Angela Bisso

Synthesis of GDP-L-fucose by the Human FX Protein

FX is a homodimeric NADP(H)-binding protein of 68 kDa, first identified in human erythrocytes, from which it was purified to homogeneity. Its function has been unrecognized despite partial structural and genetic characterization. Recently, on the basis of partial amino acid sequence, it proved to be the human homolog of the murine protein P35B, a tumor rejection antigen. In order to address the biochemical role of FX, its primary structure was completed by cDNA sequencing. This sequence revealed a significant homology with many proteins from different organisms. Specifically, FX showed a remarkable similarity with a putative Escherichia coli protein, named Yefb, whose gene maps in a region of E. coli chromosome coding for enzymes involved in synthesis and utilization of GDP-D-mannose. Accordingly, a possible role of FX in this metabolism was investigated. The data obtained indicate FX as the enzyme responsible for the last step of the major metabolic pathway resulting in GDP-L-fucose synthesis from GDP-D-mannose in procaryotic and eucaryotic cells. Specifically, purified FX apparently catalyzes a combined epimerase and NADPH-dependent reductase reaction, converting GDP-4-keto-6-D-deoxymannose to GDP-L-fucose. This is the substrate of several fucosyltranferases involved in the correct expression of many glyconjugates, including blood groups and developmental antigens.

A Self-restricted CD38-connexin 43 Cross-talk Affects NAD+ and Cyclic ADP-ribose Metabolism and Regulates Intracellular Calcium in 3T3 Fibroblasts

Connexin 43 (Cx43) hexameric hemichannels, recently demonstrated to mediate NAD+ transport, functionally interact in the plasma membrane of several cells with the ectoenzyme CD38 that converts NAD+ to the universal calcium mobilizer cyclic ADP-ribose (cADPR). Here we demonstrate that functional uncoupling between CD38 and Cx43 in CD38-transfected 3T3 murine fibroblasts is paralleled by decreased [Ca2+] i levels as a result of reduced intracellular conversion of NAD+ to cADPR. A sharp inverse correlation emerged between [Ca2+] i levels and NAD+ transport (measured as influx into cells and as efflux therefrom), both in the CD38+ cells (high [Ca2+] i , low transport) and in the CD38− fibroblasts (low [Ca2+] i , high transport). These differences were correlated with distinctive extents of Cx43 phosphorylation in the two cell populations, a lower phosphorylation with high NAD+ transport (CD38− cells) and vice versa (CD38+ cells). Conversion of NAD+-permeable Cx43 to the phosphorylated, NAD+-impermeable form occurs via Ca2+-stimulated protein kinase C (PKC). Thus, a self-regulatory loop emerged in CD38+ fibroblasts whereby high [Ca2+] i restricts further Ca2+mobilization by cADPR via PKC-mediated disruption of the Cx43-CD38 cross-talk. This mechanism may avoid: (i) leakage of NAD+from cells; (ii) depletion of intracellular NAD+ by CD38; (iii) overproduction of intracellular cADPR resulting in potentially cytotoxic [Ca2+] i .

The metabolism of 6-deoxyhexoses in bacterial and animal cells

L-fucose and L-rhamnose are two 6-deoxyhexoses naturally occurring in several complex carbohydrates. In prokaryotes both of them are found in polysaccharides of the cell wall, while in animals only L-fucose has been described, which mainly participates to the structure of glycoconjugates, either in the cell membrane or secreted in biological fluids, such as ABH blood groups and Lewis system antigens. L-fucose and L-rhamnose are synthesized by two de novo biosynthetic pathways starting from GDP-D-mannose and dTDP-D-glucose, respectively, which share several common features. The first step for both pathways is a dehydration reaction catalyzed by specific nucleotide-sugar dehydratases. This leads to the formation of unstable 4-keto-6-deoxy intermediates, which undergo a subsequent epimerization reaction responsible for the change from D- to L-conformation, and then a NADPH-dependent reduction of the 4-keto group, with the consequent formation of either GDP-L-fucose or dTDP-L-rhamnose. These compounds are then the substrates of specific glycosyltransferases which are responsible for insertion of either L-fucose or L-rhamnose in the corresponding glycoconjugates. The enzyme involved in the first step of GDP-L-fucose biosynthesis in E. coli, i.e., GDP-D-mannose 4,6 dehydratase, has been recently expressed as recombinant protein and characterized in our laboratory. We have also cloned and fully characterized a human protein, formerly named FX, and an E. coli protein, WcaG, which display both the epimerase and the reductase activities, thus indicating that only two enzymes are required for GDP-L-fucose production. Fucosylated complex glycoconjugates at the cell surface can then be recognized by specific counter-receptors in interacting cells, these mechanisms initiating important processes including inflammation and metastasis. The second pathway starting from dTDP-D-glucose leads to the synthesis of antibiotic glycosides or, alternatively, to the production of dTDP-L-rhamnose. While several sets of data are available on the first enzyme of the pathway, i.e., dTDP-D-glucose dehydratase, the enzymes involved in the following steps still need to be identified and characterized.

Expression, purification and characterization of GDP-d-mannose 4,6-dehydratase from Escherichia coli

GDP‐d‐mannose dehydratase (GMD) catalyzes the first step of the pathway that converts GDP‐d‐mannose to GDP‐l‐fucose in bacteria, plants and mammals. Recently, the gene coding for GMD has been identified and sequenced in E. coli. Based on this sequence, we have expressed and purified GMD in E. coli as a glutathione transferase (GST) fusion protein. The fused GST‐GMD protein and the thrombin‐cleaved GMD were then characterized. The catalytically active form of both enzyme species seems to be a hexamer of 410 and 250 kDa, respectively. The GST‐GMD fusion protein has a K m of 0.22±0.04 mM and a specific activity of 2.3±0.2 μmol/h/mg. Ca2+ and Mg2+ activate GMD, while GDP‐l‐β‐fucose, the end‐product of the pathway, inhibits it specifically. The GST‐GMD fusion protein contains one mole of tightly bound NADP+ per mole of hexamer. Apparently, this NADP+ is involved in the catalytic mechanism of GMD.

Defective intracellular activity of GDP-D-mannose-4,6-dehydratase in leukocyte adhesion deficiency type II syndrome.

Leukocyte adhesion deficiency type II (LAD II) is a rare genetic disease characterized by severe immunodeficiency which is related to defective expression in leukocytes of sialyl‐Lewis X (SLeX), a fucosylated ligand for endothelial selectins. The molecular basis of LAD II is still unknown, but has been tentatively localized in the de novo pathway of GDP‐l‐fucose biosynthesis from GDP‐d‐mannose. Here, we demonstrate that in cell lysates from a LAD II patient, GDP‐d‐mannose‐4,6‐dehydratase (GMD), the first of the two enzymes of the pathway has a defective activity compared to control subjects. GMD in cell lysates from both parents showed intermediate activity levels. Cloning of GMD from patient and control lymphocytes ruled out any mutation affecting the amino acid GMD sequence and the purified recombinant proteins from both controls and the patient showed identical specific activities. Since the levels of immunoreactive GMD in cell lysates were comparable in the patient and in controls, the biochemical deficiency of intracellular GMD activity in LAD II seems to be due to mutation(s) affecting some still unidentified GMD‐regulating protein.

Structural and enzymatic characterization of human recombinant GDP-D-mannose-4,6-dehydratase

GDP‐D‐mannose‐4,6‐dehydratase (GMD) is the key enzyme in the ‘de novo’ pathway of GDP‐L‐fucose biosynthesis. The reported cDNA sequences for human GMD predict three forms of different length, whose ‘in vivo’ occurrence and molecular properties are completely undefined. Here, we report the expression in Escherichia coli and the properties of each native recombinant GMD form. Only the 42 kDa long GMD (L‐GMD) and the 40.2 kDa (M‐GMD) forms were recovered as soluble functional proteins, while the 38.7 kDa form, short GMD (S‐GMD), lacking an N‐terminal domain critical for dinucleotide binding, was inactive and formed a precipitate. Both L‐GMD and M‐GMD are homodimers and contain 1 mol of tightly bound NADP+. Their kinetic properties (K m, K cat) are apparently identical and both forms are non‐competitively feedback‐inhibited by GDP‐L‐fucose to a similar extent. M‐GMD is the predominant enzyme form expressed in several human cell lines. These data seem to suggest that modulation of the ‘de novo’ pathway of GDP‐L‐fucose biosynthesis involves mechanisms other than differential ‘in vivo’ expression of GMD forms.

PRELIMINARY CRYSTALLOGRAPHIC INVESTIGATIONS OF RECOMBINANT GDP-4-KETO-6-DEOXY-D-MANNOSE EPIMERASE/REDUCTASE FROM E. COLI

The GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GM_ER) isolated from E. coli has been overexpressed as a GST-fusion protein and purified to homogeneity. The enzyme, an NADP+(H)-binding homodimer of 70 kDa, is responsible for the production of GDP-L-fucose. GM_ER shows significant structural homology to the human erythrocyte protein FX, which is involved in blood-group glycoconjugate biosynthesis, displaying 3,5 epimerase/reductase activity on GDP-4-keto-6-deoxy-D-mannose. GM_ER has been crystallized in a trigonal crystalline form, containing one molecule per asymmetric unit, suitable for high-resolution crystallographic investigations.