Glycobiology Advance Access originally published online on May 25, 2005
Glycobiology 2005 15(10):924-934; doi:10.1093/glycob/cwi081
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Core fucosylation of N-linked glycans in leukocyte adhesion deficiency/congenital disorder of glycosylation IIc fibroblasts
2 Department of Experimental Medicine and Center of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 1, 16132, Genova, Italy; 3 Department of Biochemistry, Osaka University, Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan; and 4 Department of Internal Medicine, University of Genova, Viale Benedetto XV, 6, 16132, Genova, Italy
1 To whom correspondence should be addressed; e-mail: tonetti{at}unige.it
Received on December 28, 2004; revised on May 17, 2005; accepted on May 19, 2005
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Abstract |
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Leukocyte adhesion deficiency/congenital disorder of glycosylation IIc (LAD II/CDG IIc) is a genetic disease characterized by a decreased expression of fucose in glycoconjugates, resulting in leukocyte adhesion deficiency and severe morphological and neurological abnormalities. The biochemical defect is a reduced transport of guanosine diphosphate-L-fucose (GDP-L-fucose) from cytosol into the Golgi compartment, which reduces its availability as substrate for fucosyltransferases. The aim of this study was to determine the effects of a limited supply of GDP-L-fucose inside the Golgi on core fucosylation (a1,6-fucose linked to core N-acetylglucosamine [GlcNAc]) of N-linked glycans in LAD II fibroblasts. The results showed that, although [3H]fucose incorporation was generally reduced in LAD II cells, core fucosylation was affected to a greater extent compared with other types of fucosylation of N-linked oligosaccharides. In particular, core fucosylation was found to be nearly absent in biantennary negatively charged oligosaccharides, whereas other types of structures, in particular triantennary neutral species, were less affected by the reduction. Expression and activity of a1,6-fucosyltransferase (FUT8) in control and LAD II fibroblasts were comparable, thus excluding the possibility of a decreased activity of the transferase. The data obtained confirm that the concentration of GDP-L-fucose inside the Golgi can differentially affect the various types of fucosylation in vivo and also indicate that core fucosylation is not dependent only on the availability of GDP-L-fucose, but it is significantly influenced by the type of oligosaccharide structure. The relevant reduction in core fucosylation observed in some species of oligosaccharides could also provide clues for the identification of glycans involved in the severe developmental abnormalities observed in LAD II.
Key words:
congenital disorder of glycosylation
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fibroblasts
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fucose
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leukocytes adhesion deficiency
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1,6-fucosyltransferase
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataReferences |
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Leukocyte adhesion deficiency/congenital disorder of glycosylation IIc (LAD II/CDG IIc) is a rare autosomal recessive disease characterized by a reduced expression of fucose in glycoconjugates, including blood group H and Lewis antigens. The main features of this syndrome include recurrent infections, severe psychomotor and growth retardation, and skeletal abnormalities (Etzioni et al., 1992)
. The immunodeficiency has been related to a reduced leukocyte rolling on endothelial cells. This process is mediated by the selectins, which, in LAD II, are unable to recognize their oligosaccharide ligands that lack fucose, thus impairing the recruitment of leukocytes to inflamed tissues (Etzioni and Tonetti, 2000). Conversely, the specific causes that lead to the severe neurological and developmental defects are unknown at present.
The molecular basis of the LAD II/CDG IIc syndrome has been identified as a defect in GDP-L-fucose transport into the Golgi system (Lübke et al., 2001; Sturla et al., 2001). The decreased availability of the substrate for the several fucosyltransferases inside the Golgi explains the lower fucose content of glycoconjugates. However, an analysis of the residual fucose content in different classes of glycoproteins revealed that the reduction largely affects certain specific classes of glycans (Sturla et al., 2003). In particular, the fucose content of N-linked oligosaccharides was severely reduced, whereas the O-fucosylation levels of the total and specific proteins, such as notch, were reported to be comparable to control cells (Sturla et al., 2003). A potential explanation for the differences between the addition of fucose in N-linked oligosaccharides and O-fucosylation could be due to a different affinity of the transferases for GDP-L-fucose or to differences in the localization of these enzymes in the Golgi compartments. These data provide further support for a scenario in which glyconjugate composition can be determined not only by glycosyltransferase activities but also by topological availability of the nucleotide–sugar substrates (Toma et al., 1996). The issue of whether a limited supply of GDP-L-fucose inside the Golgi is also able to differentially affect the conversion into products of the various fucosyltransferases and whether specific types of fucose linkages are more affected than others in LAD II fibroblasts is currently unknown.
Recent data by Noda et al. indicated that an increase in GDP-L-fucose levels contributes to an elevation in the 1,6 fucosylation of glycoproteins in human hepatocellular carcinoma (Noda et al., 2003), suggesting that differences in substrate concentration significantly affect the activity of 1,6-fucosyltransferase (FUT8). The presence of fucose 1,6-linked to N-acetylglucosamine (GlcNAc) (core fucose) in N-linked glycoproteins has been shown to be important in glycoprotein processing and recognition (Miyoshi et al., 1999). Core-fucosylated N-linked glycans have been observed in several soluble and membrane glycoproteins and are particularly abundant in the brain (Shimizu et al., 1993). Increased core fucosylation has been reported in certain pathological conditions, cancer in particular (Noda et al., 1998a), and following cell transformation (Noda et al., 1998b). The presence of a core fucose in N-linked oligosaccharides of the fragment crystallizable region of immunoglobulin G greatly reduces antibody-dependent cellular cytotoxicity (ADCC), and defucosylated recombinant antibodies show an increased binding to Fc
RIIIa and an enhanced ADCC against tumour cells (Shields et al., 2002; Mori et al., 2004; Niwa et al., 2004; Yamane-Ohnuki et al., 2004). Cultured skin fibroblasts have been shown to have a high 1,6 fucosyltransferase activity (Voynow et al., 1991) and to contain a high proportion of core fucosylated biantennary and triantennary structures (Scanlin et al., 1985; Wang et al., 1990). Interestingly, core fucosylation has been reported to be significantly increased in fibroblasts from cystic fibrosis patients (Scanlin et al., 1985), but the mechanisms responsible for this alteration have not been clearly identified.
In this study we analyzed N-linked glycans from LAD II skin fibroblasts with the aim of determining the effects of a reduced availability of GDP-L-fucose inside the Golgi on the relative amount of core 1,6 fucose compared to other type of fucosylation. The identification of a class of fucosylated N-glycans which may be affected more than others in LAD II patients is particularly important, because it could give clues to the role of this form of fucosylation in the severe developmental defects observed in this syndrome.
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Results |
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The presence of
1,6-fucose in LAD II fibroblasts compared to controls was initially analyzed using a specific lectin that preferentially recognizes a core fucose unit. Total homogenates of fibroblasts from controls and an LAD II patient were subjected to lectin blotting using Lens culinaris agglutinin (LCA), which interacts with -mannosyl residues of N-linked sugar chains, but which requires the presence of a fucose residue bound to the at C-6 hydroxyl group of the GlcNAc at the reducing end for strong binding (Kornfeld et al., 1981). Samples treated with peptide N-glycosidase F (PNGase F) (to remove N-linked glycans) were used as controls to identify nonspecific binding of the lectin. Significant differences were observed between the control and LAD II samples (Figure 1A), with the total core fucosylation being severely reduced in the LAD II fibroblasts. Comparable results were also obtained by flow cytometry, which demonstrated an approximate 10- to 12-fold reduction in LCA reactivity in LAD II cells compared to controls (Figure 1B). Collectively, these data indicate that 1,6 fucose was indeed significantly reduced in patient cells and that the residual fucose detected in LAD II glycoproteins (Sturla et al., 2003) was not primarily due to core fucosylation.
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To better determine the relative amount of fucose in
1,6-linkages in total glycoproteins, cells were metabolically labelled with [3H]fucose. The extent of labelling of the macromolecules was comparable to that previously reported (Sturla et al., 2003), resulting in an approximate 5-fold reduction in [3H]fucose incorporation in the total glycoproteins compared with the controls. All subsequent chromatographic separations were performed using comparable amounts of either proteins or glycopeptides for control and LAD II samples. As a consequence, the radioactivity used for the LAD II samples was about five times less compared with controls. However, allow the data to be compared more easily, the results were normalized by expressing them as percent of the total radioactivity used in the analysis. Graphs expressing the results as absolute radioactivity counts are reported in supplementary data (Supplementary Figures 1 and 2).
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Total proteins were extracted as described previously (Sturla et al., 2003) and subjected to treatment with 1,6-fucosidase from Chryseobacterium meningosepticum, which specifically cleaves core fucose; separation of free fucose from the macromolecules (which contained both N- and O-linked glycoproteins, including O-fucosylated species) was achieved by G-50 chromatography. The enzymatic treatment released about 50% of the total radioactivity as free fucose for both controls, as shown in Figure 2. Conversely, fucosidase treatment released a smaller fraction (about 15%) of the label from the LAD II glycoproteins, indicating that the amount of core fucose is further reduced compared with other types of fucosylation.
A similar result was obtained for LCA affinity chromatography of the glycopeptides obtained after pronase treatment (Figure 3). The analysis was carried out on the glycopeptides, because, in our experimental conditions, the detergents that were used to solubilize the proteins were found to interfere with lectin binding. Approximately 50 and 15% of the total radioactivity for the control and LAD II, respectively, bound to the columns, confirming the data previously obtained by fucosidase treatment.
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To determine the relative amount of fucose in N-linked oligosaccharides, total [3H]-fucose labelled glycoproteins were then subjected to PNGase F treatment and the released oligosaccharides were separated from residual macromolecular material by G-50 chromatography. The amount of label incorporated into the N-linked glycans was about six times less in the LAD II samples compared to the controls (not shown), in agreement with previously published results (Sturla et al., 2003). The increased difference in fucose incorporation observed in N-linked oligosaccharides between LAD II and controls, compared to that observed for total glycoproteins, is mainly due to the removal of O-linked glycans, in particular O-linked fucose, which is normal or even slightly increased in LAD II cells (Sturla et al., 2003).
The released [3H]fucose-labelled oligosaccharides were then fractionated by gel filtration, using a Superdex column. The elution profiles are shown in Figure 4A. Each point was expressed as the percent of injected radioactivity, to allow an easier comparison between the elution profiles of the LAD II and controls. Three main peaks were detected in the elution profiles from the control samples, the first between fractions 3 and 6, the second between fractions 7 and 9, and the third in fractions 10–15. The elution profile for LAD II samples was markedly different, the second peak being greatly reduced, with a corresponding relative increase in the third peak. Further analyses performed on the species collected in the second peak, using Canavalia ensiformis (Con A) affinity chromatography and quaternary aminoethyl (QAE)-anion exchange chromatography (see below), indicated the presence of mainly biantennary charged oligosaccharides (not shown).
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Because sialic acid can have significant effects on the elution behaviour of oligosaccharides in size exclusion chromatography, the samples were subjected to a treatment with a mixture of two different neuraminidases. The removal of negative charges was confirmed by anion exchange chromatography, using QAE-sepharose (not shown). The elution profiles after neuraminidase treatment (Figure 4B) showed an almost complete disappearance of the first and second peaks and the profiles for the controls and LAD II samples became quite comparable. These data indicate that the first two peaks contain mainly sialylated species and that the incorporation of residual fucose in N-linked oligosaccharides of LAD II is relatively lower in sialylated glycans than in neutral ones, in particular in species that elute in the second peak.
To analyze the relative amount of core fucose compared with other types of linkages in N-linked glycans, we performed initial experiments using two different fucosidases. Treatment with -fucosidase from bovine kidney, which has broad specificity (Kobata and Takasaki, 1993), induced the release of about 90–95% of the label from N-linked oligosaccharides of controls and LAD II samples, as determined by size exclusion chromatography, indicating that almost all the radioactivity associated with N-linked oligosaccharides is because of [3H]fucose. Conversely, in our experimental conditions, the efficiency of cleavage of 1,6-linked fucose from either free or peptide-bound N-linked oligosaccharides by the specific fucosidase from C. meningosepticum was very low and poorly reproducible. For this reason, to discriminate core-linked fucose from other type of linkages, we decided to treat [3H]fucose-labelled N-linked oligosaccharides with endoglycosidase H (endo H) and endoglycosidase F3 (endo F3). Endo H cleaves between the GlcNAc residues of the chitobiose core of N-linked high mannose and hybrid-type glycans, including those which are core fucosylated, whereas complex types are not hydrolyzed (Trimble et al., 1978). Endo F3 cleaves between the GlcNAc residues of bi- and triantennary complex type N-linked glycans, and the presence of a core fucose increases the rate of hydrolysis by up to 40-fold (Tarentino and Plummer, 1994). The enzyme is not active towards tetrantennary oligosaccharides; however, it has been reported that in cultured skin fibroblasts, core fucose is mainly found in biantennary and triantennary complex oligosaccharides (Scanlin et al., 1985; Wang et al., 1990).
Extensive treatment of [3H]fucose-labelled N-linked oligosaccharides with endo H/F3 resulted, as expected, in the appearance of a peak with an elution time comparable with that observed for the standard disaccharide Fuc-1,6-GlcNAc (fractions 19–20) and a relative reduction in both negatively charged and neutral high molecular weight oligosaccharides (fractions 5–15) (Figure 4C and D). The percentages of total injected radioactivity recovered in the disaccharide form, which corresponds to core 1,6-fucose, were 70, 66, and 40% for C1, C2 and LAD II, respectively.
When the peak corresponding to the disaccharide Fuc-1,6-GlcNAc was incubated in the presence of bovine kidney fucosidase, the retention time was shifted to that of a monosaccharide, as determined by Superdex analysis (which is able to discriminate between mono- and disaccharides) (Sturla et al., 2003), indicating the release of free fucose and thus confirming the identity of the disaccharide (not shown). The higher molecular weight N-glycans, which were resistant to the extensive digestion with endo H/F3, were then subjected to a combined treatment with 1,2-fucosidase (from Xanthomonas sp.) and 1,3/4-fucosidases (from almond meal), to confirm the presence also of these types of linkages. Because the cleavage efficiency of these enzymes can be highly influenced by the presence of other residues, the 1,2- and 1,3/4-fucosidase treatment was performed in the presence of other glycosidases (see Materials and Methods for details). About 15% of total initial radioactivity associated to N-linked oligosaccharides could be released as monosaccharide by the combined 1,2- and 1,3/4-fucosidase treatment in control samples. Conversely, the released radioactivity represented 30% of the total initial radioactivity for LAD II oligosaccharides. Although a detailed characterization of fucosylation of outer arms of N-linked oligosaccharides is beyond the scope of this study, 1,2- and 1,3/4-fucosidase treatment indicated that significant amounts of these types of linkages are indeed contained in fibroblast glycoproteins.
Figure 5 summarizes the amount of labelled fucose expressed as cpm associated with total N-linked oligosaccharides, with residual high molecular weight oligosaccharides after endoglycosidase cleavage, with the released disaccharide containing the core fucose and with [3H]fucose released by 1,2- and 1,3/4-fucosidase treatment of the residual high molecular weight oligosaccharides. As mentioned above (G-50 chromatography data), the total [3H] fucose incorporation was about six times less for the N-linked oligosaccharides from LAD II as compared to those from controls (Figure 5, sample 1), whereas the reduction was 3.5-fold in residual high molecular weight N-linked oligosaccharides (Figure 5, sample 2), 10-fold in the disaccharide peak obtained after endo H/F3 treatment (Figure 5, sample 3) and 3-fold after 1,2- and 1,3/4-fucosidase treatment (Figure 5, sample 4). Taken together all these data indicate that, in cultured skin fibroblasts, the majority of fucose is 1,6-linked to the core GlcNAc in both neutral and negatively charged N-linked complex and hybrid/high mannose oligosaccharides. Moreover, the core fucosylation is affected to a significantly greater extent than other types of linkages in LAD II cells, particularly in the case of sialylated oligosaccharides.
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To better characterize the species that contain core fucose, the [3H]fucose-labelled N-linked oligosaccharides were subjected to Con A affinity chromatography. Con A chromatography allows the separation of complex tri- and tetrantennary oligosaccharides, which are not retained by the column, from biantennary oligosaccharides, which bind with low affinity, and high mannose/hybrid types, which exhibit strong binding (Cummings, 1993). In the controls, most of the radioactivity was recovered in the complex tri- and tetrantennary and in biantennary fractions (Figure 6A, fractions 1 and 2). After endo H/F3 cleavage of the same amount of oligosaccharides and the removal of the radioactivity associated with the released disaccharide, the [3H]fucose in fraction 1 was reduced by about 40–50%, while the reduction was 90% in fraction 2 and was almost complete in fraction 3, which corresponds to the high mannose/hybrid species (Figure 6B). In the LAD II samples, [3H]fucose was reduced, as expected, in all the Con A–Sepharose fractions; however, fraction 2, corresponding to the biantennary glycans, was relatively more affected (Figure 6A). After endoglycosidase treatment, both fractions 1 and 3 were reduced, whereas fraction 2 was unchanged (Figure 6B). Thus, while in control cells, most of the label in biantennary glycans is associated with 1,6-linked [3H]fucose, the core fucose is virtually absent in the LAD II biantennary species.
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In conclusion, these data indicate that the core fucose is present in several classes of N-linked glycans and it is particularly abundant in biantennary oligosaccharides. In control skin fibroblasts, the core fucose represents about 70% of the total incorporated [3H]fucose: about 20% is contained by complex oligosaccharides, excepting for biantennary species, 40% in biantennary, and 10% in high mannose/hybrid types. Conversely, in LAD II fibroblasts, the core fucose represents about 40% of the total incorporated radioactivity and is equally distributed among triantennary complex and high mannose/hybrid types, although it is nearly absent from biantennary oligosaccharides. This class of glycans is, as a consequence, the most heavily affected structure in the reduction in core fucosylation in LAD II N-linked oligosaccharides.
Because the data obtained by Superdex analyses before and after treatment with neuraminidase suggested that the reduction in fucose incorporation mainly affects sialylated N-linked oligosaccharides, charge fractionation of the [3H]fucose-labelled glycans was performed by anion exchange chromatography, before and after endoglycosidase treatment (Figure 7A and B). An analysis of the controls before endoglycosidase treatment indicated that most of the [3H]fucose is equally distributed between neutral, mono-, and dicharged species (Figure 7A). Conversely, in the LAD II oligosaccharides core fucosylation is decreased, particularly in mono- and disialylated species, corresponding to fractions 1 and 2 (Figure 7A). Endo H/F3 treatment resulted in a significant decrease in radioactivity associated with neutral species in both the controls and LAD II. Moreover, although charged species from controls were significantly reduced by this treatment, the [3H]fucose-labelled charged species that do exist in LAD II were relatively resistant to endo H/F3 treatment (Figure 7B). These results lead to the conclusion that the reduction in core fucosylation in LAD II occurs mainly in mono- and disialylated species.
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The incorporation of [3H]glucosamine, which was used as control in all experiments, was comparable in all samples, indicating that no major differences in glycan compositions exist between the control and LAD II samples (not shown) thus confirming previously reported data (Sturla et al., 2003). Superdex profiles of the [3H]glucosamine-labelled samples before and after neuraminidase treatment are shown in supplementary Figure 3A and B and showed identical profiles for the control and LAD II, indicating that the differences observed are restricted to fucosylated glycans and do not reflect major alterations between samples. Endo H treatment [3H]glucosamine-labelled samples released comparable amounts of the label in control and LAD II samples, mainly corresponding to the monosaccharide (fraction 20–21) (supplementary data, Figure 3C). Conversely, after endo F3 treatment, a peak corresponding mainly to the disaccharide (fraction 19) (supplementary data, Figure 3D) was found in the control, which is consistent with the significantly higher cleavage efficiency of endo F3 in the presence of a core fucose. Moreover, the disaccharide peak was almost undetectable in LAD II, suggesting that the level of core fucose is greatly reduced in this sample (supplementary data, Figure 3D). Con A–Sepharose profiles and QAE–Sepharose charge distribution of [3H]glucosamine-labelled oligosaccharides are reported in the supplementary data, Figures 4 and 5, and had results comparable to those between the control and LAD II oligosaccharides.
The expression and activity of FUT8 are shown in Figure 8. No significant differences were observed between controls and LAD II fibroblasts, thus excluding the argument that the relative more pronounced decrease in core fucosylation in LAD II could be related to a difference in the expression or activity of this enzyme.
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Discussion |
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The findings of this study suggest that, although all types of terminal fucose in N-linked glycans are decreased in LAD II fibroblasts, core fucosylation is to a greater extent affected than other types of fucosylation of the outer arms. While a 6-fold reduction in the total incorporation of [3H]fucose in N-linked oligosaccharides from LAD II cells compared to controls was observed, confirming previously reported data (Sturla et al., 2003)
, the incorporated radioactivity associated with 1,6-linked fucose was decreased by 10-fold. Preliminary analyses using 1,2- and 1,3/4-fucosidases indicated that these type of fucose linkages are also present in fibroblasts and that they are less affected by GDP-L-fucose reduced availability (by 3-fold), compared to core fucosylation. The decrease in core fucosylation observed in LAD II fibroblasts compared to controls cannot be related to a difference in the expression or activity of FUT8, because they were found to be comparable between control and LAD II cells.
The glycan species containing core fucosylation in normal fibroblasts were neutral and mono-/disialylated oligosaccharides in equal proportions, as determined by both Superdex and anion exchange analyses. Con A affinity chromatography after endo H/F3 treatment revealed that the majority of 1,6-fucosylated glycans was of biantennary type, but a significant amount was also found in the form of complex oligosaccharides which do not bind to Con A. These species include tri- and tetrantennary glycans (Cummings, 1993). These data are in agreement with findings reported by Scanlin et al. (1985) who concluded that, in cultured skin fibroblasts, a large fraction of the core fucosylated species are mono- and disialylated biantennary and triantennary glycans (Scanlin et al., 1985; Wang et al., 1990). The level of core fucosylation of species that bind strongly to Con A–Sepharose, indicating the presence of high-mannose and hybrid-type oligosaccharides, was significantly lower than that of the other glycans.
N-linked oligosaccharides from LAD II fibroblasts showed significantly different patterns of 1,6-fucosylation. The core fucose was nearly absent in biantennary glycans, although the decrease was less pronounced in triantennary (about a 5-fold reduction compared to controls) and in the high mannose/hybrid fraction (about 3-fold reduction). These results are consistent with the reported biochemical characterization of FUT8 purified from human platelets (Kaminska et al., 1998). In fact, the purified enzyme showed the highest activity with respect to a triantennary glycopeptide and two times less activity for a biantennary substrate, the asialo-agalactotransferrin glycopeptide. A biantennary oligosaccharide substrate was also used to analyze the activity of FUT8 purified from human skin fibroblasts, but a triantennary substrate was not used for comparison (Voynow et al., 1991). However, it can not be presently excluded that further branching can occur after addition of core fucose. Moreover, core fucose was almost undetectable in negatively charged species containing one or two residues of sialic acid, whereas the neutral species were less affected.
We previously demonstrated that the decreased availability of GDP-L-fucose inside the Golgi of LAD II has important effects on the fucosylated glycan composition (Sturla et al., 2003). In particular, the O-fucosylation of epidermal growth factorlike and thrombospondin type 1 repeats was unaffected. A possible explanation for this finding could depend on different Km values of the fucosyltransferases, for example, that protein O-FucTs have higher affinities for the substrate GDP-L-fucose as compared to those that catalyze the addition of a terminal fucose (Wang et al., 2001). However, another likely reason can be a different localization of the fucosyltransferases (e.g., endoplasmic reticulum vs. Golgi cisternae). In fact, it has been recently reported that O-FucT-1 is a soluble enzyme localized in ER and it is retained in the ER by a KDEL-like sequence at its C terminus (Luo and Haltiwanger, 2005; Okajima et al., 2005). This finding, together with normal levels of O-fucosylation of notch in LAD II fibroblasts (Sturla et al., 2003), also suggests that a different ER-localized GDP-fucose transporter may exist.
Conclusive evidence to explain the high reduction of core fucosylation, in particular, in biantennary, negatively-charged N-linked oligosaccharides is not presently available. It could be because of different Km values of FUT8 for the oligosaccharide acceptors, in different localization of the fucosyltransferases or to a general perturbation of fucosylated glycan production because of the deficiency in GDP-L-fucose transport into the Golgi. It also cannot be excluded that posttranslational events and alternative splicing of FUT8 could affect enzyme activity in different tissues. Further studies are currently underway to characterize fucosylation of N-linked outer arms.
The formation of fucosylated glycans depends on the concerted activity of GDP-L-fucose synthesis, transport into the Golgi and expression of the different fucosyltransferases. Available evidence indicates that modifications at any of these steps can have important consequences on the resulting fucosylated glycans. The data obtained in this study indicate that core fucosylation in fibroblasts is greatly affected by the availability of GDP-L-fucose and that the decreased concentration of this nucleotide-sugar inside the Golgi affects manly some species, in particular biantennary charged glycans.
It is interesting to note that that genetic ablation of FUT8 (responsible for 1,6-fucosyltransferase activity) in the mouse leads to a semilethal phenotype and that the mice die within a few weeks after birth with morphological abnormalities which resemble the non-immunological aspects of the LAD II-like phenotype (Taniguchi, personal communication). Although it is not yet possible to generate a complete picture of the complicated interactions of the various glycosylation-related events to other cell types, the present data obtained using fibroblasts should serve as the basis for developing a better understanding of the mechanisms leading to the severe impairment of growth and development observed in LAD II patients.
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Materials and methods |
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Materials
L-[6–3H]Fucose (2.2 TBq/mmol) was obtained from Biotrend (Köln, Germany) and D-[3H]glucosamine (814 GBq/mmol) was from Amersham Biosciences (Milano, Italy). Biotinylated and agarose conjugates of LCA lectin was purchased from Vector Laboratories (Burlingham, CA, USA). Sepharose-conjugated Con A lectin was from Sigma (Milano, Italy). SuperdexTM peptide HR 10/30 columns, streptavidin-conjugated horseradish peroxidase (HRP) and enhanced chemiluminescence (ECL)-plus Western Blotting Detection System were acquired from Amersham Biosciences. Sephadex-G25, Sephadex-G10, QAE-Sephadex were from Sigma. PNGase F from Flavobacterium meningosepticum, neuraminidase from Arthrobacter ureafaciens, neuraminidase from Vibrio cholerae, and endo H from Streptomyces plicatus were obtained from Roche (Monza, Italy). Endo F3 from Chryseobacterium meningosepticun,
1,6-fucosidase from C. meningosepticum, 1,2-fucosidase from Xanthomonas sp., asialo-galactosylated tetraantennary oligosaccharide, trimannosyl core oligosaccharide, and Fuc-1,6-GlcNAc disaccharide were from Calbiochem (Milano). Canavalia ensiformis -N-acetylglucosaminidase, -galactosidase from bovine testis and -galactosidase from green coffee beans were obtained from Sigma; 1,3/4-fucosidases from almond meal was from Prozyme (San Leandro, CA, USA). The DC Protein Assay and nitrocellulose membrane were obtained from Biorad (Milano, Italy). Streptavidin-AlexaTM 488 conjugate was purchased from Molecular Probes (Eugene, OR, USA). The protease inhibitor cocktail for mammalian cells, pronase and maltoheptaose were obtained from Sigma.
Lectin blotting
Fibroblasts from a LAD II patient of Middle Eastern origin (Etzioni et al., 2002) and two unrelated, age- and passage-matched controls were cultured as described previously (Sturla et al., 2003). Confluent cells, seeded in 10 cm-diameter culture dishes, were washed three times with ice-cold phosphate buffered saline (PBS) and were incubated in 50 mM Tris–HCl pH 8 containing 0.5% sodium dodecyl sulphate (SDS) in the presence of protease inhibitor cocktail (see above) for 30 min at 4°C with agitation. After collecting the homogenates by scraping, they were further incubated for 30 min at 4°C with an orbital rotator and then centrifuged at 10,000 xg for 10 min at 4°C. The protein concentrations of the supernatants were determined by a DC Protein Assay (Biorad). Hydrolysis of N-linked glycans was carried out by enzymatic treatment with PNGase F as described previously (Lin et al., 1994). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) was performed according to the method of Laemmli (Laemmli, 1970). Total proteins, either untreated or treated with PNGase, were separated by SDS–PAGE using a 4–20% (w/v) linear gradient (35 µg per lane) and transferred overnight to a nitrocellulose membrane. The membrane was blocked overnight at 4°C in tris buffered saline (TBS) (50 mM Tris–HCl, 150 mM NaCl, pH 7.0) containing 3% bovine serum albumin (BSA) and incubated for 1 h at room temperature with 0.2 µg/mL biotinylated LCA lectin in TBS buffer containing 0.1% Tween 20 (TBST). After washing in TBST, the blot was incubated for 1 h at room temperature with streptavidin-conjugated peroxidase, diluted 1:10,000 in TBST, and developed with ECL plus Western Blotting Detection System.
Flow cytometry
Cultured fibroblasts were harvested by treatment with trypsin /EDTA, washed once with ice-cold PBS, and suspended in 100 µL of PBS or PBS containing 0.2 M methyl -D-mannopyranoside and 0.2 M methyl -D-glucopyranoside to inhibit specific binding. Biotinylated LCA (2 µg/mL) was added to 2.5 x 105 cells for each sample and incubated on ice for 45 min. The cells were washed twice with 1 mL of PBS and incubated for 30 min on ice in PBS containing 10% BSA, to reduce nonspecific binding. A streptavidin-AlexaTM. 488 conjugate (1 µg/mL) was added to the cell suspensions, followed by incubation for an additional 45 min on ice. The binding of the second reagent alone was performed as a negative control. After two washes with PBS, the cells were analyzed on an EPICS Elite flow cytometer (Beckman Coulter, Milan, Italy).
Labelling of fibroblast and isolation of N-linked oligosaccharides from total glycoproteins
Labelling of the fibroblasts with [3H]fucose and [3H]glucosamine, the purification of total radiolabelled glycoproteins by cell fractionation and the treatment of glycoproteins with PNGase F to release N-linked oligosaccharides were performed as described previously (Sturla et al., 2003). The released N-linked oligosaccharides were desalted on a Sephadex G-25 column (1 x 50 cm) in 10% ethanol in H2O, lyophilized, and suspended in water. Oligosaccharide concentrations were determined by 2,2’–bicinchoninate assay as reported previously (Doner and Irwin, 1992).
Enzymatic digestions
The cleavage of core fucose from the total glycoproteins was achieved by treatment with 1,6-fucosidase from C. meningosepticum. The labelled proteins were incubated in 50 mM sodium phosphate buffer, pH 5.0, in the presence of 0.03% SDS, 0.3% NP-40, 1 mg/mL BSA and 10 mU of the enzyme at 37°C for 48 h. The release of 1,6-fucose from the disaccharide Fuc- 1,6-GlcNAc was achieved by treatment with bovine kidney fucosidase, using 2 mU of the enzyme in 50 mM sodium phosphate buffer, pH 6.0. The removal of sialic acid was achieved by the combined treatment of N-linked [3H]fucose- and 3[H]glucosamine-labelled oligosaccharides with Arthrobacter ureafaciens neuraminidase (0.1 U/mL) plus Vibrio cholerae neuraminidase (0.05 U/mL). Endo H from Streptomyces plicatus (0.15 U/mL) and endo F3 from Chryseobacterium meningosepticun (0.1 U/mL) were used to release the core GlcNAc at the reducing terminal of the N-linked oligosaccharides. All incubations were performed in a 50 mM sodium acetate buffer at pH 5.0 for 24 h at 37°C and the reactions were quenched by heat inactivation for 5 min at 100°C. The higher molecular weight oligosaccharides, which were resistant to the extensive digestion with endo H/F3, were subjected to treatment with 1,2-fucosidase from Xanthomonas sp. (7.5 mU/mL) and 1,3/4-fucosidases from almond meal (0.3 mU/mL); to eliminate all the residues that could negatively influence the cleavage by fucosidases, the oligosaccharides were treated simultaneously with Arthrobacter ureafaciens neuraminidase (0.1 U/mL), Vibrio cholerae neuraminidase (0.05 U/mL), Canavalia ensiformis ß-N-acetylglucosaminidase (1.5 U/mL), ß-galactosidase from bovine testis (0.18 U/mL) and -galactosidase from green coffee beans (1.8 U/mL). This incubation was performed in a 50 mM sodium acetate buffer pH 5 for 48 h at 37°C.
Lectin affinity chromatography
The preparation of [3H]fucose-labelled glycopeptides was performed as described previously (Finne and Krusius, 1982). Briefly, total glycoproteins, suspended in 1% SDS and 1% 2-mercaptoethanol, were diluted 10-fold in 100 mM Tris–HCl, pH 8.0, containing 1 mM CaCl2 and treated with 20 µg/mL of Pronase for 48 h at 60°C. An equal amount of Pronase was added after 24 h of incubation. The digestion was stopped by heat inactivation at 95°C for 5 min. Glycopeptides were desalted by gel filtration using Sephadex G-10 in 0.1 M pyridine-acetate, pH 5.6, lyophilized, suspended in water, and subjected to LCA lectin affinity chromatography as described previously (Cummings, 1994), using a 2 mL column of LCA-agarose. Bound [3H]fucose-labelled glycopeptides were eluted with 10 mL of 100 mM methyl -D-mannopyranoside and 1 mL fractions were analyzed by liquid scintillation counting.
Con A lectin affinity chromatography of N-linked [3H]fucose- and [3H]glucosamine-labelled oligosaccharides was performed as described previously (Cummings, 1993), using a 2 mL column of Con A–Sepharose, at a flow rate 380 µL/min. Bound oligosaccharides were eluted in two steps, first with 13 mL of 10 mM methyl -D-glucopyranoside (to elute biantennary complex-type oligosaccharides) and then 13 mL of 100 mM methyl -D-mannopyranoside (to elute high mannose-type and hybrid-type oligosaccharides) at 60°C. Fractions were collected at 3 min intervals and analyzed by scintillation counting.
Superdex analyses of labelled oligosaccharides
Purified and desalted [3H]fucose- and [3H]glucosamine- labelled N-linked oligosaccharide obtained after PNGase F digestion were analyzed by gel filtration analysis on a Superdex peptide column, using water (0.5 mL/min) as the mobile phase. One minute fractions were collected starting from 14.5 min after the injection and the eluted radioactivity was analyzed by scintillation counting. All samples contained markers for the void volume (dextran) and included volume (fucose), which were analyzed by the phenol sulphuric acid assay (Dubois et al., 1956). Elution of standard markers (asialo, galactosylated tetrantennary oligosaccharide, trimannosyl core oligosaccharide, maltoheptaose and Fuc-1,6-GlcNAc disaccharide) was also determined by a phenol sulfuric acid assay of each fraction.
Charge analyses
Charge analyses were performed on QAE-Sephadex A25 columns using a stepwise salt elution as described previously (Cummings et al., 1989). The oligosaccharides, either digested or mock-digested with neuraminidase, were dissolved in 2 mM Tris base, passed over 1 mL columns of QAE-Sephadex that had been equilibrated in 2 mM Tris base. The unbound fractions represent the neutral species. Elution was performed with 6 mL of Tris containing increasing amounts of NaCl : 20 mM NaCl for the release of oligosaccharides containing one negative charge, 70 mM NaCl for two negative charges, 100 mM NaCl for three negative charges, and 140 mM NaCl for four negative charges. A final elution with 1M NaCl in 0.1 N HCl was carried out to elute compounds with five or more negative charges.
Western blot analysis and enzymatic activity of 1,6-fucosyltransferase (FUT8)
Western blot analyses were performed as described previously (Ito et al., 2003) using a mouse monoclonal antibody against FUT8 (15C6) provided by Dr. S. Ito (Fujirebio, Tokyo, Japan). Confluent fibroblasts from an LAD II patient and two controls, seeded in 10-cm culture dishes, were washed three times with ice-cold PBS, collected by scraping, and suspended, after centrifugation at 500 xg for 5 min, in PBS containing a protease cocktail inhibitor (Sigma) diluted 1:1000. The cells were disrupted by mild sonication and centrifuged at 2000 xg for 15 min at 4°C. The supernatants were quantified for protein concentration using a BCA kit (Pierce, Rockford, Illinois). For a western blot of FUT8, 15 µg of proteins were subjected to SDS–PAGE on a 10% acrylamide gel. After blotting onto a polyvinylidene difluoride (PVDF) membrane, the membrane was incubated for 1.5 h at room temperature with the monoclonal antibody diluted 1/1500, washed three times with TBS containing 0.05% Tween 20 and then incubated with a peroxidase-conjugated secondary antibody diluted 1/1500 (Promega, Milan, Italy). After washing, detection was achieved by using the ECL Western Blotting Detection System (Amersham Biosciences). 1,6-fucosyltransferase activity was determined using 10 µg of proteins and a fluorescent pyridylaminated acceptor sugar substrate (PABA (4-(2-pyridylamino butylamine) sugar), as described previously (Uozumi et al., 1996).
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Supplementary data |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataReferences |
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Supplementary data are available at Glycobiology online (http://glycob.oxfordjournals.org/).
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Acknowledgements |
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This work was supported by the Italian MIUR-FIRB 2002 and MIUR-PRIN 2003 (to M.T) and by a Grant-in aid for Scientific Research(S) 13854010 and the 21st Century Center for Excellence program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to N.T.).
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Abbreviations |
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BSA, bovine serum albumin; Con A, Canavalia ensiformis lectin; ECL, enhanced chemiluminescence; endo F3, endoglycosidase F3; endo H, endoglycosidase H; FUT8,
1,6-fucosyltransferase; GDP-L-fucose, guanosine diphosphate-L-fucose; GlcNAc, N-acetylglucosamine; HRD, horseradish peroxidase; LAD II/CDG IIc, leukocyte adhesion deficiency type II/congenital defect of glycosylation IIc; LCA, Lens culinaris agglutinin; PBS, phosphate buffered saline; PNGase F, Peptide N-glycanase F; TBS, tris buffered saline |
References |
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