3 School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
Received on February 21, 2003; revised on May 7, 2003; accepted on May 15, 2003
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Abstract |
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Key words: glycosyl-phosphatidylinositol anchor / membrane dipeptidase / N-glycosylation / oligosaccharyltransferase / prion protein
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Introduction |
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Apart from the sequence in and around the sequon, the efficiency of N-glycosylation is greatly influenced by the accessibility of the sequon to the lumenally orientated active site of OST. The presence of disulfide bonds and the conformation of the nascent polypeptide chain in the vicinity of the sequons can influence N-glycosylation (Helenius and Aebi, 2001; Imperiali and Rickert, 1995
). For example, in tissue-type plasminogen activator (tPA), disulfide bond formation during the folding of the nascent polypeptide inhibits N-glycosylation, most likely by sterically hindering the accessibility of the sequons to OST (Allen et al., 1995
). Additionally, studies utilizing in vitro transcription/translocation systems have shown that the physical parameters of the transloconOST complex also restrict sequon utilization. In such studies N-glycosylation efficiency was seen to rapidly decrease when the sequon was fewer than 60 residues from the C-terminus (Nilsson and von Heijne, 2000
; Whitley et al., 1996
). Because the distance between the ribosome P-site and OST active site is estimated to be around 65 residues, this inefficient N-glycosylation is believed to be due to the sequon failing to reach the active site of OST prior to the dissociation of the polypeptide from the ribosome (Nilsson and von Heijne, 2000
; Whitley et al., 1996
).
Recently, we have shown that the N-glycosylation of a nonanchored form of PrP in neuronal cells is abolished when the sequons are fewer than 60 residues from the C-terminus (Walmsley and Hooper, 2003; Walmsley et al., 2001
). These observations led us to conclude that sequon utilization in PrP is a cotranslational process. In the present study, we have investigated whether sequon distance from the C-terminus is a general determinant of N-glycosylation in living cells by introducing the sequons of PrP into another glycosylphosphatidylinositol (GPI)-anchored protein, membrane dipeptidase (MDP), and expressing the protein in the same neuronal cells as used to study the glycosylation of PrP. In the same cell type the introduced sequons in MDP were fully utilized even when they were fewer than 60 residues from the C-terminus, indicating that in two different proteins the same sequons in similar positions near the C-terminus can be differentially utilized by OST.
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Results |
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To assess whether the N-glycosylation of the introduced Asn-Ile/Phe-Thr sequons was dependent on their distance to the C-terminus, the GPI signal sequence of the MDP constructs was deleted so that both sequons were fewer than 60 residues from the C-terminus (Figure 3). When stably expressed in SH-SY5Y cells, MDPN
GPI, MDP
GPIN332, and MDP
GPIN358 were detected in the medium (Figure 4A), consistent with these constructs lacking a mode of membrane anchorage and thus behaving as secretory proteins. The presence of a proportion of the MDP
N
GPI construct in the cell lysate, along with some lower-molecular-weight fragments (Figure 4A, lane 2) may reflect an impaired rate of trafficking through the secretory pathway of this unglycosylated, non-GPI anchored protein. The mobility of these secreted constructs was not altered by incubation with PI-PLC (Figure 4B), confirming that they lacked a GPI anchor. Following PNGase F treatment (Figure 4C), the molecular weight of secreted MDP
N
GPI was unaltered as expected, whereas the molecular weights of secreted MDP
GPIN332 and secreted MDP
GPIN358 were reduced to that of MDP
N
GPI, demonstrating the presence of N-glycans on these proteins. Therefore, the Asn-Ile/Phe-Thr sequons were fully utilized in MDP even when their distance from the C-terminus was fewer than 60 residues.
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Discussion |
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Unlike in PrP, the introduced sequons in both the GPI-anchored and nonanchored forms of MDP were efficiently N-glycosylated even when they were fewer than 60 residues from the C-terminus. For example, even though it would be inside the ribosome on chain termination (Matlack and Walter, 1995), the Asn-Phe-Thr sequon 26 residues from the C-terminus of MDP
GPIN358 was efficiently utilized. These findings suggest that the Asn-Xaa-Thr sequons near the C-terminus of MDP, in contrast to those in PrP, can be N-glycosylated following detachment of the polypeptide chain from the ribosome. Such posttranslational glycosylation of proteins has been reported in only a limited number of cases (Hessa et al., 2003
; Kolhekar et al., 1998
; Olivares et al., 2003
).
The lack of N-glycosylation of PrP may be due to the sequons becoming inaccessible to the active site of OST during the folding of the nascent polypeptide, as appears to be the case for tPA (Allen et al., 1995). In PrP the two sequons (Asn180 and Asn196) lie between the two cysteine residues (Cys178 and Cys213) that form the only intrachain disulfide bond in the protein. Interestingly, this bond can only form posttranslationally as Cys213, being only 41 residues from the C-terminus of the nascent polypeptide chain, would not be accessible to the lumen of the ER during translation (Whitley et al., 1996
). The formation of the intrachain disulfide bond following chain termination may compromise the accessibility of the Asn180-Ile-Thr and Asn196-Phe-Thr sequons to the OST active site and thus prevent their N-glycosylation. In agreement with this is the finding that treatment of neuronal cells with the thiol reductant dithiothreitol resulted in an increase in the N-glycosylation efficiency of PrP, as did mutation of one of the cysteine residues contributing to the disulfide bond (Capellari et al., 1999
). In contrast, the two introduced sequons in MDP (Asn332-Ile-Thr and Asn358-Phe-Thr) lie C-terminal to the cysteine residues involved in the two intrachain disulfide bonds (Cys87-Cys154 and Cys242-Cys274) (Nitanai et al., 2002
).
In conclusion, these results clearly show that in two different proteins the same sequons in similar positions near the C-terminus can be differentially utilized in the same cell line. This not only reveals the problems in extrapolating from in vitro systems with model proteins to the complexity of living cells but also highlights the difficulty involved in predicting N-glycosylation sequon occupancy in silico (Gavel and von Heijne, 1990).
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Materials and methods |
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Generation of constructs
MDP in pCIneo has been previously described (Pang et al., 2001). All mutants were generated using the Quickchange site-directed mutagenesis kit (Stratagene, LaJolla, CA) according to the manufacturer's instructions and the mutations confirmed by sequencing on both strands (DNA sequencing service, University of Durham, U.K.). MDP
GPI was generated from MDP in pCIneo using the following mutagenic primers: sense primer 5'- ACGGCTACTCATGAGCCCCCAGCCT-3' and antisense primer 5'-AGGCTGGGGGCTCATGAGTAGCCGT-3'. MDP
N and MDP
N
GPI were generated from MDP and MDP
GPI, respectively, by two rounds of site-directed mutagenesis using the following primers: sense primer 5'-ACCCGGGGGCCCAACTCTCCAGCCT-3' and antisense primer 5'-AGGCTGGAGAGTTGGGCCCCCGGGT-3' for mutation of Asn41 to Gln; sense primer 5'-CGGCCAAGGCCCAATTGTCCCAAGT-3' and antisense primer 5'-ACTTGGGACAATTGGGCCTTGGCCG-3' for mutation of Asn263 to Gln. MDPN332 and MDP
GPIN332 were generated from MDP
N and MDP
N
GPI, respectively, using the following mutagenic primers: sense primer 5'-GCTCAGGAGGAACATCACGGAGGCCG-3' and antisense primer 5'-CGGCCTCCGTGATGTTCCTCCTGAGC-3'. MDPN358 and MDP
GPIN358 were generated from MDP
N and MDP
N
GPI, respectively, using the following mutagenic primers: sense primer 5'-GGCCAGCAATTTCACTCAGGTTCCAG-3' and antisense primer 5'-CTGGAACCTGAGTGAAATTGCTGGCC-3'.
Cell culture, transfection, and lysis
SH-SY5Y cells were maintained at 37°C in Dulbecco's modified Eagle's medium with Glutamax-I, sodium pyruvate, 4.5 mg/ml glucose, and pyridoxine supplemented with 10% (v/v) fetal calf serum (Gibco, Paisley, Scotland) in a humidified atmosphere of 5% CO2/95% air. Cells were electroporated with linearized DNA and selected with G-418 as described elsewhere (Walmsley et al., 2001). Briefly, cells at midconfluency were harvested with trypsin and resuspended in complete medium at a concentration of
1 x 106 cells/ml. An 800-µl aliquot of cell suspension was placed in a 4-mm electroporation cuvette and incubated for 1 min with 30 µg linearized DNA. Cells were pulsed at 1650 µF/250 V using the Easy-Ject electroporator (Flowgen, Leicestershire, UK) and immediately transferred to fresh complete medium. Selection for antibiotic resistance was imposed 48 h after electroporation by incubating the cells with complete medium containing 1 mg/ml G-418. Because pCIneo is a bicistronic vector, all G-418-resistant cells have similar levels of target protein expression, and thus cloning is not required (Rees et al., 1996
). Each stably expressing cell line represented a pooled population of G-418-resistant colonies (<50 colonies/cell line) that expressed their target protein at levels sufficient for detection by western blot analysis.
Cells at confluency were rinsed twice with phosphate buffered saline (PBS; 1.5 mM KH2PO4, 2.7 mM Na2HPO4, 150 mM NaCl, pH 7.4), scraped into the same buffer, and harvested by centrifugation for 3 min at 500 x g. The cells were resuspended in lysis buffer (10 mM TrisHCl, pH 7.8, 0.5% [w/v] sodium deoxycholate, 0.5% [v/v] Nonidet P40, 100 mM NaCl, 10 mM ethylenediamine tetra-acetic acid [EDTA], 0.1 mM phenylmethanesulfonylfluoride) and incubated for 30 min at room temperature. The lysates were clarified by centrifugation for 1 min at 13,000 x g. Medium samples were concentrated by the addition of five volumes of methanol and incubated for 30 min at -20°C. The precipitate was harvested by centrifugation for 5 min at 13,000 x g, dried, and resuspended in lysis buffer.
SDSPAGE and western blot analysis
Samples containing 10 µg of total protein were mixed with an equal volume of SDS dissociation buffer (125 mM TrisHCl, pH 6.8, 2% [w/v] SDS, 20% [v/v] glycerol, 100 mM dithiothreitol, bromophenol blue) and boiled for 5 min. Immunoprecipitates were resuspended in SDS dissociation buffer and boiled. Proteins were resolved by electrophoresis through 15% polyacrylamide gels. For western blot analysis, resolved proteins were transferred to a Hybond-P poly(vinylidene)difluoride membrane (Amersham, Little Chalfont, U.K.). The membrane was blocked by incubation for 1 h with PBS containing 0.1% (v/v) Tween 20 and 5% (w/v) dried milk powder. Incubations with primary and peroxidase-conjugated secondary antibodies were performed for 1 h in the same buffer. MDP was detected with the polyclonal antibody RP209 (Littlewood et al., 1989
). Bound peroxidase conjugates were visualized using an enhanced chemiluminescence detection system (Amersham).
PI-PLC and PNGase F digestion
For cleavage of the GPI anchor with PI-PLC, cell lysates or concentrated media samples at a total protein concentration of 100 µg/µl were incubated for 3 h at 37°C with 1 U/ml Bacillus thuringiensis PI-PLC prepared as described (Low, 1992
). Enzymatic deglycosylation was performed with PNGase F according to the supplier's instructions (Glyko, Novato, CA). Briefly, samples of cell lysate or concentrated media were made 20 mM with respect to sodium phosphate, pH 7.6, 50 mM with respect to EDTA, 5% (w/v) with respect to SDS, and 5% (v/v) with respect to ß-mercaptoethanol. Samples were boiled for 5 min, diluted fivefold with 1.25% (v/v) Triton X-100, and incubated for 16 h at 37°C with 1 U peptide N-glycosidase F/
100 µg total protein.
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; e-mail: n.m.hooper{at}leeds.ac.uk
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Abbreviations |
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References |
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