2Oxford Glycobiology Institute, Department of Biochemistry, South Parks Road, Oxford, OX1 3QU, United Kingdom; and 3Molecular Sciences Division, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 9DU, United Kingdom
Received on July 16, 2001; revised on January 7, 2002; accepted on January 7, 2002.
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Abstract |
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Key words: CD59/CHO cells/MALDI MS/N-acetyllactosamine extensions/N-linked glycosylation
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Introduction |
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The amino acid sequence of CD59, predicted from its cDNA nucleotide sequence, consists of 128 amino acids (Sugita et al., 1989), including an amino-terminal signal peptide of 25 residues and a carboxyl-terminal of 26 residues. This C-terminal sequence is a signal that directs the attachment of a GPI membrane anchor (Sugita et al., 1993
) The mature glycoprotein, after removal of the signal peptide, has 77 amino acids (sequence mass = 8707.8; Figure 1). The soluble form of CD59 used in this study was truncated at Asn70, giving a sequence mass of 8076.2. It was expressed in Chinese hamster ovary (CHO) cells (Rudd et al., 1999
).
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The N-linked glycosylation of soluble (urinary) human CD59 (shCD59) has been analyzed by several investigators (Meri et al., 1996; Nakano et al., 1994
). Rudd et al. (1999)
analyzed the glycosylation of the soluble form expressed in CHO cells. The glycans identified in all three studies consisted mainly of core-fucosylated biantennary compounds. Less than 1% of the total were triantennary complex structures. Small amounts of glycans with N-acetyllactosamine extensions to the antennae were also found. All complex glycans were variably sialylated. Rudd et al. (1997)
additionally analyzed the N-glycosylation of human erythrocyte CD59 containing the GPI anchor and identified more than 100 glycans, including a wide range of biantennary complex structures with multiple lactosamine extensions and several outer arm fucose residues. In addition, two potential O-glycosylation sites at Thr51 and Thr52 (Rudd et al., 1997
) adjacent to the active site residues, Arg53 and Glu56 (Bodian et al., 1997
) were found to be partially occupied with the O-glycans, Neu5Ac
2
3Galß1
3GalNAc and Galß1
3[Neu5Ac
2
6]GalNAc.
This article presents a more detailed description of the N-linked glycosylation of the soluble form expressed in CHO cells than that reported earlier (Rudd et al., 1999) and compares this with the glycosylation of the GPI-anchored form of the glycoprotein, again expressed in CHO cells. The aim was to investigate whether the glycan processing of CD59 is affected by the presence of a GPI anchor.
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Results and discussion |
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Figure 2 shows the positive ion matrix-assisted laser desorption/ionization (MALDI) mass spectrum of the methyl ester fraction from a 12.5% aliquot of the total glycan pool obtained from 3 µg of rhCD59. An identical profile was obtained from the solution-released sample. Glycans released by automated hydrazinolysis (Rudd et al., 1999) and examined as 2-aminobenzamide (2-AB) derivatives by either high-performance liquid chromatography (HPLC) or MALDI MS showed a similar profile.
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A parallel sequential digestion of the total glycan pool with A. ureafaciens sialidase and S. pneumoniae ß-galactosidase removed only the terminal ß14-linked galactose residues; those forming integral parts of N-acetyllactosamine repeat units remained intact on their respective core structures. Thus the number of antennae in each glycan could be determined by the number of galactose residues removed. The resulting mass spectrum (Figure 5) contained varying amounts of complex glycans consisting of biantennary structures with up to three N-acetyllactosamine extensions, triantennary glycans with up to three N-acetyllactosamine units, and tetraantennary glycans with two extensions. Only a small percentage of tri- and tetraantennary sugars without N-acetyllactosamine groups was present. Thus in Figure 5, the ion at m/z 1485.5 originates from the biantennary glycan, the ion at m/z 1688.6 from a triantennary compound (addition of 203 mass units), and the ion at m/z 1891.7 from a native tetraantennary structure. Ions at m/z 1850.7, 2215.9, and 2581.0, all of which lost two galactose residues, are from compounds containing a biantennary core (I) and one, two, and three N-acetyllactosamine extensions, respectively. The ions at m/z 2053.7, 2419.0, and 2784.0 arise from similarly extended triantennary glycans. N-acetyllactosamine extensions to the tetraantennary glycan gave the ions at m/z 2256.9 and 2621.0. It would appear that the more highly branched glycans were more likely to be present with N-acetyllactosamine extensions, although the parent tri- and particularly the tetraantennary compounds were less abundant than the biantennary glycans. This observation correlates with that reported by others (Fukuda, 1994
) who have also observed that tetraantennary structures are preferable acceptors for poly-N-acetyllactosamine extensions. The relative percentages of the bi-, tri-, and tetraantennary glycans (with and without N-acetyllactosamine extensions) measured in this experiment were 62%, 32%, and 6%, respectively.
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Distribution of the N-acetyllactosamine extensions
The distribution of the N-acetyllactosamine extensions among the antennae was probed by incubation with sialidase and endo-ß-galactosidase. The latter enzyme will reduce N-acetyllactosamine-containing antennae to GlcNAc but leave unextended antennae as Galß14GlcNAc. Thus in Figure 6, the biantennary glycans are represented by ions at m/z 1809.6 (two terminal galactose, no extensions, compound I), 1647.6 (one antennae extended), and 1485.5 (two antennae extended) in the relative proportions of 38%, 44%, and 18%, respectively, showing that most biantennary glycans that carried N-acetyllactosamine extensions did so on only one antenna. Corresponding figures for the tri- and tetraantennary glycans are listed in Table II.
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Soluble CD59
The soluble form of rhCD59 migrated as two broad bands on SDSPAGE with the apparent molecular weights of 1622 kDa (band 1) and 614 kDa (band 2). Each band was excised and digested in-gel with PNGase F, followed by either esterification of the sialic acidcontaining sugars or by exoglycosidase digestions. Figure 8 shows the positive-ion MALDI mass spectra of the total glycan pool obtained from band 1. No N-linked glycans were recovered from band 2, indicating that, in contrast to GPI-anchored CD59, the soluble form of CD59 can be secreted in an unglycosylated form, similar to Thy-1 (Devasahayam et al., 1999). Exoglycosidase digestion showed that the glycans were essentially similar to those found in the GPI-anchored sample but that the extent of poly-N-acetyllactosamine extensions to the antennae was much less. The molecular weights and compositions of the glycans are compiled in Table I. Simultaneous treatment of the sugars with A. ureafaciens sialidase and S. pneumoniae ß-galactosidase showed that approximately 97% of the sample consisted of biantennary structures of which 81% contained no N-acetyllactosamine extensions, 17% contained one N-acetyllactosamine extension, and 2% had two of these units. The presence of N-acetyllactosamine extensions was confirmed by incubation with a mixture of endo-ß-galactosidase, A. ureafaciens sialidase, and S. pneumoniae galactosidase. The remaining 3% of the glycans were triantennary. Approximately 50% of these were unmodified, and the remainder carried one or two N-acetyllactosamine chains.
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A possible reason for the difference between the glycosylation of the glycoprotein with and without a GPI anchor is that the glycoprotein with the GPI anchor passes through the Golgi at a slower rate than the soluble form allowing longer exposure to the glycosyltransferases (Nabi and Dennis, 1998). A similar result has been reported by Fukuda et al. (1988)
for the
-chain of human chorionic gonadotropin fused to the transmembrane and cytoplasmic domains of vesicular stomatitis virus-G protein, making a chimeric membrane glycoprotein. The N-glycans attached to this protein were extensively modified by poly-N-acetyllactosamines, whereas the native secretory hCG-
protein contained typical complex-type oligosaccharides. It is also possible that GPI-anchored proteins are more efficiently processed because they are held closer to the glycan-processing enzymes, which are also membrane-bound. The results presented herein, therefore, clearly show that great care should be taken in extrapolating from the analysis of the glycans of soluble proteins to those from the anchored state.
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Materials and methods |
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Gel electrophoresis and in-gel PNGase F digestion
SDSPAGE gels (80 x 80 x 0.75 mm) were run using a Mini PROTEAN II cell (Bio-Rad) at 200 V constant voltage in 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3, as previously described (Küster et al., 1997). Approximately 120 pmole of the GPI-anchored form of CD-59 (3 µg) and 900 pmole of the soluble form (10 µg) were loaded onto the gel and visualized by Coomassie staining for 15 min. After destaining, the glycoprotein bands were excised from the gel, reduced, alkylated, and digested with PNGase F as described previously (Küster et al., 1997
). The glycans were extracted from the gel pieces by three changes of 200 µl water with sonication for 30 min followed by extraction with 200 µl of freshly distilled acetonitrile. All extracts were combined, incubated with 30 µl AG50-X12 (hydrogen form) for 5 min, then filtered (Millipore syringe filter type FH, 0.45 µm) and dried in vacuo. The sample was split into two aliquots. In one aliquot, the sialic acids were then converted into their methyl esters using the procedure described by Powell and Harvey (1996)
, and the other was passed through a microcolumn of 5 µl each of AG3-X4 (free base form), AG50-X12 (hydrogen form), and C18 resins that had been prewashed with 3 x 50 µl water. Glycans were eluted with 3 x 50 µl water, collected, and dried in vacuo.
PNGase F solution digest of CD-59 (GPI anchor form)
Approximately 3 µg of rhCD-59 (GPI anchor form), in 20 mM PBS, pH 7.4, containing 10 mM CHAPS surfactant, was denatured at 100°C with 0.1% SDS for 5 min and diluted with 24 mM phosphate buffer, pH 8.6, containing 34 mM octylglucoside (160 µl). A solution of 200 U/ml PNGase F (50 µl) was added to the sample and incubated at 37°C for 24 h. Complete digestion was confirmed by SDSPAGE from an aliquot (70 µl) of the digest mixture. Glycans were recovered by applying to a microcolumn packed with 30 µl C18 resin that had been washed with 200 µl water. Sugars were eluted with 4 x 60 µl of water and dried in vacuo. The sample was split into two; half was digested with 2 U/ml A. ureafaciens sialidase (5 µl), and the remainder was converted to methyl esters as already described.
Glycan release by automated hydrazinolysis
Glycans were released with hydrazine at 95°C for 5 h and re-N-acetylated using a Glycoprep 1000 (Oxford GlycoSystems). Samples were filtered and rotary-evaporated to dryness before 2-AB labeling.
Fluorescent labeling of glycans
Glycans were fluorescently labeled with 2-AB by reductive amination according to the procedure described by Bigge et al. (1995).
Normal phase HPLC
2-AB-Labeled glycans were separated on a 4.6 x 250 mm GlycoSep N column (Oxford GlycoSciences) using a Waters 2690 Alliance separation module with a column heater at 30°C and a Waters 474 scanning fluorescence detector. The gradient used was as described by Guile et al. (1996): solvent A, 50 mM ammonium formate, pH 4.4; solvent B, acetonitrile. Initial conditions were 20% A at a flow rate of 0.4 ml/min followed by a linear gradient of 2058% A over 152 min followed by 58100% A in the next 3 min. The column was washed with 100% A for 5 min at a flow rate of 1 ml/min before being re-equilibrated in 20% A for the next sample.
Exoglycosidase digestions
The unmethylated glycans obtained from in-gel PNGase F digestion were incubated with A. ureafaciens sialidase and then split into seven aliquots (1 µl) and incubated overnight at 37°C in 20 mM sodium acetate at pH 5.5, in parallel with 1 µl of each of the following exoglycosidase arrays: (1) S. pneumoniae ß-galactosidase (1 U/ml); (2) ß-galactosidase and bovine epididymis -fucosidase (1 U/ml); (3) ß-galactosidase,
-fucosidase, and jackbean ß-N-acetylhexosaminidase (30 U/ml); (4) ß-galactosidase,
-fucosidase. and S. pneumoniae N-acetyl-ß-D-glucosaminidase (6 mU/ml); (5) ß-galactosidase,
-fucosidase, jackbean ß-N-acetylhexosaminidase, and jackbean
-mannosidase (100 U/ml); and (6) ß-galactosidase and B. fragilis endo-ß-galactosidase (0.8 U/ml). Each reaction mixture was prepared for MALDI MS by using the neutral glycan microcolumn clean-up procedure described herein.
MALDI MS
An aliquot (12 µl) of the soluble CD59 solution was drop-dialyzed against water for 10 min using a Spectra/Por CE 2000 molecular weight cut-off membrane (Spectrum Medical Industries, TX). The solution was then transferred to the MALDI target plate, mixed with 1 µl of a solution of sinapinic acid (10 mg/ml in 70:30 v/v acetonitrile:0.1% trifluoroacetic acid) and allowed to dry. Released glycans (50100 pmoles) were loaded onto the mass spectrometer target in 1 µl of water, mixed with a fresh solution of 2,5-DHB (1 µl of 10 mg/ml in acetonitrile), and allowed to dry. The mixture was then recrystallized from the minimum amount of alcohol (Harvey, 1993).
All glycan mass spectra were acquired in positive ion mode with a PerSeptive Biosystems Voyager Elite reflectron time-of-flight mass spectrometer equipped with a delayed extraction MALDI ion source. The delay time was 75 ns. Grid wire and guide wire voltages were 65% and 0.1% of the accelerating voltage (20 kV), respectively. Between 150 and 256 scans were averaged for each spectrum. The instrument was externally calibrated with a mixture of dextran oligomers. All molecular weights are quoted as monoisotopic masses. Neutral and derivatized sialic acidcontaining oligosaccharides were observed as [M+Na]+ ions accompanied by lower amounts of [M+K]+ ions.
For glycoprotein samples, the delay time was 100 ns, and the grid wire and guide wire voltages were 90% and 0.3% of the accelerating voltage (20 kV), respectively. The instrument was externally calibrated with horse heart cytochrome C. Glycoproteins were observed as [M+H]+ ions in the positive ion mode, and all molecular weights are quoted as average (chemical) masses.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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