2Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, 3Institute of Biotechnology, University of Helsinki, and 4Helsinki University Central Hospital, Laboratory Diagnostics, Helsinki, Finland
Received on March 25, 2000; revised on May 5, 2000; accepted on May 20, 2000.
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Abstract |
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Key words: glycans/GDP/E.coli/synthesis
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Introduction |
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In eukaryotic cells GDP-L-fucose can be synthesized via two different pathways, either by the more prominent de novo pathway or by the minor salvage pathway (Becker and Lowe, 1999). The de novo pathway starts from GDP-D-mannose; the first step is dehydration reaction catalyzed by specific nucleotide-sugar dehydratase, GDP-mannose-4,6-dehydratase (GMD). This leads to the formation of an unstable GDP-4-keto-6-deoxy-D-mannose, which undergoes a subsequent 3,5 epimerization and then a NADPHdependent reduction with the consequent formation of GDP-L-fucose (Figure 1). These two last steps are catalyzed by a single, bifunctional enzyme GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GMER, also known as FX in man). The salvage pathway instead includes two enzymatic reactions and takes advantage of fucose released from decomposed carbohydrates. Fucose is first phosphorylated by fucokinase to fucose-1-P, which then acts further as a substrate for GDP-L-fucose pyrophosphorylase resulting in formation of GDP-L-fucose (Becker and Lowe, 1999
) (Figure 1).
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The yeast S.cerevisiae is an ideal host to express the de novo pathway enzymes for the synthesis of GDP-L-fucose (Hirschberg et al., 1998). In the yeast cells glycosylation is largely restricted to mannosylation and they are not known to have fucose metabolism of their own (Hashimoto et al., 1997
; Romanos et al., 1992
). Thus, the cytoplasm of yeast cells is a relatively rich source of GDP-D-mannose, the starting material for the de novo pathway. In the present study, we transformed E.coli genes coding for the de novo pathway enzymes dehydratase (gmd) and epimerase/reductase (wcaG) to S.cerevisiae and showed that these genes can be expressed in a functionally active form thus synthesizing GDP-L-fucose from the inherent, yeast-borne GDP-D-mannose.
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Results |
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Expression of gmd and wcaG in S.cerevisiae
In Northern blot analysis a 1.3 kb transcript was detected for gmd in both yeast strains, YPH 499 and YPH 501, transformed with either only E.coli gmd (data not shown) or both gmd and wcaG (Figure 3A). When the presence of the corresponding enzyme, GMD was assayed with the c-myc antibody in Western blots, a 48 kDa protein band was detected in the single transfectants containing only the gmd gene (data not shown) or double transfectants containing both the gmd and wcaG genes (Figure 4A). Concomitantly the mock transformants showed no signals in either Northern or Western blots.
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Enzymatic activity of E.coli GMD and FX in yeast
The intermediate products of the de novo pathway from GDP-D-mannose to GDP-L-fucose (GDP-4-keto-6-deoxy-D-mannose and GDP-4-keto-6-deoxy-L-galactose) are known to be labile and not feasible to monitor in a high throughput screening approach. Thus, we used a microwell plate assay developed in our laboratory for measuring the 1,3fucosylation. In this assay biotinylated polyacrylamide conjugated sLN and recombinant
1,3fucosyltransferase VI were added to yeast cell lysates either not containing (mock- or only single transformants with either gmd or wcaG) or containing GDP-L-fucose, i.e., double transformants with both the gmd and wcaG genes. The readout of this assay was the turnover of sialyllactosamine to sLex detected by specific antibodies and time-resolved immunofluorometry. As the relevant acceptor and enzyme were always present in this assay, the only limiting factor in the generation of sLex from sLN was the presence or absence of GDP-L-fucose. This is shown in Figure 5a where the presence of exogenous GDP-L-fucose directly dictated the formation of sLex from sLN.
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Purification and MALDI-TOF MS of GDP-L-fucose
To further confirm the synthesis and to quantitate the amount of GDP-L-fucose produced in the gmd and wcaG expressing yeast cells, Aleuria aurantia lectin affinity chromatography was used to purify the product. As monitored with the enzymatic 1,3fucosyltransferase assay, most of the GDP-L-fucose bound to the lectin column and was eluted with addition of exogenous L-fucose. The peak fraction was further purified by size-exclusion HPLC, in which the retention times of both the purified product and commercial GDP-L-fucose was 16.2 min. As compared to external standard (GDP-L-fucose), the amount of GDP-L-fucose after purification was 3 µg per 1 ml of the original yeast cell lysate, corresponding to 0.2 mg/l of GDP-L-fucose in the original yeast cell culture.
This HPLC-purified product was then subjected to a MALDI-TOF MS analysis. The product purified from the double transfected yeast cells comigrating with the commercial GDP-L-fucose gave a single peak at m/z 588.04 (calculated m/z for [M-H] of GDP-L-fucose is 588.08). A single peak seen in the appropriate area in MALDI-TOF MS not only indicates that the product is GDP-L-fucose, but also shows that it was free of other nucleotide sugars (Figure 7).
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Discussion |
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There are two major strategies for the synthesis of GDP-L-fucose, the chemical and the enzymatical pathways. Various approaches have been used in the relatively complex chemical synthesis starting from L-fucose the endproduct being GDP-L-fucose (Adelhorst and Whitesides, 1993; Murray et al., 1997
). On the other hand, enzymatic synthesis of GDP-L-fucose can also be divided into two pathways, the predominant de novo route starting from GDP-D-mannose and the minor salvage pathway using L-fucose as the starting material (Becker and Lowe, 1999
). The latter pathway has been successfully performed with purified enzymes in a recycling one-pot approach, but not yet with recombinant enzymes (Ichikawa et al., 1992
, 1994). Furthermore this salvage pathway can be utilized with the help of enzymes from a nonpathogenic protozoa Crithidia fasciculata (Mengeling and Turco, 1999
). This parasite expresses cytosolic D-arabinose-1-kinase and D-arabinose-1-P-pyrophosphorylase activities. Besides of synthesizing GDP-D-arabinose from D-arabinose these enzymes can also use L-fucose as a substrate leading to the synthesis of GDP-L-fucose even though under normal conditions C. fasciculata is not known to have fucose-containing molecules at all (Mengeling and Turco, 1999
).
In prokaryotic cells GDP-L-fucose is synthesized only via the de novo pathway starting from GDP-D-mannose. The genes participating into this pathway have been identified in a broad range of organisms, including Gram-negative bacteria, such as E.coli, as well as Salmonella and Pseudonomas species (Tonetti et al., 1998). S.cerevisiae is the host of choice to generate GDP-L-fucose by the de novo pathway since it is rich in cytosolic GDP-D-mannose as it requires this donor for the extensive mannosylation of its glycoproteins. N-glycans in S.cerevisiae are composed of GlcNAc- and Man-residues, while the O-glycans contain only mannose (Romanos et al., 1992
). Furthermore, the yeast itself lacks the relevant enzymes to generate GDP-L-fucose per se, thus making the monitoring of the functional expression of the transformed genes feasible (Romanos et al., 1992
; Hashimoto et al., 1997
).
Previous work has shown that gmd and wcaG can be overexpressed in E.coli and that when exogenous GDP-D-mannose is provided to the reaction, GDP-L-fucose is synthesized (Sturla et al., 1997; Sullivan et al., 1998
). However, our work offers a major improvement into this approach as no exogenous expensive GDP-D-mannose needs to be added to the yeast cell lysates expressing GMD and GMER (FX) enzymes.
The availability of GDP-D-mannose was shown to be a limiting factor for our yeast transformant expressing gmd as well as wcaG genes. In yeast cells GDP-D-mannose is synthesized in the cytoplasm and further transported to the lumenal space of Golgi apparatus by specific antiporter system that involves exchange with guanosine 5'-monophosphate (GMP). Thus, as the recombinant enzymes are expressed in the cytosolic compartment of the yeast, it might be beneficial to use mutant yeast cells, which have been characterized to have a defect in the GMP antiporter function leading to increased cytosolic GDP-D-mannose levels.
A very rare human disease with hallmarks of leukocytosis, increased incidence of infections, and mental retardation has been described previously (Etzioni et al., 1998; Becker and Lowe, 1999
). Screening of the leukocytes of these LAD II patients revealed lack of all fucosylated glycoproteins and -lipids, thus suggesting for a common defect in the GDP-L-fucose synthesis and/or its availability in the Golgi (Karsan et al., 1998
). There are most probably at least two types of modifications in the LAD II patients. The first cases were shown to have impaired function of GMD activity (Sturla et al., 1998
). However, a third patient representing clinical features similar or identical to the two first LAD II patients has recently been identified (Korner et al., 1999
; Marquardt et al., 1999a
). The defect in this individual is not in the synthesis of GDP-L-fucose, but rather in the transportation of GDP-L-fucose to Golgi (Korner et al., 1999
; Lubke et al., 1999
). However, the patient has been successfully treated with dietary fucose, which is metabolized via the salvage pathway to GDP-L-fucose and finally transported to Golgi via a putatively impaired, yet partially functional GDP-fucose transporter system (Marquardt et al., 1999b
). This impaired route offers a possibility to further analyze the regulation of GDP-L-fucose synthesis and transportation into the Golgi (Puglielli and Hirschberg, 1999
).
Taken together, we have shown here that bacterial gmd and wcaG genes can be expressed as functional enzymes in S.cerevisiae and due to the inherent GDP-D-mannose synthesis in yeast cells they synthesize GDP-L-fucose. This approach was shown to be relatively effective, as >0.2 mg/l GDP-L-fucose was produced by specifically transfected yeast cells. It should also be noted that no optimization of the yeast cell culture systems has been performed so far to increase the yield. The GDP-L-fucose generated by this rapid route can be further converted to bioactive fucosylated glycans with relevant recombinant fucosyltransferases.
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Material and methods |
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Transformation into S.cerevisiae
The pESC-leu vectors not containing any genes, containing either gmd or wcaG or both of the genes were transformed into YPH 499 and YPH 501 yeast host strains by lithium acetate method following the instructions of the manufacturer (Stratagene). Transformants were selected using leucine dropout plates.
Selection of yeast transformants
Several transformants growing on leucine dropout plates were picked up and grown in 25 ml of dextrose containing selective synthetic dropout (SD) media overnight. The GAL1 and GAL10 promoters were repressed when transformed yeast cells were grown in dextrose containing SD media and induced when the cells are changed to grow in galactose containing SG media. In a typical experiment the yeast cell mass was increased by growing them in SD media overnight and the expression of transformed genes was induced for another 24 h by chancing the carbon source of the media from dextrose to galactose. After centrifugation the yeast cells were grown another 24 h in the same volume in synthetic galactose containing dropout (SG) media and the OD600 was measured; 1x109 cells were spun down and suspended in 0.5 ml of breaking buffer containing 1% TX-100 and 10% glycerol, and the cells were lysed mechanically by vortexing with glass beads (1/3 volume). After centrifugation the cell lysates were subjected to protein analysis as well as to enzymatic activity assays; 25 µg of total protein was used in Western blots and 0.30.4 µg/µl in the activity assays.
Expression of E.coli gmd and wcaG in yeast
Expression of recombinant proteins was detected on RNA and protein levels. Total RNA (15 µg) was extracted from double transformant yeast cells as well as from negative controls and broken mechanically with glass beads and subjected to Northern blot analysis as described previously (Mattila et al., 1996). The blots were probed with PCR products amplified from E.coli K-12 wca gene cluster. The expression of GMD and GMER (FX) was studied in Western blot using the antibodies against c-myc and FLAG-epitopes, respectively. Chemiluminescence (ECL, Amersham) was used as a detection method according to manufacturers instructions.
Determination of GDP-L-fucose synthesis
The presence of GDP-L-fucose was assayed by using yeast cell lysate as a source of GDP-L-fucose in a fucosyltransferase reaction converting sialyl-N-acetyllactosamine (sLN) to sLex. The reaction mixture included fucosyltransferase VI (FucTVI 25 µU, Calbiochem; San Diego, CA), yeast cell lysate diluted 1:20 as a fucose donor, sLN-polyacrylamide-biotin conjugate (Syntesome; Moscow, Russia) as a fucose acceptor, 50 mM MOPS-NaOH (pH 7.5), 6 mM MnCl2, 0.5% Triton X-100, 0.1% BSA, and 1 mM ATP. After 1 h incubation in +37°C, 50 µl aliquots of the reaction mixtures were transferred to microtitration strips coated with streptavidin (Wallac; Turku, Finland) to immobilize the biotinylated glycoconjugate. The fucosylated reaction product sLex was then detected and quantified by time-resolved fluorometry (Wallac) using anti-sLex primary antibody KM-93 (Calbiochem) and europium-labeled (DELFIA Eu-labeling kit: Wallac) anti-mouse IgM secondary antibody (Sanbio; Uden, The Netherlands) as described in this laboratory before (Rabina et al., 1997).
Purification of GDP-L-fucose
For large scale GDP-L-fucose synthesis one transformant containing both gmd and wcaG genes was grown 24 h in 750 ml of selective SD media and another 24 h in selective SG media. The cells were collected when OD600 was 3.7, and the pellet was suspended in concentration of 2 x 109 cells/ml (total volume 45 ml) of breaking buffer containing 1% TX-100 and 10% glycerol and lysed with glass beads. After vigorous 30 min vortexing the glass beads and cell debris were centrifuged and the clear lysate was filtrated through YM-10 Centricon column (Millipore Corporation, Bedford, MA) o/n +4°C according to manufacturers instructions.
GDP-L-fucose synthesized in yeast cells was then purified by two chromatographic steps. Lectin affinity chromatography was performed on a small column (diameter 0.5 cm) of agarose-bound Aleuria aurantia lectin (2 ml; Vector Laboratories, Burlingame, CA). The column was equilibrated with 10 mM HEPES buffer, pH 7.5, containing 0.15 M NaCl and 0.02% NaN3. After application of yeast cell lysate (4 ml) into the column, the elution was performed with 4 ml of the equilibration buffer followed by 4 ml of the buffer containing 25 mM L-fucose. Fractions of 1 ml were collected and assayed for the presence of GDP-L-fucose.
For desalting GDP-L-fucose and removal of the haptenic sugar, size-exclusion HPLC on a Superdex Peptide HR 10/30 column (Pharmacia, Sweden) was used. The elution was performed at 1 ml/min using 50 mM NH4HCO3 and the effluent was monitored with a UV detector at 254 nm. The amount of GDP-L-fucose was calculated from peak areas by reference to external standard (GDP-L-fucose, Calbiochem).
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Maldi-TOF mass spectrometry was performed with a Biflex mass spectrometer (Bruker Daltonics, Germany). Analysis was performed in the negative-ion linear delayed-extraction mode, using 2,4,6trihydroxyacetophenone (THAP, Fluka Chemica) as the matrix as described (Nyman et al., 1998). External calibration was performed with THAP matrix dimer and sialyl Lewis x ß-methylglycoside (Toronto Research Chemicals, Canada).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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