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PubMed1987

L-fucose, D-mannose, L-galactose, and their BSA conjugates stimulate macrophage migration.

The effect of selected monosaccharides on the random migration of normal adult rabbit alveolar macrophages (AM) was investigated. It was observed that 10 mM of L-fucose, L-galactose, or D-mannose stimulated AM migration 1.5-2.0 times. In addition, derivatives of L-fucose and D-mannose occupying the carbon-6 position such as L-fucosyl-lactose, D-mannose-6-phosphate, D-mannitol, and mannan enhanced the migration of AM, whereas derivatives of L-fucose and D-mannose in the carbon-1 position produced no migration enhancement. Macrophage migration enhancement activity that was produced spontaneously by spleen cell cultures from normal young rabbits was destroyed by treatment with L-fucosidase. Accordingly, the migration enhancement factor (MEF) found in spleen cell culture supernatants appeared to depend on L-fucose conjugated to some protein carrier because MEF was non-dialyzable. When normal adult AM were treated with L-fucosidase, they lost their responsiveness to migration inhibitory factor (MIF) but retained their responsiveness to MEF. We have interpreted this to mean that the MIF and MEF receptors are distinct. Synthetic MEFs were prepared by conjugating L-fucose, D-mannose, of L-galactose to bovine serum albumin (BSA). It was noted that these sugar-BSA conjugates were about 200 times more effective than the corresponding free sugars in producing migration enhancement. In addition, these sugar-BSA conjugates neutralized MIF activity in a migration inhibition test.

Animals↗
PubMed1993

Fucose expression in skeletal muscle: a lectin histochemical study.

Binding sites for three fucose specific lectins, Aleuria aurantia agglutinin (AAA), Lotus tetragonolobus agglutinin (LTA) and Ulex europeus I agglutinin (UEA I), were investigated in sections from normal human and rat muscles, in muscle from patients with Duchenne muscular dystrophy (DMD) and in denervated and devascularized rat muscle. In normal human and rat muscle AAA detected fucosylated glycocompounds in the sarcoplasm, sarcolemma, interfibre connective tissue and vascular structures. In normal human muscle addition of fucose to the AAA incubation medium or treatment of the sections with formaldehyde followed by periodic oxidation before lectin incubation strongly inhibited the staining at all sites other than endothelial cells. In normal rat muscle the same staining procedures strongly inhibited the AAA binding at all sites other than the sarcolemma. Incubation with LTA resulted in a diffuse reaction around the vascular structures in rat muscle, while in human muscle a moderate, homogeneous staining was present in all muscle fibres. Treatment of the sections with formaldehyde and periodic acid before incubation with LTA resulted in strongly labelled muscle capillaries in both human and rat muscle. The only elements in the muscle tissues that were stained with UEA I were human endothelial cells. In denervated and devascularized rat muscle incubation with AAA revealed a novel fucose expression that appeared intracellularly in some necrotic fibres. The AAA-positive fucose residues in the sarcolemma of normal muscle fibres that were resistant to periodic acid oxidation could not be shown by AAA in denervated muscle. In DMD muscle a cryptic sarcolemmal fucose expression could be shown with AAA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1985

Passive avoidance training increases fucose incorporation into glycoproteins in chick forebrain slices in vitro.

When day old chicks are trained to avoid pecking at a bright bead coated with methyl anthranilate, many neurochemical changes, both transient and longer lasting, have been found. These include an increased fucose incorporation in vivo into particulate glycoproteins, which persists for at least 24 hrs after training. We have now developed an in vitro method for studying fucose incorporation and have been able to replicate effects of training found in vivo. Chick forebrain slices incubated at 42 degrees in a glucose containing-medium incorporate L-[U14C]fucose linearly for up to 3 hrs at rates of 30-35 nmol/g prot/hr. Incorporation was only 60% inhibited by cycloheximide indicating that some fucosylation is occurring on pre-existing proteins. Fucose incorporation was compared in slices from trained and control chicks and, as in vivo, a 16% increase in incorporation into the right forebrain base of trained birds was found. This increase was confined to the microsomal fraction. When cycloheximide was added to the incubation medium, the enhanced fucose incorporation in slices from trained birds was still observed.

Animals↗
PubMed1995

Chemical structure of the carbohydrate moiety of fucose-rich glycopeptides from human pancreatic juice.

Human pancreatic juice, obtained from nine patients after partial excision of the pancreas for bile duct cancer, was fractionated in order to isolate its glycopeptides. Three glycopeptides were purified employing ion-exchange chromatography and gel filtration. All the glycopeptides were found to be free of sialic acid and galactosamine but to have an unusually high content of L-fucose. The chemical structures of the three glycopeptides were determined using 500-MHz [1H]-NMR spectroscopy. One of them, glycopeptide, GP-4, possessed a biantennary structure with three L-fucose residues. The second glycopeptide, GP-3, had a triantennary structure with four L-fucose residues, and the third one, G-2, had a tetra-antennary structure with five L-fucose residues. The chemical compositions of these glycopeptides, including the absence of sialic acid and the high L-fucose content, indicate that they represent a new class of glycopeptide present in the normal human pancreas.

Carbohydrate Sequence↗
PubMed2006

Search for fucose binding domains in recently sequenced hypothetical proteins using molecular modeling techniques and structural analysis.

The crystal structure of a fucose-binding lectin from the bacteria Pseudomonas aeruginosa in complex with alpha-L-fucose has been recently determined. It is a tetramer; each monomer displays a nine-stranded, antiparallel, beta-sandwiched arrangement and contains two calcium ions that mediate the binding of fucose in a recognition mode unique among protein-carbohydrate interactions. In search of this type of unique interactions in other newly discovered protein sequences, we have used molecular modeling techniques to predict and analyze the 3-D structures of some proteins, which exhibited reasonable degree of homology with the amino acid sequence of the bacterial protein. A BLAST search with the sequence of Pseudomonas aeruginosa as query in the non-redundant sequence database identified four proteins from different species, three organisms from bacteria and one from archaea. We have modeled the structures of these proteins as well as those of the complexes with carbohydrates and studied the nature of physicochemical forces involved in the complex formation both in presence and absence of calcium. The calcium-binding loops have been found to be highly conserved both in terms of primary and tertiary structures in these proteins, although a less acidic character is observed in Photorhabdus lectin due to the absence of two aspartic acid residues on the calcium-binding loop which also resulted in lower binding affinity. All these structures exhibited highly negative electrostatic environment in the vicinity of the calcium-binding loops which was essential for neutralizing the positive charges of two closely situated Ca(+2) ions. The comparison of the binding affinities of some monosaccharides other than fucose, e.g. mannose and fructose, showed higher binding energies confirming the fucose specificity of these proteins.

Amino Acid Sequence↗
PubMed1990

A spectrophotometric method for simultaneous determination of protein-bound hexoses and fucose with a mixture of L-cysteine and phenol.

A very suitable spectrophotometric method for simultaneous determination of protein-bound hexoses and fucose is presented. A mixture of L-cysteine and phenol in sulfuric acid was used as reagent, whereas absorption measurements were carried out at two wavelengths, namely at 398 nm for fucose and 490 nm for hexoses determination. Optimum conditions for the application of the method were established, special attention being paid to the possible interference of fucose determination with that of hexoses, and vice versa. The proposed method was applied to the determination of hexoses and fucose in serum glycoproteins and seromucoids. The method was found to be very practical enabling a simultaneous determination of both kinds of carbohydrates; moreover, it was proved to be more sensitive and specific in comparison with methods commonly used for individual determination of fucose with L-cysteine and hexoses with phenol.

Cysteine↗
PubMed1986

Two Chinese hamster ovary glycosylation mutants affected in the conversion of GDP-mannose to GDP-fucose.

A biochemical basis for the pea and lentil lectin resistance of two Chinese hamster ovary (CHO) cell mutants, Lec13 and Lec13A, was investigated. Studies of the G glycopeptides of vesicular stomatitis virus grown in the mutants indicated that Lec13 cells essentially lack the ability to add fucose to complex carbohydrates while Lec13A cells synthesize significant proportions of fucosylated, complex moieties. However, both mutants were known to be reverted to lectin sensitivity by growth in L-fucose, making them similar to the mouse lymphoma mutant, PLR1.3, which is defective in the conversion of GDP-mannose to GPD-fucose [M. L. Reitman, I. S. Trowbridge, and S. Kornfeld (1980) J. Biol. Chem. 255, 9900-9906]. Optimal conditions for the production of GDP-fucose from GDP-mannose by CHO cytosol were found to occur at pH 8 in the presence of 7.5 microM GDP-mannose, 15 mM Mg2+, 0.2 mM NAD+, 0.2 mM NADPH, 10 mM niacinamide, 5 mM ATP, and 50 mM Tris-HCl. Under these conditions, Lec13 cytosol produced no detectable GDP-fucose nor GDP-sugar intermediates while Lec13A cytosol produced significant quantities of both. Mixing experiments with Lec13 cytosol identified the first enzyme of the conversion pathway (GDP-mannose 4,6-dehydratase, EC 4.2.1.47) as the site of the block. In addition to being markedly reduced, the Lec13A 4,6-dehydratase activity was relatively insensitive to changes in pH in comparison to the activity in parental cytosol, suggesting that Lec13A cells might possess a structurally altered GDP-mannose 4,6-dehydratase enzyme.

Animals↗
PubMed1975

Calcium-carbohydrate bridges composed of uncharged sugars. Structure of a hydrated calcium bromide complex of alpha-fucose.

X-ray diffraction data were used to determine the crystal structure of a hydrated CaBr2 complex of alpha-fucose, a common terminal sugar of oligosaccharide chains on glycoproteins. Crystals of C6H12O5-CaBr2-3H2O are orthorhombic, space group P212121, with A equals 14.360(2), B equals 12.896(3), and C equals 8.043(1) A. Intensity data for 1442 independent reflections were measured with an automated diffractometer. A trial structure, obtained by the heavy-atom method, was refined by least-squares to R equals 0.052. Ca-2+ is chelated by a pair of hydroxyl groups from each of tow symmetry-related fucose molecules and is coordinated to three water molecules. Thus the structure consists of hydrated fucose-calcium-fucose bridges. The bridge geometry, which is dictated by the coordination requirements of Ca-2+, is like that of other calcium-carbohydrate complexes. Our results indicate that calcium-fucose interactions can provide an effective, sterospecific mechanism for cross-linking carbo hydrate chains. Similar calcium-carbohydrate bridges may be involved in a variety of Ca-2+-dependent agglutination and adhesion processes.

Binding Sites↗
PubMed1979

Affinity chromatography and separation of the molecular forms of monkey brain alpha-L-fucosidase on fucose-linked sepharose.

A simple affinity system which required coupling of alpha-L-fucose to Sepharose 4B by epichlorohydrin treatment of Sepharose 4B in the presence of alpha-L-fucose under alkaline conditions has been described. A partially purified preparation of monkey brain alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) was resolved at pH 5.0 into two major fractions: one bound and one retarded. The enzyme bound to the affinity column and specifically eluted by 2 mM alpha-L-fucose at pH 5.0 appeared to be homogeneous by polyacrylamide gel electrophoresis and was constituted mainly by the tetrameric form of the enzyme. The enzyme fraction retarded by the affinity column was found to contain mainly the monomeric form of the enzyme. Additional evidence for the different molecular forms of the enzyme in the bound and retarded fractions came from pH activity profiles and heat inactivation studies. The fucose-Sepharose appeared to bind the tetrameric form of the enzyme specifically and, further, alpha-L-fucose helped to retain the molecular integrity of the tetrameric enzyme.

Animals↗
PubMed1990

Carbohydrate composition and presence of a fucose-protein linkage in recombinant human pro-urokinase.

A new post-translational modification site in the growth factor domain of urinary type plasminogen activator has been identified. A glycopeptide containing the monosaccharide, fucose, covalently linked directly to the peptide backbone has been isolated from the tryptic digest of pro-urokinase expressed in a mouse hybridoma cell line Sp 2/0 Ag 14. The glycopeptide was isolated by semi-preparative reversed phase high performance liquid chromatography. The identity of a fucose containing peptide was confirmed by carbohydrate analysis, amino acid analysis and plasma desorption mass spectrometry (PDMS). A combination of these methodologies showed an equimolar ratio of peptide and fucose in the glycopeptide. This modification is not detected without mass spectrometry because the fucose residue is hydrolyzed under standard acidic conditions of amino acid composition and N-terminal sequence analysis. The site of attachment of fucose to the peptide has been localized towards the N-terminus (within first 23 amino acids) of the protein. Also, the carbohydrate composition of recombinant pro-urokinase is reported.

Amino Acid Sequence↗
PubMed1976

Time course and disposition of fucose radioactivity in rat hippocampus. A biochemical and microautoradiographic study.

Male adult rats were injected intraventricularly with L-[1-3H]fucose. At various intervals, ranging between 30 min and 11 days, one-half of the brain was prepared for microautoradiography, and the hippocampus from the other side was prepared for biochemical investigations. The TCA-precipitable proteins from the hippocampus homogenate were maximally labeled at between 8 and 24 h and remained at a high radioactivity level even 11 days after [3H]-fucose injection, the labeling being predominantly present in the solubilized insoluble proteins. Using gel electrophoretic separation, study of Tris-soluble material indicated a rapid turnover of soluble fucose-containing glycoproteins, whereas several slow-migrating bands of solubulized proteins revealed a time course suggesting the presence of fucose-containing glycoproteins with slower turnover rates. Using microautoradiography, a rapid labeling of neuronal cell bodies of the hippocampus was found, whereas the nuclei were not labeled. Perikarya were maximally labeled 4 h after [3H]fucose application. The radioactive material was continuously transported from the soma into the corresponding fiber layers, the latter being maximally labeled at a pulse interval of one day; even 10 days later a considerable amount of radioactivity could be detected in the neuropil.

Animals↗
PubMed1979

Axonal transport and metabolism of [3H]fucose- and [35S]-sulfate-labeled macromolecules in the rat visual system.

The axonal transport of labeled macromolecules in retinal ganglion cells of rats was investigated from 1 to 20 days following intraocular injection of [3H]fucose and [35S]sulfate. Maximal incorporation of [3H]fucose into acid insoluble material in the retina was at 8 h, followed by a biphasic decline. Transported [3H]fucose (98% as glycoprotein) was in the optic nerve at 1 h, the optic tract and lateral geniculate body by 2 h, and the superior colliculus by 3 h after injection, indicating a rate of transport of approximately 200 mm/day. Radioactivity continued to accumulate in the superior colliculus for at least 8 h and began to decline rapidly by 24 h. Between 3 and 6 days levels rose again in both optic tract and superior colliculus before starting a gradual decline, indicating that a wave of rapidly transported material was delayed in leaving the retina. When proteins in the superior colliculus were fractionated by gel electrophoresis, the composition of the two fucosylated protein transport phases could be partially resolved. Radioactivity in individual gel peaks represented primarily in the first phase decayed with an average half-life of one day, althouth that in one prominent protein of molecular weight 280,000 turned over with a half-life of the order of 12 h. Radioactive peaks primarily in the second phase decayed with an average half-life of more than a week. Incorporation of [35S]sulfate into acid insoluble material in the retina was maximal at 1-2 h, after which there was a rapid loss of label. The appearance of [35S]sulfate in the optic tract, lateral geniculate body and superior colliculus preceded by a short time that of the [3H]fucose; indicating a shorter retinal processing time for this label. The total transported [35S]sulfate in the superior colliculus peaked by 4-8 h and had fallen by 65% at one day; no prominent second wave of transport was observed as was the case for [3H]fucose. Acid insoluble [35S]sulfate in the superior colliculus was equally divided between glycopeptides and glycosaminoglycans at all times examined, indicating that these macromolecules are transported at the same rate. [35S]Sulfate incorporated into various proteins fractionated by gel electrophoresis had heterogeneous turnover rates, the average being around 12 h. Radioactivity in one group of proteins, of molecular weight around 90,000, decayed with a half-life of only a few hours.

Animals↗
PubMed1985

Specificity of macrophage-mediated cytotoxicity: role of target and effector cell fucose.

In this study, we have examined the role cell surface carbohydrates play in the activation of macrophages to kill tumor cells as well as the effect removal of tumor cell surface fucose has on the susceptibility of the treated target cells to the cytotoxic macrophages. After incubation of human monocyte-derived macrophages with alpha-L-fucosidase, a glycosidase which splits terminal alpha-L-fucose from oligosaccharides, the macrophages were no longer able to respond to lipopolysaccharide (LPS). These experiments strongly suggest that alpha-L-fucose probably comprises an essential part of the macrophage membrane receptor for LPS. In another series of experiments tumor cells were treated with alpha-L-fucosidase prior to co-cultivation with macrophages to determine whether the presence of alpha-L-fucose on the tumor cell surface had any effect on macrophage mediated cytotoxicity. It was found that the MA-160 sensitive tumor target, after alpha-L-fucosidase treatment, became resistant to the effects of the cyto-toxic macrophages whereas the effect of the alpha-L-fucosidase treatment was blocked by addition of alpha-L-fucose to the incubation mixture.

Cell Line↗
PubMed2006

Structural investigation of a fucoidan containing a fucose-free core from the brown seaweed, Hizikia fusiforme.

A fucoidan, obtained from the hot-water extract of the brown seaweed, Hizikia fusiforme, was separated into five fractions by DEAE Sepharose CL-6B and Sepharose CL-6B column chromatography. All five fractions contained predominantly fucose, mannose and galactose and also contained sulfate groups and uronic acid. The fucoidans had MWs from 25 to 950 kDa. The structure of fraction F32 was investigated by desulfation, carboxyl-group reduction, partial hydrolysis, methylation analysis and NMR spectroscopy. The results showed that the sugar composition of F32 was mainly fucose, galactose, mannose, xylose and glucuronic acid; sulfate was 21.8%, and the MW was 92.7 kDa. The core of F32 was mainly composed of alternating units of -->2)-alpha-D-Man(1--> and -->4)-beta-D-GlcA(1-->, with a minor portion of -->4)-beta-D-Gal(1--> units. The branch points were at C-3 of -->2)-Man-(1-->, C-2 of -->4)-Gal-(1--> and C-2 of -->6)-Gal-(1-->. About two-thirds of the fucose units were at the nonreducing ends, and the remainder were (1-->4)-, (1-->3)- and (1-->2)-linked. About two-thirds of xylose units were at the nonreducing ends, and the remainder were (1-->4)-linked. Most of the mannose units were (1-->2)-linked, and two-thirds of them had a branch at C-3. Galactose was mainly (1-->6)-linked. The absolute configurations of the sugar residues were alpha-D-Manp, alpha-L-Fucp, alpha-D-Xylp, beta-D-Galp and beta-D-GlcpA. Sulfate groups in F32 were at C-6 of -->2,3)-Man-(1-->, C-4 and C-6 of -->2)-Man-(1-->, C-3 of -->6)-Gal-(1-->, C-2, C-3 or C-4 of fucose, while some fucose had two sulfate groups. There were no sulfate groups in either the GlcA or xylose residues.

Carbohydrate Sequence↗
PubMed2004

Identification and molecular cloning of a functional GDP-fucose transporter in Drosophila melanogaster.

Nucleotide sugar transporters play a central role in the process of glycosylation. They are responsible for the translocation of nucleotide sugars from the cytosol, their site of synthesis, into the Golgi apparatus where the activated sugars serve as substrates for a variety of glycosyltransferases. We and others have recently identified and cloned the first GDP-fucose transporters of H. sapiens and C. elegans. Based on sequence similarity, we could identify a putative homolog in Drosophila melanogaster showing about 45% identity on protein level. The gene (CG9620) encodes a highly hydrophobic, multi-transmembrane spanning protein of 38.1 kDa that is localized in the Golgi apparatus. In order to test whether this protein serves as a GDP-fucose transporter, we performed complementation studies with fibroblasts from a patient with LADII (leukocyte adhesion deficiency II) which exhibit a strong reduction of fucosylation due to a point mutation in the human GDP-fucose transporter gene. We show that transient transfection of these cells with the Drosophila CG9620 cDNA corrects the GDP-fucose transport defect and reestablishes fucosylation. This study gives experimental proof that the product of the in silico identified Drosophila gene CG9620 serves as a functional GDP-fucose transporter.

Animals↗
PubMed1997

Structure of fucose branches in the glycosaminoglycan from the body wall of the sea cucumber Stichopus japonicus.

Fucose-branched chondroitin sulfate E was prepared from the body wall of sea cucumber Stichopus japonicus. The purified glycosaminoglycan (GAG) was chemically desulfated, followed by carboxyl reduction. Intact, desulfated, and desulfated/carboxyl-reduced GAG fractions were subjected to per-O-methylation. GC-MS analyses of the resultant partially methylated alditol acetates demonstrated that the fucose branch is formed by two fucopyranosyl residues linked glycosidically through position (1-->3), and that the fucose branch and glucuronic acid are almost equimolar. In addition, it was elucidated that about 20% of the branches stretch from O-3 position of a glucuronic acid moiety of the core chondroitin sulfate polymer, while remaining fucose branches are postulated to protrude from O-4 and/or O-6 position(s) of a N-acetylgalactosamine moiety. This fucose branch was also confirmed to be highly sulfated according to six kinds of substitution pattern in methylation analysis.

Acetylgalactosamine↗
PubMed2000

A fucose-containing epitope is shared by keyhole limpet haemocyanin and Schistosoma mansoni glycosphingolipids.

The glycolipids of Schistosoma mansoni adult worms, cercariae and eggs are recognised by schistosome infection serum and the monoclonal antibody M2D3H. The haemocyanin of the keyhole limpet, Megathura crenulata, is known to be immunoreactive to schistosomal infection sera and is, therefore, under investigation for the diagnosis of and vaccination against schistosomiasis. By dot-blot, inhibition-ELISA and inhibition-HPTLC immunostaining we have demonstrated that the M2D3H epitope is shared by both S. mansoni glycolipids and keyhole limpet haemocyanin (KLH). Analogously to the established epitopic importance of fucose to the immunorecognition of S. mansoni glycolipids, we have similarly defined the significance of the fucose residue(s) for the immunoreactivity between KLH and schistosomal infection serum and the monoclonal antibody M2D3H. Fucose was specifically removed from KLH by partial hydrolysis, monitored by ultrafiltration and carbohydrate component analysis. On removal of the fucose residue(s) the serological and immunological reactivity of KLH was greatly diminished, which implied that the fucose-containing M2D3H antigenic determinant was common to both S. mansoni glycolipids and KLH.

Animals↗
PubMed2000

Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose.

BACKGROUND: GDP-mannose 4,6 dehydratase (GMD) catalyzes the conversion of GDP-(D)-mannose to GDP-4-keto, 6-deoxy-(D)-mannose. This is the first and regulatory step in the de novo biosynthesis of GDP-(L)-fucose. Fucose forms part of a number of glycoconjugates, including the ABO blood groups and the selectin ligand sialyl Lewis X. Defects in GDP-fucose metabolism have been linked to leukocyte adhesion deficiency type II (LADII). RESULTS: The structure of the GDP-mannose 4,6 dehydratase apo enzyme has been determined and refined using data to 2.3 A resolution. GMD is a homodimeric protein with each monomer composed of two domains. The larger N-terminal domain binds the NADP(H) cofactor in a classical Rossmann fold and the C-terminal domain harbors the sugar-nucleotide binding site. We have determined the GMD dissociation constants for NADP, NADPH and GDP-mannose. Each GMD monomer binds one cofactor and one substrate molecule, suggesting that both subunits are catalytically competent. GDP-fucose acts as a competitive inhibitor, suggesting that it binds to the same site as GDP-mannose, providing a mechanism for the feedback inhibition of fucose biosynthesis. CONCLUSIONS: The X-ray structure of GMD reveals that it is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins. We have modeled the binding of NADP and GDP-mannose to the enzyme and mutated four of the active-site residues to determine their function. The combined modeling and mutagenesis data suggests that at position 133 threonine substitutes serine as part of the serine-tyrosine-lysine catalytic triad common to the SDR family and Glu 135 functions as an active-site base.

Amino Acid Sequence↗