N-linked oligosaccharides of cobra venom factor contain novel {alpha}(1-3)galactosylated Lex structures

D. Channe Gowda1,5, John Glushka6, Herman van Halbeek2,6, Rao N. Thotakura7, Reinhard Bredehorst3,5 and Carl-Wilhelm Vogel4,5

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.


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cobra venom factor (CVF), a nontoxic, complement-activating glycoprotein in cobra venom, is a functional analog of mammalian complement component C3b. The carbohydrate moiety of CVF consists exclusively of N-linked oligosaccharides with terminal {alpha}1-3-linked galactosyl residues, which are antigenic in human. CVF has potential for several medical applications, including targeted cell killing and complement depletion. Here, we report a detailed structural analysis of the oligosaccharides of CVF. The structures of the oligosaccharides were determined by lectin affinity chromatography, antibody affinity blotting, compositional and methylation analyses, and high-resolution 1H-NMR spectroscopy. Approximately 80% of the oligosaccharides are diantennary complex-type, ~12% are tri- and tetra-antennary complex-type, and ~8% are oligomannose type structures. The majority of the complex-type oligosaccharides terminate in Gal{alpha}1-3Galß1-4(Fuc{alpha}1-3)GlcNAcß1, a unique carbohydrate structural feature abundantly present in the glycoproteins of cobra venom.

Key words: Cobra venom factor/complement-activating glycoprotein/N-linked oligosaccharides/ structure determination/{alpha}-galactosylated Lewis X antigens


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cobra venom factor (CVF) is a 146-kDa complement-activating glycoprotein (Vogel, 1991Go; Vogt, 1990Go). The activity of CVF is due to the formation of CVF,Bb, a C3/C5 convertase of the alternative pathway. The formation and function of CVF,Bb closely resembles that of C3b,Bb, the physiologic C3/C5 convertase of the mammalian complement (Vogel, 1991Go; Vogt, 1990Go). Although CVF,Bb and C3b,Bb are functionally similar to one another, the two enzymes differ significantly in several important properties. Unlike C3b,Bb, CVF,Bb is a stable complex and completely resistant to the actions of complement regulatory factors H and I. Therefore, CVF continuously activates complement resulting in the depletion of complement activity. CVF has been used to study the complement pathways and to investigate the role of complement in disease pathophysiology (Vogel, 1991Go). CVF has also been used to consume complement to prevent the hyperactive rejection of organs in xenotransplantation (Leventhal et al., 1993Go; Candinas et al., 1996Go; Kobayashi et al., 1996Go) and for targeted complement-mediated cell killing (Juhl et al., 1990Go, 1997; Fu and Gowda, 2001Go).

Based on compositional analysis, it has been shown that CVF contains three N-linked oligosaccharide chains per molecule, two in the {alpha}-chain and one in the ß-chain (Gowda et al., 1994Go). 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., 1994Go). 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., 1994Go); however, it was found that the oligosaccharides of CVF contain terminal {alpha}-galactosylated LeX antigenic structures (Gowda et al., 1992Go) and are reactive to the naturally occurring human anti-{alpha}-Gal antibody (Gowda et al., 1994Go). This finding has important implications in using CVF for medical applications. Here, we report the detailed structures of the oligosaccharides of CVF.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CVF glycopeptides
Purified CVF was exhaustively digested with pronase, and the resulting glycopeptides were isolated by gel filtration on Sephadex G-50 (Figure 1). The glycopeptides eluted as a single peak at a position corresponding to a molecular weight slightly lower than that of sialylated diantennary transferrin glycopeptides (Mr ~2340). The carbohydrate moieties of the glycopeptides were found to consist of Fuc, Gal, GlcNAc, and Man in the molar ratios of 3.1:4.3:4.2:3.8. These results agree with the previous finding that CVF contains predominantly complex-type, N-linked oligosaccharides with terminal {alpha}-Fuc and {alpha}-Gal residues and lacks O-linked carbohydrates (Gowda et al., 1992Go).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1. Preparation of CVF glycopeptides. CVF purified from the venom of cobra, Naja naja kaouthia, was digested with pronase and then chromatographed on Sephadex G-50. Glycopeptide-containing fractions were pooled as indicated, lyophilized, and the N-terminal amino groups were acetylated with either [3H]acetic anhydride (for radiolabeling) or acetic anhydride. The elution positions of blue dextran (BD), glucose (Glc), and the sialylated N-linked glycopeptides obtained by pronase digestion of fetuin (F) and transferrin (T) are indicated.

 
Oligosaccharide size
The oligosaccharides were released by hydrazinolysis of either intact CVF or the total glycopeptides obtained by pronase digestion of CVF, N-acetylated, and analyzed by gel filtration on an analytical Bio-Gel P-4 column (Yamashita et al., 1982Go). The majority of the oligosaccharides eluted as a broad peak corresponding to an apparent size of 16.7 GU with a shoulder on the leading edge (Figure 2). A minor proportion of the oligosaccharides eluted as two smaller peaks corresponding to 24 GU and 15.7 GU, partially resolved from the major peak (16.7 GU); several minor peaks at 8.5, 9.5, 10.5, 11.5, and 12.5 GU were also present. The components eluted from the column were pooled into three fractions as shown in Figure 2 and their sugar composition was determined. Fraction I represented a noncarbohydrate contaminant and was not further investigated. Fractions II and III consisted of Fuc, Gal, GlcNAc, and Man in the ratios of 3.2:4.2:4.2:3.0 and 0.4:0.7:2.0:7.0, respectively. The sizes and sugar compositions suggested that Fraction II consists of predominantly diantennary oligosaccharides and Fraction III has mainly oligomannose type structures.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 2. Bio-Gel P-4 chromatography of CVF oligosaccharides. Oligosaccharides released by hydrazinolysis of CVF were analyzed on a calibrated Bio-Gel P-4 column and monitored with an in-line differential refractometer. Fractions were pooled as shown and analyzed for sugar compositions. The void volume (Vo) and elution positions of glucose oligomers are indicated.

 
Lectin affinity analysis and fractionation of CVF glycopeptides
The CVF glycopeptides were radiolabeled by N-acetylation using [3H]acetic anhydride and purified by gel filtration on Sephadex G-25. The 3H-labeled glycopeptides were analyzed for oligosaccharide structural features by serial lectin affinity chromatography (Figures 3 and 4) (Merkle and Cummings, 1987Go). Approximately 30% of the radiolabel in the glycopeptides did not bind to Con A (Figure 3). When digested with pronase, the fraction not bound to Con A released about 60% of the radioactivity as amino acids, indicating that this fraction contained a significant amount of peptide contaminants. Thus, only about 12% of CVF oligosaccharides are multiantennary complex-type structures (Fraction A in Figure 4). Approximately 90% of the glycopeptides that bound to Con A were eluted with 20 mM {alpha}-Me Glc (Figure 3; Fraction B in Figure 4) and the remainder of the glycopeptides were eluted with 0.5 M {alpha}-Me Man (Figure 3; Fraction C in Figure 4). Together, these results suggested that CVF contains about 80% diantennary complex-type oligosaccharides, 12% multiantennary complex-type oligosaccharides, and 8% oligomannose type structures.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3. Analysis of CVF glycopeptides by lectin affinity chromatography. The 3H-labeled CVF glycopeptides were chromatographed on a Con A-Sepharose column and the bound glycopeptides were eluted with 20 mM {alpha}-Me Glc and 0.5 M {alpha}-Me Man. The bound and unbound fractions were separately pooled, lyophilized, and desalted on a Sephadex G-15 column. Aliquots of the Con A-bound, 20 mM {alpha}-Me Glc-eluted glycopeptide fraction were then chromatographed either on a lentil lectin-agarose column or on a GSI-agarose column, before (closed circles) and after (open circles) treatment with {alpha}-fucosidase (in the case of lentil lectin) or coffee bean {alpha}-galactosidase (in the case of GSI).

 


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4. Fractionation of the CVF glycopeptides. The 3H-labeled and nonradiolabeled glycopeptides, obtained as described in Figure 1, were separately chromatographed on lectin columns (see Figure 3), and the proportions of the nonbound and bound glycopeptides are indicated. The structures of the oligosaccharides in the nonradiolabeled glycopeptides were determined by methylation and 1H-NMR analyses.

 
Approximately 85% of the 3H-labeled glycopeptides containing diantennary complex-type oligosaccharides (Fraction B in Figure 4) were found to bind to lentil lectin (Figure 3). Treatment of these glycopeptides with bovine epididymis {alpha}-fucosidase completely abolished the binding to lentil lectin (Figure 3). Together, the data suggested that about 85% of the diantennary complex-type oligosaccharides in CVF contain Fuc residues {alpha}(1-6)-linked to the core GlcNAc residue which is linked to Asn. About 50% of the glycopeptides not bound to Con A (Fraction A in Figure 4) bound to lentil lectin (not shown), suggesting that ~50% of the oligosaccharides in Fraction A (glycopeptides with multiantennary oligosaccharides) consist of tri- or tetra-antennary complex-type structures with a 2,6-disubstituted {alpha}-Man residue and {alpha}-Fuc residues attached to the core ß-GlcNAc residue.

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 {alpha}-Gal residues by coffee bean {alpha}-galactosidase abolished the binding of the glycopeptides to GSI (Figure 3, and data not shown). Because N-linked oligosaccharides containing two or more terminal {alpha}-Gal residues are known to bind to GSI, and those with one terminal {alpha}-Gal residue are retarded on the column due to a weak interaction with the lectin (Elices and Goldstein, 1989Go), these results suggested that ~60% and ~25% of the diantennary complex oligosaccharides (in the glycopeptide Fraction B) contain two and one terminal {alpha}-Gal, respectively, and the remaining ~15% of the oligosaccharides lack terminal {alpha}-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 {alpha}-Gal residues, respectively.

The 3H-labeled glycopeptides containing {alpha}-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 {alpha}-Gal residues, and ~88% of Fraction B2, the glycopeptides with one terminal {alpha}-Gal residue, bound to lentil lectin (not shown). In contrast, only 57% of Fraction B1, the glycopeptides that lack terminal {alpha}-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 {alpha}-galactosylated, mono {alpha}-galactosylated structures and those lacking terminal {alpha}-Gal residues, respectively, are substituted with an {alpha}(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 {alpha}-Fuc substitution on the core ß-GlcNAc residue, because the triantennary oligosaccharides with 2,4-disubstituted {alpha}-Man residues and tetraantennary structures did not bind to lentil lectin, regardless of whether they contain {alpha}-Fuc substitution or not (Merkle and Cummings, 1987Go). 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 {alpha}-Gal residues are substituted with {alpha}-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 {alpha}-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.


View this table:
[in this window]
[in a new window]
 
Table I. Sugar compositions of CVF glycopeptide fractionsa separated by Con A- and GSI-affinity chromatography.
 
Methylation analysis of the glycopeptides
The linkage positions of the various sugars in the glycopeptide fractions were determined by methylation analysis, and the results are given in Table II. In glycopeptides that interacted with GSI (Fractions A2, A3, B2, and B3, see Figure 4), Gal residues are present as 3-substituted and nonreducing terminal residues, whereas in glycopeptides that did not interact with GSI (Fractions A1 and B1), Gal residues are present almost exclusively as nonreducing terminal residues (Table II). These data, together with the results of affinity chromatography on GSI before and after treatment with {alpha}-galactosidase (see Figure 3), indicated that the oligosaccharide chains of Fractions A3 and B3 are predominantly substituted with terminal nonreducing {alpha}-Gal residues, which are linked to the penultimate ß-Gal residue by (1-3)-glycosidic bonds. The oligosaccharide chains of Fractions A2 and B2 terminate in {alpha}-Gal or ß-Gal residues, whereas those of glycopeptide Fractions A1 and B1 lack {alpha}-Gal and thus, terminate in nonreducing ß-Gal residues. In all the glycopeptide fractions (Fractions A1, A2, A3, B1, B2, and B3), Fuc is present exclusively as a nonreducing terminal residue, (1-3)-linked to ß-GlcNAc residues of the antennae and (1-6)-linked to the core GlcNAc (Table II). The results agree with the lentil lectin chromatography data, which suggested the presence of {alpha}-Fuc residues linked to proximal ß-GlcNAc residues of the chitobiose core. The presence of 2,6- and 2,4-disubstituted Man residues in Fraction A (Table II) indicated that the glycopeptides not bound to Con A contain tri- and/or tetra-antennary oligosaccharides.


View this table:
[in this window]
[in a new window]
 
Table II. Methylation analysis of CVF glycopeptides.a
 
1H-NMR analysis of glycopeptide fractions
The major glycopeptide fractions purified by Con A- and GSI-affinity chromatography were further analyzed by 1D and 2D 1H-NMR spectroscopy. Although these fractions were heterogeneous as indicated by the presence of multiple signals for sugar residues, the structures of several major oligosaccharides were established based on their structural reporter groups (Vliegenthart et al., 1983Go; Van Halbeek, 1994Go). The representative 1H-NMR data for Fractions B1, B2 and B3 are shown in Table III and Figures 5, 6, 7, 8, and 9.


View this table:
[in this window]
[in a new window]
 
Table III . 1H Chemical shiftsa for structural reporter groups of glycosyl residues in CVF glycopeptide fractions B1, B2, and B3.
 


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5. 1-D 500-MHz 1H-NMR spectrum of CVF glycopeptide Fraction B1. A, Overall spectrum. B, Anomeric proton region (expanded). The numbering of the glycosyl residues (Man4, GlcNAc1, etc.) is as in Figure 11.

 


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6. Regions from the 2D 600-MHz 1H,1H ROESY and TOCSY spectra of CVF glycopeptide Fraction B1. In the ROESY spectrum (left panel), an interglycosidic NOE was observed between the {alpha}-Fuc H1 (at 5.125 p.p.m.) and the ß-GlcNAc5,5' H3 protons (at 3.83 p.p.m.); this through-space interaction corroborates the linkage position between {alpha}-Fuc and ß-GlcNAc5,5' to be (1-3). The TOCSY spectrum (right panel) illustrates the assignment of the signal at 3.83 p.p.m. as H3 of ß-GlcNAc5,5', based on (among others) the presence of a scalar cross-peak with H1 at 4.59 p.p.m..

 


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 7. 1D 500-MHz 1H-NMR spectrum of CVF glycopeptide Fraction B3 (only the anomeric proton region and the Man H2 region are shown). The {alpha}-Gal H1 signals are virtually overlapped with those of the (1-3)-linked {alpha}-Fuc H1 (at 5.14–5.15 p.p.m.). The anomeric proton signals of ß-Gal6,6' are found at 4.525 p.p.m.; their downfield shift compared to {delta} 4.45 for H1 of terminal ß-Gal6,6' in Fraction B1 (see Figure 5) suggests the attachment of {alpha}-Gal in (1-3)-linkage (Dorland et al., 1984Go).

 


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 8. Pertinent regions from the 2D 600-MHz 1H,1H ROESY spectrum of CVF glycopeptide Fraction B3. Interglycosidic NOE cross-peaks were observed between the anomeric protons of the terminal {alpha}-Gal residues (at 5.15 p.p.m.) and the H3 and H4 atoms of the ß-Gal-6,6' residues at 3.79 and 4.18 p.p.m., respectively. Note that the intra-ring NOE cross-peak between ß-Gal6,6' H1 at 4.525 p.p.m. and H3 is strong, whereas the H1-H4 cross-peak is weak. Indeed, Gal{alpha}(1-3)Galß moieties are known to show NOE cross peaks between H1 of {alpha}-Gal and both H3 and H4 of ß-Gal, whereas a Gal{alpha}(1-4)Galß moiety would only show an NOE peak between H1 of {alpha}-Gal and H4 of ß-Gal (Van Halbeek and Poppe, 1992Go).

 


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 9. 1D 500-MHz 1H-NMR spectrum of CVF glycopeptide Fraction B2 (only the anomeric proton region and the Man H2 region are shown). The anomeric proton signals of ß-Gal6,6' were found at 4.525 and 4.45 p.p.m., in intensity ratio 3:1, indicating that 75% of the oligosaccharide chains in Fraction B3 are capped with {alpha}-Gal.

 
Analysis of glycopeptide fraction B1 with non-{alpha}-galactosylated diantennary structures
The 1-D 1H-NMR spectrum of Fraction B1, the diantennary glycopeptide fraction not bound to GSI (see Figure 4), is shown in Figure 5A. Because the oligosaccharides of this glycopeptide fraction do not contain terminal, nonreducing {alpha}-Gal residues, as indicated by the results of methylation analysis and GSI-affinity chromatography, the signals at {delta} 5.124 and {delta} 5.128 were assigned to anomeric protons of {alpha}-Fuc residues (1-3)-linked to the ß-GlcNAc5,5' residues (Figure 5B; for numbering of sugar residues of oligosaccharides see structure B3 in Figure 11 below). These assignments were confirmed from 2-D TOCSY and ROESY spectra (Figure 6). In the ROESY spectrum, the boxed crosspeak corresponds to the interglycosidic NOE between the {alpha}-Fuc H1 and ß-GlcNAc5,5' H3; the indicated box in the TOCSY spectrum represents the crosspeak between ß-GlcNAc5,5' H1 and ß-GlcNAc5,5' H3.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 11. Structures of N-linked oligosaccharides in CVF.

 
Figure 5A shows multiple signals at {delta} 1.177 and {delta} 1.200 that correspond to the methyl protons of (1-3)- and (1-6)-linked {alpha}-Fuc residues, respectively. The resonance at {delta} 4.873 represents H1 of {alpha}-Fuc (1–6)-linked to ß-GlcNAc1 (Figure 5B). The signals at {delta} 4.444 and {delta} 4.451 are due to ß-Gal6,6' H1 of oligosaccharides that contain {alpha}-Fuc residues (1-3)-linked to ß-GlcNAc5,5' and the resonance at {delta} 4.470 is due to ß-Gal6,6' H1 of oligosaccharide chains in which the ß-GlcNAc5,5' residues are not substituted with {alpha}-Fuc. The assignments for the reporter group signals are summarized in Table III. These chemical shifts match those previously described for diantennary N-linked oligosaccharides with {alpha}-Fuc residues (1-3)-linked to ß-GlcNAc residues and those with nonfucosylated GlcNAc5,5' residues (Vliegenthart et al., 1983Go; Michalski et al., 1991Go).

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 {delta} 1.810 and {delta} 2.770 were observed, and these were assigned to the H3 axial and H3 equatorial protons, respectively, of NeuAc {alpha}(2-3)-linked to ß-Gal6,6'. The spectrum also contained multiple signals at {delta} 5.153 and {delta} 1.180, which were assigned to (1-3)-linked {alpha}-Fuc H1 and methyl protons of (1-3)-linked {alpha}-Fuc residues, respectively. Thus, this minor glycopeptide fraction contains diantennary oligosaccharides terminating in (2-3)-linked {alpha}-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, 1991Go), 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-{alpha}-galactosylated diantennary structures; a major population of these oligosaccharides terminates in ß-Gal residues and a minor population terminate in (2-3)-linked {alpha}-NeuAc. The major proportions of the nonsialylated and sialylated oligosaccharides contain (1-3)-linked {alpha}-Fuc substitutions on the ß-GlcNAc5,5' residues and a minor but significant proportion lacks (1-3)-linked {alpha}-Fuc substitution on one or both ß-GlcNAc5,5' residues. Although the exact proportions of (1-6)-linked {alpha}-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 {alpha}-galactosylated diantennary structures
The majority of the diantennary oligosaccharides of CVF contains one or two terminal {alpha}-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 {alpha}-Gal residues on both antennae. Based on compositional analysis and lectin affinity chromatography, the oligosaccharides should contain Fuc residues {alpha}(1-3)- and {alpha}(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 {delta} 5.152 account for four protons relative to the Man4 H1 resonance at {delta} 5.119 and were assigned to {alpha}(1-3)-linked Gal H1 and {alpha}(1-3)-linked Fuc H1, two protons each. The chemical shifts of the ß-Gal6,6' H1 protons ({delta} 4.520, 4.527) are compatible with the presence of both the terminal {alpha}-Gal residues, which usually cause downfield shifts (Van Halbeek et al., 1983Go; Bergwerff et al., 1995Go), and the (1-3)-linked {alpha}-Fuc residues, which cause upfield shifts (Vliegenthart et al., 1983Go; Michalski et al., 1991Go), relative to the unsubstituted diantennary glycan (Vliegenthart et al., 1983Go). The chemical shift of {delta} 4.175 for ß-Gal H4 is characteristic of substitution with an {alpha}(1-3)-linked Gal residue (Van Halbeek et al., 1983Go; Dorland et al., 1984Go; Debray et al., 1991Go). The absence of a resonance at {delta} ~4.45 indicates that all the ß-Gal residues of Fraction B3 are substituted with {alpha}-Gal residues. The (1-3) type of linkage of the {alpha}-Gal residue was confirmed by DQF-COSY and ROESY spectra. The ROESY spectrum (Figure 8) showed a strong NOE cross-peak between {alpha}-Gal H1 and ß-Gal H3 and a weaker cross-peak between {alpha}-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, 1992Go). Thus, in agreement with lectin affinity chromatography and methylation analyses data, the results of the 1H-NMR study demonstrated that Fraction B3 contains fully {alpha}-galactosylated diantennary oligosaccharides in which more than 95% of ß-GlcNAc5,5' are substituted with {alpha}(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 {alpha}-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 {delta} 4.449 and {delta} 4.456 (compare Figure 9 with Figure 7, and see Table III). These resonances correspond to ß-Gal6,6' H1 lacking {alpha}-Gal substituents. If all the oligosaccharides of Fraction B2 contained only one terminal {alpha}-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 {alpha}-Gal and those due to ß-Gal6,6' H1 in oligosaccharides without {alpha}-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-{alpha}-galactosylated (as in Fraction B3) and mono-{alpha}-galactosylated oligosaccharides in the ratios of ~1:1. Thus, a significant amount of glycopeptides with fully {alpha}-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 {delta} 4.449 and {delta} 4.456 in the spectrum of Fraction B2 (Figure 9), it can be deduced that the terminal {alpha}-Gal is present almost in equal amounts on both antennae of the mono-{alpha}-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 {alpha}-Gal residues. Although the 1H-NMR spectrum showed resonances characteristic of oligosaccharides with {alpha}-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 {delta} ~5.15 due to the anomeric protons of {alpha}-Gal and {alpha}(1-3)-Fuc residues and multiple broad peaks at {delta} ~4.59 and {delta} ~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 {alpha}-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 {alpha}-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.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 10. Immunoreactivity of CVF with anti-Lex antibody. The indicated amounts of CVF and {alpha}-galactosidase-treated CVF were dot blotted onto PVDF membranes. The membranes were blocked with BSA and probed with a monoclonal antibody specific for LeX structures. Lane 1, untreated CVF; lane 2, CVF treated with {alpha}-galactosidase.

 
Taken together, the results of compositional and methylation analyses, size determination on Bio Gel P-4, lectin affinity chromatography, and NMR analysis demonstrated that CVF contains a complex mixture of oligosaccharides, with structures and abundances as shown in Figure 11.

Mass spectrometry of oligomannose-type oligosaccharides
The glycopeptides that eluted with 0.5 M {alpha}-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).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 12. Mass spectrometry of oligomannose type oligosaccharides from CVF. CVF glycopeptides containing oligomannose type structures (Fraction C in Figure 4) were subjected to hydrazinolysis, reduction, and permethylation, and analyzed by electrospray-ionization mass spectrometry. The doubly charged molecular ions of the various oligosaccharide structures are indicated. Note: M52+ = [Man5GlcNAc2+2Na] 2+; M62+ = [Man6GlcNAc2+2Na]2+; M72+ = [Man7GlcNAc2+2Na]2+; M82+ = [Man8GlcNAc2+2Na]2+; M92+ = [Man9GlcNAc2+2Na]2+.

 
Oligosaccharides containing terminal (1-3)-linked {alpha}-Gal residues are known to be expressed in the glycoproteins of most animals except those of old world monkeys, apes, and humans (Galili et al., 1987aGo,b, 1988; Thall and Galili, 1990Go; Langeveld et al., 1991Go; Yoshida et al., 1991Go). However, oligosaccharides that terminate in {alpha}-galactosylated LeX structures have so far been found only in glycoproteins of cobra (Gowda et al., 1992Go, 1994) and glycolipids of porcine kidney (Bouhours et al., 1997Go, 1998). In cobra, these structures are abundantly expressed in the venom gland (Gowda and Davidson, 1992Go). The venom glycoproteins of several other snakes that have so far been studied do not contain terminal {alpha}-Gal residues; rather they appear to terminate in sialic acid residues (Gowda and Davidson, 1992Go). Thus, the N-linked oligosaccharides of CVF have unusual structural features, and the biological relevance of the species- and tissue-specific expression of these oligosaccharides in glycoproteins of cobra venom is not known.

The presence of immunoreactive carbohydrate structures in CVF is likely to affect its usefulness for clinical applications in humans. Although the anti-{alpha}-Gal immunoreactivity of CVF can be abolished by de-{alpha}-galactosylation using {alpha}-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., 1997Go). However, the terminal {alpha}-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, 1998Go). 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-{alpha}-Gal immunoreactivity (Fu and Gowda, unpublished results).


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
[3H]Acetic anhydride (8.2 Ci/mmol), NaB[3H]4 (17.5 Ci/mmol) and Con A-Sepharose (16 mg lectin/ml gel) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ); lentil lectin-agarose (4–5 mg lectin/ml gel) and GSI-agarose (4 mg lectin/ml gel) were from E-Y Laboratories (San Mateo, CA); {alpha}-Me Glc, {alpha}-Me Man, {alpha}-Me Gal, bovine fetuin, human serum transferrin, and all exoglycosidases were from Sigma (St. Louis, MO); pronase was from Calbiochem (San Diego, CA), alkaline phosphatase conjugated goat anti-mouse IgG and goat anti-mouse IgM were from Southern Biotechnology Associates, Inc. (Birmingham, AL); PVDF membranes from Millipore Corp. (Bedford, MA); and NBT/BCIP reagent was from Promega (Madison, WI). Murine anti-Lex mAb (SH1, IgG3) and anti-Lea mAb (CA3F4, IgM) were provided by Dr. A. K. Singhal, University of Washington (Seattle, WA).

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, 1984Go). The purified CVF was analyzed by SDS-PAGE under nonreducing (7% gel) and reducing (10% gel) conditions according to Laemmli (1970)Go. The purity was found to be >98%.

Preparation of CVF glycopeptides
Purified CVF (300 mg) in 15 ml of 100 mM Tris–HCl, 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, 1962Go). 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, 1982Go). 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, 1982Go).

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, 1978Go; Merkle and Cummings, 1987Go). Briefly, glycopeptides (5 x 105 c.p.m.) were applied on Con A-Sepharose; unbound glycopeptides were washed with 10 mM Tris–HCl/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 {alpha}-Me Glc and 0.5 M {alpha}-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 {alpha}-Me Glc-eluted fractions (5–15 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 Tris–HCl/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 {alpha}-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 {alpha}-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 {alpha}-Me Glc and 0.5 M {alpha}-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 {alpha}-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 {alpha}-fucosidase and {alpha}-galactosidase
The 3H-labeled glycopeptides (5–15 x 103 c.p.m. in 50–100 µl of 100 mM sodium phosphate, pH 6.5) were separately treated with coffee bean {alpha}-galactosidase (5 units) and bovine epididymis {alpha}-fucosidase (200 milliunits) at 37°C for 6 h and 48 h, respectively. The solutions were diluted to 1 ml with 10 mM Tris–HCl/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-{alpha}-galactosylated CVF were dot blotted (0.08–10 µg per dot) onto PVDF membranes. The membranes were blocked with 0.5% casein (w/v) in 10 mM Tris–HCl, 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 manufacturer’s automated program for quantitative release of N-linked oligosaccharides (Juhl, 1997Go). 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 (5–20 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, 1994Go). 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 (50–100 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, 1984Go). 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., 1980Go) 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 (5–10 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, 1994Go).

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 {alpha}-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., 1982Go), TOCSY (Braunschweiler and Ernst, 1983Go; Bax and Davis, 1985Go), and ROESY (Bothner-By et al., 1984Go) data sets were collected in phase-sensitive mode using the time proportional phase incrementation (TPPI) method (Marion and Wüthrich, 1983Go). 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, 1985Go), and the ROESY experiments used a 200 ms CW spin-lock pulse flanked by two 90° pulses for offset compensation (Griesinger and Ernst, 1987Go). Data were processed offline on an SGI Iris computer using Felix 95 software (Molecular Simulations Inc., San Diego, CA).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by NIH grants CA61210 (to DCG), HL29523, AI26821, and CA01039 (to C.-W.V.). We thank Dr. Anil Singhal, Washington University, for providing anti-LeX and anti-Lea antibodies; Dr. Bruce Reinhold, University of New Hampshire, Durham, NH, for mass spectrometry of the oligomannose type oligosaccharide fraction; and Dr. Roberta Merkle, Complex Carbohydrate Research Center, Athens, GA, for composition and methylation analyses by GLC and GLC-MS.


    Abbreviations
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CVF, cobra venom factor; Gal, galactose, GlcNAc, N-acetylglucosamine; Man, mannose; Fuc, fucose; NeuAc, N-acetylneuraminic acid; GU, glucose units; {alpha}-Me Glc; {alpha}-methyl glucoside, {alpha}-Me Man, {alpha}-methyl mannoside; {alpha}-Me Gal, {alpha}-methyl galactoside; Con A, concanavalin A; GSI, Griffonia simplicifolia agglutinin I; mAb, monoclonal antibody; PVDF, polyvinylidene difluoride; NBT/BCIP, nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; GLC, gas-liquid chromatography; GLC-MS, gas-liquid chromatography coupled with mass spectrometry; DQF-COSY, double quantum-filtered correlated spectroscopy; ROESY, rotating frame nuclear Overhauser enhancement spectroscopy; NOE, nuclear Overhauser enhancement; TOCSY, total correlation spectroscopy; TPPI, time proportional phase incrementation.


    Footnotes
 
1 To whom correspondence should be addressed Back

2 Present address: Glycobiology Research and Training Center, School of Medicine, University of California at San Diego, La Jolla, CA 92093-0687, USA. Back

3 Present address: Department of Biochemistry and Molecular Biology, University of Hamburg, Hamburg 20146, Germany. Back

4 Present address: Cancer Research Center of Hawaii, University of Hawaii, 1236 Lauhala Street, Honolulu, HI 96813, USA. Back


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bax, A. and Davis, D.G. (1985) MLEV-17 based 2-D homonuclear magnetization transfer spectroscopy. J. Magn. Reson., 65, 355–360.[ISI]

Bergwerff, A.A., Stroop, C.J.M., Murray, B., Holtorf, A-P., Pluschke, G., Van Oostrum, J., Kamerling, J.P., and Vliegenthart,J .F.G. (1995) Variations in N-linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones. Glycoconj. J., 12, 318–330.[ISI][Medline]

Bothner-By, A.A., Stevens, R.L., Lee, J., Warren, C.D., and Jeanloz, R.W. (1984) Structure determination of a tetrasaccharide: transient nuclear Overhauser effects in the rotating frame. J. Am. Chem. Soc., 106, 811–813.[ISI]

Bouhours, D., Liaigre, J., Naulet, J., Maume, D., and Bouhours, J.F. (1997) A novel glycosphingo-lipid expressed in pig kidney: Gal{alpha}1-3Lewis(x) hexaglycosylceramide. Glycoconj. J., 14, 29–38.[ISI][Medline]

Bouhours, D., Liaigre, J., Lemoine, J., Mayer-Posner, F., and Bouhours, J.F. (1998) Two novel isoneolacto-undecaglycosylceramides carrying Gal{alpha}1-3LewisX on the 6-linked antenna and N-acetylneuraminic acid{alpha}2-3 or galactose{alpha}1-3 on the 3-linked antenna, expressed in porcine kidney. Glycoconj. J., 15, 1001–1016.[ISI][Medline]

Braunschweiler, L. and Ernst, R.R. (1983) Coherence transfer by isotropic mixing. Application to proton correlation spectroscopy. J. Magn. Reson., 53, 521–528.[ISI]

Candinas, D., Lesnikoski, B.A., Robson, S.C., Scesney, S.M., Otsu, I., Myiatake, T., Marsh, H.C., Ryan, U.S., Hancock, W.W., and Bach, F.H. (1996) Soluble complement receptor type 1 and cobra venom factor in discordant xenotransplantation. Transplant Proc., 28, 581.[Medline]

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

Davis, D.G. and Bax, A. (1985). Assignment of complex 1H NMR spectra via two-dimensional homonuclear Hartmann-Hahn spectroscopy. J. Am. Chem. Soc., 107, 2820–2821.[ISI]

Debray, H., Dus, D., Wieruszeski, J.-M., Strecker, G., and Montreuil, J. (1991) Structure of the {alpha}(1-3)galactose-containing asparagine-linked glycans of a Lewis lung carcinoma cell subline resistant to Aleuria aurantia agglutinin: Elucidation by 1H-NMR spectroscopy. Glycoconj. J., 8, 29–37.[ISI][Medline]

Dorland, L., Van Halbeek, H., and Vliegenthart, J.F.G. (1984) The identification of terminal {alpha}(1-3)-linked galactose in N-acetyllactosamine type of glycopeptides by means of 500-MHz 1H-NMR spectroscopy. Biochem. Biophys. Res. Commun., 122, 859–866.[ISI][Medline]

Elices, M.J. and Goldstein, I.J. (1989) Biosynthesis of bi- tri-, and tetra-antennary oligosaccharides containing {alpha}-D-galactosyl residues at their nonreducing termini. Branch specificity of the Ehrlich tumor cell {alpha}(1-3)-galactosyltransferase. J. Biol. Chem., 264, 1375–1380.[Abstract/Free Full Text]

Finne, J. and Krusius, T. (1982) Preparation and fractionation of glycopeptides. Methods Enzymol., 83, 269–277.[ISI][Medline]

Fournet, B., Dhalluin, J.-M., Strecker, G., and Montreuil, J. (1980) Gas-liquid chromatography and mass spectrometry of oligosaccharides obtained by partial acetolysis of glycans of glycoproteins. Anal. Biochem., 108, 35–56.[ISI][Medline]

Fritzinger, D.C., Bredehorst, R., and Vogel, C.-W. (1994) Molecular cloning and derived primary structure of cobra venom factor. Proc. Natl Acad. Sci. USA, 91, 12775–12779.[Abstract/Free Full Text]

Fu, Q., Satyaswaroop, P.G., and Gowda, D.C. (1997) Tissue targeting and plasma clearance of cobra venom factor. Biochem. Biophys. Res. Commun., 231, 316–320.[ISI][Medline]

Fu, Q., and Gowda, D.C. (2001) Carbohydrate-directed conjugation of cobra venom factor to antibody by selective derivatization of the terminal galactose residues. Bioconjugate Chem. In press.

Galili, U., Buehler, J., Shohet, S.B., and Macher, B.A. (1987a) The human natural anti-Gal IgG3: The subtlety of immune tolerance in man as demonstrated by crossreactivity between natural anti-Gal and anti-B antibodies. J. Exp. Med., 165, 693–704.[Abstract]

Galili, U., Clark, M.R., Shohet, S.B., Buehler, J., and Macher, B.A. (1987b) Evolutionary relationship between the natural anti-Gal antibody and the Gal1-3Gal epitope in primates. Proc. Natl Acad. Sci. USA, 84, 1369–1373.[Abstract]

Galili, U., Shohet, S.B., Kobrin, E., Stults, C.L.M., and Macher, B.A. (1988) Man, Apes, and Old World monkeys differ from other mammals in the expression of {alpha}-galactosyl epitope on the nucleated cells. J. Biol. Chem., 263, 17755–17762.[Abstract/Free Full Text]

Gowda, D.C. (1998) Modification at C6 of the terminal galactosyl residues of cobra venom factor abolishes anti-{alpha}-Gal antibody immunoreactivity without affecting functional activity. Biochem. Biophys. Res. Commun., 245, 28–32.[ISI][Medline]

Gowda, D.C. and Davidson, E.A. (1992) Structural features of carbohydrate moieties in snake venom glycoproteins. Biochem. Biophys. Res. Commun., 182, 294–307.[ISI][Medline]

Gowda, D.C., Schultz, M., Bredehorst, R., and Vogel, C.-W. (1992) Structure of the major oligosaccharides of cobra venom factor. Mol. Immunol., 29, 335–442.[ISI][Medline]

Gowda, D.C., Petrella, E.C., Raj, T.T., Bredehorst, R., and Vogel, C.-W. (1994) Immunoreactivity and function of oligosaccharides in cobra venom factor. J. Immunol., 152, 2977–2986.[Abstract/Free Full Text]

Griesinger, C. and Ernst, R.R. (1987) Frequency offset effects and their elimination in NMR rotating-frame cross-relaxation spectroscopy. J. Magn. Reson., 75, 261–271.[ISI]

Hardy, M.R. and Townsend, R.R. (1994) High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates. Methods Enzymol., 230, 208–225.[ISI][Medline]

Hodge, J.E. and Hofreiter, B.T. (1962) Determination of reducing sugars and carbohydrates. In Whistler, R.L., Wolfrom, M.L., BeMiller, J.N., and Shafizadeh, F. (eds.) Methods in Carbohydrate Chemistry, Vol. I, Academic Press, New York, pp. 380–394.

Juhl, H., Petrella, E.C., Cheung, N.-K.V., Bredehorst, R., and Vogel, C.-W. (1990) Complement killing of human neuroblastoma cells: A cytotoxic monoclonal antibody and its F(ab')2-cobra venom factor conjugate are equally cytotoxic. Mol. Immunol., 27, 957–964.[ISI][Medline]

Juhl, H., Petrella, E.C., Cheung, N.-K.V., Bredehorst, R., and Vogel, C.-W. (1997) Additive cytotoxicity of different monoclonal antibody cobra venom factor conjugates for human neuroblastoma cells. Immunobiology, 197, 444–459.[ISI][Medline]

Kobayashi, T., Taniguchi, S., Ye, Y., Niekrasz, M., Kosanke, S., Neethling, F.A., Wright, L.J., Rose, A.G., White, D.J.G., and Cooper, D.K.C. (1996) Delayed xenograft rejection in C3-depleted discordant (pig-to-baboon) cardiac xenografts treated with cobra venom factor. Transplant Proc., 28, 560.[Medline]

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature, 227, 680–685.[ISI][Medline]

Langeveld, J.P.M., Noelken, M.E., Hård, K., Todd, P., Vliegenthart, J.F.G., Rouse, J., and Hudson, B.G. (1991) Bovine glomerular basement membrane: location of the asparagine-linked oligosaccharides units and their potential role in the assembly of the 7S collagen IV tetramer. J. Biol. Chem., 266, 2622–2631.[Abstract/Free Full Text]

Leventhal, J.R., Dalmasso, A.P., Cromwell, J.W., Platt, J.L., Manivel, C.J., Bolman, R.M. III, and Matas, A.J. (1993) Prolongation of cardiac xenograft survival by depletion of complement. Transplantation, 55, 857–865.[ISI][Medline]

Marion, D. and Wüthrich, K. (1983) Application of phase sensitive 2D COSY for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem. Biophys. Res. Commun., 113, 967–974.[ISI][Medline]

Merkle, R.K. and Cummings, R.D. (1987) Lectin affinity chromatography of glycopeptides. Methods Enzymol., 138, 232–259.[ISI][Medline]

Merkle, R.K. and Poppe, I. (1994) Carbohydrate composition analysis of glycoconjugates by gas-liquid chromatography/mass spectrometry. Methods Enzymol., 230, 1–15.[ISI][Medline]

Michalski, J.C., Wieruszeski, J.M., Alonso, C., Cache, P., Montreuil, J., and Strecker, G. (1991) Characterization and 400-MHz 1H-NMR analysis of urinary fucosyl glycoasparagines in fucosidosis. Eur. J. Biochem., 201, 439–458.[Abstract]

Murphy, L.A. and Goldstein, I.J. (1978) Bandeiraea simplicifolia I isolectins. Methods Enzymol., 50, 345–349.[Medline]

Patel, T.P. and Parekh, R.B. (1994) Release of oligosaccharides from glycoproteins by hydrazinolysis. Methods Enzymol., 230, 57–66.[ISI][Medline]

Piantini, U., Sørensen, O., and Ernst, R.R. (1982) Multiple quantum filters for elucidating NMR coupling networks. J. Am. Chem. Soc., 104, 6800–6801.[ISI]

Thall, A. and Galili, U. (1990) Distribution of Gal{alpha}1–3Galß1–4GlcNAc residues on secreted mammalian glycoproteins (thyroglobulin, fibrinogen, and immunoglobulin D) as measured by a sensitive solid-phase radioimmunoassay. Biochemistry, 29, 3959–3965.[ISI][Medline]

Van Halbeek, H. (1994) 1H Nuclear magnetic resonance spectroscopy of carbohydrate chains of glycoproteins. Methods Enzymol., 230, 132–168.[ISI][Medline]

Van Halbeek, H. and Poppe, L. (1992) Conformation and dynamics of glycoprotein oligosaccharides as studied by 1H-NMR spectroscopy. Magn. Reson. Chem., 30, S74–S86.[ISI]

Van Halbeek, H., Vliegenthart, J.F.G., Winterwerp, H., Blanken, W.M., and Van den Eijnden, D.H. (1983) {alpha}-D-Galactosyltransferase activity in calf thymus; a high resolution of 1H-NMR study. Biochem. Biophys. Res. Commun., 110, 124–131.[ISI][Medline]

Vliegenthart, J.F.G., Dorland, L., and Van Halbeek, H. (1983) High-resolution 1H-nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins. Ad. Carbohydr. Chem. Biochem., 41, 209–374.

Vogel, C.-W. (1991) Cobra venom factor: the complement-activating protein of cobra venom. In Tu, A.T. (ed.) Handbook of natural toxins, vol. 5: Reptile and amphibian venoms. Marcel Dekker, NY, pp. 147–188.

Vogel, C.-W. and Müller-Eberhard, H.J. (1984) Cobra venom factor: Improved method for purification and biochemical characterization. J. Immunol. Methods, 73, 203–220.[ISI][Medline]

Vogt, W. (1990) Snake venom constituents affecting the complement system. In Stocker, K.F. (ed.) Medical use of snake venom proteins. CRC Press, Boca Raton, FL, pp. 79–96.

Yamashita, K., Mizuochi, T., and Kobata, A. (1982) Analysis of oligosaccharides by gel filtration. Methods Enzymol., 83, 105–137.[ISI][Medline]

Yoshida, T., Takahashi, N., and Nakashima, I. (1991) Cell type and maturation stage-dependent polymorphism of N-linked oligosaccharides on murine lymphocytes and lymphoma cells. Mol. Immunol., 28, 1121–1130.[ISI][Medline]