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It has been proposed that abnormal myo-inositol metabolism may be a factor in the development of diabetic complications. Studies with animal models of diabetes and cultured cells have suggested that hyperglycemia by an unknown mechanism may alter myo-inositol metabolism and content. Recently, we have shown that L-fucose, a 6-deoxy sugar whose content has been reported to be increased in diabetes, is a potent inhibitor of myo-inositol transport. To examine the effect of L-fucose on myo-inositol metabolism, neuroblastoma cells were cultured in medium supplemented with L-fucose. L-Fucose is a competitive inhibitor of Na(+)-dependent, high-affinity myo-inositol transport. The Ki for inhibition of myo-inositol transport by L-fucose is about 3 mM. L-Fucose is taken up and accumulates in neuroblastoma cells. The uptake of L-fucose is inhibited by Na+ depletion, D-glucose, glucose analogues, phloridzin, and cytochalasin B. In contrast, neither myo-inositol nor L-glucose inhibits L-fucose uptake. Chronic exposure of neuroblastoma cells to 1-30 mM L-fucose causes a decrease in myo-inositol accumulation and incorporation into inositol phospholipids, intracellular free myo-inositol content, and phosphatidylinositol levels. Na+,K(+)-ATPase transport activity is decreased by about 15% by acute or chronic exposure of neuroblastoma cells to L-fucose. Similar defects occur when neuroblastoma cells are exposed chronically to 30 mM glucose. Cell myo-inositol metabolism and Na+/K(+)-pump activity are maintained when 250 microM myo-inositol is added to the L-fucose-supplemented medium. Unlike the effect of chronic exposure of neuroblastoma cells to medium containing 30 mM glucose, the resting membrane potential of neuroblastoma cells is not altered by chronic exposure of the cells to 30 mM L-fucose. The effect of L-fucose on cultured neuroblastoma cell properties occurs at concentrations of L-fucose which may exist in the diabetic milieu. These data suggest that increased concentrations of L-fucose may have a role in myo-inositol-related defects in mammalian cells.
Two mutants of the BW5147 mouse lymphoma cell line have been selected for their resistance to the toxic effects of pea lectin. These cell lines, termed PLR1.3 and PHAR1.8 PLR7.2, have a decreased number of high affinity pea lectin-binding sites (Trowbridge, I.S., Hyman, R., Ferson, T., and Mazauskas, C. (1978) Eur. J. Immunol. 8, 716-723). Intact cell labeling experiments using [2-3H]mannose indicated that PLR1.3 cells have a block in the conversion of GDP-[3H]mannose to GDP-[3H]fucose whereas PHAR1.8 PLR7.2 cells appear to be blocked in the transfer of fucose from GDP-[3H]fucose to glycoprotein acceptors. In vitro experiments with extracts of PLR1.3 cells confirmed the failure to convert GDP-mannose to GDP-fucose and indicated that the defect is in GDP-mannose 4,6-dehydratase (EC 4.2.1.47), the first enzyme in the conversion of GDP-mannose to GDP-fucose. The block in the PLR1.3 cells could be bypassed by growing the cells in the presence of fucose, demonstrating that an alternate pathway for the production of GDP-fucose presumably via fucose 1-phosphate is functional in this line. PLR1.3 cells grown in 10 mM fucose showed normal high affinity pea lectin binding. PHRA1.8 PLR7.2 cells synthesize GDP-fucose and have normal or increased levels of GDP-fucose:glycoprotein fucosyltransferase when assayed in vitro. The fucosyltransferases of this clone can utilize its own glycoproteins as fucose acceptors in in vitro assays. These findings indicate that this cell line fails to carry out the fucosyltransferase reaction in vivo despite the fact that it possesses the appropriate nucleotide sugar, glycoprotein acceptors, and fucosyltransferase. The finding of decreased glycoprotein fucose in two independent isolates of pea lectin-resistant cell lines and the restoration of high affinity pea lectin binding to PLR1.3 cells following fucose feeding strongly implicates fucose as a major determinant of pea lectin binding.
L-fucose is a monosaccharide which is present in low concentrations in normal serum but is increased in diabetes, cancer, and inflammatory diseases. The contribution that abnormal L-fucose levels make to the progression of these disorders is unknown. In a previous study we showed that increased L-fucose concentration reduced proliferation and proteoglycan production by cultured cerebral microvessel endothelial cells. In the present study we show that exposing cerebral microvessel endothelial cells for 2 weeks to medium containing an increased concentration of L-fucose causes a significant decrease in collagen and to a lesser extent noncollagen protein production. The effect of L-fucose on collagen and noncollagen protein production is concentration-dependent: 1 mM L-fucose causes a significant decrease in collagen production but has no effect on noncollagen protein production; a 5 mM L-fucose concentration causes a maximum decrease in both collagen and noncollagen protein production. This defect is unrelated to the reduction in myo-inositol uptake caused by L-fucose and is not prevented by aminoguanidine. Collagen production can be improved by restoring L-fucose-conditioned cells to normal medium. Culturing cells for 2 weeks in medium containing 10 mM L-fucose resulted in a 50% decrease in collagen production, which was restored to 75% of control after cells were transferred to normal medium for 7 days. In contrast, noncollagen protein production was totally restored after 3 days in normal medium. Increasing levels of L-fucose in serum of rats also resulted in a decrease in collagen production. Collagenase digestible incorporation of L-[2,3,4,5-3H]proline into protein of the articular cartilage from rats fed a diet containing 20% L-fucose for 3 weeks was reduced by about 40% compared to rats fed a normal diet. The decrease in collagen production in L-fucose fed rats was less than the reduction that occurred in streptozotocin-induced diabetic rats. These data suggest that changes in L-fucose concentration itself may be a factor in the regulation of collagen production.
The effects of monosaccharides on various lymphocyte functions have provided useful probes for the study of cell-cell interactions. In this report, we show that a monosaccharide, alpha-L-fucose, significantly enhances the cytolytic capacity of MLC-induced or preincubated effector cells. The increase in activity was seen against cytotoxic T lymphocyte (CTL) targets (:relevant PHA blasts), natural killer cell (NK) targets (:K562), and natural cytotoxic cell (NC) targets (:MA-160). In addition, traditionally NK-insensitive targets (Raji cells, irrelevant and autologous PHA blasts) were lysed after preincubation of effector cells with fucose. Conversely, ADCC activity was not significantly increased with fucose induction. The addition of fucose directly to assay cultures did not enhance NK or CTL activity, whereas other sugars, such as alpha-methyl-D-mannoside and D-fructose, were inhibitory. The proportion of target-binding cells was not affected by preincubation with fucose, but the percentage of lytic conjugates was doubled. Significant augmentation of NK activity could be observed within 24 hr of incubation with alpha-L-fucose. Conversely, when fucose was added more than 24 hr after initiation of the culture, the increase in cytolytic activity was not observed. Parallel to the increase in cytolytic activity, after preincubation with alpha-L-fucose, an increase in the expression of a newly defined human NC cell marker, HNC-1A3, was observed. The HNC-1A3+ cells were not the major subpopulation responsible for fucose-induced activity, as ascertained by the use of positively sorted cells. The populations expressing antigens defined by the antibodies OKT8 and Leu-7 showed no quantitative change. The treatment of cells with OKM1 and complement (C) before culture eliminated fucose-enhanced killing, whereas similar treatment with OKT8 and C had no significant effect. The induction of fucose-activated killers (FAK) does not result in higher concentrations of interferon (IFN) in culture supernatants, in contrast to poly I:C, which induced both higher cytolytic activity and high titers of IFN. In addition, the induction of FAK was not sensitive to 100 ng/ml of cyclosporin A, suggesting that IL 2 did not play a major role in fucose activation of killing. These results provide strong evidence that alpha-L-fucose is capable of augmenting nonspecific activity by acting on OKM1+ precursors of cytotoxic cells and influencing a postbinding event.
Our previous studies have shown that the monosaccharide alpha-L-fucose significantly enhances the cytolytic capacity of peripheral blood mononuclear leukocytes (PBMLs). To examine possible mechanisms through which fucose affects cytolytic activity, we studied the production of cytokines after alpha-L-fucose stimulation. In this report, we show that fucose induced a minor but significant augmentation of production of interleukin-2 (IL-2), but anti-IL-2 antibodies did not completely inhibit fucose-activated cytolysis. Fucose induced significantly higher secretion of TNF-alpha by both lymphocytes and monocytes. The nature of the lytic molecule detected in the TNF bioassay was verified with specific neutralizing antibodies. In addition, fucose induced the accumulation of TNF-alpha mRNA in a time-dependent manner with a peak at 8 h and a return to baseline values at 20 h after stimulation. In vitro nuclear transcription assays determined that fucose augmented the rate of transcription of the TNF-alpha gene, and inhibition of de novo transcription with actinomycin D indicated that the turnover rate of the TNF-alpha mRNA was not affected by fucose stimulation. We also determined that fucose did not modulate the mRNA expression of the pore-forming protein, a major lytic protein involved in lymphocyte cytotoxicity. Specific neutralizing antibodies indicated that TNF-alpha was not an effector molecule in fucose-activated killing of K562 or Raji target cells but that this cytokine had an essential role in the induction of the augmented killing by alpha-L-fucose.
The levels of fucosylated glycoproteins in various cancers and inflammatory processes have been a subject of intense study. The level of fucosyltransferases and intracellular GDP-L-fucose, a sugar nucleotide and a common donor substrate for all fucosyltransferases, may regulate the level of fucosylated glycoproteins. This study reports on the determination of GDP-L-fucose levels in human hepatocellular carcinoma (HCC) and surrounding tissues, using a recently established high-throughput assay system. Levels of GDP-L-fucose in HCC tissues were significantly increased compared with adjacent nontumor tissues or normal livers. The mean +/- SD for GDP-L-fucose level was 3.6 +/- 0.2 micro mol/mg in control liver, 4.6 +/- 0.9 micro mol/mg in adjacent noninvolved liver tissues (chronic hepatitis, 4.4 +/- 0.7 micro mol/mg; liver cirrhosis, 4.8 +/- 0.9 micro mol/mg), and 7.1 +/- 2.5 micro mol/mg in HCC tissues. The level of GDP-L-fucose in HCC decreased in proportion with tumor size (r = -0.675, P = 0.0002). When expression of the series of genes responsible for GDP-L-fucose synthesis was investigated, the gene expression of FX was found to be increased in 70% (7 of 10) of the HCC tissues examined compared with that in their surrounding tissues. The levels of GDP-L-fucose were positively correlated with the expression of FX mRNA (r = 0.599, P = 0.0074). The levels of FX gene expression in some human hepatoma and hepatocyte cell lines were determined. FX mRNA production was strongly increased in HepG2 and Chang liver, moderately increased in Hep3B and HLF, and, in HLE, was similar to that of a normal human liver tissue. To investigate the effect of GDP-L-fucose on core fucosylation, FX cDNA was transfected into Hep3B cells, which express a relatively low level of GDP-L-fucose:N-acetyl-beta-D-glucosaminide alpha1-6 fucosyltransferase (alpha1-6 FucT) and FX mRNA. Transfection of this gene caused an increase in GDP-L-fucose levels as well as the extent of fucosylation on glycoproteins, including alpha-fetoprotein, as judged by reactivity to lectins. Collectively, the results herein suggest that the high level of fucosylation in HCC is dependent on a high expression of FX followed by increases in GDP-L-fucose, as well as an enhancement in alpha1-6 FucT expression. Thus, an elevation in GDP-L-fucose levels and the up-regulation of FX expression represent potential markers for HCC.
Since various experimental findings point towards an important role of cell surface carbohydrates - in particular sialic acid - in cancer metastasis, the rationale of this study was to look for possible differences in carbohydrate metabolism and glycoprotein expression in well-defined related tumor lines of different metastatic capacity. The tumor lines analyzed were L5178Y E (= Eb), a low-metastasizing, methylcholanthrene-induced lymphoma of a DBA/2 mouse, and L5178Y ES (= ESb), a spontaneous high-metastatic variant thereof. A non-related, highly metastasizing tumor, MDAY-D2, and ConA-stimulated spleen cells were included in the study. These cell lines were compared for incorporation rates of various labelled carbohydrates and for glycoprotein patterns in SDS-polyacrylamide gels. Marked differences were observed in the incorporation of 3H-fucose, while the incorporation of 3H-galactose and 3H-mannose was similar in the different cell lines studied. Only the metastatic variant ESb incorporated 3H-fucose at a rate similar to that of ConA-stimulated T-cell blasts. Eb cells did not incorporate 3H-fucose while MDAY-D2 had a significantly lower 3H-fucose incorporation rate. Separation and purification of the intracellular products of 3H-fucose by gel filtration and high-voltage electrophoresis revealed in Eb cells a block in the synthesis of fucosylated glycoproteins at the step of the fucose-I-P-guanylyltransferase. No apparent defect in the fucose pathway was detectable in MDAY-D2 cells. An Eb----ESb shifted cell line regained the ability to incorporate 3H-fucose. All tumors displayed unique glycoprotein patterns in SDS-PAGE. Labelling with 3H-mannose revealed the most distinct bands, while labelling with 3H-galactose gave fewer and broader bands. Although clonal instability of metastasizing tumor variants has been frequently reported, subclones of Eb and ESb showed characteristics similar to those of the original cell lines with regard to metastatic capacity, fucose metabolism and glycoprotein expression. These results will be discussed in relation to differences in fucose metabolism and in surface expression of fucose as observed in other tumor systems consisting of high- and low-metastatic lines.
Mutants of Klebsiella aerogenes W70 were isolated that had gained the ability to utilize the uncommon pentose D-arabinose as their sole source of carbon and energy. In contrast to the D-arabinose-negative, parent strain, these mutants were found to be either constitutive for certain enzymes of the L-fucose catabolic pathway or inducible for such enzymes when incubated in the presence of D-arabinose. The mutants used L-fucose isomerase to convert D-arabinose to D-ribulose, which is an intermediate and inducer of the ribitol catabolic pathway. The D-ribulokinase of the ribitol pathway was then induced. This enzyme catalyzed the phosphorylation of D-ribulose at the 5-carbon position. Mutants that were negative for D-ribulokinase could still dissimilate D-arabinose slowly by using all three enzymes, the isomerase, kinase, and aldolase, of the L-fucose pathway. Using condition negative mutants, we were able to demonstrate that the natural induction of the L-fucose pathway enzymes by L-fucose required the activity of a functional L-fucose isomerase and a functional L-fuculokinase but not an L-fuculose-1-phosphate aldolase. A metabolic intermediate, L-fuculose-1-phosphate, was thereby shown to be a probable inducer of at least the isomerase and kinase of the L-fucose catabolic pathway. Similar experiments, with D-arabinose-positive mutants, which were induced for the L-fucose pathway enzymes upon incubation with D-arabinose, revealed that the activities of the L-fucose isomerase and the L-fuculokinase were also required for the induction of the L-fucose enzymes. These D-arabinose-positive mutants apparently produced an altered regulatory protein that accepted both L-fuculose-1-phosphate and D-ribulose-1-phosphate as inducers. Examination of constitutive mutants revealed that L-fucose isomerase and L-fuculokinase were both synthesized constitutively, with the aldolase apparently under separate control.
L-Fucose is a monosaccharide normally present at low concentrations in serum and is the only levorotatory sugar utilized by mammalian systems. The metabolism of L-fucose is only partially understood. In this report, we characterize the uptake of L-fucose by four widely varying mammalian cell lines (murine neuroblastoma, bovine aortic endothelial, murine cerebral microvessel endothelial, and Madin-Darby canine kidney cells). Based on the criteria of saturability and specificity of L-fucose uptake, we conclude that L-fucose is accumulated via a specific recognition mechanism. Accumulation of L-fucose at 4 degrees C and in the presence of colchicine and cytochalasin D rules out receptor-mediated endocytosis as an uptake mechanism. Thus, the accumulation appears to be via a carrier system. Using a variety of criteria, we determined that L-fucose is not taken up by a glucose transporter system. Accumulation of L-[5,6-3H]fucose is Na(+)-independent and reduced by loading cells with L-fucose or depleting the cell of its phosphorylation capability, suggesting that the uptake of L-fucose is by passive facilitative diffusion. A significant amount of the L-fucose taken up by each of the four cell types was incorporated into protein and secreted into the medium.
In the salvage pathway of GDP-L-fucose, free cytosolic fucose is phosphorylated by L-fucokinase to form L-fucose-L-phosphate, which is then further converted to GDP-L-fucose in the reaction catalyzed by GDP-L-fucose pyrophosphorylase. We report here the cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. Murine L-fucokinase is expressed as two transcripts of 3057 and 3270 base pairs, encoding proteins of 1019 and 1090 amino acids with predicted molecular masses of 111 kDa and 120 kDa respectively. Only the longer splice variant of L-fucokinase was enzymatically active when expressed in COS-7 cells. Murine GDP-L-fucose pyrophosphorylase has an open reading frame of 1773 base pairs encoding a protein of 591 amino acids with a predicted molecular mass of 65.5 kDa. GDP-L-fucose, the reaction product of GDP-L-pyrophosphorylase, was identified by HPLC and MALDI-TOF MS analysis. The tissue distribution of murine L-fucokinase and GDP-L-fucose pyrophosphorylase was investigated by quantitative real time PCR, which revealed high expression of L-fucokinase and GDP-L-fucose pyrophosphorylase in various tissues. The wide expression of both enzymes can also be observed from the large amount of data collected from a number of expressed sequence tag libraries, which indicate that not only the de novo pathway alone, but also the salvage pathway, could have a significant role in the synthesis of GDP-L-fucose in the cytosol.