The compartmentalisation of phosphorylated free oligosaccharides in cells from a CDG Ig patient reveals a novel ER-to-cytosol translocation process

Abstract

Background: Biosynthesis of the dolichol linked oligosaccharide (DLO) required for protein N-glycosylation starts on the cytoplasmic face of the ER to give Man(5)GlcNAc(2)-PP-dolichol, which then flips into the ER for further glycosylation yielding mature DLO (Glc(3)Man(9)GlcNAc(2)-PP-dolichol). After transfer of Glc(3)Man(9)GlcNAc(2) onto protein, dolichol-PP is recycled to dolichol-P and reused for DLO biosynthesis. Because de novo dolichol synthesis is slow, dolichol recycling is rate limiting for protein glycosylation. Immature DLO intermediates may also be recycled by pyrophosphatase-mediated cleavage to yield dolichol-P and phosphorylated oligosaccharides (fOSGN2-P). Here, we examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I) in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation.

Methods and principal findings: In EBV-transformed lymphoblastoid cells from CDG I patients and normal subjects a correlation exists between the quantities of metabolically radiolabeled fOSGN2-P and truncated DLO intermediates only when these two classes of compounds possess 7 or less hexose residues. Larger fOSGN2-P were difficult to detect despite an abundance of more fully mannosylated and glucosylated DLO. When CDG Ig cells, which accumulate Man(7)GlcNAc(2)-PP-dolichol, are permeabilised so that vesicular transport and protein synthesis are abolished, the DLO pool required for Man(7)GlcNAc(2)-P generation could be depleted by adding exogenous glycosylation acceptor peptide. Under conditions where a glycotripeptide and neutral free oligosaccharides remain predominantly in the lumen of the ER, Man(7)GlcNAc(2)-P appears in the cytosol without detectable generation of ER luminal Man(7)GlcNAc(2)-P.

Conclusions and significance: The DLO pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that pyrophosphatase-mediated cleavage of DLO intermediates yields recyclable dolichol-P. The kinetics of cytosolic fOSGN2-P generation from a luminally-generated DLO intermediate demonstrate the presence of a previously undetected ER-to-cytosol translocation process for either fOSGN2-P or DLO.

Figure 1. The dolichol cycle and protein N-glycosylation.

The dolichol cycle consists of a series of reactions (heavy dashed blue lines) involved in the construction of the oligosaccharide precursor (Glc₃Man₉GlcNAc₂) on a dolichol carrier. The cycle is completed by reactions (heavy dashed pink lines) involved in the recycling of dolichol-phosphate (dol-P). Mature lipid linked oligosaccharide (DLO) is generated by the transfer of two residues of N-acetylglucosamine (blue squares), 9 residues of mannose (green circles) and 3 residues of glucose (blue circles) onto the lipid carrier dolichol-P (zig zag line). The monosaccharides are added sequentially, in the order indicated by their numbers, by glycosyltransferases whose gene names are shown in yellow ovals and whose order of action is also indicated. The first seven sugars are added by cytoplasmically orientated UDP-GlcNAc- and GDP-Man-requiring glycosyltransferases. The growing DLO is then flipped into the lumen of the ER by a process thought to involve the RFT1 gene product. Subsequently dolichol-P-Man (DPM)- and dolichol-P-Glc (DPG)-requiring glycosyltransferases complete DLO biosynthesis. The addition of the last glucose residue to the growing DLO allows efficient oligosaccharyltransferase (OST)-mediated transfer of the oligosaccharide from lipid onto nascent polypeptides (-N-X-T/S-) in the ER. As indicated by the heavy dashed pink lines, a series of reactions carried out by gene products indicated in the yellow ovals, ensure that the lumenally orientated dol-P and dol-PP molecules that are generated during the construction of mature DLO are reorientated towards the cytosolic face of the ER. The different type I congenital disorders of glycosylation subtypes (CDG Ia-p, indicated by letters in red circles on the gene name) are caused by mutations in genes encoding enzymes involved in either the construction of mature DLO or dolichol recycling.

Figure 2. Identification of negatively charged oligosaccharide-like material in EBV-transformed lyphoblasts and murine lymphoma cells.

A. EBV lymphoblastoid cells derived from a normal subject (EBV Ctrl1) and a patient diagnosed with CDG Ig (ALG12 deficiency, see Fig. 1: EBV CDG Ig) were pulse radiolabeled with [2-³H]mannose and after extraction with organic solvents as described in Materials and Methods, water soluble components were applied to Biogel P2 columns. Radioactive components, except those eluted in the total inclusion volume (Vi) of the column, were pooled and subjected to ion-exchange chromatography on AG-1(acetate) and AG-50(H⁺) resins. Neutral species passed through both columns (Neutral). Subsequently, the AG-50 column was washed with 2 M pyridine acetate pH, 5.0 (PyrAc) and the AG-1 column was eluted with 3 M formic acid (FA). Fractions were collected and assayed for radioactivity by scintillation counting. B. In addition to the above described cells, EBV CDG Ig cells transduced with wild type ALG12 (EBV CDG Ig + wtALG12) and the parental (BW5147.3) and DPM1-deficient (Thy^-1) mouse lymphoma cells were radiolabeled as described above. After extraction with organic solvents radioactivity associated with lipid linked oligosaccharides ([³H]DLO), glycoproteins ([³H]GP) and the oligosaccharide-like materials described above was quantitated by scintillation counting. Radioactivity associated with neutral and FA eluted components is expressed as a percentage of total [2-³H]mannose incorporation ([³H]DLO + [³H]GP + [³H]neutral oligosaccharide-like material + [³H]FA-eluted oligosaccharide-like material) into the different cell lines.

Figure 3. Characterisation of negatively charged oligosaccharide-like material derived from different cell lines.

A. Equal amounts of radioactivity associated with material that was eluted from AG-1 columns with 3M FA as described for Fig. 2A were subjected to QAE-Sephadex chromatography as described in Material and Methods. The column was eluted with increasing concentrations of NaCl (indicated on the right hand y axis). Fractions were collected and assayed for radioactivity by scintillation counting. B. Aliquots of the negatively charged oligosaccharide-like material derived from EBV CDG Ig cells were analysed by thin layer chromatography (TLC) before and after treatment with either alkaline phosphatase (P'ase) or endo-β-N-acetylglucosaminidase H (EndoH). Abbreviations: lines to the left of the TLC fluorograms indicate the migration position of the oligosaccharide (Man₇GlcNAc₂; M₇GN₂) that was derived by mild acid hydrolysis of Man₇GlcNAc₂-PP-dolichol isolated from CDG Ig cells. This oligosaccharide was also treated with endoH to yield Man₇GlcNAc (M₇GN). The structure of the oligosaccharide moiety known to occur in the Man₇GlcNAc₂-PP-dolichol that accumulates in cells from CDG Ig is shown to the right of the TLC (mannose; green circles, N-acetylglucosamine; blue squares). The di-N-acetylchitobiose moiety of this oligosaccharide is sensitive to endoH. C. Aliquots of the negatively charged oligosaccharide-like material derived from DPM synthase-deficient Thy^-1 mouse lymphoma cells were analysed by thin layer chromatography (TLC) before and after treatment with either alkaline phosphatase (P'ase) or 20 mM HCl. The structure of the oligosaccharide moiety known to occur in the Man₅GlcNAc₂-PP-dolichol that accumulates in these cells is shown to the right of the TLC (mannose; green circles, N-acetylglucosamine; blue squares). The di-N-acetylchitobiose moiety of this oligosaccharide is not sensitive to endoH. The line to the left of the fluorograph indicates the migration position of Man₅GlcNAc₂ (M₅GN₂) that was released by mild acid acid treatment of Man₅GlcNAc₂-PP-dolichol derived from Thy^-1 cells. D. [¹⁴C]glucose-1-phosphate (Glc1P) and [¹⁴C]glucose-6-phosphate (Glc6P) were subjected to ion-exchange chromatography on AG-1(acetate) before and after either alkaline phosphatase or mild acid treatment as described in Materials and Methods. Neutralised material was assayed by scintillation counting and expressed as a percentage of input radioactivity.

Figure 4. Comparison of oligosaccharide structures generated from DLO and fOSGN2-P isolated from cells of different CDG patients.

EBV Ctrl1, EBV CDG Ia, EBV CDG Ie, EBV CDG Ig, and EBV CDG Ih cells were metabolically radiolabelled with [2-³H]mannose for 30 min prior to being extracted with organic solvents. DLO and fOSGN2-P were isolated and treated with 20 mM HCl as described in Materials and Methods. Oligosaccharides were subjected to HPLC and resolved components were detected with an on-line flow through scintillation counter. The HPLC traces for DLO- and fOSGN2-P-derived oligosaccharides are blue and red, respectively. The solid arrow heads indicate the elution times of oligosaccharides containing 1–9 residues of mannose (Man_nGN₂) and those containing 9 residues of mannose and 1–3 residues of glucose (Glc_nMan₉GN₂). In EBV CDG Ie and Ig cells, glucosylated oligosaccharides containing 5 (Glc_nMan₅GN₂) and 7 (Glc_nMan₇GN₂) residues of mannose, respectively, are also known to occur and their migration positions are indicated with open arrow heads.

Figure 5. Computation of the ratio of fOSGN-P to DLO for different oligosaccharide structures observed in the different cell lines cultivated in the absence or presence of glucosidase and mannosidase inhibitors.

A. Oligosaccharides derived from DLO and fOSG2-P isolated from EBV Ctrl1, EBV Ctrl2, EBV CDG Ia, EBV CDG Ie, EBV CDG Ig, EBV CDG Ih, BW5417.3 and Thy^-1 cells were prepared and resolved by HPLC as described in Fig. 4. Peak areas were recorded and used to derive the ratio fOSGN2-P/DLO for each oligosaccharide structure. These values were multiplied by 1000 and imposed on a logarithmic scale. Abbreviations: M_1-9; Man_1-9GlcNAc₂, G_1-3M_1-9; Glc_1-3Man_1-9GlcNAc₂, G_1-3M_1-7; Glc_1-3Man_1-7GlcNAc₂, G_1-3M_1-5; Glc_1-3Man_1-5GlcNAc₂. In a separate experiment EBV Ctrl2 (B) and EBV CDG Ig (C) cells were preincubated and then radiolabeled as described above, in the presence of the mannosidase inhibitors swainsonine (SW) and kifunensin (KIF) or the glucosidase inhibitor castanospermine (CST). Oligosaccharides derived from DLO and fOSGN2-P were resolved by TLC and, after elution of radioactive components from the chromatography plates followed by scintillation counting, the ratio fOSGN2-P/DLO for the oligosaccharide structures M₇–G₃M₉ were generated and presented as described above. D. Using data from the experiment described in B and C the percentage of total DLO species occurring as triglucosylated species was computed for EBV Ctrl2 and EBV CDG Ig cells radiolabeled in either the absence or presence of CST. E. Using data from the experiment described in B and C, DLO-, and fOSGN2-P-derived oligosaccharides possessing 7 residues of mannose were quantitated. The amounts of these components that were generated in cells treated with CST have been expressed as a percentage of those generated in cells radiolabeled in the absence of this reagent.

Figure 6. fOSGN2-P are generated in streptolysin O-permeabilised cells.

A. EBV CDG Ig cells were pulse radiolabeled for 30 min with [2-³H]mannose and then permeabilised with streptolysin O (SLO) at 4°C in permeabilisation buffer as described in Materials and Methods. After centrifugation fOSGN2-P and neutral fOS were recovered from both the supernatant containing cytosolic components (Cyt) and the permeabilised cell pellet containing intact membrane bound compartments (MBC). After dephosphorylation with mild acid treatment fOSGN2-P and fOS were examined by TLC. The migration position of Man₇GlcNAc₂ (M₇GN₂), derived by mild acid hydrolysis of Man₇GlcNAc₂-PP-dolichol isolated from CDG Ig cells, is indicated to the left of each pair of chromatograms. B. EBV CDG Ig cells were pulse radiolabeled for 30 min with [2-³H]mannose and then permeabilised with SLO in incubation buffer as described in Materials and Methods. After incubation of permeabilised cells in the presence of 20 µM each of UDP-Glc, GDP-Man, and UDP-GlcNAc for various times at 37°C, Cyt and MBC fractions were generated as described above. Neutral fOS (C) and fOSGN2-P (D) were recovered from the Cyt and MBC fractions and assayed by scintillation counting. E. DLO (L) and fOSGN2-P (P) recovered from the incubations described in C and D were hydrolysed using mild acid treatment and the resulting oligosaccharides were analysed by TLC. Pyrophosphate 10 mM was added to the indicated reaction mixture. The migration positions of standard oligosaccharides are indicated by the solid lines to the left of the chromatograms. The oligosaccharide migrating slightly slower than Man₉GlcNAc₂ (indicated with the dotted line) was not characterised but migrates as Glc₁Man₉GlcNAc₂ or Glc₃Man₇GlcNAc₂. The TLC plate on which DLO-derived oligosaccharides were resolved was exposed to film for 7 days whereas that on which fOSGN2-P-derived oligosaccharides were resolved was exposed for 14 days.

Figure 7. A tripeptide containing the N-glycosylation concensus sequence inhibits fOSGN2-P generation in permeabilised cell incubations.

Permeabilised EBV CDG Ig cells were prepared as described for Fig. 6B and incubated in the absence (−NYT) or presence of 1 µM Ac-Asn-Tyr-Thr-NH₂ (+ NYT) for 60 min. The resulting [2-³H]mannose-labelled glycotripeptide (A) and fOSGN2-P (B) were isolated from both the MBC (MBC) and cytosolic (Cyt) fractions as described in Materials and Methods and assayed by scintillation counting. C. In a different experiment permeabilised cells were incubated in the absence (NYT: 0) or the indicated concentrations of the tripeptide before isolation and quantitation of MBC- or cytosol-situated neutral fOS (fOS) and fOSGN2-P. The quantity of the two components recovered from each cellular compartment is expressed as a percentage of that occurring in the absence of tripeptide. D. Data shown in Figs. 6 and 7 indicate that Man₇GlcNAc₂-P is either generated in the lumen of the ER and then transported into the cytosolic compartment by a highly efficient process (left panel), or is liberated on the cytosolic face of the ER. Although Man₇GlcNAc₂-PP-dol is thought to be synthesised on the luminal face of the ER, in vitro experiments suggest that this structure can be potentially flipped onto the cytosolic face of the ER (right panel).