Increased fucosylation and reduced branching of serum glycoprotein N-glycans in all known subtypes of congenital disorder of glycosylation I

Nico Callewaert2, Els Schollen3, Annelies Vanhecke2, Jaak Jaeken4, Gert Matthijs3 and Roland Contreras1,2

2 Department of Molecular Biomedical Research, Ghent University and Flanders Interuniversity Institute for Biotechnology, K.l.-ledeganckstraat 35, B-9000 Ghent, Belgium
3 Center for Human Genetics, Campus Gasthuisberg, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium
4 Center for Metabolic Disease, Campus Gasthuisberg, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium

Received on October 31, 2002; revised on December 5, 2002; accepted on December 5, 2002


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The N-glycans present on the total mixture of serum glycoproteins (serum N-glycome) were analyzed in 24 subjects with congenital disorder of glycosylation type I (CDG-I) and 7 healthy, age-matched individuals. No new N-glycan structures were observed in the sera of CDG-I patients as compared with normal sera. However, we observed in all subtypes a significantly increased degree of core {alpha}-1,6-fucosylation of the biantennary glycans as compared to normal, as well as a significant decrease in the amount of triantennary glycans. These serum N-glycome changes appear to be a milder manifestation of some of the changes observed in adult liver cirrhosis patients, which is compatible with the reported steatosis and fibrosis in CDG-I patients. In the CDG-Ia subgroup, the extent of the serum N-glycome changes correlates with the aberration of the serum transferrin isoelectric focusing pattern, which measures the severity of the lack of entire N-glycan chains (primary consequence of CDG-I) in the liver and is the standard diagnostic test for this category of inherited diseases.

Key words: congenital disorders of glycosylation / DNA sequencer / glycopathology / N-glycan structure


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Congenital disorders of glycosylation (CDGs) are a group of inherited disorders characterized by alterations in protein glycosylation (Jaeken et al., 2001Go; Matthijs, 2000Go; Marquardt and Freeze, 2001Go). CDG-I is hallmarked by the absence of one or more N-glycan chains on glycoproteins. The penetrance of the defect is different for different tissues (Dupre et al., 2000Go), possibly because of differences in the rate of glycoprotein production between these tissues. Several subtypes have been described, of which the most common one is CDG-Ia, caused by mutations in the PMM2 gene (Matthijs et al., 1997Go), which results in deficiency in phosphomannomutase (PMM) activity (Pirard et al., 1999Go). This enzyme is essential for the synthesis of GDP-mannose, a substrate that is indispensable in the biosynthesis of N-linked glycans.

The other known CDG-I subtypes (Ib to Ig) have been characterized on the genetic and enzymological level only recently; consequently, the number of reported patients is still low (Niehues et al., 1998Go; Burda et al., 1998Go; Korner et al., 1998Go, 1999Go; Imbach et al., 2000Go; Chantret et al., 2002Go). CDG-I is commonly diagnosed using isoelectric focusing (IEF) of serum proteins, followed by immunodetection of transferrin isoforms (Lof et al., 1993Go). However, this technique does not give detailed structural information on the N-glycans present.

In this study, we report on the quantitative analysis of these N-glycans present on the total mixture of serum proteins of CDG-I patients as compared to those of individuals with normal transferrin IEF patterns. We discuss the relation between these results and those obtained in an accompanying study for adult patients with liver cirrhosis.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Profiling and structural analysis of the N-glycans on total serum proteins using DNA-sequencer- adapted-FACE
First, we wanted to get a global picture of the N-glycan structures that were present on serum glycoproteins of healthy children and detectable using our recently developed technology (Callewaert et al., 2001Go). Therefore, we profiled the N-glycans of a control group of seven healthy children's sera. A surprisingly simple fingerprint arose after desialylation of the glycans using the broad-specificity Arthrobacter ureafaciens sialidase (see Figure 1, panel 2 for an example). From comparison with oligosaccharide standards and from the analysis of the profiles after digestion with different exoglycosidase arrays, we could conclude that the main peaks represented biantennary and triantennary glycans, either unfucosylated or substituted with one fucose residue. Analytical results leading to this conclusion are described in the following paragraphs. (The seemingly odd numbering of the peaks stems from our concern to achieve consistency with our study on the use of serum N-glycome profiling for the diagnosis of liver cirrhosis in adults [Callewaert et al., in preparation]. In Figure 2, panel 2, of the current study, we give a complete peak numbering.)



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Fig. 1. Sequencing of the N-glycans present on serum glycoproteins. GU: glucose units. The symbols used in the structural formulas are: ß-linked N-acetylglucosamine residues, {circ}; ß-linked mannose, {blacksquare}; {alpha}-linked mannose, {square}; ß-linked galactose, {bullet}; and {alpha}-linked fucose, {triangleup}. Panel 1: malto-oligosaccharide ladder. The number of glucose units of the 5-mer and the 10-mer is indicated above the figure. Panels 2–7: profiles obtained after digestion of serum glycoprotein N-glycans with different exoglycosidase mixtures (see Materials and methods for the details of the enzymes and the digestion conditions and Results for a detailed description of the figure). Panel 2: profile of the desialylated serum glycoprotein N-glycans. Panel 3: sialidase + {alpha}-1,3/4/6-fucosidase. Panel 4: sialidase + {alpha}-1,3/4-fucosidase. Panel 5: sialidase + ß-1,4-galactosidase. Panel 6: sialidase + ß-1,4-galactosidase + {alpha}-1,3/4/6-fucosidase. Panel 7: same as panel 6 but with extra ß-N-acetylhexosaminidase digestion. Panels 8–10 provide the profiles obtained after the following digestions of the A2F reference glycan, which is the bisialylated derivative of the glycan structure shown in panel 8: Panel 8, sialidase; panel 9, sialidase + ß-1,4-galactosidase; and panel 10, sialidase + ß-1,4-galactosidase + {alpha}-1,3/4/6-fucosidase. Panel 11 shows the profile obtained from the reference glycan of the structure shown (NA2). Panel 12 represents the profile of the same glycan after ß-1,4-galactosidase digestion. In panels 13 and 14, the profiles of the A3 reference glycan are shown, which is the trisialylated derivative of the glycan structure shown in panel 13, with sialidase digestion and sialidase + ß-1,4-galactosidase digestion, respectively.

 


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Fig. 2. Representative profiles of the desialylated glycans of CDG-Ia patients. GU: glucose units. Structural formula symbols are the same as in Figure 1. Panel 1: malto-oligosaccharides. Panels 2–5: profiles of desialylated serum glycoprotein N-glycans of four CDG-Ia patients. The profiles have been arranged according to decreasing extent of deviation from the normal profile, which is shown in the bottom panel. Panel 2 is the profile of the patient most extremely deviating from the normal pattern, panel 5 represents the mildest glycophenotype observed in the population of 14 CDG-Ia patients, and panels 3 and 4 are representative of the 12 intermediately aberrant profiles observed. In panel 2, the complete numbering of desialylated serum N-glycans is given, as used both in this study as well as in the companion paper (Callewaert et al., in preparation).

 
Peak 3
This is the most abundant N-glycan on total serum glycoproteins. After sialidase digestion (Figure 1, panel 2), its size is estimated to be 9 monosaccharide units by comparison to the malto-oligosaccharide reference ladder. After sialidase/ß-1,4-galactosidase digestion (Figure 1, panel 5), this glycan loses two galactose residues and further loses two GlcNAc residues on digestion with ß-N-acetylhexosaminidase (Figure 1, panel 7). The residual glycan migrates at the position of the Man3GlcNAc2 core N-glycan structure. We conclude that the structure of glycan 3 is biantennary, bi-ß-1,4-galactosylated. This conclusion is corroborated by its exact comigration with a reference glycan of this structure, both undigested (compare panels 2 and 11) and digested with ß-1,4-galactosidase (compare panels 5 and 12).

Peak 6
This glycan is about 1 monosaccharide unit larger than the glycan corresponding to peak 3. On bovine kidney fucosidase digestion (panel 3), this glycan must have been converted to glycan 3, because no new peak appears after fucosidase digestion that could account for a glycan with the large abundance of peak 6. After removal of the two ß-1,4-linked galactose residues, the same conclusion can be drawn from comparison of panels 5 and 6, where the peak marked as 6' is converted in the peak marked with 3'. Peak 6 is resistant to digestion with the almond meal {alpha}-1,3/4-fucosidase, indicating that the fucose residue is of the {alpha}-1,6 core type. Thus, peak 6 represents the biantennary, bi-ß-1, 4-galactosylated core {alpha}-1,6-fucosylated glycan. This is corroborated by the exact comigration of a reference glycan of this structure with peak 6, both undigested and after ß-1, 4-galactosidase and ß-1,4-galactosidase/bovine kidney fucosidase double digestion (compare panels 2 and 8, panels 5 and 9, and panels 6 and 10, respectively).

Peak 8
The glycan corresponding to peak 8 is about 2 monosaccharide units longer than glycan 3, is not digestable by bovine kidney fucosidase (panel 3), and comigrates with a triantennary fully ß-1,4-galactosylated reference glycan (compare panels 2 and 13). Moreover, ß-1,4-galactosidase removes 3 galactose residues from the glycan (shift of 3 glucose units between panels 2 and 6), after which the glycan is 1 monosaccharide unit longer than the remnant of glycan 3, in accordance with the one extra GlcNAc residue that is expected for a triantennary glycan when compared to a biantennary structure.

Peak 9
This glycan is 1 glucose unit longer than the triantennary unfucosylated glycan of peak 8 and is sensitive to both bovine kidney (panel 3) and almond meal fucosidase (panel 4), after which digestions the glycan is converted to peak 3. Thus the fucose residue present on this glycan is {alpha}-1,3/4 linked. We conclude that the glycan of peak 9 is a branch-fucosylated derivative of glycan 8. The exact position of the branch fucose residue cannot be determined using exoglycosidase digestions.

Peak 1
See Figure 2. This peak is barely visible in Figure 1 but was more abundant in most sera. In our study on liver disease in adults (Callewaert et al., in preparation), we determined that this peak represents the agalacto-biantennary, core-{alpha}-1,6-fucosylated glycan.

Analysis of CDG-I patient sera
CDG-Ia
Sera from 14 genetically characterized CDG-Ia patients (aged 28.9 ± 34.6 months) were used for the analysis of desialylated N-glycans, and their pattern was compared to the ones obtained from 7 nonaffected age-matched individuals (average age 46.3 ± 31.3 months). The patient data are summarized in Table I. Representative profiles obtained after desialylation of the glycans are shown in Figure 2, panels 2–5. For comparison, the bottom panel is a representative profile obtained from a normal serum. The profile in panel 2 was the one deviating the most from normal, whereas the one in panel 5 was the least severely aberrant. Profiles 3 and 4 are representative for the intermediately aberrant ones.


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Table I. Summary of CDG-Ia patient data and results of serum N-glycan profiling

 
A qualitatively similar profile is obtained in patient sera as for the normal serum, but important quantitative differences were observed, as shown in Figure 3. Two main changes are apparent. First, the extent of core fucosylation on the fully galactosylated biantennary glycans is increased ( p = 0.02), as peak 6 becomes more abundant. Second, the amount of the fully galactosylated triantennary glycan peak 8 was decreased ( p = 0.001). The percentage changes in these parameters as compared to the average profile of the nonaffected control group are shown for all 14 investigated patients in Table I.



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Fig. 3. Statistical analysis of the height of the major individual N-glycan peaks in the serum protein N-glycan profile of healthy controls, CDG-Ia patients, and a pool of non-Ia CDG-I subtypes. The error bars represent the 95% confidence interval for the mean. Differences in the means were assessed using the two-tailed Student t-test, and significant differences at the 0.05 confidence level are flagged with an asterisk, whereas those significant at the 0.005 confidence level are flagged with a double asterisk. The structures of the respective glycans are given below the statistics, and the symbols used in drawing these structures are the same as clarified in the legend to Figure 1.

 
Finally, it is remarkable that a gradient is observed over the patient population in the extent of these changes. (In Table I, the patients are arranged in decreasing order of aberrance of the glycophenotype, determined by adding the % change in the two measured parameters described.) Because of this gradient, we tried to correlate the observed quantitative changes to other known and measurable disease-related parameters. An obvious parameter would be the residual PMM activity (values can be found in Table I), with more residual activity producing a less severe glycophenotype. However, we did not find a significant correlation (p>0.05) with residual skin fibroblast PMM activity. The reasons for this can be many. For example, the standard deviation for the residual PMM activity measurements in fibroblasts is reportedly very large (Jaeken et al., 1997Go), which may hamper correlation analysis, and fibroblasts may also differ in passage number and so on, perhaps masking a potentially significant correlation. Alternatively, skin fibroblast residual PMM activity may be a bad indicator for the functioning of the liver, which is the organ producing most of the abundant glycoproteins in serum (Dupre et al., 2000Go).

We also compared the extent of the serum protein N-glycosylation alterations with the clinical presentation of the patients. The symptoms of CDG-Ia can vary from a severely debilitating disease to a relatively mild mental retardation. The clinical presentation of the CDG-Ia patients studied here is indicated in Table I in terms of severe, moderate to severe, mild, and very mild, as explained in Materials and methods. For two affected siblings, both with a low score for their serum glycoprofiles, no detailed clinical information could be obtained. Comparison of the extent of the glycophenotype with the clinical evaluation showed no obvious concordance. It has also been observed before that the severity of the disease does not correlate well with residual PMM activity (Grunewald et al., 2001Go), and this noncorrelation of the severity of the clinical presentation with biochemical disease markers remains a puzzle in the field of CDG-Ia.

The routine diagnostic assay for the CDGs is IEF of serum transferrin, which basically measures the degree of sialylation of transferrin. In CDG-I, the loss of sialic acid in transferrin is due to the loss of complete N-glycan chains (Yamashita et al., 1993Go). In this respect, densitometric scanning of the IEF patterns, together with knowledge of the N-glycan structures present on transferrin, can give an approximation of the degree of underoccupation of transferrin N-glycosylation sites. This parameter is included in Table I as the % of serum transferrin N-glycans missing (as compared to a nonaffected control population's transferrin). Within the CDG-Ia group, the abundance of the bigalacto, core-{alpha}-1,6-fucosylated biantennery glycan (peak 6) was positively correlated in a linear way with this degree of transferrin N-glycan site underoccupation (Figure 4A; Pearson r = 0.76; p = 0.003). The abundance of the trigalacto triantennary glycan (peak 8) was negatively correlated in an exponential way with the transferrin N-glycan site underoccupation (Figure 4B shows the linearized plot; Pearson r = –0.92; p = 0.00001). Thus the decrease in abundance of this triantennary glycan is more sensitive to the N-glycosylation site underoccupancy than is the increase in abundance of the core fucosylation.



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Fig. 4. The serum glycoprotein N-glycan profile alterations in CDG-Ia correlate with the degree of underoccupation of serum transferrin N-glycosylation sites. In (A) the % of the total glycans contributed by the core-fucosylated peak 6 is plotted against the % underoccupation of transferrin N-glycosylation sites for the 13 CDG-Ia patients for whom full data were available. A positive correlation is evident from this graph (Pearson's r = 0.76; p = 0.003). (B) shows the linearized plot of the negative exponential correlation between the abundance of the trigalactosylated triantennary glycan peak 8 and the % underoccupation of transferrin N-glycosylation sites (Pearson's r = –0.92; p = 0.00001). The horizontal lines in both plots represent the average value for the respective parameter over the healthy control group.

 
The other known CDG-I subtypes
CDG-I is the group of disorders with malfunctioning of steps in the biosynthesis of dolichyl-PP-linked Glc3Man9GlcNAc2, resulting in a less efficient occupation of the N-glycosylation sites on nascent proteins in the endoplasmic reticulum. Because this biochemical deficiency is the same for all of these subtypes, we wanted to evaluate whether the serum N-glycome changes already reported for CDG-Ia would also occur for these other CDG-I subtypes. Therefore, we analyzed the serum glycoprotein N-glycans of one patient with CDG-Ib (phosphomannose isomerase deficiency; Niehues et al., 1998Go), two with CDG-Ic ({alpha}-1,3-glucosyltransferase deficiency; Burda et al., 1998Go; Korner et al., 1998Go), two with CDG-Id ({alpha}-1,3-mannosyltransferase deficiency; Korner et al., 1999Go), three with CDG-Ie (dolichol-phosphate-mannose synthase deficiency; Imbach et al., 2000Go), and two with CDG-If (Schenk et al., 2001Go; Kranz et al., 2001Go). (CDG-Ig [dolichyl-P-mannose: Man7GlcNAc2-PP-dolichyl mannosyltransferase deficiency] was only very recently characterized, and the publication by Chantret et al. [2001] appeared during the review process of the current study. Consequently, no patient samples could be included.) The resulting profiles are shown in Figure 5. For most of these disorders, the limited number of known patients precludes sound statistical analysis, so we were forced to compare the pooled group of profiles of the non-CDG-Ia disorders (N = 10) with the control group (N = 7). As in CDG-Ia, this analysis revealed increased core fucosylation of the biantennary bigalacto glycans ( p = 0.01) and a lower abundance of the trigalacto triantennary glycan (peak 8; p<10E-6).



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Fig. 5. Analysis of the desialylated serum protein N-glycans of a number of patients with other subtypes of the CDG-I group of syndromes. GU: glucose units. The abbreviation of the subtype is given in the top left corner of the panels. The genes that are deficient in these CDG-I subtypes are as follows: for CDG-Ib, phosphomannose isomerase; CDG-Ic, {alpha}-1,3-glucosyltransferase; CDG-Id, {alpha}-1,3-mannosyltransferase; CDG-Ie, dolichol-phosphate-mannose synthase; and CDG-If, MPDU gene (human homolog of Chinese hamster ovary Lec 35). As in CDG-Ia (Figure 2), one observes the increased fucosylation and the decreased ratio between triantennary and biantennary glycans.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In an accompanying study, we show that adults with liver cirrhosis can be reliably differentiated from patients with noncirrhotic or precirrhotic liver disease based on differences in the N-glycan profile of total serum proteins. With these data in hand, a clinically applicable technology was developed to measure these glycomics profiles (Callewaert et al., in preparation).

Here, we report that the same assay used in the pediatric context of CDG-I also detects changes in the serum N-glycome. Moreover, these changes seem to be a subset of what we observed in liver cirrhosis: Both increased core fucosylation of the biantennary glycans and reduced abundance of the trigalacto triantennary glycan were observed, as in cirrhosis. However, in adult's liver cirrhosis, we also observed strong undergalactosylation of the biantennary glycans and a strong increase in the abundance of glycans with a bisecting GlcNAc residue (GnT-III product). In contrast, for CDG-I, the agalacto core-fucosylated biantennary glycan (peak 1) had no significantly increased presence ( p>0.05), although there was a trend toward higher values, especially for the non-CDG-Ia subtypes ( p = 0.07). In adult's liver cirrhosis, the bisecting GlcNAc residue is mainly present on peak 7 (see Figure 2), but the abundance of this peak in healthy children's sera and in almost all CDG-I cases was low (average <3% of the total N-glycans), and this peak was not overrepresented in CDG-I ( p>0.1).

The function of the liver is severely affected by the CDG-I syndromes. In the rare studies where this could be assessed using liver biopsy, the histological spectrum includes steatosis and severe fibrosis (Kristiansson et al., 1998Go). Therefore, we suggest that the serum N-glycome changes that we describe here are a milder manifestation of the glycome changes we observed in adult's cirrhosis. We hope to be able to perform a biopsy-controlled study (which is difficult to organize in this pediatric population) or to obtain serum samples from previous biopsy-controlled studies to confirm this. The liver insufficiency in CDG-I patients is a cause of death contributing to the 30% lethality before age 5 in these patients and probably contributes to the hemorrhaging and thromboembolic complications by reduced production of especially factors V and XI, protein C, and antithrombin. Therefore, a serum marker for the routine noninvasive assessment of the degree of chronic liver damage might contribute to the clinical management of these patients.

In CDG-Ia, where a sufficiently large patient population could be studied, there was a significant correlation of the extent of the observed serum protein N-glycan changes with the severity of the change in the serum transferrin IEF pattern. This latter assay measures the extent of underoccupation of transferrin's two N-glycosylation sites. Therefore it is a direct measure for the effect that the CDG-I causing mutations have on the hepatocyte's N-glycosylation pathway efficiency.

As to the glycosyltransferases that are implicated in the observed N-glycosylation changes, the up-regulation of the core-{alpha}-1,6-fucosyltransferase has been documented in chronic liver disease (cirrhosis and hepatocellular carcinoma; Noda et al., 1998Go). Increases in fucosylation have also been observed on transferrin, {alpha}1-antitrypsin (Mills et al., 2001Go), and {alpha}1-acid glycoprotein (Van Dijk et al., 2001Go) in CDG-I sera.

The reduction in the amount of triantennary serum glycoprotein N-glycans has not been reported in chronic liver disorders but seems to be a general hallmark of these diseases because it was also one of the most reliable changes observed in our adult's study. The obvious cause for this reduced branching pattern would be a down-regulation of hepatocyte N-acetyl-glucosaminyltransferase IV activity, but this could not be confirmed due to the difficulties involved in obtaining patient liver biopsies. In contrast to the reduced amount of triantennary serum glycoprotein N-glycans on total serum proteins that we observed, Mills et al. (2001)Go reported that the branching of {alpha}1-antitrypsin and transferrin was actually increased in CDG-I, as measured by mass spectrometry. Therefore, different serum glycoproteins' branching patterns can apparently react differently to the primary CDG-I biochemical defects.

Another factor that could contribute to the observed serum N-glycome changes is that the serum protein spectrum produced by the diseased liver is quantitatively shifted somewhat from normal. The final glycosylation pattern of a protein is determined both by the characteristics of each protein and by the nature and condition of the secreting cell.

That a reduction in the extent of N-glycosylation site occupancy due to CDG-I has a change in the proportion of serum protein N-glycans as a consequence might be relevant in the framework set by a recent hypothesis that attempts to explain the relatively high carrier frequency of CDG-I-causing mutations (as high as 1/70 for the most frequent R141H amino acid substitution in PMM2) (Freeze and Westphal, 2001Go). The hypothesis is that this high carrier frequency reflects a certain degree of competitive advantage caused by a slightly reduced level of N-glycosylation in carriers of the mutation, especially with respect to viral resistance. Indeed, the viral envelope assembly of some viruses seems to be exquisitely sensitive to the glycosylation status of the envelope glycoproteins, with just a slight disturbance being sufficient to disrupt the orderly packing of the envelope and thus effectively blocking virus replication. Experimental evidence that at least transferrin can be underglycosylated in healthy carriers of a CDG-Ia mutation was recently obtained (Helander et al., 2001Go). Our results open the possibility that underoccupation of N-glycan sites due to the carrying of a CDG-Ia-causing mutation could also cause changes in the proportion of different serum N-glycans, potentially further modulating glycan-structure dependent functions of certain serum glycoproteins. We will make efforts to study whether the N-glycan changes in CDG-I patients observed here are also present (albeit with an expected reduced penetrance) in the sera of carriers of a single CDG-Ia-causing mutation.

Currently, we are applying the described methodology in the analysis of sera of genetically still uncharacterized CDG subtypes. We expect that the N-glycan structural information obtained there will be valuable in pinpointing the genetic defects in this growing family of disorders.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Reference glycans (NA2, NA3, and A2F) were obtained from Glyko (Novato, CA). All other chemicals were of the highest purity available.

N-glycan analytical methods
Sample preparation procedure
Five microliters of sera of genetically confirmed CDG-Ia (N = 14), CDG-Ib (N = 1), CDG-Ic (N = 2), CDG-Id (N = 2), CDG-Ie (N = 3), CDG-If (N = 2) patients and individuals with normal serum transferrin IEF pattern (N = 8) were incubated with 50 µl of RCM buffer (8 M urea, 360 mM Tris, pH 8.6, 3.2 mM ethylenediaminetetra-acetate [EDTA]) at 50°C for 1 h to denature the serum proteins. Subsequently, these mixtures were loaded in the wells of a Multiscreen-IP plate (Millipore, Bedford, MA), prepared as described previously (Papac et al., 1998Go). Reduction, iodoalkylation, and deglycosylation steps were performed according to reported procedures (Papac et al., 1998Go).

APTS derivatization reaction and cleanup
N-glycan derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) and removal of excess free label were as described recently (Callewaert et al., 2001Go). Briefly, the deglycosylation mixture was evaporated to dryness, and a 1 µl 1:1 mixture of 20 mM APTS (Molecular Probes, Eugene, OR) in 1.2 M citric acid and 1 M NaCNBH3 in dimethyl sulfoxide was added. The derivatization was allowed for 18 h at 37°C. After this, the reaction was quenched by the addition of 10 µl deionized water. Excess not reacted APTS was removed using a bed of Sephadex G10 packed in a Multiscreen filterplate (Millipore). After sample application, the resin beds were eluted three times by addition of 10 µl of water and a 10-s centrifugation at 750xg in a tabletop centrifuge equipped for handling 96-well plates (Eppendorf, Hamburg, Germany). The eluate was evaporated to dryness. After evaporation, the derivatized glycans were reconstituted in 5 µl deionized water.

Exoglycosidase digestions
One-microliter batches of the cleaned up derivatized N-glycans were transferred to 250-µl polymerase chain reaction tubes or tapered-well microtiter plates for treatment with exoglycosidase arrays. In this study all digestions were done by overnight incubation at 37°C in 10 µl 20 mM sodium acetate, pH 5.0. The enzymes used in this study are: A. ureafaciens sialidase (2 U/ml, Boehringer Mannheim, Germany); D. pneumoniae ß-1,4-galactosidase (1 U/ml, Boehringer Mannheim); jack bean ß-N-acetylhexosaminidase (30 U/ml, Glyko); jack bean {alpha}-mannosidase (100 U/ml, Sigma Biochemicals, Bornem, Belgium); bovine epididymis {alpha}-fucosidase (0.5 U/ml, Glyko); and almond fucosidase (3 mU/ml, Glyko). Unit definitions are as specified by the enzyme suppliers. After completion of the digestions, the samples were evaporated to dryness and reconstituted in 1 µl deionized water.

Analysis by DNA-sequencer-adapted FACE
To each sample, 0.5 µl of the rhodamine-labelled Genescan 500 standard mixture (Perkin Elmer, Foster City, CA) and 1 µl deionized formamide was added for internal referencing and to facilitate sample loading, respectively.

All experiments were performed on an Applied Biosystems 377A DNA sequencer (Perkin Elmer) adapted for cooling as described (Callewaert et al., 2001Go). The 36-cm gel contained 10% of a 19:1 mixture of acrylamide:bisacrylamide (89 mM Tris, 89 mM borate, 2.2 mM EDTA). Prerunning was done at 3000 V for 1 h. The electrophoresis voltage during separation was 3500 V, and data were collected for 3 h (separation of glycans up to 15 glucose units in size). Data analysis was performed using the Genescan 3.1 software (Applied Biosystems, Foster City, CA). Using the positions of the peaks of the internal rhodamine-oligonucleotide standard, all lanes on the same gel were aligned with the lane containing the APTS-labeled malto-oligosaccharide standard. After this alignment, samples on different gels can be easily and reliably compared by aligning the positions of the malto-oligosaccharides present on both gels. For clarity and to allow black-and-white reproduction of the figures, the peaks corresponding to the rhodamine-labeled internal standards have been omitted after the alignment procedure.

Measurement of PMM activity in fibroblasts
PMM enzymatic activity was measured in fibroblasts as described (Jaeken et al., 1997Go).

Serum transferrin IEF and estimation of the degree of underoccupancy of transferrin N-glycosylation sites
Routine serum transferrin IEF was performed as described (Wada et al., 1992Go), and the patterns were quantified using densitometry. Serum transferrin is glycosylated mainly (>93% according to Yamashita et al., 1993Go) with biantennary bisialylated oligosaccharides, with a much lower abundance of the trisialylated triantennary and tetrasialylated tetraantennary glycans. This knowledge allows researchers to approximate the degree of underglycosylation of transferrin from the IEF pattern, for comparison purposes. So, for example, for the bisialylated IEF band, the approximation is that this species can only arise by having one fully sialylated biantennary N-glycan (neglecting the small amount of protein that will have two monoantennary glycans, each contributing one sialic acid charge).

Formally, this approximation is expressed as: Mol glycans/mol transferrin = {0[%0] + 1[%1] + 1[%2] + 2[%3] + 2[%4] + 2[%5] + 2[%6] + 2[%7]}/100, with [%X] for 0xXx7 the intensity of the IEF band with X sialic acid derived charges, in % of total transferrin measured in the sample. Ratioing the obtained value for the patient to the average of the thus calculated values of the control individuals gives a quantitative measure for the degree of underoccupation of serum transferrin N-glycan sites in the patients as compared to a nonaffected control population.

Statistical analysis
All statistical calculations were performed in the SPSS 10.0 software package (SPSS, Chicago, IL). Normal distribution of the measured parameters over the tested populations was assessed using the Kolmogorov-Smirnov test with Lillieford correction at the 0.05 significance level. No evidence for deviations from a normal distribution were found, and thus, means comparisons were done using the appropriate Student t-tests and correlations were tested according to bivariate Pearson correlation analysis.

Clinical assessment of disease severity
Patients with CDG-Ia have a variable phenotype (de Lonlay et al., 2001Go). Therefore, the patients were stratified, as proposed by Grunewald et al. (2001)Go. In practice, the clinical outcome was graded as mild, moderate, or severe on the basis of the predominant clinical symptoms. Patients with the classical CDG-Ia picture including failure to thrive, recurrent infections, multiorgan involvement, convulsions, retinitis pigmentosa, severe developmental delay and inability to walk, peripheral neuropathy, or scoliosis were labeled as severe. This group includes patients who died early in childhood. Patients with mild developmental delay but who learned to sit and walk, had no failure to thrive, no feeding difficulties, no visceral organ problems, and no disturbance of clotting factors and transaminases were classified as mild. Between these extremes were patients with a moderate clinical expression. In all patients, psychomotor retardation to a very variable degree, strabismus, hypotonia, and cerebellar hypoplasia were documented.


    Acknowledgements
 
We wish to thank Dr. H. Carchon for support in the statistical analysis of the data and for providing the serum transferrin IEF data. The members of the Carchon, Jaeken, and Matthijs labs are acknowledged for their support. Nico Callewaert is a research assistant of the Fund for Scientific Research Flanders. Research partially funded by grants from Ghent University (GOA No. 12052299) and by the Euroglycan project of the European Commission.

1 To whom correspondence should be addressed; e-mail: roland.contreras{at}dmb.rug.ac.be Back


    Abbreviations
 
APTS, 8-amino-1,3,6-pyrenetrisulfonic acid; CDG, congenital disorder of glycosylation; EDTA, ethylenediaminetetra-acetate; IEF, isoelectric focusing; N-glycosylation, asparagine-linked glycosylation; PMM, phosphomannomutase.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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