A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis.

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 alpha1,3-mannosyltransferase. The resulting Manalpha1,3-ManGlcNAc2-PP dolichol is further elongated by a yet unknown alpha1,6-mannosyltransferase.

Congenital disorders of glycosylation (CDG) 1 compose a rapidly growing group of inherited multisystemic disorders in man, which are commonly associated with severe psychomotor and mental retardation (1). 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 co-and 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 (2,3). The complex process of protein glycosylation requires more than a hundred glycosyltransferases, glycosidases, and transport proteins. CDG are classified into two groups. Defects of the assembly of lipid-linked oligosaccharides or their transfer onto nascent glycoproteins compose CDG type I, whereas CDG type II includes all defects of trimming and elongation of N-linked oligosaccharides (4). In the past 7 years the molecular nature of eight CDG-I and four CDG-II types could be identified (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24).
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 Man 1 GlcNAc 2 -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 Man 1 GlcNAc 2 -PP-dolichol and Man 2 GlcNAc 2 -PP-dolichol pointing to an involvement of ALG2 in mannose addition (25,26). 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 ␣1,3-mannosyltransferase that catalyzes the transfer of mannose residues onto Man 1 GlcNAc 2 -PP-dolichol.

MATERIALS AND METHODS
Cell Lines and Cell Culture-The fibroblasts from patient M. S., her father, and the controls were maintained at 37°C under 5% CO 2 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% CO 2 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 (7).
Analysis of Dolichol-linked Oligosaccharides-Fibroblasts derived from controls and the patient were cultured and metabolically labeled with [2-3 H]mannose for 30 min, and dolichol-linked oligosaccharides carrying more than four mannose residues were extracted and analyzed by HPLC as described previously (27). Man 1-2 GlcNAc 2 -PP-dolichol oligosaccharides were extracted with chloroform/methanol (3:2) and analyzed by TLC as described (27).
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 (20).
Preparation of GlcNAc 2 -PP-dolichol-The reaction contained in a final volume of 0.06 ml: Dol-P (3.5 g), UDP-GlcNAc (0.05 Ci; specific activity 305 mCi/mmol), 30 mM Tris-HCl, pH 7.5, 19 mM MgCl 2 , 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 M 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 (28). As enzyme source a solubilized extract from yeast membranes, prepared according to Ref. 29 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 ϫ g for 40 min.
Preparation of Man 1 GlcNAc 2 -PP-dolichol-The reaction contained in a final volume of 0.06 ml: [ 14 C]GlcNAc 2 -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 MgCl 2 , 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 (28). Both lower and interphase were collected and combined. As enzyme source a solubilized extract from yeast membranes prepared according to Ref. 29 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.
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 g of total RNA with Omniscript reverse transcriptase (Qiagen) and the primer R1 (5Ј-GTGGCTCACATTCAAGACT-CAA-3Ј). In a first round of PCR the cDNA was amplified using the primers F1 (5Ј-GGAGCTTGCGCAGAAGACCC-3Ј) 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 (5Ј-GTGCAGTTGCGGCTCCAG-3Ј) and R2 (5Ј-CAAAACT-GGGTCTACATACCATA-3Ј). 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).
Genomic DNA was prepared from control and patient fibroblasts and from peripheral blood leukocytes of the parents by standard procedures (31). PCR was carried out as described above with the primers intron A (5Ј-GGACATTCTTATGTATCAATATTAG-3Ј) and R1 in a first round of PCR and primers intron B (5Ј-GTGGCCAGAAAATCCACTTTTG-3Ј) 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 (5Ј-GGCATATGGTACTGGGTGAGAG-3Ј) was used for sequencing for the G393T mutation and primer Ex2-L was used for the ⌬1040G mutation.
Site-directed Mutagenesis-A 1.3-kb fragment of a wild type ALG2-cDNA of the isoform representing the coding sequence (nucleotides Ϫ22 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 ⌬1040G was inserted into the cDNA using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions with the primers Mut-A (5Ј-GCTGT-TAATTCGGTGGACCCTTGGAG-3Ј) and Mut-B (5Ј-CTCCAAGGGTC-CACCGAATTAACAGC-3Ј) 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 ϫ 10 6 FNX-Eco cells were seeded onto 60-mm dishes 1 day before transfection. Transient transfection by FuGENE6 reagent was carried out according to the manufacturerЈs protocol (Roche Applied Science) with 1 g of LNCX2 vector (mock), LNCX-wild type (wild type), and LNCX-Patient (⌬1040G), respectively. The further processing was carried out as described (23). 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 g/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 (32) under the control of the PMA1 promoter to give pNEV-ManTwt and pNEV-ManTpat, respectively. Plasmids were transformed into the alg2-1 strain (MAT␣ ura3-52) using standard techniques (33). Yeast cells were grown in YNBD medium (0.67% yeast nitrogen dropout ura, 2% glucose).

RESULTS
Case 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.
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 Glc 3 Man 9 GlcNAc 2 from dolichol-PP onto newly synthesized glycoproteins in the endoplasmic reticulum by the oligosaccharyltransferase, [2-3 H]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 Glc 1 Man 9 GlcNAc 2 and Man 9 GlcNAc 2 standards, respectively (Fig. 3, B and D). In the patient, the amount of 3 H 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).
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 (7). Fibroblasts from control and patient were metabolically labeled with [2-3 H]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 Glc 3 Man 9 GlcNAc 2 standard (Fig. 3,  A and C), although the amount of Glc 3 Man 9 GlcNAc 2 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 [2-3 H]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 Man 1 GlcNAc 2 -PP-dolichol and Man 2 GlcNAc 2 -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) (Fig. 6C). In contrast, when using the microsomal extract from the patient as an enzyme source, hardly any elongation was detectable (Fig. 6A) indicating that in the patient the biosynthesis of dolichol-linked oligosaccharides is defective at the step adding the second mannose residue.
The elongation of Man 1 GlcNAc 2 -PP-dolichol to Man 2 GlcNAc 2 -PP-dolichol involves the addition of either an ␣1,3or an ␣1,6mannosyl residue (Fig. 7). In order to define the sequence in which the ␣1,3and ␣1,6-mannosyl residues are added, [ 3 H]Man-(␣1,6)- Man-GlcNAc 2 -PP-dolichol was prepared by partial enzymatic digestion from [ 3 H]Man 5 GlcNAc 2 -PP-dolichol and used as acceptor substrate. As shown in Fig. 6B a microsome extract from patient fibroblasts fails to elongate Man(␣1,6)Man-GlcNAc 2 -PP-dolichol in the presence of GDP-mannose, whereas a microsome extract from control fibroblasts elongates the acceptor to Man 5 GlcNAc 2 -PP-dolichol (Fig. 6D). The rate of elongation of the Man 2 GlcNAc 2 acceptor, however, is much slower than that of the Man 1 GlcNAc 2 acceptor (compare Fig. 6, C and D, note the different incubation times). These results demonstrate that Man 1 GlcNAc 2 -PP-dolichol is the preferred substrate of the ␣1,3-mannosyltransferase and that it is this activity which is deficient in the patient.
Genetic Analysis of hALG2-Due to the similar glycosylation phenotypes in the patient fibroblasts and the alg2-1 strain of S. cerevisiae, we performed an NCBI Blast search for the human ortholog of the yeast ALG2 gene (accession number NP_011450). A human homolog (accession number CAC07999.1) with 37% sequence identity to the yeast Alg2 protein was identified. The hALG2 gene is located on chromosome 9q22 and encodes a polypeptide of 416 amino acids.
Sequencing of the ALG2 gene of the patient revealed heterozygosity for a ⌬1040G deletion and a single nucleotide substitution (G393T). The ⌬1040G mutation causes a frameshift with altering the sequence after amino acid 346 of hALG2 and a premature translation stop after amino acid 372 (Fig. 8). At the level of RNA the patient was homozygous for this mutation indicating that the transcript carrying the G393T substitution is unstable. The mother was heterozygous for the ⌬1040G mutation on the genomic and the RNA level. The father was heterozygous for the G393T mutation at the genomic level, whereas no transcripts with the G393T substitution were detectable.
Complementation for hALG2 Activity in CDG-Ii Fibroblasts-To confirm the deficiency of hALG2 as the primary cause of the glycosylation defect in the patient, we expressed the human ALG2 wild type enzyme as well as the ⌬1040G mutant in patient-derived fibroblasts using a retroviral expression system (Fig. 9, lanes 4 and 5). As control the vector alone was transduced into control and patient fibroblasts (Fig. 9,  lanes 1 and 3). In mock-transduced control fibroblasts Transduction of the wild type hALG2 led to the disappearance of Man 1 GlcNAc 2 -PP-dolichol and Man 2 GlcNAc 2 -PP-dolichol in patient fibroblasts (Fig. 9, lane 4). In contrast, transduction of the ⌬1040G mutant had no effect (Fig. 9, lane 5).
These results indicate that the accumulation of shortened dolichol-linked oligosaccharides in the patient is caused by the reduced activity of the truncated hALG2 and that expression of wild type hALG2 restores the biosynthesis of dolichol-linked oligosaccharides.
Complementation of the Glycosylation and Growth Defects in a S. cerevisiae alg2-1 Mutant Strain by hALG2-To demonstrate that the human hALG2 gene is the ortholog of yeast ALG2 and to further confirm that the ⌬1040G deletion is the disease causing mutation in our patient, we introduced the cDNAs encoding wild type hALG2 or the ⌬1040G mutant into the temperature-sensitive alg2-1 yeast strain. Only transformation with the wild type but not with the mutant form of hALG2 cDNA restored the formation of lipid-linked oligosaccharides in alg2-1 cells (Fig. 10A). Also the growth defect of the alg2-1 strain at 30°C was only corrected by transformation with wild type hALG2 cDNA but not with the ⌬1040G form ( Fig. 10B). We also assessed the glycosylation status of the vacuolar glycoprotein carboxypeptidase Y. In the alg2-1 strain carboxypeptidase Y is underglycosylated at the non-permissive temperature of 36°C because of a reduced transfer of truncated oligosaccharides to the protein (Fig. 10C). Transformation of the alg2-1 strain with the wild type but not with the ⌬1040G hALG2 cDNA normalized the glycosylation of carboxypeptidase Y. Altogether these results demonstrate that the hALG2 gene is the ortholog of the yeast ALG2 gene. DISCUSSION A new disorder affecting one of the early steps of dolichollinked oligosaccharide biosynthesis was identified in a patient who was born healthy but developed in the 1st year of life a multisystemic disorder with mental retardation, defective myelinization, seizures, coloboma of the iris, hepatomegaly, and coagulation abnormalities. The molecular defect in this disorder, termed CDG-Ii, is a deficiency of hALG2 that catalyzes the elongation of Man 1 GlcNAc 2 -PP-dolichol.
Biosynthesis of N-glycans starts at the cytoplasmic side of the endoplasmic reticulum membrane with the stepwise addition of two N-acetylglucosamine and five mannose residues onto the lipid carrier dolichol-phosphate. Donors are the respective nucleotide sugars. The product is Man 5 GlcNAc 2 -PPdolichol, which is translocated to the luminal face of the endoplasmic reticulum membrane by a reaction that involves RFT1  (34). Here it is elongated to Glc 3 Man 9 GlcNAc 2 -PP-dolichol by a set of glycosyltransferases that utilize Dol-P-Man and Dol-P-Glc as donors. The precursor oligosaccharide Glc 3 Man 9 Glc-NAc 2 is finally transferred by the oligosaccharyltransferase complex onto selected asparagine residues in nascent glycoproteins (35,36).
All known defects in the biosynthesis of dolichol-linked oligosaccharides affect glycosyltransferases acting at the luminal side of the endoplasmic reticulum (8 -10, 21-24). Deficiency of hALG2 represents the first defect of a glycosyltransferase catalyzing the transfer of monosaccharide residues from a nucleotide sugar donor onto the nascent dolichol-linked oligosaccharide chain at the cytosolic side of the endoplasmic reticulum.
Computer-assisted protein structure analysis for hALG2 predicts for hALG2 the existence of two closely adjacent putative transmembrane domains close to the N terminus. It remains to be determined whether these hydrophobic sequences mediate a peripheral attachment of hALG2 to the outer leaflet of the endoplasmic reticulum membrane or whether they form a short loop with two transmembrane domains placing the Nand C-terminal parts into the cytosol. Irrespective of that, the topology of hALG2 is clearly different from that of glycosyltransferases that catalyze at the inner leaflet of the endoplasmic reticulum the transfer of mannose or glucose residues from dolichol-linked donors onto dolichol-linked oligosaccharides. These glycosyltransferases are highly hydrophobic transmembrane proteins predicted to have 8 -13 transmembrane helices (37).
The accumulation of the short dolichol-linked oligosaccharides Man 1 GlcNAc 2 -PP-dolichol and Man 2 GlcNAc 2 -PP-dolichol gave the first hint for identification of the defect. The accumulation pattern in patient-derived fibroblasts resembled that of the S. cerevisiae alg2-1 strain at the restrictive temperature (25,38).
Two different mutations were detected in the human ortholog of the yeast ALG2 gene: a substitution mutation inherited from the father, which did not give rise to stable transcripts, and a single nucleotide deletion inherited from the mother, which did not affect the stability of the transcripts. The disease-causing nature of the mutations in the hALG2 gene could be demonstrated by complementation of the mannosylation defect in patient-derived fibroblasts by transduction of the wild type hALG2 cDNA, whereas the hALG2 cDNA carrying the maternal mutation did not restore the defect.
The orthologous nature of hALG2 and yeast ALG2 was further substantiated by expression of the hALG2 cDNA in the yeast alg2-1 strain that led to phenotypic complementation. The biosynthesis of dolichol-linked oligosaccharides, the growth behavior, and the glycosylation of the yeast vacuolar aspartylproteinase carboxypeptidase Y were normalized. Again, transfection with a hALG2 cDNA carrying the maternal single nucleotide deletion did not complement the alg2 phenotypes. Although the biochemical phenotype of the patient fibroblasts and of yeast alg2-1 strain at the non-permissive temperature resemble each other closely, there is one difference. Whereas in the alg2-1 strain Man 1 GlcNAc 2 -PP-dolichol and Man 2 GlcNAc 2 -PP-dolichol are transferred from dolichol to newly synthesized glycoproteins (38), no shortened oligosaccharides could be detected in newly synthesized proteins of the patient. A possible explanation might be a somewhat different substrate specificity of the oligosaccharyltransferase in yeast and man.
Under the conditions of the in vitro assay, the ␣1,6-mannosyltransferase was inactive toward Man␤1,4GlcNAc 2 -PP-dolichol. Fibroblasts of the patient, however, accumulated also Man 2 GlcNAc 2 -PP-dolichol. This indicates that under conditions such as ALG2 deficiency, some of the accumulating Man␤1,4GlcNAc 2 -PP-dolichol can be elongated by the ␣1,6-mannosyltransferase.
The normal size pattern of dolichol-linked oligosaccharides in the chloroform/methanol/H 2 O (10:10:3) extract of fibroblasts and the residual N-glycosylation clearly demonstrate that the defect in the patient is leaky as observed in other CDG-type I forms. Moreover, the defect would have been missed, if the analysis of dolichol-linked oligosaccharides would have been restricted to the chloroform/methanol/H 2 O (10:10:3) extract. The early intermediates such as Man 1-2 GlcNAc 2 -PP-dolichol are enriched in the chloroform/methanol (3:2) extract. Analysis of the latter fraction should therefore routinely be performed and may help to elucidate further defects among the group of CDG type I patients still awaiting molecular identification (40).