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Abnormal synthesis of mannose 1-phosphate derived carbohydrates in carbohydrate-deficient glycoprotein syndrome type I fibroblasts with phosphomannomutase deficiency
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Abnormal synthesis of mannose 1-phosphate derived carbohydrates in carbohydrate-deficient glycoprotein syndrome type I fibroblasts with phosphomannomutase deficiency
In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] mannose into mannose phosphates, GDP-mannose, GDP-fucose, dolichol-P-mannose, lipid-linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into mannose 1-phosphate, GDP-mannose, GDP-fucose, dolichol-P-mannose, and nascent glycoproteins. The incorporation of radioactivity into mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected ([le]20%), but their size was severely reduced, mostly containing five or fewer residues. As a consequence, truncated oligosaccharides were transferred to newly synthesized glycoproteins. The metabolic changes can be explained by a deficiency of phosphomannomutase activity, which was reduced to [le]10% of control. Key words: phosphomannomutase deficiency/CDGS type I/mannose metabolites
Introduction
Carbohydrate-deficient glycoprotein syndromes (CDGS) are a group of autosomal recessively transmitted disorders, in which abnormally glycosylated proteins are formed. The biochemical diagnosis of CDGS is based on the abnormal isoelectric focusing pattern of serum glycoproteins such as transferrin, which generally show higher isoelectric points (Jaeken and Carchon, 1993; Jaeken et al., 1993). In the more rare CDGS type II this is fully explained by a deficiency of the Golgi enzyme N-acetylglucosaminyltransferase II (Jaeken et al., 1994; Tan et al., 1996). This enzyme is required for the synthesis of biantennary complex type oligosaccharides in glycoproteins. Its deficiency leads to the formation of undersialylated glycoproteins. The biochemical defects in CDGS type III (Stibler et al., 1993) and CDGS type IV (Stibler et al., 1995), which so far have both been described in two patients, are unknown.
In CDGS type I, the most common form of the CDGS, a higher isoelectric point of glycoproteins is observed that is due to incomplete usage of N-glycosylation sites, which normally carry sialylated complex type oligosaccharides (Wada et al., 1992; Yamashita et al., 1993). Incomplete usage of N-glycosylation sites points to a defect in the oligosaccharyl transferase or in the assembly of the lipid-linked oligosaccharide (LLO) precursor that serve as a donor for the oligosaccharides transferred onto nascent glycoproteins. While the activity of oligosaccharyl transferase was found to be normal (Knauer et al., 1994), a partial deficiency of dehydrodolichol reductase was observed in three CDGS type I of Japanese origin (Ohkura et al., 1997). The defect was accompanied by an accumulation of dehydrodolichol and decreased levels of dolichol and LLO and proposed to be the cause of CDGS type I. On the other hand, deficiency of phosphomannomutase (PMM) has recently been reported in fibroblasts, leukocytes, and liver from four CDGS typed patients. The intermediate PMM activity in parents of CDGS type I patients (van Schaftingen and Jaeken, 1995) and the identification of mutations in the PMM gene on chromosome 16p13 (Matthijs et al., 1997) strongly suggested that PMM deficiency is the primary cause of the disease.
The significance of the enzymatic abnormalities found in CDGS type I for the synthesis of glycoproteins has been difficult to evaluate due to conflicting data on the metabolic alterations in CDGS type I. Moreover, in most studies only a selected number of mannose metabolites and or enzyme activities have been determined, making it difficult to compare these studies. In three Japanese CDGS type I patients the levels of mannose 6-phosphate, mannose 1-phosphate and GDP-mannose were found to be normal in spite of a deficient PMM activity. Furthermore, the composition of the oligosaccharides in LLOs was normal in these patients (Ohkura et al., 1997). This is in contrast to other patients with CDGS type 1, in which truncated oligosaccharides were observed in LLOs and nascent glycoproteins (Powell et al., 1994; Krasnewich et al., 1995; Panneerselvam and Freeze, 1996). While the observations in the Japanese patients would point to a secondary nature of the PMM deficiency, those in the other patients may indicate a decreased availability of GDP-mannose resulting from a deficiency of PMM.
While the present study was in progress, reports on truncated oligosaccharides in LLOs and glycoproteins and the deficiency of PMM and of dehydrodolichol reductase in CDGS type I appeared (Powell et al., 1994; Krasnewich et al., 1995; van Schaftingen and Jaeken, 1995; Panneerselvam and Freeze, 1996; Ohkura et al., 1997). For the five patients with CDGS type I, two of which were sibs, of which the fibroblasts were investigated, we have tried to obtain a comprehensive analysis of mannose derived metabolites. Part of these results have been reported as an abstract (Körner et al., 1995). In all fibroblasts we observed a profound deficiency of PMM, which was associated with a reduction in mannose 1-phosphate, GDP-mannose, GDP-fucose, dolichol phosphate mannose (Dol-P-Man), and the occurrence of truncated oligosaccharides in LLOs and nascent glycoproteins. Unexpectedly, however, the level of [3H] mannose 6-phosphate was normal and [3H] mannose labeled LLOs showed only a slight reduction up to 20% when compared to controls. The biochemical alterations in these patients can sufficiently be explained by a deficiency of PMM activity.
Table Synthesis of LLOs with truncated oligosaccharide moieties in CDGS type 1 fibroblasts
In an earlier study we could exclude a deficiency of the N-oligosaccharyltransferase as a cause for the incomplete usage of N-glycosylation sites in glycoproteins of CDGS type 1 fibroblasts (Knauer et al., 1994). We therefore analyzed the formation of LLOs, which serve as the glycosyl donor in N-glycosylation. Cells were metabolically labeled for 30 min with [2-3H] mannose and sequentially extracted with chloroform-methanol (3:2) and chloroform-methanol-H2O (10:10:3). In control fibroblasts LLOs were recovered exclusively in the chloroform-methanol-H2O extract, while in CDGS type 1 fibroblasts a variable fraction of LLOs (containing truncated oligosaccharides) was recovered in the chloroform-methanol extract. The LLOs of both extracts were subjected to mild acid hydrolysis and the released oligosaccharides were separated by RP-HPLC (Figure 1 and Table I). While the [3H] mannose radioactivity incorporated into LLOs was only slightly reduced ([le]20%) in two CDGS type 1 fibroblasts lines, the structural composition of the oligosaccharides was strikingly different when compared to two control fibroblast lines. In the LLO fraction from controls, oligosaccharides with nine mannose and three glucose residues predominate, while truncated oligosaccharides with five or fewer mannose residues represent less than 10% of the [3H] oligosaccharides. In contrast in CDGS type 1 fibroblasts, oligosaccharides with five or fewer mannose residues represented about half of the LLO-associated radioactivity. While in the four CDGS type 1 fibroblast lines tested truncated oligosaccharides with [le]5 mannose residues were consistently found, their frequency varied considerably, extremes being 13% and 84%. This variation was observed also for each of the fibroblast lines when examined in independent experiments.
Figure
K.) fibroblast line. M5 and G3 refer to the elution of GlcNac2Man5 and GlcNac2Man9Glc3 standards.
Fibroblast line
3H-GDP-mannose
(c.p.m./mg protein)3H-GDP-fucose
(c.p.m./mg protein)
Control
O. K. (n = 2)
152.1 ± 15.3
99.5 ± 3.1
A. K.
166.7
103.6
CDGS type 1
K. K. (n = 2)
41.5 ± 9.3
32.5 ± 9.1
G. M.
52.6
19.6
P. A.
31.7
11.0
Results
Truncated oligosaccharides are transferred onto nascent glycoproteins in CDGS type 1 fibroblasts
Next we examined whether CDGS type 1 fibroblasts transfer truncated oligosaccharides from LLO to nascent glycoproteins. Fibroblasts were metabolically labeled for 30 min with [2-3H] mannose and cells extracts were digested with the endoglycosidases PNGaseF and EndoH, respectively. The released oligosaccharides were subjected to size fractionation. The major oligosaccharide fraction released by PNGaseF from extracts of control fibroblasts had a size corresponding to an oligosaccharide standard containing two core N-acetylglucosamine and 9 mannose residues (Figure 2A). In addition, oligosaccharides with one hexose unit less (M8) or more (G1) were found. Among the oligosaccharides released by PNGaseF from extracts of CDGS type 1 fibroblasts truncated species with five to seven mannose residues were prominent and on a molar basis, assuming the same radioactive specificity, they amount to about 75% (Figure 2B). The size distribution of the oligosaccharides released by EndoH was comparable for control and CDGS type 1 fibroblasts (Figure 2C,D). Oligosaccharides with one core N-acetylglucosamine and nine mannose residues were the most abundant species. Altogether, these data show that truncated oligosaccharides are transferred onto nascent glycoproteins in CDGS type 1 fibroblasts. These are resistant to EndoH, indicating that they lack the [alpha]1,3 mannose residue linked to the [alpha] 1,6 mannose of high mannose oligosaccharides (Kobata, 1979).
Figure
K.) fibroblast line. M5, M9, and G3 refer to the elution of GlcNAc2Man5, GlcNAc2Man9 and GlcNAc2Man9Glc3 standards. Oligosaccharides with nine mannose or nine mannose plus one glucose residues released by EndoH elute at a slightly earlier position than the corresponding standard oligosaccharides, since the former carry only a single core N-acetylglucosamine residue.
Figure
Deficiency of phosphomannomutase in CDGS type 1 is associated with decreased synthesis of mannose 1-phosphate, GDP-Man, GDP-Fuc and Dol-P-Man
After labeling fibroblasts for 30 min with [2-3H] mannose the amount of radioactivity in Dol-P-Man was decreased to 17-20% in three CDGS type 1 fibroblasts compared to controls (Figure 4). When membranes from CDGS type 1 and control fibroblasts were assayed for Dol-P-Man synthase activity, comparable values were measured. This holds true for assays following the transfer of mannose from GDP-[14C] mannose to endogenous or to exogenous and endogenous (not shown) dolicholphosphate.
Figure
K.) fibroblast cell line, respectively. The bar indicates the migration of a Dol-P-Man standard and the arrow the start point. The radioactive material recovered in the extract from CDGS type 1 fibroblasts at the start point represents LLOs with small size oligosaccharides. The table at the bottom gives the radioactivity incorporated into Dol-P-Man (c.p.m./mg cell protein) of three CDGS type 1 fibroblast lines as percentage of control.
While this study was in progress, deficiency of PMM was reported in fibroblasts from four patients with CDGS type 1 (van Schaftingen and Jaeken, 1995). Two of their patients (P.A. and P.S.) were also included in the present study. The deficiency of PMM activity could be confirmed in the fibroblasts of the five CDGS type 1 patients studied here using two different assays. The activities measured with an assay following the conversion of mannose 6-phosphate into mannose 1,6-bisphosphate in the presence of an excess of glucose 1,6-bisphosphate are shown in Table II. The residual PMM activity in CDGS type 1 fibroblasts was [le]10% of controls. Table Deficiency of PMM is expected to result in an accumulation of mannose 6-phosphate and a decrease of mannose 1-phosphate. After metabolic labeling fibroblasts for 30 min with [2-3H] mannose, the incorporation of radioactivity into mannose 6-phosphate was comparable in control and CDGS type 1 fibroblasts, while the incorporation into mannose 1-phosphate was decreased in the CDGS type 1 fibroblast lines to about 25% of controls (Table III).
The present study provides a comprehensive investigation on alterations of metabolites involved in protein glycosylation in CDGS type 1. The data support recent results which described the deficiency of PMM to be the primary defect in CDGS type 1 by localizing mutations in the gene encoding the PMM activity on chromosome 16p13 (Matthijs et al.,1997).
All biochemical alterations observed in the five CDGS type 1 fibroblast lines investigated in this study can be explained by the profound deficiency of PMM. The residual PMM activity measured by two independent assays was below 10% of control. Thisl activity, which was consistently observed in all CDGS type 1 cell lines, could originate from an unrelated phosphohexomutase. The deficiency of PMM led to a decrease in mannose 1-phosphate and metabolites such a GDP-mannose, Dol-P-Man and also of GDP-fucose, the synthesis of which depends on mannose 1-phosphate. One of the predictions for deficiency of PMM would be an accumulation of mannose 6-phosphate, which was not observed, however. This may indicate that phosphomannoisomerase maintains an equilibrium between fructose 6-phosphate and mannose 6-phosphate that prevents accumulation of mannose 6-phosphate. TABLE
In this as in other studies (Powell et al., 1994; Krasnewich et al., 1995; Panneerselvam and Freeze, 1996) a considerable variability was observed for the composition of oligosaccharides in LLOs from CDGS type 1 fibroblasts. This variability cannot be ascribed to variations in the genetic background, since it was observed for most of the cell lines when examined repeatedly. Apparently, the availability of GDP-mannose and Dol-P-Man for the synthesis of full length LLOs is borderline in CDGS type 1 fibroblasts. When it is limiting to the extent that LLOs with truncated oligosaccharides accumulate, the truncated oligosaccharides are transferred onto nascent glycoproteins.
The lower incorporation of [2-3H] mannose into nascent glycoproteins can be explained in part by the transfer of truncated oligosaccharides, which on average contain five to six mannose residues compared to nine mannose residues in control fibroblasts. While this can explain a reduction of incorporation of [2-3H] mannose to about two-thirds of control, the incorporation was reduced to about one-third. This implies that the specific activity of [2-3H] mannose is lower in CDGS type 1 fibroblasts and/or that only a fraction of the N-glycosylation sites that are utilized in control fibroblasts are glycosylated in CDGS type 1 fibroblasts due to suboptimal, truncated glycosyl donor oligosaccharides. Either of the two possibilities is conceivable, and the latter has a precedent in the underglycosylation of transferrin in CDGS type 1 (Yamashita et al., 1993).
Since trimming reactions during oligosaccharides processing remove the glucose residues and the six outer of the nine mannose residues, the structural defect of the oligosaccharide moieties of nascent glycoproteins in CDGS type 1 fibroblasts will mostly be corrected in the mature glycoproteins that have passed the endoplasmic reticulum and the Golgi and carry complex type N-linked oligosaccharides. Mature glycoproteins, however, will still show the underglycosylation due to defective usage of N-glycosylation sites. Preliminary results in CDGS type 1 fibroblasts show that the mature secretory glycoproteins contain only half of the [2-3H] mannose that is found in controls (C. Körner, unpublished observations). This observation lends further support to the notion that not only in liver, but also in fibroblasts deficiency of PMM is associated with defective usage of N-glycosylation sites.
As noted previously (van Schaftingen and Jaeken, 1995) limited availability of GDP-mannose in PMM-deficient cells may not only affect synthesis of N-linked glycoproteins but also that O-mannosylated glycoconjugates and glycosylphosphatidyl inositol anchors. The deficiency of GDP-fucose, a metabolic product of GDP-mannose, may result in an underfucosylation of N- and O-linked fucosylated carbohydrates which serve as blood group determinants (Watkins, 1980) and are implicated in recognition events during fertilization (Rosati and De Santis, 1980; Ahuja, 1982), development (Bird and Kimben, 1984), inflammation (Lowe et al., 1990; Ramphal et al., 1994), and tumor and metastasis (Rice and Bevilacqua, 1989; Sawada et al., 1993). While the metabolic alterations of the mannose metabolism in CDGS type 1 mostly fulfill what would be expected from a deficiency of PMM, the understanding of functional consequences and the clinical symptoms is only beginning.
Chemicals, enzymes, and isotopes
d-[2-3H]mannose (18 Ci/mmol), [35S]methionine, and GDP-D-[U-14C] mannose (267mCi/mmol) were from Amersham Buchler (Braunschweig). Recombinant endoglycosidase H (EndoH) from Streptomyces plicatus and N-glycosidase F (PNGase F) from Flavobacterium meningosepticum were from Boehringer (Mannheim). Dolicholphosphate was obtained from Sigma (Deisenhofen). [14C]Dol-P-Man, [2-3H]mannose-6-phosphate (Ohkura et al., 1997) and [2-3H]oligosaccharide standards of known size were prepared as described previously (Lehle, 1980).
Cell culture
Human primary fibroblast cultures established from skin biopsies were kindly provided by Prof. F. Hanefeld (Department of Pediatrics, University Hospital Gsttingen, Gsttingen, Germany) and Prof. J. Jaeken (Department of Pediatrics, University Hospital Gasthuisberg, Leuven, Belgium). Cells were grown at 37°C in the presence of 5% CO2 on Dulbecco's modified Eagles medium (DMEM, GibcoBRL) supplemented with 10% fetal calf serum (PANSYSTEMS) and passaged by trypsinization.
Metabolic labeling with [2-3H]mannose and [35S]methionine
Fibroblasts (6.6 × 104/cm2) were plated onto culture dishes and grown for 60 h in DMEM. Cells were metabolically labeled for 30 min in MEM free of methionine and glucose reduced to 0.5 mM, supplemented with either 20 to 125 µCi [2-3H]mannose or 2 µCi [35S] methionine. Cells were then washed three times with ice-cold 10 mM phosphate-buffered saline pH 7.4 (PBS), scraped into 10 mM PBS/0.1% Triton X-100 or buffers as indicated and lysed by sonification. Aliquots were used for protein determination following the method of Lowry et al. (1951).
Determination of radioactivity incorporated into proteins
Aliquots of cell lysates were spotted onto Whatman 3MM paper filters and dried for 1 h. The filters were fixed for 10 min in 10% ice-cold TCA, boiled for 5 min in 5% TCA, and subsequently washed in 5% ice-cold TCA followed by a short wash in ethanol/diethyl ether (1:1). Radioactivity was determined by liquid scintillation counting.
Digestion with EndoH and PNGaseF
Cells metabolically labeled with [2-3H] mannose as above were suspended in water supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), lysed by sonification, and dialyzed overnight against water. An aliquot of the dialyzed material was used for protein determination. For EndoH treatment 20 µg of protein lysate was dried under vacuum; resuspended in 50 µl 50 mM 2-mercaptoethanol, 0.1%SDS; sonified; and heated for 5 min at 95°C. The sample was supplemented with 15 µl 0.5 M sodium citrate, pH5.5, 5 µl 10% PMSF, 5 µl 0.5 U/ml EndoH, and 75 µl of water. After incubation for 12 h at 30°C the sample was heated for 5 min at 95°C. For PNGase F digestion 100 µg of protein was dried under vacuum, resuspended in 25 µl of 0.1 M 2-mercaptoethanol, 0.5% SDS and heated for 5 min at 95°C. After incubation in 25 µl 0.5 M Tris-HCl, pH 8.0, 10 µl 0.1 M 1,10-phenanthroline, 10 µl 10% Triton X-100, and 5 µl of PNGase F (250 mU/ml), the sample was incubated and heated as above. Released oligosaccharides were separated from protein bound oligosaccharides by elution with water from C18 Sep Pak cartridges (Waters), dried under vacuum, resuspended in 100 µl of water, and analyzed by HPLC (see below).
Metabolic labeling and extraction of Dol-P-Man and LLO
Cells were labeled with [2-3H] mannose as described, scraped into 2 ml of ice-cold methanol, and lysed by sonification. After addition of 4 ml chloroform, the material was again sonified, followed by centrifugation for 10 min at 5000 r.p.m. at 4°C. Supernatants were collected and the pellets extracted two more times with chloroform/methanol (3:2). The combined supernatants containing Dol-P-Man and LLOs of small size were dried under nitrogen, dissolved in 3 ml chloroform/methanol (3:2), washed, and analyzed by thin layer chromatography on Silica gel 60 aluminum sheets in a running buffer containing chloroform/methanol/water (65:25:4) as described previously (Lehle, 1980). The remaining pellet containing the large size LLOs were washed and extracted with chloroform/methanol/water (10:10:3) as described previously (Lehle, 1980). Corresponding aliquots of the chloroform/methanol and chloroform/methanol/H2O extracts were combined and dried under nitrogen and resuspended in 35 µl 1-propanol. To release the oligosaccharides by mild acid hydrolysis 500 µl 0.02N HCl were added followed by an incubation for 30 min at 100°C. The hydrolyzed material was dried under nitrogen, resuspended by sonification in 200µl H2O and cleared by centrifugation. The supernatant containing the released oligosaccharides was used for HPLC analysis (see below).
Size fractionation of oligosaccharides by HPLC
The separation of LLOs was performed on a Supelcosil LC-NH2 column (25 cm × 4.6 mm; 5 µm; Supelco) including a LC-NH2 (2 cm × 4.6mm) precolumn. A linear gradient of acetonitril from 70% to 50% in H2O (total volume 75 ml) was applied at a flow rate of 1 ml/min. Eluate fractions were analyzed by liquid scintillation counting.
Determination of Dol-P-Man synthase activity
Cells grown as described above were harvested by scraping into 1 ml 10 mM PBS and collected by centrifugation at 1000 × g at 4°C. The cells were suspended in 500 µl PBS and passed 20 times through a 22 gauge needle. After centrifugation for 15 min at 100,000 × g the supernatant was discarded. The pellet was resuspended in 200 µl H2O and Dol-P-Man synthase activity was measured as described previously (Lehle, 1980).
Preparation of mannose 6-phosphate, mannose 1-phosphate, GDP-mannose, and GDP-fucose
After labeling with [2-3H]mannose cells were harvested by scraping into 2 ml of ice-cold 50% methanol, 10 mM Tris, pH 7.4, 1 mM EDTA. The suspension was boiled for 3 min and sonified. After centrifugation the supernatant was collected. The pellet was extracted two more times. Combined supernatants were dried under N2, suspended in methanol/water (1:1), and desalted by descending paper chromatography on Whatman no.1 paper in methanol/1 M ammonium acetate, pH 7.5 (5:2) for 16 h. The radioactivity was analyzed by flat screen scanning. Mannose-phosphates were separated from nucleotide sugars (GDP-mannose, GDP-fucose) and free mannose. [3H]Mannose-phosphates were eluted with water, dried by lyophilization and subjected to high voltage electrophoresis at 65 V/cm for 50 min on Whatman 3MM paper in a buffer containing 80 mM pyridine adjusted to pH 5.5 with acetic acid. [3H]Mannose-phosphates were eluted with water, dried by lyophilization, and subjected to descending paper chromatography on Whatman No.1 paper in butanol/acetic acid /water (3:3:2) for 16 h. [3H]Mannose-phosphates were eluted with water, dried by lyophilization and subjected to mild acid hydrolysis in 0.02N HCl at 95°C for 30min. [2-3H]Mannose released from [2-3H]mannose 1-phosphate and [2-3H]mannose 6-phosphate were separated by high voltage electrophoresis as above, eluted, and quantified by liquid scintillation counting.
The nucleotide sugars were eluted from the paper chromatogram (see above) and analyzed by high voltage electrophoresis, paper chromatography, and acid hydrolysis as the mannose phosphates except that acid hydrolysis products ([3H]mannose and [3H]fucose) were separated by thin layer chromatography in acetone/butanol/water (70:15:15).
Analysis of phosphomannomutase
Phosphomannomutase activity was determined by two different methods. The first one followed the protocol of van Schaftingen and Jaeken (1995). Cells were grown as described above, scraped into 20 mM HEPES/150 mM NaCl, and collected by centrifugation. Pellets were resuspended in homogenization buffer (20 mM HEPES, 25 mM KCl, 1 mM dithiothreitol, 10 µg/ml each of leupeptin and antipain). Homogenization was achieved by one freeze-thaw cycle followed by sonification. Phosphomannomutase activity was assayed in a reaction mixture containing 50 mM HEPES pH 7.1, 5 mM MgCl2, 0.25 mM NADP, 0.1 mM mannose 1-phosphate, 1 µM mannose 1,6-bisphosphate, 10 µg/ml yeast glucose 6-phosphate dehydrogenase, 10 µg/ml phosphoglucose isomerase, 3.5 µg/ml phosphomannose isomerase, and varying concentrations of cell extract as described. All incubations were carried out at 23°C over a time period of 3 h with continuous recording of absorbance at 340 nm. In a two channel spectrophotometer a parallel assay without substrate was recorded.
In a second test, phosphomannomutase activity was determined by following the conversion of mannose 6-phosphate into mannose 1,6-bisphosphate in the presence of glucose 1,6-bisphosphate. The assay mixture contained 50 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 50,000 d.p.m. [2-3H]mannose 6-phosphate, 1 mM glucose 1,6-bisphosphate, and varying amounts of cell extracts in a total volume of 25 µl. After incubation for 30 min at 37°C the supernatants were collected by centrifugation and [2-3H]mannose 6-phosphate was separated from [2-3H]mannose 1,6-bisphosphate by high voltage paper electrophoresis as described above.
This work was supported by the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft (SFB 521).
CDGS, carbohydrate-deficient glycoprotein syndrome; LLO, lipid linked oligosaccharide; PMM, phosphomannomutase; EndoH, endoglycosidase H; PNGaseF, N-glycosidase F; Dol-P-Man, dolichol phosphate mannose.
Cell line
Phosphomannomutase (mU/mg protein)a
Control
O.K. (n = 6)
0.87 ± 0.086
M. J.
0.99
S. M.
1.01
A.K.
0.75
CDGS type 1
K .K. (n = 4)
0.12 ± 0.05
G. M.
0.02
P. A.
0.03
P. S.
0.04
W.N.
0.07
Discussion
Cell line
3H-Man-6-P (c.p.m./mg protein)
3H-Man-1-P (c.p.m./mg protein)
RatioMan-1-P/Man-6-P
Control
O. K. (n = 3)
9974 ± 1600
1787 ± 560
0.179
S. M.
12819
3607
0.281
A. K. (n = 2)
9168 ± 2309
2318 ± 481
0.252
CDGS type 1
K. K. (n = 3)
10003 ± 2734
512 ± 187
0.051
G. M.
18241
809
0.044
P. A. (n = 2)
7796 ± 1028
625 ± 81
0.080
Materials and methods
Acknowledgments
Abbreviations
References
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T. Lubke, T. Marquardt, K. von Figura, and C. Korner A New Type of Carbohydrate-deficient Glycoprotein Syndrome Due to a Decreased Import of GDP-fucose into the Golgi J. Biol. Chem., September 10, 1999; 274(37): 25986 - 25989. [Abstract] [Full Text] [PDF]
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C. Korner, R. Knauer, U. Holzbach, F. Hanefeld, L. Lehle, and K. von Figura Carbohydrate-deficient glycoprotein syndrome type V: Deficiency of dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl glucosyltransferase PNAS, October 27, 1998; 95(22): 13200 - 13205. [Abstract] [Full Text] [PDF]
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