FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2022190919> ?p ?o ?g. }

• W2022190919 endingPage "14587" @default.
• W2022190919 startingPage "14582" @default.
• W2022190919 abstract "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 α-1,3-fucosyltransferase and α-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 anArabidopsis 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. 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 α-1,3-fucosyltransferase and α-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 anArabidopsis 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. 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 (1Fukuda M. Fukuda M. Hindsgaul O. Molecular Glycobiology. Oxford University Press, Oxford, United Kingdom1994: 1-52Google Scholar). In particular, sialyl Lex, 1The abbreviations used are: Le, Lewis; GMD, GDP-d-mannose-4,6-dehydratase; α-1,2-FT, α-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. 1The abbreviations used are: Le, Lewis; GMD, GDP-d-mannose-4,6-dehydratase; α-1,2-FT, α-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.NeuNAcα2→3Galβ1→4(Fucα1→3)GlcNAc→R, discovered in granulocytes (2Fukuda M. Spooncer E. Oates J.E. Dell A. Klock J.C. J. Biol. Chem. 1984; 259: 10925-10935Abstract Full Text PDF PubMed Google Scholar), was found to be a ligand for E- and P-selectin (3Lowe J.B. Stoolman L.M. Nair R.P. Larsen R.D. Berhend T.L. Marks R.M. Cell. 1990; 63: 475-484Abstract Full Text PDF PubMed Scopus (668) Google Scholar, 4Phillips M.L. Nudelman E. Gaeta F.C.A. Perez M. Singhal A.K. Hakomori S.-I. Paulson J.C. Science. 1990; 250: 1130-1132Crossref PubMed Scopus (1295) Google Scholar, 5Walz G. Aruffo A. Kolanus W. Bevilacqua M. Seed B. Science. 1990; 250: 1132-1135Crossref PubMed Scopus (881) Google Scholar, 6Polley M.J. Phillips M.L. Wayner E. Nudelman E. Singhal A.K. Hakomori S.-I. Paulson J.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6224-6228Crossref PubMed Scopus (453) Google Scholar). The isomer of sialyl Lex, sialyl Lea, NeuNAcα2→3Galβ1→3(Fucα1→4)GlcNAc→R, is also a ligand for E- and P- selectin (7Berg E.L. Robinson M.K. Mansson O. Butcher E.C. Magnani J.L. J. Biol. Chem. 1991; 266: 14869-14872Abstract Full Text PDF PubMed Google Scholar, 8Takada A. Ohmori K. Takahashi N. Tsuyuoka K. Yago A. Zenita K. Hasegawa A. Kannagi R. Biochem. Biophys. Res. Commun. 1991; 179: 713-719Crossref PubMed Scopus (324) Google Scholar). Moreover, sulfated derivatives of sialyl Lex, were found to be ligands for l-selectin (9Imai Y. Lasky L.A. Rosen S.D. Nature. 1993; 361: 555-557Crossref PubMed Scopus (330) Google Scholar, 10Tsuboi S. Isogai Y. Hoda N. King J.K. Hindsgaul O. Fukuda M. J. Biol. Chem. 1996; 271: 27213-27216Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), although sialyl Lex serves as an inefficientl-selectin ligand (10Tsuboi S. Isogai Y. Hoda N. King J.K. Hindsgaul O. Fukuda M. J. Biol. Chem. 1996; 271: 27213-27216Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 11Imai Y. Lasky L.A. Rosen S.D. Glycobiology. 1992; 2: 373-381Crossref PubMed Scopus (67) Google Scholar). Expression of these fucosylated oligosaccharides is dependent on fucosyltransferases and donor substrate GDP-l-fucose (12Natsuka S. Lowe J.B. Curr. Opin. Struct. Biol. 1994; 4: 683-691Crossref Scopus (63) Google Scholar). So far at least four fucosyltransferases, FucTIV, -V, -VI, and -VII were found to be capable of forming sialyl Lex (13Goelz S. Kumar R. Potvin B. Sundaram S. Brickelmaier M. Stanley P. J. Biol. Chem. 1994; 269: 1033-1040Abstract Full Text PDF PubMed Google Scholar, 14Sasaki K. Kurata K. Funayama K. Nagata M. Watanabe E. Ohta S. Hanai N. Nishi T. J. Biol. Chem. 1994; 269: 14730-14737Abstract Full Text PDF PubMed Google Scholar, 15Natsuka S. Gersten K.M. Zenita K. Kannagi R. Lowe J.B. J. Biol. Chem. 1994; 269: 16789-16794Abstract Full Text PDF PubMed Google Scholar, 16Gersten K.M. Natsuka S. Trinchera M. Petryniak B. Kelly R.J. Hiraiwa N. Jenkins N.A. Gilbert D.J. Copeland N.G. Lowe J.B. J. Biol. Chem. 1995; 270: 25047-25056Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 17Smith P.L. Gersten K.M. Petryniak B. Kelly R.J. Rogers C. Natsuka Y. Alford III, J.A. Scheidegger E.P. Natsuka S. Lowe J.B. J. Biol. Chem. 1996; 271: 8250-8259Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). On the other hand, FucTIII was found to be responsible for the expression of sialyl Lea as well as sialyl Lex (12Natsuka S. Lowe J.B. Curr. Opin. Struct. Biol. 1994; 4: 683-691Crossref Scopus (63) Google Scholar). Among these α1→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 (14Sasaki K. Kurata K. Funayama K. Nagata M. Watanabe E. Ohta S. Hanai N. Nishi T. J. Biol. Chem. 1994; 269: 14730-14737Abstract Full Text PDF PubMed Google Scholar, 15Natsuka S. Gersten K.M. Zenita K. Kannagi R. Lowe J.B. J. Biol. Chem. 1994; 269: 16789-16794Abstract Full Text PDF PubMed Google Scholar, 17Smith P.L. Gersten K.M. Petryniak B. Kelly R.J. Rogers C. Natsuka Y. Alford III, J.A. Scheidegger E.P. Natsuka S. Lowe J.B. J. Biol. Chem. 1996; 271: 8250-8259Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Recent report on gene knockout of mouse FucTVII clearly demonstrated the role of FucTVII in selectin ligand presentation (18Maly P. Thall A.D. Petryniak B. Rogers C.E. Smith P.L. Marks R.M. Kelly R.J. Gersten K.M. Cheng G. Saunders T.L. Camper S.A. Camphausen R.T. Sullivan F.F. Isogai Y. Hindsgaul O. von Andrian U.H. Lowe J.B. Cell. 1996; 86: 643-653Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar). On the other hand, FucTIII, -V, and -VI are present in a wide variety of cells, and more than one α1→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 (19Etzioni A. Frydman M. Pollack S. Avidor I. Phillips M.L. Paulson J.C. Gershoni-Baruch R. N. Engl. J. Med. 1992; 327: 1789-1792Crossref PubMed Scopus (433) Google Scholar). 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 (19Etzioni A. Frydman M. Pollack S. Avidor I. Phillips M.L. Paulson J.C. Gershoni-Baruch R. N. Engl. J. Med. 1992; 327: 1789-1792Crossref PubMed Scopus (433) Google Scholar). 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 (20Ginsburg V. J. Biol. Chem. 1960; 235: 2196-2201Abstract Full Text PDF PubMed Google Scholar, 21Liao T.H. Barber G.A. Biochim. Biophys. Acta. 1971; 230: 64-71Crossref PubMed Scopus (21) Google Scholar). 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 (22Ripka J. Adamany A. Stanley P. Arch. Biochem. Biophys. 1986; 249: 533-545Crossref PubMed Scopus (69) Google Scholar). 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 (23Bonin C.P. Potter I. Vanzin G.F. Reiter W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2085-2090Crossref PubMed Scopus (190) Google Scholar), 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. α-1,2-Fucosyltransferase (α-1,2-FT) cDNA was excised from pcDNA I-α-1,2-FT (24Kelly R.J. Rouquier S. Giorgi D. Lennon G.G. Lowe J.B. J. Biol. Chem. 1995; 270: 4640-4649Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar) byEcoRI and XbaI digestion and cloned into the same sites in pcDNA3, resulting in pcDNA3-α-1,2-FT. α-1,2-FT was shown to add α-1,2-fucose to both type 1 (Galβ1→3GlcNAc) and type 2 (Galβ1→4GlcNAc) oligosaccharides, forming Leb and H structure, respectively (24Kelly R.J. Rouquier S. Giorgi D. Lennon G.G. Lowe J.B. J. Biol. Chem. 1995; 270: 4640-4649Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). pcDNA I-FucTIII was prepared as described previously (25Kukowska-Latallo J.F. Larsen R.D. Nair R.P. Lowe J.B. Genes Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (467) Google Scholar). Anti-H antibody was prepared from a hybridoma cell line obtained from American Type Culture Collection. Lec13 cells were found to be negative for H antigen after pcDNA3-α-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 (26Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar), 7 μg of pcDNA3-α-1,2-FT, and 7 μg of pPSVE1-PyE harboring polyoma large T antigen cDNA (27Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar), using LipofectAMINE™ (Life Technologies, Inc.) as described previously (26Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar). 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 (28Hirt B. J. Mol. Biol. 1967; 26: 365-369Crossref PubMed Scopus (3338) Google Scholar) from those transfected cells strongly positive for H antigen expression. Plasmid DNA was amplified in the host bacteria Escherichia coliDH10B/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-α-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 (26Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar), 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 (23Bonin C.P. Potter I. Vanzin G.F. Reiter W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2085-2090Crossref PubMed Scopus (190) Google Scholar) and one sequence (AA065072) was identified. This human EST cDNA, obtained from Genome Systems, was digested withEcoRI 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 (23Bonin C.P. Potter I. Vanzin G.F. Reiter W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2085-2090Crossref PubMed Scopus (190) Google Scholar). 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. The nucleotide sequence of the obtained clone indicated that the 5′-end was missing. To obtain a complete cDNA, 5′-RACE (Life Technologies, Inc.) was carried out using poly(A)+ RNA from human fetal brain (CLONTECH) as a template and an oligonucleotide 5′-TCAGTGAGATCGCCATAGTGCAAC-3′ complementary to nucleotides 144–167. Using the cDNA formed as a template, 5′-region of human GMD cDNA was amplified by PCR using 5′-anchor primer provided in the RACE kit and 3′-PCR nested primer, 5′-TGTTTCCTTCAATGTGAGCCTGGG, which is complementary to nucleotides 116–139. The 241-base pair PCR product was cloned into pBluescript II/SK+ by T-A cloning. HindIII-NcoI fragment of 5′-RACE product was then ligated into NcoI andEcoRI fragment of GMD at a common NcoI site to form a cDNA encoding a full length of the coding region. This cDNA was cloned into pcDNA I, resulting in pcDNA I-hGMD. Lec13 cells were transfected with pcDNA3-FucTIII and pcDNA I-hGMD using LipofectAMINE as described (26Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar). After selection with G418 (Life Technologies, Inc.), the transfected cells were selected by immunofluorescent staining for their expression of Lexusing anti-Lex antibody (Immunotech, Marseille, France) or sialyl Lex using CSLEX-1 antibody (Becton Dickinson), as described previously (10Tsuboi S. Isogai Y. Hoda N. King J.K. Hindsgaul O. Fukuda M. J. Biol. Chem. 1996; 271: 27213-27216Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 26Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar). 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 FastTrack™ 2.0 kit (Invitrogen). These blots were hybridized with a gel-purified cDNA insert of pcDNA I-hGMD after labeling with [α-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) (29Tonetti M. Sturla L. Bisso A. Benatti U. De Flora A. J. Biol. Chem. 1996; 271: 27274-27279Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). This cDNA encoding FX protein was obtained as an EST cDNA (AA115440) and purchased from Genome Systems. 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 (22Ripka J. Adamany A. Stanley P. Arch. Biochem. Biophys. 1986; 249: 533-545Crossref PubMed Scopus (69) Google Scholar). 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 (20Ginsburg V. J. Biol. Chem. 1960; 235: 2196-2201Abstract Full Text PDF PubMed Google Scholar, 21Liao T.H. Barber G.A. Biochim. Biophys. Acta. 1971; 230: 64-71Crossref PubMed Scopus (21) Google Scholar). This keto intermediate accumulates because its subsequent conversion to GDP-l-fucose by the action of the FX protein is an NADPH-dependent reaction (29Tonetti M. Sturla L. Bisso A. Benatti U. De Flora A. J. Biol. Chem. 1996; 271: 27274-27279Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). 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 (22Ripka J. Adamany A. Stanley P. Arch. Biochem. Biophys. 1986; 249: 533-545Crossref PubMed Scopus (69) Google Scholar). 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 (22Ripka J. Adamany A. Stanley P. Arch. Biochem. Biophys. 1986; 249: 533-545Crossref PubMed Scopus (69) Google Scholar). 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 α-d-rhamnose), or 6-deoxytalose standard generously provided by Dr. James Paulson and Katherine Ketchum (Cytel Corp.). The parent CHO (Pro−5) and Lec13 cells were kindly provided by Dr. Pamela Stanley, Albert Einstein College of Medicine. CHO mutant Lec13 cells were co-transfected with a human fetal brain cDNA library in pcDNA I, pcDNA3-α-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). We then screened the plasmid pool right after the cell sorting with a human cDNA fragment which contains a sequence highly homologous toA. thaliana GMD (23Bonin C.P. Potter I. Vanzin G.F. Reiter W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2085-2090Crossref PubMed Scopus (190) Google Scholar) and isolated four positive clones. Comparing A. thaliana GMD to human cDNA sequences cloned, the cloned DNA contained almost all of the coding sequence but lacks the extreme 5′-region. The sequence obtained by 5′-RACE reaction was ligated to the common NcoI site of the cDNA clone to form a cDNA encoding the entire GMD, resulting in pcDNA I-hGMD. The cDNA sequence of hGMD contains 1466 nucleotides encoding 339 amino acids (Fig. 1). Because poly(A)n tail is present in nucleotides 1383–1409, ATTAAA at nucleotides 1355–1360 may have functioned as a polyadenylation signal. The amino acid and nucleotide sequence of human GMD are similar to those of the A. thaliana GMD (55.0% and 55.4% identity, respectively). The sequence is highly conserved from bacteria to human (see also Ref. 23Bonin C.P. Potter I. Vanzin G.F. Reiter W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2085-2090Crossref PubMed Scopus (190) Google Scholar). 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-α-1,2-FT. As shown in Fig. 2 (G andH), 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-α-1,2-FT (Fig. 2 D). 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 α-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 Fucα1→2Galβ1→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-α-1,2-FT. 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 (29Tonetti M. Sturla L. Bisso A. Benatti U. De Flora A. J. Biol. Chem. 1996; 271: 27274-27279Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). 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.Figure 4Separation of intermediate monosaccharides and fucose by paper chromatography. Reduced intermediate monosaccharides, released from GDP-bound forms, were separated by paper chromatography, and radioactivity in each 3-cm strip was determined by a scintillation counter. Rhamnose and 6-deoxytalose, derived from GDP-4-keto-6-deoxy-d-mannose, migrated at the positions of 25–30 cm and 31–36 cm from the origin, respectively. Fucose and 6-deoxyglucose, derived from GDP-4-keto-6-deoxy-l-galactose, migrated together at the positions of 15–21 cm from the origin. The majority of fucose was produced from the final product, GDP-fucose. The cell lysates from Lec13 stably transfected with FucTIII (A and B) and Lec13 cells stably transfected with pcDNA I-GMD and FucTIII (C and D) and the parent CHO Pro−5cells (E and F) were assayed in the absence (A, C, and E) or the presence (B, D, and F) of NADPH. The radioactivity in the origin may be phosphorylated sugars (22Ripka J. Adamany A. Stanley P. Arch. Biochem. Biophys. 1986; 249: 533-545Crossref PubMed Scopus (69) Google Scholar). The parent Lec13 cells produced the same results as Lec13-FTIII cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Lec13-FTIII cells produced almost no detectable amount of rhamnose, 6-deoxygalactose or fucose, in the absence (Fig. 4 A) or presence (Fig. 4 B) of NADPH, indicating that no GDP-4-keto-6-deoxy-d-mannose was formed. In contrast, Lec13-FTIII cells transfected with pcDNA I-GMD produced a substantial amount of rhamnose and 6-deoxytalose in the absence of NADPH (Fig. 4 C) as did the wild-type CHO Pro−5cells (Fig. 4 E). In the presence of NADPH, GDP-4-keto-6-deoxy-d-mannose was converted to GDP-l-fucose by the FX protein present in Lec13 and wild-type CHO cells, inasmuch as fucose was detected from reduced-acid hydrolyzed product (Fig. 4, D and F; see also Table I). These results establish that pcDNA I-hGMD encodes GDP-d-mannose-4,6-dehydratase, capable of correcting the defect in fucose metabolism in Lec13 cells.Table IQuantitation of products formed from GDP-[14C]mannose using cytosolic extracts derived from different cell linesGDP-4-keto-6-deoxy-d-mannoseGDP-l-fucose−NADPH+NADPH−NADPH+NADPHCHO(Pro−5)27.136.980.9928.37Lec13<0.5<0.5<0.5<0.5Lec13+FucTIII0.62<0.5<0.5<0.5Lec13+FucTIII+GMD10.40.88<0.56.81The numbers correspond to the amount of products formed in picomoles/90 min of incubation/96 μg of cytosolic proteins. Open table in a new tab The numbers correspond to the amount of products formed in picomoles/90 min of incubation/96 μg of cytosolic proteins. 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. 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. 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 (23Bonin C.P. Potter I. Vanzin G.F. Reiter W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2085-2090Crossref PubMed Scopus (190) Google Scholar, 30Currie H.L. Lightfoot J. Lam J.S. Clin. Diag. Lab. Immunol. 1995; 2: 554-562Crossref PubMed Google Scholar). This situation differs completely from Golgi-associated glycosyltransferases. For example, the amino acid sequences of mammalian polysialyltransferases (26Nakayama J. Fukuda M.N. Fredette B. Ranscht B. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7031-7035Crossref PubMed Scopus (218) Google Scholar, 31Livingston B.D. Paulson J.C. J. Biol. Chem. 1993; 268: 11504-11507Abstract Full Text PDF PubMed Google Scholar, 32Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar, 33Kojima N. Yoshida Y. Kurosawa N. Lee Y.C. Tsuji S. FEBS Lett. 1995; 360: 1-4Crossref PubMed Scopus (104) Google Scholar, 34Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. J. Biol. Chem. 1995; 270: 22685-22688Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) differ entirely from bacterial polysialyltransferase (35Troy F.A. Glycobiology. 1992; 2: 5-23Crossref PubMed Scopus (316) Google Scholar). Similarly, human β-1,3-N-acetylglucosaminyltransferase has no homology withNisseria gonorrhoeae β-1,3-N-acetylglucosaminyl transferase (36Sasaki K. Kurata-Miura K. Ujita M. Angata K. Nakagawa S. Sekine S. Nishi T. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14294-14299Crossref PubMed Scopus (133) Google Scholar, 37Gotschlich E.C. J. Exp. Med. 1994; 180: 2181-2190Crossref PubMed Scopus (168) Google Scholar). On the other hand, the amino acid sequences of Golgi-associated α-mannosidases are conserved from yeast to humans (38Jarvis D.L. Bohlmeyer D.A. Lian Y.-F. Lomax K.K. Merkle R.K. Weinkauf C. Moremen K.W. Glycobiology. 1997; 7: 113-127Crossref PubMed Scopus (36) Google Scholar). 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 (39Puthalakath H. Burke J. Gleeson P.A. J. Biol. Chem. 1996; 271: 27818-27822Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar,40Weinstein J. Sundaram S. Wang X. Delgado D. Basu R. Stanley P. J. Biol. Chem. 1996; 271: 27462-27469Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In carbohydrate-deficient glycoprotein syndrome type II, a point mutation was discovered in the nucleotide sequence encoding the catalytic domain of β-1,2-N-acetylglucosaminyltransferase II (41Tan J. Dunn J. Jaeken J. Schachter H. Am. J. Hum. Genet. 1996; 59: 810-817PubMed Google Scholar). Such a point mutation leads to inactivation of the enzyme, causing defective brain development (41Tan J. Dunn J. Jaeken J. Schachter H. Am. J. Hum. Genet. 1996; 59: 810-817PubMed Google Scholar). 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 α-mannosidase II is substantially reduced (42Fukuda M.N. Masri K.A. Dell A. Luzzato L. Moremen K.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7443-7447Crossref PubMed Scopus (93) Google Scholar). These results strongly suggest that a defect in the genomic structures encoding GMD and α-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 (43Maquat L.E. RNA. 1995; 1: 453-465PubMed Google Scholar). 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 (19Etzioni A. Frydman M. Pollack S. Avidor I. Phillips M.L. Paulson J.C. Gershoni-Baruch R. N. Engl. J. Med. 1992; 327: 1789-1792Crossref PubMed Scopus (433) Google Scholar) 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 (23Bonin C.P. Potter I. Vanzin G.F. Reiter W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2085-2090Crossref PubMed Scopus (190) Google Scholar). Although an additional gene for GMD is suggested in A. thaliana (23Bonin C.P. Potter I. Vanzin G.F. Reiter W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2085-2090Crossref PubMed Scopus (190) Google Scholar), 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. 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." @default.
• W2022190919 created "2016-06-24" @default.
• W2022190919 creator A5006762365 @default.
• W2022190919 creator A5013168337 @default.
• W2022190919 creator A5018918465 @default.
• W2022190919 creator A5059157188 @default.
• W2022190919 creator A5059962014 @default.
• W2022190919 creator A5076273759 @default.
• W2022190919 date "1998-06-01" @default.
• W2022190919 modified "2023-10-10" @default.
• W2022190919 title "Molecular Cloning and Expression of GDP-d-mannose-4,6-dehydratase, a Key Enzyme for Fucose Metabolism Defective in Lec13 Cells" @default.
• W2022190919 cites W139175307 @default.
• W2022190919 cites W1546312651 @default.
• W2022190919 cites W1565007202 @default.
• W2022190919 cites W1568834538 @default.
• W2022190919 cites W1587213486 @default.
• W2022190919 cites W1647336621 @default.
• W2022190919 cites W1720344590 @default.
• W2022190919 cites W1964709514 @default.
• W2022190919 cites W1967446284 @default.
• W2022190919 cites W1968104539 @default.
• W2022190919 cites W1985534693 @default.
• W2022190919 cites W1986150938 @default.
• W2022190919 cites W1986611378 @default.
• W2022190919 cites W1995579116 @default.
• W2022190919 cites W1995702637 @default.
• W2022190919 cites W1996413800 @default.
• W2022190919 cites W1996912052 @default.
• W2022190919 cites W1997993215 @default.
• W2022190919 cites W2001057012 @default.
• W2022190919 cites W2004070658 @default.
• W2022190919 cites W2007181246 @default.
• W2022190919 cites W2016596101 @default.
• W2022190919 cites W2018749935 @default.
• W2022190919 cites W2022408153 @default.
• W2022190919 cites W2044434051 @default.
• W2022190919 cites W2044501103 @default.
• W2022190919 cites W2055090102 @default.
• W2022190919 cites W2067762612 @default.
• W2022190919 cites W2079657455 @default.
• W2022190919 cites W2085402247 @default.
• W2022190919 cites W2087866237 @default.
• W2022190919 cites W2087881430 @default.
• W2022190919 cites W2089974771 @default.
• W2022190919 cites W2109522940 @default.
• W2022190919 cites W2109784589 @default.
• W2022190919 cites W2130561621 @default.
• W2022190919 cites W2145458242 @default.
• W2022190919 cites W2156613209 @default.
• W2022190919 cites W2332565014 @default.
• W2022190919 cites W4323905076 @default.
• W2022190919 doi "https://doi.org/10.1074/jbc.273.23.14582" @default.
• W2022190919 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9603974" @default.
• W2022190919 hasPublicationYear "1998" @default.
• W2022190919 type Work @default.
• W2022190919 sameAs 2022190919 @default.
• W2022190919 citedByCount "87" @default.
• W2022190919 countsByYear W20221909192012 @default.
• W2022190919 countsByYear W20221909192013 @default.
• W2022190919 countsByYear W20221909192014 @default.
• W2022190919 countsByYear W20221909192015 @default.
• W2022190919 countsByYear W20221909192016 @default.
• W2022190919 countsByYear W20221909192017 @default.
• W2022190919 countsByYear W20221909192018 @default.
• W2022190919 countsByYear W20221909192019 @default.
• W2022190919 countsByYear W20221909192021 @default.
• W2022190919 countsByYear W20221909192022 @default.
• W2022190919 countsByYear W20221909192023 @default.
• W2022190919 crossrefType "journal-article" @default.
• W2022190919 hasAuthorship W2022190919A5006762365 @default.
• W2022190919 hasAuthorship W2022190919A5013168337 @default.
• W2022190919 hasAuthorship W2022190919A5018918465 @default.
• W2022190919 hasAuthorship W2022190919A5059157188 @default.
• W2022190919 hasAuthorship W2022190919A5059962014 @default.
• W2022190919 hasAuthorship W2022190919A5076273759 @default.
• W2022190919 hasBestOaLocation W20221909191 @default.
• W2022190919 hasConcept C108625454 @default.
• W2022190919 hasConcept C121050878 @default.
• W2022190919 hasConcept C181199279 @default.
• W2022190919 hasConcept C185592680 @default.
• W2022190919 hasConcept C199360897 @default.
• W2022190919 hasConcept C2775887612 @default.
• W2022190919 hasConcept C2776841590 @default.
• W2022190919 hasConcept C2781374117 @default.
• W2022190919 hasConcept C41008148 @default.
• W2022190919 hasConcept C44538786 @default.
• W2022190919 hasConcept C55493867 @default.
• W2022190919 hasConcept C62231903 @default.
• W2022190919 hasConcept C86803240 @default.
• W2022190919 hasConceptScore W2022190919C108625454 @default.
• W2022190919 hasConceptScore W2022190919C121050878 @default.
• W2022190919 hasConceptScore W2022190919C181199279 @default.
• W2022190919 hasConceptScore W2022190919C185592680 @default.
• W2022190919 hasConceptScore W2022190919C199360897 @default.
• W2022190919 hasConceptScore W2022190919C2775887612 @default.
• W2022190919 hasConceptScore W2022190919C2776841590 @default.
• W2022190919 hasConceptScore W2022190919C2781374117 @default.
• W2022190919 hasConceptScore W2022190919C41008148 @default.

• next