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At least 127 records · Page 7Linked to original sources
PubMed1984

Monoclonal antibodies that block cell adhesion in Polysphondylium pallidum: reaction with L-fucose, a terminal sugar in cell-surface glycoproteins.

By permethylation analysis the linkages of L-fucose and D-mannose in the oligosaccharide residues of cell surface glycoproteins of Polysphondylium pallidum were determined. Mannose was found in terminal positions, in 1,2-, 1,3- and 1,6-intra-chain linkages, and at branch points. Fucose was exclusively located at nonreducing ends. Fab of a monoclonal antibody, mAb 293, has been previously shown to inhibit cell adhesion in P. pallidum completely. Binding of this antibody to glycoprotein was blocked by L-fucose, and at very high concentrations also by D-mannose. The dissociation constant for the antibody-fucose complex was Kd = 70 microM, which was two orders of magnitude higher than estimated for the natural oligosaccharide. Antibody-glycoprotein complexes dissociated in the presence of 100 mM L-fucose with a half-time of about 56 s. The blockage by L-fucose is taken as evidence that the adhesion-blocking antibody binds to oligosaccharide end groups containing L-fucose as the terminal sugar.

Adhesiveness↗
PubMed2002

Insights into leukocyte adhesion deficiency type 2 from a novel mutation in the GDP-fucose transporter gene.

Leukocyte adhesion deficiency type 2 (LADII) is characterized by defective selectin ligand formation, recurrent infection, and mental retardation. This rare syndrome has only been described in 2 kindreds of Middle Eastern descent who have differentially responded to exogenous fucose treatment. The molecular defect was recently ascribed to single and distinct missense mutations in a putative Golgi guanosine diphosphate (GDP)-fucose transporter. Here, we describe a patient of Brazilian origin with features of LADII. Sequencing of the GDP-fucose transporter revealed a novel single nucleotide deletion producing a shift in the open-reading frame and severe truncation of the polypeptide. Overexpression of the mutant protein in the patient's fibroblasts did not rescue fucosylation, suggesting that the deletion ablated the activity of the transporter. Administration of oral L-fucose to the patient produced molecular and clinical responses, as measured by the appearance of selectin ligands, normalization of neutrophil counts, and prevention of infectious recurrence. The lower neutrophil counts paralleled improved neutrophil interactions with activated endothelium in cremasteric venules of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. However, fucose supplementation induced autoimmune neutropenia and the appearance of H antigen on erythrocytes, albeit without evidence of intravascular hemolysis. The robust response to fucose despite a severely truncated transporter suggests alternative means to transport GDP-fucose into the Golgi complex.

Abnormalities, Multiple↗
PubMed1986

Subcellular localization of fucose incorporation into mouse thyrotropin and free alpha-subunits: studies employing subcellular fractionation and inhibitors of the intracellular translocation of proteins.

To determine the subcellular sites of fucose incorporation into TSH subunits, pituitaries from hypothyroid mice were incubated with [3H]fucose and fractionated by sucrose gradient centrifugation. To assess potential molecular cross-contamination between subcellular fractions enriched in rough endoplasmic reticulum (RER) or Golgi elements, trace amounts of exogenous [35S]methionine-labeled proteins or [125I]rat TSH were added before tissue homogenization. Particulate contamination of fractions was monitored by electron microscopy. TSH subunits were immunoprecipitated from fractions and analyzed by gel electrophoresis. After both a 2-h pulse incubation and a 2-h pulse, 3-h chase incubation, about half (range, 44-71%) of the [3H]fucose-labeled TSH subunit precursors present in microsomes were in the RER (amounts in excess of estimated contamination by nonspecific readsorption of molecules to vesicles or the presence of Golgi vesicles in the RER fractions); [3H]fucose-labeled free alpha-subunits were also detected in RER as well as in Golgi fractions. During chase incubations, both monensin and carboxyl cyanide m-chlorophenylhydrazone inhibited the appearance of [35S]methionine- or [3H]fucose-labeled TSH subunits in medium in a dose-dependent manner, suggesting that [3H] fucose was added to subunits, in part, early in the secretory pathway. Free alpha-subunits were more fucosylated than was TSH; in TSH heterodimers, beta-subunits were richer in fucose than were alpha-subunits. Thus, the fucosylation of TSH and free alpha-subunits in pituitaries of hypothyroid mice appears to begin at an unusually early stage of intracellular transport and may represent an adaptation to special posttranslational processing requirements.

Acetylglucosaminidase↗
PubMed1976

Clinical value of protein-bound fucose in patients with carcinoma and other diseases.

Protein-bound fucose content in sera from normal persons and patients with various malignant and non-malignant diseases was measured and statistically analyzed. Normal serum gave a mean value of 6.84 +/- 0.13 mg/100 ml, and rarely exceeded 9 mg/100 ml. Although no significant difference was found between sexes, there was a tendency of fucose content to decrease in older persons. It was noted that more than 90% of cancer-bearing patients have significantly higher level than critical value (9 mg/100 ml), while only 8.7% of patients with benign tumor showed positive result. These results were not limited to special organs but in common to all cases studied. The elevation of serum fucose content in malignant tumor was well correlated with its stages of progression, though the levels were less significant in early and in rather locally restricted breast and thyroid cancer. Serial postoperative follow-up study showed that the levels in serum fucose content was a useful parameter for judging the effectiveness of therapy and the prognosis of the patient. The fucose content in malignant tumor tissue and metastasized lymph node appeared to be significantly elevated than that in normal tissue. The practical usage and limitation of the fucose value in various diseases, together with a possible source of serum fucose were discussed.

Adenocarcinoma↗
PubMed1991

[A study of the determination of serum fucose concentrations].

We examined the biochemical properties of reaction products during the procedure for serum fucose determination (Dische & Shettles). The optical density at 396 nm (OD396) of the reaction products increased linearly with the increment of fucose concentrations, and was stable for at least 210-240 min at room temperature. The reaction products were destroyed with the addition of distilled water. Sephadex G-200 gel chromatography of the mixed serum samples revealed a single peak of fucose in the void volume fractions. Of interest was the presence of trace amounts of fucose detected in the low-molecular weight fractions less than 66,000, suggesting the presence of free fucose in the serum. Based on these observations, the method for serum fucose determination was modified so it was possible to use 100 microliters of the serum sample. The mean serum fucose concentration, measured by this modified method, was 561.0 +/- 191.1 mumol/l (9.1 +/- 3.1 mg/dl) in normal healthy children. In patients with idiopathic nephrotic syndrome, the values were significantly higher both at relapse and in remission than those in normal healthy children.

Chemical Fractionation↗
PubMed1987

The GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferases of LEC11 and LEC12 Chinese hamster ovary mutants exhibit novel specificities for glycolipid substrates.

Previous studies have shown that the GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferase (alpha (1,3) fucosyltransferase (Fuc-T)) activities expressed by the Chinese hamster ovary cell mutants LEC11 (Fuc-TI) and LEC12 (Fuc-TII) are different enzymes and indicated that Fuc-TI might act on sialylated lactosamine sequences (Campbell, C., and Stanley, P. (1984) J. Biol. Chem. 259, 11208-11214). In this paper we show that CSLEX-1, a monoclonal antibody specific for NeuNac alpha (2,3)Gal beta (1,4)(Fuc alpha (1,3))GlcNAc beta 1 sequences, bound to LEC11 cells but not to LEC12 cells. Direct evidence that Fuc-TI could act on sialylated substrates was sought with a series of glycolipid acceptors. Optimal assay conditions in crude cell extracts were determined with nLc4, a glycolipid which accepted fucose with both Fuc-TI and Fuc-TII to generate the Lex antigenic determinant. The two enzymes differed in their detergent sensitivities, pH optima, Mn2+ requirements, and apparent Km values for nLc4. When sialylated glycolipids were examined as substrates, Fuc-TI added fucose to IV3NeuNAcnLc4 but not to IV6NeuNAcnLc4, whereas Fuc-TII was unable to utilize either glycolipid as a substrate. Further studies showed that Fuc-TI and Fuc-TII possess novel specificities for glycolipids containing two lactosamine sequences as potential fucose acceptors. Fuc-TI exhibited good activities with VI3NeuNAcnLc6 and VI6NeuNAcnLc6 whereas Fuc-TII had very low activity with both substrates. Glycosidase digestions of the labeled products showed that Fuc-TI added fucose primarily to the internal N-acetylglucosamine of both glycolipids. The same preference for the internal N-acetylglucosamine was shown by Fuc-TI when nLc6 was the acceptor. In contrast, Fuc-TII preferred to transfer fucose to the external acceptor site of nLc6, consistent with the low activities of Fuc-TII with sialylated nLc6 derivatives. Thus the two enzymes preferentially add fucose to different N-acetylglucosamines in the same substrate, nLc6. This indicates that the biosynthetic pathway for fucosylation of polylactosamine sequences in glycolipids and glycoproteins will vary depending upon the particular alpha (1,3)fucosyltransferase present.

Animals↗
PubMedDate not supplied

Fucose concentrations in sera from patients with rheumatoid arthritis.

The clinical significance of L-fucose was investigated in serum of a well characterized cohort of 135 RA patients and 60 healthy controls. In RA patients, serum L-fucose was significantly decreased to a mean of 5.57 +/- 0.97 mg%, (range 2.2-7.5 mg%), compared with 7.5 +/- 1.04 mg% (range 5.7-9.8 mg%) (p < 0.001) in normal controls. In early RA serum L-fucose was significantly depressed to a mean of 6.08 mg%, a value which was significantly higher than the mean concentration of L-fucose in patients with advanced RA. Serum L-fucose was significantly correlated with the rheumatoid disease activity, duration, number of involved joints, and bone erosions. No statistically significant difference was found between seropositive and seronegative RA patients. However, a significant difference (p < 0.001) between female (5.57 mg%) and male (6.59 mg%) patients with advanced RA, and between male (6.08 mg%) and female (5.99 mg%) patients with early RA was found. These findings suggest that serum L-fucose is depressed in patients with rheumatoid arthritis, a promising observation since L-fucose is a safe and simple natural sugar. It may be used as an additional parameter and as an indicator for the disease activity in the follow up of patients with rheumatoid arthritis.

Adolescent↗
PubMed1993

Studies on the origin of the glycoproteins of the rabbit vitreous body using a protein-synthesis inhibitor and radioactive fucose and amino acids.

Rabbits were injected intravitreally with [3H]-fucose and were killed at 0.5, 4, 24, and 40 h postinjection. The radiometric measurements and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus fluorography performed on the vitreous bodies of these rabbits were compared with those carried out on excised vitreous bodies of noninjected rabbits incubated with [3H]-fucose for the same periods. The results of these in vitro experiments demonstrated that at all test intervals there were only two lightly labeled bands in the fluorograms, and this was interpreted as nonenzymatic binding of [3H]-fucose to proteins (glycation), occurring in the extracellular environment. In contrast, intravitreal injections resulted in about 14 conspicuously labeled bands, mainly after the longer time intervals, and the nature of these bands was revealed by additional experiments. In another series of experiments, rabbits were injected intravitreally with either [3H]-fucose or [3H]-amino acids and their vitreous bodies were processed as described above. The specific activity (counts per minute per microgram of protein) of the glycoproteins labeled with [3H]-fucose was always higher than that of the proteins labeled with any of the amino acids. In fluorograms, eight bands unequivocally labeled with the [3H]-amino acids were detected, and corresponding bands almost invariably appeared in the [3H]-fucose lane. Cycloheximide, a protein-synthesis inhibitor, blocked almost completely the incorporation of both [3H]-proline and [3H]-fucose into the vitreous proteins as revealed by the radiometric measurements and fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1977

Clinical significance of fucose level in glycoprotein fraction of serum in patients with malignant tumors.

Serum fucose content in the glycoprotein fraction was determined in various patients with malignant and benign diseases. The results showed that, in contrast to benign diseases, malignant diseases were characterized by an increased fucose content in the glycoprotein fraction. However, no significant difference was noted in the fucose levels in the mucoprotein fraction. The increased fucose level in glycoprotein in malignant diseases was parallel to the increment in total fucose content in serum, which suggests that the increased levels in total fucose in malignant diseases, reported previously, are primarily due to the increase in fucose-containing glycoprotein.

Female↗
PubMed2006

Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC.

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.

Animals↗
PubMed2005

Synthesis and evaluation of 5-thio-L-fucose-containing oligosaccharide.

5-Thio-L-fucose-containing trisaccharide H-type II was synthesized. The 3',4'-O-isopropylidene-2-azido-2-deoxylactoside derivative, which was prepared from lactose by azidonitration of lactal, was used as a starting material. By regio- and stereoselective 5-thio-L-fucosylation of the 6,6'-dibenzoate 5 with 5-thiofucosyl trichloroacetimidate 6 and subsequent deprotection gave the 5-thio-L-fucose-containing H-type II 1. Conformational analysis of the 5-thio-L-fucose-containing H-type II and the native H-type II was carried out through NOESY experiments. The observed NOE values between N-acetylglucosamine and galactose, and galactose and fucose were same for these two trisaccharides. However, NOE values between fucose and N-acetylglucosamine were significantly different. Binding of the 5-thio-L-fucose-containing H-type II to lectins and antibodies were in some case stronger and in some case weaker than those of the native trisaccharide.

Carbohydrate Sequence↗
PubMed1989

Colorimetric assay for free and bound L-fucose.

A novel, rapid, and reliable colorimetric method for measuring L-fucose has been developed. This method utilizes NADH formed from the interaction of L-fucose with fucose dehydrogenase and NAD to generate color in a reaction involving CuSO4 and neocuproine. NADH reduces Cu2+ to Cu1+ and the latter interacts with neocuproine to yield a complex with a maximal absorption at 455 nm. The reaction of NADH with copper-neocuproine is immediate and under the conditions of the assay the color formed remains stable for at least 2 h. When the assay is used to determine levels of L-fucose, the absorbance is found to be linearly proportional to exogenously added fucose concentrations from 16 to 179 nmol with resulting molar extinction coefficient of 13,660. Using this procedure, L-fucose released by acid hydrolysis from porcine submaxillary mucin, and by alpha-L-fucosidase from p-nitrophenyl-alpha-L-fucopyranoside, was quantitated.

Animals↗
PubMed1989

L-fucose blocks MIF/MAF priming of rabbit alveolar macrophages for a PMA-induced oxidative response.

The effect of L-fucose on the priming of AM from normal adult rabbits and their subsequent chemiluminescent (CL) responses to phorbol myristate acetate (PMA) was investigated. It was observed that 12.5 mM L-fucose, but not D-fucose, blocked the "spontaneous" priming of normal AM during an 18-hr incubation period in serum-free RPMI 1640 medium by about 40% (P less than 0.01) as detected by their CL responses to a PMA challenge. In addition, the optimal concentration of L-fucose (12.5 mM) blocked MIF/MAF priming during the 3- or 18-hr incubation period by 71 or 93% (P less than 0.05), respectively, as determined by their CL responses following PMA challenge. It is of particular significance that D-fucose was inactive. These results, together with previously published data, indicate that L-fucose (a) blocks priming of AM for an oxidative response, (b) stimulates random migration of AM, and (c) reverses migration inhibition produced by migration inhibition factor.

Animals↗
PubMed2005

Fucose removal from complex-type oligosaccharide enhances the antibody-dependent cellular cytotoxicity of single-gene-encoded antibody comprising a single-chain antibody linked the antibody constant region.

Fucose removal from complex-type oligosaccharide of human IgG1-type antibody results in a great enhancement of antibody-dependent cellular cytotoxicity (ADCC). The aim of this study was to clarify the effect of fucose removal on effector functions of a single-gene-encoded antibody with an scFv used as the binding domain. We generated both a fucose-negative anti-tumor associated glycoprotein (TAG)-72 scFv-Fc using alpha-1,6-fucosyltransferase knock-out CHO cells and a highly fucosylated scFv-Fc from parental CHO cells. Expression, assembly and antigen binding activity of the scFv-Fcs were not influenced by fucose removal. The scFv-Fc lacking fucose exhibited significantly more potent FcgammaRIIIa binding and ADCC compared to highly fucosylated scFv-Fc. These results prove that ADCC enhancement by fucose-removal is effective in not only whole IgG1, but also scFv-Fc, and thus increases the potential of Fc-fusion proteins as therapeutic candidates.

Animals↗
PubMedDate not supplied

Pharmacological properties of fucose. Applications in age-related modifications of connective tissues.

Fucose is the only component of glycoconjugates of vertebrates in the L-configuration. It exhibits a number of unique and interesting biological properties reviewed briefly in this article. Its constant end-standing position on glycan chains predisposes fucose to play a key role in cell-cell and cell-matrix interactions, mediated by several receptors such as those recognising the Lewis-type blood group substances, fucose-recognising lectines and the mannose-fucose receptors. Some of the as yet unstudied or less well understood properties of L-fucose were explored in the present study, as its non-enzymatic interaction with amine-groups on macromolecules, its cellular uptake attributed to specific transport mechanisms and its effect on fibroblast cell cultures. We could document the stimulation of cell-proliferation and the inhibition of MMP-expression and activation, both for MMP-2 and MMP-9. These and the other shortly reviewed properties of L-fucose may play an important role in its biological applications and actions.

Aging↗
PubMed1980

Halogenated L-fucose and D-galactose analogues: synthesis and metabolic effects.

Several new analogues of L-fucose modified in the 2 position and the 5-methyl group have been synthesized as potential plasma-membrane glycoconjugate inhibitors or modifiers, and their biological effects have been studied. 2-Chloro-, 2-bromo-, and 2-iodo-2-deoxy-L-fucose (9a, 9b, and 13, respectively) have been prepared by addition of the appropriate halogen to 3,4-di-O-acetyl-L-fucal, followed by hydrolysis of the anomeric halogen and the acetyl groups. A series of four halogenated 5-methyl analogues of L-fucose (4, X = F, Cl, Br, and I) have been obtained starting from 1,2:3,4-di-O-isopropylidene-L-galactose. The synthesis of this latter compound has been improved. A corresponding series of 6-deoxy-6-halo-D-galactose analogues, which are enantiomers of the 5-(halomethyl)-L-fucose analogues, has also been synthesized. Analogues 4b, 4c, and 9b at 1 x 10(-3) M specifically inhibited the incorporation of L-[3H]fucose into macromolecular components of SW613 human mammary tumor cells. Analogue 13 inhibited the growth of L1210 murine leukemic cells with an IC50 of 6 x 10(5) M in culture. 6-Deoxy-6-fluoro-D-galactose and its enantiomer 4a were found to be effective inhibitors of D-[3H]galactose and L-[3H]fucose incorporation, respectively, into macromolecular components of human mammary tumor cells. The effectiveness of inhibition was reduced with an increase in size of the halogen atom. Analogue 4a and its enantiomer have been tritiated at C-1 and both were found to be activated to a nucleotide sugar, which was followed by incorporation into the macromolecular fraction of SW613 human mammary tumor cells in vitro.

Animals↗
PubMed1998

Studies on selectin blockers. 6. Discovery of homologous fucose sugar unit necessary for E-selectin binding.

We describe a mimic of the sugar unit of the E-selectin ligand, sialyl Lewis X (sLeX). Carbohydrates are entering the realm of rational drug design, aided by the growing understanding of the structure-function relationships. We investigated a new methodology of preparing sLeX mimetics and developed a potent E-selectin blocker characterized by beta-turn dipeptides. Another characteristic point of this E-selectin blocker is that the six-membered fucose ring was replaced with a five-membered fucose ring. Interestingly, it was found that the five-membered fucose ring could also bind to a calcium ion on the E-selectin, which could be an important role of the six-membered fucose ring. Especially, the L-Ser-D-Glu and D-Ser-L-Glu derivatives 3a,b showed 65-90-fold more potent inhibitory activities than the sulfated LeX analogue 1. In addition, molecular dynamics (MD) studies indicated that the 2- and 3-OH groups of the six-membered fucose ring, which were necessary for the calcium binding, overlapped well with the 2- and 3-OH groups of the five-membered fucose ring. These new findings could be useful for the design of new types of selectin blockers.

Carbohydrate Sequence↗
PubMed1979

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of L-[1,5,6-3H]fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo.

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-(3)H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.

Animals↗