Cloning and Functional Expression of UGT Genes
Encoding Sterol Glucosyltransferases from Saccharomyces
cerevisiae, Candida albicans, Pichia pastoris, and
Dictyostelium discoideum*
Dirk
Warnecke
§,
Ralf
Erdmann¶,
Annette
Fahl
,
Bernhard
Hube
,
Frank
Müller
,
Thorsten
Zank
,
Ulrich
Zähringer
, and
Ernst
Heinz
From the
Universität Hamburg, Institut
für Allgemeine Botanik, 22609 Hamburg, ¶ Freie
Universität Berlin, Institut für Biochemie,
Limonenstrasse 7, 12203 Berlin, and
Forschungszentrum Borstel,
23845 Borstel, Germany
 |
ABSTRACT |
Sterol glucosides, typical membrane-bound lipids
of many eukaryotes, are biosynthesized by a UDP-glucose:sterol
glucosyltransferase (EC 2.4.1.173). We cloned genes from three
different yeasts and from Dictyostelium discoideum, the
deduced amino acid sequences of which all showed similarities with
plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from
Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris
(UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum
(ugt52) were expressed in Escherichia coli. In
vitro enzyme assays with cell-free extracts of the transgenic
E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols
such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An
S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either
UGT51 or UGT51B1 in this null mutant under the
control of a galactose-induced promoter restored sterol glucoside
synthesis in vitro. Lipid extracts of these cells contained
a novel glycolipid. This lipid was purified and identified as
ergosterol-
-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode
sterol-
-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.
 |
INTRODUCTION |
Sterol glycosides are widespread membrane lipids, occurring in all
plants, several algae (1-3), some fungi (4-9), slime molds (10-12),
Dictyostelium (13), a few bacteria (14-19), and even
animals (20-23). The knowledge base for sterol glycosides is rather
limited compared with free sterols and sterol esters, where the
synthesis, transport, and functions have been studied extensively in
animals (24-29), plants, (30-35), and yeast (36-40). The basis for
studies on the functions of sterol glycosides is the assumption that
free sterols and sterol glycosides differ physiologically. It is
obvious that the attachment of a glycosyl moiety to the sterol backbone
alters the physical properties of this lipid. As a result, there are
changes in the properties of membranes containing different proportions
of free sterols and sterol glycosides. Such changes have been studied
with artificial membranes in terms of membrane fluidity, permeability,
hydration, and phase behavior (41-44). However, we still do not know
how free sterols and sterol glycosides differ physiologically in
biological membranes and why many eukaryotic organisms synthesize
sterol glycosides. One of the main reasons for our limited knowledge in
this field is the lack of a genetic approach. The objective of the
present work was the isolation and characterization of sterol
glycosyltransferase genes from eukaryotic organisms. We expect that
genetic manipulation of these genes will facilitate the elucidation of
sterol glycoside functions in these organisms.
The predominating sugar moiety in sterol glycosides is glucose. Besides
plants, UDP-glucose:sterol glucosyltransferase activity was determined
by in vitro enzyme assays in Saccharomyces
cerevisiae (45-48), Candida bogoriensis (49), other
fungi (50), and Physarum polycephalum (51, 11). Although the
lipid composition of S. cerevisiae was studied in detail
during the last decades, there are only rare reports on the actual
isolation of sterol glucoside (SG)1 from this yeast (8, 9).
The expression level of sterol glucosyltransferase gene(s) seems to be
so low that very sensitive radioactive assays are required to detect
enzyme activity in vitro. However, expression is too low for
accumulation of a significant amount of SG in vivo suitable
for lipid analysis. Another possibility is the existence of a sterol
glucoside hydrolase that counteracts the sterol glucosyltransferase
keeping the amount of SG very low. Such a hydrolase has been detected
in plants (52, 53) but not in S. cerevisiae.
In contrast to S. cerevisiae, significant amounts of SG were
occasionally found in other fungi such as C. bogoriensis (4) and Phytium sylvaticum (5), whereas sterol mannosides were detected in the human pathogen Candida albicans (6, 7).
Here we describe the identification and characterization of sterol
glucosyltransferases from three different yeasts as well as from
Dictyostelium discoideum. Since the genes cloned in this work all belong to a superfamily of UDP-glycosyltransferases (UGTs) (54, 55), we use the gene symbol UGT for each new member. The UGT51 gene of S. cerevisiae was identified by
a homology probing approach with the cDNA sequences of
UDP-glucose:sterol glucosyltransferases from plants that were
recently cloned from oat
(ugt80A1,2
GenBankTM accession number Z83832) and
Arabidopsis (ugt80A2, GenBankTM
accession number Z83833) (56, 57). The UGT51 genes from C. albicans, Pichia pastoris, and UGT52 from
D. discoideum were cloned by either homology probing or
PCR-based strategies. Moreover, we identified ergosterol as an in
vivo substrate for the sterol glucosyltransferases from S. cerevisiae and P. pastoris. Furthermore, a new glycolipid was
identified in S. cerevisiae as the product of the in
vivo action of the yeast sterol glucosyltransferase from P. pastoris.
 |
EXPERIMENTAL PROCEDURES |
Bacterial and Saccharomyces Strains and Plasmids
Escherichia coli strains XL1-Blue (MRF')
(Stratagene), BL21(DE3) (Novagen), TOP10 (Invitrogen), and the vectors
pGEM-T (Promega), pBluescript (Stratagene, La Jolla, CA), pET19b
(Novagen), and pBAD-TOPO (Invitrogen) were used for cloning and
expression of different fragments of the sterol glucosyltransferase
genes. Yeast strains used in this study were UTL-7A
(MATa, ura3-52, trp1, leu2-3,112) and
ugt51 (MATa, ura3-52, trp1, leu2-3, 112, ugt51::kanMX4; this study). Expression
of different UGT genes in S. cerevisiae was performed with
pYES2 (Invitrogen) and pGAL4 (this study). For the construction of
pGAL4, the XbaI/BamHI fragment of pTerm1 (58)
which contains the CYC1 terminator (59) was subcloned into
plasmid pRS316 (60), resulting in pRS316t. The 0.5-kb
SpeI/HindIII fragment of pYES2.0 which contains
the GAL1 promoter (61) was subcloned into pBluescript KS+,
resulting in pGAL1. The GAL1 promoter was excised with
XbaI/PvuII and subcloned into
XbaI/HincII-restricted pBluescript KS+. The
resulting plasmid was designated pGAL2. Subcloning of the
XhoI/SacI fragment of pGAL2 into pYES2.0 resulted
in pGAL3. The KpnI/XhoI fragment of pGAL3
comprised the GAL1 promoter, and it was subcloned in front of the CYC1 terminator of pRS316t resulting in pGAL4.
Media and Growth of Bacteria and Saccharomyces
E. coli was grown in Luria-Bertani broth (LB,
Duchefa, Haarlem, The Netherlands). Ampicillin (100 mg/liter) and 1.5%
agar (Difco) was included for solid LB media. Yeast synthetic dextrose medium (SD) was prepared according to Ausubel et al.
(62).
Cloning of Sterol Glucosyltransferases from S. cerevisiae, C. albicans, P. pastoris, and D. discoideum
S. cerevisiae--
UGT51 (YLR189C,
GenBankTM accession number U17246) is a gene located on
chromosome XII of S. cerevisiae. We isolated a genomic 6359-bp NdeI/SpeI DNA fragment containing
UGT51 by restriction digestion of cosmid 9470 DNA that we
received from the American Type Culture Collection. This fragment was
cloned into pBluescript II KS giving the plasmid pUGT51g. This plasmid
was used to clone the open reading frame (ORF) UGT51 into
expression vectors for E. coli (pET19b
pUGT51x) and
S. cerevisiae (pGAL4
pUGT51xy). In addition, PCR
fragments of UGT51 corresponding to polypeptides with
N-terminal or C-terminal deletions of amino acids were cloned into the
following plasmids: pN690x, pN722x, pN799x, pC1043x, and pN722xy.
C. albicans--
PCR with genomic DNA of C. albicans
was performed with the primer DW3 5'-GSI WCI VGI GGI GAY GTH CAR CC-3'
and WA3 5'-GTI GTI CCI SHI CCI SCR TGR TG-3'. The resulting PCR
fragment was used to synthesize a digoxigenin-labeled probe for the
screening of a fosmid library of genomic DNA of C. albicans
strain 1161.3 Three positive
fosmid clones were isolated: 2H6, 3E6, and 8C12. An 8.2-kb
HindIII fragment of 2H6 was identified by agarose gel separation of the digestion fragments and hybridization with the probe.
This fragment contained the ORF UGT51C1 and was ligated with
pUC18/HindIII (
pUGT51C1g). A PCR fragment corresponding to the ORF UGT51C1 was cloned into an expression vector for
E. coli (pBAD-TOPO
pUGT51C1x).
P. pastoris--
PCR with genomic DNA of P. pastoris
was performed with the primer DW5 5'-TTY ACI ATG CCI TGG ACI MSI AC-3'
and DW30 5'-YKI GRI SHI GCI SCI GTI GTN CC-3'. The resulting PCR
fragment was used to synthesize a digoxigenin-labeled probe for the
screening of a genomic DNA library of P. pastoris strain
GS115 (63, 64). An SphI/EcoRV fragment of a
positive clone was isolated according to the method described above for
Candida. This fragment contained the ORF UGT51B1
and was ligated with pUC19/SphI/SmaI (
pUGT51B1g). A PCR fragment corresponding to the ORF UGT51B1
was cloned into expression vectors for E. coli (pBAD-TOPO
pUGT51B1x) and S. cerevisiae (pYES2
pUGT51B1xy).
D. discoideum--
We used the cDNA clone FC-AZ07
(GenBankTM Accession numbers C25752 and C25753 from H. Urushihara, University of
Tsukuba)4 from the cDNA
library of D. discoideum strain CAX3 for the PCR synthesis
of a digoxigenin-labeled probe. With this probe, we screened a
-ZAP
cDNA library of D. discoideum strain AX4 (from W. F. Loomis, University of California). In vivo excision of a positive clone resulted in the plasmid pUGT52c that had a 3331-bp cDNA insert within the vector pBluescript I SK. A PCR fragment corresponding to the ORF ugt52 was cloned into an expression
vector for E. coli (pBAD-TOPO
pUGT52x). All
plasmids used in this study are listed in Table I.
Disruption of the Genomic S. cerevisiae UGT51 Gene
To construct a ugt51 null mutant, the entire
UGT51 open reading frame was replaced by the
kanMX4 marker gene (65). The PCR-derived construct for
disruption comprised the kanMX4 gene flanked by short (45 bp) homology regions to the UGT51 3'- and 5'-noncoding region. Plasmid pFA6a-kanMX4 (65) served as template, and PCR primers
were as follows:
5'-TTGCACTTTATGCTTTGGTGAAAATCCGTATAACTTAAAAGAATGCGTACGCTGCAGGTCGAC-3' (S1/ham1) and
5'-TTACGCTTTTTTATAAAAGTGAGAGTGATACTCGGTTTAAATCATATCGATGAATTCGAGCTCG-3' (S2/ham1). The resulting amplification construct was introduced into
S. cerevisiae wild-type UTL-7A. Geneticin-resistant clones were selected by growth on YPD plates containing 200 mg/liter G418
(65). The correct replacement of UGT51 by the
kanMX4 gene was confirmed by PCR.
Expression of Sterol Glucosyltransferases
E. coli--
Cells of BL21(DE3) and TOP 10 were transformed with
plasmids derived from pET19b and pBAD TOPO, respectively. Cells were
grown at 30 °C in 25 ml of LB medium containing 100 mg/liter
ampicillin to an absorbance between 0.5 and 0.8 at 600 nm. Induction
was performed by adding 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside (for BL21(DE3)
with pET19b plasmids) or 0.1% arabinose (for TOP10 with pBAD TOPO
plasmids) and further incubation for 2-4 h at 30 °C. Cells were
harvested by centrifugation (10 min, 2000 × g, 20 °C), resuspended in 1-2 ml of buffer (100 mM
Tris/HCl, pH 7.5, 15% glycerol, 5 mM 1,4-dithiothreitol,
200 µM Pefabloc (Serva), 0.5 mg/ml lysozyme), and
incubated for 5 min at 20 °C. Cells were cooled in an ice bath, and
cell disruption was performed by ultrasonication (probe tip, 6 × 10 s). If necessary, a crude membrane fraction was prepared by
centrifugation of the cell homogenate at 4 °C for 20 min at
25,000 × g and resuspension of the sediment with buffer. This procedure was repeated once.
S. cerevisiae--
Yeast cells were grown at 30 °C in 100 ml
of the defined medium described above to an absorbance
(A600) between 2 and 6. The medium was
supplemented with 2% raffinose and 1% galactose (instead of glucose)
when the vectors pGAL4 or pYES2 were used. Cells were harvested by
centrifugation (10 min, 2000 × g, 20 °C),
resuspended in 2 ml of buffer (100 mM Tris/HCl, pH 7.5, 15% glycerol, 5 mM 1,4-dithiothreitol, 200 µM Pefabloc (Serva), 0.5 mg/ml lyticase from Sigma), and
incubated for 30 min at 25 °C. 2-3 g of glass beads (inner
diameter, 0.4 mm) were added, and cells were disrupted by vortexing for
16 × 30 s.
Sterol Glucosyltransferase Assay
The assay mixture contained in a total volume of 100 µl the
following: 80 µl of E. coli or S. cerevisiae
cell homogenate or membrane fraction, respectively, either 10 µl of a
solution of 4 mM cholesterol in ethanol (400 µM final concentration) or 8 µl of 500,000 dpm
[4-14C]cholesterol in ethanol (final concentration 45 µM, specific activity 1.85 GBq/mmol), and either 100,000 dpm UDP-[U-14C]glucose (final concentration 1.5 µM, specific activity 10.8 GBq/mmol) or 360 µM unlabeled UDP-glucose. After incubating for 2 h
at 30 °C, the reaction was terminated by the addition of 0.9 ml of
0.45% NaCl solution and 4 ml of chloroform/methanol, 2:1. The
extracted lipids were separated by thin layer chromatography with
chloroform/methanol (85:15, v/v). The radioactivity on the silica gel
plate was detected by radioscanning with a BAS-1000 BioImaging Analyzer
(Raytest, Straubenhardt, Germany).
Lipid Extraction and Analysis
S. cerevisiae cells were grown at 30 °C on
defined, lipid-free media described above. Harvesting of the cells by
centrifugation was followed by the extraction of lipids with
chloroform/methanol, 1:2, and chloroform/methanol, 2:1. The extracted
lipids were separated by thin layer chromatography on silica gel 60 (Merck, Darmstadt, Germany) with chloroform/methanol, 85:15.
Glycolipids were visualized with a spray of
-naphthol/sulfuric acid
mixture. For nuclear magnetic resonance spectroscopy and mass
spectrometry, the novel glycolipid was purified by column
chromatography on silica gel by elution with acetone. The lipid was
acetylated with acetic anhydride in pyridine and subjected to
preparative thin layer chromatography in diethyl ether.
Mass Spectrometry
Mass spectrometric analysis of the peracetylated sterol
glycoside was carried out with a Hewlett-Packard model 5989 spectrometer using the direct insert probe mode. Temperature was raised
from 80 to 325 °C at a rate of 30 °C/min. EI-mass spectra were
recorded at 70 eV with an ion source temperature of 200 °C.
Nuclear Magnetic Resonance (NMR) Spectroscopy
NMR spectra of the peracetylated glycoside (200 µg) were
recorded at 300 K in CDCl3 (99.96%, Cambridge Isotope
Laboratories, Andover, MA) for 1H NMR at 600 MHz (Avance
DRX-600 spectrometer) and for 13C NMR at 90.6 MHz (DPX 360)
(Bruker, Germany). Signals were referenced to internal
tetramethylsilane (
H =
C = 0.000). One-
and two-dimensional homonuclear (1H, 1H COSY,
relayed COSY) and 1H-detected heteronuclear
1H, 13C multiple quantum coherence (HMQC)
experiments were performed using standard Bruker software (XWINNMR,
version 1.3).
The nucleotide sequences of the sterol glucosyltransferases have been
deposited in the GenBankTM data base under accession
numbers AF091398 (C. albicans), AF098916 (D. discoideum), and AF091397 (P. pastoris).
 |
RESULTS |
Isolation and Cloning of Putative Sterol
Glycosyltransferases--
A BLAST (66) data base search with the amino
acid sequences of plant sterol glucosyltransferases revealed
similarities to Ugt51 (Ylr189c, PIR: locus S51434) which was described
as a hypothetical protein from S. cerevisiae with unknown
function. The first aim of the present work was to elucidate the
function of this protein. UGT51 contains an open reading
frame (ORF) of 3594 bp encoding a polypeptide of 1198 amino acids with
a calculated molecular mass of 136 kDa. We isolated a 6359-bp
NdeI/SpeI fragment of the cosmid 9470 (accession
number U17246) from the chromosome XII of S. cerevisiae that
contained UGT51. This fragment was cloned into pBluescript
(
pUGT51c) and used to subclone the ORF UGT51 into
expression vectors for E. coli and S. cerevisiae.
Furthermore, we wanted to isolate and clone homologous genes from other
organisms. The identification of novel sterol glycosyltransferase genes
will provide the basis for genetic manipulation of these genes and
contribute to the elucidation of sterol glycoside functions that may
differ in these organisms. We cloned the homologous genes by a
PCR-based strategy with degenerated oligonucleotide primers that were
derived from conserved regions of Ugt51 and the plant sterol
glucosyltransferases. PCR products obtained from C. albicans and P. pastoris were sequenced, and the deduced amino acid
sequences of these fragments showed similarities with Ugt51 and the
plant sterol glucosyltransferases. These PCR products were labeled and used to isolate the complete genes from genomic libraries of these organisms. An 8.2-kb fragment of C. albicans that contained
the incomplete MDL1 gene and an unknown ORF was cloned into
pUC18 (
pUGT51C1c). Both strands of the unknown ORF were sequenced, and these data were published in nucleotide data bases under the accession number AF091398. The ORF of 4548 bp, called
UGT51C1, encoded a polypeptide of 1516 amino acids with a
calculated molecular mass of 171 kDa.
From P. pastoris we cloned and sequenced a genomic fragment
of 6777 bp (accession no. AF091397). It contained an ORF
(UGT51B1) of 3633 bp encoding a polypeptide of 1211 amino
acids with a calculated molecular mass of 136 kDa.
A BLAST search (66) with Ugt51 revealed sequence similarity with a
cDNA clone from D. discoideum CAX3 (clone FC-AZ07,
accession numbers C25752 and C25753). This clone was used to label a
PCR fragment that was suitable for the isolation of longer fragments from a D. discoideum AX4 cDNA library. The longest clone
(pUGT52c) had an insert of 3331 bp that was sequenced on both strands
(accession number AF098916). It contained an ORF from bp 244-3312
(ugt52) that encoded a polypeptide of 1023 amino acids with
a calculated molecular mass of 114 kDa.
Fig. 1 shows a comparison of the deduced
amino acid sequences of the plant sterol glucosyltransferases with
those of Ugt51p and the gene products of the homologous genes from
Pichia, Candida, and Dictyostelium. The
polypeptides showed only a few similarities in their N-terminal parts,
but within a C-terminal region of about 360 amino acids (residues
1030-1425) similarities were significant as follows: 69% identity
between the three yeasts, 36-39% between the yeasts and
Dictyostelium, 34-37% identity between the yeasts and the
plants, 86% between the plants, and 33% between the plants and
Dictyostelium. Near their C-terminal ends all sequences
showed a 29-amino acid region of striking homology (box 4 in
Fig. 1). This region was very similar to a "signature sequence"
that is characteristic for a superfamily of nucleoside diphospho-sugar glycosyltransferases and that was suggested to represent a UDP-sugar binding domain (54). For this reason, all open reading frames mentioned
in this work were classified as UGTs (UDP-glycosyltransferases) in
agreement with the UGT Nomenclature Committee (54) and the Saccharomyces Genome Data
base.5 Most of the members of
this superfamily, but not all, use UDP-sugars as substrates. The
conserved boxes 1 and 3 of the sequences shown in Fig. 1 are also
present in some prokaryotic members of the UGT superfamily,
e.g. a rhamnosyltransferase from Pseudomonas aeruginosa (GenBankTM accession number L28170), but
are absent in the eukaryotic members of the superfamily. The region
named psbd in Fig. 1 corresponds to a putative
steroid-binding domain found in a solanidine glucosyltransferase from
potato (see Ref. 67, GenBankTM accession number U82367) and
mammalian steroid UDP-glucuronosyltransferases (e.g. UGT2B7,
GenBankTM accession number J05428). The UGTs cloned in the
present work showed only a few similarities with this putative
steroid-binding domain. These data suggest that the novel UGTs cloned
in the present work were more related to bacterial glycosyltransferases
than to other eukaryotic glycosyltransferases, although some of them use structurally similar substrates. The conserved box 2 in
Fig. 1 is not present in other members of the UGT superfamily and
seemed to be characteristic for sterol glucosyltransferases.


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Fig. 1.
Sequence alignment of sterol
glucosyltransferases from Arabidopsis thaliana
(A.t.), A. sativa
(A.s.), C. albicans
(C.a.), S. cerevisiae
(S.c.), P. pastoris
(P.p.), and D. discoideum
(D.d.). Black boxes indicate
identical amino acids in all six sequences. Gray boxes indicate identical amino acids in four or five sequences. The
polypeptides of the three yeasts and D. discoideum contain
long N-terminal extensions that do not exist in the plant polypeptides
(residues 1-820). Within this N-terminal extension there are only a
few sequence similarities between the four species. In contrast to this
N-terminal region, all sequences show significant similarity in their
C-terminal parts (residues 1030-1425). Within this region four highly
conserved boxes are underlined and numbered. The conserved
box 4 corresponds to the signature sequence of a protein
superfamily of UDP-glycosyltransferases (54). The region named
psbd corresponds to a putative steroid-binding domain found
in solanidine glucosyltransferase (67) and steroid
UDP-glucuronosyltransferases. Clusters of asparagine, glutamine, or
threonine in the sequence from D. discoideum are indicated
by dotted lines.
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Functional Expression of the UGTs in E. coli and in Vitro
Determination of Sterol Glycosyltransferase Activity--
The ORFs of
the DNA fragments cloned in this work were expressed in E. coli to examine the function of the expression products. This was
done by in vitro determination of sterol glycosyltransferase activity in cell-free extracts using radiolabeled substrates. E. coli is suitable for such experiments since untransformed cells do
not contain a sterol glycosyltransferase activity. The ORF UGT51 from S. cerevisiae was cloned into the
expression vector pET19b (pUGT51x). The other UGTs from
C. albicans, P. pastoris, and D. discoideum were
amplified by PCR and subcloned into the expression vector pBAD-TOPO
(pUGT51C1x, pUGT51B1x, and pUGT52x). Transformed and induced E. coli cells were disrupted, and the homogenates or crude membrane
fractions were used for in vitro enzyme assays. Membrane
fractions had the advantage that most of the intracellular UDP-glucose
was eliminated. It turned out that more than 50% of the expressed
protein was not in the soluble fraction but was found in the sediment
of membranes and inclusion bodies (data not shown).
To measure the sterol glycosyltransferase activity, we performed
in vitro assays with various radiolabeled sugar donors
(NDP-sugars) and acceptors (sterols). After thin layer chromatography
(TLC), the radioactivity in the lipophilic reaction products was
determined. Table II shows a comparison
of the substrate specificities of the recombinant enzymes from S. cerevisiae, C. albicans, P. pastoris, D. discoideum, and
Avena sativa. These data should not be considered as a
quantitative determination of substrate affinities, since the assays
were not performed under linearized conditions. These results rather
suggest which substrates were accepted or discriminated against by each
of the heterologously expressed proteins. With UDP-[U-14C]glucose as the donor, all expressed UGT
proteins used various sterols as sugar acceptors, whereas there was no
background SG synthesis of a control E. coli homogenate. The
UGT proteins glucosylated sterols with a planar backbone such as
cholesterol, ergosterol,
-sitosterol, stigmasterol, and even the
steroidal alkaloid tomatidine. However, other lipids, e.g.
ceramide and dioleoyl glycerol, were not accepted. Regarding the sugar
moiety of the donor, all enzymes exhibited a distinct specificity for
glucose (UDP-glucose), whereas UDP-galactose, UDP-glucuronic acid,
UDP-mannose, and GDP-mannose were not accepted. This was of particular
relevance in the case of the Ugt51C1 from C. albicans in
view of the identification of sterol mannoside in this organism (see
"Discussion"). UDP-xylose, as a pyranose, mimics UDP-glucose except
for the lacking CH2OH moiety. It was incorporated into
cholesterol xyloside by the Avena, Saccharomyces, and
Pichia enzymes at a low rate. UDP served as the best
nucleotide moiety of the glucose donors, but CDP-glucose and
GDP-glucose were also used, although at significantly lower rates.
TDP-glucose was not accepted. These in vitro data gave strong evidence that all UGT genes cloned in this work
encode specific sterol glucosyltransferases using UDP-glucose as sugar donor and different planar sterols as sugar acceptors.
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Table II
Substrate specificities of different recombinant sterol
glucosyltransferases expressed in E. coli
Reaction mixtures contained in a total volume of 100 µl: 80 µl of
E. coli cell-free homogenate or membrane preparation,
NDP-U-14C-sugar or unlabeled NDP-sugar and unlabeled acceptor
or [4-14C]cholesterol. E. coli expressed the
sterol glucosyltransferases from A. sativa (A.s.), C. albicans (C.a.), D. discoideum (D.d.), P. pastoris (P.p.) or S. cerevisiae (S.c.). Incorporation
with UDP-glucose and cholesterol was taken as 100%. Results are
averages of two determinations. ND, not determined.
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Only a C-terminal Conserved Region of UGT51 Was Essential for
Enzyme Activity--
An alignment of the different sterol
glucosyltransferases (Fig. 1 and Fig. 2)
showed that the sequences shared significant similarities only at their
C-terminal parts and that the polypeptides from the three yeasts and
from Dictyostelium had large N-terminal extensions that did
not exist in the plant polypeptides. This phenomenon raised the
question which parts of the polypeptides were necessary for their
function. We selected one of the proteins, Ugt51p from S. cerevisiae, to investigate this phenomenon. Therefore, various
fragments of Ugt51p, obtained by deleting different N- or C-terminal
sequences, were expressed in E. coli (Fig. 2). The cell-free
homogenates of the transgenic E. coli were used for in
vitro enzyme assays performed with cholesterol and UDP-glucose. Ugt51p fragments with N-terminal deletions of either 690 or 722 amino
acids synthesized SG (Fig. 3). These
fragments contained the conserved region indicated in Fig. 2. In
contrast, elimination of parts of the conserved region by deletion of
either 799 N-terminal or 155 C-terminal amino acids resulted in
complete loss of enzyme activity. These data indicate that at least 722 N-terminal amino acids of Ugt51p are not required for in
vitro enzyme function. Ugt51p fragments containing the complete
conserved region of the sterol glucosyltransferases are sufficient for
in vitro SG synthesis.

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Fig. 2.
Schematic alignment of amino acid sequences
of the sterol glucosyltransferases Ugt80A1p from A. sativa
and Ugt51p from S. cerevisiae. The
gray boxes indicate a region of significant amino acid
similarity between these glucosyltransferases (region
1030-1425 in Fig. 1) The black boxes indicate the
signature sequence of UGTs (54) that contains highly conserved amino
acids. In the lower part of the figure four fragments of
Ugt51p are shown resulting from the deletion of sequences with a length
of either 690, 722, and 799 N-terminal amino acids (N690, N722,
and N799) or 1043 C-terminal amino acids (C1043). These
fragments were expressed in E. coli to determine which parts
of the polypeptide were essential for enzyme function. A indicates
in vitro sterol glucosyltransferase activity and a indicates a lack of activity. The detailed results of these experiments
are given in Fig. 3.
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Fig. 3.
In vitro activity of recombinant
S. cerevisiae sterol glucosyltransferase (Ugt51p)
expressed in E. coli as full-length or as N- or
C-terminally shortened fragments. Cell-free homogenates from
transgenic E. coli were used for in vitro enzyme
assays. The assay mixtures contained UDP-glucose and cholesterol with
either radiolabeled glucose (A) or radiolabeled cholesterol
(B). The labeled lipophilic reaction products were separated
by thin layer chromatography with subsequent analysis by a BAS 1000 BioImaging Analyzer. SG was identified by co-chromatography with an
authentic standard. The transgenic E. coli harbored the
control plasmid pET19b (lanes 1, 1'), pUGT51x (encoding the
complete polypeptide Ugt51p, lanes 2, 2'), pN690x (encoding
Ugt51p that lacked 690 N-terminal amino acids, lanes 3, 3'),
pN722x (encoding Ugt51p that lacked 722 N-terminal amino acids,
lanes 4, 4'), pN799x (encoding Ugt51p that lacked 799 N-terminal amino acids, lanes 5, 5'), and pC1043x (encoding
Ugt51p that lacked 155 C-terminal amino acids, lanes 6, 6').
|
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Deletion and Overexpression of UGT51 in S. cerevisiae--
To
address the question whether the in vitro synthesis of SG
reflects the in vivo function of Ugt51p, we deleted
UGT51 in S. cerevisiae strain UTL7A. The null
mutant was still viable and grew like the parental strain on complex
and minimal media, at low and elevated temperatures, under different
conditions of osmotic stress and in the presence of nystatin. Fig.
4 shows that cell-free homogenates of the
parental strain UTL7A synthesized low amounts of SG in vitro
(1700 dpm of radiolabeled product), whereas the enzyme activity was
completely lost in the null mutant. The labeled compound near the front
probably was sterol ester. Then we transformed the null mutant with a
plasmid that led to the expression of complete UGT51 under
the control of a galactose-induced promoter. The in vitro SG
synthesis was restored in these cells and was more than 10-fold higher
(22,000 dpm of labeled product) than with UTL7A cells (Fig. 4). These
data prove that the expression of UGT51 in S. cerevisiae resulted in the biosynthesis of an enzymatically active
sterol glucosyltransferase. The expression of the N722 fragment in the
null mutant also resulted in a detectable enzyme activity (1500 dpm
of labeled product). However, the activity resulting from the
expression of the truncated enzyme was much lower than that observed
for the expression of the wild-type enzyme.

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Fig. 4.
In vitro determination of the
sterol glucosyltransferase Ugt51p from S. cerevisiae. Cell-free homogenates of wild-type and
transgenic S. cerevisiae cells were used for in
vitro enzyme assays. The assays contained UDP-glucose and
radiolabeled cholesterol. The labeled lipophilic reaction products were
separated by thin layer chromatography with subsequent analysis by a
BAS 1000 BioImaging Analyzer. SG was identified by co-chromatography
with an authentic standard. Lane 0, radiolabeled cholesterol
(the spot with slightly lower Rf value represents a
degradation/oxidation product of cholesterol, which is only absent in
freshly purified cholesterol); lane 1, S. cerevisiae strain
UTL7A; lane 2, UTL7A ugt51 (sterol
glucosyltransferase deletion mutant); lane 3, UTL7A
ugt51 harboring the control plasmid pGAL4; lane
4, UTL7A ugt51 harboring pN722xy (encoding Ugt51p
that lacked 722 N-terminal amino acids); lane 5, UTL7A
ugt51 harboring pUGT51xy (encoding the complete
polypeptide Ugt51p).
|
|
Isolation of a Novel Glycolipid from S. cerevisiae and Its
Identification as
Ergosterol-
-D-Glucopyranoside--
The lipids of
S. cerevisiae were extracted and separated by thin layer
chromatography to address the question whether SG was accumulated
in vivo (Fig. 5A).
The total lipid extracts of the S. cerevisiae wild-type
cells and the null mutant did not contain detectable amounts of SG.
Also, expression of the N722 fragment in null mutant cells did not
result in the biosynthesis of significant amounts of SG, whereas cells
expressing the complete UGT51 contained a new glycolipid
that co-chromatographed with an authentic SG standard.

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Fig. 5.
Search for in vivo accumulation
of SG in wild-type and transgenic strains of S. cerevisiae. Lipid extracts were separated by thin layer
chromatography. A, lane 1, S. cerevisiae strain UTL7A; lane 2, UTL7A
ugt51 (sterol glucosyltransferase deletion mutant);
lane 3, UTL7A ugt51 harboring the control
plasmid pGAL4; lane 4, UTL7A ugt51 harboring
pN722xy (encoding Ugt51p that lacked 722 N-terminal amino acids);
lane 5, UTL7A ugt51 harboring pUGT51xy
(encoding the complete polypeptide Ugt51p). Authentic SG was
chromatographed as control (at right). B,
expression of the P. pastoris Ugt51B1p in S. cerevisiae. Lane 1, UTL7A ugt51 harboring
pUGT51B1xy (encoding the complete polypeptide Ugt51B1p from P. pastoris). A lipid extract of this strain was used to isolate SG
for subsequent analysis by spectroscopy (see Figs. 6 and 7).
|
|
We found that a heterologous expression of UGT51B1 from
P. pastoris in the S. cerevisiae knock-out mutant
led to the accumulation of larger amounts of the novel glycolipid (Fig.
5B) as compared with the homologous expression of
UGT51. We made use of this large accumulation of the
glycolipid for its purification from lipid extracts of S. cerevisiae expressing UGT51B1 from P. pastoris. This was done by column chromatography on silica gel,
acetylation of the isolated glycolipid, and subsequent thin layer
chromatography. The peracetylated glycolipid was subjected to
structural analysis by mass spectrometry and nuclear magnetic resonance
(NMR) spectroscopy.
Electron impact (EI) mass spectrometry analysis of the peracetylated
glycolipid was performed using the direct insert probe mode. Two
characteristic fragments were obtained, one (m/z = 331) from a terminal single tetra-O-acetylated hexosyl residue
and the other (m/z = 378) from the steroid (Fig.
6). The molecular ion [M
H]
(m/z = 726) was found to be in
agreement with the calculated mass for
tetra-O-acetylhexosyl-ergosterol
(C42H62O10;
Mr = 726.94).

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Fig. 6.
Structure and electron-impact (EI) mass
spectrum of per-O-acetylated
-glucosyl-ergosterol isolated from transgenic
S. cerevisiae expressing the sterol
glucosyltransferase from P. pastoris.
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|
The structure of the purified
tetra-O-acetylhexosyl-ergosterol was further investigated by
1H NMR spectroscopy at 600 MHz. The 1H NMR
spectrum revealed signals for four O-acetyl groups (
2.011, 1.984, 1.954, and 1.937), indicating one terminal hexose and no additional OAc group derived from a putative OH group of the steroid. The anomeric signal of the hexose (H-1,
4.551) expressed a coupling constant J1, 2 of 8 Hz
(Table III), thus showing
-configuration. All other hexose ring protons showed high coupling
constant values (J2, 3 9.5, J3, 4 9.6, and J4, 5
9.8) characteristic for the gluco configuration in a
pyranoside that was further supported by the chemical shift data (Table
III). These data are in full agreement with a terminal
tetra-O-acetyl
-glucopyranose (68).
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Table III
NMR spectroscopic identification of
ergosterol- -D-glucoside
600-MHz 1H and 90.6-MHz 13C NMR data of
3 -O-(2,3,4,6-tetra-O-acetyl- -D-glucopyranosyloxy)-ergosta-5,7,22E-trien
that was isolated in its nonacetylated form from S. cerevisiae
expressing UGT51B1 from P. pastoris (see "Experimental Procedures";
CDCl, 300 K; internal tetramethylsilane, H C = 0.000). Published data for 3-OAc-ergosterol are given in
parentheses; 1H (69); 13C (70). Signals for four OAc
are as follows: H 2.011, 1.984, 1.954, 1.937; C
169.3, 169.2, 168.4, 168.3; 19.6 (2×), 19.7, and 19.8 ppm.
|
|
Characteristic signals for the steroid were found at
5.501 and
5.312 and were diagnostic for the conjugated diene system in ring B
(H-6 and H-7) of ergosterol (69). Signals for H-22 and H-23 at the
isolated double bond between C-22 and C-23 (
5.156 and 5.105, respectively) expressed a high coupling constant value of
J22, 23 15.2 Hz, thus indicating E
configuration (Fig. 7). Signals for H-3,
H-4
, and H-4
were at
3.531, 2.367, and 2.173, respectively.
All other signals could be assigned and part of the coupling constant
values determined based on relay COSY and
1H,13C HMQC experiments (Fig. 7 and Table
III).

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Fig. 7.
Two-dimensional 1H,
13C HMQC spectrum of
3 -(2,3,4,6-tetra-O-acetyl- -D-glucopyranosyloxy)ergosta-5,7,22E-trien
which was isolated in its nonacetylated form from S. cerevisiae expressing UGT51B1 from P. pastoris. The spectrum was measured in a solution of
CDCl3 at 300 K. Assignments of protons to the glycosyl and
ergosterol part (in italics) are indicated. The
corresponding 1H NMR spectrum (600 MHz) is displayed along
the horizontal (F2) axis and the 13C
NMR spectrum (90.6 MHz) along the vertical (F1)
axis.
|
|
In the HMQC spectrum (Fig. 7) the positions of the signals for sugar
carbons, including the anomeric carbon (C-1,
98.6), were in
agreement with a
2,3,4,6-tetra-O-acetyl-
-D-glucopyranoside (Table III). The steroid moiety expressed diagnostic resonances for the
isolated double bond (C-23,
134.4; C-22,
131.1) and for the two
conjugated double bonds (C-6,
119.3; C-7,
115.2), further
confirming ergosterol to be the aglycon in the glycolipid. Other carbon
resonances were assigned as well and found to be in agreement with
published data (70).
Based on the EI-mass spectrometry and 1H and
13C NMR spectroscopy data, the per-O-acetylated
product of the UDP-glucose:sterol glucosyltransferase Ugt51B1 from
P. pastoris, as expressed in transgenic S. cerevisiae, could be unambiguously identified as 3
-(2, 3, 4, 6-tetra-O-acetyl-
-D-glucopyranosyloxy)ergosta-5,7,22E-trien (Fig. 6) thus demonstrating the enzyme to be a
-glucosyltransferase with ergosterol as acceptor.
 |
DISCUSSION |
This work reports the cloning, identification, and
characterization of novel UDP-glucose:sterol glucosyltransferases from S. cerevisiae, C. albicans, P. pastoris, and D. discoideum. We also isolated a sterol
glucoside from transgenic S. cerevisiae expressing
UGT51B1 from P. pastoris. This is the first
report on the structural analysis of a glycosylated sterol from
S. cerevisiae which we identified as
ergosterol-
-D-glucoside. These data prove that
UGT51B1 encodes a UDP-glucose:sterol
-D-glucosyltransferase. Combined with in
vitro data on the substrate specificities of the identified
glucosyltransferases from different organisms, we assume that all the
recombinant enzymes that we characterized in this work are sterol
-D-glucosyltransferases. The enzymes show a narrow
specificity for the sugar donor using predominantly UDP-glucose. In
contrast, the in vitro specificity for the sterol acceptor
is broad, pointing to the biosynthesis of glucosyl sterols with
different sterol moieties depending on the individual sterol composition of each organism.
The isolation of novel sterol glucosyltransferases represents one step
in our approach to elucidate the functions of sterol glucosides in
these organisms. Amino acid sequence comparisons showed that these
genes represent a gene family with members in plants, fungi, and other
eukaryotic organisms (Fig. 1). This gene family is part of a
superfamily of UDP-glycosyltransferases (UGTs) (54), which is
characterized by a C-terminal signature amino acid sequence (Fig.
1).
Glucose is the main sugar moiety in the sterol glycosides of many
organisms, but other sugars have also been found (1). This raises the
question whether there are physiological differences between sterol
glycosides having different hydrophilic head groups. Since C. albicans was reported to contain mainly cholesterol mannoside (6,
7), it was our intention to isolate the gene encoding the corresponding
mannosyltransferase. Our data on the substrate specificity of Ugt51C1p
from C. albicans showed that it is not a mannosyltransferase
as expected but a glucosyltransferase. The discrepancy between the
glucose preference of Ugt51C1p and the presence of mainly sterol
mannoside in C. albicans points to the possibility that this
organism may contain two sterol glycosyltransferases with different
sugar specificities. The ongoing C. albicans genome sequencing project should answer the question whether there is a gene
homologous to UGT51C1 which may encode a mannosyltransferase.
The expression of different fragments of UGT51 in E. coli and S. cerevisiae showed that at least 722 N-terminal amino acids of the polypeptide are not required for the
in vitro glucosyltransferase activity of the protein (Fig. 3
and Fig. 4). No significant difference in enzyme activity was observed
between the wild-type and the truncated protein upon expression in
E. coli. Interestingly, upon expression in S. cerevisiae, a significantly lower sterol glycosyltransferase activity was observed for the truncated form (Fig. 4). In line with
this observation, expression in S. cerevisiae of the
wild-type form but not of the truncated form led to the biosynthesis of detectable amounts of SG (Fig. 5). These observations might suggest that the N-terminal region contains yeast-specific regulatory elements
for the activity, expression, or stability of the protein. However, it
remains to be investigated what function this large N-terminal
extension of about 700 amino acids may have. There are only low
sequence similarities between these N-terminal extensions of the three
yeasts and D. discoideum and they do not show significant similarity to any sequence from the data bases. They do not contain known membrane targeting motifs nor membrane spanning domains. Interestingly, this N-terminal extension is not present in the plant
enzymes (Fig. 1).
By deletion of UGT51 in S. cerevisiae we showed
for the first time that SG is non-essential for growth of S. cerevisiae. In addition, no differences in growth were observed
under various stress conditions. This is consistent with three
genome-wide expression studies of S. cerevisiae which showed
no alterations in UGT51 expression during the sporulation
process (71), shift from anaerobic to aerobic conditions (72), and when
grown in rich or minimal media (73). The expression level of
UGT51 seems to be very low, and we do not know whether a low
amount of SG is sufficient to fulfill its physiological function or
whether larger amounts need to be biosynthesized, e.g. under
special growing conditions, that are sufficient for altering membrane
properties. Lipid analyses of other yeasts and fungi showed that some
of them contain significantly larger amounts of sterol glycosides than
Saccharomyces and that an increase in its synthesis was
induced under stress
conditions.6
In this context, it should be mentioned that insertional mutagenesis
experiments with the rice blast fungus Magnaporthe grisea and a subsequent screening procedure revealed mutants with reduced pathogenicity (74). One of these mutants identifies the gene PTH8 (GenBankTM accession number AF027983), the
partial sequence of which shows striking similarity to the sterol
glucosyltransferases from yeasts. These data suggest that SG may be
involved in the pathogenicity of phytopathogenic fungi.
Another interesting observation concerns some regions of the cDNA
of the D. discoideum sterol glucosyltransferase which are rich in AAC codons pointing to clusters of asparagine, glutamine, or
threonine (see Fig. 1). Such clusters were reported to be
characteristic for developmentally regulated genes (75-77). A
hypothetical change of SG synthesis during development would be
consistent with the observations that SG synthesis alters during
development of D. discoideum (13) and that SG accumulation
was induced by the process of differentiation from amoeboid stage to
plasmodium in P. polycephalum (78, 51).
In summary, we have shown that SG synthesis is not restricted to plants
but that it is also a common feature of yeasts, fungi, and other
eukaryotes. We have identified and characterized four novel sterol
glucosyltransferases from yeasts and D. discoideum. Our work
provides the basis for new approaches to elucidate the functions of SG
in these organisms.
 |
ACKNOWLEDGEMENTS |
We thank J. M. Cregg for providing us
with a DNA library from P. pastoris; D. Fuller and W. F. Loomis for a Dictyostelium cDNA library; H. Urushihara for the Dictyostelium cDNA clone FC-AZ07; M. Strathman and S. Scherer for the C. albicans fosmid library; R. Swoboda for replicating a copy of it; D. Hess and M. Bossenz for
preparing dot-blots of this library; and P. Mackenzie for help
regarding gene nomenclature.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft, Sonderforschungsbereich 470, and Fonds der
Chemischen Industrie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF091398 (C. albicans), AF098916 (D. discoideum), and AF091397 (P. pastoris).
§
To whom correspondence should be addressed: University of Hamburg,
Institut fuer Allgemeine Botanik, Ohnhorststrasse 18, 22609 Hamburg,
Germany. Tel.: 49 40 42816 364; Fax: 49 40 42816 254; E-mail:
warnecke{at}botanik.uni-hamburg.de.
2
We use lowercase italics for the gene symbols of
plants and Dictyostelium due to the gene nomenclature rules
which differ from that practiced with S. cerevisiae. It must
be emphasized that in contrast to yeasts, lowercase italics do not
indicate recessive alleles but mean the same as uppercase italics for
S. cerevisiae.
3
Information available from M. Strathmann
and S. Scherer at the following on-line address:
http://alces.med.umn.edu.candida/fosmidinfo/html.
4
Information available on-line at the following
address:
http: //www.csm.biol.tsukuba.ac.jp/Sexual-cDNA/SexualcDNA/html.
5
Saccharomyces Genome Data base
available at the following on-line address:
http://genome-www.stanford.edu/.
6
T. Sakaki, D. Warnecke, U. Zaehringer, and E. Heinz, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
SG, sterol glucoside(s);
ORF, open reading frame;
PCR, polymerase chain reaction;
UGT, UDP-glycosyltransferases;
kb, kilobase pair(s);
bp, base pair(s);
EI, electron impact.
 |
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