From the Glycobiology Program, La Jolla Cancer
Research Center, The Burnham Institute, La Jolla, California 92037 and
the § Howard Hughes Medical Institute, University of
Michigan Medical Center, Ann Arbor, Michigan 40109
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ABSTRACT |
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Subsets of mammalian cell
surface oligosaccharides contain specific fucosylated moieties
expressed in lineage- and/or temporal-specific patterns. The functional
significance of these fucosylated structures is incompletely defined,
although there is evidence that subsets of them, represented by the
sialyl Lex determinant, are important participants in
leukocyte adhesion and trafficking processes. Genetic deletion of these
fucosylated structures in the mouse has been a powerful tool to address
functional questions about fucosylated glycans. However, successful use
of such approaches can be problematic, given the substantial redundancy in the mammalian -1,3-fucosyltransferase and
-1,2-fucosyltransferase gene families. To circumvent this problem,
we have chosen to clone the genetic locus encoding a mammalian
GDP-D-mannose-4,6-dehydratase (GMD). This enzyme generates
GDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which
is then converted by the FX protein
(GDP-4-keto-6-D-deoxymannose epimerase/GDP-4-keto-6-L-galactose reductase) to
GDP-L-fucose. GMD is thus imperative for the synthesis of
all fucosylated oligosaccharides. An expression cloning approach and
the GMD-deficient CHO host cell line Lec13 were used to generate a
population of cDNA molecules enriched in GMD cDNAs. This
enriched plasmid population was then screened using a human expressed
sequence tag (EST AA065072) with sequence similarity to an
Arabidopsis thaliana GMD cDNA. This approach, together
with 5'-rapid amplification of cDNA ends, yielded a human cDNA
that complements the fucosylation defect in the Lec13 cell line.
Northern blot analyses indicate that the GMD transcript is absent in
Lec13 cells, confirming the genetic deficiency of this locus in these
cells. By contrast, the transcript encoding the FX protein, which forms
GDP-L-fucose from the ketosugar intermediate produced by
GMD, is present in increased amounts in the Lec13 cells. These results
suggest that metabolites generated in this pathway may participate in
the transcriptional regulation of the FX protein and possibly the GMD
protein. The results also suggest that the genomic structure encoding
GMD in Lec13 cells likely has a defect different from a point mutation
in the coding region.
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INTRODUCTION |
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Fucose is one of the critical carbohydrates in membrane-associated
glycoproteins and glycolipids. Carbohydrates containing fucose are
often characteristic of different cell types and are determinants for
carbohydrate antigens (1). In particular, sialyl
Lex,1
NeuNAc2
3Gal
1
4(Fuc
1
3)GlcNAc
R, discovered in
granulocytes (2), was found to be a ligand for E- and P-selectin
(3-6). The isomer of sialyl Lex, sialyl Lea,
NeuNAc
2
3Gal
1
3(Fuc
1
4)GlcNAc
R, is also a ligand for
E- and P- selectin (7, 8). Moreover, sulfated derivatives of sialyl
Lex, were found to be ligands for L-selectin
(9, 10), although sialyl Lex serves as an inefficient
L-selectin ligand (10, 11).
Expression of these fucosylated oligosaccharides is dependent on
fucosyltransferases and donor substrate GDP-L-fucose (12). So far at least four fucosyltransferases, FucTIV, -V, -VI, and -VII
were found to be capable of forming sialyl Lex (13-17). On
the other hand, FucTIII was found to be responsible for the expression
of sialyl Lea as well as sialyl Lex (12). Among
these 1
3/4 fucosyltransferases, FucTVII is present in
granulocytes, memory T cells, and high endothelia venules and directs
the synthesis of selectin ligands in these cells (14, 15, 17). Recent
report on gene knockout of mouse FucTVII clearly demonstrated the role
of FucTVII in selectin ligand presentation (18). On the other hand,
FucTIII, -V, and -VI are present in a wide variety of cells, and more
than one
1
3/4 fucosyltransferase is present in a given tissue or
cell.
To address the roles of sialyl Lex and fucosylated oligosaccharides in general, a mouse lacking each gene encoding a fucosyltransferase must first be established and then such a mutant mouse must breed with another mutant mouse. Such a step has to be repeated three or possibly four times to obtain null mouse which completely lacks sialyl Lex in all tissues. In order to overcome this problem, we decided to clone a cDNA encoding an enzyme that is critically involved in fucose metabolism in general. This direction was also prompted by the report on patients with recurrent pneumonia and skin infections (19). These patients lack sialyl Lex in neutrophils and are defective in the recruitment of neutrophils to sites of inflammation. Because fucose-containing antigens such as ABO blood group antigens are also absent in these patients, it is assumed that a step in the fucose metabolism is defective in these patients (19).
Donor substrate GDP-L-fucose is synthesized from GDP-D-mannose via three steps; GDP-D-mannose is first converted to GDP-4-keto-D-deoxymannose, and then to GDP-4-keto-6-L-deoxygalactose, which is further converted to GDP-L-fucose (20, 21). It has been demonstrated that GDP-D-mannose-4,6-dehydratase, catalyzing the first reaction in the above metabolic pathway, is defective in a mutant CHO cell line Lec13 (22). However, no successful correction of Lec13 phenotype has been reported.
In this report, we first describe the molecular cloning of the human GDP-D-mannose-4,6-dehydratase (GMD). For this cloning, cDNA was initially enriched by expression cloning strategy using Lec13 as recipient cells. Plasmids rescued from those Lec13 cells expressing fucose were then screened by a human expressed sequence tag (EST) sequence, which has a strong similarity to cDNA encoding GDP-D-mannose-4,6-dehydratase cloned from Arabidopsis thaliana (23), resulting in the isolation of a plasmid DNA containing the human GMD. Introduction of the cloned cDNA into Lec13 cells resulted in the expression of fucosylated oligosaccharides in Lec13 cells, correcting the Lec13 phenotype.
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EXPERIMENTAL PROCEDURES |
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Vectors and Antibody--
-1,2-Fucosyltransferase
(
-1,2-FT) cDNA was excised from pcDNA I-
-1,2-FT (24) by
EcoRI and XbaI digestion and cloned into the same
sites in pcDNA3, resulting in pcDNA3-
-1,2-FT.
-1,2-FT was
shown to add
-1,2-fucose to both type 1 (Gal
1
3GlcNAc) and type
2 (Gal
1
4GlcNAc) oligosaccharides, forming Leb and H
structure, respectively (24). pcDNA I-FucTIII was prepared as
described previously (25). Anti-H antibody was prepared from a
hybridoma cell line obtained from American Type Culture Collection.
Cloning of GDP-D-mannose-4,6-dehydratase--
Lec13
cells were found to be negative for H antigen after
pcDNA3--1,2-FT was transiently expressed. Lec13 cells were thus co-transfected with 7 µg of a human fetal brain cDNA library in pcDNA I (26), 7 µg of pcDNA3-
-1,2-FT, and 7 µg of
pPSVE1-PyE harboring polyoma large T antigen cDNA (27), using
LipofectAMINETM (Life Technologies, Inc.) as described previously (26).
Sixty-two h after the transfection, the transfected cells were
dispersed into single cells by the cell dissociation solution (Cell & Molecular Technologies, Levellete, NJ), and then incubated with mouse
anti-H antibody followed by FITC-conjugated goat affinity-purified
(Fab')2 fragment specific to mouse IgM. The cells were then
sorted by fluorescence-activated cell sorting using FACStar (Becton
Dickinson). Plasmids were rescued by the Hirt procedure (28) from those transfected cells strongly positive for H antigen expression. Plasmid
DNA was amplified in the host bacteria Escherichia coli DH10B/P3 in the presence of ampicillin and tetracycline. The pcDNA I vector contains the supF suppressor tRNA gene, so that DH10B/P3 cells
containing pcDNA I are resistant to both ampicillin and tetracycline. In contrast, DH10B/P3 cells having pcDNA3-
-1,2-FT or pPSVE1-PyE are resistant only to ampicillin. By selection with ampicillin and tetracycline, only bacteria containing pcDNA I were
rescued and amplified (26), allowing the isolation of plasmids responsible for fucose expression assessed by the anti-H antibody. From
this initial pool of 2 × 104 plasmids, sibling
selection was carried out to isolate a plasmid clone that encodes a
human GMD. However, this attempt was not successful. We then decided to
clone GMD by hybridization method. Human EST data bases were searched
with the nucleotide sequence reported for GMD cloned from A. thaliana (23) and one sequence (AA065072) was identified. This
human EST cDNA, obtained from Genome Systems, was digested with
EcoRI and the resultant 5'-half cDNA fragment (450 base
pairs), having high homology with A. thaliana GMD cDNA,
was used as a probe. This was necessary because the nucleotide sequence
after 301 base pairs from the 5'-end in the EST cDNA differ
entirely from that of A. thaliana GMD cDNA (23). After
screening approximately 2 × 104 clones of plasmids
derived from the sorted cells as described above, four positive clones
were isolated. One of them was found to have the largest cDNA
insert, the nucleotide sequence of which is very similar to that
reported for A. thaliana GMD and was tentatively designated
as a partial human GMD cDNA.
Transfection of Lec13 cells with pcDNA I-hGMD-- Lec13 cells were transfected with pcDNA3-FucTIII and pcDNA I-hGMD using LipofectAMINE as described (26). After selection with G418 (Life Technologies, Inc.), the transfected cells were selected by immunofluorescent staining for their expression of Lex using anti-Lex antibody (Immunotech, Marseille, France) or sialyl Lex using CSLEX-1 antibody (Becton Dickinson), as described previously (10, 26).
Northern Blot Analysis--
Northern blots of
poly(A)+ RNA from human fetal and adult multiple tissues
were purchased from CLONTECH. Northern blots were also made using poly(A)+ RNA isolated from CHO parent
cells, Lec13, HeLa, and HepG2 cells using a FastTrackTM 2.0 kit
(Invitrogen). These blots were hybridized with a gel-purified cDNA
insert of pcDNA I-hGMD after labeling with
[-32P]dCTP by random oligonucleotide priming (Prime
It-II labeling kit, Stratagene). The blots, made in an identical
manner, were hybridized with a gel-purified cDNA encoding
GDP-4-keto-6-D-deoxymannose epimerase/NADPH-depen-dent reductase (FX protein) (29). This cDNA encoding FX protein was obtained as an EST cDNA (AA115440) and purchased from Genome Systems.
In Vitro Assay for Conversion of GDP-D-mannose to GDP-L-fucose-- Enzymatic activity of GMD and formation of the GDP-L-fucose from GDP-D-mannose were assayed using a slight modification of procedures published previously (22). The incubation mixture in 100 µl contained 0.1 µmol of GDP-[14C]mannose (0.08 µCi, NEN Life Science Products), 100 mM Hepes, pH 7.0, 5 mM ATP, 10 mM nicotinamide, 2% glycerol, 0.28 mg/ml phenylmethylsulfonyl fluoride, and 96 µg of cytosolic proteins. In some instances, assays were supplemented with 0.2 mM NADPH. Assays completed in the absence of added NADPH measure the generation of GDP-4-keto-6-D-deoxymannose intermediate from GDP-D-mannose via the action of GDP-D-mannose-4,6-dehydratase activity (20, 21). This keto intermediate accumulates because its subsequent conversion to GDP-L-fucose by the action of the FX protein is an NADPH-dependent reaction (29). Consequently, assays supplemented with NADPH measure the concerted actions of both steps in this pathway.
The unstable GDP-ketosugar intermediates generated in the assays were converted to their reduced forms and acid-hydrolyzed as described before (22). Free mannose liberated by this hydrolysis procedure was re-phosphorylated with yeast hexokinase and removed by passing through a Dowex 1X8(-200) column (phosphate counter-ion) as described (22). The eluate was dried by rotary evaporation, resuspended in a small volume of water, and subjected to descending paper chromatography for 6 h on Whatman No. 1 paper in the upper phase of pyridine:ethyl acetate:water (1.0:3.6:1.15, v/v/v). The paper was cut into 3-cm strips, and the radioactivity in each strip was quantitated by scintillation counting. Sugars were identified by their mobilities relative to commercially available standards (mannose, fucose, 6-deoxyglucose, and ![]() |
RESULTS |
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Cloning of cDNA Encoding Human GMD--
CHO mutant Lec13 cells
were co-transfected with a human fetal brain cDNA library in
pcDNA I, pcDNA3--1,2-FT, and pPSVE1-PyE. The transfected
cells highly positive for H antigen expression were isolated by
fluorescence-activated cell sorting. Plasmid DNAs, recovered from the
above H-antigen positive Lec13 cells, were initially subjected to
sibling selection with sequentially smaller, active pools, attempting
to isolate a single clone that directs the synthesis of fucose in Lec13
cells. However, this attempt was not successful (see below).
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Expression of Various Fucosylated Antigens on Lec13 Cells after
Transfection with pcDNA I-hGMD--
To confirm that pcDNA
I-hGMD directs the expression of fucosylated oligosaccharides on Lec13
cells, Lec13 cells were co-transfected with pcDNA I-hGMD and
pcDNA3-FucTIII or pcDNA I-hGMD and pcDNA3--1,2-FT. As
shown in Fig. 2 (G and
H), Lec13 cells were highly positive for sialyl
Lex and Lex after transfection with pcDNA
I-hGMD and pcDNA I-FucTIII. In contrast, Lec13 cells were barely
positive for H-antigen expression after transfection with pcDNA
I-GMD and pcDNA3-
-1,2-FT (Fig. 2D). On the other
hand, wild-type CHO cells were highly positive for H-antigen expression
as well as Lex and sialyl Lex expression after
transfection with
-1,2-FT or FucTIII (Fig. 2, B,
E, and F). These results indicate that the fucose
metabolism of Lec13 was corrected by the expression of pcDNA
I-hGMD. The above results also indicate that Lec13 cells can barely
express Fuc
1
2Gal
1
4GlcNAc structure, possibly because the
high degree of sialylation prevents the addition of fucose at terminal
galactose residues. This was probably the reason why the cloning of GMD was not successful by detecting H-antigen after transfection of Lec13
cells with fractionated plasmids and pcDNA-
-1,2-FT.
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Formation of GDP-4-keto-6-deoxy-D-mannose by hGMD-- To confirm that the cloned cDNA encodes GDP-D-mannose-4,6-dehydratase, the enzymatic assay was carried out on the Lec13 cells and Lec13 cells stably transfected with pcDNA I-hGMD. GDP-D-mannose is converted to GDP-4-keto-6-deoxy-D-mannose by GMD. This keto intermediate will be converted to GDP-L-fucose by an epimerase and GDP-4-keto-6-L-deoxygalactose reductase (Fig. 3). As shown previously, the last two reactions are carried out by a single enzyme, FX protein (29). In the absence of NADPH, however, the second and the third reactions do not take place, thus allowing us to measure the activity of the dehydratase by determining the amount of GDP-4-keto-6-deoxy-D-mannose formed (Fig. 3). GDP-4-keto-6-D-deoxymannose would be converted by NaBH4 reduction to GDP-6-deoxy-D-talose and GDP-rhamnose, whereas GDP-4-keto-6-deoxy-L-galactose would be converted to GDP-L-fucose and GDP-6-deoxy-L-glucose by the same treatment. These products can be then released by acid hydrolysis and resultant monosaccharides can be separated by paper chromatography as shown in Fig. 4.
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Expression of GMD and FX mRNAs in Human Tissues-- Northern blots of poly(A)+ RNA derived from various human tissues were examined. A GMD transcript of ~1.7 kilobases was detected in all tissues examined. However, it was more prominent in fetal kidney than in fetal brain, lung, and liver. Among various adult tissues, the strongest signal was detected in colon and pancreas and a moderately strong signal was detected in testis and small intestine (data not shown).
An ~1.4-kilobase transcript of FX protein, on the other hand, was more prominent in fetal liver than in fetal brain, lung, and kidney. Among adult tissues, pancreas, testis, colon, and skeletal muscle expressed more prominently the transcript for the FX protein than the other tissues.Expression of GMD and FX mRNAs in Lec13, Wild-type CHO, HeLa, and HepG2 Cells-- Northern blot analysis of poly(A)+ RNA derived from Lec13 and CHO cells demonstrated that the transcript for GMD was not detectable in Lec13 cells whereas it was detected in CHO cell (Fig. 5). In contrast, Lec13 cells express more transcripts for FX protein than CHO. The same analysis also showed that the transcript for FX protein is less in HepG2 cells than HeLa cells whereas the transcript for GMD is more in HepG2 cells than HeLa cells (Fig. 5). These results indicate that the transcript for GMD is absent in Lec13 cells. The results also suggest that the transcript for FX protein is increased when GMD is not sufficiently expressed.
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DISCUSSION |
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In the present study, we have isolated a human cDNA encoding
GMD using expression cloning strategy and then screening the obtained
plasmid pool by EST sequence. The expression of cloned GMD in Lec13
cells corrected the phenotype of Lec13 cells, acquiring fucosylated
oligosaccharides. The amino acid sequence of human GMD is highly
homologous to those isolated from other organisms including bacteria
(23, 30). This situation differs completely from Golgi-associated
glycosyltransferases. For example, the amino acid sequences of
mammalian polysialyltransferases (26, 31-34) differ entirely from
bacterial polysialyltransferase (35). Similarly, human
-1,3-N-acetylglucosaminyltransferase has no homology with Nisseria gonorrhoeae
-1,3-N-acetylglucosaminyl
transferase (36, 37). On the other hand, the amino acid sequences of
Golgi-associated
-mannosidases are conserved from yeast to humans
(38). These results suggest that enzymes forming donor sugars in the
cytoplasm evolved early in very primitive organisms and were conserved
during evolution. In contrast, Golgi-associated glycosyltransferases most likely evolved only after organisms reached eukaryotes, acquiring the Golgi-apparatus.
Previously, it has been shown that a point mutation in the coding
region of N-acetylglucosaminyltransferase I and V leads into
the glycosylation defect in Lec1 and Lec4A cells, respectively (39,
40). In carbohydrate-deficient glycoprotein syndrome type II, a point
mutation was discovered in the nucleotide sequence encoding the
catalytic domain of -1,2-N-acetylglucosaminyltransferase II (41). Such a point mutation leads to inactivation of the enzyme,
causing defective brain development (41). In contrast, Lec13 cells lack
the transcript for GMD as shown in the present study. This defect is
similar to that discovered in one of HEMPAS (congenital
dyserythropoietic anemia type II) patients, where the transcript for
-mannosidase II is substantially reduced (42). These results
strongly suggest that a defect in the genomic structures encoding GMD
and
-mannosidase II leads to glycosylation anomaly in Lec13 and
HEMPAS patients, respectively. These defects can be due to a defect in
the transcription regulatory element, a deletion of part or all of the
locus or dramatically decreased mRNA stability caused by nonsense
mutation (43). Moreover, the amount of the transcript for the FX
protein, which converts the intermediate formed by GMD to
GDP-L-fucose, is increased in Lec13 cells as if Lec13 cells
try to compensate the low activity of GMD (Fig. 5). These results
suggest that metabolites generated in this pathway may participate in
the transcriptional regulation of the FX protein and possibly the GMD
protein. It will be significant to determine if patients with the
defect in fucose metabolism (19) is due to a genomic defect in GMD or
FX protein, and in parallel to determine the genetic defect in Lec13
cells.
In the present study, we have demonstrated that the defect of fucose metabolism in Lec13 cells can be corrected by the expression of GMD. Similarly, the mutant of A. thaliana cells regained normal fucose metabolism by expressing GMD in the mutant cell line. A point mutation was identified in the coding sequence of GMD gene, MUR1 in the latter studies (23). Although an additional gene for GMD is suggested in A. thaliana (23), these results strongly suggest that only one gene may be dominant for expressing GMD in A. thaliana, and most likely in Lec13 cells. Northern blot analysis indicated that cloned human GMD is expressed in all tissues so far examined. However, we do not know whether the cloned enzyme is solely responsible for fucose metabolism in hematopoietic and endothelial cells. These issues need to be dissolved before attempts for generating knock-out mice defective in the GMD gene will be carried out.
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ACKNOWLEDGEMENTS |
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We thank Drs. Pamela Stanley, James Paulson, and Katherine Ketchum for their kind gifts of cells and reagents; Dr. Edgar Ong for critical reading of the manuscript; and Susan Greaney for organizing the manuscript.
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FOOTNOTES |
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* This work was supported by Grants R37 CA33000 and P01 CA71932 from the National Cancer Institute.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) AF040260.
¶ To whom correspondence should be addressed: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3144; Fax: 619-646-3193; E-mail: minoru{at}burnham-inst.org.
1
The abbreviations used are: Le, Lewis; GMD,
GDP-D-mannose-4,6-dehydratase; -1,2-FT,
-1,2-fucosyltransferase; FITC, fluorescein isothiocyanate;
RACE, rapid amplification of cDNA ends; PCR, polymerase chain
reaction; FX protein, GDP-4-keto-6-D-deoxymannose
epimerase/GDP-4-keto-6-L-galactose reductase; CHO, Chinese
hamster ovary; FucT, fucosyltransferase; EST, expressed sequence
tag.
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REFERENCES |
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