3(a), lane 1). A parallel cell sample was subjected
to removal of the cell walls and the remaining sphero-
plasts were immunoprecipitated (lane 2). The isolated
cell walls were extracted with SDS, and the
extract was immunoprecipitated (lane 3). A parallel
SDS- extract of released cell walls was digested with
b-1,3-glucanase, and the released material was immuno-precipitated with Hsp150 antiserum (lane 4).
SDS–PAGE analysis showed that cell-associated
346 H. Salo et al. / FEMS Yeast Research 5 (2005) 341–350
Hsp150D–FucTe (lane 1) could be released with SDS
only (lane 3), and was thus bound non-covalently to
the cell wall. Authentic Hsp150 (Fig. 1(a)) expressed in
normal cells lacking recombinant genes (H1) served as
a control. We have shown before that part of Hsp150
is bound covalently to the cell wall, though most of itis secreted to the culture medium [33]. SDS was not able
to remove Hsp150 from the cell walls (Fig. 3(b), lane 3),
whereas b-1,3-glucanase was (lane 4).
3.3. Pichia pastoris strain construction
The methylotrophic yeast Pichia pastoris can be
grown to high cell density in inexpensive growth media,and it can produce heterologous proteins in high yields.
The heterologous gene expression in P. pastoris is based
on a strong and regulatable alcohol oxidase promoter,
and a number of proteins have been successfully ex-
pressed using P. pastoris as a host organism [41,42]. P.
pastoris strain P714 was transformed with an expression
vector coding for Hsp150 D-FucTe, which harbored a
recognition site for the Golgi-located Kex2 protease be-tween the Hsp150D carrier and the FucTe portion (Fig.
1(d)). No tags were added to the FucTe portion, because
tagging of the C-terminus of ST3Ne inactivated
Hsp150D–ST3Ne (Sievi, E., and Makarow, M., unpub-
lished data) as well as fucosyltransferase of Arabidopsis
thaliana [43]. The HSP150D–FUCTe gene was expressed
Fig. 3. Location of Hsp150D–FucTe in S. cerevisiae cells. (a) S.
cerevisiae strain H649 was metabolically labeled for 1 h at 24 �C and
chased for 30 min. One cell sample was lyzed and immunoprecipitated
(lane 1). Another cell sample was subjected to removal of cell walls,
and the remaining spheroplasts were immunoprecipitated (lane 2). The
released cell wall material was extracted with SDS, and the extract was
immunoprecipitated (lane 3). A parallel SDS extract of cell walls was
digested with b-1,3-glucanase and the released material was immuno-
precipitated (lane 4). All samples consisted of the same amount of
yeast cells. (b) The above experiment was repeated with S. cerevisiae
strain H1 expressing only authentic Hsp150. The immunoprecipitates
were subjected to SDS–PAGE analysis.
under the methanol-inducible AOX1/2 promoter, and
transformants were screened after 12 days of growth
on methanol by Western blotting using Hsp150 antise-
rum (not shown). The transformant giving the strongest
signal was chosen and named P1755 (Table 1).
3.4. FucT activity in S. cerevisiae and P. pastoris
Next we studied whether Hsp150D–FucTe expressed
in S. cerevisiae and P. pastoris was catalytically active.
S. cerevisiae strain H649 was cultivated overnight, and
the cell wall-located FucT activity was determined by
incubating intact cells with sialyl-a-2,3-N-acetyllactos-
amine and GDP-[14C]Fuc as described in Section 2.According to ion exchange chromatography, 32600
cpm of [14C]Fuc had been transferred to sialyl-a-2,3-N-acetyllactosamine, yielding NeuNAca-2,3-Galb-1,4([14C]Fuca1, 3)GlcNAc (designated radioactive sLex
from hereon). The Km value of yeast cell wall-immobi-
lized Hsp150D–FucTe for sialyl-a-2,3-N-acetyllactos-
amine was 1.7 mM (not shown). In the case of the
parental strain H23, only 970 cpm was detected in theeluate. Thus, S. cerevisiae strain H649 appeared to har-
bor catalytically active FucTe in the cell wall.
Next we cultivated strain P1755 in methanol for
three, six and nine days to induce the expression of the
HSP150D–FUCTe gene. The FucT activity of culture
medium (Fig. 4, columns A), spheroplast lysates (Fig.
4, columns B), as well as intact cells (Fig. 4, columns
C) was determined. Most of the FucT activity was foundin the intact cells, i.e. in the cell wall, and only little in
the medium or inside the cells. The FucT activity
reached a maximum after six days of induction.
Whether the activity was due to Hsp150D–FucTe or
the FucTe portion released by Kex2p cleavage is not
known.
3.5. Team work of FucT and ST3N in the yeast cell wall
Human a-1,3-fucosyltransferase VII can transfer
fucose only to sialylated N-acetyllactosamine
[30,31,44,45]. Next we studied whether Hsp150D–ST3Ne
(Fig. 1(f)) and Hsp150D–FucTe (Fig. 1(c)) could work in
tandem, while present in the cell wall of two separate S.
cerevisiae strains, which had been mixed in the same test
tube. Thus, the two strains were cultivated separatelyovernight and thereafter combined to the same test tube.
Then, N-acetyllactosamine, CMP-NeuNAc and GDP-
[14C]Fuc were added, with the anticipation that
Hsp150D–ST3Ne would transfer NeuNAc to N-acetyl-
lactosamine, and thereafter the product, sialyl-a-2,3-N-
acetyllactosamine, would be [14C]fucosylated by
Hsp150D–FucTe. Indeed, the reaction produced more
than 11,000 cpm of radioactive saccharide, i.e. sLex(Fig. 5(A), column a). Thereafter, we transformed S.
cerevisiae strain H626 expressing Hsp150D–ST3Ne (see
Fig. 4. FucT activity in P. pastoris. P. pastoris strain P1755 was grown
overnight, whereafter induction of the HSP150D–FUCTe gene was
started by shifting the cells to methanol-containing medium. The
cultivation was continued at 30 �C for nine days. The amount of
NeuNAca-2,3Galb-1,4([14C]Fuc1,3)GlcNAc produced by parallel
samples (black and white columns) of culture medium (A; secreted
activity), spheroplast lysates (B; intracellular activity) and intact cells
(C; cell walls) was determined on days 3, 6, and 9.
Fig. 5. ST3N and FucT activities in the yeast cell wall. (A) S. cerevisiae
strains H626 and H649 expressing Hsp150D–ST3Ne and Hsp150D–FucTe, respectively, were cultivated overnight. Cells of both strains
were pelleted and resuspended together to the same test tube. N-
acetyllactosamine, CMP-NeuNAc and GDP-[14C]Fuc were added to
the reaction mixture, and the amount of NeuNAca-2,3Galb-1,4([14C]Fuca-1,3)GlcNAc was determined after 18 h of incubation
at 24C (column a). The same experiment was repeated using S.
cerevisiae strain H970 co-expressing Hsp150-ST3Ne and Hsp150D–FucTe (column b), the parental strain H23 (column c), and strain H649
expressing Hsp150D–FucTe only (column d). (B) The P. pastoris
strains were cultivated for seven days on methanol before harvesting
the cells for the experiments. Strains P1402 and P1755 (expressing
Hsp150D–ST3Ne and Hsp150D–FucTe, respectively) were combined
to the same test tube, and N-acetyllactosamine, CMP-NeuNAc and
GDP-[14C]Fuc were provided for 18 h like above (column a). P1402
cells were resuspended into a concentrate of culture medium of P1755
cells (see Section 2), and the experiment was repeated as above (column
b). The parental cells P714 (column c), and P1755 cells (column d) were
subjected to the same experiment. The amount of NeuNAca-2,3Galb-1,4([14C]Fuca-1,3)GlcNAc produced in each experiment is plotted.
H. Salo et al. / FEMS Yeast Research 5 (2005) 341–350 347
Fig. 1(f)) [18] with cDNA encoding Hsp150D–FucTe(see Fig. 1(c)), creating strain H970. When these cellswere provided with N-acetyllactosamine, CMP-Neu-
NAc and GDP-[14C]Fuc, nearly the same amount of
radioactive sLex was produced (Fig. 5(A), column b)
as in the previous experiment. The parental strain lack-
ing recombinant genes was not able to produce this
radioactive saccharide (Fig. 5(A), column c). Neither
could the strain expressing only Hsp150D–FucTe alone
synthesize radioactive sLex (Fig. 5(A), column d), be-cause a-1,3-fucosyltransferase VII is able to fucosylate
only a-2,3-sialylated Galb1-4GlcNAc. We conclude that
Hsp150D–ST3Ne and Hsp150D–FucTe were able to
work in tandem whether co-expressed in one strain, or
expressed in two separate strains.
Finally, we verified that Hsp150D–ST3Ne (Fig. 1(g))
and Hsp150D-FucTe (Fig. 1(d)) were able to perform
teamwork also in the cell walls of P. pastoris. StrainP1402 expressing Hsp150D–ST3Ne (Table 1) [19] and
strain P1755 expressing Hsp150D–FucTe were induced
for seven days with methanol, combined to the same test
tube and incubated with N-acetyllactosamine, CMP-
NeuNAc and GDP-[14C]Fuc like above. 32,000 cpm of
radioactive sLex was obtained (Fig. 5(B), column a). Be-
cause some of the FucT activity was found to diffuse to
the culture medium of P1755 cells (see Fig. 4, column a),this was concentrated for activity assays. P1402 cells
were then resuspended into the concentrate, followed
by the incubation with N-acetyllactosamine, CMP-Neu-
NAc and GDP-[14C]Fuc like above. Much less radioac-
tive sLex was produced (Fig. 5(B), column b) than in the
case of intact P1755 cells, consistent with the notion that
most of the fusion proteins remained in the cell wall. The
parental strain P714 produced negligible amounts of
radioactive sLex (Fig. 5(B), column c). The same was
true for strain P1755 alone, as N-acetyllactosaminecould not be sialylated (Fig. 5(B), column d).
We conclude that the catalytic domains of human
fucosyltransferase VII and rat a-2,3-sialyltransferasecan function in tandem in the cell wall of intact yeast
348 H. Salo et al. / FEMS Yeast Research 5 (2005) 341–350
cells, and synthesize sLex from CMP-sialic acid, GDP-
fucose, and N-acetyllactosamine.
4. Discussion
Complex carbohydrates of cell surface glycoconju-
gates are known to take part in numerous biological
functions such as tumorigenesis, tissue differentiation
and leukocyte adhesion during inflammatory processes
[46]. The development of pharmaceuticals based on
e.g. sialyl Lewis X antigen requires large-scale synthesis
of oligosaccharides in vitro. The drawbacks of chemical
synthesis, which include tedious protection and depro-tection steps, can be avoided by using glycosyltransfe-
rases. The advantages of glycosyltransferases in
oligosaccharide synthesis include regioselectivity and
stereospecificity [47]. However, a major shortcoming is
the lack of availability of glycosyltransferases. For this
reason, a great deal of effort has been directed towards
cloning and heterologous expression of glycosyltransfe-
rases. Here we report the expression of the catalytic do-main of human FucTVII (FucTe) in S. cerevisiae and P.
pastoris. Several mammalian Golgi glycosyltransferases,
b-1,4-galactosyltransferase, a-2,6-sialyltransferase and
a-2,3-sialyltransferase, have been shown to misfold in
the yeast ER when expressed as full length proteins or
soluble catalytic domains [22,29,48,49]. Thus, we fused
FucTe to the Hsp150D carrier derived form the secre-
tory S. cerevisiae glycoprotein Hsp150. We have shownthat the Hsp150D carrier promotes proper folding of
several heterologous proteins in the yeast ER, and con-
fers them secretion competence both in S. cerevisiae and
P. pastoris [18,19,23–27,29,50]. The newly synthesized
fusion protein Hsp150D–FucTe was translocated into
the yeast ER, and the FucTe portion acquired a catalyt-
ically active conformation. Instead of being transported
all the way to the culture medium, most of Hsp150D–FucTe remained attached to the porous cell wall in the
case of both S. cerevisiae and P. pastoris. Incubation
of intact S. cerevisiae or P. pastoris cells with GDP-
[14C]fucose and sialyl-a-2,3-N-acetyllactosamine resulted
in synthesis of radioactive sLex, which diffused to the
medium. Abe et al. [51] fused the catalytic domain of hu-
man a-1,3-fucosyltransferase VI to full-length Hsp150
(also called Pir2p), and observed FucTVI activity in theS. cerevisiae cell wall. The catalytic domain of FucTVI
has been expressed in P. pastoris with the aid
of the S. cerevisiae-derived MFa signal peptide, and 1 l
of shake-flask culture produced 3 U of the enzyme to
the medium [52]. The catalytic domain of FucTIII fused
to the prepro fragment of MFa was expressed in P. pas-
toris. First the activity was found in the cell wall, [53], but
after seven days of fed-batch fermentation themajority ofthe enzymatic activity was in the culture medium, and
11.3 U of the enzyme could be harvested from 1 l [54].
Full-length human FucTVII has been expressed in
mammalian cells (CHO and COS-7) and in insect cells
(Trichoplusia ni) [30,44,55,56]. Due to membrane
anchoring of the transferase, cell lysates were used as en-
zyme source. This was problematic because of interfer-
ing enzyme activities and difficulties in purification ofthe product from the lysates. The human FucTVII cata-
lytic domain has been fused to protein-A and expressed
in B cell lymphoma (Namalwa KJM-1) cell line and in
insect cells (Spodoptera frugiperda). In both cases the fu-
sion protein was secreted to the culture medium and
affinity-purified with IgG-Sepharose [31,45,57]. Twenty
five litres of Namalwa KJM-1 cell culture yielded 2.6
mg of purified fusion protein, and 250 ml culture ofSf9 cells 2.4 mg. The purified fusion protein specifically
fucosylated a-2,3-sialylated type-2 oligosaccharide
acceptors catalyzing the formation of sLex antigen
[31,45,57].
In this study, Hsp150D–FucTe was bound to the
yeast cell wall non-covalently via the FucTe portion.
Immobilization of Hsp150D-FucTe to the cell wall al-
lowed us to use intact yeast cells as an enzyme source,as shown before for b-1,4-galactosyltransferase [21]
and a-2,3-sialyltransferase [18,29]. sLex was synthesized
simply by incubating the recombinant yeast cells with
sialyl-a-2,3-N-acetyllactosamine and GDP-[14C]Fuc.
We determined the Km value of yeast cell wall
Hsp150D–FucTe to be 1.7 mM for sialyl-a-2,3-N-acetyl-
lactosamine. The Km value of FucTVII expressed in
Trichoplusia ni insect cells for sialyl-a-2,3-N-acetyllac-tosamine has been reported to be 1.6 mM [55]. The sub-
strates diffused into the yeast cell wall, and the product
to the medium. Our data show that the recombinant
yeast cells provide an inexpensive, self-perpetuating
source of fucosyltransferase activity immobilized in the
cell wall, useful for in vitro synthesis of sLex.
We have shown earlier that the catalytic domain of
rat a-2,3-sialyltransferase (ST3Ne) can be expressed asan Hsp150 fusion protein in the cell walls of S. cerevisiae
and P. pastoris [18,29]. Finally, we constructed here a S.
cerevisiae strain co-expressing Hsp150D–FucTe and
Hsp150D–ST3Ne in the S. cerevisiae cell wall. When pro-
vided with N-acetyllactosamine, CMP-sialic acid and
GDP-[14C]fucose, radioactive sLex was produced to the
medium. This demonstrates that the glycosyltransferases
functioned in tandem and were able to perform team-work in the yeast cell wall in the synthesis of sLex.
Acknowledgements
This work was supported by grants 38017 and 178444
of the Academy of Finland, and grant 1211/401/99 of
the Technology Development Center TEKES, respec-tively. Ms. A. L. Nyfors is acknowledged for excellent
technical assistance. H.S. is Ph.D. student of Viikki
H. Salo et al. / FEMS Yeast Research 5 (2005) 341–350 349
Graduate School in Biosciences. M.M. is a Biocentrum
Helsinki fellow.
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