Detection of a high affinity binding site in recombinant <Emphasis Type="Italic">Aleuria aurantia</Emphasis> lectin

Lectins are carbohydrate binding proteins that are involved in many recognition events at molecular and cellular levels. Lectin-oligosaccharide interactions are generally considered to be of weak affinity, however some mushroom lectins have unusually high binding affinity towards oligosaccharides with K (d) values in the micromolar range. This would make mushroom lectins ideal candidates to study protein-carbohydrate interactions. In the present study we investigated the properties of a recombinant form of the mushroom lectin Aleuria aurantia (AAL). AAL is a fucose-binding lectin composed of two identical 312-amino acid subunits. Each subunit contains five binding sites for fucose. We found that one of the binding sites in rAAL had unusually high affinities towards fucose and fucose-containing oligosaccharides with K (d) values in the nanomolar range. This site could bind to oligosaccharides with fucose linked alpha1-2, alpha1-3 or alpha1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with alpha1-6 linked fucose. This binding site is not detected in native AAL (nAAL) one possible explanation may be that this site is blocked with free fucose in nAAL. Recombinant AAL was produced in E. coli as a His-tagged protein, and purified in a one-step procedure. The resulting protein was analyzed by electrophoresis, enzyme-linked lectin assay and circular dichroism spectroscopy, and compared to nAAL. Binding properties were measured using tryptophan fluorescence and surface plasmon resonance. Removal of the His-tag did not alter the binding properties of recombinant AAL in the enzyme-linked lectin assay. Our study forms a basis for understanding the AAL-oligosaccharide interaction and for using molecular techniques to design lectins with novel specificities and high binding affinities towards oligosaccharides.     

Carbohydrate binding specificity of a fucose-specific lectin from Aspergillus oryzae: a novel probe for core fucose

The alpha1,6-fucosyl residue (core fucose) of glycoproteins is widely distributed in mammalian tissues and is altered under pathological conditions. A probe that specifically detects core fucose is important for understanding the role of this oligosaccharide structure. Aleuria aurantia lectin (AAL) and Lens culimaris agglutinin-A (LCA) have been often used as carbohydrate probes for core fucose in glycoproteins. Here we show, by using surface plasmon resonance (SPR) analysis, that Aspergillus oryzae l-fucose-specific lectin (AOL) has strongest preference for the alpha1,6-fucosylated chain among alpha1,2-, alpha1,3-, alpha1,4-, and alpha1,6-fucosylated pyridylaminated (PA)-sugar chains. These results suggest that AOL is a novel probe for detecting core fucose in glycoproteins on the surface of animal cells. A comparison of the carbohydrate-binding specificity of AOL, AAL, and LCA by SPR showed that the irreversible binding of AOL to the alpha1,2-fucosylated PA-sugar chain (H antigen) relative to the alpha1,6-fucosylated chain was weaker than that of AAL, and that the interactions of AOL and AAL with alpha1,6-fucosylated glycopeptide (FGP), which is considered more similar to in vivo glycoproteins than PA-sugar chains, were similar to their interactions with the alpha1,6-fucosylated PA-sugar chain. Furthermore, positive staining of AOL, but not AAL, was completely abolished in the cultured embryo fibroblast (MEF) cells obtained from alpha1,6-fucosyltransferase (Fut8) knock-out mice, as assessed by cytological staining. Taken together, these results suggest that AOL is more suitable for detecting core fucose than AAL or LCA.     

Epithelial repair is inhibited by an alpha(1,6)-fucose binding lectin

The effective repair of damage to the airway epithelium is essential to maintain the ability to exclude airborne particulates and protect against potential pathogens. Carbohydrates on the cell surface have an important role in cell-cell and cell substrate interactions. Using a model of repair with airway epithelial-derived cells of the 16HBE 14o(-) cell line, we have examined the effect of the Aleuria aurantia lectin (AAL), which binds very selectively to alpha(1,6)-linked fucose residues. Addition of unconjugated or FITC-labeled AAL reduced the rate of epithelial repair to approximately one-third of control values as measured by image analysis while cell viability was maintained. Pulse labeling with AAL-FITC for 30 min followed by incubation in AAL-free medium caused similar inhibition of repair but could be reversed by addition of fucose up to 7 h after AAL removal. By confocal microscopy, AAL binding was found to be on the apical, but not basolateral, surfaces of cells, and internalization of the labeled lectin was seen. Preincubation of the lectin with fucose prevented this effect. Ulex europeaus I lectin, which is also fucose specific, resulted in similar binding to the cells and internalization, but it did not affect the speed of the repair process. We conclude that alpha(1,6)-fucose binding sites play an important role in epithelial repair. Better understanding of this process will provide a deeper insight into the crucial mechanisms of epithelial repair.     

Molecular cloning and characterization of a plant alpha1,3/4-fucosidase based on sequence tags from almond fucosidase I

Our work with almond peptide N-glycosidase A made us interested also in the alpha1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences. A homology search retrieved the hypothetical protein Q8GW72 from Arabidopsis thaliana. This protein has recently been described as being specific for alpha1,2-linkages. However, cDNA cloning and expression in Pichia pastoris of the A. thaliana fucosidase showed that it hydrolyzed fucose in 3- and 4-linkage to GlcNAc in Lewis determinants whereas neither 2-linked fucose nor fucose in 3-linkage to the innermost GlcNAc residue were attacked. This first cloning of a plant alpha1,3/4-fucosidase also confirmed the identity of the purified almond enzyme and thus settles the notorious uncertainty about its molecular mass. The alpha1,3/4-fucosidase from Arabidopsis exhibited striking sequence similarity with an enzyme of similar substrate specificity from Streptomyces sp. (Q9Z4I9) and with putative proteins from rice.     

Crystal structure of fucose-specific lectin from Aleuria aurantia binding ligands at three of its five sugar recognition sites

Aleuria aurantia possesses a fucose-specific lectin (AAL) that is widely used as a specific probe for fucose. Fucosylated sugars often play pivotal roles in many cellular processes. We have determined the crystal structure of AAL at 2.24 A resolution in complex with only three fucose molecules in its five sugar binding sites of a six-fold beta-propeller structure. Very recently, the structure of AAL has been independently determined, showing that all the five binding sites were occupied by fucose molecules [Wimmerova, M., et al. (2003) J. Biol. Chem. 278, 27059-27067]. Stabilization of the arginine conformation bound to fucose molecules plays an essential role in generating the difference in the affinity in the five binding sites. Binding models with a couple of saccharides based on biochemical assays suggest that hydrophobic contacts also play important roles in AAL recognizing its ligand.     

Primary structure determination of five sialylated oligosaccharides derived from bronchial mucus glycoproteins of patients suffering from cystic fibrosis. The occurrence of the NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)] GlcNAc beta(1----.) structural element revealed by 500-MHz 1H NMR spectroscopy

The structure of sialylated carbohydrate units of bronchial mucins obtained from cystic fibrosis patients was investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar analysis. After subjecting the mucins to alkaline borohydride degradation, sialylated oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high performance liquid chromatography. Five compounds could be obtained in a rather pure state; their structures were established as the following: A-1, NeuAc alpha(2----3)Gal beta(1----4) [Fuc alpha(1----3)]GlcNAc beta(1----3)Gal-NAc-ol; A-2, NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)-[GlcNAc beta (1----3)]GalNAc-o1; A-3, NeuAc alpha(2----3)Gal beta-(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----3)Gal beta(1----3) GalNAc-o1; A-4, NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)]Glc-NAc NAc beta(1----6)[GlcNAc beta(1----3)]GalNAc-o1; A-6,NeuAc alpha-(2----3) Gal beta(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----6)[Gal beta-(1----4) GlcNAc beta(1----3)]GalNAc-o1. The simultaneous presence of sialic acid in alpha(2----3)-linkage to Gal and fucose in alpha(1----3)-linkage to GlcNAc of the same N-acetyllactosamine unit could be adequately proved by high resolution 1H NMR spectroscopy. This sequence constitutes a novel structural element for mucins.     

Development of recombinant Aleuria aurantia lectins with altered binding specificities to fucosylated glycans

Changes in glycosylation have long been associated with disease. While there are many methods to detect changes in glycosylation, plant derived lectins are often used to determine changes on specific proteins or molecules of interest. One change in glycosylation that has been observed by us and by others is a disease or antigen associated increase in fucosylation on N-linked glycans. To measure this change, the fucose binding Aleuria aurantia lectin (AAL) is often utilized in plate and solution based assays. AAL is a mushroom derived lectin that contains five fucose binding sites that preferentially bind fucose linked (α-1,3, α-1,2, α-,4, and α-1,6) to N-acetyllactosamine related structures. Recently, several reports by us and by others have indicated that specific fucose linkages found on certain serum biomarker glycoprotein’s are more associated with disease than others. Taking a site-directed mutagenesis approach, we have created a set of recombinant AAL proteins that display altered binding affinities to different analytes containing various fucose linkages.     

HPLC method for the determination of Fuc to Asn-linked GlcNAc fucosyltransferases

Mammalian cells often contain an enzyme which transfers fucose onto the reducing terminal GlcNAc (GlcNAc-1) of N-glycans with an α1,6-linkage. In plants, on the other hand, the fucose is transferred to GlcNAc-1 with an α1,3-linkage. Insect cells can exhibit both enzymatic activities. Hitherto, the activity of these fucosyltransferases has been determined by the incorporation of radioactively labelled fucose into an acceptor glycopeptide. This assay, however, cannot discriminate these two activities. Here we report on the use of dansylated glycoasparagine for the specific determination of 1,3- and 1,6-fucosyltransferases. The two possible products and the substrate are separated on a reversed phase column and detected by fluorescence.     

Conformational analysis of the sialyl alpha(2----3/6)N-acetyllactosamine structural element occurring in glycoproteins, by two-dimensional NOE 1H-NMR spectroscopy in combination with energy calculations by hard-sphere exo-anomeric and molecular mechanics force-field with hydrogen-bonding potential

The conformation is described of the sialyl alpha(2----3/6)N-acetyllactosamine structural element, frequently occurring in glycoproteins. NOE spectroscopy of NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----N)Asn and NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta(1----N)Asn is presented and for each glycosidic linkage, except for the alpha(2----6)-linkage, a number of interglycosidic NOEs are measured. The analysis of these effects is performed using a full relaxation matrix. Analysis of intraresidue NOEs provides a calibration of the calculation method. Hard-sphere exo-anomeric (HSEA) energy calculations indicate a single conformation for the beta(1----4)-linkage in both compounds, both being consistent with the NOE data. HSEA and molecular-mechanics force-field with hydrogen-bonding potential energy calculations both indicate the existence of three preferred conformations for the alpha(2----3)-linkage. The analysis of the NOE spectra are consistent with a distribution over two or three of these conformations; by combination with the energy diagram for this linkage the existence of onyl a single conformation can be excluded. The NOE spectrum of the compound with the alpha(2----6)-linkage indicates a gt orientation for the Gal C-6 hydroxymethyl group. On this basis, the HSEA energy calculations for the alpha(2----6)-linkage indicate an extended low-energy surface with a number of preferred conformations. The absence of NOEs across this linkage is interpreted in terms of a non-rigid, but overall folded conformation of the NeuAc alpha(2----6)Gal beta(1----4)GlcNAc beta structural element. This provides an explanation for the shift effects induced by alpha(2----6) attachment of NeuAc to the N-acetyllactosamine unit.     

A single amino acid in the hypervariable stem domain of vertebrate alpha1,3/1,4-fucosyltransferases determines the type 1/type 2 transfer. Characterization of acceptor substrate specificity of the lewis enzyme by site-directed mutagenesis

Alignment of 15 vertebrate alpha1,3-fucosyltransferases revealed one arginine conserved in all the enzymes employing exclusively type 2 acceptor substrates. At the equivalent position, a tryptophan was found in FUT3-encoded Lewis alpha1,3/1,4-fucosyltransferase (Fuc-TIII) and FUT5-encoded alpha1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in alpha1, 4-linkage. The single amino acid substitution Trp111 --> Arg in Fuc-TIII was sufficient to change the specificity of fucose transfer from H-type 1 to H-type 2 acceptors. The additional mutation of Asp112 --> Glu increased the type 2 activity of the double mutant Fuc-TIII enzyme, but the single substitution of the acidic residue Asp112 in Fuc-TIII by Glu decreased the activity of the enzyme and did not interfere with H-type 1/H-type 2 specificity. In contrast, substitution of Arg115 in bovine futb-encoded alpha1, 3-fucosyltransferase (Fuc-Tb) by Trp generated a protein unable to transfer fucose either on H-type 1 or H-type 2 acceptors. However, the double mutation Arg115 --> Trp/Glu116 --> Asp of Fuc-Tb slightly increased H-type 1 activity. The acidic residue adjacent to the candidate amino acid Trp/Arg seems to modulate the relative type 1/type 2 acceptor specificity, and its presence is necessary for enzyme activity since its substitution by the corresponding amide inactivated both Fuc-TIII and Fuc-Tb enzymes.     

alpha-L-fucosidase from aspergillus niger: demonstration of a novel alpha-L-(1----6)-fucosidase acting on glycopeptides

An alpha-L-fucosidase which hydrolyzes fucose from alpha-(1----6)-linkage to N-acetylglucosamine was found in Aspergillus niger. The enzyme was purified by affinity chromatography with bovine IgG glycopeptide-Sepharose 4B. The enzyme preparation released fucose from bovine IgG glycopeptide and fucosylated asialoagalactofetuin, but failed to cleave 1----2, 1----3 or 1----4 linkages of alpha-L-fucosides.     

Structures of neutral oligosaccharides isolated from the respiratory mucins of a non-secretor (O, Le(a+b-)) patient suffering from chronic bronchitis.

Mucin glycopeptides were prepared from the respiratory mucus of a non-secretor, chronic bronchitis patient with blood group O, Le(a+b-). Oligosaccharides were released by alkaline-borohydride treatment and purified by anion-exchange chromatography, size exclusion chromatography, and HPLC on a silica-bonded alkylamine column. Structural studies employed 500-MHz 1H-NMR spectroscopy, fast-atom-bombardment mass spectrometry and methylation analysis. Twenty-four neutral oligosaccharides, ranging in size from disaccharides to hexasaccharides, were fully characterized in this study. None of the structures contained an alpha(1----2)-linked fucose residue, in keeping with the non-secretor status of the patient. Seven of the structures had fucose present in alpha(1----3)-linkage in the X-determinant, while only one oligosaccharide (compound 14b) was seen with fucose alpha(1----4)-linked in the Le(a) determinant. The following eight structures isolated from the mucins of the non-secretor patient had not been found previously in the mucins of secretor individuals: [formula: see text] This study confirms that the blood group status of an individual is reflected in the carbohydrate structures of the secreted mucins. Furthermore, it clearly illustrates the need for detailed carbohydrate structural studies of mucins from different individuals before any attempt can be made to correlate observed differences in oligosaccharide profiles to disease status.     

Structural studies of sialylated oligosaccharides of human midcycle cervical mucin

It was previously shown that reductive alkali treatment of purified human cervical mucin releases a heterogeneous population of reduced neutral, sialylated, and sulfated oligosaccharides (Yurewicz, E. C., and Moghissi, K. S. (1981) J. Biol. Chem. 256, 11895-11904). Four major sialylated oligosaccharide fractions were isolated with approximate compositions of Fuc:GlcNac:Gal:NeuAc:N-acetylgalactosaminitol (GalNAcol) = 0:0:0:1:1 (B1a), 0:0:1:1:1 (B2b), 0:1:2:1:1 (B3a), and 1:1:2:1:1 (B4a), where Fuc is fucose. They comprised roughly 3, 11, 7, and 6% of recovered oligosaccharide chains, respectively. On the basis of periodate oxidations, methylation analyses, and sequential degradations with glycosidases, the following structures were determined. (Formula: see text) Oligosaccharides 1 and 2 are characterized by the presence of N-acetylneuraminic acid in alpha 2,6-linkage to N-acetylgalactosaminitol. The remaining oligosaccharides contain N-acetylneuraminic acid in alpha 2,3-linkage to galactose residues. Oligosaccharides 3 and 4 and oligosaccharides 5 and 6 were isolated as unresolved isomeric mixtures in fractions B3a and B4a, respectively. Oligosaccharides 3 and 4 were distinguished on the basis of susceptibility to digestion with Aspergillus niger beta-galactosidase whereas oligosaccharides 5 and 6 were distinguished on the basis of differential rates of digestion with beef kidney alpha-fucosidase. The structural data indicate the presence of at least two sialyltransferases in human cervical epithelium and further suggest a potential physiologically significant competition between sialyltransferase and beta-N-acetylglucosaminyltransferase for C-6 of the N-acetylgalactosamine residue O-glycosidically linked to serine/threonine of the polypeptide core.     

Comparative analysis of oligosaccharide specificities of fucose-specific lectins from Aspergillus oryzae and Aleuria aurantia using frontal affinity chromatography

Aleuria aurantia lectin (AAL) is widely used to estimate the extent of α1,6-fucosylated oligosaccharides and to fractionate glycoproteins for the detection of specific biomarkers for developmental antigens. Our previous studies have shown that Aspergillus oryzae lectin (AOL) reflects the extent of α1,6-fucosylation more clearly than AAL. However, the subtle specificities of these lectins to fucose linked to oligosaccharides through the 2-, 3-, 4-, or 6-position remain unclear, because large amounts of oligosaccharides are required for the systematic comparative analysis using surface plasmon resonance. Here we show a direct comparison of the dissociation constants (K_d) of AOL and AAL using 113 pyridylaminated oligosaccharides with frontal affinity chromatography. As a result, AOL showed a similar specificity as AAL in terms of the high affinity for α1,6-fucosylated oligosaccharides, for smaller fucosylated oligosaccharides, and for oligosaccharides fucosylated at the reducing terminal core GlcNAc. On the other hand, AOL showed 2.9-6.2 times higher affinity constants (K_a) for α1,6-fucosylated oligosaccharides than AAL and only AAL additionally recognized oligosaccharides which were α1,3-fucosylated at the reducing terminal GlcNAc. These results explain why AOL reflects the extent of α1,6-fucosylation on glycoproteins more clearly than AAL. This systematic comparative analysis made from a quantitative viewpoint enabled a clear physical interpretation of these fucose-specific lectins with multivalent fucose-binding sites.     

Aleuria aurantia lectin exhibits antifungal activity against Mucor racemosus

Aleuria aurantia lectin (AAL) is an L-fucose-specific lectin produced in the mycelia and fruit-bodies of the widespread ascomycete fungus Aleuria aurantia. It is extensively used in the detection of fucose, but its physiological role remains unknown. To investigate this, we analyzed the interaction between AAL and, a zygomycete fungus Mucor racemosus, which is assumed to contain fucose in its cell wall. AAL specifically bound to the hyphae of M. racemosus, because binding was inhibited by L-fucose but not by D-fucose. It inhibited the growth of the fungus at 1 μM, and the M. racemosus cells were remarkably disrupted at 7.5 μM. In contrast, two other fucose-specific lectins, Anguilla anguilla agglutinin and Ulex europaeus agglutinin, did not inhibit the growth of M. racemosus. These results suggest that the growth inhibition activity is unique to AAL, and that AAL could act as an antifungal protein in natural ecosystems.     

Identification of Auxin Activity Like 1, a chemical with weak functions in auxin signaling pathway

Key message

A new synthetic auxin AAL1 with new structure was identified. Different from known auxins, it has weak effects. By AAL1, we found specific amino acids could restore the effects of auxin with similar structure.

Abstract

Auxin, one of the most important phytohormones, plays crucial roles in plant growth, development and environmental response. Although many critical regulators have been identified in auxin signaling pathway, some factors, especially those with weak fine-tuning roles, are still yet to be discovered. Through chemical genetic screenings, we identified a small molecule, Auxin Activity Like 1 (AAL1), which can effectively inhibit dark-grown Arabidopsis thaliana seedlings. Genetic screening identified AAL1 resistant mutants are also hyposensitive to indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). AAL1 resistant mutants such as shy2-3c and ecr1-2 are well characterized as mutants in auxin signaling pathway. Genetic studies showed that AAL1 functions through auxin receptor Transport Inhibitor Response1 (TIR1) and its functions depend on auxin influx and efflux carriers. Compared with known auxins, AAL1 exhibits relatively weak effects on plant growth, with 20 µM and 50 µM IC50 (half growth inhibition chemical concentration) in root and hypocotyl growth respectively. Interestingly, we found the inhibitory effects of AAL1 and IAA could be partially restored by tyrosine and tryptophan respectively, suggesting some amino acids can also affect auxin signaling pathway in a moderate manner. Taken together, our results demonstrate that AAL1 acts through auxin signaling pathway, and AAL1, as a weak auxin activity analog, provides us a tool to study weak genetic interactions in auxin pathway.

     

A new fungal lectin recognizing alpha(1-6)-linked fucose in the N-glycan

In this report, we describe a new lectin from the fungus Rhizopus stolonifer that agglutinates rabbit red blood cells. Agglutinating activity was detected in the extract of mycelium-forming spores cultured on agar plates but not in the mycelium-forming no spores from liquid medium. This lectin, which we designated R. stolonifer lectin (RSL), was isolated by affinity chromatography with porcine stomach mucin-Sepharose. SDS-polyacrylamide gel electrophoresis and mass spectral analysis showed that RSL is approximately 4.5 kDa, whereas gel filtration indicated a mass of 28 kDa. This indicates that the lectin is a hexamer of noncovalently associated RSL monomers. RSL activity was very stable, since it was insensitive to heat treatment at 70 degrees C for 10 min. Analysis of RSL binding specificity by both microtiter plate and precipitation assays showed that N-glycans with l-fucose linked to the reducing terminal GlcNAc residues are the most potent inhibitors of RSL binding, whereas N-glycans without alpha(1-6)-linked fucose residues are approximately 100-fold weaker inhibitors of binding. Oligosaccharides with alpha(1-2, -3, and -4) linkages showed no inhibition of binding in these assays. In a mirror resonance biosensor assay, high affinity binding was observed between RSL and the glycopeptide of bovine gamma-globulin, which has N-glycans with alpha(1-6)-linked fucose residues. However, RSL showed only a weak interaction with the glycopeptide of quail ovomucoid, which lacks fucose residues. Finally, capillary affinity electrophoresis studies indicated that RSL binds strongly to N-glycans with alpha(1-6)-linked fucose residues. Together, these results show that RSL recognizes the core structure of N-glycans with alpha(1-6)-linked l-fucose residues. This specificity could make RSL a valuable tool for glycobiological studies.     

The identification of terminal alpha (1----3)-linked galactose in N-acetyllactosamine type of glycopeptides by means of 500-MHz 1H-NMR spectroscopy

500-MHz 1H-NMR spectroscopy was employed to study two N-acetyllactosamine-type glycopeptide fractions which were derived from a bovine thyroglobulin preparation (Cummings, R.D., and Kornfeld, S. (1982) J. Biol. Chem. 257, 11230-11234). By this method, their branches were found to be terminated either by NeuAc in alpha (2----6)-linkage or by Gal in alpha (1----3)-linkage. For the first time, the Gal alpha (1----3) Gal beta (1----4) GlcNAc beta (1----.) sequence is characterized by 1H-NMR to occur in N-glycosidic carbohydrate chains of glycoproteins. Moreover, this approach made possible the branch localization of such a unit. Microheterogeneity with respect to the presence of alpha-linked Gal or NeuAc in terminal position of a certain branch in one of the preparations, could be adequately assessed in terms of structures by 1H-NMR.     

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 alpha 1,2-fucosidase from Aspergillus niger. Using this method, it was shown that 2-deoxy-D-galactose markedly inhibits alpha 1,2-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 alpha 1,2-fucosylation. In addition, a hitherto unreported compensatory increase of alpha 1,3/alpha 1,4-fucosylation was found to occur when alpha-1,2-fucosylation was inhibited by treatment with 2-deoxy-D-galactose.     

Occurrence of alpha 1-2-fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver. Aberrant type of fucosylation in a malignant tissue.

A comparative study was undertaken to characterize the linkages of L-fucose in N-glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum. After in-vivo radiolabelling of rats with L-[6-3H]fucose, the asparagine-linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride. Two specific alpha-L-fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of L-fucose in the oligosaccharide chains. Fucose alpha 1-2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total L-[3H]fucose in N-glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose alpha 1-3/4 linked to N-acetyl-D-glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained alpha-fucosyl residues linked at the C6 position of the proximal N-acetyl-D-glucosamine.