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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: 1-antitrypsin/CDG/ GLUT-1/ immunoglobulin G/ protein glycosylation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1997), 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., 1992; Yuasa et al., 1995) and of liver proteins (Henry et al., 1997
) 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., 1997
; 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., 1995
; Bergmann et al., 1998
; 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Although serum AAT is essentially of hepatic origin, many other cell types are able to produce this protein (Ikuta et al., 1982; Perlmutter et al., 1989
). 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).
|
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).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1994). We have also observed such alterations in the early steps of glycoprotein glycan biosynthesis (Dupré et al., 1999
) 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., 1995; Seta et al., 1996
). 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., 1993; Stibler and Skovby, 1994
). 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., 1993
). Moreover, the pattern of liver glycoproteins from a CDG Ia patient showed abnormally glycosylated glycoprotein precursors compared to non-CDG (Henry et al., 1997
). 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., 1997; 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., 1998), 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., 1999
).
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., 1999). The only mutation for which the corresponding recombinant enzyme has no activity at all (Pirard et al., 1999
), the R141H mutation, has never been observed in a homozygote situation (Matthijs et al., 1998
) and is considered to be lethal (Matthijs et al., 1998
; Pirard et al., 1999
). 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., 1997
; Metzler et al., 1994
), 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 1-antitrypsin,
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, 1995
). 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., 1999). Keratinocytes were cultivated in RPMI 1640 exactly as described above.
Enterocytes, obtained from intestinal biopsy, were cultured for 48 h (Molmenti et al., 1993) 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 manufacturers 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., 1994).
Isoelectric focusing
Isoelectric focusing of serum transferrin was carried out as described previously (Stibler et al., 1991)
Western blot
Glycoproteins in samples were resolved by sodium dodecyl sulfatepolyacrylamide 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., 1996).
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bergmann,M., Gröss,H.-J., Abdelatty,F., Möller,P., Jaeken,J. and Schwartz-Albeit,R. (1998) Abnormal surface expression of sialoglycans on B lymphocyte cell lines from patients with carbohydrate deficient glycoprotein syndrome IA (CDGS IA). Glycobiology, 8, 963972.
Clayton,P., Winchester,B., Di Tomaso,E., Young,E., Keir,G. and Rodeck,C. (1993) Carbohydrate-deficient glycoprotein syndrome: normal glycosylation in the fetus. Lancet, 341, 956.[ISI][Medline]
Dupré,T., Ogier-Denis,E., Moore,S.E.H., Cormier-Daire,V., Dehoux,M., Durand,G., Seta,N. and Codogno,P. Alteration of mannose transport in fibroblasts from type I carbohydrate-deficient glycoprotein syndrome patients. (1999) Biochim. Biophys. Acta, 1453, 369377.[ISI][Medline]
Grunewald,S., Huyben,K., de Jong,J.G., Smeitink,J.A., Rubin,E., Boers,G.H., Conradt,H.S., Wendel,U. and Wevers,R.A. (1999) Beta-trace protein in human cerebrospinal fluid: a diagnostic marker for N-glycosylation defects in brain. Biochim. Biophys. Acta., 1455, 5460.[ISI][Medline]
Harrison,H., Miller,K., Harbison,M. and Slonim,A. (1992) Multiple serum protein abnormalities in carbohydrate-deficient glycoprotein syndrome: pathognomonic finding of two-dimensional electrophoresis? Clin. Chem., 38, 13901392.[ISI][Medline]
Henry,H., Tissot,J.D., Messerli,B., Markert,M., Muntau,A., Skladal,D., Sperl,W., Jaeken,J., Weidinger,S., Heyne,K. and Bachmann,C. (1997) Microheterogeneity of serum glycoproteins and their liver precursors in patients with carbohydrate-deficient glycoprotein syndrome type I: apparent deficiencies in clusterin and serum amyloid P. J. Lab. Clin. Med., 129, 412421.[ISI][Medline]
Ichisaka,S., Ohno,K., Yuasa,I., Nanba,E., Sakuraba,H. and Suzuki,Y. (1998) Increased expression of beta-hexosaminidase alpha chain in cultured skin fibroblasts from patients with carbohydrate-deficient glycoprotein syndrome type 1. Brain Dev., 20, 302306.[ISI][Medline]
Ikuta,T., Okubo,H., Kudo,J., Ishibashi,H. and Inoue,T. (1982) Alpha 1-antitrypsin synthesis by human lymphocytes. Biochem. Biophys. Res. Commun., 104, 15091516.[ISI][Medline]
Imbach,T., Burda,P., Kuhnert,P., Wevers,R.A., Aebi,M., Berger,E.G. and Hennet,T. (1999) A mutation in the human ortholog of the Saccharomyces cerevisiae ALG6 gene causes carbohydrate-deficient glycoprotein syndrome type-Ic. Proc. Natl. Acad. Sci. USA, 96, 69826987.
Ioffe,E., Liu,Y. and Stanley,P. (1997) Complex N-glycans in Mgat1 null preimplantation embryos arise from maternal Mgat1 RNA. Glycobiology, 7, 913919.[Abstract]
Jaeken,J., van Eijk,H.G., van der Heul,C., Corbeel,L., Eeckels,R. and Eggermont,E. (1984) Sialic acid-deficient serum and cerebrospinal fluid transferrin in a newly recognized genetic syndrome. Clin. Chim. Acta, 144, 245247.[ISI][Medline]
Korner,C., Knauer,R., Holzbach,U., Hanefeld,F., Lehle,L. and von Figura,K. (1998) Carbohydrate-deficient glycoprotein of dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl glucosyltransferase. Proc. Natl. Acad. Sci USA, 95, 1320013205.
Krasnewich,D., Holt,G.H., Brantly,M., Skovby,F., Redwine,J. and Gahl,W. (1995) Abnormal synthesis of dolichol-linked oligosaccharides in carbohydrate-deficinet glycoprotein syndrome. Glycobiology, 5, 503510.[Abstract]
Marquardt,T., Ullrich,K., Zimmer,P., Hasilik,A.,Deufel,T. and Harms,E. (1995) Carbohydrate-deficient glycoprotein syndrome (CDGS)glycosylation, folding and intracellular transport of newly synthesized glycoproteins. Eur. J. Cell Biol., 66, 268273.
Matthijs,G., Schollen,E., Pardon,E., Veiga-Da-Cunha,M., Jaeken,J., Cassiman,J.J. and van Schaftingen,E. (1997) Mutations in PMM2, a phosphomannomutase gene on chromosome 16p13, in carbohydrate-deficient glycoprotein type I syndrome. Nature Genet., 16, 8892.
Matthijs,G., Schollen,E., Van Schaftingen,E., Cassiman,J.J. and Jaeken,J. (1998) Lack of homozygotes for the most frequent disease allele in carbohydrate-deficient glycoprotein syndrome type 1A. Am. J. Hum. Genet., 62, 542550.[ISI][Medline]
Metzler,M., Gertz,A., Sarkar,M., Schachter,H., Schrader,J.W. and Marth,J.D. (1994) Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development. EMBO J., 13, 20562065.[Abstract]
Molmenti,E., Perlmutter,D. and Rubin,D. (1993) Cell-specific expression of alpha 1-antitrypsin in human intestinal epithelium. J. Clin. Invest., 92, 20222034.[ISI][Medline]
Perlmutter,D., Daniels,J., Auerbach,H., De Schryver-Kecskemeti,K., Winter,H. and Apers,D. (1989) The alpha1-antitrypsin gene is experssed in a human intestinal epithelial cell line. J. Biol. Chem., 264, 94859490.
Pirard,M.,Matthijs,G.,Heykants,L.,Schollen,E.,Grunewald,S.,Jaeken,J. and van Schaftingen,E. (1999) Effect of mutations found in carbohydrate-deficient glycoprotein syndrome type IA on the activity of phosphomannomutase 2. FEBS Lett., 452, 319322.[ISI][Medline]
Pohl,S.,Hoffmann,A.,Rudiger,A.,Nimtz,M.,Jaeken,J. and Conradt,H.S. (1997) Hypoglycosylation of a brain glycoprotein (ß-trace protein) in CDG syndromes due to phosphomannomutase deficiency and N-acetylglucosaminyl-transferase II deficiency. Glycobiology, 7, 10771084.[Abstract]
Powell,L.D.,Paneerselvam,K.,Vij,R.,Diaz,S.,Manzi,A.,Buist,N.,Freeze,H. and Varki,A. (1994) Carbohydrate-deficient glycoprotein syndrome: not an N-linked oligosaccharide processing defect, but an abnormality in lipid-linked oligosaccharide biosynthesis? J. Clin. Invest., 94, 19011909.[ISI][Medline]
Seta,N., Barnier,A., Hochedez,F., Besnard,M. and Durand,G. (1996) Diagnostic value of Western blotting in carbohydrate-deficient glycoprotein syndrome. Clin. Chim. Acta, 254, 131140.[ISI][Medline]
Stibler,H. and Skovby,F. (1994) Failure to diagnose carbohydrate-deficient glycoprotein syndrome prenatally. Pediatr. Neurol., 11, 71.[ISI][Medline]
Stibler,H., Jaeken,J. and Kristiansson B. (1991) Biochemical characteristics and diagnosis of the carbohydrate deficient glycoprotein syndrome. Acta Pediatr. Scand., 375, 2131.
Van Schaftingen,E. and Jaeken,J. (1995) Phosphomannomutase deficiency is a cause of carbohydrate-deficient glycoprotein syndrome type I. FEBS Lett., 377, 318320.[ISI][Medline]
Venembre,P., Boutten,A., Seta,N., Dehoux,M., Crestani,B., Aubier,M. and Durand,G. (1994) Secretion of alpha 1-antitrypsin by alveolar epithelial cells. FEBS Lett., 346, 171174.[ISI][Medline]
Wada,Y.,Nishikawa,A.,Okamoto,N.,Inui,K.,Tsukamoto,H.,Okada,S. and Taniguchi,N. (1992) Structure of serum transferrin in carbohydrate-deficient glycoprotein syndrome. Biochem. Biophys. Res. Commun., 189, 832836.[ISI][Medline]
Yamashita,K., Ideo,H., Ohkura,T., Fukushima,K., Yuasa,I., Ohno,K. and Takeshita K. (1993) Sugar chains from serum transferrin from patients with carbohydrate-deficient glycoprotein syndrome. J. Biol.Chem., 268, 57835789.
Yuasa,I., Ohno,K., Hashimoto,K., Iijima,K., Yamashita,K. and Takeshita,K. (1995) Carbohydrate-deficient glycoprotein syndrome: electrophoretic study of multiple serum glycoproteins. Brain Dev., 17, 1319.[ISI][Medline]