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

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Sialic acids of cell surface glycoproteins and glycolipids play a pivotal role in the structure and function of animal tissues. The pattern of cell surface sialylation is speciesand tissue-specific, is highly regulated during embryonic development, and changes with stages of differentiation. A prerequisite for the synthesis of sialylated glycoconjugates is the activated sugar-nucleotide cytidine 5-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac), which provides a substrate for Golgi sialyltransferases. Although a mammalian enzymatic activity responsible for the synthesis of CMP-Neu5Ac has been described and the enzyme has been purified to near homogeneity, sequence information is restricted to bacterial CMP-Neu5Ac synthetases. In this paper, we describe the molecular characterization, functional expression, and subcellular localization of murine CMPNeu5Ac synthetase. Cloning was achieved by complementation of the Chinese hamster ovary lec32 mutation that causes a deficiency in CMP-Neu5Ac synthetase activity. A murine cDNA encoding a protein of 432 amino acids rescued the lec32 mutation and also caused polysialic acid to be expressed in the capsule of the CMP-Neu5Ac synthetase negative Escherichia coli mutant EV5. Three potential nuclear localization signals were found in the murine synthetase, and immunofluorescence studies confirmed predominantly nuclear localization of an N-terminally Flag-tagged molecule. Four stretches of amino acids that occur in the N-terminal region are highly conserved in bacterial CMP-Neu5Ac synthetases, providing evidence for an ancestral relationship between the sialylation pathways of bacterial and animal cells.

A previously isolated endocytic trafficking mutant (TRF1) isolated from HuH-7 cells is defective in the distribution of subpopulations of cell-surface receptors for asialoorosomucoid (asialoglycoprotein receptor (ASGR)), transferrin, and mannose-terminating glycoproteins. The pleiotropic phenotype of TRF1 also includes an increased sensitivity to Pseudomonas toxin and deficient assembly and function of gap junctions. HuH-7؋TRF1 hybrids exhibited a normal subcellular distribution of ASGR, consistent with the TRF1 mutation being recessive. A cDNA expression library derived from HuH-7 mRNA was transfected into TRF1 cells, which were subsequently selected for resistance to Pseudomonas toxin. Sequence analysis of a recovered cDNA revealed a unique isoform of casein kinase 2 (CK2), CK2␣؆. Western blot analysis of TRF1 proteins revealed a 60% reduction in total CK2␣ expression. Consistent with this finding, the hybrids HuH-7؋HuH-7 and HuH-7؋TRF1 expressed equivalent amounts of total CK2␣. Immunoblots using antibodies against peptides unique to the previously described CK2 isoforms CK2␣ and CK2␣ and the novel CK2␣؆ isoform showed that, although TRF1 and parental HuH-7 cells expressed comparable amounts of CK2␣ and CK2␣, the mutant did not express CK2␣؆. Based on the genomic DNA sequence, RNA transcripts encoding CK2␣؆ apparently originate from alternative splicing of a primary transcript. Protein overexpression following transfection of TRF1 cells with cDNAs encoding either CK2␣ or the newly cloned CK2␣؆ restored the parental HuH-7 phenotype, including Pseudomonas toxin resistance, cell-surface ASGR binding activity, phosphorylation, and the assembly of gap junctions. This study suggests that HuH-7 cells express at least three CK2␣ isoforms and that the pleiotropic TRF1 phenotype is a consequence of a reduction in total CK2 expression.Receptor-mediated endocytosis, a universal mechanism for the uptake of macromolecules by cells, is initiated by the binding of ligand to specific cell-surface receptors, followed by a complex series of intracellular vesicular transfers (1). The asialoglycoprotein receptor (ASGR) 1 is a prototype of the class of receptors that constitutively enter cells via clathrin-coated pits and traffic the endocytic pathway, recycling between early endosomal compartments and the cell surface (2). Two subpopulations of ASGR, States 1 and 2, were originally identified based on the kinetics of ligand transport and the identification of parallel endocytic pathways (3). Changes in both the phosphorylation (4) and acylation (5) status of the receptor have been suggested to affect the transition between these states. The existence of receptor subpopulations has also been suggested for other class II recycling receptors, including the low density lipoprotein, mannose 6-phosphate, and ␣ 2 -macroglobin receptors (6). A mutant affecting endocytic traffic (TRF1) was previously isolated from HuH-7 cells using a dual selection protocol (7). To avoid the selection of receptor-minus mutants, selection pressure was ap...

To isolate a mutant liver cell defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was devised. Ovalbumin-gelonin and asialoorosomucoid (ASOR)-gelonin were incubated with mutagenized HuH-7 cells, and a rare survivor termed trafficking mutant 1 (Trf1) was isolated. Trf1 cells were stably 3-fold more resistant than the parental HuH-7 to both toxic conjugates. The anterograde steps of intracellular endocytic processing of ASOR, including internalization, endosomal acidification, and ligand degradation, were unaltered in Trf1 cells. In contrast, retrograde diacytosis of asialoglycoprotein receptor (ASGR).ASOR complex back to the cell surface was enhanced by about 250%. Selective labeling revealed an approximately 46% reduction in cell surface-associated ASGR in Trf1 cells, although their total cellular ASGR content was essentially equivalent to that in HuH-7. Similar results were obtained with the transferrin receptor. Binding of 125I-ASOR and 125I-transferrin was reduced in Trf1 cells to 49 +/- 2.5% and 30 +/- 2%, respectively, of HuH-7 cells. The methionine transporter was also reduced in Trf1 cells, as revealed by a 2-fold reduction in Vmax with no change in apparent Km. Pretreatment with monensin, sodium azide, or colchicine reduced surface binding of 125I-ASOR in HuH-7 cells by 50% but had no effect on binding to Trf1 cells. This result is predicted for a cell that expresses only State 1 ASGRs, which are resistant to modulation by metabolic and cytoskeletal inhibitors in contrast to State 2, which are responsive to these agents (Weigel, P. H., and Oka, J. A. (1984) J. Biol. Chem. 259, 1150-1154). The Trf1 mutant, having lost the ability to express State 2 receptors, provides genetic evidence for the existence of these two receptor subpopulations and an approach to identifying the biochemical mechanism by which they are generated.