We have cloned the cDNA encoding human
GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting
GDP-mannose to GDP-fucose. The message is expressed in all tissues and
cell lines examined, and the cDNA complements Lec13, a Chinese
Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase
activity. The human GDP-mannose 4,6-dehydratase polypeptide shares 61%
identity with the enzyme from Escherichia coli, suggesting
broad evolutionary conservation. Purified recombinant enzyme utilizes
NADP+ as a cofactor and, like its E. coli
counterpart, is inhibited by GDP-fucose, suggesting that this aspect of
regulation is also conserved. We have isolated the product of the
dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its
structure by electrospray ionization-mass spectrometry and high field
NMR. Using purified recombinant human GDP-mannose 4,6-dehydratase and
FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we
show that the two proteins alone are sufficient to convert GDP-mannose
to GDP-fucose in vitro. This unequivocally demonstrates
that the epimerase and reductase activities are on a single
polypeptide. Finally, we show that the two homologous enzymes from
E. coli are sufficient to carry out the same enzymatic
pathway in bacteria.
 |
INTRODUCTION |
Fucose is found as a component of glycoconjugates such as
glycoproteins and glycolipids in a wide range of species from humans to
bacteria. For example, fucose is a component of the capsular polysaccharides and antigenic determinants of bacteria, while in
mammals fucose is present in many glycoconjugates, the most widely
known being the human blood group antigens. Fucose-containing glycoconjugates have been implicated as playing key roles in embryonic development in the mouse (1) and more recently in the regulation of the
immune response, specifically as a crucial component of the selectin
ligand sialyl Lewis X (reviewed in Refs. 1 and 2). In all cases, fucose
is transferred from GDP-fucose to glycoconjugate acceptors by specific
transferases. Thus, defects in GDP-fucose biosynthesis will affect all
fucosylation within the cell. Recently, individuals deficient in the
biosynthesis of GDP-fucose have been identified (3, 4) and suffer from the immune disorder leukocyte adhesion deficiency type II
(LADII).1 These patients fail
to synthesize fucosylated blood groups, and their leukocytes do not
express the fucose containing carbohydrate sialyl Lewis X. The
patient's leukocytes do not extravasate normally, which leads to
recurrent infections.
In his pioneering work in the early 1960s, Ginsberg (5, 6) elucidated
the enzymatic pathway converting GDP-mannose to GDP-fucose. Later,
Yurchenco and Atkinson (7) showed that this was the primary
biosynthetic route to GDP-fucose. As shown in Fig.
1, GDP-mannose is converted to GDP-fucose
by GDP-mannose 4,6-dehydratase via the oxidation of mannose at C-4
followed by the reduction of C-6 to a methyl group, yielding
GDP-4-keto-6-deoxymannose. The reaction has been reported to proceed
with transfer of a hydride from C-4 to C-6 (8) by a tightly bound
cofactor, thought to be NADP+ or NAD+, which is
regenerated during the reaction. This intermediate is then epimerized
at C-3 and C-5 to yield GDP-4-keto-6-deoxy-glucose and finally reduced
by NADH or NADPH at C-4 to produce GDP-fucose. In the initial studies,
it was not certain if the last two steps, the epimerizations and
reduction, were performed by one enzyme or two. One potential
regulatory mechanism in the pathway was first revealed in the studies
of Kornfeld and Ginsberg (9), who demonstrated that GDP-mannose
4,6-dehydratase was inhibited by the final product in the biosynthetic
pathway, GDP-fucose.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1.
GDP-fucose biosynthetic pathway. The
pathway for the conversion of GDP-mannose to GDP-fucose is shown.
GDP-mannose 4,6-dehydratase catalyzes the oxidation of C-4 of mannose
to the ketone and the reduction of C-6 to the methyl group to yield
GDP-4-keto-6-deoxymannose. The NADP+ cofactor is reduced
and then oxidized during these two steps. GDP-4-keto-6-deoxy-mannose
3,5-epimerase, 4-reductase catalyzes the epimerization at C-3 and C-5
to yield GDP-4-keto-6-deoxyglucose, followed by the reduction of C-4 by
NADPH or NADH, yielding GDP-fucose. Chemical reduction of the
GDP-4-keto-6-deoxymannose intermediate produces GDP-6-deoxytalose and
GDP-rhamnose.
|
|
Recent studies have addressed several open questions about the enzymes
in this pathway. Two studies have shown that GDP-mannose 4,6-dehydratase utilizes NADP+ and not NAD+ as
a cofactor. Yamamoto et al. demonstrated this with the
enzyme from Klebsiella pneumoniae (10) and more recently
Sturla et al. detected NADP+ bound to the
Escherichia coli enzyme (11). This requirement for
NADP+ differentiates GDP-mannose 4,6-dehydratase from the
two other well characterized sugar nucleotide 4,6-dehydratases,
dTDP-glucose 4,6-dehydratase and CDP-glucose 4,6-dehydratase, both
which require NAD+ (12, 13). Additionally, recent studies
have addressed the question of whether the epimerase and reductase
activities are present in one protein or are two separate proteins as
is the case in dTDP-rhamnose biosynthesis (14, 15). Serif and
co-workers (16) suggested that the 3,5-epimerase and 4-reductase
activities were present on a single polypeptide when they purified
small amounts of the enzyme from pig thyroids (16). This was confirmed by Tonetti et al. (17) when they cloned the human protein
FX. Sequencing the gene revealed homology to the bacterial sugar
nucleotide reductases. Using antibody depletion experiments, purified
protein, and cell extracts as a source for the
GDP-4-keto-6-deoxymannose, they demonstrated that FX combined both the
epimerase and reductase activities in one polypeptide.
To understand better the human enzymes involved in this pathway, their
role in selectin-mediated cell adhesion, and the LADII defect, we have
undertaken the molecular cloning of the human gene encoding GDP-mannose
4,6-dehydratase. Furthermore, utilizing purified recombinant enzymes
expressed in E. coli, we have reconstituted the GDP-fucose
biosynthetic pathway in vitro. We demonstrate that two
enzymes, GDP-mannose 4,6-dehydratase and GDP-4-keto-6-deoxymannose 3,5-epimerase, 4-reductase, are sufficient to synthesize GDP-fucose from GDP-mannose, confirming earlier studies suggesting that both epimerase and reductase activities are encoded in a single polypeptide. Additionally, we show that human GDP-mannose 4,6-dehydratase has a
strict specificity for NADP+ over NAD+. Using
the homologous E. coli enzymes, GDP-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3,5-epimerase, 4-reductase (WCAG),
we demonstrate that the same is true in bacteria. We also show that
human GDP-mannose 4,6-dehydratase is subject to feedback inhibition by
GDP-fucose and that this, along with its differential levels of gene
expression, provides potential mechanisms for regulating its
activity.
 |
MATERIALS AND METHODS |
Data Base Searching and Sequence Alignments--
The
National Center for Biotechnology Information (NCBI) EST data
base was searched with BLAST on the NCBI server. The human and E. coli dehydratase peptides were aligned with the program GAP
of the Genetics Computer Group analysis package. Amino terminal peptides of the GDP-mannose 4,6-dehydratase from different species were
aligned using the GeneWorks program from IntelliGenetics, Inc.
Cloning Human GDP-mannose 4,6-Dehydratase--
A cDNA
library was constructed from HL-60 cells as described by Sako et
al. (18). This plasmid-based cDNA expression library was
assembled into 19 pools, each representing 60,000-100,000 individual
clones/pool. The pools were screened by PCR using DNA primers based on
both the mouse EST (accession number W29220) and the published E. coli sequence: 5'-TGATGAGCCAGAGGACTTTGTCATAGCTAC-3' and
5'-CAGAAAGTCCACTTCAGTCGGTCGGTAGTA-3'.
Two pools gave the expected 200-base pair fragment. The 200-base pair
PCR product was reamplified by PCR, random primer-labeled with
32P, and used to identify positive clones from the two
library pools by colony hybridization. This approach yielded plasmid
pMT-hGMD containing the cDNA of human GDP-mannose dehydratase in a
eukaryotic expression vector.
Cloning of Human GDP-4-keto-6-deoxymannose 3,5-Epimerase,
4-Reductase--
A similar approach was taken to isolate the human
epimerase reductase genes using following oligonucleotides based upon
the published sequence (17): 5'-CTGACATGGGTGAACCCCAGGGATCCATGC-3' and 5'-TGGCCATCCTCGATGTTGAAGTTGTCGTGG-3'.
The positive pools were probed with the 32P-labeled PCR
primers. This yielded plasmid pMT-hFX, containing the cDNA of human GDP-mannose epimerase-reductase in a eukaryotic expression vector.
Transfection of Lec13 and Cell Staining--
The Chinese hamster
ovary (CHO) cell line Lec13, was obtained from Professor P. Stanley at
Albert Einstein Collage of Medicine. This line was first transfected
with pMTNeoFTIV, a vector expressing human fucosyltransferase IV and
the neomycin resistance genes, by the calcium phosphate method as
described previously (19). To make this cell line capable of
replicating vectors containing the polyoma virus origin of replication,
one of the Fuc-T IV-positive cell lines, clone 9E9A, was again
transfected with pCDNA3.1 ZeoPyLT, a plasmid expressing the early
region of polyoma virus including large T, by the lipofectamine method
according to the manufacturer's instructions (Invitrogen). One
zeocin-resistant clone had Fuc-T IV activity and was
replication-competent. This cell line, 9E9A LT2.9, was transfected with
pMT-hGMD or pMT-hFX by the lipofectamine method. After 48 h, cells
were analyzed for Lewis X expression by immunofluorescence after
staining with CD15 antibody (Immunotech) and goat anti-mouse
fluorescein isothiocyanate secondary antibody (Boehringer Manheim).
In Vitro Assays of CHO Cell Extracts--
Mutant CHO cells
transfected with human dehydratase cDNA, human epimerase-reductase
cDNA, or wild type CHO cells were lysed under nitrogen pressure in
0.75 ml of 25 mM Hepes, pH 7.4, 100 mM NaCl, 10 mM EDTA, 10 mM DTT for 5 min in a Parr Bomb at
900 p.s.i. on ice. The cell debris was pelleted at 50,000 × g for 1 h, and soluble extracts were assayed in 25 mM Hepes, pH 7.4, 100 mM NaCl, 15 mM MgCl2, 10 mM DTT, 10 µM GDP-mannose, with 100,000 cpm of
14C-labeled GDP-mannose for 2 h at 37 °C. The
reactions were stopped by boiling for 5 min followed by centrifugation
for 5 min at 15,000 rpm in a microcentrifuge. Unlabeled GDP-mannose and
GDP-fucose were added as standards. GDP-mannose, GDP-fucose and the
4-keto 6-deoxy intermediate were separated as described by Yamamoto
et al. (10) except the amide-80 column (Tosohaas) was run in
66% acetonitrile and 7.5 mM citric
acid/Na2HPO4 buffer, pH 4.0. The 14C-labeled sugar nucleotides were detected with a flow
through scintillation counter Beta-1 (Packard) run with a solid
scintillant cell. The unlabeled sugar nucleotides were detected at 254 nm.
Expression and Purification of Human Enzymes from E. coli--
The human dehydratase and epimerase-reductase genes were
cloned by PCR into the EcoRI and HindIII sites of
vector pRSETB (Invitrogen) for expression in E. coli. This
yielded vectors pRSEThGMD and pRSEThFX. The inserts in the resulting
vectors were sequenced in their entirety. The resulting dehydratase
fusion protein had the sequence
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSPSAGTMEF added to
amino acid 20 of the predicted sequence, the position homologous to the
start of the E. coli enzyme. For the epimerase-reductase, the same 43-amino acid fusion peptide was added to amino acid 2 of the
published sequence. The two expression vectors were transformed into
E. coli strain BL21/DE3 and the bacteria were grown in LB media containing ampicillin and chloramphenicol. Both the
dehydratase-expressing cells and epimerase-reductase-expressing cells
were grown at room temperature to an A600 of 0.5 and induced with 0.3 mM
isopropyl-1-thio-
-D-galactopyranoside for 3-4 h. The
cells were resuspended in Tris-buffered saline containing DNase I and
lysed in a French press at 12,000 p.s.i. After clarification, the
dehydratase was purified by successive chromatography over
nitrolotriacetic acid-Ni2+-agarose (Qiagen) eluting with
200 mM imidazole, chromatography over ceramic
hydroxyapatite (Bio-Rad) eluting with a phosphate gradient (0.0-0.5
M), and chromatography over MonoQ (Pharmacia) eluting with
a NaCl gradient (0.0-0.5 M). The epimerase-reductase was
purified by successive chromatography over nitrolotriacetic acid-Ni2+-agarose eluting with 200 mM imidazole
and chromatography over 2'-5' ADP-Sepharose eluting with 10 mM NADP+. The resulting proteins were greater
than 90% pure as judged by SDS-PAGE and staining with Coomassie Blue.
Amino terminal sequencing of the first 15 amino acids of the proteins
confirmed their identity as fusion protein expressed from the pRSET
vector.
A second expression construct was made for each gene, which had a
20-amino acid amino-terminal leader, MRGSHHHHHHGSDYKDDDDK, added to the
Met19 of hGMD or Met1 of hFX. To construct
these expression plasmids, synthetic DNA was used to rebuild the hGMD
gene from the AgeI site near the 5'-end of the gene. The
synthetic DNA was hybridized and ligated to the
AgeI-HindIII fragment of pRSEThGMD into the
NdeI and HindIII sites of pRSETB to yield
pT7hGMD. A similar approach was taken to construct pT7hFX using the
BamHI-HindIII fragment of pRSEThFX. The
resulting vectors pT7hMGD and pT7hFX were transformed into E. coli strain BL21/DE3, and protein was expressed and purified as
above. No significant differences were observed in the activity, substrate, or cofactor preference for both the human dehydratase or
epimerase-reductase having either the longer or shorter N-terminal fusion.
In Vitro Assays of Purified Dehydratase and
Epimerase-Reductase--
Purified proteins were assayed in 25 mM MOPS, pH 7.0, 100 mM NaCl, 5 mM
EDTA, 10 mM DTT, 25 µM GDP-mannose with
100,000 cpm of 14C-labeled GDP-mannose for 1 h at
37 °C. When sequential reactions were performed, the mix was
incubated for an additional 1 h at 37 °C after adding second
enzyme or cofactor. The reactions were stopped by boiling for 2 min
followed by centrifugation for 1 min at 15,000 rpm in a
microcentrifuge. Unlabeled GDP-mannose and GDP-fucose were added at
this point as internal standards. Kinetic constants were determined
graphically using S/V versus S plots with
GDP-mannose concentrations ranging from 1/2 to 5 Km. The reaction conditions were as above with 1 mM NADP+ added to reactions.
Paper Chromatography--
14C-Labeled GDP-mannose,
GDP-fucose, and reaction products were incubated in 0.2 M
NaBH4 for 15 min at room temperature to reduce keto
intermediates and then were cleaved from the sugar by incubating in 1 M trichloroacetic acid for 10 min in boiling water. This mixture was chromatographed on Whatman 3MM paper in descending mode
using three different solvent systems (solvent I, water-saturated methyl ethyl ketone for 24 h; solvent II, ethyl
acetate/pyridine/water (3.6:1.0:1.15) for 7 h; solvent III, ethyl
acetate/pyridine/water (10:2.5:1.5) for 17 h. Free sugar standards
were localized by staining with AgNO3 in acetone followed
by NaOH in methanol (20). Radioactivity was detected by cutting the
paper into strips, and 1-cm sections of each strip were counted in 1 ml
of water plus 10 ml of formula 989 (Packard).
Synthesis and Characterization of
GDP-4-keto-6-deoxymannose--
GDP-4-keto-6-deoxymannose was
synthesized from GDP-mannose using the purified human and purified
E. coli dehydratases. Typical reaction conditions for human
dehydratase were 2.5 mM GDP-mannose, 0.1 mg/ml human
dehydratase, 25 mM MOPS, pH 7.0, 100 mM NaCl, 10 mM DTT, 5 mM EDTA, 10 µM
NADPH, and 10 µM NADP+ at 37 °C for 3-6
h. Typical reaction conditions for E. coli dehydratase were
10 mM GDP-mannose, 0.1 mg/ml E. coli
dehydratase, 10 mM MOPS, pH 6.5, 100 mM NaCl, 2 mM DTT, 1 mM EDTA, 10 µM NADPH,
and 100 µM NADP+ at 37 °C for 3-6 h. The
reactions were allowed to proceed to completion as judged by HPLC, and
the protein was removed by ultrafiltration on a Centricon-10. The
resulting mix was desalted on Sephadex G-10 run in water and
lyophilized. GDP-4-keto-6-deoxymannose prepared this way was stable
frozen at
20 °C either as a dried powder or in water and was
judged essentially pure by HPLC and ESI-MS analysis. For ESI-MS
analysis, 500 mM citric acid-sodium phosphate buffer was
added to a 20 pmol/µl solution of GDP-4-keto-6-deoxymannose (in 50%
acetonitrile) to give a final concentration of 0.5 mM at pH
4. Flow injection at 10 µl/min was used to introduce this sample into
the Micromass Platform II electrospray ionization-equipped mass
spectrometer, which was operated in the negative ion mode with a cone
voltage of 40 V. The intermediate was further characterized by high
field NMR. All NMR experiments were performed on a Varian Unity plus
600-MHz spectrometer. Samples were dissolved in D2O, and
HDO was used as an internal reference, set to 4.76 ppm at 25 °C.
Two-dimensional total correlation spectroscopy, nuclear Overhauser
effect spectroscopy, and heteronuclear multiple-quantum coherence were
performed with standard Varian pulse sequences.
Cloning of E. coli GMD and E. coli WCAG--
The gmd
and wcaG genes were cloned from the E. coli K-12
genome using PCR oligonucleotides that created an NcoI site
overlapping the initiating methionine of each gene and a
HindIII site following the termination codon of each gene.
The PCR products were cloned into the NcoI and
HindIII sites of pSE380 (Invitrogen), yielding pSEGMD and
pSEWCAG. Sequences were confirmed by DNA sequencing. During the
sequencing of the wcaG gene, it was discovered that multiple
clones contained two nucleotide differences from the published sequence
(21). The last nucleotide in codon 255 and the first nucleotide in
codon 256 changed from a CG in the published sequence to a GC in the
cloned gene. This changed the predicted amino acids in those positions
from an aspartate at amino acid 255 and a valine at amino acid 256 in
the published sequence to glutamate and leucine in the cloned gene.
Expression and Purification of E. coli Enzymes from E. coli--
E.
coli strain GI934 harboring either pSEGMD or pSEWCAG were grown in
LB media containing ampicillin at 370 °C to an
A600 of 0.5. The cells were induced with 1 mM isopropyl-1-thio-
-D-galactopyranoside and
grown for an additional 4 h. Cell pellets were broken in a French
press at 12,000 p.s.i. After clarification, the E. coli dehydratase was purified by successive chromatography over Toyopearl DEAE (TosoHaas) eluting with a NaCl gradient (0.0-0.5 M),
chromatography over ceramic hydroxyapatite eluting with a phosphate
gradient (0.0-0.5 M), and chromatography over MonoQ
eluting with a NaCl gradient (0.0-0.5 M). The E. coli epimerase-reductase was purified by successive chromatography
over Toyopearl DEAE eluting with a NaCl gradient (0.0-0.5
M), followed by chromatography over heparin-Toyopearl eluting with 200 mM NaCl. The resulting proteins were
greater than 90% pure as judged by SDS-PAGE and staining with
Coomassie Blue. Amino-terminal sequencing of the first 15 amino acids
of the proteins confirmed their identities.
 |
RESULTS |
Molecular Cloning of Human GDP-mannose 4,6-Dehydratase--
To
clone human GDP-mannose 4,6-dehydratase, we first performed a TBLASTN
search of the NCBI EST data base using the sequence of the E. coli enzyme. This identified a mouse EST, accession number W29220,
with a high degree of homology to the E. coli enzyme. We
designed two oligonucleotide primers based on conserved amino acids
present in both the E. coli sequence and the partial sequence of the mouse gene. Using the oligonucleotides as primers, we
obtained a 200-base pair PCR fragment from a human promyelocytic cell
line HL-60 cDNA library. This fragment was then used as a probe to
isolate two apparently full-length cDNA clones for the putative
human GDP-mannose 4,6 dehydratase, the sequence of which is shown in
Fig. 2. There are two potential initiator
methionines located at nucleotides 76 and 130 of this sequence. As
shown in Fig. 3A, the
downstream methionine of the human protein more closely aligns with the
initiating methionine of the E. coli protein. However,
alignment of the human sequence to the recently cloned arabidopsis
GDP-mannose 4,6-dehydratase gene and a putative C. elegans
translation product (Fig. 3B) suggests that translation of
the human protein initiates at the first methionine, at nucleotide 76, and that dehydratases from nonbacterial sources contain an amino-terminal extension. The human and E. coli proteins are
61% identical over their entire lengths, and both contain an extended consensus sequence, GXXGXXG, identifying the


fold found in many NAD+- and
NADP+-binding proteins (22). This sequence is found between
amino acids 9 and 15 of the E. coli enzyme. Fig.
4 shows that the human dehydratase gene
encodes a single mRNA transcript of about 1.7 kilobase pairs that
is expressed in all tissue and cell types examined, albeit at varying
levels. The mRNA levels are highest in pancreas followed by small
intestine, liver, colon, and prostate and lowest in ovary, brain, lung,
spleen, and peripheral blood lymphocytes. Likewise, a varied level of
expression was seen in the human cell lines examined (Fig.
4C).

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 2.
cDNA and amino acid sequence of human
GDP-mannose 4,6-dehydratase. The sequence of the cDNA
for human GDP-mannose 4,6-dehydratase is shown above the putative
peptide sequence.
|
|

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 3.
Alignment of GDP-mannose 4,6-dehydratases.
A, the peptide sequence for human GDP-mannose 4,6 dehydratase is shown above the sequence of the E. coli
protein. This alignment illustrates that the second methionine in the
human sequence aligns more closely with the start of the E. coli peptide. B, alignment of amino termini of
GDP-mannose 4,6-dehydratases, showing the extension relative to the
bacteria enzyme. The translated peptide sequences of human enzyme,
putative C. elegans translation product (accession number
Z68215), Arabidopsis cDNA clone (accession number
U81805), and E. coli gmd (accession number P32054) are
shown.
|
|

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 4.
Northern analysis of human GDP-mannose
4,6-dehydratase gene expression. Autoradiogram of Northern blots
probed with 32P-labeled human GDP-mannose
dehydratase-specific probe is shown. The 1.5-kilobase pair
EcoRI fragment of plasmid fragment pMT-HGMD, containing the
complete hGMD cDNA insert, was gel-purified, random primer-labeled,
and used to probe poly(A)+ RNA blots from
CLONTECH (human I (catalog number 7760-1; human II
(catalog number 7759-1); and human cancer cell line (catalog number
7757-1)) under high stringency conditions. A and
B show expression in human tissues, and C shows
expression in human cell lines. Analysis of the -actin messages are
shown at the bottom.
|
|
GDP-mannose 4,6-Dehydratase cDNA Complements the Dehydratase
Defect in Lec13--
To demonstrate the isolated cDNA encoded
GDP-mannose 4,6-dehydratase activity, we transiently transfected it
into Lec13, a CHO cell line that previously has been identified as
lacking GDP-mannose 4,6-dehydratase activity (23). To monitor the
GDP-mannose 4,6-dehydratase activity, we first stably transfected Lec13
with human fucosyltransferase IV (19), an enzyme that utilizes
GDP-fucose to synthesize the Lewis X (CD15) epitope. This gave a
readily identifiable cell surface marker dependent upon the
biosynthesis of GDP-fucose. Restoration of dehydratase activity in the
mutant cells should restore GDP-fucose biosynthesis and produce Lewis X
antigen on the cell surface. As demonstrated in Fig.
5A, culture of these Fuc-T
IV-expressing Lec13 cells in media containing fucose allowed the
synthesis of GDP-fucose through the salvage pathway and generated CD15-positive cells. Transient transfection of the same cell line with
the vector expressing the human GDP-mannose 4,6-dehydratase, in media
lacking fucose, also causes the cells to stain positive for Lewis X,
demonstrating that the dehydratase gene complements the CHO cell defect
(Fig. 5B). By contrast, transfection with the same vector
expressing the human epimerase-reductase gene, again in media lacking
fucose, shows no staining by CD15 (Fig. 5C). As further
evidence, lysates of Lec13 cells transiently transfected with human
GDP-mannose 4,6-dehydratase cDNA readily converted 14C-labeled GDP-mannose to GDP-fucose (Fig.
6B), whereas lysates of Lec13
cells transfected with the human epimerase-reductase cDNA in the
same expression vector did not (Fig. 6A). The level of
activity in dehydratase-transfected cells (Fig. 6B, 25 µg) is higher than that of wild type CHO cells (Fig. 6C, 125 µg). Based on this data, we conclude that the cDNA encodes
GDP-mannose 4,6-dehydratase.

View larger version (84K):
[in this window]
[in a new window]
|
Fig. 5.
Transfection with human GDP-mannose
4,6-dehydratase cDNA complements the defect in Lec13 cells.
The top parts of panels A-C show fluorescent
micrographs of Fuc-T IV-transformed 9E9A LT2.9 Lec13 cells stained with
CD15 antibody and fluorescein isothiocyanate-conjugated secondary
antibody. The lower parts show phase contrast micrographs of
the same fields. A, the cells fed fucose, as a positive
control, stain positive for Lewis X, the product of the Fuc-T IV gene
and GDP-fucose. B, the cells transfected with the cDNA
for human GDP-mannose 4,6-dehydratase also stain positive for Lewis X,
demonstrating the complementation of the dehydratase defect in Lec13.
C, the cells transfected with the cDNA for human FX do
not stain at all. Fuc-T IV activity in 9E9A LT2.9 cells was unstable
and gradually decreased with passage in culture. Thus, not all of the
cells in panel A stain positive. The experiment shown above
was done on the same day using the same starting cells to make the
comparison in CD15 staining meaningful.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6.
In Vitro assay of dehydratase and
epimerase-reductase activity in wild type CHO and transfected Lec13 CHO
cell lines. HPLC analysis of reactions of 14C-labeled
GDP-mannose with cell extracts are shown. A, 125 µg of
extract of 9E9A LT2.9 Lec13 cells transfected with epimerase-reductase
cDNA. B, 25 µg of extract of 9E9A LT2.9 Lec13 cells
transfected with dehydratase cDNA. C, 125 µg of
extract of CHO Dukx cells (wild type for GDP-mannose 4,6-dehydratase).
The positions of 14C-labeled GDP-mannose and GDP-fucose
standards are shown above.
|
|
Chromosomal Localization of Human GDP-mannose 4,6-Dehydratase and
FX Gene--
To determine if the two genes for GDP-fucose biosynthesis
are linked on the human genome we mapped their location using
fluorescence in situ hybridization. Full-length cDNA
inserts encoding the human dehydratase (pMT-hGMD) and epimerase
reductase (pMT-hFX) genes were used to probe a human genomic PAC (hGMD)
or P1 (hFX) libraries (24, 25) (Genome Systems, Inc., St. Louis, MO).
Two genomic clones were obtained for each probe. We confirmed that the
genomic clones contained the hGMD and hFX genes by subcloning and
sequencing (data not shown). The hGMD and hFX genes were mapped using
two-color fluorescence in situ hybridization utilizing the
genomic clone for each gene and a centromere-specific probe (26)
(Genome Systems, Inc., St. Louis, MO). The two genes are not linked in
the human genome. The human dehydratase gene was localized to the p
terminus of chromosome 6, an area corresponding to band 6p25. A total
of 80 metaphase cells were analyzed, with 64 exhibiting specific labeling. In a similar fashion, human epimerase-reductase was mapped to
the q terminus of chromosome 8, an area corresponding to band 8q24.3. A
total of 80 metaphase cells were analyzed, with 70 exhibiting specific
labeling (data not shown).
Human GDP-mannose Dehydratase and Epimerase-Reductase Are
Sufficient to Convert GDP-mannose to GDP-fucose--
To further
characterize GDP-mannose 4,6-dehydratase, and to reconstitute
GDP-fucose biosynthesis in vitro, we needed to obtain purified proteins for both the dehydratase and epimerase-reductase enzymes. To this end, we expressed both the human GDP-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-mannose 3,5-epimerase
4-reductase in E. coli as fusion proteins. The fusion
proteins were purified (Fig.
7A), and their identities were
confirmed by sequencing the first 15 amino acids of each peptide (data
not shown). The human dehydratase protein migrated on SDS-PAGE near the
position expected based upon its calculated molecular mass (42.7 kDa),
but the epimerase-reductase migrated more slowly than expected (38.3 kDa), as previously reported by Tonetti et al. (17). We
confirmed that recombinant hFX had the mass predicted from its cDNA
sequence by ESI-LC-MS (data not shown).

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 7.
SDS-PAGE of recombinant enzymes
purified from E. coli. A, hGMD and hFX.
B, GMD and WCAG. Molecular masses of markers, in kDa, are
shown in the center. Gels are stained with Coomassie
Blue.
|
|
To characterize the reactions of the purified dehydratase and
epimerase-reductase, we incubated the enzymes with
14C-labeled GDP-mannose and identified the reaction
products by HPLC and paper chromatography. As shown in Fig.
8A, purified GDP-mannose 4,6-dehydratase converts 14C-labeled GDP-mannose to a new
species that runs at the position reported for
GDP-4-keto-6-deoxymannose (10). We confirmed the identity of
GDP-4-keto-6-deoxymannose by descending paper chromatography. As shown
in Fig. 1, the expected monosaccharides resulting from reduction of
GDP-4-keto-6-deoxymannose by borohydride and cleavage from the
nucleotide with acid would be rhamnose and 6-deoxytalose. Fig.
9A (filled
triangles) shows that when the reaction products obtained by
incubating GDP-mannose, human dehydratase, and NADP+ were
reduced, cleaved, and run on paper, four spots resulted. The major
species, running at 16 cm, co-migrates with an unlabeled standard for
rhamnose. This component also co-migrates with rhamnose in solvent
systems II and III (data not shown). The species at 5 cm co-migrates
with mannose and was presumably derived from the unreacted starting
material. The species at 24 cm runs faster than rhamnose in this
solvent, as expected for 6-deoxytalose, but has an
RF that differs from the published value for 6-deoxytalose (6, 27). This is also true in solvents II and III. An
additional species was observed in this sample, running near 37 cm.
Without authentic 6-deoxytalose, we have not been able to confirm the
identity of the two faster running spots. Using the purified enzyme, we
were able to determine the cofactor utilized by human GDP-mannose
4,6-dehydratase. Comparing panels A and B of Fig.
8, we see that the human dehydratase utilizes NADP+ as a
cofactor and cannot utilize NAD+ even at a 10-fold higher
concentration.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 8.
In vitro assay of human and E. coli dehydratase and epimerase reductase activity. HPLC
analysis of reactions of 14C-labeled GDP-mannose with
purified recombinant enzymes are shown. A, 1 µg of human
dehydratase plus 100 µM NADP+; B,
1 µg of human dehydratase plus 1 mM NAD+;
C, 1 µg of human dehydratase plus 100 µM
NADP+, followed by 1 µg of human epimerase-reductase plus
100 µM NADPH; D, 1 µg of E. coli
dehydratase plus 1 mM NADP+; E, 1 µg of E. coli dehydratase plus 1 mM
NAD+; F, 1 µg of E. coli
dehydratase plus 1 mM NADP+, followed by 1 µg
of E. coli epimerase-reductase plus 100 µM
NADPH. The arrows show the position of
14C-labeled standards GDP-mannose and GDP-fucose
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 9.
Analysis of the reaction products of human
and E. coli dehydratase and epimerase-reductase by
descending paper chromatography. 14C-Labeled reaction
products were reduced with NaBH4, and the resulting sugar
was cleaved with acid and spotted on Whatman 3MM paper developed in
water-saturated methyl ethyl ketone for 24 h. The paper was cut
into strips, and 1-cm sections of each strip were counted.
A, the reactions contained GDP-mannose, human dehydratase,
and NADP+, followed by human epimerase-reductase plus NADPH
(open squares) or GDP-mannose, human dehydratase, and
NADP+ (filled triangles). B, the
reactions contained GDP-mannose, E. coli dehydratase,
NADP+, E. coli epimerase-reductase, and NADPH
(open squares) or GDP-mannose, E. coli
dehydratase, and NADP+ (filled triangles). The
position of 14C-labeled GDP-mannose and GDP-fucose that had
been treated identically to the reaction mixtures is shown
above labeled as mannose and fucose, respectively. The
position of unlabeled free rhamnose is also shown at the top
of the trace.
|
|
Using the purified human dehydratase and epimerase-reductase, we could
demonstrate that these two enzymes alone were sufficient to convert
GDP-fucose to GDP-mannose. Sequential incubation of GDP-mannose with
human dehydratase and NADP+ followed by human
epimerase-reductase and NADPH converts the GDP-mannose to GDP-fucose,
based upon co-chromatography with authentic GDP-fucose (Fig.
8C). We confirmed the identify of the product as GDP-fucose
by descending paper chromatography of the free monosaccharide after
reduction with NaBH4 and cleavage from GDP with acid. Fig. 9A (open squares) shows the free monosaccharide
co-migrates with an identically treated 14C-labeled
GDP-fucose standard. Identical results were obtained in solvents
systems II and III (data not shown). Unlike the human dehydratase,
which shows a strict cofactor preference for NADP+ over
NAD+, the human epimerase-reductase can utilize either
NADPH or NADH, although NADPH is used more efficiently (data not
shown).
Characterization of GDP-4-keto-6-deoxymannose by ESI-MS and High
Field NMR--
To confirm that the reaction product of human
dehydratase and GDP-mannose was GDP-4-keto-6-deoxymannose, we isolated
the product and analyzed it by mass spectrometry and NMR. The major
peaks in the ESI-MS spectrum of the isolated intermediate were at 586.1 and 292.6, corresponding to [M
H]
ion and
[M
H]2
, respectively for
GDP-4-keto-6-deoxymannose. A partial spectrum is shown in Fig.
10, where the [M
H]
peak at 586.1, the [M + Na+
2H]
peak at 608.1, and the [M + 2Na+
3H]
peak at 630.1 for GDP-4-keto-6-deoxymannose are
clearly visible. A [M
H]
peak for residual
GDP-mannose at 604.0 is also present in the spectra. A combination of
one-dimensional homonuclear, two-dimensional homonuclear, and
two-dimensional heteronuclear NMR experiments revealed that the
isolated reaction product was a mixture of at least two related sugar
nucleotides. The observed chemical shifts and coupling constants are
listed in Table I. From these data, we
have identified the two major species as GDP-4-keto-6-deoxymannose and
GDP-3-keto-6-deoxymannose. NMR analysis of the isolated product of
E. coli dehydratase also showed a similar mixture of
GDP-4-keto-6-deoxymannose and GDP-3-keto-6-deoxymannose.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 10.
High mass region of the ESI-MS spectrum for
the reaction products of GDP-mannose and human dehydratase.
Spectrum shows the [M H] peak at 586.1, the
[M Na+ 2H] peak at 608.1, and
the [M 2Na+ 3H] peak at 630.1 for GDP-4-keto-6-deoxymannose. Also present is the [M H] peak for GDP-mannose at 604.0. No other peaks are
evident that would correspond to any other derivatives of GDP-mannose
having a different mass.
|
|
E. coli GDP-mannose Dehydratase and Epimerase-Reductase Are
Sufficient to Convert GDP-mannose to GDP-fucose--
To address
whether the bacterial enzymes catalyze the same reactions as the human
dehydratase and epimerase-reductase, we cloned, expressed, and purified
the E. coli enzymes (Fig. 7B). We examined the
reactions of the E. coli enzymes in the same way we
characterized the human enzymes. Fig. 8E shows that
incubation of E. coli dehydratase with GDP-mannose and
NADP+ resulted in production of the reaction intermediate
GDP-4-keto-6-deoxymannose. This reaction product produced only two
spots in paper chromatography after reduction and cleavage. One
migrated with the rhamnose standard at 17 cm, and one migrated faster
at 37 cm (Fig. 9B, filled triangles). The spot
seen at 25 cm in the reaction with human dehydratase is missing (Fig.
9, compare A and B (filled
triangles)). The E. coli dehydratase used
NADP+ as a cofactor and could not substitute
NAD+ at concentrations up to 1 mM (Fig. 8,
D and E).
As with the human enzymes, E. coli dehydratase and
epimerase-reductase were sufficient to convert GDP-mannose to
GDP-fucose. Incubation of GDP-mannose with E. coli
dehydratase and E. coli epimerase-reductase in the presence
of NADP+ and NADPH converted the GDP-mannose to GDP-fucose
(Fig. 8F). We confirmed the identity of GDP-fucose by paper
chromatography (Fig. 9B, open squares). The
E. coli epimerase-reductase can use NADH but not as
efficiently as NADPH (data not shown).
We performed a preliminary characterization of the two dehydratases,
and the results are shown in Table II.
The E. coli enzyme has a significantly higher
Km for GDP-mannose than the human enzyme but also
has a significantly higher Vmax. Additionally, we monitored inhibition of the dehydratase by GDP-fucose, which has
been proposed as a mechanism to regulate the enzyme's activity. Both
human and E. coli dehydratases were inhibited by GDP-fucose with IC50 values lower than the IC50 values for
inhibition by GDP, suggesting a specific effect and a potential role in
regulation of the enzyme's activity. Quite unexpectedly, both enzymes
are stimulated by NADPH at micromolar concentrations, although this co-factor does not play a role in catalysis. It is not clear if this
stimulation by NADPH is relevant to the in vivo regulation of the enzyme.
 |
DISCUSSION |
We have cloned the gene encoding human GDP-mannose 4,6-dehydratase
using homology between the E. coli enzyme and a mouse EST. The cloned gene complemented the previously identified GDP-mannose 4,6-dehydratase defect in the CHO cell line Lec13, demonstrating that
the cDNA encodes a functional protein. The dehydratase gene shows
high levels of identity between bacteria and human and indeed across
the spectrum of species examined (for a comparison of dehydratases from
a variety of bacterial and nonmammalian sources see Bonin et
al. (28). The message for human GDP-mannose 4,6-dehydratase is
expressed in all tissues examined, albeit at varying levels. The
varying levels of expression of the dehydratase message suggest the
enzyme may be regulated at the level of transcription, and in fact
there is evidence for developmental regulation in rat and nereids (29,
30). It also appears that, in both humans and E. coli,
GDP-fucose biosynthesis is regulated by feedback inhibition of the
dehydratase by GDP-fucose, the final product in the pathway. This
mechanism of inhibition was noted for Aerobacter aerogenes
by Kornfeld and Ginsberg (9) and was suggested for the porcine enzyme
by Serif and co-workers (33).
The cloning of the human dehydratase gene, along with the recent
cloning of the human epimerase-reductase by Tonetti et al. (17) has allowed us to reconstitute GDP-fucose biosynthesis in
vitro using purified, recombinant enzymes. Thus, we have
definitively shown that two enzymes, a dehydratase and an
epimerase-reductase, are sufficient to convert GDP-mannose to
GDP-fucose. In doing so, we demonstrated, using purified recombinant
proteins, that in humans and in E. coli, both 3,5-epimerase
and 4,6-reductase activities are present in a single protein. This
confirms the earlier studies of Serif and co-workers (16) on the enzyme
purified from porcine thyroids and the recent work of Tonetti et
al. (17) with the human FX protein. Additionally, we find that
human dehydratase has a strict cofactor requirement for
NADP+ for which NAD+ cannot substitute. This is
consistent with earlier reports demonstrating that GDP-mannose
4,6-dehydratase from K. pneumoniae requires
NADP+ (10) as well as the recent work of Sturla et
al., who reported that purified E. coli dehydratase
contains NADP+.
We have characterized the product of the dehydratase reaction,
GDP-4-keto-6-deoxy-mannose. This product was first reported by Ginsberg
(6) for the bacterial enzyme and later by Overton and Serif (27) for
the porcine enzyme. Using purified dehydratase, we have analyzed the
reaction products by both HPLC and paper chromatography and find
results consistent with the expected product. For unambiguous
structural confirmation, we isolated and analyzed the intermediate by
ESI-MS and high field NMR. Mass spectrometric analysis yielded a
molecular mass consistent with GDP-4-keto-6-deoxymannose but no
evidence of any other GDP-mannose derivatives of different masses. High
field NMR revealed the presence of two compounds, one being the
expected product GDP-4-keto-6-deoxymannose and the other the related
GDP-3-keto-6-deoxymannose. This was the case for the product of both
the human and E. coli dehydratases. The presence of both the
4-keto- and 3-keto-6-deoxy-sugars was also seen in the dTDP-rhamnose
pathway where a 4,6-dehydratase converts dTDP-glucose to
dTDP-4-keto-6-deoxy-glucose (31, 32). The biological significance of
both GDP-4-keto-6-deoxymannose and GDP-3-keto-6-deoxymannose
intermediates is unclear, although apparently both are epimerized and
reduced to GDP-fucose by the epimerase reductase (Fig. 8, C
and F). It is possible that the 3-keto-sugar arose during
work-up of the isolated reaction product. However, the isolated,
unlabeled intermediate also was converted to GDP-fucose by purified
human epimerase-reductase (data not shown).
Cells from two patients having LADII do not fucosylate their cell
surfaces (4) and as such lack both blood group antigens and the sialyl
Lewis X and related epitopes that function as selectin ligands. The
molecular basis of this disorder is still unknown. As with Lec13, this
phenotype can be rescued in cell lines derived from these patients by
culturing them in the presence of fucose, suggesting that GDP-fucose
transport and the complement of fucosyltransferases are intact in these
cells.2 This would imply that
the defect in the LADII patients lies in the pathway converting
GDP-mannose to GDP-fucose, i.e. either the dehydratase or
epimerase-reductase. With the human genes for both enzymes now cloned,
we can determine if either is responsible for the LADII phenotype.
There are suggestions from E. coli that there may be an
additional gene that plays a role in GDP-fucose biosynthesis in
vivo. Sequencing of the capsular polysaccharide operon in E. coli led to the identification of an open reading frame
(wcaH) immediately downstream of the dehydratase,
gmd, and epimerase-reductase genes, wcaG (21).
This putative protein has been assigned to the GDP-fucose biosynthetic
pathway, yet it is clearly not necessary for conversion of GDP-mannose
to GDP-fucose in vitro. WcaH may play a role in
GDP-fucose biosynthesis in vivo or play another, yet
unidentified, role in capsular polysaccharide biosynthesis. The cloning
of the GDP-mannose 4,6-dehydratase gene provides a valuable tool to
address outstanding questions in the regulation, biosynthesis, and role
of GDP-fucose in vivo.
We thank Professor Pamala Stanley of the
Albert Einstein Collage of Medicine for the gift of the Lec13 cell
line; Drs. Diane Sako and Monique Davies of Genetics Institute for the
gifts of plasmids pMTNeoFTIV and pEDPyLT, respectively; Drs. Elliott
Nickbarg and Robert Gassaway of Genetics Institute for ESI-LC-MS
analysis and peptide sequencing; Kevin Bean and Mark Proia of
Genetics Institute for DNA sequencing and library screening. We
thank Drs. John Lowe and Peter Smith of Univeristy of Michigan for
sharing data on the LADII cell lines prior to publication. We thank
Drs. Simon Jones and John Knopf of Genetics Institute for critical reading of the manuscript.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF 042377.