2 315 Riverbend Road, Complex Carbohydrate Research Center, Athens, GA 30602; and 3 Centro de Investigacion en Alimentacion y Desarrollo Ac. Km 0.6. carr a La Victoria, Hermosillo, Sonora, Mexico, 83000
Received on October 4, 2003; revised on February 13, 2004; accepted on February 13, 2004
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Abstract |
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Key words: glycoforms / LC-MS / MALDI MS / N-acetylglucosaminyltransferase V / N-glycosylation
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Introduction |
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Given the evidence that GnT-V activity can increase the rate of progression of some tumors, it is a logical target for rational drug design. To produce for X-ray crystallographic studies the smallest fragment of active GnT-V, a recent study suggested that the minimal catalytic region for GnT-V consisted of amino acids 213740 (Korczak et al., 2000). A comparison of this sequence relative to the full-length sequence of 740 amino acids showed that this fragment of GnT-V contained only three of the six total putative N-glycosylation sites. Peptide N-glycosidase F (PNGase F) treatment of GnT-V resulted in precipitation (Kamar et al., unpublished data), indicating that N-glycans are likely critical for the stability of this glycoprotein. Thus to design strategies to produce large quantities of recombinant GnT-V for structural studies, it is important to understand in detail its N-linked glycosylation. Therefore these studies focused on the three putative glycosylation sites that are present in the C-terminal portion of GnT-V, which is responsible for catalytic activity.
The aims of the experiments described in the present article were to characterize the N-linked oligosaccharides of GnT-V, to determine which of the three potential N-linked glycosylation sites in the molecule are occupied, and to assign to each occupied site its associated oligosaccharides. Very few studies have determined the occupied N-glycosylation sites of glycosyltransferases, and most of these use site-directed mutagenesis and western blotting methods to identify them (Baboval et al., 2000). Reports from Macher's laboratory have described the mapping of N-glycosylation sites of two fucosyltransferases by mass spectrometric analysis (de Vries et al., 2001
; Holmes et al., 2000
). The present article uses mass spectrometric analyses to assign specific oligosaccharide structures to each site that expresses an N-linked glycan.
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Results and discussion |
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In Figure 8b (fraction 9), the complexity of the mass spectra was much greater due to the presence of several glycoforms (b18), all of which contained a 5+ and 6+ charge. Species b14 (5+, 6+) differed by a m/z of 58 or 48, respectively, corresponding to the mass of NeuAc, and this trend was also observed in species b57 (5+, 6+). When the experimental masses were compared to theoretical masses, the glycoforms b18 corresponded to a glycopeptide with a glycosylation at site Asn 433. Analysis of the glycoforms with the mass of the peptide removed indicated that the glycans attached to the peptide ranged from a biantennary structure with two sialic acids to a tetraantennary structure with four sialic acids. A loss in m/z of 58 (5+) or 48 (6+) between glycoform b1 and b2 showed the presence of both a tetraantennary glycan with four sialic acids and a tetraantennary glycan with three sialic acids. Ions were also present that would account for a loss of one or two sialic acids from b2 to give ions b3 or b4. A mass difference of 73 (5+) or 61 (6+), which was seen between b2 to b5, would result from the removal of Gal and GlcNAc. Species b5 represented a triantennary glycan with three sialic acids, which was by far the most predominant species for both the 5+ and 6+ ions in the mass spectra. A loss of one or two sialic acids from b5 would give rise to b6 or b7, triantennary structures with two or one sialic acids. Unlike site Asn 334, site Asn 433 contained bi-, tri-, and tetraantennary structures. All structures were core fucosylated and sialylated.
In Figure 8c, a large amount of heterogeneity was observed in this chromatogram, although this mass spectrum was less complicated than Figure 8b, due to the presence of only triply charged ions. A mass increment of 97 (3+) between ions c14 and c57 indicated differences in degrees of sialic acid substitution. An m/z difference of 122 (3+) between c2 and c5, c3 and c6, c4 and c7, and c5 and c8 corresponded to the absence of one antennary branch containing a Gal and GlcNAc. A comparison of theoretical masses compared to the observed masses indicated that this spectrum was that of glycopeptide containing site Asn 447. Analysis of the variety of masses observed showed the presence of various glycoforms of Asn 447, with structures ranging from a biantennary with two sialic acids (c8) to a tetraantennary with four sialic acids (c1). By far the most abundant species was the triantennary with three sialic acids, as seen in ion c5.
To determine accurately the total ratios of complex N-linked glycans, GnT-V (0.1 mg) was treated with PNGase F, the free N-glycans hydrolyzed under mild acid conditions to remove the terminal sialic acids, and the reducing termini were fluorescently labeled with 2-aminopyridine (2-AP; Hase, 1994), followed by high-pressure liquid chromatography (HPLC) separation on an amide-based resin. The ratio of GnT-V N-glycans was determined by integrating the fluorescence intensity from the various peaks observed (data not shown). Tri- and tetraantennary structures made up 35.5% and 30.1% of the glycans, respectively, and polylactosamine structures composed only 9.4%. Although polylactosamine structures were detected by MALDI-TOF analysis, none were found by LC-MS, likely due to the relatively small amount of polylactosamine present and the complexity of fraction 9 and 10 and would be undetectable due to the high background.
Interestingly, the biantennary structures made up 25.0% of the total N-glycan pool. Because tri- and tetraantennary structures composed such a large percentage (75%) of the N-glycans, GnT-V most likely underwent autocatalysis during its transit through the Golgi. Moreover, only biantennary structures were detected at site Asn 334, whereas the other two Asn sites showed bi-, tri-, and tetraantennary glycans. For Asn 334 to express only biantennary structures, unlike the other two sites, it is reasonable to suggest that during biosynthesis the biantennary structure at Asn 334 was inaccessible as an acceptor for GnT-V. Because Asn 334 is found galactosylated and sialylated, it must, however, have been accessible to the galactosyltransferase and sialyltransferase. Do et al. (1994) determined that many endogenous proteins expressed in CHO Lec 8 cells were not acceptors for GnT-V in vivo until after in vitro denaturation. The GnT-V glycan site-mapping data extend these results to demonstrate that on a single protein a particular N-linked site can be restricted in its ability to be branched by GnT-V while still being accessible to other glycosyltransferases. These data also imply that there are significant differences in the accessibility of the GnT-V acceptor binding site compared to those of the sialyltransferase and galactosyltransferase.
There have been several studies analyzing the requirement of N-glycosylation for activity of particular glycosyltransferases. In experiments performed on (2,6) sialyltransferase, PNGase F treatment caused a significant decrease in enzyme activity (Fast et al., 1993
). In other studies, the putative Asn glycosylation sites of recombinant glycosyltransferases were altered individually and in combinations using site-directed mutagenesis and expression in animal cells to study these effects on the enzymatic activities. The removal of putative N-glycosylation sites decreased the activity of ß1,4-N-acetylgalactosaminyltransferase (Haraguchi et al., 1995
), N-acetylglucosaminyltransferase III (Nagai et al., 1997
), and
1,3/4-fucosyltransferase III, V, and VI (Christensen et al., 2000
). The removal of these sites also inhibited the ability of N-acetylglucosaminyltransferase III (Nagai et al., 1997
) and fucosyltransferase III (Morais et al., 2003
) to transit from the endoplasmic reticulum to the Golgi. Because PNGase F treatment of GnT-V caused precipitation of the enzyme from solution and because we now know that sites Asn 334, 433, and 447 are N-glycosylated, we can conclude that at least one of these three glycosylation sites is required for the stability of GnT-V. Large-scale production of GnT-V for structural studies must therefore take into account this requirement.
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Materials and methods |
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Purification of GnT-V
GnT-V was initially purified from the concentrated media on a phenyl Sepharose high-performance column. Ammonium sulfate was added to the concentrated minimal medium to bring the final concentration to 1 M. The sample was centrifuged for 30 min at 9500 rpm to remove any debris. The solvents used were buffer A (1 M ammonium sulfate and 25 mM 2-morpholinoethanesulfonic acid [MES], pH 6.5) and buffer B (25 mM MES, pH 6.5). For all purification steps, flow rate was maintained at 5 ml/min, and the sample was detected by ultraviolet at 280 nm. After injection of sample, the column was maintained at 0% B for 20 min followed by a linear gradient to 90% B for 2 min. The column was eluted at 90% B for 12 min, followed by another linear gradient to 100% B over 2 min; 100% B was maintained for 10 min, followed by another linear gradient to 0% B for 2 min. Finally, the column was washed for 10 min at 0% B.
The second purification step for GnT-V was performed on two 5-ml HiTrap chelating columns coupled together and charged with copper sulfate. The eluted sample fractions from the phenyl Sepharose column were pooled and brought to a final concentration equivalent to buffer C. The solvents used were buffer C (2 5 mM MES, pH 6.5 and 100 mM NaCl) and buffer D (25 mM MES, pH 6.5, 10 0 mM NaCl, and 50 mM imidazole). After injection of sample, the column was maintained at 0% D for 10 min followed by a linear gradient to 60% D for 1 min. The column was eluted at 60% D for 8 min, followed by another linear gradient to 100% B over 1 min; 100% D was maintained 8 min, followed by a linear gradient to 0% D for 1 min. Finally, the column was washed in 0% D for 10 min.
The third purification step for GnT-V was performed on an SP Sepharose column. The eluted sample fractions from the copper chelating column were pooled and brought to a final concentration equivalent to 25 mM MES, pH 6.5, and 100 mM NaCl. The solvents used were buffer E (25 mM MES, pH 6.5) and buffer B. Prior to injection of the sample, the column was equilibrated in 10% F. After injection of the sample, the column remained at 10% F for 10 min, followed by a linear gradient to 46% F for 2 min. The column was maintained at 46% F for 10 min, followed by a linear gradient to 100% F for 2 min. The column was washed in 100% F for 10 min, followed by a linear gradient to 10% F. Finally, the column was washed for 10 min at 10% F.
In-solution PNGase F digestion of GnT-V
One milligram of GnT-V in 25 mM MES, pH 6.5, and 100 mM NaCl was denatured at 100°C in 0.1% sodium dodecyl sulfate (SDS) for 10 min. The sample was cooled on ice for 5 min and then mixed with Nonidet P-40 to a final concentration of 0.5%. After addition of PNGase F, the sample was incubated at 37°C for 16 h. The released glycans were purified from the protein and SDS by elution on a C18 Sep-pak. The C18 cartridge was attached to a 10-ml syringe and washed with 5 ml methanol, followed by 10 ml 5% acetic acid/H2O by use of gentle air pressure. The digest was loaded onto the C18 cartridge and the glycans were eluted in 5 ml 5% acetic acid/H2O. The eluted glycan fraction was split in to two aliquots, which were then dried overnight in a Speed Vac Concentrator (Savant). One-half of the eluted glycans was used for 2-AP labeling and the other half for analysis by MALDI-TOF MS and exoglycosidase experiments.
2-AP labeling of glycans
The glycans were fluorescently labeled with twice-crystallized 2-AP by reductive amination according to the method of Hase (1994). The excess 2-AP was removed by gel filtration using 50 ml G-15 sephadex with a Beckman (Fullerton, CA) 110B solvent delivery module HPLC, and fluorescence was detected on a Perkin Elmer (Wellesley, MA) 65015 fluorescence spectrophotometer. Excitation wavelength was at 320 nm, and emission wavelength was at 400 nm. The sample was run at a flow rate of 1 ml/min for 180 min with a 10 mM ammonium bicarbonate buffer. The fractions containing the desalted 2-AP-labeled N-glycans were pooled and dried down overnight in a Speed Vac concentrator.
Mild acid hydrolysis of 2-AP labeled glycans
To remove the sialic acids from GnT-V glycans, samples were resuspended in 200 µl 2 M acetic acid and heated at 100°C for 1 h. This was followed by addition of 500 µl 50% methanol and dried under N2. The sample was resuspended and dried under N2 three times.
Separation of 2-AP-labeled glycans on TSK Amide-80 column
2-AP-labeled glycans were size fractionated on a 4.6 x 250 mm TSK Amide-80 column and detected by fluorescence. The column was heated at 30°C in a column oven. The gradient and solvents used were according to Hase (1994): buffer A (3% acetic acid/H2O, pH 7.3, with triethylamine) and buffer B (80% acetonitrile, 3% acetic acid, ph 7.3, with triethylamine). After applying the sample, elution is carried out with a linear gradient from 0% to 10% of A over 2 min at a flow rate of 1 ml/min. This is followed by a linear gradient from 10% to 30% A for 28 min and then 30% to 100% A for another 25 min, at which point the column can be reequilibrated in 0% A for injection of the next sample.
Exoglycosidase digestions
The released unmethylated N-glycans were resuspended in 100 µl H2O, and 10 µl was removed for immediate permethylation and analysis by MALDI-TOF MS. The remaining material was split into four aliquots and incubated at 37°C overnight in 50 mM sodium phosphate, pH 5.0. One microliter of each of the following exoglycosidases was used: (1) A. ureafaciens sialidase (10 U/ml); (2) A. ureafaciens sialidase and E. coli ß-galactosidase (7 U/ml); (3) A. ureafaciens sialidase, ß-galactosidase, and bovine kidney ß-N-acetylglucosaminidase (52 U/ml); and (4) A. ureafaciens sialidase and Xanthomonas sp. -1
(2,3,4)-fucosidase (0.1 U/ml). Each sample was then permethylated and run on MALDI-TOF MS.
Permethylation of N-glycans
All N-glycan samples were permethylated according the method of Ciucanu and Kerek (1984). The dried permethylated samples were then dissolved in 20 µl of methanol prior to MALDI-TOF analysis.
MALDI-TOF MS
One microliter of permethylated glycans was added to 1 µl DHB (5 mg/ml in acetonitrile) and mixed; 0.3 µl was applied to the surface of the MS probe. The probe was then placed under vacuum to remove the organic solvents and cocrystallize the material with the matrix.
MALDI-TOF-MS was performed with a Bruker Reflex (Bremen, Germany) retrofitted with a delayed extraction MALDI ion source. Samples were analyzed using reflex mode with delayed extraction in positive ion mode. Prior to acquiring mass spectra for the permethylated glycans, the instrument was calibrated using human angiotensin I and human insulin. All masses are listed as monoisotopic.
Denaturation, reduction, alkylation, and trypsinization of GnT-V
An aliquot of 4.5 mg of lyophilized GnT-V was completely resuspended in 200 ml 45 mM dithiothreitol and 8 M urea in 40 mM TrisHCl, pH 8.5. This solution was incubated at 55°C for 1 h. Enough 500 mM iodoacetamide was added to the sample to bring the final concentration to 80 mM. The solution was then incubated in the dark at room temperature for 30 min. The 8 M urea in the sample was diluted to 1 M before addition of porcine trypsin was added in a 1:50 ratio of trypsin to sample. This sample was then incubated overnight at 37°C.
Separation of GnT-V glycopeptides on Superdex Peptide column
Trysinized GnT-V was run on a Superdex Peptide column under isocratic conditions for 90 min at 0.5 ml/min using a 100 mM ammonium bicarbonate and 1% butanol buffer. The sample was detected by ultraviolet at 230 nm with an online detector, and 1-ml fractions were collected.
Phenol sulfuric assay
Each fraction collected from the Superdex Peptide column was tested for the presence of carbohydrates using a phenol sulfuric assay according to the method of Dubois et al. (1956). Fractions positive for the presence of glycans were dried down in a Speedvac and run on tandem MS.
Analysis of the glycopeptides by online ESI-MS using Q-TOF
The GnTV samples after reduction and carboxymethylation and tryptic digest were introduced into the Q-TOF 2 (Micromass) mass spectrometer using a Waters CapLc and an autosampler. The original CapLc solvent delivery configuration was modified to minimize the dead volume between the mixer and the nanocapillary column (75 µm ID; LC packings). The mobile phases used for gradient elution consisted of water (A) with 0.1% formic acid and acetonitrile (B) with 0.1% formic acid. The gradient conditions were 10% B to 40% B in 60 min and then up to 80% B in 120 min and a flow rate of 1 µl/min was used to elute the glycopeptides. The Q-TOF 2 was operated in a data-dependent scan mode. The survey MS spectra were acquired from 450 to 3500.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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