Analysis of the site-specific N-glycosylation of ß1,6 N-acetylglucosaminyltransferase V

Maria Kamar2, Gerardo Alvarez-Manilla2,3, Trina Abney2, Parastoo Azadi2, V.S. Kumar Kolli2, Ron Orlando2 and Michael Pierce1,2

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


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a ß1,6-linked GlcNAc to the {alpha}1,6 mannose of the trimannosyl core to form tri- and tetraantennary N-glycans and contains six putative N-linked sites. We used mass spectrometry techniques combined with exoglycosidase digestions of recombinant human GnT-V expressed in CHO cells, to identify its N-glycan structures and their sites of expression. Release of N-glycans by PNGase F treatment, followed by analysis of the permethylated glycans using MALDI-TOF MS, indicated a range of complex glycans from bi- to tetraantennary species. Mapping of the glycosylation sites was performed by enriching for trypsin-digested glycopeptides, followed by analysis of each fraction with Q-TOF MS. Predicted tryptic glycopeptides were identified by comparisons of theoretical masses of peptides with various glycan masses to the masses of the glycopeptides determined experimentally. Of the three putative glycosylation sites in the catalytic region, peptides containing sites Asn 334, 433, and 447 were identified as being N-glycosylated. Asn 334 is glycosylated with only a biantennary structure with one or two terminating sialic acids. Sites Asn 433 and 447 both contain structures that range from biantennary with two sialic acids to tetraantennary terminating with four sialic acids. The predominant glycan species found on both of these sites is a triantennary with three sialic acids. The appearance of only biantennary glycans at site Asn 433, coupled with the appearance of more highly branched structures at Asn 334 and 447, demonstrates that biantennary acceptors present at different sites on the same protein during biosynthesis can differ in their accessibility for branching by GnT-V.

Key words: glycoforms / LC-MS / MALDI MS / N-acetylglucosaminyltransferase V / N-glycosylation


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a ß1,6-linked GlcNAc to the {alpha}1,6-linked Man of the trimannosyl core of N-linked glycans to form tri- or tetraantennary branches (Brockhausen et al., 1988Go, Cummings et al., 1982Go). Several studies have linked increased GnT-V activity with augmented cancer cell invasiveness and tumor progression (Fernandes et al., 1991Go; Ito et al., 2001Go; Yamamoto et al., 2000Go; Yao et al., 1998Go). GnT-V expression is transcriptionally regulated by oncogenes, such as ras (Yamashita, K., et al. 1984Go), src (Buckhaults et al., 1997Go), and her2/neu (Chen et al., 1998Go), through the Ets pathway (Kang et al., 1996Go). In a more recent study using a mouse model of mammary tumor formation and progression, lack of GnT-V expression in homozygous null mice resulted in a decrease in the rate of tumor progression (Granovsky et al., 2000Go).

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 213–740 (Korczak et al., 2000Go). 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., 2000Go). Reports from Macher's laboratory have described the mapping of N-glycosylation sites of two fucosyltransferases by mass spectrometric analysis (de Vries et al., 2001Go; Holmes et al., 2000Go). The present article uses mass spectrometric analyses to assign specific oligosaccharide structures to each site that expresses an N-linked glycan.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
To characterize the N-linked glycans of GnT-V, recombinant soluble human GnT-V was expressed in Chinese hamster ovary (CHO) cells and purified from concentrated minimal media as described (Chen et al., 1995Go). Initially, GnT-V (1 mg) was denatured and treated with PNGase F to release its N-linked glycans, which were then subjected to different combinations of exoglycosidases prior to permethylation. The permethylated glycans were then analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) in positive ion mode. To determine their structures, the N-glycan masses obtained by MALDI-TOF were compared to molecular masses calculated from common N-linked oligosaccharide structures found in vertebrate cells. Figure 1 shows a chromatogram of the MALDI-TOF data, which is compiled in Table I, showing the observed masses of N-glycans from GnT-V, their theoretical masses, and the corresponding predicted structures. All of the masses detected could be assigned a composition made up of bi-, tri-, and tetraantennary branches with core fucosylation. No theoretical masses for high-mannose or hybrid type structures were matched to observed masses, indicating that all structures were complex type. All of the glycans analyzed appeared to terminate with sialic acid to varying degrees, but those species that were completely sialylated were the most abundant. Also of note was the presence of polylactosamine structures, with up to three repeating units, which were observed only on the tetraantennary structures.



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Fig. 1. Positive-ion MALDI spectrum of permethylated glycans released from GnT-V. Peaks are identified in Table I. Triangles = fucose, squares = N-acetylglucosamine, gray circles = mannose, black circles = galactose, diamonds = sialic acid.

 

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Table I. Carbohydrate structure and composition for I–XI in Fig. 1 and their predicted and measured m/z values. Masses include sodium adduct ions.

 
To confirm the structures proposed from the initial MALDI-TOF profile, aliquots of intact oligosaccharides released from GnT-V were subjected to various combinations of glycosidase treatments. The N-glycans were then permethylated after each treatment and analyzed by MALDI-TOF MS. Initial exoglycosidase experiments began with the treatment of GnT-V glycans with sialidase from Arthrobacter ureafaciens, a broad-range enzyme that recognizes sialic acid. Figure 2 shows the MALDI-TOF spectrum of the GnT-V glycans after sialidase digestion. The number of peaks was significantly reduced compared to those seen in the MALDI-TOF spectrum of the intact GnT-V glycans with no sialidase digestion, demonstrating that most of the heterogeneity in the N-glycans was due to the varying degrees to which sialic acid terminated each structure. Sialic acids were assumed to be {alpha}(2,3)-linked because CHO cells do not express {alpha}(2,6) sialyltransferase activity (Svensson et al., 1990Go), and the analysis of N-glycans of other proteins expressed in CHO cells demonstrated only {alpha}(2,3)-linked sialic acid (Bergwerff et al., 1993Go; Conradt et al., 1987Go; Sasaki et al., 1987Go; Watson et al., 1994Go). With the decrease in the number of species observed in the MALDI-TOF profile after sialidase treatment, bi-, tri-, and tetraantennary species that were incompletely galactosylated were then resolved (Figure 2). Each antennary structure now appeared to be either fully galactosylated or, in much less abundance, to be missing one terminal galactose.



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Fig. 2. Positive-ion MALDI spectrum of permethylated glycans released from GnT-V following digestion with A. ureafaciens sialidase. Triangles = fucose, squares = N-acetylglucosamine, gray circles = mannose, black circles = galactose, diamonds = sialic acid.

 
The measured masses from the MALDI-TOF profile confirmed that the released N-glycans were composed solely of complex type structures. To verify this conclusion, prior to permethylation and MALDI-TOF analysis, N-glycans from GnT-V were treated with A. ureafaciens sialidase and a ß-galactosidase from Escherichia coli. Figure 3 shows the MALDI-TOF chromatogram after this treatment, verifying that galactose was removed during the sialidase and ß-galactosidase treatments. All galactose was assumed to be ß1,4-linked because CHO cells do not normally express type I structures (Lowe et al., 1991Go). Following sialidase and ß-galactosidase digestion, some of the glycans were treated further with ß-N-acetylhexosaminidase. Figure 4 is the MALDI-TOF spectrum showing that five species were found as a result of this glycosidase treatment, indicating that the digestion was not complete. Based on the masses observed, the structure corresponding to m/z 1345.4 was completely digested with the ß-N-acetylhexosaminidase, which represent the core N-glycan structure composed of two GlcNAc and three mannoses. The next largest species corresponds to a structure with one GlcNAc residue, and each mass increase shows an addition of one GlcNAc. The largest mass observed corresponds to the core structure with four GlcNAc residues or a degalactosylated tetraantennary structure. These data confirm that the oligosaccharides from GnT-V are composed solely of complex type structure.



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Fig. 3. Positive-ion MALDI spectrum of permethylated glycans released from GnT-V following digestion with A. ureafaciens sialidase and E. coli ß-galactosidase. Triangles = fucose, squares = N-acetylglucosamine, gray circles = mannose, black circles = galactose, diamonds = sialic acid.

 


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Fig. 4. Positive-ion MALDI spectrum of permethylated glycans released from GnT-V following digestion with A. ureafaciens sialidase, E. coli ß-galactosidase, and bovine kidney ß-N-acetylglucosaminidase. Triangles = fucose, squares = N-acetylglucosamine, gray circles = mannose, black circles = galactose, diamonds = sialic acid.

 
The masses of the permethylated oligosaccharides from GnT-V, measured by MALDI-TOF MS, suggest that all the structures contain one fucose residue. To verify whether these fucose residues were linked to the chitobiosyl core through an {alpha}1,6 linkage, the oligosaccharides were treated simultaneously with sialidase and Xanthomonas sp. fucosidase, which cleaves fucose linked to reducing GlcNAc or Gal in all linkages except {alpha}(1,6). If the fucose present were other than that of {alpha}1,6-linked core fucose, a loss of a mass of 174 from the permethylated structural profile should be observed. The fucosidase was confirmed to be active using paranitrophenol-{alpha}-fucose. As shown in Figure 5, there was no change in the masses when compared with the sample treated only with sialidase (see Figure 2), suggesting that the fucose present was indeed a core fucose.



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Fig. 5. Positive-ion MALDI spectrum of permethylated glycans released from GnT-V following digestion with A. ureafaciens sialidase and Xanthomonas sp. {alpha}-1->(2,3,4)-fucosidase. Triangles = fucose, squares = N-acetylglucosamine, gray circles = mannose, black circles = galactose, diamonds = sialic acid.

 
To determine the site-specific glycosylation of GnT-V, we performed glycopeptide mapping of a tryptic digest using liquid chromatography (LC)-MS. Figure 6 shows the amino acid sequence of human GnT-V, and the pipe marks above the amino acids indicate the potential tryptic cleavage sites and the putative N-glycosylation sites are underscored. Attempts to identify glycopeptides using LC-MS analysis directly after proteolytic digestion were unsuccessful due to the complexity of the MS spectrum because of the relatively large number of fragments produced from trypsinizing GnT-V, six potential N-glycosylation sites, and the range of N-glycan structures observed. Therefore, to enrich for N-linked glycopeptides, the tryptic digest was applied to a Superdex Peptide size-exclusion chromatography column from which the larger peptides and glycopeptides would elute first. To determine which of the fractions from this column contained the N-linked glycopeptides, each was tested for the presence of carbohydrate using the phenol sulfuric acid assay. Figure 7 shows the chromatogram of trypsinized GnT-V (monitored at 230 nm) and overlaid with the phenol sulfuric acid assay values for each fraction (monitored at 490 nm). Based on the values obtained from the phenol sulfuric assay, fractions 8–11 contained the N-linked glycopeptides. These fractions were dried and analyzed by Q-TOF MS using an online LC-MS. Information from trypsin cleavage sites and N-glycan masses determined by MALDI-TOF MS allowed us to determine which sites were glycosylated and which glycan structures were observed at each site.



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Fig. 6. Amino acid sequence of human GnT-V. Tryptic cleavage sites are indicated by pipe marks above the amino acids. Putative N-glycosylation sites are underscored.

 


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Fig. 7. Chromatogram of trypsinized GnT-V run on a Superdex Peptide column, with absorbance measured at 230 nm (solid black line). Overlaid is the result from phenol sulfuric assay, measured at 490 nm (dashed black line). Numbers 8–11 indicates those fractions positive for phenol sulfuric assay. V0 is the excluded volume and Vi is the included volume for the column used.

 
Figure 8 shows the mass spectra of fractions 9, 10, and 11, which were each confirmed to contain glycopeptides based on matching theoretical masses of glycopeptides to masses obtained experimentally (Table II). In summary, based on these comparisons, all three of the putative sites within the catalytic region of GnT-V were N-glycosylated. Glycopeptides in fractions 9, 10, and 11 were assigned sites Asn 433, 447, and 334, respectively. The carbohydrate composition of each glycopeptide was assigned (Table II), and each composition corresponds to a structure already identified by MALDI-TOF (Table I). Fraction 8 eluted near the excluded volume of the Superdex Peptide column, indicating the presence of high-molecular-weight species, such as incompletely digested protein, that may have given a positive signal on the phenol sulfuric acid assay. These high-molecular-weight fragments could not be detected by electrospray ionization (ESI) MS.



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Fig. 8. Mass spectra of glycopeptides detected by Q-TOF MS. Fractions refer to those from Fig. 7; fraction 11 (a), fraction 9 (b), and fraction 10 (c). The observed m/z value of each ion is described in Table II.

 

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Table II. Compositional assignments of glycopeptides in Figure 8 and their theoretical and observed m/z values

 
Shown in Figure 8a, the predominant ion observed in fraction 11 was found as both a triply charged and doubly charged species (a1). An increment m/z of 96.7 between a1 (3+) and a2 (3+) showed the presence of two glycoforms with a difference of one NeuAc. Comparisons of these masses with theoretical masses of the six potential glycosylation sites indicated that a1–2 is the glycopeptide glycosylated at site Asn 334. By calculating the mass of the glycan alone for a1, it was determined that Asn 334 was glycosylated with a biantennary structure with two sialic acids. Observed at much smaller intensity, the a2 species was a biantennary structure with one sialic acid. The smaller relative abundance of a2 compared to a1 is consistent with the glycan analysis by MALDI-TOF MS (Figure 1). Interestingly, masses corresponding to higher branched N-glycans were not observed at this site.

In Figure 8b (fraction 9), the complexity of the mass spectra was much greater due to the presence of several glycoforms (b1–8), all of which contained a 5+ and 6+ charge. Species b1–4 (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 b5–7 (5+, 6+). When the experimental masses were compared to theoretical masses, the glycoforms b1–8 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 c1–4 and c5–7 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, 1994Go), 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)Go 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 {alpha}(2,6) sialyltransferase, PNGase F treatment caused a significant decrease in enzyme activity (Fast et al., 1993Go). 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., 1995Go), N-acetylglucosaminyltransferase III (Nagai et al., 1997Go), and {alpha}1,3/4-fucosyltransferase III, V, and VI (Christensen et al., 2000Go). The removal of these sites also inhibited the ability of N-acetylglucosaminyltransferase III (Nagai et al., 1997Go) and fucosyltransferase III (Morais et al., 2003Go) 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Materials
All biochemical and chemical reagents were purchased from Sigma (St. Louis, MO). All solvents were HPLC-grade and purchased from Fisher Scientific (Silver Spring, MD). The other enzymes and reagents obtained from Sigma included G-15 Sephadex, phenyl sepharose, SP sepharose, 2-AP, dextran, 2,5-dihydroxybenzoic acid (DHB), ß-galactosidase recombinantly expressed in E.coli, {alpha}-1->(2,3,4)-fucosidase from Xanthomonas sp., and ß-hexosaminidase from bovine kidney. Neuraminidase from A. ureafaciens was purchased from Roche (Indianapolis, IN). C18 Sep-Pak cartridges were obtained from Waters (Milford, MA). The TSK-Amide-80 column (4.6 x 250 mm) was purchased from Tosoh Biosciences (Montgomeryville, PA). The 10 mm x 300 mm column was purchased from Biorad (Hercules, California). PNGase F was purified according to the method described (Plummer and Tarentino, 1991Go). The 5-ml HiTrap chelating columns, phenyl Sepharose high-performance column, Superdex Peptide, and SP Sepharose high-performance column were purchased from Amersham Biosciences (Upsala, Sweden).

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)Go. 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) 650–15 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)Go: 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. {alpha}-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)Go. 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 Tris–HCl, 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)Go. 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.


    Acknowledgements
 
This work was supported by National Cancer Institute grant CA 064462 to M.P. and NCRR P41RR018502.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: hawkeye{at}uga.edu


    Abbreviations
 
2-AP, 2-aminopyridine; CHO, Chinese hamster ovary; DHB, 2,5-dihydroxybenzoic acid; ESI, electrospray ionization; GnT-V, N-acetylglucosaminyltransferase V; HPLC, high-pressure liquid chromatography; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; MES, 2-morpholinoethanesulfonic acid; PNGase F, peptide N-glycosidase F; SDS, sodium dodecyl sulfate


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Baboval, T., Koul, O., and Smith, F.I. (2000) N-glycosylation site occupancy of rat alpha-1,3-fucosyltransferase IV and the effect of glycosylation on enzymatic activity. Biochim. Biophys. Acta, 1475, 383–389.[ISI][Medline]

Bergwerff, A.A., van Oostrum, J., Asselbergs, F.A., Burgi, R., Hokke, C.H., Kamerling, J.P., and Vliegenthart, J.F. (1993) Primary structure of N-linked carbohydrate chains of a human chimeric plasminogen activator K2tu-PA expressed in Chinese hamster ovary cells. Eur. J. Biochem., 212, 639–656.[Abstract]

Brockhausen, I., Carver, J.P., and Schachter, H. (1988) Control of glycoprotein synthesis. The use of oligosaccharide substrates and HPLC to study the sequential pathway for N-acetylglucosaminyltransferases I, II, III, IV, V, and VI in the biosynthesis of highly branched N-glycans by hen oviduct membranes. Biochem. Cell Biol., 66, 1134–1151.[ISI][Medline]

Buckhaults, P., Chen, L., Fregien, N., and Pierce, M. (1997) Transcriptional regulation of N-acetylglucosaminyltransferase V by the src oncogene. J. Biol. Chem., 272, 19575–19581.[Abstract/Free Full Text]

Chen, L., Zhang, N., Adler, B., Browne, J., Freigen, N., and Pierce, M. (1995) Preparation of antisera to recombinant, soluble N-acetylglucosaminyltransferase V and its visualization in situ. Glycoconj. J., 12, 813–823.[ISI][Medline]

Chen, L., Zhang, W., Fregien, N., and Pierce, M. (1998) The her-2/neu oncogene stimulates the transcription of N-acetylglucosaminyltransferase V and expression of its cell surface oligosaccharide products. Oncogene, 17, 2087–2093.[CrossRef][ISI][Medline]

Christensen, L.L., Jensen, U.B., Bross, P., and Orntoft, T.F. (2000) The C-terminal N-glycosylation sites of the human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, adn -VI) are necessary for the expression of full enzyme activity. Glycobiology, 10, 931–939.[Abstract/Free Full Text]

Ciucanu, I. and Kerek, F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res., 131, 209–217.[CrossRef][ISI]

Conradt, H.S., Egge, H., Peter-Katalinic, J., Reiser, W., Siklosi, T., and Schaper, K. (1987) Structure of the carbohydrate moiety of human interferon-beta secreted by a recombinant Chinese hamster ovary cell line. J. Biol. Chem., 262, 14600–14605.[Abstract/Free Full Text]

Cummings, R.D., Trowbridge, I.S., and Kornfeld, S. (1982) A mouse lymphoma cell line resistant to the leukoagglutinating lectin from Phaseolus vulgaris is deficient in UDP-GlcNAc: alpha-D-mannoside beta 1,6 N-acetylglucosaminyltransferase. J. Biol. Chem., 257, 13421–13427.[Free Full Text]

de Vries, T., Yen, T.Y., Joshi, R.K., Storm, J., van Den Eijnden, D.H., Knegtel, R.M., Bunschoten, H., Joziasse, D.H., and Macher, B.A. (2001) Neighboring cysteine residues in human fucosyltransferase VII are engaged in disulfide bridges, forming small loop structures. Glycobiology, 11, 423–432.[Abstract/Free Full Text]

Do, K.Y., Fregien, N., Pierce, M., and Cummings, R.D. (1994) Modification of glycoproteins by N-acetylglucosaminyltransferase V is greatly influenced by accessibility of the enzyme to oligosaccharide acceptors. J. Biol. Chem., 269, 23456–23464.[Abstract/Free Full Text]

Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., and Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350–356.[ISI]

Fast, D.G., Jamieson, J.C., and McCaffrey, G. (1993) The role of the carbohydrate chains of Gal beta-1,4-GlcNAc alpha 2,6-sialyltransferase for enzyme activity. Biochim. Biophys. Acta, 1202, 325–330.[ISI][Medline]

Fernandes, B., Sagman, U., Auger, M., Demetrio, M., and Dennis, J.W. (1991) Beta 1-6 branched oligosaccharides as a marker of tumor progression in human breast and colon neoplasia. Cancer Res., 51, 718–723.[Abstract]

Granovsky, M., Fata, J., Pawling, J., Muller, W.J., Khokha, R., and Dennis, J.W. (2000) Suppression of tumor growth and metastasis in Mgat5-deficient mice. Nat. Med., 6, 306–312.[CrossRef][ISI][Medline]

Haraguchi, M., Yamashiro, S., Furukawa, K., Takamiya, K., and Shiku, H. (1995) The effects of the site-directed removal of N-glycosylation sites from beta-1,4-N-acetylgalactosaminyltransferase on its function. Biochem. J., 312(1), 273–280.[ISI][Medline]

Hase, S. (1994) High-performance liquid chromatography of pyridylaminated saccharides. Methods Enzymol., 230, 225–237.[ISI][Medline]

Holmes, E.H., Yen, T.Y., Thomas, S., Joshi, R., Nguyen, A., Long, T., Gallet, F., Maftah, A., Julien, R., and Macher, B.A. (2000) Human alpha 1,3/4 fucosyltransferases. Characterization of highly conserved cysteine residues and N-linked glycosylation sites. J. Biol. Chem., 275, 24237–24245.[Abstract/Free Full Text]

Ito, Y., Miyoshi, E., Sakon, M., Takeda, T., Noda, K., Tsujimoto, M., Ito, S., Honda, H., Takemura, F., Wakasa, K., and others. (2001) Elevated expression of UDP-N-acetylglucosamine: alphamannoside beta1,6 N-acetylglucosaminyltransferase is an early event in hepatocarcinogenesis. Int. J. Cancer, 91, 631–637.[CrossRef][ISI][Medline]

Kang, R., Saito, H., Ihara, Y., Miyoshi, E., Koyama, N., Sheng, Y., and Taniguchi, N. (1996) Transcriptional regulation of the N-acetylglucosaminyltransferase V gene in human bile duct carcinoma cells (HuCC-T1) is mediated by Ets-1. J. Biol. Chem., 271, 26706–26712.[Abstract/Free Full Text]

Korczak, B., Le, T., Elowe, S., Datti, A., and Dennis, J.W. (2000) Minimal catalytic domain of N-acetylglucosaminyltransferase V. Glycobiology, 10, 595–599.[Abstract/Free Full Text]

Lowe, J.B., Stoolman, L.M., Nair, R.P., Larsen, R.D., Behrend, T.L., and Marks, R.M. (1991) A transfected human fucosyltransferase cDNA determines biosynthesis of oligosaccharide ligand(s) for endothelial-leukocyte adhesion molecule I. Biochem. Soc. Trans., 19, 649–653.[ISI][Medline]

Morais, V.A., Costa, M.T., and Costa, J. (2003) N-glycosylation of recombinant human fucosyltransferase III is required for its in vivo folding in mammalian and insect cells. Biochim. Biophys. Acta, 1619, 133–138.[ISI][Medline]

Nagai, K., Ihara, Y., Wada, Y., and Taniguchi, N. (1997) N-glycosylation is requisite for the enzyme activity and Golgi retention of N-acetylglucosaminyltransferase III. Glycobiology, 7, 769–776.[Abstract]

Plummer, T.H. Jr. and Tarentino, A.L. (1991) Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum. Glycobiology, 1, 257–263.[Abstract]

Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987) Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J. Biol. Chem., 262, 12059–12076.[Abstract/Free Full Text]

Svensson, E.C., Soreghan, B., and Paulson, J.C. (1990) Organization of the beta-galactoside alpha 2,6-sialyltransferase gene. Evidence for the transcriptional regulation of terminal glycosylation. J. Biol. Chem., 265, 20863–20868.[Abstract/Free Full Text]

Watson, E., Bhide, A., and van Halbeek, H. (1994) Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. Glycobiology, 4, 227–237.[Abstract]

Yamamoto, H., Swoger, J., Greene, S., Saito, T., Hurh, J., Sweeley, C., Leestma, J., Mkrdichian, E., Cerullo, L., Nishikawa, A., and others. (2000) Beta1,6-N-acetylglucosamine-bearing N-glycans in human gliomas: implications for a role in regulating invasivity. Cancer Res., 60, 134–142.[Abstract/Free Full Text]

Yamashita, K., Ohkura, T., Tachibana, Y., Takasaki, S., and Kobata, A. (1984) Comparative study of the oligosaccharides released from baby hamster kidney cells and their polyoma transformant by hydrazinolysis. J. Biol. Chem., 259, 10834–10840.[Abstract/Free Full Text]

Yao, M., Zhou, D.P., Jiang, S.M., Wang, Q.H., Zhou, X.D., Tang, Z.Y., and Gu, J.X. (1998) Elevated activity of N-acetylglucosaminyltransferase V in human hepatocellular carcinoma. J. Cancer Res. Clin. Oncol., 124, 27–30.[CrossRef][ISI][Medline]





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