A New Type of Congenital Disorders of Glycosylation (CDG-Ii) Provides New Insights into the Early Steps of Dolichol-linked Oligosaccharide Biosynthesis
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m302850200
ISSN1083-351X
AutoresChristian Thiel, Markus Schwarz, Jianhe Peng, Michal Grzmil, Martin Hasilik, Thomas Braulke, Alfried Kohlschütter, Kurt Von Figura, Ludwig Lehle, Christian Körner,
Tópico(s)Genomics and Phylogenetic Studies
ResumoDeficiency of GDP-Man:Man1GlcNAc2-PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii. The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities. An accumulation of Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol was observed in skin fibroblasts of the patient. Incubation of patient fibroblast extracts with Man1GlcNAc2-PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man1GlcNAc2-PP dolichol. Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog. Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2. Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells. hALG2 was shown to act as an α1,3-mannosyltransferase. The resulting Manα1,3-ManGlcNAc2-PP dolichol is further elongated by a yet unknown α1,6-mannosyltransferase. Deficiency of GDP-Man:Man1GlcNAc2-PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii. The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities. An accumulation of Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol was observed in skin fibroblasts of the patient. Incubation of patient fibroblast extracts with Man1GlcNAc2-PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man1GlcNAc2-PP dolichol. Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog. Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2. Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells. hALG2 was shown to act as an α1,3-mannosyltransferase. The resulting Manα1,3-ManGlcNAc2-PP dolichol is further elongated by a yet unknown α1,6-mannosyltransferase. Congenital disorders of glycosylation (CDG) 1The abbreviations used are: CDG, congenital disorders of glycosylation; HPLC, high pressure liquid chromatography; DTT, dithiothreitol. 1The abbreviations used are: CDG, congenital disorders of glycosylation; HPLC, high pressure liquid chromatography; DTT, dithiothreitol. compose a rapidly growing group of inherited multisystemic disorders in man, which are commonly associated with severe psychomotor and mental retardation (1Freeze H.H. Glycobiology. 2001; 11: 2364-2369Crossref Scopus (145) Google Scholar). The characteristic biochemical feature of CDG is defective glycosylation of proteins due to mutations in genes required for the biosynthesis of N-linked oligosaccharides.The attachment of oligosaccharide chains onto newly synthesized proteins is one of the most widespread forms of coand post-translational modifications and is found in animals, plants, and bacteria. Glycoproteins are located inside cells predominantly in subcellular organelles and in cellular membranes and most abundantly in extracellular fluids and matrices. The oligosaccharide moiety of the glycoproteins can affect their folding, their transport, as well as their biological activity and stability (2Spiro R. Glycobiology. 2002; 12: 43-56Crossref PubMed Scopus (1041) Google Scholar, 3Helenius A. Aebi A. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1955) Google Scholar). The complex process of protein glycosylation requires more than a hundred glycosyltransferases, glycosidases, and transport proteins. 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Moore S.E.H. J. Biol. Chem. 2002; 277: 9962-9971Abstract Full Text Full Text PDF Scopus (82) Google Scholar).Here we describe for the first time a molecular defect in glycoprotein biosynthesis in man which affects at the cytosolic side of the endoplasmic reticulum the transfer of mannosyl residues from GDP-Man to Man1GlcNAc2-PP-dolichol by the enzyme hALG2. We show that the affected mannosyltransferase is the human ortholog to the yeast ALG2 gene, an enzyme that has so far not been characterized in higher eukaryotes. In the Saccharomyces cerevisiae alg2-1 mutant a defect caused accumulation of Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol pointing to an involvement of ALG2 in mannose addition (25Huffaker C. Robbins P. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7466-7470Crossref PubMed Scopus (187) Google Scholar, 26Jackson B. Kukuruzinska M. Robbins P. Glycobiology. 1993; 3: 357-364Crossref PubMed Scopus (55) Google Scholar). However, the precise biochemical defect was not known. The characterization of the human ALG2 deficiency described here has helped to define ALG2, both from man and yeast, as the α1,3-mannosyltransferase that catalyzes the transfer of mannose residues onto Man1GlcNAc2-PP-dolichol.MATERIALS AND METHODSCell Lines and Cell Culture—The fibroblasts from patient M. S., her father, and the controls were maintained at 37 °C under 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum (PAN Biotech GmbH). The ecotropic packaging cell line FNX-Eco (ATCC) and the amphotropic packaging cell line retroPack PT67 (Clontech) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, which was heat-inactivated at 56 °C for 30 min, at 37 °C under 5% CO2 unless otherwise stated.Isoelectric Focusing and SDS-PAGE of Serum Transferrin—Isoelectric focusing and SDS-PAGE of serum transferrin was carried out as described previously (7Niehues R. Hasilik M. Alton G. Körner C. Schiebe-Sukumar M. Koch H.G. Zimmer K.P. Wu R. Harms E. Reiter K. von Figura K. Freeze H. Harms H.K. Marquardt T. J. Clin. Invest. 1998; 101: 1414-1420Crossref PubMed Google Scholar).Analysis of Dolichol-linked Oligosaccharides—Fibroblasts derived from controls and the patient were cultured and metabolically labeled with [2-3H]mannose for 30 min, and dolichol-linked oligosaccharides carrying more than four mannose residues were extracted and analyzed by HPLC as described previously (27Körner C. Lehle L. von Figura K. Glycobiology. 1998; 8: 165-171Crossref PubMed Scopus (40) Google Scholar). Man1–2GlcNAc2-PP-dolichol oligosaccharides were extracted with chloroform/methanol (3:2) and analyzed by TLC as described (27Körner C. Lehle L. von Figura K. Glycobiology. 1998; 8: 165-171Crossref PubMed Scopus (40) Google Scholar).Mass Spectrometry—Matrix-assisted laser desorption/ionizationtime of flight analysis of oligosaccharides released from dolichol-pyrophosphate by mild acid hydrolysis with 20 mm HCl at 95 °C for 30 min was performed on a Bruker Reflex III (Bruker Daltonik GmbH) as described previously (20Hansske B. Thiel C. Lübke T. Hasilik M. Höning S. Peters V. Heidemann P. Hoffmann G. von Figura K. Körner C. J. Clin. Invest. 2002; 109: 725-733Crossref PubMed Google Scholar).Preparation of GlcNAc2-PP-dolichol—The reaction contained in a final volume of 0.06 ml: Dol-P (3.5 μg), UDP-GlcNAc (0.05 μCi; specific activity 305 mCi/mmol), 30 mm Tris-HCl, pH 7.5, 19 mm MgCl2, 0.7 mm DTT, 2.8% Nonidet P-40, 26% glycerol, and solubilized enzyme (equivalent to 1 mg of membrane protein). The reaction was carried out for 35 min at 24 °C followed by addition of unlabeled UDP-GlcNAc (83 μm final concentration) and incubated for another 10 min. The reaction was stopped by addition of 1 ml of chloroform/methanol (3:2, by volume) and processed by phase partitioning as described (28Sharma C.B. Lehle L. Tanner W. Eur. J. Biochem. 1982; 126: 319-325Crossref PubMed Scopus (43) Google Scholar). As enzyme source a solubilized extract from yeast membranes, prepared according to Ref. 29Knauer R. Lehle L. FEBS Lett. 1994; 344: 83-86Crossref PubMed Scopus (59) Google Scholar was used. Solubilization was carried out by incubation of membranes on ice for 20 min at a protein concentration of 17 mg/ml in the presence of 2.5% Nonidet P-40. The solubilized extract was separated from the insoluble material by centrifugation at 150,000 × g for 40 min.Preparation of Man1GlcNAc2-PP-dolichol—The reaction contained in a final volume of 0.06 ml: [14C]GlcNAc2-PP-dolichol (18,000 cpm), 29 mm Tris-HCl, pH 7.2, 11 mm NaCl, 14% glycerol, 0.6% Nonidet P-40, 0.5 mm DTT, 11 mm MgCl2, 1.5 mm GDP-Man, and solubilized enzyme (equivalent to 0.65 mg of membrane protein). Incubation was carried out for 50 min at 24 °C, stopped by addition of 1 ml of chloroform/methanol (3:2, by volume), followed by phase partitioning (28Sharma C.B. Lehle L. Tanner W. Eur. J. Biochem. 1982; 126: 319-325Crossref PubMed Scopus (43) Google Scholar). Both lower and interphase were collected and combined. As enzyme source a solubilized extract from yeast membranes prepared according to Ref. 29Knauer R. Lehle L. FEBS Lett. 1994; 344: 83-86Crossref PubMed Scopus (59) Google Scholar was used. Solubilization was carried out at a protein concentration of 6.5 mg/ml in the presence of 1.5% Nonidet P-40 as described above.Preparation of Man(α1–6)Man(β1–4)GlcNAc2-PP-dolichol—Man(α1–6)Man(β1–4)GlcNAc2-PP-dolichol was produced from [2-3H]Man5GlcNAc2-PP-dolichol (Man5-LLO) subjected to limited jack bean α-mannosidase (Glyco) digestion. Man5-LLO was isolated from Δalg3 yeast cells metabolically labeled with [2-3H]mannose as described previously (30Knauer R. Lehle L. J. Biol. Chem. 1999; 274: 17249-17256Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Man5-LLO (18,000 cpm) was dispersed by sonication in 100 mm sodium acetate, pH 5.0, containing 2 mm ZnCl2 and incubated with 3.75 milliunits of enzyme at 37 °C for 50 min under shaking in a final volume of 0.015 ml. The reaction was stopped by addition of chloroform/methanol to give a ratio of chloroform/methanol/water of 2:1:1 (by volume) and processed further by phase separation (28Sharma C.B. Lehle L. Tanner W. Eur. J. Biochem. 1982; 126: 319-325Crossref PubMed Scopus (43) Google Scholar) using an upper phase of chloroform/methanol/water of 1:32:48 (by volume) and collecting both lower and interphase. The (α1–6)-linkage in the Man(α1–6)Man(β1–4)GlcNAc2-PP-dolichol product formed was determined by incubation of the tetrasaccharide, released from the lipid by mild acid hydrolysis (30Knauer R. Lehle L. J. Biol. Chem. 1999; 274: 17249-17256Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), with recombinant α1–6-mannosidase (Calbiochem) or recombinant α1–3-mannosidase (Calbiochem) and HPLC analysis. Whereas in the case of α1–6-mannosidase treatment (1.1 milliunits of enzyme, 3.5 h of incubation), Man(α1–6)Man(β1–4)GlcNAc2 was converted to Man(β1–4)GlcNAc2, and no digestion was observed using α1–2,3-mannosidase (10 units of enzyme, 24 h of incubation). Incubation conditions were as suggested by the manufacturer. Control tests, to verify the correct function of the mannosidases used, were carried out with α1–6-mannobiose, α1–2-mannobiose, and mannose(α1–3) mannose(α1–2) mannotriose as substrates, obtained by acetolysis from yeast mannan.Elongation of Man1[14C]GlcNAc2-PP-dolichol and [2-3H]Man2GlcNAc2-PP-dolichol—The reactions contained the following in a final volume of 0.06 ml: Man1[14C]GlcNAc2-PP-dolichol (3.000 cpm) or [2-3H]Man2GlcNAc2-PP-dolichol (7.000 cpm), 0.13% Nonidet P-40, 10 mm MgCl2, 0.9 mm DTT, 0.14 mm Na-EDTA, 19 mm Tris-HCl, pH 7.2, 1 mm GDP-Man, and solubilized enzyme (equivalent to 0.05 mg of membrane protein). Incubation was performed at 37 °C for the times indicated, stopped with chloroform/methanol to give a ratio of chloroform/methanol/water of 2:1:1 (by volume), and processed further as described above. The solubilized extract was obtained from a particulate fibroblast fraction prepared as described (23Thiel C. Schwarz M. Hasilik M. Grieben U. Hanefeld F. Lehle L. von Figura K. Körner C. Biochem. J. 2002; 367: 195-201Crossref PubMed Scopus (50) Google Scholar), except that membranes were suspended in 20 mm Tris-HCl, pH 7.2, 10 mm MgCl2,1mm DTT. Solubilization was carried out at a protein concentration of 7 mg/ml and 1% Nonidet P-40 as described above.Mutation Analysis—Total RNA was extracted from control and patient fibroblasts as well as from peripheral blood leukocytes of the parents using the RNAeasy kit (Qiagen). First strand cDNA was synthesized from 0.5 μg of total RNA with Omniscript reverse transcriptase (Qiagen) and the primer R1 (5′-GTGGCTCACATTCAAGACTCAA-3′). In a first round of PCR the cDNA was amplified using the primers F1 (5′-GGAGCTTGCGCAGAAGACCC-3′) and R1 using the HotStar-Taq-Polymerase kit (Qiagen) with a preincubation at 95 °C for 15 min followed by 28 cycles with 1 min at 94 °C, 0.5 min at 55 °C, and 3 min at 72 °C. Further amplification was carried out with the nested primers F2 (5′-GTGCAGTTGCGGCTCCAG-3′) and R2 (5′-CAAAACTGGGTCTACATACCATA-3′). Reverse transcriptase-PCR products were run on 1% agarose gels. The PCR fragments were prepared with the QIAquick PCR purification kit (Qiagen) and subcloned into the pGEM-T-easy vector (Promega GmbH).Sequence analysis of the PCR products and the plasmids was done by dye-determined cycle sequencing with the primers pUC M13 forward, pUC M13 reverse (Stratagene Europe), F2, Ex1-O (5′-TCCTGGCGCTCTACGTGCTGTT-3′), Ex2-L (5′-GGAGAATGTGGAACATTATCAGG-3′), R2 on a Applied Biosystems model 373A automated sequencer.Genomic DNA was prepared from control and patient fibroblasts and from peripheral blood leukocytes of the parents by standard procedures (31Maniatis N.T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbour Laboratory Press, Cold Spring Harbor, NY1989: 9.16-9.21Google Scholar). PCR was carried out as described above with the primers intron A (5′-GGACATTCTTATGTATCAATATTAG-3′) and R1 in a first round of PCR and primers intron B (5′-GTGGCCAGAAAATCCACTTTTG-3′) and R2 in a second round of PCR. The PCR products were run on a 1% agarose gel and prepared as described above. Primer intron C (5′-GGCATATGGTACTGGGTGAGAG-3′) was used for sequencing for the G393T mutation and primer Ex2-L was used for the Δ1040G mutation.Site-directed Mutagenesis—A 1.3-kb fragment of a wild type ALG2-cDNA of the isoform representing the coding sequence (nucleotides –22 to 1310) was amplified by PCR using primers F2 und R2. The resulting fragment was purified and cloned into pGEM-T-easy vector (wild type). The mutation Δ1040G was inserted into the cDNA using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions with the primers Mut-A (5′-GCTGTTAATTCGGTGGACCCTTGGAG-3′) and Mut-B (5′-CTCCAAGGGTCCACCGAATTAACAGC-3′) to obtain plasmid pGEM-T-Easy-Pat. Wild type and patient ALG2-cDNA was subcloned into the Moloney mouse leukemia virus-derived vector pLNCX2 (Clontech).Production of Retroviruses—0.5 × 106 FNX-Eco cells were seeded onto 60-mm dishes 1 day before transfection. Transient transfection by FuGENE6 reagent was carried out according to the manufacturer′s protocol (Roche Applied Science) with 1 μg of LNCX2 vector (mock), LNCX-wild type (wild type), and LNCX-Patient (Δ1040G), respectively. The further processing was carried out as described (23Thiel C. Schwarz M. Hasilik M. Grieben U. Hanefeld F. Lehle L. von Figura K. Körner C. Biochem. J. 2002; 367: 195-201Crossref PubMed Scopus (50) Google Scholar). The supernatant with the amphotropic retroviral particles was used to transfect the fibroblasts of the patient and the control. 24 h after the final infection of the fibroblasts, medium was changed to Dulbecco's modified Eagle's medium, 10% heat-inactivated fetal calf serum with geneticin (335 μg/ml; Invitrogen). Selection was carried out for 10 days to obtain stable cell lines.Yeast Genetics—The cDNAs of the hALG2 from a control and patient M. S., cloned into the pBS-SK vector (Stratagene), were isolated as EcoRI 1260-bp fragments and ligated into the EcoRI site of the yeast shuttle vector pNEV-E (32Sauer N. Stolz J. Plant J. 1994; 6: 67-77Crossref PubMed Scopus (296) Google Scholar) under the control of the PMA1 promoter to give pNEV-ManTwt and pNEV-ManTpat, respectively. Plasmids were transformed into the alg2-1 strain (MATα ura3-52) using standard techniques (33Gietz R.D. Schiestl R.H. Yeast. 1991; 7: 253-263Crossref PubMed Scopus (363) Google Scholar). Yeast cells were grown in YNBD medium (0.67% yeast nitrogen dropout ura, 2% glucose).RESULTSCase Report—Patient M. S. is now a 3-year-old girl, the first child of healthy German parents. The family history was unremarkable on the paternal side, and on the maternal side there were several cases of a retarded psychomotor development, several early infantile deaths, cases with electroencephalogram abnormalities or seizures, and with migraine, as shown in the family tree (Fig. 1).The patient was born spontaneously without perinatal complications in the 36th week of an uneventful pregnancy. The birth weight was 3230 g. There were no postnatal complications, and the infant was thought to be normal up to the age of 2 months when her vision was suspected to be abnormal. An ophthalmologic examination revealed bilateral colobomas of the iris and a unilateral cataract, which was removed and replaced by an artificial lens. Vision, however, seemed to remain very poor, as there was no fixation of objects or faces, and an irregular nystagmus was frequently present. From the age of 4 months, a seizure disorder appeared with infantile spasms and hypsarrhythmia on electroencephalogram tracings. At the age of 5 months, a cranial magnetic resonance tomography showed a severely retarded myelinization. A follow-up MRT at the age of 8 months showed that myelin formation had come to a standstill and that the volume of white matter was markedly reduced. Mental and motor development were both severely delayed, and tendon reflexes were brisk without distinct spasticity. Hearing was not impaired. With the exception of a coccygeal dimple, a faint cardiac murmur, and borderline enlargement of the liver, the remainder of physical findings were unremarkable.Extensive laboratory investigations failed to reveal any significant metabolic or hematological abnormality with the exception of a prolonged activated partial thromboplastin time and a strongly reduced level of clotting factor XI.Because abnormalities of these parameters had been described in cases of CDG, investigations on the glycosylation state of serum transferrin by isoelectric focusing and SDS-PAGE were carried out.Isoelectric Focusing and SDS-PAGE of Serum Transferrin— The isoelectric focusing of serum transferrin, the standard diagnostic procedure for CDG, showed an increased amount of di- and asialo-transferrin at the expense of tetrasialo-transferrin, a pattern characteristic of CDG-I (Fig. 2, upper panel). Size determination of transferrin by SDS-PAGE revealed the presence of faster migrating forms suggesting the absence of either one or both of the two N-glycan chains that are normally present in transferrin (Fig. 2, lower panel). The activity of phosphomannomutase and phosphomannose isomerase, which are missing in two of the most common forms of CDG, CDG-Ia and Ib, were found to be normal.Fig. 2Isoelectric focusing pattern and SDS-PAGE of serum transferrin. Samples from a control, a CDG-Ia patient, and patient M. S. (Pat M.S.) were analyzed by isoelectric focusing (upper panel) and SDS-PAGE (lower panel) followed by Western blotting and immunodetection of transferrin. Tetrasialo, disialo, and asialo on the upper panel indicate transferrin forms with four, two, or no sialic acid residues. 2, 1, and 0 on the lower panel indicate transferrin forms with two, one, or zero oligosaccharide chains.View Large Image Figure ViewerDownload (PPT)Analysis of Protein- and Dolichol-derived Oligosaccharides—To determine whether the loss of complete N-glycan chains in transferrin molecules of the patient was due to a reduced transfer of the oligosaccharide Glc3Man9GlcNAc2 from dolichol-PP onto newly synthesized glycoproteins in the endoplasmic reticulum by the oligosaccharyltransferase, [2-3H]mannose-labeled oligosaccharides were released from the total glycoprotein fraction by peptide:N-glycosidase F digestion and analyzed by HPLC. N-Glycans from control and patient fibroblasts eluted mainly at the positions of Glc1Man9GlcNAc2 and Man9GlcNAc2 standards, respectively (Fig. 3, B and D). In the patient, the amount of 3H radioactivity in N-glycans was consistently reduced to about 70% of controls, although the oligosaccharyltransferase activity as well as the activities of N-acetylglucosaminyltransferase I and II and dolichol-P-mannose synthase were not significantly altered (data not shown). Furthermore, the size of the oligosaccharides in the N-glycan fraction released from newly synthesized glycoproteins was normal (Fig. 3D).Fig. 3Analysis of dolichol- and protein-derived oligosaccharides in CDG-Ii. Fibroblasts from a control person (A) and the patient (C) were metabolically labeled with [2-3H]mannose for 30 min. 2-3H-Oligosaccharides were released from the dolichol-PP moiety by mild acid hydrolysis and size-fractionated by HPLC. M9G3 refers to the position of a Glc3Man9GlcNAc2 standard. Glycoprotein-derived oligosaccharides from the control (B) and the patient (D) were prepared after metabolic labeling with [2-3H]mannose for 30 min. 2-3H-Oligosaccharides were released from the glycoprotein fraction by peptide:N-glycosidase F digestion and size-fractionated by HPLC. M9 and M9G1 refer to the positions of Man9GlcNAc2 and Glc1Man9GlcNAc2 standards, respectively.View Large Image Figure ViewerDownload (PPT)Further investigations focused on the analysis of dolichollinked oligosaccharides, which so far have been observed to be truncated in all known CDG-I types, except CDG-Ib (7Niehues R. Hasilik M. Alton G. Körner C. Schiebe-Sukumar M. Koch H.G. Zimmer K.P. Wu R. Harms E. Reiter K. von Figura K. Freeze H. Harms H.K. Marquardt T. J. Clin. Invest. 1998; 101: 1414-1420Crossref PubMed Google Scholar). Fibroblasts from control and patient were metabolically labeled with [2-3H]mannose, and the dolichol-linked oligosaccharides were extracted with a mixture of chloroform/methanol/water (10:10: 3). The glycan moiety was released by mild acid hydrolysis and analyzed by HPLC. The main peak fraction in control and the patient cells eluted with a Glc3Man9GlcNAc2 standard (Fig. 3, A and C), although the amount of Glc3Man9GlcNAc2 was slightly reduced in case of the patient.In order to examine the more hydrophobic dolichol-linked saccharides with short sugar chains that in part may have escaped the extract with chloroform/methanol/water (10:10:3), we also analyzed the chloroform/methanol (3:2) extract of [2-3H]mannose-labeled fibroblasts by thin layer chromatography (Fig. 4). Besides radioactivity that comigrated with dolichol-P-mannose and non-migrating material at the origin, we observed in extracts from the patient fibroblasts two additional spots, which were supposed to be Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol. For further characterization of the dolichol-linked oligosaccharides from the chloroform/methanol (3:2) extract, the oligosaccharide moieties were released by mild acid hydrolysis and analyzed by HPLC (Fig. 5). In case of the patient two [2-3H]mannose-labeled oligosaccharides were observed. Their masses, 917.2 and 1079.2 Da, as determined by matrix-assisted laser desorption/ionization time of flight, corresponded to that of Man1GlcNAc2-1-phenyl-3-methyl-5-pyrazolon and Man2GlcNAc2-1-phenyl-3-methyl-5-pyrazolon, respectively.Fig. 4Thin layer chromatography analysis of short dolichol-linked oligosaccharides. Fibroblasts derived from a control person and patient M. S., respectively, were metabolically labeled for 30 min with [2-3H]mannose. Short lipid-linked oligosaccharides were extracted with chloroform/methanol (3:2) and further analyzed by TLC in a running buffer containing chloroform/methanol/H2O (65:25:4). The position of the origin, a [3H]mannose-P-dolichol standard, the assumed positions of [3H]Man1GlcNAc2-PP-dolichol, and [3H]Man2GlcNAc2-PP-dolichol are indicated.View Large Image Figure ViewerDownload (PPT)Fig. 5High performance liquid chromatography and mass spectrometric analysis of short dolichol-linked [3H]mannose oligosaccharides. Short dolichol-linked oligosaccharides were extracted from control and patient fibroblasts after metabolic labeling with [2-3H]mannose for 30 min. The oligosaccharide moieties were released by mild acid hydrolysis, separated by HPLC, and subsequently analyzed by liquid scintillation counting. The peak fractions were further investigated by mass spectrometry. The values beside the HPLC peaks indicate the detected masses. The α-1,3-linkage of Man2GlcNAc2 is inferred from the enzymatic studies presented in Fig. 6. ▪, N-acetyl-glucosamine; •, mannose.View Large Image Fi
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