5Department of Biochemistry and Molecular Biology, and Vincent T. Lombardi Cancer Center, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20007, USA, 6Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA, and 7Molecular and Cellular Endocrinology Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA
Received on July 24, 2000; revised on October 16, 2000; accepted on October 25, 2000.
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Abstract |
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Key words: Cobra venom factor/complement-activating glycoprotein/N-linked oligosaccharides/ structure determination/-galactosylated Lewis X antigens
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Introduction |
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Based on compositional analysis, it has been shown that CVF contains three N-linked oligosaccharide chains per molecule, two in the -chain and one in the ß-chain (Gowda et al., 1994
). These results are consistent with the predictions made based on the amino acid sequences of CVF determined by the molecular cloning of cDNA (Fritzinger et al., 1994
). To test whether the carbohydrate moieties are responsible for the unique functional properties of CVF-dependent C3/C5 convertase, we carried out a detailed structure/functional analysis of the oligosaccharide chains. The carbohydrate moieties are not involved in the functions of CVF (Gowda et al., 1994
); however, it was found that the oligosaccharides of CVF contain terminal
-galactosylated LeX antigenic structures (Gowda et al., 1992
) and are reactive to the naturally occurring human anti-
-Gal antibody (Gowda et al., 1994
). This finding has important implications in using CVF for medical applications. Here, we report the detailed structures of the oligosaccharides of CVF.
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Results and discussion |
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Approximately 60% of the 3H-labeled glycopeptides that contain diantennary complex oligosaccharides (Fraction B in Figure 4) bound to Griffonia simplicifolia agglutinin I (GSI) (Fraction B3), 25% of the glycopeptides were retarded (but these eluted from the column with buffer in the absence of hapten) (Fraction B2), and
15% of the glycopeptides did not bind to the lectin (Fraction B1) (Figures 3 and 4). Of the glycopeptides that contain multiantennary complex-type structures (Fraction A in Figure 4),
70% were found to bind to GSI (Fraction A3),
20% were retarded (Fraction A2), and
10% did not bind (Fraction A1) (Figure 4). Removal of the terminal
-Gal residues by coffee bean
-galactosidase abolished the binding of the glycopeptides to GSI (Figure 3, and data not shown). Because N-linked oligosaccharides containing two or more terminal
-Gal residues are known to bind to GSI, and those with one terminal
-Gal residue are retarded on the column due to a weak interaction with the lectin (Elices and Goldstein, 1989
), these results suggested that
60% and
25% of the diantennary complex oligosaccharides (in the glycopeptide Fraction B) contain two and one terminal
-Gal, respectively, and the remaining
15% of the oligosaccharides lack terminal
-Gal residues. The results also suggested that, in the case of Fraction A, that is, the glycopeptides with multiantennary oligosaccharides,
70%,
20%, and
10% of the oligosaccharides contain two or more, one, and no terminal
-Gal residues, respectively.
The 3H-labeled glycopeptides containing -galactosylated, diantennary complex-type oligosaccharides (the glycopeptides that interacted with GSI; Fractions B2 and B3 in Figure 4) were further analyzed on lentil lectin columns. Over 95% of Fraction B3, the glycopeptides with two terminal
-Gal residues, and
88% of Fraction B2, the glycopeptides with one terminal
-Gal residue, bound to lentil lectin (not shown). In contrast, only 57% of Fraction B1, the glycopeptides that lack terminal
-Gal residues, bound to lentil lectin (not shown). These results suggested that, in the diantennary oligosaccharides of CVF, >95%,
88%, and
57% of the core ß-GlcNAc residues of the fully
-galactosylated, mono
-galactosylated structures and those lacking terminal
-Gal residues, respectively, are substituted with an
(1-6)-linked Fuc residue. Although similar studies were carried out for the 3H-labeled glycopeptides bearing multiantennary oligosaccharides (Fractions A2 and A3), the results could not be interpreted to assess the level of
-Fuc substitution on the core ß-GlcNAc residue, because the triantennary oligosaccharides with 2,4-disubstituted
-Man residues and tetraantennary structures did not bind to lentil lectin, regardless of whether they contain
-Fuc substitution or not (Merkle and Cummings, 1987
). However, based on results of methylation analysis (see below), >80% of the core ß-GlcNAc residues of tri- and tetra-antennary oligosaccharides containing two or more terminal
-Gal residues are substituted with
-Fuc residues.
The nonradiolabeled CVF glycopeptides, used for the oligosaccharide structural analysis (see below), were also fractionated on a preparative Con A column as outlined in Figure 4. The sugar compositions of the glycopeptide fractions (Table I) agree with the oligosaccharide structures predicted based on the affinity interactions of the glycopeptides with the lectins (see above). Thus, the glycopeptides that bound to GSI (Fractions A3 and B3) and those retarded on GSI (Fractions A2 and B2) have higher contents of galactose compared with glycopeptides not bound to GSI (Fractions A1 and B1) due to the presence of terminal -Gal residues in the glycopeptides (Table I). Fraction C, the glycopeptide that strongly bound to Con A (see Figures 3 and 4) exclusively consists of GlcNAc and Man (see Table I), suggesting that this glycopeptide fraction contains oligomannose type oligosaccharides.
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During desalting of Fraction B1 (Figure 4) on a Sephadex G-50 column, a minor glycopeptide fraction eluted at the void volume as a peak partially resolved from the major glycopeptides. The 1H-NMR spectrum (not shown) of this minor glycopeptide fraction showed structural features characteristic of sialylated diantennary oligosaccharides. Two clearly resolved multiplets with chemical shifts at 1.810 and
2.770 were observed, and these were assigned to the H3 axial and H3 equatorial protons, respectively, of NeuAc
(2-3)-linked to ß-Gal6,6'. The spectrum also contained multiple signals at
5.153 and
1.180, which were assigned to (1-3)-linked
-Fuc H1 and methyl protons of (1-3)-linked
-Fuc residues, respectively. Thus, this minor glycopeptide fraction contains diantennary oligosaccharides terminating in (2-3)-linked
-NeuAc. However, the proportion of these oligosaccharides is approximately 1% of the total complex-type oligosaccharides of CVF. Previously, conflicting results have been reported regarding the occurrence of sialic acid in CVF (Vogel, 1991
), which appears to be due to the very low abundance of sialylated oligosaccharides in CVF.
Together, the above data indicate that the glycopeptide fraction not bound to GSI (Fraction B1) contains non--galactosylated diantennary structures; a major population of these oligosaccharides terminates in ß-Gal residues and a minor population terminate in (2-3)-linked
-NeuAc. The major proportions of the nonsialylated and sialylated oligosaccharides contain (1-3)-linked
-Fuc substitutions on the ß-GlcNAc5,5' residues and a minor but significant proportion lacks (1-3)-linked
-Fuc substitution on one or both ß-GlcNAc5,5' residues. Although the exact proportions of (1-6)-linked
-Fuc substitution on the ß-GlcNAc1 residue of these oligosaccharides could not be assessed by 1H-NMR studies, lectin chromatographic analysis (see above) suggests that approximately 60% of the structures in Fraction B1 contain such a substitution.
Analysis of glycopeptide Fractions B2 and B3 with -galactosylated diantennary structures
The majority of the diantennary oligosaccharides of CVF contains one or two terminal -Gal residues as suggested by affinity chromatography of the glycopeptides on GSI and by the methylation analysis data (see above). Because the glycopeptide Fraction B3 bound to both Con A and GSI, it should contain diantennary oligosaccharides terminating in
-Gal residues on both antennae. Based on compositional analysis and lectin affinity chromatography, the oligosaccharides should contain Fuc residues
(1-3)- and
(1-6)-linked to the ß-GlcNAc55' and core ß-GlcNAc1 residues, respectively. The 1-D 1H NMR spectrum of Fraction B3 is shown in Figure 7, and chemical shifts for structural reporter groups are listed in Table III. The multiplets at
5.152 account for four protons relative to the Man4 H1 resonance at
5.119 and were assigned to
(1-3)-linked Gal H1 and
(1-3)-linked Fuc H1, two protons each. The chemical shifts of the ß-Gal6,6' H1 protons (
4.520, 4.527) are compatible with the presence of both the terminal
-Gal residues, which usually cause downfield shifts (Van Halbeek et al., 1983
; Bergwerff et al., 1995
), and the (1-3)-linked
-Fuc residues, which cause upfield shifts (Vliegenthart et al., 1983
; Michalski et al., 1991
), relative to the unsubstituted diantennary glycan (Vliegenthart et al., 1983
). The chemical shift of
4.175 for ß-Gal H4 is characteristic of substitution with an
(1-3)-linked Gal residue (Van Halbeek et al., 1983
; Dorland et al., 1984
; Debray et al., 1991
). The absence of a resonance at
4.45 indicates that all the ß-Gal residues of Fraction B3 are substituted with
-Gal residues. The (1-3) type of linkage of the
-Gal residue was confirmed by DQF-COSY and ROESY spectra. The ROESY spectrum (Figure 8) showed a strong NOE cross-peak between
-Gal H1 and ß-Gal H3 and a weaker cross-peak between
-Gal H1 and ß-Gal H4, as is typically seen for the (1-3) but not for the (1-4) type of linkage between two Gal residues (Van Halbeek and Poppe, 1992
). Thus, in agreement with lectin affinity chromatography and methylation analyses data, the results of the 1H-NMR study demonstrated that Fraction B3 contains fully
-galactosylated diantennary oligosaccharides in which more than 95% of ß-GlcNAc5,5' are substituted with
(1-3)-linked Fuc residues.
Based on lectin affinity chromatography and methylation analyses, Fraction B2 is predicted to contain diantennary oligosaccharides terminating in a single -Gal terminal residue. The 1D 1H-NMR spectrum (Figure 9) of Fraction B2 is very similar to that of Fraction B3 except that the diagnostic resonance for ß-Gal6,6' H1 has an additional set of doublet signals at
4.449 and
4.456 (compare Figure 9 with Figure 7, and see Table III). These resonances correspond to ß-Gal6,6' H1 lacking
-Gal substituents. If all the oligosaccharides of Fraction B2 contained only one terminal
-Gal residue (on either one of the antennae), as suggested by lectin affinity chromatography, the signals due to ß-Gal 6,6' H1 in oligosaccharides with
-Gal and those due to ß-Gal6,6' H1 in oligosaccharides without
-Gal should have equal intensities. However, in the spectrum (Figure 9), the intensities of these peaks are
3:1. These data are compatible with Fraction B2 containing glycopeptides with di-
-galactosylated (as in Fraction B3) and mono-
-galactosylated oligosaccharides in the ratios of
1:1. Thus, a significant amount of glycopeptides with fully
-galactosylated diantennary oligosaccharides eluted as a retarded component during preparative fractionation on GSI, apparently due to overloading of the column. Furthermore, from the 1:1 intensity ratio of the doublets at
4.449 and
4.456 in the spectrum of Fraction B2 (Figure 9), it can be deduced that the terminal
-Gal is present almost in equal amounts on both antennae of the mono-
-galactosylated oligosaccharide chains.
Analysis of tri- and tetra-antennary glycopeptides in Fraction A3
Fraction A3, the glycopeptide fraction that contains tri- and tetra-antennary oligosaccharides, was also analyzed by 1H-NMR (spectrum not shown). Based on lectin affinity and methylation analyses, this glycopeptide fraction was predicted to contain two or more terminal -Gal residues. Although the 1H-NMR spectrum showed resonances characteristic of oligosaccharides with
-galactosylated LeX epitopes, the signals were not resolved well enough to integrate peak areas and confirm the relative amounts of various residues. The spectrum had intense, unresolved signals in the regions of
5.15 due to the anomeric protons of
-Gal and
(1-3)-Fuc residues and multiple broad peaks at
4.59 and
4.47, which correspond to the anomeric protons of ß-GlcNAc and ß-Gal residues of N-acetyllactosaminyl units, respectively.
Analysis of CVF for LeX epitope
On dot blot analysis, CVF reacted with anti-LeX antibody (Figure 10). Treatment with coffee bean -galactosidase increased the reactivity of CVF to anti-LeX antibody by five- to tenfold (Figure 10). Anti-Lea antibody was not reactive with CVF. Together, these results agree with the presence of terminal LeX and
-galactosylated LeX structures in CVF. The results further suggest that the oligosaccharide chains of CVF have type 2 but not type 1 Gal-GlcNAc structures. This agrees with structures determined by 1H-NMR and methylation analyses.
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Mass spectrometry of oligomannose-type oligosaccharides
The glycopeptides that eluted with 0.5 M -Me Man from the Con A column (Fraction C in Figure 4; see also Figure 3) were subjected to hydrazinolysis, followed by reduction with NaBH4 and permethylation. The derivatized oligosaccharides were analyzed by electrospray-mass spectrometry (Figure 12). The mass spectrum indicated that glycopeptide Fraction C contains oligosaccharides with two HexNAc and five, six, seven, eight, and nine Hex residues, respectively. Based on composition analysis data (Fraction C in Table I), these oligosaccharides correspond to Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 in the approximate molar rations of 1.2:1.8:2.6:1.4:1.0 (Figure 12). The data agree with the results of Con A-affinity chromatography (Figure 3) and of gel filtration on Bio-Gel P-4 (see Figure 2).
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The presence of immunoreactive carbohydrate structures in CVF is likely to affect its usefulness for clinical applications in humans. Although the anti--Gal immunoreactivity of CVF can be abolished by de-
-galactosylation using
-galactosidase, this treatment significantly increases the rate of plasma clearance due to the recognition of exposed LeX structures by the liver lectins (Fu et al., 1997
). However, the terminal
-Gal residues of CVF can be quantitatively derivatized by selective oxidation at the C-6 position using galactose oxidase in the presence of hydrazides, and this procedure completely abolishes the immunoreactivity of CVF without affecting its activity (Gowda, 1998
). This derivatization method can be used for the carbohydrate site-directed conjugation of CVF to antibodies to obtain immunoconjugates with retention of CVF functional activity while simultaneously abolishing anti-
-Gal immunoreactivity (Fu and Gowda, unpublished results).
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Materials and methods |
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Purification of CVF
CVF was purified from freeze-dried Naja naja kaouthia venom (Miami Serpentarium Laboratories, Salt Lake City, UT) as previously described (Vogel and Müller-Eberhard, 1984). The purified CVF was analyzed by SDS-PAGE under nonreducing (7% gel) and reducing (10% gel) conditions according to Laemmli (1970)
. The purity was found to be >98%.
Preparation of CVF glycopeptides
Purified CVF (300 mg) in 15 ml of 100 mM TrisHCl, 1 mM CaCl2, pH 8.0, was digested with pronase (40 mg) at 55°C in the presence of toluene (200 µl). Pronase was added in 10-mg aliquots at 12-h intervals. After 48 h, the enzyme digest was chromatographed on a Sephadex G-50 column (1.5 x 105 cm) in 50 mM pyridine/50 mM acetic acid, pH 5.2. Fractions (2.8 ml) were collected and 100-µl aliquots were assayed for hexoses using the anthrone reagent (Hodge and Hofreiter, 1962). Glycopeptide-containing fractions were pooled and lyophilized; yield, 24 mg. About 0.7 mg of the glycopeptides were 3H-labeled in their peptide moiety by N-acetylation using [3H]acetic anhydride in sodium bicarbonate (Finne and Krusius, 1982
). The solution was chromatographed on a Sephadex G-15 column (1 x 105 cm) in 50 mM pyridine/50 mM acetic acid, pH 5.2. Fractions (1 ml) were collected, and aliquots (5 µl) were monitored for radioactivity by scintillation counting. Fractions containing the 3H-labeled glycopeptides were pooled and lyophilized, specific activity, 5.8 x 105 c.p.m./300 nmol glycopeptide. The nonlabeled glycopeptides were N-acetylated with acetic anhydride in saturated, aqueous sodium bicarbonate and purified by gel filtration on Sephadex G-50 (1.5 x 105 cm) in 50 mM pyridine/50 mM acetic acid, pH 5.2 as above (Finne and Krusius, 1982
).
Lectin affinity chromatography of CVF glycopeptides
3H-labeled CVF glycopeptides were analyzed on immobilized Con A, lentil lectin, and GSI analytical columns (0.7 x 6.8 cm, 2.6 ml gel) at room temperature (Murphy and Goldstein, 1978; Merkle and Cummings, 1987
). Briefly, glycopeptides (5 x 105 c.p.m.) were applied on Con A-Sepharose; unbound glycopeptides were washed with 10 mM TrisHCl/0.15 M NaCl/1 mM CaCl2/1 mM MgCl2/1 mM MnCl2, pH 8.0; and the bound glycopeptides were eluted step-wise with 20 mM
-Me Glc and 0.5 M
-Me Man in the above buffer. Fractions (0.5 ml) were collected and 10-µl aliquots were monitored for radioactivity using a liquid scintillation counter. Unbound and bound fractions were separately pooled, lyophilized, and desalted on a Sephadex G-15 column (1 x 105 cm) in 50 mM pyridine/50 mM acetic acid, pH 5.2. The Con A-unbound fraction was redigested with pronase and chromatographed on Sephadex G-50 column (1 x 105 cm) in 50 mM pyridine/50 mM acetic acid, pH 5.2. Fractions (2.8 ml) were collected, and glycopeptide-containing fractions were pooled and lyophilized.
The Con A-bound 20 mM -Me Glc-eluted fractions (515 x 103 c.p.m.) were further analyzed on lentil lectin-agarose and GSI-agarose columns. The lentil lectin columns were washed with 10 mM TrisHCl/0.15 M NaCl/ 1 mM CaCl2/1 mM MgCl2/1 mM MnCl2, pH 8.0, and the bound glycopeptides were eluted with 0.5 M
-Me Man in the same buffer. The GSI columns were washed with 10 mM sodium phosphate/0.15 M NaCl/1 mM CaCl2, pH 7.4, and the bound glycopeptides were eluted with 0.1 M
-Me Gal in the same buffer. The recovery of glycopeptides from the above lectin columns was >80%.
Preparative fractionation of the CVF glycopeptides
The nonradiolabeled glycopeptides (12 mg) obtained by pronase digestion of CVF and gel filtration on Sephadex G-50 were applied onto a Con A-Sepharose column (1.5 x 25 cm) and chromatographed as described above. Unbound glycopeptides, as well as those bound and eluted with 20 mM -Me Glc and 0.5 M
-Me Man were separately pooled, lyophilized, and desalted on a Sephadex G-15 column (1.5 x 100 cm) using 5% (v/v) propanol in water. The Con A unbound glycopeptide fraction and that bound and eluted with 20 mM
-Me Glc were then fractionated on a GSI-agarose column (1.5 x 21 cm) as described above for the analytical column. The unbound, retarded, and bound glycopeptide fractions from GSI-agarose columns were desalted on a Sephadex G-15 column (1.5 x 100 cm) by eluting with 5% propanol in water. The glycopeptides were further chromatographed on columns of Sephadex G-50 (1.5 x 110 cm) using 5% propanol in water. The glycopeptide-containing fractions were pooled and lyophilized. The elution of glycopeptides was monitored by measuring absorption at 214 nm.
Treatment of CVF glycopeptides with -fucosidase and
-galactosidase
The 3H-labeled glycopeptides (515 x 103 c.p.m. in 50100 µl of 100 mM sodium phosphate, pH 6.5) were separately treated with coffee bean -galactosidase (5 units) and bovine epididymis
-fucosidase (200 milliunits) at 37°C for 6 h and 48 h, respectively. The solutions were diluted to 1 ml with 10 mM TrisHCl/0.15 M NaCl/1 mM CaCl2/1 mM MgCl2/1 mM MnCl2, pH 8.0, and analyzed by lectin affinity chromatography.
Antibody affinity blotting
CVF and de--galactosylated CVF were dot blotted (0.0810 µg per dot) onto PVDF membranes. The membranes were blocked with 0.5% casein (w/v) in 10 mM TrisHCl, 150 mM NaCl, pH 8.0, and then treated with anti-LeX mAb (culture supernatant, 1:20 diluted) or with anti-Lea mAb (culture supernatant, 1:4 diluted). The immunoreactivity was detected with alkaline phosphatase-conjugated goat anti-mouse IgG or IgM and the NBT/BCIP color developing reagent.
Release of CVF oligosaccharides by hydrazinolysis and fractionation by Bio-Gel P-4 chromatography
Lyophilized CVF (2 mg) was treated with hydrazine in a GlycoPrep 1000 (Oxford Glycosciences, Rosedale, NY) using the manufacturers automated program for quantitative release of N-linked oligosaccharides (Juhl, 1997). The released oligosaccharides were processed, re-N-acetylated, and purified online using the glycan processing reagent kit (Oxford Glycosciences). The purified oligosaccharides were analyzed on a Bio-Gel P-4 column (1 x 48 cm) using a GlycoMap 1000 HPLC system. The column was eluted with water at 55°C, and elution of oligosaccharides was monitored by an in-line differential refractometer coupled to a GlycoLink computer data processing system (Oxford Glycosciences). The Bio-Gel P-4 column was calibrated with a mixture of glucose oligomers ranging in size from 2 to 25 GU.
Carbohydrate composition analysis
The oligosaccharide or glycopeptide fractions (520 µg) were hydrolyzed with 2.5 M trifluoroacetic acid at 100°C for 5 h. The hydrolysates were dried in a Speed Vac. The residues were dissolved in water and analyzed for neutral sugars and hexosamines using a Dionex HPLC system (Dionex Corp., Sunnyvale, CA) with pulsed amperometric detection on a CarboPac PA1 column (4 x 250 mm) (Hardy and Townsend, 1994). Elution was with 20 mM NaOH at a flow rate of 0.9 ml/min. Retention times were calibrated using standard solutions of monosaccharides, and molar responses were calculated by analyzing a standard fucosylated diantennary complex oligosaccharide (Oxford Glycosystems) hydrolyzed with 2.5 M trifluoroacetic acid at 100°C for 5 h.
Methylation analysis
Glycopeptides (50100 µg) were dissolved in DMSO (0.5 ml) in a 2-ml reactivial. Finely powdered solid NaOH (100 µg) and methyl iodide (250 µl) were added, and the mixture was stirred at room temperature for 2 h (Ciucanu and Kerek, 1984). Excess methyl iodide in the reaction mixture was removed by a stream of nitrogen. Water (0.5 ml) was added, and the solution was extracted with CHCl3 (3 x 1 ml). The combined CHCl3 extract was washed with water (2 x 0.5 ml) and dried with a stream of nitrogen. The permethylated oligosaccharides were purified on silica gel (Fournet et al., 1980
) and then hydrolyzed with 2.5 M trifluoroacetic acid (0.4 ml) at 100°C for 5 h. Acid was removed in a Speed Vac, and the residue was dissolved in water (100 µl) and reduced with NaBD4 (510 mg) at room temperature for 4 h. Excess NaBD4 was removed by acidification with MeOH/HOAc (95:5, v/v) in an ice bath. Boric acid was removed by evaporation with 1% (v/v) methanolic acetic acid (3 x 0.5 ml). The residue was dried over P2O5 and acetylated with acetic anhydride (0.5 ml) at 100°C for 1 h. The solution was dried under nitrogen, and the residue was dissolved in CHCl3, washed with water (2 x 0.5 ml), and dried. The partially methylated alditol acetates thus obtained were analyzed by gas liquid chromatography using DB-23 (0.54 mm x 15 m) and DB-17 (0.54 mm x 15 m) capillary columns. The methylated alditol acetates obtained from Fraction B3 were also analyzed by combined gas chromatography-mass-spectrometry (Merkle and Poppe, 1994
).
Analysis of permethylated oligomannose-type oligosaccharides by electrospray mass spectrometry
The oligomannose-type oligosaccharides (10 µg) released by hydrazinolysis of the CVF glycopeptides eluted with 0.5 M -Me Man from the Con A column were permethylated as described above. The permethylated oligosaccharides were extracted with chloroform, back washed with 30% aqueous acetic acid, and then analyzed on a TSQ 700 triple quadrupole mass spectrometer (Finnigan MAT, San Jose, CA) equipped with an electrospray ion source (Analytica Inc., Branford, CT).
1H-NMR spectroscopic analysis of CVF glycopeptides
Hydroxyl hydrogens in the glycopeptides were exchanged with deuterium by repeated dissolution in 99.9% D2O with intermittent lyophilization. The deuterium-exchanged glycopeptides were dissolved in 0.6 ml of 99.96% D2O (Cambridge Isotope Labs, Andover, MA) and subjected to 1H-NMR analysis. 1D spectra were recorded on a Bruker AM 500 MHz spectrometer at 23°C, and 2D spectra were collected on a Bruker AMX 600 MHz spectrometer at 25°C. 2D DQF-COSY (Piantini et al., 1982), TOCSY (Braunschweiler and Ernst, 1983
; Bax and Davis, 1985
), and ROESY (Bothner-By et al., 1984
) data sets were collected in phase-sensitive mode using the time proportional phase incrementation (TPPI) method (Marion and Wüthrich, 1983
). In all experiments, low power presaturation was applied to the residual HDO signal.
For the 2D analyses, 512 free induction decays of 1024 complex data points were collected, with 32 scans per free induction decay. The spectral width was set to 5000 Hz, and the carrier was placed at the residual HDO peak. The TOCSY pulse program contained an 80 ms MLEV17 spin-lock pulse (Davis and Bax, 1985), and the ROESY experiments used a 200 ms CW spin-lock pulse flanked by two 90° pulses for offset compensation (Griesinger and Ernst, 1987
). Data were processed offline on an SGI Iris computer using Felix 95 software (Molecular Simulations Inc., San Diego, CA).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 Present address: Glycobiology Research and Training Center, School of Medicine, University of California at San Diego, La Jolla, CA 92093-0687, USA.
3 Present address: Department of Biochemistry and Molecular Biology, University of Hamburg, Hamburg 20146, Germany.
4 Present address: Cancer Research Center of Hawaii, University of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813, USA.
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References |
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