Defect in N-glycosylation of proteins is tissue-dependent in Congenital Disorders of Glycosylation Ia

Thierry Dupré1,2, Anne Barnier2, Pascale de Lonlay3, Valérie Cormier-Daire4, Geneviève Durand2,6, Patrice Codogno5 and Nathalie Seta2,7

2Biochimie A, Hôpital Bichat, 75877 Paris cedex 18, France, 3Service de Pédiatrie et maladies métaboliques, 4Service de Génétique Médicale, INSERM U393, Hôpital Necker, 75743 Paris cedex 15, France, 5INSERM U504, 94807 Villejuif cedex, France, 6Faculté de Pharmacie, Université Paris XI, 92260 Châtenay-Malabry cedex, France, and 7Faculté de Pharmacie, Université Paris V, 75270 Paris cedex 06, France

Received on March 17, 2000; revised on July 27, 2000; accepted on July 28, 2000.


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
The biochemical hallmark of Congenital Disorders of Glycosylation (CDG) including type Ia is a defective N-glycosylation of serum glycoproteins. Hypoglycosylated forms of {alpha}1-antitrypsin have been detected by Western blot in serum from CDG Ia patients. In contrast we were not able to detect hypoglycosylation in {alpha}1-antitrypsin synthesized by fibroblasts, keratinocytes, enterocytes, and leukocytes. Similarly no hypoglycosylation was detectable in a membrane-associated N-linked glycoprotein, the facilitative glucose transporter GLUT-1 and also in serum immunoglobulin G isolated from sera of CDG Ia patients. We conclude that the phenotypic expression of CDG Ia is tissue-dependent.

Key words: {alpha}1-antitrypsin/CDG/ GLUT-1/ immunoglobulin G/ protein glycosylation


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Congenital Disorders of Glycosylation, formerly called carbohydrate-deficient glycoprotein syndromes (CDG), are a recently described group of inherited disorders (Jaeken et al., 1984Go) characterized by abnormal N-glycosylation of proteins. CDG I is characterized by the lack of one or more N-glycan chains on serum N-glycoproteins (Wada et al., 1992Go). This glycosylation abnormality is used for biological diagnosis purpose, which is usually performed by isoelectrofocusing of serum transferrin (Stibler et al., 1991Go), or by Western blot of different serum glycoproteins (Seta et al., 1996Go). The transferrin remaining N-glycan chains, when they exist, have been shown to have a normal structure (Wada et al., 1992Go; Yamashita et al., 1993Go).

Among the different types of CDG I, CDG Ia, by far the most frequent, is due to mutations in the PMM2 gene (Matthijs et al., 1997Go), which encodes phosphomannomutase (PMM), a key enzyme that controls the synthesis of GDP-mannose and is essential for the generation of N-glycans.

A lot of data are available on hypoglycosylation of liver-derived serum glycoproteins (Harrison et al., 1992Go; Yuasa et al., 1995) and of liver proteins (Henry et al., 1997Go) in CDG Ia patients. But very little is known about N-glycoproteins from other origins. On one hand, a N-glycosylation defect of cerebrospinal fluid ß-trace protein, a glycoprotein synthesized by glia-rich fractions of the brain, was evidenced in CDG Ia patients (Pohl et al., 1997Go; Grünewald et al., 1999). On the other hand, a few studies have shown that some membrane-bound and secreted N-glycoproteins were fully glycosylated in fibroblasts or lymphoblastoid cells from more or less defined CDG I patients (Marquardt et al., 1995Go; Bergmann et al., 1998Go; Ichisaka et al., 1998).

Thus, the aim of our study was to investigate the relationship between PMM deficiency and hypoglycosylation of glycoproteins in different tissues from well-defined CDG Ia patients. Our results show that no hypoglycosylated glycoproteins could be detected among the extrahepatic glycoproteins investigated. This suggests that the defect in N-glycosylation of protein is tissue-dependent in CDG Ia.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Our study was performed using cells and serum from 8 CDG Ia patients, with typical clinical features and biological abnormalities, i.e., typical serum transferrin isoelectric focusing and Western blot patterns (Figure 1) and a deficiency in leukocyte PMM activity (CDG Ia: 0.7 ± 0.7 mU/mg protides; control: 4.7 ± 1.5 ).



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Fig. 1. Isoelectric focusing pattern of serum transferrin (A) and Western blot patterns of serum transferrin (B) and {alpha}1-antitrypsin (C) from control and eight CDG Ia patients.

 
As shown in Figure 1, the Western blot analysis of {alpha}1-antitrypsin (AAT) from control serum shows a unique band corresponding to the fully glycosylated form of the glycoprotein which contains three N-glycan chains per mole of AAT. As expected, an additional band with a lower molecular weight corresponding to the hypoglycosylated form of AAT is observed in the serum of all the CDG Ia studied patients.

Although serum AAT is essentially of hepatic origin, many other cell types are able to produce this protein (Ikuta et al., 1982Go; Perlmutter et al., 1989Go). Surprisingly, AAT secreted by skin fibroblasts from either control donors or CDG Ia patients (n = 5) was fully glycosylated (Figure 2), although with an apparent molecular weight slightly higher than in serum. A similar result, fully glycosylated AAT, was obtained when keratinocytes (n = 2), enterocytes (n = 2) and peripheral blood mononuclear cells (PBMC) (n = 7) were considered (data not shown). After N-glycanase treatment, an identical molecular weight was observed for AAT secreted by CDG Ia and control fibroblasts corresponding to polypeptide, which thus demonstrates that N-glycans are as well represented in control AAT as in CDG Ia AAT (Figure 2) and that the increase in apparent molecular weight is due to more highly branched glycans at the three glycosylation sites. In addition AAT secreted by both control and CDG Ia fibroblasts has normal biological properties as determined by its ability to bind to neutrophile elastase (data not shown).



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Fig. 2. Western blot of AAT synthesized by hepatocytes and fibroblasts from control and a CDG Ia patient. All other patients patterns were similar. (A) Serum AAT which is mainly secreted by hepatocytes presents a characteristic pattern in a CDG Ia patient. In contrast no difference between control and CDG Ia patients is observed for AAT synthesized by fibroblasts either in cell culture medium or in cell homogenates. (B) Western blot pattern of AAT with (+) or without (-) N-glycanase treatment indicates that the differences observed in (A) are due to differences of glycosylation.

 
However, we cannot exclude that hypoglycosylated synthesized forms of AAT are not secreted in CDG Ia cells. In order to explore this possibility we analyzed the AAT profile in cell homogenates. We failed to detect differences in the electrophoretic mobility of AAT present in control and CDG Ia fibroblasts (Figure 2) and other cell types studied (data not shown).

Next we considered another major class of serum glycoproteins, immunoglobulin G (IgG), which is synthesized by lymphocytes but not by hepatocytes. Two bands corresponding to heavy and light chains were detected by Western blotting in both control and CDG Ia sera. Only the heavy chain is N-glycosylated with a single N-glycan chain per mole. No difference in the mobility of heavy chains was detected. A similar shift in the mobility of control and CDG Ia IgG was observed after N-glycanase treatment. An identical result was found when PBMC homogenate was tested (Figure 3).



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Fig. 3. Western blot of IgG with (+) or without (-) N-glycanase treatment in serum and in peripheral blood mononuclear cells (PBMC) homogenate present no difference between control and one of the CDG Ia patients. All other patients patterns were similar.

 
Finally, we investigated the electrophoretic profile of a plasma membrane-associated N-linked glycoprotein, GLUT 1, the ubiquitous facilitative glucose transporter. It is a monoglycosylated glycoprotein with an apparent molecular weight of 55,000 Da. The loss of the N-glycan chain decreases its apparent molecular weight to 38,000 Da (Asano et al., 1991Go). Whatever the origin of GLUT-1, control or CDG Ia cells, only one band corresponding to the glycosylated form of the protein was observed (Figure 4).



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Fig. 4. Western blot of GLUT-1 from cultivated skin fibroblast homogenate with (+) or without (-) N-glycanase treatment present no difference between control and one of the CDG Ia patients. All other patients patterns were similar.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Reduced N-glycosylation of liver-derived serum glycoproteins is the central diagnostic feature of CDG I. Strikingly, we show in the present work that secreted and membrane-bound glycoproteins with extrahepatic origins (beside the ß-trace protein) do not present the same defects in N-glycosylation. Our results are in agreement with the ones of other authors. Secreted glycoprotein, X31 influenza hemagglutinin, analyzed by SDS-PAGE, was found to be normally glycosylated and folded in X31 transfected fibroblasts from three CDG I patients (Marquardt et al., 1995Go). In the same way, Ichisaka et al. (1998) showed that no abnormal secreted forms of ß-hexosaminidase and {alpha}-fucosidase were detected in media from cultured CDG I fibroblasts. In another study, no apparent defects in the gross glycosylation process of defined complex glycosylated proteins such as the surface-expressed major histocompatibility complex class I glycoprotein or secreted immunoglobulin were identified in three CDG Ia patients lymphoblastoid cell lines (Bergmann et al., 1998Go). Although we have studied a limited number of tissues (fibroblasts, enterocytes, keratinocytes, leukocytes), our study is the first one to bring together evidence from more than one cell type, and for miscellaneous glycoproteins.

Nevertheless, PMM-deficient fibroblasts from patients with CDG Ia, labeled with tritiated mannose, show reduced protein glycosylation and make smaller sized lipid-linked oligosaccharides (Powell et al., 1994Go). We have also observed such alterations in the early steps of glycoprotein glycan biosynthesis (Dupré et al., 1999Go) in those fibroblasts in which no major modifications of the glycoproteins are observed.

In fact, in addition to lower molecular weight bands seen in CDG I, a band with normal molecular weight is always seen in Western blot pattern of liver-derived serum N-glycoproteins (Krasnewich et al., 1995Go; Seta et al., 1996Go). This indicates that a fraction of the serum N-glycoproteins of hepatic origin is normally glycosylated, confirming that the genetic defect, i.e., PMM deficiency, is leaky. It seems that defects in N-glycosylation will only occur in tissues or organs in which the need for glycosylation substrates is greater than the supply. We hypothesize that only metabolically active cells, as such post-natal hepatocytes, or cells with a specific sensitivity, as such fetal brain cells, are affected.

Indeed, two different reports showed that serum N-glycoproteins from fetus with CDG I present a normal electrophoresis pattern, thereby preventing CDG I antenatal diagnosis (Clayton et al., 1993Go; Stibler and Skovby, 1994Go). One of these report shows, just before, and during the first few weeks after birth, the gradual evolution from normal to pathological transferrin and AAT profiles (Clayton et al., 1993Go). Moreover, the pattern of liver glycoproteins from a CDG Ia patient showed abnormally glycosylated glycoprotein precursors compared to non-CDG (Henry et al., 1997Go). These results could be interpreted by the rapid hepatic maturation that occurs after birth.

On the other hand, the same glycosylation abnormality was found for the beta-trace protein, a glycoprotein with brain origin, in the cerebrospinal fluid of CDG I patients (Pohl et al., 1997Go; Grünewald et al., 1999). The central nervous system has a very high level of anabolism during fetal life and, in our opinion, thus could be related to the clinical features of CDG Ia patients, which are dominated by central nervous system disorders.

However this unaffected glycoprotein synthesis observed in CDG Ia cells other than hepatocytes and brain cells might be specific to type Ia. It could not be the case in CDG Ic which is associated with a reticular glucosyltransferase deficiency (Korner et al., 1998Go), since in yeasts transformed with the mutated glucosyltransferase gene observed in CDG Ic, the newly synthesized CPY protein, a vacuolar protein which carries four N-linked oligosaccharides, is abnormally glycosylated, lacking one or two oligosaccharide chains (Imbach et al., 1999Go).

In the CDG Ia cell types studied, PMM activity is deficient but this deficiency does not affect the structure of all the synthesized glycoproteins. The existence of an as yet uncharacterized secondary metabolic pathway able to produce GDP-mannose bypassing PMM can be hypothesized. Residual PMM activity, which is sufficient for normal glycosylation in cells with low metabolism, could also fit our results. This hypothesis seems more likely, since all except one mutated PMM recombinant enzymes have a residual activity (Pirard et al., 1999Go). The only mutation for which the corresponding recombinant enzyme has no activity at all (Pirard et al., 1999Go), the R141H mutation, has never been observed in a homozygote situation (Matthijs et al., 1998Go) and is considered to be lethal (Matthijs et al., 1998Go; Pirard et al., 1999Go). This observation has to be related to others, such as the N-acetylglucosaminyl transferase I knockout mouse model which is not viable (Ioffe et al., 1997Go; Metzler et al., 1994Go), indicating that the total absence of some enzyme activities involved in the glycoprotein biosynthesis may have severe consequences.

In summary, in CDG Ia patients, bearing a deficiency in PMM activity, the abnormality in glycoprotein glycosylation, i.e., the lack of one or more N-glycan chain on N-glycoproteins, cannot be generalized, since our results suggest that it is limited to serum glycoproteins synthesized by hepatocytes and to the cerebrospinal fluid ß-trace protein.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Reagents
Peptide-N4-(N-acetyl-glucosaminyl) asparagine amidase F (EC 3.5.1.52) (N-glycanase) from Flavobacterium meningosepticum was from Boehringer (Meylan, France). Donkey anti-rabbit IgG antibody conjugated to horseradish peroxidase, Ficoll-Paque Plus, enhanced chemiluminescent (ECL) substrate and Hyperfilm-ECL were from Amersham (Les Ulis, France). Rabbit anti-human AAT and anti-human IgG antisera were from Behring (Rueil-Malmaison, France). Anti-human GLUT 1 was from Valbiotech (Paris, France). Dulbecco’s modified essential medium (DMEM), RPMI 1640 medium, and fetal calf serum (FCS) were obtained from Gibco (Cergy-Pontoise, France). All other chemicals were of analytical grade.

Patients
Samples (serum and cells) were collected from eight patients diagnosed as having CDG I based on clinical presentation and biochemical analysis: serum transferrin isoelectric focusing and serum glycoprotein (transferrin, {alpha}1-antitrypsin, {alpha}1-acid glycoprotein, haptoglobin) Western blotting. The CDG Ia diagnosis was confirmed by measuring phosphomannomutase activity in leukocytes, as described elsewhere (Van Schaftingen and Jaeken, 1995Go). Control samples were also obtained from healthy adults or pediatric patients with chromosomic abnormalities (other than on chromosomes 22 and 16) and without CDG.

Cell populations
Fibroblasts and keratinocytes were isolated from outgrowths of forearm skin specimens. Fibroblasts, used between the 5th and 15th passage, were cultured to confluence in DMEM containing 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (complete medium) supplemented with 10% (v/v) heat-inactivated FCS, at 37°C under 5% CO2 (Dupré et al., 1999Go). Keratinocytes were cultivated in RPMI 1640 exactly as described above.

Enterocytes, obtained from intestinal biopsy, were cultured for 48 h (Molmenti et al., 1993Go) in DMEM under 10% CO2.

To collect serum-free medium, cells were rinsed three times with sterile saline and cultured in FCS-free complete medium for 24 h. The supernatant was collected, centrifuged, and stored frozen until analysis. The cells were submitted to trypsin treatment and washed before homogenization and stored frozen until analysis.

AAT secretion in fibroblast and keratinocyte supernatants was studied over a 24 h period.

PBMC were isolated using a Ficoll-Paque Plus gradient, according to the manufacturer’s instructions.

Enzymatic procedure
Before N-glycanase treatment, glycoproteins were denatured by boiling samples for 5 min in the presence of 0.1% SDS and 1% ß-mercaptoethanol. SDS was next neutralized by Triton X-100 (10-fold excess). N-Glycanase treatment was performed at 37°C for 24 h in a total volume of 100 µl. N-glycanase was used in 0.2 M sodium phosphate, pH 8.6, 13 mM EDTA at 20 U/ml (Venembre et al., 1994Go).

Isoelectric focusing
Isoelectric focusing of serum transferrin was carried out as described previously (Stibler et al., 1991Go)

Western blot
Glycoproteins in samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in reducing conditions, using 7% (for transferrin) or 9% (for other glycoproteins) homogenous separating gel and 4% homogenous stacking gel. Proteins in the gel were electrotransferred to a nitrocellulose membrane and Western blot was developed using the ECL procedure, as previously reported (Seta et al., 1996Go).


    Acknowledgments
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
We acknowledge the collaboration of the patients and their families and of the referring clinicians. This work was supported by a Contrat de Recherche Clinique CRC 97–139 (Assistance Publique-Hôpitaux de Paris).


    Footnotes
 
1 To whom correspondence should be addressed at: Laboratoire de Biochimie A Hôpital Bichat–Claude Bernard, 46 rue H. Huchard, 75877 Paris cédex 18, France Back


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