Incorporation Of L-fucose Research Articles

Human Milk Oligosaccharides And Site-Specific Glycosylation Heterogeneity Of Filipino Human Milk Glycoproteins Exhibit Dynamic Variations During Lactation As Influenced By Secretor Status And Coastal Environmental Condition – A Preliminary Study

Human Milk Oligosaccharides (HMOs) have a role in the immune system development of infants. HMOs and the N-glycans of human milk glycoproteins, especially highly fucosylated ones, function as prebiotics to the bifidogenic gut microbiota. This study reports the first longitudinal profile of HMOs and human milk glycoproteins from coastal, noncoastal, secretor and nonsecretor Filipino mothers analyzed and annotated using QTOF LC/MS and QqQ LC/MS - MRM, respectively. A milk glycan profile from coastal mothers revealed higher (P<0.05) presence of fucosylated branched HMOs, such as DFLNHa, LDFT, LNDFH I, and LNFP II. Moreover, secretor mothers expressed the highest 2’FL from colostrum (Day 5) until the end of the study (Month 6). Interestingly, the predominance of secretory IgA (64%) on the total milk glycoproteins from Filipino mothers supported their biological adaptation in conferring immune protection to their breastfeeding infant. Glycoform variations on milk glycoproteins, sIgA, IgG, Lactoferrin, and a1-Antitrypsin, in coastal mothers were observed. Moreover, lactoferrin was found to be highly glycosylated with 20 fucosylated and sialylated glycoforms. These results suggest that the coastal mother’s unique long-term seafood diet may have influenced their milk glycan profile and the potential incorporation of L-fucose derived from seafood in the biosynthesis of their milk glycans.

Relationship Between Increased Fucosylation and Metastatic Potential in Colorectal Cancer.

Fucose is utilized for the modification of different molecules involved in blood group determination, immunological reactions, and signal transduction pathways. We have recently reported that enhanced activity of the fucosyltransferase 3 and/or 6 promoted TGF-ß-mediated epithelial mesenchymal transition and was associated with increased metastatic potential of colorectal cancer (CRC), suggesting that fucose is required by CRC cells. With this in mind, we examined requirement of L-fucose in CRC cells and developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specific to CRC. In this study, we first examined the expression of fucosylated proteins in 50 cases of CRC by immunochistochemical staining with biotinylated Aleuria aurantia lectin (AAL). Then we carried out an L-fucose uptake assay using three CRC cell lines. Finally, we developed fucose-bound nanoparticles as vehicles for the delivery of an anticancer drug, SN38, and examined tumor growth inhibition in mouse xenograft model (n = 6 mice per group). All statistical tests were two-sided. We found a statistically significant relationship between vascular invasion, clinical stage, and intensity score of AAL staining (P ≤ .02). L-fucose uptake assay revealed that L-fucose incorporation, as well as fucosylated protein release, was high in cells rich in fucosylated proteins. L-fucose-bound liposomes effectively delivered Cy5.5 into CRC cells. The excess of L-fucose decreased the efficiency of Cy5.5 uptake through L-fucose-bound liposomes, suggesting an L-fucose receptor dependency. Intravenously injected, L-fucose-bound liposomes carrying SN38 were successfully delivered to CRC cells, mediating efficient tumor growth inhibition (relative tumor growth ratio: no treatment group [NT], 8.29 ± 3.09; SN38-treated group [SN38], 3.53 ± 1.47; liposome-carrying, SN38-treated group [F0], 3.1 ± 1.39; L-fucose-bound, liposome-carrying, SN38-treated group [F50], 0.94 ± 0.89; F50 vs NT, P = .003; F50 vs SN38, P = .02, F50 vs F0, P = .04), as well as prolonging survival of mouse xenograft models (log-rank test, P < .001). Thus, fucose-bound liposomes carrying anticancer drugs provide a new strategy for the treatment of CRC patients.

Relationship Between Increased Fucosylation and Metastatic Potential in Colorectal Cancer.

Fucose is utilized for the modification of different molecules involved in blood group determination, immunological reactions, and signal transduction pathways. We have recently reported that enhanced activity of the fucosyltransferase 3 and/or 6 promoted TGF-ß-mediated epithelial mesenchymal transition and was associated with increased metastatic potential of colorectal cancer (CRC), suggesting that fucose is required by CRC cells. With this in mind, we examined requirement of L-fucose in CRC cells and developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specific to CRC. In this study, we first examined the expression of fucosylated proteins in 50 cases of CRC by immunochistochemical staining with biotinylated Aleuria aurantia lectin (AAL). Then we carried out an L-fucose uptake assay using three CRC cell lines. Finally, we developed fucose-bound nanoparticles as vehicles for the delivery of an anticancer drug, SN38, and examined tumor growth inhibition in mouse xenograft model (n = 6 mice per group). All statistical tests were two-sided. We found a statistically significant relationship between vascular invasion, clinical stage, and intensity score of AAL staining (P≤ .02). L-fucose uptake assay revealed that L-fucose incorporation, as well as fucosylated protein release, was high in cells rich in fucosylated proteins. L-fucose-bound liposomes effectively delivered Cy5.5 into CRC cells. The excess of L-fucose decreased the efficiency of Cy5.5 uptake through L-fucose-bound liposomes, suggesting an L-fucose receptor dependency. Intravenously injected, L-fucose-bound liposomes carrying SN38 were successfully delivered to CRC cells, mediating efficient tumor growth inhibition (relative tumor growth ratio: no treatment group [NT], 8.29 ± 3.09; SN38-treated group [SN38], 3.53 ± 1.47; liposome-carrying, SN38-treated group [F0], 3.1 ± 1.39; L-fucose-bound, liposome-carrying, SN38-treated group [F50], 0.94 ± 0.89; F50 vs NT,P= .003; F50 vs SN38,P= .02, F50 vs F0,P= .04), as well as prolonging survival of mouse xenograft models (log-rank test,P< .001). Thus, fucose-bound liposomes carrying anticancer drugs provide a new strategy for the treatment of CRC patients.

Inhibition of L-Fucokinase from Rat Liver by L-Fucose AnaloguesIn Vitro

By investigating the effects of more than 15 different L-fucose analogues on the activity of L-fucokinase (EC 2.7.1.52) from rat liver in vitro, certain structural requirements for potent inhibition of this enzyme were established. Of the novel compounds, 4,6-dideoxy-L-xylo-hexopyranose (4) and methyl 4,6-dideoxy-4-iodo-L-glucopyranose (9) were found to be competitive inhibitors with Ki-values of 0.5 mM and 5.0 mM respectively. Thus 4,6-dideoxy-L-xylo-hexopyranose is a better inhibitor of L-fucokinase than methyl-alpha-L-fucoside (1). Uptake of L-fucose into rat hepatoma cells is reduced by 52% in the presence of the deoxy derivative (4), leading to a decrease of 45% in the incorporation of L-fucose into total cellular glycoproteins.

Incorporation of the hexose analogue 2-deoxy- d-galactose into membrane glycoproteins in HepG2 cells

The incorporation of 2-deoxy- d-galactose into the oligosaccharide moieties of glycoproteins and the consequences of 2-deoxy- d-galactose treatment on the fucosylation of glycoproteins were investigated in the human hepatoma cell line HepG2. Using different methods, it was shown that treatment of HepG2 cells with 2-deoxy- d-galactose leads to an incorporation of 2-deoxy- d-galactose and a decrease of l-fucose incorporation into the oligosaccharides of glycoproteins. The extent of labeling by l-[ 3H]fucose was determined by removing l-[ 3H]fucose from labeled cells with the aid of a purified α1,2-fucosidase from Aspergillus niger. Using this method, it was shown that 2-deoxy- d-galactose markedly inhibits α1,2-fucosylation. Measurement of the amount of 2-deoxy- d-galactose incorporated, however, showed that replacement of d-galactose by 2-deoxy- d-galactose does not entirely account for the decrease in α1,2-fucosylation. In addition, a hitherto unreported compensatory increase of α1,3/α1,4-fucosylation was found to occur when α-1,2-fucosylation was inhibited by treatment with 2-deoxy- d-galactose.

Apomorphine and glycoprotein synthesis during consolidation

The dopamine agonist, apomorphine, was injected intrahippocampally immediately after acquisition of a brightness discrimination task, which was motivated by footshock in rats. This led to an increase in the incorporation of L-fucose into total proteins which were measured in the hippocampus 7–9 hours later. In behavioral experiments, the same application improved the retention of a learned task. A possible linkage between increased glycoprotein synthesis and improvement of the retention of a new learned behavior due to the action of apomorphine is discussed.

Dopamine induced changes in L-fucose incorporation into proteins of rat hippocampus and corpus striatum during postnatal development

In 60 day old (adult) male Wistar rats dopamine caused a dose-dependent increase of L-fucose incorporation into total proteins of both hippocampus and corpus striatum slices up to +47.8±6.0% (n=6) and +53.2±8.5% (n=20), respectively, when compared to corresponding controls. Under these conditions the dopamine concentration leading to a maximum stimulation of fucose incorporation was 5×10 −4 M in hippocampus and 1×10 −3 M in corpus striatum. In the latter tissue the range of dopamine concentrations causing a significant elevation in incorporation rates was larger than in hippocampal tissue. In the corpus striatum of 9 day old rats dopamine was ineffective, but by 30 days the transmitter stimulated fucose incorporation rate reached the maximum observed for any age studied. This developmental pattern seems to be related to the ontogenesis of dopamine receptor sites or dopamine sensitive adenylate cyclase formation in this brain structure. In the hippocampus the postnatal development of dopamine induced augmentation of glycoprotein synthesis showed a longer latency, but the maximum effect was also seen in 30 day old animals. These results support our assumption that at the end of the postnatal differentiation period the glycoprotein synthesis in brain tissue may be controlled (at least to some extent) by the state of dopaminergic receptors and/or of dopamine sensitive adenylate cyclase.

2-Deoxy-D-galactose impairs the fucosylation of glycoproteins of rat liver and Morris hepatoma.

The consequences of injected 2‐deoxy‐d‐galactose on the biosynthesis of glycoproteins have been studied in rat liver and Morris hepatoma 9121. The incorporation of 2‐deoxy‐d‐galactose into glycoprotein (a), the Subsequent glycosylation with l‐fucose and N‐acetylneuraminic acid (b), and the impairment of the rat uracil nucleotide levels (c) have been determined.a) The rates of uptake and incorporation of 2‐deoxy‐d‐galactose into glycoprotein were evaluated in various tissues of the rat. When compared to d‐galactose they were found to be decreased in most tissues, especially in liver and hepatoma. After labeling with 2‐deoxy‐d‐galactose in vivo the identification of the label in glycoprotein fractions mainly yielded 2‐deoxy‐d‐galactose and no 2‐deoxy‐d‐glucose. Autoradiographic analysis of plasma membranes and serum, using polyacrylamide gel electrophoresis for separation, indicates the presence of the same bands with either 2‐deoxy‐d‐galactose or with d‐galactose as the label.b) 1 h after a single dose of 200 mg 2‐deoxy‐d‐galactose/kg body weight a 25–50% decrease of the subsequent l‐fucose incorporation into the acid‐precipitable fraction of serum, liver and hepatoma was observed compared to untreated controls. Time‐courses and dose‐dependences of the inhibitory effect were obtained in these tissues including the hepatoma plasma membrane fraction. A simultaneous increase in the acid‐soluble l‐fucose concentration was found in both liver and serum. The specificity of this effect was shown by an unchanged incorporation of N‐acetyl‐d‐mannosamine as the precursor of N‐acetylneuraminic acid. Protein synthesis, as measured by l‐leucine incorporation, was also not impaired after 2‐deoxy‐d‐galactose treatment. Injection of 2‐dcoxy‐d‐galactose as followed by labeling with l‐[14C] fucose in vivo and autoradiographic analysis of plasma membrane, cytosol and serum lead to shifts of bands. In contrast, only minimal changes were seen in hepatoma tissue.c) Uracil 5′‐nucleotide concentrations were determined in livers and tumors of ACI rats bearing the Morris hepatoma 9121. 1 h after the injection of 200 mg 2‐deoxy‐d‐galactose/kg body weight the levels of UDP‐glucose and UDP‐galactose in host liver were depressed by 50%. However, no fall was found in hepatoma tissue. Supplementation experiments with uridine showed that the decreased concentrations of uracil nucleotides are not responsible for the reduced l‐fucose incorporation into glycoprotein.From the data presented it can be concluded that the incorporation of 2‐deoxy‐d‐galactose specifically modifies the of oligosaccharide moiety of glycoprotein in vivo. By blocking the C‐2 for the attachment of l‐fucose this d‐galactose analog may serve as an experimental tool for studying the significance of terminal l‐fucose in secreted and membrane‐bound glycoproteins.

The influence of dopamine on the incorporation of different sugars into total proteins of hippocampal slices

Dopamine increases the incorporation of L-fucose and of D-mannose to a similar significant degree, whereas the incorporation of D-galactose as well as of N-acetyl-D-glucosamin into the total proteins of hippocampal slices was only slightly enhanced. The incorporation of N-acetyl-neuraminic acid was not influenced by dopamine. The results suggest that the effect of dopamine on glycoprotein formation seems mainly to depend on the kind of nucleotides necessary for activation of sugars and not on the sugar's final position in the glycan chain.

Enzymic synthesis of a new type of fucose-containing glycolipid with fucosyltransferase of rat ascites hepatoma cell, ah 7974f

An α-fucosyltransferase activity has been demonstrated in rat ascites hepatoma AH 7974F cells catalyzing the transfer of l-fucose to asialo-GM 1 prepared from bovine brain GM 1 ganglioside to form a fucolipid in the presence of Triton X-100. The radioactive fucolipid was shown to be Fuc-(α 1→ 2)-Gal-(β1 → 3)-GalNAc-(β1→ 4)-Gal-(β1 → 4)-Glc-ceramide from the following results. The radioactive product coincided with authentic blood group H-active fucolipid from AH 7974F cell on thin-layer chromatography. The product formed a precipitation line not only with Ulex europeus lectin but also with eel anti-H serum on agarose gel plates. The terminal 14C-labeled fucose was released by Bacillus fulminans α (1→2)fucosidase as well as Charonia lampas α-fucosidase. The optimum pH value for the incorporation of l-fucose into asialo-GM 1 was 5.8 in sol|cacodylate/HCl buffer. The fucosyltransferase was highly specific for asialo-GM 1.

Biosynthesis of plasma-membrane proteins during myogenesis of skeletal muscle in vitro.

1. Surface labelling of plasma-membrane proteins with 125I, catalysed by lactoperoxidase, and radioactive l-fucose incorporation into glycoprotein were used as plasma-membrane markers for skeletal-muscle cells in culture. 2. Plasma membranes were prepared at various stages of myogenesis in vitro and rates of synthesis and accumulation of proteins in the membranes were compared. 3. Increased synthesis and accumulation of a protein of apparent mol.wt. 70000 occurred in the plasma-membrane fraction concomitant with the onset of myoblast fusion. 4. In cultures in which fusion of myoblasts was inhibited by 5'-bromo-2-deoxyuridine, synthesis and accumulation of the protein of apparent mol.wt. 70000 was selectively inhibited. 5. It is suggested the protein of apparent mol.wt. 70000 may be involved in the process of myoblast fusion.

A Comparative Analysis of the Protein Components of Plasma Membranes Isolated from Differentiated and Undifferentiated Mouse Neuroblastoma Cells in Tissue Culture

The plasma membranes of the cells of mouse neuroblastoma clone NB41A, were isolated without fixation by hardening procedures and were characterised by their enzyme activities and by their morphology in the light and electron microscopes. The membranes were prepared from two kinds of differentiated monolayer cultures; one in which the cells were induced to form long axon-like processes by the addition of N6,O2'-dibutyryladenosine 3':5'-monophosphate to the culture medium, and the other in which the cells were induced to form processes by the addition of 5-bromo-2'-deoxyuridine. The proteins from the solubilised membranes were compared with similar preparation from the membranes of undifferentiated cells, grown in suspension, by sodium dodecylsulphate polyacrylamide gel electrophoresis, using the incorporation of radioactive amino acids and L-fucose into proteins in the cultures to follow the differences in the patterns of polypeptide synthesis and glycosylation of the membrane proteins. The differentiated cells induced with either inducer show an increased incorporation of L-fucose into two distinct components with molecular weights of 60 000 and 70 000. The two types of induced cells differ from each other in that N6,O2'-dibutyryladenosine 3':5'-monophosphate stimulates both glycosylation and protein synthesis, with a relative increase in the low molecular weight proteins, but 5-bromo-2'-deoxyuridine stimulates mostly increased glycosylation of the membrane proteins.

The control of glycoprotein synthesis: N-acetylgluocosamine linkage to a mannose residue as a signal for the attachment of L-fucose to the asparagine-linked N-acetylglucosamine residue of glycopeptide from α 1-acid glycoprotein

Rat liver microsomes catalyze the incorporation of L-fucose from GDP-L-fucose into a glycopeptide prepared from human plasma α 1 -acid glycoprotein; the glycopeptide must have terminal mannose-linked β-N-acetylglucosamine to be active as an acceptor but fucose does not become linked to this N-acetylglucosamine residue. Endo-β-N-acetyl-glucosaminidase treatment of the product of the reaction shows that the fucose is incorporated into the asparagaine-linked N-acetylglucosamine residue. Competition studies show that this focusyltransferase is the same enzyme as a GDP-flucose:β-N-acetylglucosaminide focusyltransferase previously described to be present in rat liver Golgi apparatus.

Enzymatic synthesis of a blood group H-related glycosphingolipid by an alpha-fucosyltransferase from bovine spleen.

An alpha-fucosyltransferase activity has been detected in a purified membrane preparation isolated from bovine spleen which catalyzes the transfer of L-fucose from GDP-L-[14C]-fucose to a tetraglycosylceramide (Lac-nTet-cer, Galbetal-4GlcNAcbeta1-3Galbeta1-4-Glc-cer) to form the blood group H-related glycosphingolipid. The membrane preparation contained a highly active endogenous nonlipid acceptor, which could be precipitated by 5% trichloroacetic acid or chloroform-methanol-water (6:3:5, v/v/v), whereas there was little endogenous glycosphingolipid acceptor. The optimum pH value for the incorporation of L-fucose was 6.4 in cacodylate-HCl buffer. The Km values were 0.6 mM and 0.36 mM for Lac-nTet-cer and GDP-L-fucose, respectively. The 14C-labeled product of the reaction was isolated and purified; it migrated with human erythrocyte blood group H-active pentaglycosylceramide. the terminal [14C]fucose was hydrolyzed 85% and 55% by 0.1 N trichloroacetic acid at 100 degrees for 2 hours and Charonia lampas alpha-fucosidase (19 hours at 37 degrees), respectively. The 14C-labeled product inhibited the hemagglutination reaction of O-type cells against eel anti H(O) globulin and formed a precipitin line with Ulex europeus lectin.

The incorporation of l-fucose into glycoproteins in the Golgi apparatus of rat liver and in serum

l-Fucose was used as a radioactive precursor to study the synthesis of glycoproteins in the Golgi complex isolated from rat liver using both biochemical and au toradiographic techniques. Protein-bound fucose appeared in the Golgi complex of rat liver initially in the perchloric acid-insoluble fraction (maximum specific activity at 10 min after fucose injection) of the isolated Golgi comples and then in the perchloric acid-soluble fraction (maximum at 15 min) before being released in the perchloric acid-soluble fraction of serum. The results of biochemical studies correlated well with electron microscope autradiographic studies on sections of rat liver and isolated Golgi fractions labelled with l-[ 3H]fucose. Within 2 min, fucose was incorporated into the Golgi complex with the maximum number of silver grains over the Golgi complex observed at approximately 10 min, while the maximum number over the plasma membrane and associated vesicles appeared at 30 min. In isolated Golgi membranes, autoradiographic studies showed most radioactivity localized in the cisternae (maximum at 2 min) of the Golgi complex and then in the tubules (maximum at 10 min) befor appearing in the secretory vesicles (maximum at 20 min).

Pork Liver Guanosine Diphosphate-l-Fucose Glycoprotein Fucosyltransferases

Abstract Two fucosyltransferases have been found in pork liver which transfer l-fucose from GDP-l-fucose into various derivatives of human plasma α1-acid glycoprotein. One of these enzymes incorporates l-fucose into the terminal positions of α1-acid glycoprotein prosthetic groups and into the disaccharide, galactosyl-(β,1 → 4)-N-acetylglucosamine. This enzyme does not transfer fucose to lactose, galactosyl(β,1 → 3)-N-acetylglucosamine, galactosyl-(β,1 → 6)-N-acetylglucosamine, and the α- and β-methyl-d-galactopyranosides. The substrate specificity indicates that the fucosyltransferase is responsible for the incorporation of l-fucose into plasma glycoproteins in vivo. The enzyme is membrane-bound and requires Triton X-100 and divalent cation for optimum activity. The other pork liver fucosyltransferase incorporates l-fucose into a position on the oligosaccharide prosthetic group of α1-acid glycoprotein at which l-fucose does not occur in the native glycoprotein; the function of this enzyme in pork liver is not known.