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Molecular Cloning of Human GDP-mannose 4,6-Dehydratase and Reconstitution of GDP-fucose Biosynthesis in Vitro

Molecular Cloning of Human GDP-mannose 4,6-Dehydratase and Reconstitution of GDP-fucose... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 14, Issue of April 3, pp. 8193–8202, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Molecular Cloning of Human GDP-mannose 4,6-Dehydratase and Reconstitution of GDP-fucose Biosynthesis in Vitro* (Received for publication, October 31, 1997, and in revised form, January 20, 1998) Francis X. Sullivan‡, Ravindra Kumar, Ronald Kriz, Mark Stahl, Guang-Yi Xu, Jason Rouse, Xiao-jia Chang, Amechand Boodhoo§, Barry Potvin¶, and Dale A. Cumming From Small Molecule Drug Discovery, Genetics Institute, Inc., §On-Site Biochromatography Inc., 424 Wilkinway, Edmonton, Alberta T6M 2H8, Canada, and the ¶Department of Biology, Yeshiva University, New York, New York 10033 We have cloned the cDNA encoding human GDP-man- been identified (3, 4) and suffer from the immune disorder leukocyte adhesion deficiency type II (LADII). These patients nose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose. The message is fail to synthesize fucosylated blood groups, and their leuko- expressed in all tissues and cell lines examined, and the cytes do not express the fucose containing carbohydrate sialyl cDNA complements Lec13, a Chinese Hamster Ovary cell Lewis X. The patient’s leukocytes do not extravasate normally, line deficient in GDP-mannose 4,6-dehydratase activity. which leads to recurrent infections. The human GDP-mannose 4,6-dehydratase polypeptide In his pioneering work in the early 1960s, Ginsberg (5, 6) shares 61% identity with the enzyme from Escherichia elucidated the enzymatic pathway converting GDP-mannose to coli, suggesting broad evolutionary conservation. Puri- GDP-fucose. Later, Yurchenco and Atkinson (7) showed that as a cofactor fied recombinant enzyme utilizes NADP this was the primary biosynthetic route to GDP-fucose. As and, like its E. coli counterpart, is inhibited by GDP- shown in Fig. 1, GDP-mannose is converted to GDP-fucose by fucose, suggesting that this aspect of regulation is also GDP-mannose 4,6-dehydratase via the oxidation of mannose at conserved. We have isolated the product of the dehy- C-4 followed by the reduction of C-6 to a methyl group, yielding dratase reaction, GDP-4-keto-6-deoxymannose, and con- GDP-4-keto-6-deoxymannose. The reaction has been reported firmed its structure by electrospray ionization-mass to proceed with transfer of a hydride from C-4 to C-6 (8) by a spectrometry and high field NMR. Using purified recom- 1 1 tightly bound cofactor, thought to be NADP or NAD , which binant human GDP-mannose 4,6-dehydratase and FX is regenerated during the reaction. This intermediate is then protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-re- epimerized at C-3 and C-5 to yield GDP-4-keto-6-deoxy-glucose ductase), we show that the two proteins alone are suffi- and finally reduced by NADH or NADPH at C-4 to produce cient to convert GDP-mannose to GDP-fucose in vitro. GDP-fucose. In the initial studies, it was not certain if the last This unequivocally demonstrates that the epimerase two steps, the epimerizations and reduction, were performed by and reductase activities are on a single polypeptide. one enzyme or two. One potential regulatory mechanism in the Finally, we show that the two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway was first revealed in the studies of Kornfeld and pathway in bacteria. Ginsberg (9), who demonstrated that GDP-mannose 4,6-dehy- dratase was inhibited by the final product in the biosynthetic pathway, GDP-fucose. Fucose is found as a component of glycoconjugates such as Recent studies have addressed several open questions about glycoproteins and glycolipids in a wide range of species from the enzymes in this pathway. Two studies have shown that 1 1 humans to bacteria. For example, fucose is a component of the GDP-mannose 4,6-dehydratase utilizes NADP and not NAD capsular polysaccharides and antigenic determinants of bacte- as a cofactor. Yamamoto et al. demonstrated this with the ria, while in mammals fucose is present in many glycoconju- enzyme from Klebsiella pneumoniae (10) and more recently gates, the most widely known being the human blood group Sturla et al. detected NADP bound to the Escherichia coli antigens. Fucose-containing glycoconjugates have been impli- enzyme (11). This requirement for NADP differentiates GDP- cated as playing key roles in embryonic development in the mannose 4,6-dehydratase from the two other well character- mouse (1) and more recently in the regulation of the immune ized sugar nucleotide 4,6-dehydratases, dTDP-glucose 4,6-de- response, specifically as a crucial component of the selectin hydratase and CDP-glucose 4,6-dehydratase, both which ligand sialyl Lewis X (reviewed in Refs. 1 and 2). In all cases, require NAD (12, 13). Additionally, recent studies have ad- fucose is transferred from GDP-fucose to glycoconjugate accep- dressed the question of whether the epimerase and reductase tors by specific transferases. Thus, defects in GDP-fucose bio- activities are present in one protein or are two separate pro- synthesis will affect all fucosylation within the cell. Recently, teins as is the case in dTDP-rhamnose biosynthesis (14, 15). individuals deficient in the biosynthesis of GDP-fucose have Serif and co-workers (16) suggested that the 3,5-epimerase and * The costs of publication of this article were defrayed in part by the The abbreviations used are: LADII, leukocyte adhesion deficiency payment of page charges. This article must therefore be hereby marked type II; hGMD, human GDP-mannose 4,6-dehydratase (EC 4.2.1.47); “advertisement” in accordance with 18 U.S.C. Section 1734 solely to GMD, E. coli GDP-mannose 4,6-dehydratase; hFX, human FX protein indicate this fact. (GDP-4-keto-6-deoxymannose 3,5-epimerase, 4-reductase); WCAG, E. The nucleotide sequence(s) reported in this paper has been submitted coli GDP-4-keto-6-deoxymannose 3,5-epimerase, 4-reductase; PCR, po- TM to the GenBank /EBI Data Bank with accession number(s) AF lymerase chain reaction; DTT, dithiothreitol; MOPS, 3-(N-morpholin- 042377. o)propanesulfonic acid; ESI-LC-MS, electrospray ionization-liquid chro- ‡ To whom correspondence should be addressed: Small Molecule Drug matography-mass spectrometry; ESI-MS, electrospray ionization-mass Discovery, Genetics Institute, Inc., 87 Cambridgepark Dr., Cambridge, spectrometry; EST, expressed sequence tag; CHO, Chinese hamster MA 02140. Tel.: 617-498-8936; Fax: 617-498-8993; E-mail: fsullivan@ ovary; HPLC, high pressure liquid chromatography; PAGE, polyacryl- genetics.com. amide gel electrophoresis. This paper is available on line at http://www.jbc.org 8193 This is an Open Access article under the CC BY license. 8194 Cloning of Human GDP-mannose 4,6-Dehydratase FIG.1. GDP-fucose biosynthetic pathway. The pathway for the conver- sion 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 re- duced and then oxidized during these two steps. GDP-4-keto-6-deoxy-mannose 3,5- epimerase, 4-reductase catalyzes the epi- merization 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 inter- mediate produces GDP-6-deoxytalose and GDP-rhamnose. colony hybridization. This approach yielded plasmid pMT-hGMD con- 4-reductase activities were present on a single polypeptide taining the cDNA of human GDP-mannose dehydratase in a eukaryotic when they purified small amounts of the enzyme from pig expression vector. thyroids (16). This was confirmed by Tonetti et al. (17) when Cloning of Human GDP-4-keto-6-deoxymannose 3,5-Epimerase, 4-Re- they cloned the human protein FX. Sequencing the gene re- ductase—A similar approach was taken to isolate the human epimerase vealed homology to the bacterial sugar nucleotide reductases. reductase genes using following oligonucleotides based upon the pub- Using antibody depletion experiments, purified protein, and lished sequence (17): 59-CTGACATGGGTGAACCCCAGGGATCCAT- GC-39 and 59-TGGCCATCCTCGATGTTGAAGTTGTCGTGG-39. cell extracts as a source for the GDP-4-keto-6-deoxymannose, The positive pools were probed with the P-labeled PCR primers. they demonstrated that FX combined both the epimerase and This yielded plasmid pMT-hFX, containing the cDNA of human GDP- reductase activities in one polypeptide. mannose epimerase-reductase in a eukaryotic expression vector. To understand better the human enzymes involved in this Transfection of Lec13 and Cell Staining—The Chinese hamster ovary pathway, their role in selectin-mediated cell adhesion, and the (CHO) cell line Lec13, was obtained from Professor P. Stanley at Albert LADII defect, we have undertaken the molecular cloning of the Einstein Collage of Medicine. This line was first transfected with pMT- NeoFTIV, a vector expressing human fucosyltransferase IV and the human gene encoding GDP-mannose 4,6-dehydratase. Fur- neomycin resistance genes, by the calcium phosphate method as de- thermore, utilizing purified recombinant enzymes expressed in scribed previously (19). To make this cell line capable of replicating E. coli, we have reconstituted the GDP-fucose biosynthetic vectors containing the polyoma virus origin of replication, one of the pathway in vitro. We demonstrate that two enzymes, GDP- Fuc-T IV-positive cell lines, clone 9E9A, was again transfected with mannose 4,6-dehydratase and GDP-4-keto-6-deoxymannose pCDNA3.1 ZeoPyLT, a plasmid expressing the early region of polyoma 3,5-epimerase, 4-reductase, are sufficient to synthesize GDP- virus including large T, by the lipofectamine method according to the manufacturer’s instructions (Invitrogen). One zeocin-resistant clone fucose from GDP-mannose, confirming earlier studies suggest- had Fuc-T IV activity and was replication-competent. This cell line, ing that both epimerase and reductase activities are encoded in 9E9A LT2.9, was transfected with pMT-hGMD or pMT-hFX by the a single polypeptide. Additionally, we show that human GDP- lipofectamine method. After 48 h, cells were analyzed for Lewis X mannose 4,6-dehydratase has a strict specificity for NADP expression by immunofluorescence after staining with CD15 antibody over NAD . Using the homologous E. coli enzymes, GDP-man- (Immunotech) and goat anti-mouse fluorescein isothiocyanate second- nose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose ary antibody (Boehringer Manheim). 3,5-epimerase, 4-reductase (WCAG), we demonstrate that the In Vitro Assays of CHO Cell Extracts—Mutant CHO cells transfected with human dehydratase cDNA, human epimerase-reductase cDNA, or same is true in bacteria. We also show that human GDP- wild type CHO cells were lysed under nitrogen pressure in 0.75 ml of 25 mannose 4,6-dehydratase is subject to feedback inhibition by mM Hepes, pH 7.4, 100 mM NaCl, 10 mM EDTA, 10 mM DTT for 5 min GDP-fucose and that this, along with its differential levels of in a Parr Bomb at 900 p.s.i. on ice. The cell debris was pelleted at gene expression, provides potential mechanisms for regulating 50,000 3 g for 1 h, and soluble extracts were assayed in 25 mM Hepes, its activity. pH 7.4, 100 mM NaCl, 15 mM MgCl ,10mM DTT, 10 mM GDP-mannose, with 100,000 cpm of C-labeled GDP-mannose for2hat37 °C.The MATERIALS AND METHODS reactions were stopped by boiling for 5 min followed by centrifugation Data Base Searching and Sequence Alignments—The National for 5 min at 15,000 rpm in a microcentrifuge. Unlabeled GDP-mannose Center for Biotechnology Information (NCBI) EST data base was and GDP-fucose were added as standards. GDP-mannose, GDP-fucose searched with BLAST on the NCBI server. The human and E. coli and the 4-keto 6-deoxy intermediate were separated as described by dehydratase peptides were aligned with the program GAP of the Yamamoto et al. (10) except the amide-80 column (Tosohaas) was run in Genetics Computer Group analysis package. Amino terminal peptides 66% acetonitrile and 7.5 mM citric acid/Na HPO buffer, pH 4.0. The 2 4 of the GDP-mannose 4,6-dehydratase from different species were C-labeled sugar nucleotides were detected with a flow through scin- aligned using the GeneWorks program from IntelliGenetics, Inc. tillation counter Beta-1 (Packard) run with a solid scintillant cell. The Cloning Human GDP-mannose 4,6-Dehydratase—A cDNA library unlabeled sugar nucleotides were detected at 254 nm. was constructed from HL-60 cells as described by Sako et al. (18). This Expression and Purification of Human Enzymes from E. coli—The plasmid-based cDNA expression library was assembled into 19 pools, human dehydratase and epimerase-reductase genes were cloned by each representing 60,000 –100,000 individual clones/pool. The pools PCR into the EcoRI and HindIII sites of vector pRSETB (Invitrogen) for were screened by PCR using DNA primers based on both the mouse expression in E. coli. This yielded vectors pRSEThGMD and pRS- EST (accession number W29220) and the published E. coli sequence: EThFX. The inserts in the resulting vectors were sequenced in their 59-TGATGAGCCAGAGGACTTTGTCATAGCTAC-39 and 59-CAGAAA- entirety. The resulting dehydratase fusion protein had the sequence GTCCACTTCAGTCGGTCGGTAGTA-39. MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSPSAGTM- Two pools gave the expected 200-base pair fragment. The 200-base EF added to amino acid 20 of the predicted sequence, the position pair PCR product was reamplified by PCR, random primer-labeled with homologous to the start of the E. coli enzyme. For the epimerase- P, and used to identify positive clones from the two library pools by reductase, the same 43-amino acid fusion peptide was added to amino Cloning of Human GDP-mannose 4,6-Dehydratase 8195 acid 2 of the published sequence. The two expression vectors were Micromass Platform II electrospray ionization-equipped mass spec- trometer, which was operated in the negative ion mode with a cone transformed into E. coli strain BL21/DE3 and the bacteria were grown voltage of 40 V. The intermediate was further characterized by high in LB media containing ampicillin and chloramphenicol. Both the de- field NMR. All NMR experiments were performed on a Varian Unity hydratase-expressing cells and epimerase-reductase-expressing cells plus 600-MHz spectrometer. Samples were dissolved in D O, and HDO were grown at room temperature to an A of 0.5 and induced with 0.3 600 2 was used as an internal reference, set to 4.76 ppm at 25 °C. Two- mM isopropyl-1-thio-b-D-galactopyranoside for 3– 4 h. The cells were dimensional total correlation spectroscopy, nuclear Overhauser effect resuspended in Tris-buffered saline containing DNase I and lysed in a spectroscopy, and heteronuclear multiple-quantum coherence were per- French press at 12,000 p.s.i. After clarification, the dehydratase was formed with standard Varian pulse sequences. purified by successive chromatography over nitrolotriacetic acid-Ni - Cloning of E. coli GMD and E. coli WCAG—The gmd and wcaG genes agarose (Qiagen) eluting with 200 mM imidazole, chromatography over were cloned from the E. coli K-12 genome using PCR oligonucleotides ceramic hydroxyapatite (Bio-Rad) eluting with a phosphate gradient that created an NcoI site overlapping the initiating methionine of each (0.0 – 0.5 M), and chromatography over MonoQ (Pharmacia) eluting with gene and a HindIII site following the termination codon of each gene. a NaCl gradient (0.0 – 0.5 M). The epimerase-reductase was purified by The PCR products were cloned into the NcoI and HindIII sites of successive chromatography over nitrolotriacetic acid-Ni -agarose elut- pSE380 (Invitrogen), yielding pSEGMD and pSEWCAG. Sequences ing with 200 mM imidazole and chromatography over 29–59 ADP-Sepha- were confirmed by DNA sequencing. During the sequencing of the wcaG rose eluting with 10 mM NADP . The resulting proteins were greater gene, it was discovered that multiple clones contained two nucleotide than 90% pure as judged by SDS-PAGE and staining with Coomassie differences from the published sequence (21). The last nucleotide in Blue. Amino terminal sequencing of the first 15 amino acids of the codon 255 and the first nucleotide in codon 256 changed from a CG in proteins confirmed their identity as fusion protein expressed from the the published sequence to a GC in the cloned gene. This changed the pRSET vector. predicted amino acids in those positions from an aspartate at amino A second expression construct was made for each gene, which had a acid 255 and a valine at amino acid 256 in the published sequence to 20-amino acid amino-terminal leader, MRGSHHHHHHGSDYKD- 19 1 glutamate and leucine in the cloned gene. DDDK, added to the Met of hGMD or Met of hFX. To construct these Expression and Purification of E. coli Enzymes from E. coli—E. coli expression plasmids, synthetic DNA was used to rebuild the hGMD strain GI934 harboring either pSEGMD or pSEWCAG were grown in gene from the AgeI site near the 59-end of the gene. The synthetic DNA LB media containing ampicillin at 370 °C to an A of 0.5. The cells was hybridized and ligated to the AgeI–HindIII fragment of pRSETh- were induced with 1 mM isopropyl-1-thio-b-D-galactopyranoside and GMD into the NdeI and HindIII sites of pRSETB to yield pT7hGMD. A grown for an additional 4 h. Cell pellets were broken in a French press similar approach was taken to construct pT7hFX using the BamHI– at 12,000 p.s.i. After clarification, the E. coli dehydratase was purified HindIII fragment of pRSEThFX. The resulting vectors pT7hMGD and by successive chromatography over Toyopearl DEAE (TosoHaas) elut- pT7hFX were transformed into E. coli strain BL21/DE3, and protein ing with a NaCl gradient (0.0 – 0.5 M), chromatography over ceramic was expressed and purified as above. No significant differences were hydroxyapatite eluting with a phosphate gradient (0.0 – 0.5 M), and observed in the activity, substrate, or cofactor preference for both the chromatography over MonoQ eluting with a NaCl gradient (0.0 – 0.5 M). human dehydratase or epimerase-reductase having either the longer or The E. coli epimerase-reductase was purified by successive chromatog- shorter N-terminal fusion. raphy over Toyopearl DEAE eluting with a NaCl gradient (0.0 – 0.5 M), In Vitro Assays of Purified Dehydratase and Epimerase-Reductase— followed by chromatography over heparin-Toyopearl eluting with 200 Purified proteins were assayed in 25 mM MOPS, pH 7.0, 100 mM NaCl, mM NaCl. The resulting proteins were greater than 90% pure as judged 5mM EDTA, 10 mM DTT, 25 mM GDP-mannose with 100,000 cpm of by SDS-PAGE and staining with Coomassie Blue. Amino-terminal se- C-labeled GDP-mannose for1hat37 °C. When sequential reactions quencing of the first 15 amino acids of the proteins confirmed their were performed, the mix was incubated for an additional1hat37 °C identities. 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 RESULTS a microcentrifuge. Unlabeled GDP-mannose and GDP-fucose were added at this point as internal standards. Kinetic constants were de- Molecular Cloning of Human GDP-mannose 4,6-Dehy- termined graphically using S/V versus S plots with GDP-mannose con- dratase—To clone human GDP-mannose 4,6-dehydratase, we centrations ranging from ⁄2 to 5 K . The reaction conditions were as first performed a TBLASTN search of the NCBI EST data base above with 1 mM NADP added to reactions. using the sequence of the E. coli enzyme. This identified a Paper Chromatography— C-Labeled GDP-mannose, GDP-fucose, mouse EST, accession number W29220, with a high degree of and reaction products were incubated in 0.2 M NaBH for 15 min at homology to the E. coli enzyme. We designed two oligonucleo- room temperature to reduce keto intermediates and then were cleaved from the sugar by incubating in 1 M trichloroacetic acid for 10 min in tide primers based on conserved amino acids present in both boiling water. This mixture was chromatographed on Whatman 3MM the E. coli sequence and the partial sequence of the mouse paper in descending mode using three different solvent systems (solvent gene. Using the oligonucleotides as primers, we obtained a I, water-saturated methyl ethyl ketone for 24 h; solvent II, ethyl ace- 200-base pair PCR fragment from a human promyelocytic cell tate/pyridine/water (3.6:1.0:1.15) for 7 h; solvent III, ethyl acetate/ line HL-60 cDNA library. This fragment was then used as a pyridine/water (10:2.5:1.5) for 17 h. Free sugar standards were localized probe to isolate two apparently full-length cDNA clones for the by staining with AgNO in acetone followed by NaOH in methanol (20). Radioactivity was detected by cutting the paper into strips, and 1-cm putative human GDP-mannose 4,6 dehydratase, the sequence sections of each strip were counted in 1 ml of water plus 10 ml of of which is shown in Fig. 2. There are two potential initiator formula 989 (Packard). methionines located at nucleotides 76 and 130 of this sequence. Synthesis and Characterization of GDP-4-keto-6-deoxymannose— As shown in Fig. 3A, the downstream methionine of the human GDP-4-keto-6-deoxymannose was synthesized from GDP-mannose us- protein more closely aligns with the initiating methionine of ing the purified human and purified E. coli dehydratases. Typical the E. coli protein. However, alignment of the human sequence 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 to the recently cloned arabidopsis GDP-mannose 4,6-dehy- mM DTT, 5 mM EDTA, 10 mM NADPH, and 10 mM NADP at 37 °C for dratase gene and a putative C. elegans translation product 3– 6 h. Typical reaction conditions for E. coli dehydratase were 10 mM (Fig. 3B) suggests that translation of the human protein ini- GDP-mannose, 0.1 mg/ml E. coli dehydratase, 10 mM MOPS, pH 6.5, tiates at the first methionine, at nucleotide 76, and that dehy- 100 mM NaCl, 2 mM DTT, 1 mM EDTA, 10 mM NADPH, and 100 mM 1 dratases from nonbacterial sources contain an amino-terminal NADP at 37 °C for 3– 6 h. The reactions were allowed to proceed to extension. The human and E. coli proteins are 61% identical completion as judged by HPLC, and the protein was removed by ultra- filtration on a Centricon-10. The resulting mix was desalted on Seph- over their entire lengths, and both contain an extended con- adex G-10 run in water and lyophilized. GDP-4-keto-6-deoxymannose sensus sequence, GXXGXXG, identifying the bab fold found in 1 1 prepared this way was stable frozen at 220 °C either as a dried powder many NAD - and NADP -binding proteins (22). This sequence or in water and was judged essentially pure by HPLC and ESI-MS is found between amino acids 9 and 15 of the E. coli enzyme. analysis. For ESI-MS analysis, 500 mM citric acid-sodium phosphate Fig. 4 shows that the human dehydratase gene encodes a single buffer was added to a 20 pmol/ml solution of GDP-4-keto-6-deoxyman- mRNA transcript of about 1.7 kilobase pairs that is expressed nose (in 50% acetonitrile) to give a final concentration of 0.5 mM at pH 4. Flow injection at 10 ml/min was used to introduce this sample into the in all tissue and cell types examined, albeit at varying levels. 8196 Cloning of Human GDP-mannose 4,6-Dehydratase 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. 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 biosyn- thesis 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-dehy- FIG.2. cDNA and amino acid sequence of human GDP-man- nose 4,6-dehydratase. The sequence of the cDNA for human GDP- dratase, in media lacking fucose, also causes the cells to stain mannose 4,6-dehydratase is shown above the putative peptide sequence. positive for Lewis X, demonstrating that the dehydratase gene complements the CHO cell defect (Fig. 5B). By contrast, trans- The mRNA levels are highest in pancreas followed by small fection with the same vector expressing the human epimerase- intestine, liver, colon, and prostate and lowest in ovary, brain, reductase gene, again in media lacking fucose, shows no stain- lung, spleen, and peripheral blood lymphocytes. Likewise, a ing by CD15 (Fig. 5C). As further evidence, lysates of Lec13 varied level of expression was seen in the human cell lines cells transiently transfected with human GDP-mannose 4,6- examined (Fig. 4C). dehydratase cDNA readily converted C-labeled GDP-man- GDP-mannose 4,6-Dehydratase cDNA Complements the De- nose to GDP-fucose (Fig. 6B), whereas lysates of Lec13 cells hydratase Defect in Lec13—To demonstrate the isolated cDNA transfected with the human epimerase-reductase cDNA in the encoded GDP-mannose 4,6-dehydratase activity, we tran- same expression vector did not (Fig. 6A). The level of activity in siently transfected it into Lec13, a CHO cell line that previ- dehydratase-transfected cells (Fig. 6B,25 mg) is higher than ously has been identified as lacking GDP-mannose 4,6-dehy- that of wild type CHO cells (Fig. 6C, 125 mg). Based on this dratase activity (23). To monitor the GDP-mannose 4,6- data, we conclude that the cDNA encodes GDP-mannose dehydratase activity, we first stably transfected Lec13 with 4,6-dehydratase. Cloning of Human GDP-mannose 4,6-Dehydratase 8197 FIG.4. Northern analysis of human GDP-mannose 4,6-dehydratase gene expression. Autoradiogram of Northern blots probed with P-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 b-actin messages are shown at the bottom. 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. FIG.6. In Vitro assay of dehy- dratase and epimerase-reductase ac- tivity in wild type CHO and trans- fected Lec13 CHO cell lines. HPLC analysis of reactions of C-labeled GDP- mannose with cell extracts are shown. A, 125 mg of extract of 9E9A LT2.9 Lec13 cells transfected with epimerase-reduc- tase cDNA. B,25 mg of extract of 9E9A LT2.9 Lec13 cells transfected with dehy- dratase cDNA. C, 125 mg of extract of CHO Dukx cells (wild type for GDP-man- nose 4,6-dehydratase). The positions of C-labeled GDP-mannose and GDP-fu- cose standards are shown above. 8198 Cloning of Human GDP-mannose 4,6-Dehydratase Chromosomal Localization of Human GDP-mannose 4,6-De- mapped their location using fluorescence in situ hybridization. hydratase and FX Gene—To determine if the two genes for Full-length cDNA inserts encoding the human dehydratase GDP-fucose biosynthesis are linked on the human genome we (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 ge- nome. 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 epi- merase-reductase was mapped to the q terminus of chromo- some 8, an area corresponding to band 8q24.3. A total of 80 metaphase cells were analyzed, with 70 exhibiting specific la- FIG.7. SDS-PAGE of recombinant enzymes purified from E. coli. beling (data not shown). 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. Human GDP-mannose Dehydratase and Epimerase-Reduc- FIG.8. In vitro assay of human and E. coli dehydratase and epimerase re- ductase activity. HPLC analysis of re- actions of C-labeled GDP-mannose with purified recombinant enzymes are shown. A,1 mg of human dehydratase plus 100 mM NADP ; B,1 mg of human dehy- dratase plus 1 mM NAD ; C,1 mgofhu- man dehydratase plus 100 mM NADP , followed by 1 mg of human epimerase- reductase plus 100 mM NADPH; D,1 mgof E. coli dehydratase plus 1 mM NADP ; E, 1 mgof E. coli dehydratase plus 1 mM NAD ; F,1 mgof E. coli dehydratase plus 1mM NADP , followed by 1 mgof E. coli epimerase-reductase plus 100 mM NADPH. The arrows show the position of C-labeled standards GDP-mannose and GDP-fucose Cloning of Human GDP-mannose 4,6-Dehydratase 8199 tase Are Sufficient to Convert GDP-mannose to GDP-fucose—To further characterize GDP-mannose 4,6-dehydratase, and to re- constitute GDP-fucose biosynthesis in vitro, we needed to ob- tain 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-man- nose 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 se- quence by ESI-LC-MS (data not shown). To characterize the reactions of the purified dehydratase and epimerase-reductase, we incubated the enzymes with C-la- beled GDP-mannose and identified the reaction products by HPLC and paper chromatography. As shown in Fig. 8A, puri- fied GDP-mannose 4,6-dehydratase converts C-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 chromatog- raphy. As shown in Fig. 1, the expected monosaccharides re- sulting from reduction of GDP-4-keto-6-deoxymannose by boro- hydride 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 FIG.9. Analysis of the reaction products of human and E. coli standard for rhamnose. This component also co-migrates with dehydratase and epimerase-reductase by descending paper rhamnose in solvent systems II and III (data not shown). The 14 chromatography. C-Labeled reaction products were reduced with species at 5 cm co-migrates with mannose and was presumably NaBH , and the resulting sugar was cleaved with acid and spotted on Whatman 3MM paper developed in water-saturated methyl ethyl ke- derived from the unreacted starting material. The species at 24 tone for 24 h. The paper was cut into strips, and 1-cm sections of each cm runs faster than rhamnose in this solvent, as expected for strip were counted. A, the reactions contained GDP-mannose, human 6-deoxytalose, but has an R that differs from the published 1 dehydratase, and NADP , followed by human epimerase-reductase value for 6-deoxytalose (6, 27). This is also true in solvents II plus NADPH (open squares) or GDP-mannose, human dehydratase, and NADP (filled triangles). B, the reactions contained GDP-mannose, E. and III. An additional species was observed in this sample, coli dehydratase, NADP , E. coli epimerase-reductase, and NADPH running near 37 cm. Without authentic 6-deoxytalose, we have (open squares) or GDP-mannose, E. coli dehydratase, and NADP not been able to confirm the identity of the two faster running (filled triangles). The position of C-labeled GDP-mannose and GDP- spots. Using the purified enzyme, we were able to determine fucose that had been treated identically to the reaction mixtures is shown above labeled as mannose and fucose, respectively. The position the cofactor utilized by human GDP-mannose 4,6-dehydratase. of unlabeled free rhamnose is also shown at the top of the trace. 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. and High Field NMR—To confirm that the reaction product of Using the purified human dehydratase and epimerase-re- human dehydratase and GDP-mannose was GDP-4-keto-6-de- ductase, we could demonstrate that these two enzymes alone oxymannose, we isolated the product and analyzed it by mass were sufficient to convert GDP-fucose to GDP-mannose. Se- spectrometry and NMR. The major peaks in the ESI-MS spec- quential incubation of GDP-mannose with human dehydratase trum of the isolated intermediate were at 586.1 and 292.6, 2 22 and NADP followed by human epimerase-reductase and corresponding to [M 2 H] ion and [M 2 H] , respectively for NADPH converts the GDP-mannose to GDP-fucose, based upon GDP-4-keto-6-deoxymannose. A partial spectrum is shown in 2 1 co-chromatography with authentic GDP-fucose (Fig. 8C). We Fig. 10, where the [M 2 H] peak at 586.1, the [M 1 Na 2 2 1 2 confirmed the identify of the product as GDP-fucose by de- 2H] peak at 608.1, and the [M 1 2Na 2 3H] peak at 630.1 scending paper chromatography of the free monosaccharide for GDP-4-keto-6-deoxymannose are clearly visible. A [M 2 after reduction with NaBH and cleavage from GDP with acid. H] peak for residual GDP-mannose at 604.0 is also present in Fig. 9A (open squares) shows the free monosaccharide co-mi- the spectra. A combination of one-dimensional homonuclear, grates with an identically treated C-labeled GDP-fucose two-dimensional homonuclear, and two-dimensional hetero- standard. Identical results were obtained in solvents systems II nuclear NMR experiments revealed that the isolated reaction and III (data not shown). Unlike the human dehydratase, product was a mixture of at least two related sugar nucleotides. which shows a strict cofactor preference for NADP over The observed chemical shifts and coupling constants are listed NAD , the human epimerase-reductase can utilize either in Table I. From these data, we have identified the two major NADPH or NADH, although NADPH is used more efficiently species as GDP-4-keto-6-deoxymannose and GDP-3-keto-6-de- (data not shown). oxymannose. NMR analysis of the isolated product of E. coli Characterization of GDP-4-keto-6-deoxymannose by ESI-MS dehydratase also showed a similar mixture of GDP-4-keto-6- 8200 Cloning of Human GDP-mannose 4,6-Dehydratase FIG. 10. High mass region of the ESI-MS spectrum for the reaction products of GDP-mannose and human dehydratase. Spectrum 2 1 2 1 2 shows the [M 2 H] peak at 586.1, the [M 2 Na 2 2H] peak at 608.1, and the [M 2 2Na 2 3H] peak at 630.1 for GDP-4-keto-6-deoxymannose. Also present is the [M 2 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. TABLE I way we characterized the human enzymes. Fig. 8E shows that Chemical shifts and J coupling for GDP-4-keto-6-deoxymannose incubation of E. coli dehydratase with GDP-mannose and and GDP-3-keto-6-deoxymannose NADP resulted in production of the reaction intermediate GDP-4-keto-6-deoxymannose GDP-3-keto-6-deoxymannose GDP-4-keto-6-deoxymannose. This reaction product produced 13 1 13 1 C H J C H J only two spots in paper chromatography after reduction and cleavage. One migrated with the rhamnose standard at 17 cm, Base 140.15 8.11 140.15 8.11 Ribose and one migrated faster at 37 cm (Fig. 9B, filled triangles). The 19 89.12 5.93 6.0 89.12 5.96 6.0 spot seen at 25 cm in the reaction with human dehydratase is 29 75.97 4.81 5.6 75.97 4.86 missing (Fig. 9, compare A and B (filled triangles)). The E. coli 39 72.85 4.51 72.85 4.53 dehydratase used NADP as a cofactor and could not substi- 49 86.29 4.35 86.29 4.35 tute NAD at concentrations up to 1 mM (Fig. 8, D and E). 59a,b 67.69 4.21 67.69 4.21 Mannose As with the human enzymes, E. coli dehydratase and epime- 10 98.09 5.59 2.1, 7.7 98.87 5.45 1.6, 7.7 rase-reductase were sufficient to convert GDP-mannose to 20 77.53 4.46 ;3 72.99 4.01 ;1 GDP-fucose. Incubation of GDP-mannose with E. coli dehy- 30 75.09 4.82 dratase and E. coli epimerase-reductase in the presence of 40 70.88 3.94 ,1 50 74.07 4.70 6.5 72.88 4.07 6.6 NADP and NADPH converted the GDP-mannose to GDP- CH 15.18 1.23 13.63 1.21 fucose (Fig. 8F). We confirmed the identity of GDP-fucose by The identification of the 3-keto compound was complicated by the paper chromatography (Fig. 9B, open squares). The E. coli small coupling constant of the proton on C-4 of mannose, which reduced epimerase-reductase can use NADH but not as efficiently as magnetization transfer in the total correlation spectroscopy experi- NADPH (data not shown). ment. During repeated lyophilization of the samples in D O to exchange We performed a preliminary characterization of the two de- protons, new peaks were evident in the NMR, suggesting the GDP-4- keto-6-deoxymannose and its related compounds are not entirely stable hydratases, and the results are shown in Table II. The E. coli under these conditions. enzyme has a significantly higher K for GDP-mannose than the human enzyme but also has a significantly higher V . max Additionally, we monitored inhibition of the dehydratase by deoxymannose and GDP-3-keto-6-deoxymannose. GDP-fucose, which has been proposed as a mechanism to reg- E. coli GDP-mannose Dehydratase and Epimerase-Reductase ulate the enzyme’s activity. Both human and E. coli dehydrata- Are Sufficient to Convert GDP-mannose to GDP-fucose—To ad- ses were inhibited by GDP-fucose with IC values lower than dress whether the bacterial enzymes catalyze the same reac- the IC values for inhibition by GDP, suggesting a specific tions as the human dehydratase and epimerase-reductase, we effect and a potential role in regulation of the enzyme’s activity. cloned, expressed, and purified the E. coli enzymes (Fig. 7B). Quite unexpectedly, both enzymes are stimulated by NADPH We examined the reactions of the E. coli enzymes in the same at micromolar concentrations, although this co-factor does not Cloning of Human GDP-mannose 4,6-Dehydratase 8201 TABLE II but no evidence of any other GDP-mannose derivatives of dif- Characteristics of human and E. coli GDP-mannose-4,6-dehydratase ferent masses. High field NMR revealed the presence of two 50% compounds, one being the expected product GDP-4-keto-6-de- a IC of IC of Stimulation 50 50 K V b c stimulation m max GDP GDP-fucose by NADPH oxymannose and the other the related GDP-3-keto-6-deoxym- by NADPH annose. This was the case for the product of both the human mM mmol/min/mg mM mM -fold mM and E. coli dehydratases. The presence of both the 4-keto- and Human 80 0.11 225 75 4.5 2 3-keto-6-deoxy-sugars was also seen in the dTDP-rhamnose E. coli 260 0.73 400 10 2.3 15 pathway where a 4,6-dehydratase converts dTDP-glucose to K is in mM. The S.D. values in the kinetic constants are less than dTDP-4-keto-6-deoxy-glucose (31, 32). The biological signifi- 10%. Reaction conditions were 25 mM MOPS, pH 7.0, 100 mM NaCl, 10 cance of both GDP-4-keto-6-deoxymannose and GDP-3-keto-6- mM DTT, 5 mM EDTA, and 1 mM NADP , 37 °C. deoxymannose intermediates is unclear, although apparently Reactions contained 25 mM GDP-mannose. c 1 both are epimerized and reduced to GDP-fucose by the epime- Reactions contained 100 mM NADP . rase reductase (Fig. 8, C and F). It is possible that the 3-keto- sugar arose during work-up of the isolated reaction product. play a role in catalysis. It is not clear if this stimulation by However, the isolated, unlabeled intermediate also was con- NADPH is relevant to the in vivo regulation of the enzyme. verted to GDP-fucose by purified human epimerase-reductase DISCUSSION (data not shown). We have cloned the gene encoding human GDP-mannose Cells from two patients having LADII do not fucosylate their 4,6-dehydratase using homology between the E. coli enzyme cell surfaces (4) and as such lack both blood group antigens and and a mouse EST. The cloned gene complemented the previ- the sialyl Lewis X and related epitopes that function as selectin ously identified GDP-mannose 4,6-dehydratase defect in the ligands. The molecular basis of this disorder is still unknown. CHO cell line Lec13, demonstrating that the cDNA encodes a As with Lec13, this phenotype can be rescued in cell lines functional protein. The dehydratase gene shows high levels of derived from these patients by culturing them in the presence identity between bacteria and human and indeed across the of fucose, suggesting that GDP-fucose transport and the com- spectrum of species examined (for a comparison of dehydrata- plement of fucosyltransferases are intact in these cells. This ses from a variety of bacterial and nonmammalian sources see would imply that the defect in the LADII patients lies in the Bonin et al. (28). The message for human GDP-mannose 4,6- pathway converting GDP-mannose to GDP-fucose, i.e. either dehydratase is expressed in all tissues examined, albeit at the dehydratase or epimerase-reductase. With the human varying levels. The varying levels of expression of the dehy- genes for both enzymes now cloned, we can determine if either dratase message suggest the enzyme may be regulated at the is responsible for the LADII phenotype. There are suggestions level of transcription, and in fact there is evidence for develop- from E. coli that there may be an additional gene that plays a mental regulation in rat and nereids (29, 30). It also appears role in GDP-fucose biosynthesis in vivo. Sequencing of the that, in both humans and E. coli, GDP-fucose biosynthesis is capsular polysaccharide operon in E. coli led to the identifica- regulated by feedback inhibition of the dehydratase by GDP- tion of an open reading frame (wcaH) immediately downstream fucose, the final product in the pathway. This mechanism of of the dehydratase, gmd, and epimerase-reductase genes, wcaG inhibition was noted for Aerobacter aerogenes by Kornfeld and (21). This putative protein has been assigned to the GDP-fucose Ginsberg (9) and was suggested for the porcine enzyme by Serif biosynthetic pathway, yet it is clearly not necessary for conver- and co-workers (33). sion of GDP-mannose to GDP-fucose in vitro. WcaH may play a The cloning of the human dehydratase gene, along with the role in GDP-fucose biosynthesis in vivo or play another, yet recent cloning of the human epimerase-reductase by Tonetti et unidentified, role in capsular polysaccharide biosynthesis. The al. (17) has allowed us to reconstitute GDP-fucose biosynthesis cloning of the GDP-mannose 4,6-dehydratase gene provides a in vitro using purified, recombinant enzymes. Thus, we have valuable tool to address outstanding questions in the regula- definitively shown that two enzymes, a dehydratase and an tion, biosynthesis, and role of GDP-fucose in vivo. epimerase-reductase, are sufficient to convert GDP-mannose to Acknowledgments—We thank Professor Pamala Stanley of the GDP-fucose. In doing so, we demonstrated, using purified re- Albert Einstein Collage of Medicine for the gift of the Lec13 cell line; combinant proteins, that in humans and in E. coli, both 3,5- Drs. Diane Sako and Monique Davies of Genetics Institute for the gifts epimerase and 4,6-reductase activities are present in a single of plasmids pMTNeoFTIV and pEDPyLT, respectively; Drs. Elliott protein. This confirms the earlier studies of Serif and co-work- Nickbarg and Robert Gassaway of Genetics Institute for ESI-LC-MS analysis and peptide sequencing; Kevin Bean and Mark Proia of ers (16) on the enzyme purified from porcine thyroids and the Genetics Institute for DNA sequencing and library screening. We thank recent work of Tonetti et al. (17) with the human FX protein. Drs. John Lowe and Peter Smith of Univeristy of Michigan for sharing Additionally, we find that human dehydratase has a strict data on the LADII cell lines prior to publication. We thank Drs. Simon 1 1 cofactor requirement for NADP for which NAD cannot sub- Jones and John Knopf of Genetics Institute for critical reading of the manuscript. stitute. This is consistent with earlier reports demonstrating that GDP-mannose 4,6-dehydratase from K. pneumoniae re- REFERENCES quires NADP (10) as well as the recent work of Sturla et al., 1. Feizi, T. (1991) Trends Biochem. 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Molecular Cloning of Human GDP-mannose 4,6-Dehydratase and Reconstitution of GDP-fucose Biosynthesis in Vitro

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 14, Issue of April 3, pp. 8193–8202, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Molecular Cloning of Human GDP-mannose 4,6-Dehydratase and Reconstitution of GDP-fucose Biosynthesis in Vitro* (Received for publication, October 31, 1997, and in revised form, January 20, 1998) Francis X. Sullivan‡, Ravindra Kumar, Ronald Kriz, Mark Stahl, Guang-Yi Xu, Jason Rouse, Xiao-jia Chang, Amechand Boodhoo§, Barry Potvin¶, and Dale A. Cumming From Small Molecule Drug Discovery, Genetics Institute, Inc., §On-Site Biochromatography Inc., 424 Wilkinway, Edmonton, Alberta T6M 2H8, Canada, and the ¶Department of Biology, Yeshiva University, New York, New York 10033 We have cloned the cDNA encoding human GDP-man- been identified (3, 4) and suffer from the immune disorder leukocyte adhesion deficiency type II (LADII). These patients nose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose. The message is fail to synthesize fucosylated blood groups, and their leuko- expressed in all tissues and cell lines examined, and the cytes do not express the fucose containing carbohydrate sialyl cDNA complements Lec13, a Chinese Hamster Ovary cell Lewis X. The patient’s leukocytes do not extravasate normally, line deficient in GDP-mannose 4,6-dehydratase activity. which leads to recurrent infections. The human GDP-mannose 4,6-dehydratase polypeptide In his pioneering work in the early 1960s, Ginsberg (5, 6) shares 61% identity with the enzyme from Escherichia elucidated the enzymatic pathway converting GDP-mannose to coli, suggesting broad evolutionary conservation. Puri- GDP-fucose. Later, Yurchenco and Atkinson (7) showed that as a cofactor fied recombinant enzyme utilizes NADP this was the primary biosynthetic route to GDP-fucose. As and, like its E. coli counterpart, is inhibited by GDP- shown in Fig. 1, GDP-mannose is converted to GDP-fucose by fucose, suggesting that this aspect of regulation is also GDP-mannose 4,6-dehydratase via the oxidation of mannose at conserved. We have isolated the product of the dehy- C-4 followed by the reduction of C-6 to a methyl group, yielding dratase reaction, GDP-4-keto-6-deoxymannose, and con- GDP-4-keto-6-deoxymannose. The reaction has been reported firmed its structure by electrospray ionization-mass to proceed with transfer of a hydride from C-4 to C-6 (8) by a spectrometry and high field NMR. Using purified recom- 1 1 tightly bound cofactor, thought to be NADP or NAD , which binant human GDP-mannose 4,6-dehydratase and FX is regenerated during the reaction. This intermediate is then protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-re- epimerized at C-3 and C-5 to yield GDP-4-keto-6-deoxy-glucose ductase), we show that the two proteins alone are suffi- and finally reduced by NADH or NADPH at C-4 to produce cient to convert GDP-mannose to GDP-fucose in vitro. GDP-fucose. In the initial studies, it was not certain if the last This unequivocally demonstrates that the epimerase two steps, the epimerizations and reduction, were performed by and reductase activities are on a single polypeptide. one enzyme or two. One potential regulatory mechanism in the Finally, we show that the two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway was first revealed in the studies of Kornfeld and pathway in bacteria. Ginsberg (9), who demonstrated that GDP-mannose 4,6-dehy- dratase was inhibited by the final product in the biosynthetic pathway, GDP-fucose. Fucose is found as a component of glycoconjugates such as Recent studies have addressed several open questions about glycoproteins and glycolipids in a wide range of species from the enzymes in this pathway. Two studies have shown that 1 1 humans to bacteria. For example, fucose is a component of the GDP-mannose 4,6-dehydratase utilizes NADP and not NAD capsular polysaccharides and antigenic determinants of bacte- as a cofactor. Yamamoto et al. demonstrated this with the ria, while in mammals fucose is present in many glycoconju- enzyme from Klebsiella pneumoniae (10) and more recently gates, the most widely known being the human blood group Sturla et al. detected NADP bound to the Escherichia coli antigens. Fucose-containing glycoconjugates have been impli- enzyme (11). This requirement for NADP differentiates GDP- cated as playing key roles in embryonic development in the mannose 4,6-dehydratase from the two other well character- mouse (1) and more recently in the regulation of the immune ized sugar nucleotide 4,6-dehydratases, dTDP-glucose 4,6-de- response, specifically as a crucial component of the selectin hydratase and CDP-glucose 4,6-dehydratase, both which ligand sialyl Lewis X (reviewed in Refs. 1 and 2). In all cases, require NAD (12, 13). Additionally, recent studies have ad- fucose is transferred from GDP-fucose to glycoconjugate accep- dressed the question of whether the epimerase and reductase tors by specific transferases. Thus, defects in GDP-fucose bio- activities are present in one protein or are two separate pro- synthesis will affect all fucosylation within the cell. Recently, teins as is the case in dTDP-rhamnose biosynthesis (14, 15). individuals deficient in the biosynthesis of GDP-fucose have Serif and co-workers (16) suggested that the 3,5-epimerase and * The costs of publication of this article were defrayed in part by the The abbreviations used are: LADII, leukocyte adhesion deficiency payment of page charges. This article must therefore be hereby marked type II; hGMD, human GDP-mannose 4,6-dehydratase (EC 4.2.1.47); “advertisement” in accordance with 18 U.S.C. Section 1734 solely to GMD, E. coli GDP-mannose 4,6-dehydratase; hFX, human FX protein indicate this fact. (GDP-4-keto-6-deoxymannose 3,5-epimerase, 4-reductase); WCAG, E. The nucleotide sequence(s) reported in this paper has been submitted coli GDP-4-keto-6-deoxymannose 3,5-epimerase, 4-reductase; PCR, po- TM to the GenBank /EBI Data Bank with accession number(s) AF lymerase chain reaction; DTT, dithiothreitol; MOPS, 3-(N-morpholin- 042377. o)propanesulfonic acid; ESI-LC-MS, electrospray ionization-liquid chro- ‡ To whom correspondence should be addressed: Small Molecule Drug matography-mass spectrometry; ESI-MS, electrospray ionization-mass Discovery, Genetics Institute, Inc., 87 Cambridgepark Dr., Cambridge, spectrometry; EST, expressed sequence tag; CHO, Chinese hamster MA 02140. Tel.: 617-498-8936; Fax: 617-498-8993; E-mail: fsullivan@ ovary; HPLC, high pressure liquid chromatography; PAGE, polyacryl- genetics.com. amide gel electrophoresis. This paper is available on line at http://www.jbc.org 8193 This is an Open Access article under the CC BY license. 8194 Cloning of Human GDP-mannose 4,6-Dehydratase FIG.1. GDP-fucose biosynthetic pathway. The pathway for the conver- sion 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 re- duced and then oxidized during these two steps. GDP-4-keto-6-deoxy-mannose 3,5- epimerase, 4-reductase catalyzes the epi- merization 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 inter- mediate produces GDP-6-deoxytalose and GDP-rhamnose. colony hybridization. This approach yielded plasmid pMT-hGMD con- 4-reductase activities were present on a single polypeptide taining the cDNA of human GDP-mannose dehydratase in a eukaryotic when they purified small amounts of the enzyme from pig expression vector. thyroids (16). This was confirmed by Tonetti et al. (17) when Cloning of Human GDP-4-keto-6-deoxymannose 3,5-Epimerase, 4-Re- they cloned the human protein FX. Sequencing the gene re- ductase—A similar approach was taken to isolate the human epimerase vealed homology to the bacterial sugar nucleotide reductases. reductase genes using following oligonucleotides based upon the pub- Using antibody depletion experiments, purified protein, and lished sequence (17): 59-CTGACATGGGTGAACCCCAGGGATCCAT- GC-39 and 59-TGGCCATCCTCGATGTTGAAGTTGTCGTGG-39. cell extracts as a source for the GDP-4-keto-6-deoxymannose, The positive pools were probed with the P-labeled PCR primers. they demonstrated that FX combined both the epimerase and This yielded plasmid pMT-hFX, containing the cDNA of human GDP- reductase activities in one polypeptide. mannose epimerase-reductase in a eukaryotic expression vector. To understand better the human enzymes involved in this Transfection of Lec13 and Cell Staining—The Chinese hamster ovary pathway, their role in selectin-mediated cell adhesion, and the (CHO) cell line Lec13, was obtained from Professor P. Stanley at Albert LADII defect, we have undertaken the molecular cloning of the Einstein Collage of Medicine. This line was first transfected with pMT- NeoFTIV, a vector expressing human fucosyltransferase IV and the human gene encoding GDP-mannose 4,6-dehydratase. Fur- neomycin resistance genes, by the calcium phosphate method as de- thermore, utilizing purified recombinant enzymes expressed in scribed previously (19). To make this cell line capable of replicating E. coli, we have reconstituted the GDP-fucose biosynthetic vectors containing the polyoma virus origin of replication, one of the pathway in vitro. We demonstrate that two enzymes, GDP- Fuc-T IV-positive cell lines, clone 9E9A, was again transfected with mannose 4,6-dehydratase and GDP-4-keto-6-deoxymannose pCDNA3.1 ZeoPyLT, a plasmid expressing the early region of polyoma 3,5-epimerase, 4-reductase, are sufficient to synthesize GDP- virus including large T, by the lipofectamine method according to the manufacturer’s instructions (Invitrogen). One zeocin-resistant clone fucose from GDP-mannose, confirming earlier studies suggest- had Fuc-T IV activity and was replication-competent. This cell line, ing that both epimerase and reductase activities are encoded in 9E9A LT2.9, was transfected with pMT-hGMD or pMT-hFX by the a single polypeptide. Additionally, we show that human GDP- lipofectamine method. After 48 h, cells were analyzed for Lewis X mannose 4,6-dehydratase has a strict specificity for NADP expression by immunofluorescence after staining with CD15 antibody over NAD . Using the homologous E. coli enzymes, GDP-man- (Immunotech) and goat anti-mouse fluorescein isothiocyanate second- nose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose ary antibody (Boehringer Manheim). 3,5-epimerase, 4-reductase (WCAG), we demonstrate that the In Vitro Assays of CHO Cell Extracts—Mutant CHO cells transfected with human dehydratase cDNA, human epimerase-reductase cDNA, or same is true in bacteria. We also show that human GDP- wild type CHO cells were lysed under nitrogen pressure in 0.75 ml of 25 mannose 4,6-dehydratase is subject to feedback inhibition by mM Hepes, pH 7.4, 100 mM NaCl, 10 mM EDTA, 10 mM DTT for 5 min GDP-fucose and that this, along with its differential levels of in a Parr Bomb at 900 p.s.i. on ice. The cell debris was pelleted at gene expression, provides potential mechanisms for regulating 50,000 3 g for 1 h, and soluble extracts were assayed in 25 mM Hepes, its activity. pH 7.4, 100 mM NaCl, 15 mM MgCl ,10mM DTT, 10 mM GDP-mannose, with 100,000 cpm of C-labeled GDP-mannose for2hat37 °C.The MATERIALS AND METHODS reactions were stopped by boiling for 5 min followed by centrifugation Data Base Searching and Sequence Alignments—The National for 5 min at 15,000 rpm in a microcentrifuge. Unlabeled GDP-mannose Center for Biotechnology Information (NCBI) EST data base was and GDP-fucose were added as standards. GDP-mannose, GDP-fucose searched with BLAST on the NCBI server. The human and E. coli and the 4-keto 6-deoxy intermediate were separated as described by dehydratase peptides were aligned with the program GAP of the Yamamoto et al. (10) except the amide-80 column (Tosohaas) was run in Genetics Computer Group analysis package. Amino terminal peptides 66% acetonitrile and 7.5 mM citric acid/Na HPO buffer, pH 4.0. The 2 4 of the GDP-mannose 4,6-dehydratase from different species were C-labeled sugar nucleotides were detected with a flow through scin- aligned using the GeneWorks program from IntelliGenetics, Inc. tillation counter Beta-1 (Packard) run with a solid scintillant cell. The Cloning Human GDP-mannose 4,6-Dehydratase—A cDNA library unlabeled sugar nucleotides were detected at 254 nm. was constructed from HL-60 cells as described by Sako et al. (18). This Expression and Purification of Human Enzymes from E. coli—The plasmid-based cDNA expression library was assembled into 19 pools, human dehydratase and epimerase-reductase genes were cloned by each representing 60,000 –100,000 individual clones/pool. The pools PCR into the EcoRI and HindIII sites of vector pRSETB (Invitrogen) for were screened by PCR using DNA primers based on both the mouse expression in E. coli. This yielded vectors pRSEThGMD and pRS- EST (accession number W29220) and the published E. coli sequence: EThFX. The inserts in the resulting vectors were sequenced in their 59-TGATGAGCCAGAGGACTTTGTCATAGCTAC-39 and 59-CAGAAA- entirety. The resulting dehydratase fusion protein had the sequence GTCCACTTCAGTCGGTCGGTAGTA-39. MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSPSAGTM- Two pools gave the expected 200-base pair fragment. The 200-base EF added to amino acid 20 of the predicted sequence, the position pair PCR product was reamplified by PCR, random primer-labeled with homologous to the start of the E. coli enzyme. For the epimerase- P, and used to identify positive clones from the two library pools by reductase, the same 43-amino acid fusion peptide was added to amino Cloning of Human GDP-mannose 4,6-Dehydratase 8195 acid 2 of the published sequence. The two expression vectors were Micromass Platform II electrospray ionization-equipped mass spec- trometer, which was operated in the negative ion mode with a cone transformed into E. coli strain BL21/DE3 and the bacteria were grown voltage of 40 V. The intermediate was further characterized by high in LB media containing ampicillin and chloramphenicol. Both the de- field NMR. All NMR experiments were performed on a Varian Unity hydratase-expressing cells and epimerase-reductase-expressing cells plus 600-MHz spectrometer. Samples were dissolved in D O, and HDO were grown at room temperature to an A of 0.5 and induced with 0.3 600 2 was used as an internal reference, set to 4.76 ppm at 25 °C. Two- mM isopropyl-1-thio-b-D-galactopyranoside for 3– 4 h. The cells were dimensional total correlation spectroscopy, nuclear Overhauser effect resuspended in Tris-buffered saline containing DNase I and lysed in a spectroscopy, and heteronuclear multiple-quantum coherence were per- French press at 12,000 p.s.i. After clarification, the dehydratase was formed with standard Varian pulse sequences. purified by successive chromatography over nitrolotriacetic acid-Ni - Cloning of E. coli GMD and E. coli WCAG—The gmd and wcaG genes agarose (Qiagen) eluting with 200 mM imidazole, chromatography over were cloned from the E. coli K-12 genome using PCR oligonucleotides ceramic hydroxyapatite (Bio-Rad) eluting with a phosphate gradient that created an NcoI site overlapping the initiating methionine of each (0.0 – 0.5 M), and chromatography over MonoQ (Pharmacia) eluting with gene and a HindIII site following the termination codon of each gene. a NaCl gradient (0.0 – 0.5 M). The epimerase-reductase was purified by The PCR products were cloned into the NcoI and HindIII sites of successive chromatography over nitrolotriacetic acid-Ni -agarose elut- pSE380 (Invitrogen), yielding pSEGMD and pSEWCAG. Sequences ing with 200 mM imidazole and chromatography over 29–59 ADP-Sepha- were confirmed by DNA sequencing. During the sequencing of the wcaG rose eluting with 10 mM NADP . The resulting proteins were greater gene, it was discovered that multiple clones contained two nucleotide than 90% pure as judged by SDS-PAGE and staining with Coomassie differences from the published sequence (21). The last nucleotide in Blue. Amino terminal sequencing of the first 15 amino acids of the codon 255 and the first nucleotide in codon 256 changed from a CG in proteins confirmed their identity as fusion protein expressed from the the published sequence to a GC in the cloned gene. This changed the pRSET vector. predicted amino acids in those positions from an aspartate at amino A second expression construct was made for each gene, which had a acid 255 and a valine at amino acid 256 in the published sequence to 20-amino acid amino-terminal leader, MRGSHHHHHHGSDYKD- 19 1 glutamate and leucine in the cloned gene. DDDK, added to the Met of hGMD or Met of hFX. To construct these Expression and Purification of E. coli Enzymes from E. coli—E. coli expression plasmids, synthetic DNA was used to rebuild the hGMD strain GI934 harboring either pSEGMD or pSEWCAG were grown in gene from the AgeI site near the 59-end of the gene. The synthetic DNA LB media containing ampicillin at 370 °C to an A of 0.5. The cells was hybridized and ligated to the AgeI–HindIII fragment of pRSETh- were induced with 1 mM isopropyl-1-thio-b-D-galactopyranoside and GMD into the NdeI and HindIII sites of pRSETB to yield pT7hGMD. A grown for an additional 4 h. Cell pellets were broken in a French press similar approach was taken to construct pT7hFX using the BamHI– at 12,000 p.s.i. After clarification, the E. coli dehydratase was purified HindIII fragment of pRSEThFX. The resulting vectors pT7hMGD and by successive chromatography over Toyopearl DEAE (TosoHaas) elut- pT7hFX were transformed into E. coli strain BL21/DE3, and protein ing with a NaCl gradient (0.0 – 0.5 M), chromatography over ceramic was expressed and purified as above. No significant differences were hydroxyapatite eluting with a phosphate gradient (0.0 – 0.5 M), and observed in the activity, substrate, or cofactor preference for both the chromatography over MonoQ eluting with a NaCl gradient (0.0 – 0.5 M). human dehydratase or epimerase-reductase having either the longer or The E. coli epimerase-reductase was purified by successive chromatog- shorter N-terminal fusion. raphy over Toyopearl DEAE eluting with a NaCl gradient (0.0 – 0.5 M), In Vitro Assays of Purified Dehydratase and Epimerase-Reductase— followed by chromatography over heparin-Toyopearl eluting with 200 Purified proteins were assayed in 25 mM MOPS, pH 7.0, 100 mM NaCl, mM NaCl. The resulting proteins were greater than 90% pure as judged 5mM EDTA, 10 mM DTT, 25 mM GDP-mannose with 100,000 cpm of by SDS-PAGE and staining with Coomassie Blue. Amino-terminal se- C-labeled GDP-mannose for1hat37 °C. When sequential reactions quencing of the first 15 amino acids of the proteins confirmed their were performed, the mix was incubated for an additional1hat37 °C identities. 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 RESULTS a microcentrifuge. Unlabeled GDP-mannose and GDP-fucose were added at this point as internal standards. Kinetic constants were de- Molecular Cloning of Human GDP-mannose 4,6-Dehy- termined graphically using S/V versus S plots with GDP-mannose con- dratase—To clone human GDP-mannose 4,6-dehydratase, we centrations ranging from ⁄2 to 5 K . The reaction conditions were as first performed a TBLASTN search of the NCBI EST data base above with 1 mM NADP added to reactions. using the sequence of the E. coli enzyme. This identified a Paper Chromatography— C-Labeled GDP-mannose, GDP-fucose, mouse EST, accession number W29220, with a high degree of and reaction products were incubated in 0.2 M NaBH for 15 min at homology to the E. coli enzyme. We designed two oligonucleo- room temperature to reduce keto intermediates and then were cleaved from the sugar by incubating in 1 M trichloroacetic acid for 10 min in tide primers based on conserved amino acids present in both boiling water. This mixture was chromatographed on Whatman 3MM the E. coli sequence and the partial sequence of the mouse paper in descending mode using three different solvent systems (solvent gene. Using the oligonucleotides as primers, we obtained a I, water-saturated methyl ethyl ketone for 24 h; solvent II, ethyl ace- 200-base pair PCR fragment from a human promyelocytic cell tate/pyridine/water (3.6:1.0:1.15) for 7 h; solvent III, ethyl acetate/ line HL-60 cDNA library. This fragment was then used as a pyridine/water (10:2.5:1.5) for 17 h. Free sugar standards were localized probe to isolate two apparently full-length cDNA clones for the by staining with AgNO in acetone followed by NaOH in methanol (20). Radioactivity was detected by cutting the paper into strips, and 1-cm putative human GDP-mannose 4,6 dehydratase, the sequence sections of each strip were counted in 1 ml of water plus 10 ml of of which is shown in Fig. 2. There are two potential initiator formula 989 (Packard). methionines located at nucleotides 76 and 130 of this sequence. Synthesis and Characterization of GDP-4-keto-6-deoxymannose— As shown in Fig. 3A, the downstream methionine of the human GDP-4-keto-6-deoxymannose was synthesized from GDP-mannose us- protein more closely aligns with the initiating methionine of ing the purified human and purified E. coli dehydratases. Typical the E. coli protein. However, alignment of the human sequence 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 to the recently cloned arabidopsis GDP-mannose 4,6-dehy- mM DTT, 5 mM EDTA, 10 mM NADPH, and 10 mM NADP at 37 °C for dratase gene and a putative C. elegans translation product 3– 6 h. Typical reaction conditions for E. coli dehydratase were 10 mM (Fig. 3B) suggests that translation of the human protein ini- GDP-mannose, 0.1 mg/ml E. coli dehydratase, 10 mM MOPS, pH 6.5, tiates at the first methionine, at nucleotide 76, and that dehy- 100 mM NaCl, 2 mM DTT, 1 mM EDTA, 10 mM NADPH, and 100 mM 1 dratases from nonbacterial sources contain an amino-terminal NADP at 37 °C for 3– 6 h. The reactions were allowed to proceed to extension. The human and E. coli proteins are 61% identical completion as judged by HPLC, and the protein was removed by ultra- filtration on a Centricon-10. The resulting mix was desalted on Seph- over their entire lengths, and both contain an extended con- adex G-10 run in water and lyophilized. GDP-4-keto-6-deoxymannose sensus sequence, GXXGXXG, identifying the bab fold found in 1 1 prepared this way was stable frozen at 220 °C either as a dried powder many NAD - and NADP -binding proteins (22). This sequence or in water and was judged essentially pure by HPLC and ESI-MS is found between amino acids 9 and 15 of the E. coli enzyme. analysis. For ESI-MS analysis, 500 mM citric acid-sodium phosphate Fig. 4 shows that the human dehydratase gene encodes a single buffer was added to a 20 pmol/ml solution of GDP-4-keto-6-deoxyman- mRNA transcript of about 1.7 kilobase pairs that is expressed nose (in 50% acetonitrile) to give a final concentration of 0.5 mM at pH 4. Flow injection at 10 ml/min was used to introduce this sample into the in all tissue and cell types examined, albeit at varying levels. 8196 Cloning of Human GDP-mannose 4,6-Dehydratase 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. 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 biosyn- thesis 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-dehy- FIG.2. cDNA and amino acid sequence of human GDP-man- nose 4,6-dehydratase. The sequence of the cDNA for human GDP- dratase, in media lacking fucose, also causes the cells to stain mannose 4,6-dehydratase is shown above the putative peptide sequence. positive for Lewis X, demonstrating that the dehydratase gene complements the CHO cell defect (Fig. 5B). By contrast, trans- The mRNA levels are highest in pancreas followed by small fection with the same vector expressing the human epimerase- intestine, liver, colon, and prostate and lowest in ovary, brain, reductase gene, again in media lacking fucose, shows no stain- lung, spleen, and peripheral blood lymphocytes. Likewise, a ing by CD15 (Fig. 5C). As further evidence, lysates of Lec13 varied level of expression was seen in the human cell lines cells transiently transfected with human GDP-mannose 4,6- examined (Fig. 4C). dehydratase cDNA readily converted C-labeled GDP-man- GDP-mannose 4,6-Dehydratase cDNA Complements the De- nose to GDP-fucose (Fig. 6B), whereas lysates of Lec13 cells hydratase Defect in Lec13—To demonstrate the isolated cDNA transfected with the human epimerase-reductase cDNA in the encoded GDP-mannose 4,6-dehydratase activity, we tran- same expression vector did not (Fig. 6A). The level of activity in siently transfected it into Lec13, a CHO cell line that previ- dehydratase-transfected cells (Fig. 6B,25 mg) is higher than ously has been identified as lacking GDP-mannose 4,6-dehy- that of wild type CHO cells (Fig. 6C, 125 mg). Based on this dratase activity (23). To monitor the GDP-mannose 4,6- data, we conclude that the cDNA encodes GDP-mannose dehydratase activity, we first stably transfected Lec13 with 4,6-dehydratase. Cloning of Human GDP-mannose 4,6-Dehydratase 8197 FIG.4. Northern analysis of human GDP-mannose 4,6-dehydratase gene expression. Autoradiogram of Northern blots probed with P-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 b-actin messages are shown at the bottom. 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. FIG.6. In Vitro assay of dehy- dratase and epimerase-reductase ac- tivity in wild type CHO and trans- fected Lec13 CHO cell lines. HPLC analysis of reactions of C-labeled GDP- mannose with cell extracts are shown. A, 125 mg of extract of 9E9A LT2.9 Lec13 cells transfected with epimerase-reduc- tase cDNA. B,25 mg of extract of 9E9A LT2.9 Lec13 cells transfected with dehy- dratase cDNA. C, 125 mg of extract of CHO Dukx cells (wild type for GDP-man- nose 4,6-dehydratase). The positions of C-labeled GDP-mannose and GDP-fu- cose standards are shown above. 8198 Cloning of Human GDP-mannose 4,6-Dehydratase Chromosomal Localization of Human GDP-mannose 4,6-De- mapped their location using fluorescence in situ hybridization. hydratase and FX Gene—To determine if the two genes for Full-length cDNA inserts encoding the human dehydratase GDP-fucose biosynthesis are linked on the human genome we (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 ge- nome. 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 epi- merase-reductase was mapped to the q terminus of chromo- some 8, an area corresponding to band 8q24.3. A total of 80 metaphase cells were analyzed, with 70 exhibiting specific la- FIG.7. SDS-PAGE of recombinant enzymes purified from E. coli. beling (data not shown). 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. Human GDP-mannose Dehydratase and Epimerase-Reduc- FIG.8. In vitro assay of human and E. coli dehydratase and epimerase re- ductase activity. HPLC analysis of re- actions of C-labeled GDP-mannose with purified recombinant enzymes are shown. A,1 mg of human dehydratase plus 100 mM NADP ; B,1 mg of human dehy- dratase plus 1 mM NAD ; C,1 mgofhu- man dehydratase plus 100 mM NADP , followed by 1 mg of human epimerase- reductase plus 100 mM NADPH; D,1 mgof E. coli dehydratase plus 1 mM NADP ; E, 1 mgof E. coli dehydratase plus 1 mM NAD ; F,1 mgof E. coli dehydratase plus 1mM NADP , followed by 1 mgof E. coli epimerase-reductase plus 100 mM NADPH. The arrows show the position of C-labeled standards GDP-mannose and GDP-fucose Cloning of Human GDP-mannose 4,6-Dehydratase 8199 tase Are Sufficient to Convert GDP-mannose to GDP-fucose—To further characterize GDP-mannose 4,6-dehydratase, and to re- constitute GDP-fucose biosynthesis in vitro, we needed to ob- tain 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-man- nose 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 se- quence by ESI-LC-MS (data not shown). To characterize the reactions of the purified dehydratase and epimerase-reductase, we incubated the enzymes with C-la- beled GDP-mannose and identified the reaction products by HPLC and paper chromatography. As shown in Fig. 8A, puri- fied GDP-mannose 4,6-dehydratase converts C-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 chromatog- raphy. As shown in Fig. 1, the expected monosaccharides re- sulting from reduction of GDP-4-keto-6-deoxymannose by boro- hydride 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 FIG.9. Analysis of the reaction products of human and E. coli standard for rhamnose. This component also co-migrates with dehydratase and epimerase-reductase by descending paper rhamnose in solvent systems II and III (data not shown). The 14 chromatography. C-Labeled reaction products were reduced with species at 5 cm co-migrates with mannose and was presumably NaBH , and the resulting sugar was cleaved with acid and spotted on Whatman 3MM paper developed in water-saturated methyl ethyl ke- derived from the unreacted starting material. The species at 24 tone for 24 h. The paper was cut into strips, and 1-cm sections of each cm runs faster than rhamnose in this solvent, as expected for strip were counted. A, the reactions contained GDP-mannose, human 6-deoxytalose, but has an R that differs from the published 1 dehydratase, and NADP , followed by human epimerase-reductase value for 6-deoxytalose (6, 27). This is also true in solvents II plus NADPH (open squares) or GDP-mannose, human dehydratase, and NADP (filled triangles). B, the reactions contained GDP-mannose, E. and III. An additional species was observed in this sample, coli dehydratase, NADP , E. coli epimerase-reductase, and NADPH running near 37 cm. Without authentic 6-deoxytalose, we have (open squares) or GDP-mannose, E. coli dehydratase, and NADP not been able to confirm the identity of the two faster running (filled triangles). The position of C-labeled GDP-mannose and GDP- spots. Using the purified enzyme, we were able to determine fucose that had been treated identically to the reaction mixtures is shown above labeled as mannose and fucose, respectively. The position the cofactor utilized by human GDP-mannose 4,6-dehydratase. of unlabeled free rhamnose is also shown at the top of the trace. 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. and High Field NMR—To confirm that the reaction product of Using the purified human dehydratase and epimerase-re- human dehydratase and GDP-mannose was GDP-4-keto-6-de- ductase, we could demonstrate that these two enzymes alone oxymannose, we isolated the product and analyzed it by mass were sufficient to convert GDP-fucose to GDP-mannose. Se- spectrometry and NMR. The major peaks in the ESI-MS spec- quential incubation of GDP-mannose with human dehydratase trum of the isolated intermediate were at 586.1 and 292.6, 2 22 and NADP followed by human epimerase-reductase and corresponding to [M 2 H] ion and [M 2 H] , respectively for NADPH converts the GDP-mannose to GDP-fucose, based upon GDP-4-keto-6-deoxymannose. A partial spectrum is shown in 2 1 co-chromatography with authentic GDP-fucose (Fig. 8C). We Fig. 10, where the [M 2 H] peak at 586.1, the [M 1 Na 2 2 1 2 confirmed the identify of the product as GDP-fucose by de- 2H] peak at 608.1, and the [M 1 2Na 2 3H] peak at 630.1 scending paper chromatography of the free monosaccharide for GDP-4-keto-6-deoxymannose are clearly visible. A [M 2 after reduction with NaBH and cleavage from GDP with acid. H] peak for residual GDP-mannose at 604.0 is also present in Fig. 9A (open squares) shows the free monosaccharide co-mi- the spectra. A combination of one-dimensional homonuclear, grates with an identically treated C-labeled GDP-fucose two-dimensional homonuclear, and two-dimensional hetero- standard. Identical results were obtained in solvents systems II nuclear NMR experiments revealed that the isolated reaction and III (data not shown). Unlike the human dehydratase, product was a mixture of at least two related sugar nucleotides. which shows a strict cofactor preference for NADP over The observed chemical shifts and coupling constants are listed NAD , the human epimerase-reductase can utilize either in Table I. From these data, we have identified the two major NADPH or NADH, although NADPH is used more efficiently species as GDP-4-keto-6-deoxymannose and GDP-3-keto-6-de- (data not shown). oxymannose. NMR analysis of the isolated product of E. coli Characterization of GDP-4-keto-6-deoxymannose by ESI-MS dehydratase also showed a similar mixture of GDP-4-keto-6- 8200 Cloning of Human GDP-mannose 4,6-Dehydratase FIG. 10. High mass region of the ESI-MS spectrum for the reaction products of GDP-mannose and human dehydratase. Spectrum 2 1 2 1 2 shows the [M 2 H] peak at 586.1, the [M 2 Na 2 2H] peak at 608.1, and the [M 2 2Na 2 3H] peak at 630.1 for GDP-4-keto-6-deoxymannose. Also present is the [M 2 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. TABLE I way we characterized the human enzymes. Fig. 8E shows that Chemical shifts and J coupling for GDP-4-keto-6-deoxymannose incubation of E. coli dehydratase with GDP-mannose and and GDP-3-keto-6-deoxymannose NADP resulted in production of the reaction intermediate GDP-4-keto-6-deoxymannose GDP-3-keto-6-deoxymannose GDP-4-keto-6-deoxymannose. This reaction product produced 13 1 13 1 C H J C H J only two spots in paper chromatography after reduction and cleavage. One migrated with the rhamnose standard at 17 cm, Base 140.15 8.11 140.15 8.11 Ribose and one migrated faster at 37 cm (Fig. 9B, filled triangles). The 19 89.12 5.93 6.0 89.12 5.96 6.0 spot seen at 25 cm in the reaction with human dehydratase is 29 75.97 4.81 5.6 75.97 4.86 missing (Fig. 9, compare A and B (filled triangles)). The E. coli 39 72.85 4.51 72.85 4.53 dehydratase used NADP as a cofactor and could not substi- 49 86.29 4.35 86.29 4.35 tute NAD at concentrations up to 1 mM (Fig. 8, D and E). 59a,b 67.69 4.21 67.69 4.21 Mannose As with the human enzymes, E. coli dehydratase and epime- 10 98.09 5.59 2.1, 7.7 98.87 5.45 1.6, 7.7 rase-reductase were sufficient to convert GDP-mannose to 20 77.53 4.46 ;3 72.99 4.01 ;1 GDP-fucose. Incubation of GDP-mannose with E. coli dehy- 30 75.09 4.82 dratase and E. coli epimerase-reductase in the presence of 40 70.88 3.94 ,1 50 74.07 4.70 6.5 72.88 4.07 6.6 NADP and NADPH converted the GDP-mannose to GDP- CH 15.18 1.23 13.63 1.21 fucose (Fig. 8F). We confirmed the identity of GDP-fucose by The identification of the 3-keto compound was complicated by the paper chromatography (Fig. 9B, open squares). The E. coli small coupling constant of the proton on C-4 of mannose, which reduced epimerase-reductase can use NADH but not as efficiently as magnetization transfer in the total correlation spectroscopy experi- NADPH (data not shown). ment. During repeated lyophilization of the samples in D O to exchange We performed a preliminary characterization of the two de- protons, new peaks were evident in the NMR, suggesting the GDP-4- keto-6-deoxymannose and its related compounds are not entirely stable hydratases, and the results are shown in Table II. The E. coli under these conditions. enzyme has a significantly higher K for GDP-mannose than the human enzyme but also has a significantly higher V . max Additionally, we monitored inhibition of the dehydratase by deoxymannose and GDP-3-keto-6-deoxymannose. GDP-fucose, which has been proposed as a mechanism to reg- E. coli GDP-mannose Dehydratase and Epimerase-Reductase ulate the enzyme’s activity. Both human and E. coli dehydrata- Are Sufficient to Convert GDP-mannose to GDP-fucose—To ad- ses were inhibited by GDP-fucose with IC values lower than dress whether the bacterial enzymes catalyze the same reac- the IC values for inhibition by GDP, suggesting a specific tions as the human dehydratase and epimerase-reductase, we effect and a potential role in regulation of the enzyme’s activity. cloned, expressed, and purified the E. coli enzymes (Fig. 7B). Quite unexpectedly, both enzymes are stimulated by NADPH We examined the reactions of the E. coli enzymes in the same at micromolar concentrations, although this co-factor does not Cloning of Human GDP-mannose 4,6-Dehydratase 8201 TABLE II but no evidence of any other GDP-mannose derivatives of dif- Characteristics of human and E. coli GDP-mannose-4,6-dehydratase ferent masses. High field NMR revealed the presence of two 50% compounds, one being the expected product GDP-4-keto-6-de- a IC of IC of Stimulation 50 50 K V b c stimulation m max GDP GDP-fucose by NADPH oxymannose and the other the related GDP-3-keto-6-deoxym- by NADPH annose. This was the case for the product of both the human mM mmol/min/mg mM mM -fold mM and E. coli dehydratases. The presence of both the 4-keto- and Human 80 0.11 225 75 4.5 2 3-keto-6-deoxy-sugars was also seen in the dTDP-rhamnose E. coli 260 0.73 400 10 2.3 15 pathway where a 4,6-dehydratase converts dTDP-glucose to K is in mM. The S.D. values in the kinetic constants are less than dTDP-4-keto-6-deoxy-glucose (31, 32). The biological signifi- 10%. Reaction conditions were 25 mM MOPS, pH 7.0, 100 mM NaCl, 10 cance of both GDP-4-keto-6-deoxymannose and GDP-3-keto-6- mM DTT, 5 mM EDTA, and 1 mM NADP , 37 °C. deoxymannose intermediates is unclear, although apparently Reactions contained 25 mM GDP-mannose. c 1 both are epimerized and reduced to GDP-fucose by the epime- Reactions contained 100 mM NADP . rase reductase (Fig. 8, C and F). It is possible that the 3-keto- sugar arose during work-up of the isolated reaction product. play a role in catalysis. It is not clear if this stimulation by However, the isolated, unlabeled intermediate also was con- NADPH is relevant to the in vivo regulation of the enzyme. verted to GDP-fucose by purified human epimerase-reductase DISCUSSION (data not shown). We have cloned the gene encoding human GDP-mannose Cells from two patients having LADII do not fucosylate their 4,6-dehydratase using homology between the E. coli enzyme cell surfaces (4) and as such lack both blood group antigens and and a mouse EST. The cloned gene complemented the previ- the sialyl Lewis X and related epitopes that function as selectin ously identified GDP-mannose 4,6-dehydratase defect in the ligands. The molecular basis of this disorder is still unknown. CHO cell line Lec13, demonstrating that the cDNA encodes a As with Lec13, this phenotype can be rescued in cell lines functional protein. The dehydratase gene shows high levels of derived from these patients by culturing them in the presence identity between bacteria and human and indeed across the of fucose, suggesting that GDP-fucose transport and the com- spectrum of species examined (for a comparison of dehydrata- plement of fucosyltransferases are intact in these cells. This ses from a variety of bacterial and nonmammalian sources see would imply that the defect in the LADII patients lies in the Bonin et al. (28). The message for human GDP-mannose 4,6- pathway converting GDP-mannose to GDP-fucose, i.e. either dehydratase is expressed in all tissues examined, albeit at the dehydratase or epimerase-reductase. With the human varying levels. The varying levels of expression of the dehy- genes for both enzymes now cloned, we can determine if either dratase message suggest the enzyme may be regulated at the is responsible for the LADII phenotype. There are suggestions level of transcription, and in fact there is evidence for develop- from E. coli that there may be an additional gene that plays a mental regulation in rat and nereids (29, 30). It also appears role in GDP-fucose biosynthesis in vivo. Sequencing of the that, in both humans and E. coli, GDP-fucose biosynthesis is capsular polysaccharide operon in E. coli led to the identifica- regulated by feedback inhibition of the dehydratase by GDP- tion of an open reading frame (wcaH) immediately downstream fucose, the final product in the pathway. This mechanism of of the dehydratase, gmd, and epimerase-reductase genes, wcaG inhibition was noted for Aerobacter aerogenes by Kornfeld and (21). This putative protein has been assigned to the GDP-fucose Ginsberg (9) and was suggested for the porcine enzyme by Serif biosynthetic pathway, yet it is clearly not necessary for conver- and co-workers (33). sion of GDP-mannose to GDP-fucose in vitro. WcaH may play a The cloning of the human dehydratase gene, along with the role in GDP-fucose biosynthesis in vivo or play another, yet recent cloning of the human epimerase-reductase by Tonetti et unidentified, role in capsular polysaccharide biosynthesis. The al. (17) has allowed us to reconstitute GDP-fucose biosynthesis cloning of the GDP-mannose 4,6-dehydratase gene provides a in vitro using purified, recombinant enzymes. Thus, we have valuable tool to address outstanding questions in the regula- definitively shown that two enzymes, a dehydratase and an tion, biosynthesis, and role of GDP-fucose in vivo. epimerase-reductase, are sufficient to convert GDP-mannose to Acknowledgments—We thank Professor Pamala Stanley of the GDP-fucose. In doing so, we demonstrated, using purified re- Albert Einstein Collage of Medicine for the gift of the Lec13 cell line; combinant proteins, that in humans and in E. coli, both 3,5- Drs. Diane Sako and Monique Davies of Genetics Institute for the gifts epimerase and 4,6-reductase activities are present in a single of plasmids pMTNeoFTIV and pEDPyLT, respectively; Drs. Elliott protein. This confirms the earlier studies of Serif and co-work- Nickbarg and Robert Gassaway of Genetics Institute for ESI-LC-MS analysis and peptide sequencing; Kevin Bean and Mark Proia of ers (16) on the enzyme purified from porcine thyroids and the Genetics Institute for DNA sequencing and library screening. We thank recent work of Tonetti et al. (17) with the human FX protein. Drs. John Lowe and Peter Smith of Univeristy of Michigan for sharing Additionally, we find that human dehydratase has a strict data on the LADII cell lines prior to publication. We thank Drs. Simon 1 1 cofactor requirement for NADP for which NAD cannot sub- Jones and John Knopf of Genetics Institute for critical reading of the manuscript. stitute. This is consistent with earlier reports demonstrating that GDP-mannose 4,6-dehydratase from K. pneumoniae re- REFERENCES quires NADP (10) as well as the recent work of Sturla et al., 1. Feizi, T. (1991) Trends Biochem. 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Published: Apr 1, 1998

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