Glycobiology Advance Access originally published online on July 21, 2005
Glycobiology 2005 15(12):1312-1319; doi:10.1093/glycob/cwj017
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Patients with unsolved congenital disorders of glycosylation type II can be subdivided in six distinct biochemical groups
2 Laboratory of Pediatrics and Neurology, and 3 Department of Pediatrics, Radboud University Nijmegen Medical Center, Nijmegen 6525 GA, The Netherlands; 4 Consultant Pediatric Metabolic Medicine, Great Ormond Street Hospital, London WC1N 3JH, United Kingdom; and 5 Laboratory of Diagnostics Department, The Children’s Memorial Health Institute, Warsaw 04 730, Poland
1 To whom correspondence should be addressed; e-mail: r.wevers{at}cukz.umcn.nl
Received on April 25, 2005; revised on June 20, 2005; accepted on July 13, 2005
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Abstract |
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Defects in the biosynthesis of N- and core 1 O-glycans may be found by isoelectric focusing (IEF) of plasma transferrin and apolipoprotein C-III (apoC-III). We hypothesized that IEF of transferrin and apoC-III in combination with sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of apoC-III may provide a classification for congenital disorders of glycosylation (CDG) patients. We analyzed plasma from 22 patients with eight different and well-characterized CDG subtypes and 19 cases with unsolved CDG. Transferrin IEF (TIEF) has been used to distinguish between N-glycan assembly (type 1 profile) and processing (type 2 profile) defects. We differentiated two different CDG type 2 TIEF profiles: The "asialo profile" characterized by elevated levels of asialo- and monosialotransferrin and the "disialo profile" characterized by increased levels of disialo- and trisialotransferrin. ApoC-III IEF gave two abnormal profiles ("apoC-III0" and "apoC-III1" profiles). The results for the eight established CDG forms exactly matched the theoretical expectations, providing a validation for the study approach. The combination of the three electrophoretic techniques was not additionally informative for the CDG-Ix patients as they had normal apoC-III IEF patterns. However, the CDG-IIx patients could be further subdivided into six biochemical subgroups. The robustness of the methodology was supported by the fact that three patients with similar clinical features ended in the same subgroup and that another patient, classified in the "CDG-IIe subgroup," turned out to have a similar defect. Dividing the CDG-IIx patients in six subgroups narrows down drastically the options of the primary defect in each of the subgroups and will be helpful to define new CDG type II defects.
Key words: apolipoprotein C-III SDS–PAGE / congenital disorders of glycosylation / N-glycosylation / O-glycosylation / transferrin / apolipoprotein C-III isoelectric focusing
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Introduction |
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Congenital disorders of glycosylation (CDG) form a group of autosomal recessive metabolic disorders caused by defects in the biosynthesis of protein-linked glycans. Until now, genetic defects mainly in the N-glycan biosynthesis have been classified as CDG. At present, 18 genetically distinct types of CDG have been described; CDG-Ia through -Il and CDG-IIa through -IIf (Frank et al., 2004
; Wu et al., 2004; Martinez Duncker et al., 2005). Transferrin isoelectric focusing (TIEF) is generally used as a screening for defects in the biosynthesis of N-linked glycans (Jaeken, 2003). The division of CDG into type I and type II is based on the location of the defect in the N-glycan biosynthetic pathway. CDG-I includes all defects in the early N-glycan pathway in the cytoplasm or in the endoplasmic reticulum (ER) and covers all enzymatic steps to the transfer of the glycan to the protein. A defect in this part of the N-glycan biosynthetic pathway results in a typical CDG type 1 TIEF profile with increased amounts of asialo and disialo transferrin. CDG-II includes all defects involved in the processing of N-glycans on the glycosylated protein. These are mainly situated in the Golgi compartment. A defect in this part of the N-glycan biosynthetic pathway results in an abnormal TIEF profile for CDG subtypes -IIa, -IId, and -IIe. CDG subtypes -IIb, -IIc, and –IIf do not result in a hyposialylation of transferrin and give normal TIEF profiles. In plasma, TIEF of patients with CDG type II is different from that of patients with CDG type I. This allows a preliminary assignment of cases to the CDG I or II group and thus to the underlying organelle, where the defect is located.
Isoelectric focusing (IEF) of apolipoprotein C-III (apoC-III) has recently been described as a new screening assay for defects in the biosynthesis of core 1 O-glycans (Wopereis et al., 2003). In mammals, the most common form of O-glycosylation is the mucin type of O-glycosylation in which glycans are attached to the protein via an N-acetylgalactosamine (GalNAc) residue. This mucin type O-glycosylation can be subdivided into eight core structures based on the second sugar(s) and/or sugar linkage. The core 1 O-glycan, with Galß 1-3 GalNAc
-(Ser/Thr) as the core, is the most common subtype and occurs on many membrane and secreted proteins, especially in mucus, brain, and other neural tissue (Finne, 1975; Hounsell et al., 1996; Van den Steen et al., 2000). Recently, the first genetic defect in the biosynthesis of mucin type O-glycans has been described. Mutations in N-acetylgalactosaminyltransferase 3 (GALNT3) cause familial tumoural calcinosis (FTC) (Topaz et al., 2004). Other inborn errors of metabolism that affect O-glycosylation are defects in proteoglycan biosynthesis (the progeroid variant of Ehlers Danlos syndrome [Okajima et al., 1999] and the multiple exostoses syndrome [Wuyts and van Hul, 2000]) and defects in the biosynthesis of O-mannosyl glycans (muscle-eye-brain disease [Yoshida et al., 2001] and Walker Warburg syndrome [Beltran Valero de Bernabe et al., 2002]).
Nucleotide sugars, enzymes, and transporters involved in the biosynthesis of glycans might be common to N- and O-glycans, explaining why some glycosylation defects will affect both the biosynthesis of N- and O-glycans. A congenital defect affecting both N- and O-glycans has been described previously in patients with CDG-IIe, who have a defect in subunit 7 of the conserved oligomeric Golgi complex (COG-7) (Wu et al., 2004), in CDG-IIc (Lubke et al., 2001), in a subgroup of patients with cutis laxa (Morava et al., 2005; Wopereis et al., 2005), and in a patient with a yet unsolved defect (CDG-IIx [Wopereis et al., 2003]).
In this study, we investigated 19 patients with an unsolved CDG defect found in the selective screening with TIEF. This patient group was divided into a CDG-Ix group and a CDG-IIx group based on the TIEF profile. ApoC-III IEF and apoC-III sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) made it possible to divide the CDG-IIx group into six biochemical subgroups. The strategy applied in this article narrows down the options for the primary defect in the various subgroups especially for CDG-IIx patients.
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Results |
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IEF of plasma transferrin
Plasma of 19 patients with an unsolved CDG defect were classified as CDG-Ix or as CDG-IIx based on their TIEF profile. TIEF of plasma samples from patients with CDG-Ia, -Ib, -Ic, -Ie, and -If results in a typical CDG type 1 IEF profile with increased asialo- and disialotransferrin and decreased tetra- and pentasialotransferrin (Figure 1, lane 2). Seven of the 19 patients (patients 1–7) with an unsolved CDG defect were classified as CDG-Ix.
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All abnormal TIEF profiles that are not typical CDG type 1 were classified as a CDG type 2 profile. Twelve of the 19 patients (patients 8–19) were classified as CDG-IIx. The clinical features of these 12 CDG-IIx patients are summarized in Table I. The relative amounts of the transferrin isoforms of these 12 CDG-IIx patients and of patients with CDG-IIa, -IId, and -IIe are summarized in Table II. On the basis of the ratio of the transferrin isoforms, two different TIEF profile types could be distinguished in patients with CDG type II. The "asialo TIEF profile" is characterized by highly elevated levels of the asialo- and monosialotransferrin isoforms (Figure 1, lane 3). TIEF of plasma samples from patients 9,10, 13, and CDG-IId have this asialo TIEF profile. The "disialo TIEF profile" is characterized by normal or slightly elevated levels of asialo- and/or monosialotransferrin and increasingly higher levels of the disialo- and trisialotransferrin isoforms (Figure 1, lane 4). Patients 8, 11, 12, 14–19, CDG-IIa, and CDG-IIe have this disialo TIEF profile.
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IEF of plasma apoC-III
Patients with CDG-Ia, -Ib, -Ic, -Ie, and -If had a normal apoC-III isoform distribution, just like patients 1–7 with CDG-Ix. A normal apoC-III IEF profile was found in patients with CDG-IIa and IId, whereas plasma from a patient with CDG-IIe resulted in an abnormal apoC-III isoform distribution (Table III). Of the 12 CDG-IIx patients, three had a normal apoC-III IEF profile (patients 8–10), whereas nine had an abnormal apoC-III IEF profile (patients 11–19). The relative amounts of the apoC-III isoforms of patients 8–19 are summarized in Table III. On basis of the ratio of the three apoC-III isoforms, two different IEF profile types could be distinguished. The "apoC-III0 IEF profile" is characterized by elevated levels of apoC-III0 (Figure 1, lane 6). Patients 11–15 and the patient with CDG-IIe have this apoC-III0 IEF profile. The "apoC-III1 IEF profile" is characterized by elevated levels of the monosialo apoC-III form (Figure 1, lane 7).
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SDS–PAGE of apoC-III
SDS–PAGE of apoC-III was only performed in cases with abnormal profiles for apoC-III IEF. Using SDS–PAGE, two bands of apoC-III could be distinguished; a major band containing apoC-III1 and apoC-III2 and a minor apoC-III0 band. Two-dimensional electrophoresis (IEF and SDS–PAGE) has shown that the apoC-III1 and apoC-III2 bands comigrate on SDS–PAGE (data not shown). Plasma from patients 14, 15, and the CDG-IIe showed an abnormal apoC-III SDS–PAGE pattern (Figure 2A, lanes 4–6), whereas plasma from patients 11–13 and 16–19 had a normal apoC-III SDS–PAGE profile (not shown). The apoC-III0 band of plasma from patients 14, 15, and the CDG-IIe migrated further into the gel than the apoC-III0 band of the controls.
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The samples from the patients with abnormal apoC-III SDS–PAGE profiles were sequentially treated with neuraminidase and O-glycosidase. Similar results were obtained for plasma from patients 14, 15, and the CDG-IIe. In Figure 2B, the resulting SDS–PAGE profiles are shown of plasma from patient 15 (lanes 4–6) and from a control (lanes 1–3). After the incubation of control plasma with neuraminidase, the sialic acid residues of apoC-III2 and apoC-III1 were cleaved off, and apoC-III0 with the Galß1-3GalNAc glycan remained (Figure 2B, lane 2). After neuraminidase treatment of plasma of patient 15, the lower band composed of apoC-III2 and apoC-III1 disappeared, and a new band was visible at the position of apoC-III0 with the Galß1-3GalNAc glycan (Figure 2B, lane 5). Clearly, a second band is visible on top of the Galß1-3GalNAc glycan, indicating a lower molecular mass.
After the incubation of desialylated control plasma and of desialylated plasma of patient 15 with O-glycosidase, the Galß1-3GalNAc glycan of apoC-III0 was cleaved off, and the protein part of apoC-III remained (Figure 2B, lanes 3 and 6). This deglycosylated apoC-III band appears at the same position as the "second" band in lane 5. This suggests that the apoC-III0 fraction in plasma from patients 14, 15, and CDG-IIe consists of nonglycosylated apoC-III.
Subdivision of unsolved CDG-II patients in groups
On the basis of the biochemical results, it is possible to divide the 12 unsolved CDG-II patients into six biochemical different groups (Figure 3). After TIEF, two distinct groups could be distinguished based on either the asialo or the disialo profile. Patients 8–10 had normal apoC-III IEF profiles, but because of the different TIEF profiles they form two groups in the final classification. The patient with CDG-IId is in the same group as patients 9 and 10, whereas the patient with CDG-IIa is in the same group as patient 8.
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Nine patients had abnormal apoC-III IEF profiles (cases 11–19). Therefore, they must have molecular defects affecting both N- and O-glycosylation. The apoC-III SDS–PAGE profile of plasma from patients 14 and 15 was clearly abnormal and similar to the plasma profile of the CDG-IIe case. The remaining seven patients had normal SDS–PAGE profiles and could be divided in three separate groups, based on different combinations of TIEF with apoC-III IEF profile types. Therefore, the 12 CDG-IIx cases can be subdivided in a total number of six biochemical distinct groups.
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Discussion |
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In this study, we classified 19 patients with unsolved CDG into seven biochemically distinct patient groups based on TIEF, apoC-III IEF, and apoC-III SDS–PAGE. A previous attempt to classify CDG-IIx cases was performed by Mills et al. (2003)
by glycan analysis. The TIEF profile separates the 19 patients into a CDG-Ix and a CDG-IIx group. The seven patients classified as CDG-Ix all had a normal apoC-III IEF profile. The same holds for patients with CDG-Ia, -Ib, -Ic, -Ie, and -If. The primary defect in these patients is located in the early N-glycan biosynthesis pathway, before the transfer of the glycan from the dolichol carrier to the protein by the oligosaccharyl transferase complex. This part of the N-glycan biosynthesis is not relevant for the biosynthesis of core 1 O-glycans. Therefore, the finding of normal apoC-III IEF results is as expected in CDG-Ix cases. The CDG-Ix patients present as one biochemical group in this study. The methodology used in this study is not informative for a further subclassification of this CDG-Ix group. The primary defect in patients 8–19, who had been classified as CDG-IIx, is probably localized in the processing of N-glycans, which mainly occurs in the Golgi compartment. The processing of N-linked glycans involves the removal of several monosaccharides by glycosidases in the late ER and Golgi apparatus and the addition of monosaccharides catalyzed by specific glycosyltransferases in the Golgi apparatus. The addition of monosaccharides requires the biosynthesis of the nucleotide sugars UDP-GlcNAc, UDP-Gal, and CMP-NeuAc in the cytosol and their transport into the Golgi. The biosynthesis of the core 1 O-glycan attached to apoC-III requires (1) the biosynthesis of UDP-GalNAc, UDP-Gal, and CMP-NeuAc; (2) their transport into the Golgi; and (3) the subsequent transfer by specific glycosyltransferases. CDG-IIx patients with normal apoC-III IEF results are likely to have the primary defect in an N-glycan–specific processing step, whereas CDG-IIx patients with abnormal apoC-III IEF results are likely to have the defect in an enzyme or protein, that plays a role in the biosynthesis of both N- and core 1 O-glycans.
We hypothesized that IEF profile types provide further information for the localization of the primary defect. For example, we found that a CDG-IId patient has the "asialo TIEF profile," with increased amounts of a-, mono-, and disialo and decreased amounts of tri-, tetra-, and pentasialotransferrin fractions, whereas a CDG-IIa patient has the "disialo TIEF profile," with normal or slightly elevated amounts of asialo- and monosialo-, increased amounts of disialo-, and decreased amounts of tetra- and pentasialotransferrin fractions. Both patients had normal results for apoC-III IEF. These results correspond with the biochemical defects in CDG-IIa and CDG-IId. Deficiency of ß4GalT1 affects both antennae of the N-glycan in patients with CDG-IId. Theoretically, this deficiency would result in a TIEF profile with high amounts of the asialo fraction. Our CDG-IId patient indeed shows a TIEF profile with elevated levels of asialo- and monosialotransferrin. Deficiency of GlcNAcT-II affects only the Man-1,6-antenna in patients with CDG-IIa. As transferrin contains two N-glycans, this deficiency would result in a TIEF profile with elevated levels of the disialo fraction. Our CDG-IIa patient indeed shows a TIEF profile with elevated levels of disialo transferrin. The defective transferases in CDG-IIa and -IId are not required for the biosynthesis of the apoC-III O-glycan. Therefore, the finding of normal apoC-III IEF results is as expected in the CDG-IIa and -IId cases.
Patients 8–10 have a type 2 TIEF profile and normal results for apoC-III IEF. Therefore, they are likely to have a defect in a protein involved in N-glycan biosynthesis that is not involved in core 1 O-glycan biosynthesis. A further division could be made on the basis of the type of TIEF profile. Patients 9 and 10 share the asialo TIEF profile and form biochemical group 1. These patients are likely to have the primary defect in ST6Gal I (Takashima et al., 2003), ß4GalT1, Golgi mannosidase I (Snider and Rogers, 1986), GlcNAcT-I (Chen and Stanley, 2003), or in the UDP-GlcNAc transporter. If one of these proteins carries a genetic defect, synthesis of both antennae of the N-glycan is hampered. The biosynthesis of core 1 O-glycans would not be affected. In contrast, biochemical group 3 with patient 8 has a disialo TIEF profile. This patient is likely to have the primary defect in one of the enzymes that acts specifically on one antenna of the N-glycan. Candidates for the putative defect are GlcNAcT-II and mannosidase II (Moremen, 2002).
Seventy-five per cent of the analyzed CDG-IIx patients have an abnormal distribution of the plasma apoC-III IEF isoforms. These patients have a defect affecting both the biosynthesis of N-linked glycans and the core 1 O-glycans and thus have a combined defect in N- and O-glycosylation. The defect has to be localized in one of the shared biosynthetic steps of both pathways and may involve the biosynthesis of CMP-NeuAc or UDP-Gal or their transporters. As UDP-GalNAc is mainly synthesized from UDP-GlcNAc, the primary defect could also be situated in the biosynthetic route of UDP-GlcNAc (Pastuszak et al., 1996).
For a further subdivision, we used SDS–PAGE of apoC-III. The abnormal apoC-III SDS–PAGE profile with a shift of the apoC-III0 isoform is likely to be caused by nonglycosylated apoC-III protein. A patient with CDG-IIe has such a profile. CDG-IIe is caused by mutations in one of the subunits of the COG complex, which affects the regulation, compartmentalization, transport, and activity of several Golgi enzymes. Defects in the COG complex result in abnormal protein-linked N- and O-glycans (Wu et al., 2004).
Patients 14 and 15 also have an abnormal apoC-III SDS–PAGE profile. Like the CDG-IIe patient, they both had the disialo TIEF and apoC-III0 IEF profile types and formed biochemical group 5. The abnormal apoC-III SDS–PAGE results could be caused by a defect in the biosynthesis of UDP-GlcNAc, in one of the COG subunits, in one of the proteins involved in glycoprotein traffic through the Golgi, or in proteins involved in Golgi maintenance and biogenesis. It was found very recently that patient 15 indeed has a defect in one of the COG subunits (Prof. G. Matthijs, Leuven, Belgium, personal communication). Patient 14 is still under investigation for COG subunit defects.
Patients 11–13 and 16–19 have a combined defect in N- and core 1 O-glycosylation and normal apoC-III SDS–PAGE profiles and form biochemical groups 2, 4, and 6 on the basis of their TIEF and apoC-III IEF profile types. It is likely that the underlying defect in these patients is situated in the biosynthesis or transport of CMP-NeuAc or UDP-Gal, as these monosaccharides are required for the biosynthesis of both N-glycans and core 1 O-glycans, and these deficiencies would result in normal apoC-III SDS–PAGE profiles. The differences in TIEF and apoC-III IEF profile types suggest a similar defect in patients within one biochemical group and a different defect in patients between the subgroups.
Clinically, the 12 CDG-IIx patients have a phenotype with widely variable clinical features as central nervous system involvement, hepatic dysfunction, or congenital malformations (Table I). It was not possible to make a classification on the basis of the clinical symptoms. For the robustness of our methodology, it is encouraging that in biochemical group 6, three of the four patients (patients 17–19) have a very similar and peculiar phenotype with cutis laxa, microcephaly, dysmorphy, hypotonia, mental retardation, and eye anomalies (Morava et al., 2005). These three patients may share the same primary defect. The observed cutis laxa has not been described in other CDG subtypes so far.
In conclusion, the combination of the TIEF, apoC-III IEF, and apoC-III SDS–PAGE allowed us to subdivide the CDG type II patient group into six biochemically distinct groups. This subdivision narrows down the options for the position of the primary defect, with a limited number of theoretical possibilities for each individual patient.
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Materials and methods |
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Patients
Nineteen patients with an unsolved CDG defect were used as study group. The patients were found in the selective screening with TIEF. The TIEF profile was found to be abnormal repeatedly (two or more samples). Patients 1–7 with increased asialo- and disialotransferrin fractions and decreased tetrasialotransferrin were classified as CDG type Ix after excluding CDG-Ia and Ib by the phosphomannomutase and phosphomannose isomerase assay in leucocytes or fibroblasts according to the method of Van Schaftingen and Jaeken (1995)
. CDG types Ic–Ih and CDG type Il were excluded by lipid-linked oligosaccharide (LLO) analysis according to the method of Imbach et al. (2000a). Patients 8–19 with type 2 TIEF profiles (the hyposialylation IEF profiles that are not type 1) were classified as CDG-IIx. LLO analysis was normal in these patients. The clinical features of patients 8–19 are summarized in Table I. Patients 17–19 have been described in more detail by Morava et al. (2005). Polymorphisms of transferrin and of apoC-III were excluded by the incubation of the samples with neuraminidase to remove the negatively charged sialic acid residues. After neuraminidase treatment, all transferrin and apoC-III isoforms migrated at the asialo position on IEF, indicating that changes in sialic acid content had been responsible for the abnormal profiles. All secondary causes for glycosylation abnormalities, such as galactosemia, fructosemia, alcohol abuse, and hemolytic uremic syndrome, were excluded by appropriate measures. The IEF profiles of thyroxine-binding globulin (TBG), another N-glycosylated protein, were also abnormal in the 19 patients (data not shown). This indicates that the defect is not restricted to the N-glycans of transferrin and affects the glycosylation of other N-glycosylated plasma proteins as well.
In addition, plasma samples from patients with established CDG subtypes were studied (CDG-Ia, n = 8; CDG-Ib, n = 1; CDG-Ic, n = 8; CDG-Ie [Imbach et al., 2000b], n = 1; CDG-If [Schenk et al., 2001], n = 1; CDG-IIa, n = 1 [Jaeken et al., 1994]; CDG-IId, n = 1 [Peters et al., 2002]; CDG-IIe, n = 1 [Wu et al., 2004; P2]).
Samples and sample preparation
Blood samples were obtained from children with unsolved CDG with informed parental consent. Plasma was prepared by centrifugation and stored immediately at –80°C until required for analysis.
Transferrin and apoC-III IEF
TIEF was carried out essentially as described by Van Eijk and van Noort (1992). Plasma samples were incubated for 30 min with a solution of 20 mM ferric citrate and 0.5 mM sodium hydrogen carbonate in a ratio of 10:3 (plasma to solution) to saturate the transferrin with iron. The iron-saturated plasma was diluted five times with water and applied to a hydrated immobiline gel (pH 4–7) on an Ultraphore system (Amersham Biosciences, Piscataway, NJ). Transferrin isoforms were detected after immunofixation with rabbit anti-human transferrin antibody (Dako, Glostrup, Denmark) and Coomassie blue staining. The relative amounts of the transferrin isoforms were determined by scanning the stained gel using an Image Master Labscan, Ver. 3.00 (Amersham Biosciences) and quantified using Image Master 1D gel analysis, Ver. 4.10 software (Amersham Biosciences).
IEF of apoC-III was carried out, as described by Wopereis et al. (2003). A dry IEF Phastgel was hydrated in a solution of 8 M urea and 60 mL/L of pharmalyte, pH 4.2–4.9, and ampholine, pH 3.5–5.0 (Amersham Biosciences) in a ratio of 2:1. Plasma samples were diluted 10-fold with saline and applied to the hydrated gel on a Phastsystem. After IEF, the isoforms of apoC-III were detected by western blotting using rabbit anti-human apoC-III antibody (ANAWA Biomedical Services & Products, Wangen bei Duebendorf, Switzerland) and secondary goat anti-rabbit horseradish peroxidase-coupled antibody and visualized by electrochemiluminescence. The relative amounts of the apoC-III isoforms were determined by densitometry, as described above.
SDS–PAGE of apoC-III
Plasma samples used for SDS–PAGE of apoC-III were diluted 10-fold with freshly made sample buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 2.5% SDS, 2% DTT, and 0.01% bromophenol blue). The diluted samples were heated at 100°C for 5 min and applied to a "Phastgel high density" on a Phastsystem with a separation time of 300 V h at the standard running conditions for "Phastgel high density." After SDS–PAGE, the apoC-III variants were detected by western blotting using rabbit anti-human apoC-III antibody and secondary goat anti-rabbit horseradish peroxidase-coupled antibody and visualized by electrochemiluminescence.
Neuraminidase and O-glycosidase treatment
Negatively charged terminal sialic acid residues were removed from N- and O-linked glycans using neuraminidase. Neuraminidase (cat. no. 107590; Boehringer Mannheim, Mannheim, Germany; 1 g/L in 0.1 M Tris, pH 7.0) and human plasma samples were incubated in a ratio of 2:1 overnight at room temperature.
O-Glycosidase can remove the unsubstituted Galß1-3GalNAc unit from core 1 O-glycans. For that purpose, O-glycosidase (cat. no. 1347101; Roche Applied Science, Basel, Switzerland; 0.5 mU/µL) and human plasma samples pretreated with neuraminidase were incubated in a ratio of 1:1 overnight at 37°C.
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This work was supported by the European Commission, contract no. QLG-CT2000-0047 (Euroglycan) and Euroglycanet (contract nr. 512131). We thank L. Spaapen, A. Evangeliou, P. Briones, B. Kremer, M. Wisskirchen, K. Niezen-Koning, and J. Penzien for allowing us to investigate their patients’ plasma samples.
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Abbreviations |
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ApoC-III, apolipoprotein C-III; CDG, congenital disorders of glycosylation; CMP, cytidine 5'monophospho; COG, conserved oligomeric Golgi complex; Gal, galactose; GalNAc, N-acetylgalactosamine; IEF, isoelectric focusing; NeuAc, neuraminic acid or sialic acid; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; TIEF, transferrin isoelectric focusing; UDP, uridine 5'diphospho
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References |
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