Localization and characterization of polysialic acid–containing N-linked glycans from bovine NCAM

Maren von der Ohe1,2,5, Susan F. Wheeler1,3,6, Manfred Wuhrer7, David J. Harvey6, Steffen Liedtke3,5, Martina Mühlenhoff8, Rita Gerardy-Schahn8, Hildegard Geyer7, Raymond A. Dwek6, Rudolf Geyer7, David R. Wing6 and Melitta Schachner4,5

5Zentrum für Molekulare Neurobiologie, University of Hamburg, D-20246 Hamburg, Germany; 6Oxford Glycobiology Institute, Department of Biochemistry, South Parks Road, Oxford, OX1 3QU, UK; 7Institute of Biochemistry, University of Giessen, D-35392 Giessen, Germany; and 8Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany

Received on June 12, 2001; revised on August 13, 2001; accepted on August 22, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The neural cell adhesion molecule (NCAM) plays important roles during development, plasticity, and regeneration in the adult nervous system. Its function is strongly influenced by attachment of the unusual {alpha}2-8-linked polysialic acid (PSA). Here we analyzed the N-glycosylation pattern of polysialylated NCAM from brains of newborn calves. Purified PSA-NCAM glycoprotein was digested with trypsin, and PSA-glycopeptides were separated by immunoaffinity chromatography. For determining the N-glycosylation sites, PNGase F-treated glycopeptides were analyzed by Edman degradation and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). They were found to be exclusively linked to the fifth (Asn 439) and sixth (Asn 468) N-glycosylation sites in the fifth immunoglobulin-like domain of NCAM. The chain length of PSA consisted of at least 30 sialic acid residues, as shown by anion exchange chromatography. For analysis of the core structures, endoneuraminidase N–treated PSA-NCAM was separated by SDS–PAGE and digested with PNGase F. The core structures of polysialylated glycans were characterized by MALDI-MS combined with exoglycosidase digestions and chromatographic fractionation. They include hybrid, di-, tri-, and small amounts of tetraantennary carbohydrates, which were all fucosylated at the innermost N-acetylglucosamine. For the triantennary glycans, the "2,6" arm was preferred in polysialylated structures. High levels of sulfated groups were found on polysialylated structures and to a lower extent also on nonpolysialylated glycans. In addition, high-mannose-type glycans could be detected on PSA-NCAM glycoforms ranging from (GlcNAc)2(Man)5 up to (GlcNAc)2(Man)9. In conclusion, we observed a structural variability and high regional selectivity for the PSA-glycans attached to the NCAM molecule that are most likely influencing its biological functions.

Key words: NCAM/N-glycans/polysialic acid


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Carbohydrates are important modifiers of molecular functions in cell recognition in the nervous system. Cell adhesion molecules of the immunoglobulin family may carry different glycans, such as oligomannosidic and complex-type species bearing a 3'-sulfated glucuronic acid (HNK-1) or the unusual polysialic acid (PSA), which is expressed on the neural cell adhesion molecule (NCAM; Schachner and Martini, 1995Go). NCAM plays an important role in morphogenesis, plasticity, and regeneration of the nervous system (for review, see Edelman and Crossin, 1991Go). The molecule contains five immunoglobulin (Ig)-like domains and two fibronectin type III–like repeats in the extracellular domain. Alternative splicing from a single gene generates three major isoforms of NCAM, which are called NCAM 180, 140, and 120, corresponding to their apparent molecular weight. All three isoforms contain six potential N-glycosylation sites, from which the fifth and the sixth site in the fifth Ig-like domain may be modified by PSA (Nelson et al., 1995Go; Liedtke et al., 2001Go). PSA forms an unusual structure of a helical homopolymer of {alpha}2-8-linked sialic acid residues of more than 55 monomers (Finne and Mäkelä, 1985Go; Michon et al., 1987Go; Kabat et al., 1988Go; Livingston et al., 1988Go) and generates a bulky and highly negatively charged moiety. PSA could also be detected on the coat of Gram-negative bacteria, such as Escherichia coli K1 and Neisseria meningitidis (Troy and McCloskey, 1979Go; Bitter-Suermann and Roth, 1987Go), on polysialoglycoproteins in fish (Inoue and Iwasaki, 1978Go, 1980; Nomoto et al., 1982Go; Iwasaki et al., 1990Go) and on sodium channels in the electric eel (James and Agnew, 1989Go), as well as in rat brain (Zuber et al., 1992Go).

The presence of PSA on NCAM plays an important role in its function because the glycan diminishes the interaction of NCAM with other molecules in the cis- or trans-mode, where it leads to an anti-adhesive effect, influencing a calcium-independent homophilic cell adhesion (Rao et al., 1992Go, 1994). NCAM also mediates heterophilic interactions with other cell adhesion molecules, such as L1 (Kadmon et al., 1990aGo,b) and components of the extracellular matrix (Reyes et al., 1990Go; Storms et al., 1996Go; Kiselyov et al., 1997Go). The highest PSA expression is found in young animals when nerve cells grow out and try to find their appropriate targets. In the adult, PSA is more restricted to regions that are capable of morphological changes and synaptic plasticity, such as the olfactory system and the dentate gyrus, where new cells are generated (Seki and Arai, 1993Go; Rousselot et al., 1995Go). Under conditions of regeneration and plasticity, PSA upregulation has been observed (Muller et al., 1994Go, 1996; Becker et al., 1996Go). It has remained unclear, however, whether PSA influences signal transduction events, because it modifies cell interactions and may act as a receptor itself (Joliot et al., 1991Go; Garcia-Segura et al., 1995Go; Storms and Rutishauser, 1998Go; Muller et al., 2000Go; Vutskits et al., 2001Go). The PSA-mediated modulation of these interactions may strongly influence neurite outgrowth (Doherty et al., 1990Go), synaptic plasticity (Becker et al., 1996Go; Muller et al., 1996Go; Eckhardt et al., 2000Go), and neural regeneration in the adult (Muller et al., 1994Go), which are all necessary for normal brain development and function.

To gain further insights into the roles of PSA under physiological and pathological conditions, a considerable amount of work was performed to analyze the structures of PSA and corresponding core glycans, the attachment sites of these glycans to the NCAM polypeptide, and the biosynthesis of PSA by different polysialyltransferases. First studies concerning PSA-substituted carbohydrates were performed on fetal rat brain (Finne, 1982Go), where fucosylated tri- and tetraantennary core structures could be detected. Further structural features, such as repeating N-acetyllactosamine groups, type 1 (Galß1-3GlcNAc) and type 2 (Galß1-4GlcNAc) antennae, or sulfated oligosaccharides have been described for PSA core glycans of embryonic chick brain (Kudo et al., 1996Go). In a recent study, NCAM glycosylation from newborn mice was analyzed (Liedtke et al., 2001Go) showing predominantly core-fucosylated, partially sulfated isomers of tri- and tetraantennary glycans that carry the PSA homopolymer. In the latter publication, addition of PSA to the fifth and sixth N-glycosylation sites in the fifth Ig-like domain of NCAM was verified, as shown previously for recombinantly expressed NCAM mutants in which the potential N-glycosylation sites were eliminated by point mutations (Nelson et al., 1995Go).

In the present study we immunoaffinity-purified PSA-NCAM from calf brain with a monoclonal anti-PSA antibody to allow a comparison of PSA core structures between different vertebrate species. Furthermore, experiments were performed to determine the PSA attachment sites on NCAM from calf brain tissue allowing a comparison with the polysialylated N-glycosylation sites of NCAM established in vitro by Nelson et al. (1995)Go. In addition, the chain length of polysialic acid was analyzed by partial hydrolysis.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Isolation of bovine PSA-NCAM
PSA-NCAM was isolated from extracts of calf brain by immunoaffinity chromatography using a monoclonal anti-PSA antibody as outlined in Materials and methods. The purity of the PSA-NCAM preparation was analyzed by 2D gel electrophoresis. Silver staining showed a protein smear with an acidic pH of about 3–3.5 due to the sialic acid residues and a molecular mass ranging from 120 kDa to more than 200 kDa. No other proteins could be detected (Figure 1). The protein smear was PSA- and NCAM-positive, as demonstrated by western blotting (not shown). Purified PSA-NCAM was similarly analyzed with and without digestion with endoneuraminidase N (endo N). The enzyme cleaves specifically {alpha}2-8-linked PSA (Hallenbeck et al., 1987Go) and leaves a residue of about three to six sialic acid monomers (Vimr et al., 1984Go) attached to the core glycan. The residual oligosaccharides are not recognized by the PSA antibody 735 (Frosch et al., 1985Go). Without endo N digestion, NCAM and PSA antibodies gave a signal starting around 120 kDa and 140 kDa, respectively. After endo N digestion, no PSA signal could be detected and the NCAM antibody showed more distinct bands around 120 and 140 kDa, corresponding to the two smaller NCAM isoforms (Figure 2). The remaining smear resulted from attached glycans different from PSA. The higher molecular bands may show dimers and trimers of PSA-NCAM because both are NCAM- and PSA-positive. It is clear that PSA is only linked to the isoforms 120 and 140 kDa in calves.



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Fig. 1. 2D gel analysis of purified PSA-NCAM. Purified PSA-NCAM (5 µg) was separated by isoelectric focusing in the range of pH 3–10 followed by SDS–PAGE and silver staining.

 


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Fig. 2. Endo N digestion of PSA-NCAM. PSA-NCAM (5 µg/lane) was treated without (lane 1) or with (lane 2) endo N for 5 h at 37°C and separated on a 4–8% gradient gel. Western blots using monoclonal antibodies against NCAM (A) or PSA (B) are shown.

 
Isolation of PSA-glycopeptides and characterization of peptide moieties
As in the case of murine NCAM (Liedtke et al., 2001Go), trypsin was used for generation of peptides. Resulting (glyco)peptides were fractionated by immunoaffinity chromatography using an anti-PSA monoclonal antibody (mAb) 735 column, thus yielding two fractions, termed PSA-glycopeptides and residual glycopeptides. For allocation of PSA-glycans, PSA-glycopeptides were chemically desialylated and subsequently treated with peptide-N4-(N-acetyl-ß-glucosaminyl) asparagine amidase F (PNGase F). The deglycosylated peptides were fractionated by reversed-phase (RP) high-performance liquid chromatography (HPLC) (Figure 3). Individual peptide fractions were identified by Edman degradation and matrix-assisted laser desorption/ioniszation mass spectrometry (MALDI-MS) (Table I). The results revealed that PSA-glycopeptides contained exclusively the N-glycosylation sites 5 and 6. Peptides, including other glycosylation sites, were neither detected by MALDI-MS nor identified by amino acid sequencing. As a characteristic feature, Edman sequencing revealed Asp-residues at each potential glycosylation site instead of Asn predicted from the sequence database (SWISS-PROT P31836). This is in accordance with the known conversion of N-glycosylated Asn into Asp during PNGase F-release of N-glycans. In addition to the intact tryptic peptides, truncated forms of them were observed in minor amounts. The peptides Ile445–Asp459 in fraction 4 and Ser460–Arg474 in fraction 1, for example, indicated that the tryptic precursor Ile445–Arg474 was, in part, split at Asp459–Ser460 probably during chemical desialylation.



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Fig. 3. Separation of tryptic peptides derived from PSA-containing glycopeptides after deglycosylation with PNGase F. Peptides were fractionated by RP-HPLC, monitored by their absorbance at 220 nm, and subjected to Edman degradation and MALDI-MS.

 

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Table I. Characterization and assignment of tryptic, deglycosylated PSA peptides after separation by RP-HPLC
 
Characterization of PSA chains
PSA chain fragments were released from PSA-glycopeptides by mild acid hydrolysis. The fragments obtained were analyzed by high-pH anion exchange chromatography (HPAEC; Figure 4) and compared with a colominic acid standard, which was similarly treated. Identical patterns were registered for PSA-glycopeptides and colominic acid comprising fragments with up to 30 sialic acid residues.



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Fig. 4. HPAEC of PSA chain fragments released from PSA-glycopeptides. Oligosialyl chains obtained by mild acid hydrolysis were analysed by HPAEC (A). The magnification (B) shows PSA chain fragments with up to 30 sialic acids. (C) Colominic acid after mild acid hydrolysis. The numbers of sialic acid residues are indicated above the chromatograms.

 
Neutral and sialylated glycans
Following incubation with endo N to remove much of the polysialic acid (Vimr et al., 1984Go) that would prevent analysis by HPLC or MALDI-MS, and separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), the glycans were released from within the gel according to the procedure described by Küster et al. (1997)Go. Complete glycan removal was verified by examination of the residual protein by SDS–PAGE analysis. The resulting gel showed two discrete bands after silver staining that corresponded to masses of 120 and 140 kDa with minor bands at the resolving/stacking gel interface.

Because sialylated glycans fragment significantly during analysis by MALDI-MS as seen in both positive and negative ion spectra, a sample of the released glycans was treated with methyl iodide to stabilize the sialic acids for analysis (Powell and Harvey, 1996Go). Analysis was done by positive ion MALDI-MS; the resulting profile, which is shown in Figure 5, showed several series of peaks differing by the mass of methylated sialic acid (305 mass units). Negative ion spectra were also examined but gave similar results to the positive ion spectra. Endo N has been reported (Vimr et al., 1984Go) to cleave PSA chains attached to N-linked glycans providing that at least five consecutive {alpha}2-8-linked-sialic acid residues are present. The end products of such digestions are known to contain a maximum of seven N-acetylneuraminic acids still attached to the core glycan structure (Vimr et al., 1984Go). Thus, the resulting glycan mixture following endo N treatment would be expected to contain neutral and sialylated glycans with both one and several sialic acids terminating the antennae. The observation of sialic acid numbers in excess of the number of antennae supported the existence of PSA chains.



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Fig. 5. Positive-ion MALDI mass spectrum of endo N–treated N-linked glycans from PSA-NCAM. Residual sialic acids after endo N treatment were stabilized by methyl esterification. The horizontal solid and vertical dashed lines linked to each structure show the peaks containing increasing numbers of sialic acids associated with each structure. Numbers above the horizontal lines indicate the numbers of sialic acids associated with each structure, numbers on the peaks refer to the structures of the neutral structures listed in Table II. In some cases, the masses of the neutral compounds differed by only one to three mass units from those of sialylated structures. Although these masses were clearly visible in the unsmoothed spectrum, they appear in the same peak of the smoothed spectrum (Savitsky-Golay, 20 point) shown. Symbols for the structural formulae are as defined in the footnotes to Table II. In cases where several structures are possible for each composition, only the most abundant is shown.

 
The glycans to which these sialic acids were attached were examined further by HPLC and MALDI-MS combined with exoglycosidase digestions. Neutral glycans from the solution PNGase F digest were separated from the negatively charged N-glycans by weak anion exchange (WAX)-HPLC, following labeling with 2-aminobenzamide (2-AB), and were analyzed by both normal-phase (NP)-HPLC and positive ion MALDI-MS. Twelve glycans were found (Figure 6A); their masses and derived compositions are listed in Table II. The same neutral glycans were found in the total glycan sample (Fig. 5) following methyl ester formation but without 2-AB labeling.



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Fig. 6. Positive-ion MALDI mass spectra of neutral and neutralized (desialylated) glycans. (A) 2-AB-labeled glycans present in the neutral fraction (obtained by WAX-HPLC) as released by PNGase F (in solution). (B) Neutral and neutralized (desialylated) glycans (underivatized) obtained by in-gel PNGase F release (of the endo N–treated PSA-NCAM) and treated with NDV sialidase. Structures of the numbered peaks are listed in Table II. (C) As (B) but also treated with A. ureafaciens sialidase.

 




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Table II. Structures of N-linked glycans from bovine PSA-NCAM

 
Linkage of the sialic acids to the neutral core structures was investigated by incubations with linkage-specific sialidases. Thus incubation of the in-gel-released glycans with Newcastle disease virus (NDV) sialidase and analysis by MALDI-MS gave the profile shown in Figure 6B. Masses and derived compositions are listed in Table II. The profile was almost identical to that given by the neutral glycans from the WAX-HPLC pool (Figure 6A) indicating that, with few exceptions (mainly the minor peaks corresponding to Compounds 11 and 15), there was little {alpha}2-3-linked sialylation. The peak corresponding to Compound 12, which had no terminal galactose residues, and thus could not have been sialylated, was approximately the same height in both spectra, indicating that little or no increase in peak height had occurred with the other glycans. There were, however, some peaks (Compounds 1, 2, 6, 13, and 19) that appeared in the profile after sialidase treatment and that were later shown to be high-mannose glycans and, therefore, not expected. Their apparent absence in the spectrum of the neutral fraction obtained from the solution digest (Figure 6A) was attributed to fractionation during the work-up because they were also absent from the profile of the glycans from this preparation following complete desialylation.

Further desialylation of the glycans from the in-gel release with Arthrobacter ureafaciens sialidase additionally removed all {alpha}2–6-linked sialic acids. The resulting positive ion MALDI spectrum (Figure 6C) showed an increase in the number of fully galactosylated and multiply fucosylated structures and an increase in the relative abundance of several other glycans as indicated by comparison of the peak heights with that of Compound 12. Thus, most proximal sialic acid in these glycans was in {alpha}2-6-linkage.

Structures of the core glycans
Overall, the MS analyses indicated a general similarity in the isobaric monosaccharide compositions of the neutral core structures underlying naturally neutral glycans and the acidic glycans bearing sialylation and/or sulfation. The profile of the neutral glycans was dominated by a peak at 7.70 NP glucose units (GU) and another, half its relative abundance, at 6.26 NP-GU (Figure 7A). The compounds producing these peaks had masses corresponding to the compositions (Hex)4(HexNAc)5(Fuc)2 (Compound 22, Table II) and (Hex)3(HexNAc)5(Fuc)1 (Compound 12, Table II), respectively. Sequential exoglycosidase digestions, paralleling the MALDI-MS data, showed that the difference was due to outer-arm galactose and fucose residues, forming the Lewisx epitope in the larger structure. Thus, after removal of these residues by almond meal {alpha}-fucosidase and S. pneumoniae ß-galactosidase (and, in each case, removal of a fucose from the reducing terminal by bovine epidydimis {alpha}-fucosidase) the larger structure collapsed into the smaller one with a GU value of 5.85 (Figure 7B), characteristic for the degalactosylated biantennary structure possessing a bisecting N-acetylglucosamine (Rudd et al., 1999Go). Further digestion of this glycan mixture with S. pneumoniae ß-N-acetylhexosaminidase, at an arm-specific concentration, resulted in loss of one N-acetylglucosamine (GlcNAc) residue (from the 3-arm), consistent with the bisected biantennary structure (Chen et al., 1998Go).



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Fig. 7. NP-HPLC Profiles of 2-AB-labeled glycans. (A) The neutral fraction obtained by WAX-HPLC and (B) the neutral fraction following enzymatic removal of all galactose and fucose residues. The numbers above the peaks are the NP-GU values and, in parentheses, the compound number as given in Table II.

 
Alternative core structures for these two glycans would be those based on a triantennary configuration and a peak at 6.20 NP – GU in the HPLC profile following removal of all galactose and fucose residues (Figure 7B) was consistent with the structure of a triantennary glycan containing a branched 6-antenna. Typically, the degalactosylated core of the alternative 2,4-triantennary glycan (branched on the 3-arm) would have eluted at 5.94 NP-GU (Rudd et al., 1999Go) and lost two N-acetylhexosamine residues on digestion with S. pneumoniae ß-N-acetylhexosaminidase when used at an arm-specific concentration. This behavior was not observed in the present study, supporting the presence of the triantennary glycan branched on the 6-arm, known to elute at a higher GU value on HPLC (Chen et al., 1998Go) than its isomer. Subsequent sensitivity to jackbean {alpha}-mannosidase (data not shown) of a number of residual peaks in the profile shown in Figure 7B, confirmed the presence of hybrid structures as also indicated by the MALDI-MS data. The remaining peaks appeared to be bisected tri- and tetraantennary glycans with varying numbers of fucose residues. The HPLC results indicated that glycans with lactosamine extensions were not present.

Most of the naturally neutral and desialylated glycans were found to be resistant to digestion with S. pneumoniae ß-galactosidase or bovine testes ß-galactosidase. However, after addition of almond meal {alpha}-fucosidase to the previous digestion mixture, most compounds became sensitive. A similar profile of digestion products was obtained when the bovine testes ß-galactosidase was replaced with S. pneumoniae ß-galactosidase. Because the latter galactosidase is specific for ß1-4-linked galactose residues and is known to be inactive when the adjacent GlcNAc is substituted at the 3-position, the previous resistance to both galactosidases can be explained by the presence of fucose in a Lewisx-type structure (i.e., Galß1-4({alpha}1-3Fuc)GlcNAcß1-X, where X = the rest of the glycan). The presence of one or more fucose residues was clearly revealed by the compound masses following MALDI-MS. Further confirmation of the Lewisx structure was obtained from the difference between NP-GU values before and after the digestion array. This difference was consistent with the removal of terminal galactose and outer arm fucose residues (i.e., approximately 0.8 + 0.7 = 1.5 NP-GU). After digestion of the intact glycan mixture with bovine epididymis {alpha}-fucosidase, all the glucose unit values of the major glycan structures decreased by 0.4 NP – GU, a change that was consistent with the loss of one core {alpha}1-6-linked fucose residue per glycan.

Structures of the sialylated glycans carrying PSA
More detailed interpretations have been made of the underlying structures of the sialylated compounds highlighted in Figure 5 as the strongest candidates for carrying PSA chains. All of these glycans were fucosylated at the reducing terminal.

Compound 7.
The proposed hybrid structure ((Gal)1(GlcNAc)3(Man)4(Fuc)1, Compound 7) was not observed in the neutral state (Figure 6A) and remained acidic after NDV neuraminidase digestion. It was only neutralized by A. ureafaciens neuraminidase (presence in Figure 6C) and so possessed a proximal {alpha}2-6-linked sialic acid. The compound resisted digestion with S. pneumoniae ß-galactosidase but was totally sensitive to bovine testis ß-galactosidase indicative of the outer arm galactose in ß1-3-linkage (Figure 8). This allowed possible substitution of the outer arm GlcNAc with N-acetylneuraminic acid (sialic acid) (NeuNAc) ({alpha}2-6-linked) as well as the galactose residue itself (Zamze et al., 1998Go).



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Fig. 8. Positive-ion MALDI mass spectrum of the partially digested, neutral, core structures. Glycans following digestion with either (A) bovine testis ß-galactosidase, bovine epididymis, and almond meal {alpha}-fucosidases, or (B) S. pneumoniae ß-galactosidase, bovine epididymis, and almond meal {alpha}-fucosidases.

 

The existence of the unusual Galß1-3-containing hybrid structure (Compound 7) as a carrier of PSA was further supported by the presence of an HPLC peak at 5.23 NP-GU following earlier removal of sialic acids, galactose, fucose, and N-acetylhexosamine. This peak was sensitive to jackbean {alpha}-mannosidase and was subsequently digested down to (GlcNAc)2(Man)1. Its NP-GU value was consistent with that of (GlcNAc)2(Man)4, an expected product of the earlier exoglycosidase digestions when a hybrid N-linked glycan (such as Compound 7) is indeed present. The peak at m/z 1501.5 (Hex)4(GlcNAc)4 in Figure 8A is consistent with the presence of a bisected hybrid core structure as, at this stage, the biantennary glycan had been digested to (Hex)3(GlcNAc)4 (peak at m/z 1339.6). A compound of the bisected hybrid type is given in Table II (Compound 28). It is likely that bisected hybrid structures also exist with isobaric monosaccharide compositions (Hex)5(HexNAc)4(Fuc)(1,2), consistent with the product (Hex)4(HexNAc)4 (m/z 1501.5) seen after removal of galactoses and fucoses (Figure 8A) and, therefore, also PSA-carrying.

Compounds 16 and 20.
The proposed mono- and difucosylated biantennary structures, (Gal)2(GlcNAc)4(Man)3(Fuc)1 (Compound 16) and (Gal)2(GlcNAc)4(Man)3(Fuc)2 (Compound 20) each existed in an approximate ratio of 6:1 as sialylated and naturally neutral forms, with neutralization again achieved after removal of a proximal {alpha}2-6-linked sialic acid. Evidence from exoglycosidase digestion of the more highly fucosylated member of this family (Compound 20) indicated the presence of one galactose in ß1-3-linkage (insensitivity to S. pneumoniae ß-galactosidase, but loss of one hexose with bovine testis ß-galactosidase) and one Lewisx epitope (sensitivity to S. pneumoniae ß-galactosidase in the presence of almond meal {alpha}-fucosidase) (Figure 8). Complete interpretation of these data also indicated that bisected hybrid structures were contained within the glycan families possessing these isobaric monosaccharide compositions.

Glycans containing five GlcNAc residues
As revealed by the HPLC data, the families of glycans with compositions (Gal)1(GlcNAc)5(Man)3(Fuc)1, (Gal)2(GlcNAc)5-(Man)3(Fuc)1, (Gal)1(GlcNAc)5(Man)3(Fuc)2 and (Gal)2(GlcNAc)5(Man)3(Fuc)2 (Compounds 18, 25, 22, and 29, respectively) were dominated by bisected biantennary structures, with only a small constituent 2,6-triantennary component. The major glycan of the entire preparation—(Gal)1(GlcNAc)5(Man)3(Fuc)2 Compound 22—was found to exist in a ratio of 1:3 as sialylated and naturally neutral forms, again with the proximal sialic acid in {alpha}2-6-linkage where present and with one Lewisx epitope. The relative abundance of this compound in Figure 5 (methyl ester derivatives of the sialylated forms) shows a rapid decline with increasing sialylation, and so only a minor proportion can be suggested to carry potential polysialylation. With (Gal)1(GlcNAc)5(Man)3(Fuc)1 and (Gal)2(GlcNAc)5(Man)3(Fuc)1 the ratios of sialylated to neutral forms were approximately 1:1 and 6:1, respectively (again with proximal {alpha}2-6-linked sialic acids present). Differential sensitivities to ß-galactosidases suggested a galactose content of approximately 50% in ß1-4-linkage and 50% in ß1-3-linkage, again offering, in the latter case, the possibility of {alpha}2-6-linked sialic acid to that outer-arm GlcNAc.

Glycans containing six GlcNAc residues
Full characterization of the relative proportions of bisected triantennary and nonbisected tetraantennary glycans in (Gal)1(GlcNAc)6(Man)3(Fuc)2 (Compound 27) and (Gal)2(GlcNAc)6(Man)3(Fuc)2 (Compound 39) has not been made. Only a small proportion (<10%) of (Gal)1(GlcNAc)6(Man)3(Fuc)2 was sialylated, again with a proximal {alpha}2-6-linked sialic acid and with one outer arm carrying the Lewisx epitope. In contrast, the glycan with the composition (Gal)2(GlcNAc)6(Man)3(Fuc)2 (Compound 39) was not observed in the neutral state; >90% possessed a proximal {alpha}2-6-linked sialic acid, but the remaining 10% had only {alpha}2-3/8-linked sialic acids. With one Lewisx epitope, most of the outer arm galactose was in ß1-4-linkage, a small proportion (<10%) of the non-Lewisx galactose being ß1-3-linked. The contrasting properties of the two compounds described here may reflect differences between the bisected triantennary and nonbisected tetraantennary families. The presence of the tetraantennary glycans is supported by Compounds 48–54 having isobaric monosaccharide compositions in the series (Gal)4(GlcNAc)6(Man)3(Fuc)0–4 and compounds 55–58 containing an additional bisecting GlcNAc, though these were all in minor relative abundance.

N-linked sulfated glycans
When the total glycan pool was desialylated and rerun on WAX-HPLC, a number of peaks were still present in the anionic region (not shown). NP-HPLC suggested that an additional negative charge, consistent with sulfation, remained on these oligosaccharides. These sulfated glycans were isolated by porous graphitized carbon (PGC) chromatography (Wheeler and Harvey, 2001Go). Forty-five additional structures were found in the acidified 1:3 acetonitrile:water (v/v) fraction from this separation, and Figure 9 shows the resulting negative ion MALDI mass spectrum. The compositions derived from their molecular weight indicated that they were sulfated derivatives of the same structures as those observed in the neutral and sialylated fractions. Indeed, many of the relatively abundant sulfated structures in Figure 9, namely, Compounds 7, 16, 18, 20, 22, 25, 29, and 39—were seen as PSA-carrying glycans (Figure 5). Interestingly, the most abundant of the compounds in Figure 9 not seen earlier as PSA-carrying (Compounds 32 and 34) were observed to exist also as disulfated species (Compounds 33 and 35).



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Fig. 9. Negative-ion MALDI mass spectrum of the sulfated glycans following desialylation. The glycans were recovered from a PGC column as described in the text. Numbers on the peaks refer to the structures listed in Table II.

 
The calculated compositions of each structure did not correspond to any molecular ion containing glucuronic acid; therefore, another carbohydrate motif was presumably present for these sulfated N-glycans. Unfortunately the amount of sample obtained from the in-gel PNGase F digests precluded any further analysis by methods, such as methylation analysis (Hellerqvist, 1990Go) or fragmentation by post-source decay that would provide information about the position of the sulfate group within the glycan pool. Fragmentation experiments with a quadrupole TOF (Q-TOF) mass spectrometer did not yield any useful information. However, Kudo et al. (1996)Go have reported the presence of terminal 3-O-sulfated GlcNAc residues on polysialylated tri- and tetraantennary NCAM oligosaccharides obtained from fetal chick brain. In addition, Liedtke et al. (2001)Go have reported the presence of 4,6-substituted GlcNAc in NCAM glycans, suggesting that, in some molecules, sulfate might be attached to the 6-position of GlcNAc.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In this study we have examined the carbohydrate structures of PSA-NCAM from young bovine brain. After immunoaffinity chromatography using an anti-PSA antibody we could demonstrate that PSA occurred only on two major NCAM isoforms, NCAM 140 and 120. NCAM 180 did not carry detectable levels of PSA at this stage, as could be demonstrated by gradient SDS–PAGE after complete digestion with {alpha}2-8-specific endo N.

Our results show a strong regional selectivity for PSA to the fifth and sixth N-glycosylation site in the fifth Ig-like domain of NCAM. These findings agree with previous work regarding mutated asparagine-residues and deletion mutants of chicken NCAM (Nelson et al., 1995Go) and correlates with the direct identification of polysialylated N-glycosylation sites by N-terminal sequencing of PSA-NCAM glycopeptides from newborn mouse brain (Liedtke et al., 2001Go). No PSA-containing peptide of the alpha-subunit of the voltage-dependent sodium channel protein could be detected, although the occurrence of PSA on the sodium channel in adult rat brain has been described (Zuber et al., 1992Go). This discrepancy could be due to the age of the animal or to the species that was analyzed.

The chain length of PSA was analyzed by HPAEC following partial hydrolysis. The maximal chain length detectable consisted of at least 30 sialic acid residues in our preparation from young bovine brain. This correlates with earlier experiments from developing rat and mouse brain as well as human neuroblastoma cells (Finne, 1982Go; Finne et al., 1983Go; Livingston et al., 1988Go). The question as to whether higher degrees of polymerization would have been detectable when employing different hydrolysis and chromatographic conditions (Zhang et al., 1997Go; Inoue et al., 2000Go) remains open. Due to the small amounts of material available, it could not be determined whether the sizes of these chains differed at the two glycosylation sites.

The core structures from polysialylated glycans include hybrid, di- tri-, and small amounts of tetraantennary glycans. Those of the di- and triantennary type may contain a bisected GlcNAc. A common structural feature of all PSA core glycans is a fucose residue that is attached in {alpha}1-6-linkage to the innermost GlcNAc residue. Fucose occurred to some extent also on the outer arms of the core bound to a GlcNAc residue, thus forming a Lewisx epitope, which is defined as a 3-fucosyl N-acetyllactosamine sequence (Streit et al., 1996Go). This fucose substitution was not observed for PSA-carrying N-glycans of NCAM from the brains of young mice (Liedtke et al., 2001Go). The terminal sialic acid monomers bound to the core glycans showed a slight preference for an {alpha}2-6-linkage and, to a smaller degree, an {alpha}2-3-linkage, as could be demonstrated by specific digestion with A. ureafaciens sialidase and NDV sialidase, respectively. In contrast to this result, sialic acid appeared to occur in an {alpha}2-3-linkage in glycans from young chicken and young mouse brain (Kudo et al., 1996Go; Liedtke et al., 2001Go). This unexpected finding might be special for bovines and might represent slightly different acceptor specificities for the polysialyltransferases. In the present study, a preference was observed for the "2,6"-type of triantennary N-glycans, rather than the "2,4"-type, for carrying PSA-containing carbohydrates on this class of oligosaccharides. This observation was in agreement with that of Kudo et al. (1996)Go and Liedtke et al. (2001)Go. In our preparation, glycans with N-acetyllactosamine extensions were not readily detectable, but their occurrence has been described in earlier studies on chicken NCAM-associated glycans (Kudo et al., 1996Go).

In the present study we could also show for the first time that bovine NCAM carries high-mannose-type glycans (GlcNAc)2(Man)5 to (GlcNAc)2(Man)9, as described before for other cell adhesion molecules, such as adhesion molecule on glia, L1, myelin-associated glycoprotein, and P0 (Griffith et al., 1992Go; Horstkorte et al., 1993Go; Schmitz et al., 1993Go; Heiland et al., 1998Go). Although oligomannosidic glycans are the least processed N-glycans in the biosynthetic pathway occurring in the Golgi apparatus, they can nevertheless also reach the cell surface and play important roles in cell recognition and adhesion events (Kadmon et al., 1990bGo; Horstkorte et al., 1993Go; Schmitz et al., 1993Go; Porwoll et al., 1998Go; Helenius and Aebi, 2001Go). Another novel feature was that PSA was detected on a core-fucosylated hybrid type structure possessing galactose in ß1-3-linkage on its complex arm.

As discussed before (Kudo et al., 1996Go; Liedtke et al., 2001Go) we also found a relative abundance of sulfate groups on the PSA core structures. The sulfated structures occurred mainly on polysialylated, and to a lower extent also on nonpolysialylated glycans. Four fucosylated tri- and tetraantennary compounds were found to carry two sulfate groups.

NCAM plays important roles during development, regeneration, and synaptic plasticity. The most powerful glycan attached to NCAM is PSA because it has a strong influence on the binding characteristics of NCAM by rendering it less adhesive to itself as well as to other cell adhesion molecules (Sadoul et al., 1983Go; Rutishauser et al., 1985Go; Doherty et al., 1990Go). As for PSA, the negatively charged sulfate groups play important roles during development, as described for example for the HNK-1 epitope, which contains a sulfated glucuronic acid on a lactosaminyl residue (Kruse et al., 1984Go; Schachner and Martini, 1995Go; Voshol et al., 1996Go). In the present study, the precise nature of the sulfate substitutions and their functional significance remain to be established. It is possible that O-glycosylation sites exist on NCAM. O-glycosylation is the main glycosylation type on the extracellular matrix protein tenascin-R (Zamze et al., 1999Go), but was not analyzed in this study on NCAM. The attachment of PSA to the glycan core structure takes place in the trans-Golgi compartment (Alcaraz and Goridis, 1991Go). Three different enzymes can attach PSA to NCAM, the polysialyltransferases ST8SiaII/STX (Kitagawa and Paulson, 1993Go; Kojima et al., 1996Go), ST8SiaIV/PST (Eckhardt et al., 1995Go; Nakayama et al., 1995Go)—which both show a different spatial and temporal expression (Angata et al., 1998Go; Seidenfaden et al., 2000Go; Kitazume-Kawaguchi et al., 2001Go)—and ST8SiaIII (Angata et al., 2000Go). Although different studies were done looking for an acceptor for the polysialyltransferases ST8SiaII, ST8SiaIV and ST8SiaIII (Angata et al., 2000Go) further knowledge of the PSA-glycans attached to NCAM may help determine the specific structure that enables the polysialyltransferases to bind to NCAM and to catalyze the functionally important glycosylation reaction.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
The mAbs 735 (anti-PSA) and 123C3 (anti-NCAM) have been described elsewhere (Frosch et al., 1985Go; Gerardy-Schahn and Eckhardt, 1994Go). For isolation of PSA-NCAM and PSA glycopeptides, purified mAbs were coupled to CNBr-activated Sepharose 4B following the manufacturer’s protocol (Pharmacia, Freiburg, Germany).

Milli-Q water was freshly distilled over sodium permanganate. AG3-X4 (free base form) and AG 50-X12 (hydrogen form) ion exchange resins were purchased from Bio-Rad (Hemel Hempstead, UK). C18 resin was taken from a Sep-Pak cartridge (Waters, Milford, MA). Acrylamide and bis-acrylamide were purchased from National Diagnostics (Atlanta, GA). Hypersep PGC was purchased from ThermoQuest (Runcorn, UK) and packed into microcolumns using Eppendorf GELoader pipette tips from BDH. 2,5-Dihydroxybenzoic acid (DHB) was purchased from Aldrich Chemical Company (Gillingham, Dorset, UK) and was recrystallized from methanol/chloroform. Sodium-depleted D-arabinosazone was prepared according to the method described by Chen et al. (1997)Go, dried in vacuo, and stored at –20°C.

Endo N was purified from E. coli bacteriophage PK1E (Gerardy-Schahn et al., 1995Go). Recombinant (E. coli) PNGase F from Flavobacterium meningosepticum and recombinant NDV sialidase were purchased from Boehringer Mannheim (Mannheim, Germany). Almond meal {alpha}-fucosidase and jackbean ß-N-acetylhexosaminidase were purified from source at the Glycobiology Institute by Dr. T. Butters. All other exoglycosidases and the 2-AB labeling kit were purchased from Oxford GlycoSciences (Abingdon, Oxon, UK).

Purification of PSA-NCAM from calf brain
Three hundred grams (wet weight) brain from 2- to 3-day-old calves were homogenized (Polytron homogenizer) in ice-cold acetone, transferred to a G2 glass filter (Schott, Mainz, Germany), and dried under reduced pressure. The acetone powder was washed twice for 15 min with cold 10 mM sodium phosphate (pH 7.4), 150 mM sodium chloride (phosphate buffered saline, PBS), containing complete protease inhibitor mix (Roche, Basel, Switzerland) and centrifuged for 15 min at 20,000 x g and 4°C. The pellet was then suspended in PBS containing 1% Triton X-100 (v/v) and complete protease inhibitor mix (2 ml/g brain) and incubated overnight at 4°C on a shaker. After ultracentrifugation at 100,000 x g for 60 min at 4°C, the supernatant was concentrated and the buffer was exchanged with PBS by ultrafiltration (YM100 membrane, Millipore, Eschborn, Germany).

After passing a precolumn of Sepharose 4B the concentrated supernatant was loaded onto an anti-PSA affinity column (40 mg mAb/ml) with a flow rate of 0.2 ml/min. The column was washed with 20 column volumes of PBS containing 1% Triton X-100 (v/v), 20 column volumes of PBS containing 300 mM sodium chloride, and 10 column volumes of 10 mM sodium phosphate, pH 8.0 (flow rate 0.75 ml/min). Bound PSA-NCAM was eluted with an alkaline buffer containing 100 mM diethylamine, pH 11.5, 150 mM sodium chloride, 2 mM ethylenediamine tetra-acetic acid (EDTA) and 0.05% Triton X-100 (v/v). The eluate was immediately neutralized with 1 M Tris–HCl (pH 6.8) and subjected to concentration and buffer exchange with PBS using ultrafiltration (YM100 membrane). The bovine PSA-NCAM was stored at a concentration of 0.5 mg/ml PBS at –80°C.

2D gel electrophoresis and western blot analysis
For isoelectric focusing, proteins were precipitated with chloroform/methanol, resuspended in reswelling buffer (5 M urea, 2 M thiourea, 65 mM dithiothreitol [DTT], 0.8% ampholyte/ampholine, 4% CHAPS), and incubated with ImmobilineTM dry strips pH 3–10 (Pharmacia) overnight at 4°C. Focusing was carried out at 17°C at 200 V for 1 min, followed by a voltage gradient from 200 V to 3500 V for 1.5 h and finally at 3500 V for 1 h (Multiphor II, Pharmacia). After incubation in equilibration buffer for 2 x 10 min (50 mM Tris–HCl, pH 6.8, 6 M urea, 30% glycerol [v/v], 0.1 mM DTT, 2% SDS, 0.01% bromophenol blue) the strips were run on a 10% SDS–PAGE. Proteins were detected by silver staining according to the method described by Heukeshoven and Dernick (1988)Go.

Gradient gel electrophoresis was carried out in a 4–8% polyacrylamide separating gel with a 3% stacking gel. Separated proteins were transferred to a nitrocellulose membrane (Schleicher & Schüll, Dassel, Germany) in blotting buffer (25 mM Tris, 192 mM glycine, 10% methanol) at 80 V for 90 min at 4°C (Protean II, BioRad, Munich, Germany). After blocking for 1 h at room temperature with 2% milk powder (fatty acid–free) the mAb 123C3 (anti-NCAM) and mAb 735 (anti-PSA) were used for immunostaining. The membranes were incubated with the antibodies for 1 h, washed five times with PBS, and incubated with goat-anti-mouse/horseradish peroxidase–conjugated antibodies (1:10,000, Dianova, Hamburg, Germany) for 1 h at room temperature. After washing five times with PBS the detected bands were visualized by ECL reagent (Amersham, Freiburg, Germany).

Isolation of PSA-glycopeptides
PSA-NCAM was carboxymethylated with iodoacetamide and digested with trypsin (sequencing grade; Sigma, Deisenhofen, Germany). After boiling for 3 min at 96°C the peptides were applied to an anti-PSA column, and the column was washed with five column volumes of PBS containing 0.5% Triton X-100 followed by five column volumes of PBS. Bound material was eluted with alkaline buffer containing 100 mM diethylamine, pH 11.5, 150 mM NaCl, and 2 mM EDTA. The flow rate was 0.3 ml/min for all steps. Eluted glycopeptides were immediately neutralized by addition of 1 M Tris–HCl buffer, pH 6.8. PSA-glycopeptides were desalted on a Bio-Gel P30-column (BioRad) with 50 mM ammonium bicarbonate (flow rate 0.5 ml/min). Fractions were monitored by absorption at 206 nm, and glycan-containing fractions were pooled and lyophilized.

Release and analysis of PSA chain fragments
PSA-glycopeptides and colominic acid (Sigma) were treated with 10 mM acetic acid at 60°C for 30 min. The products were directly applied to a RP-cartridge (Macherey & Nagel, Düren), the combined flow-through and wash (0.1% trifluoroacetic acid [TFA]) containing the released PSA chain fragments was lyophilized. Glycopeptides were recovered by stepwise elution with acetonitrile (20%, 40%, and 60% acetonitrile in water, 0.1% TFA), and used for the identification of peptide moieties. Released PSA chain fragments were analyzed by HPAEC at room temperature, 1 ml/min, using a CarboPac PA-100 column (4.6 x 250 mm), pulsed amperometric detection, and the equipment described elsewhere (Pfeiffer et al., 1990Go). A linear gradient (0–60 min) from 200 to 900 mM sodium acetate in 100 mM sodium hydroxide was used.

Isolation of peptide moieties
After desialylation by incubation with 1 M acetic acid for 2 h at 80°C and lyophilization, glycans were released from the different PSA-glycopeptides by treatment with PNGase F (Roche; 200 µl 20 mM sodium phosphate buffer, pH 7.5, 10 U of enzyme, 37°C, 24 h) and separated from residual peptides on an RP-cartridge. Retrieved peptides were separated by RP-HPLC (ODS-Hypersil C18; 2.1 x 250 mm; 3 µm; Shandon, UK) at 120 µl/min with a linear gradient from 0.1% TFA to 60% acetonitrile containing 0.1% TFA in 60 min at 30°C. Peptides were monitored by absorption at 220 nm.

Peptide identification
Peptides were amino-terminally sequenced by automated Edman degradation on an Applied Biosystems (Foster City, CA) pulsed liquid phase sequencer, model 477A, under standard conditions. Phenylthiohydantoin-derivatives of amino acids were identified by an online analyzer, model 120A (Applied Biosystems). MALDI-MS was performed using a Vision 2000 mass spectrometer (Finnigan MAT, Bremen, Germany), equipped with a UV-nitrogen laser ({lambda} = 337 nm). Mass spectra were recorded at an accelerating voltage of 5 kV in the positive-ion reflectron mode. Typically, obtained spectra resulted from the accumulation of 5–25 laser shots. One microliter of analyte solution was mixed on the stainless steel target with 1 µl of matrix solution (5 mg/ml of 6-aza-2-thiothymine [Sigma] in twice distilled water) and allowed to air-dry. Calibration was performed with human angiotensin and bovine insulin (both from Sigma). Given mass values represent average masses.

Endo N digestion
Bovine PSA-NCAM (50 µl of a 0.5 mg/ml solution) was incubated in 1M Tris buffer, pH 6.8, 150 mM NaCl, 2 mM EDTA, 100 mM diethylamine, and 0.1% Triton X-100, with 2 µl endo N at 37°C for 20 h.

Gel electrophoresis and in-gel PNGase F digestion
SDS–PAGE gels (80 x 80 x 0.75 mm) were prepared as described previously (Küster et al., 1997Go). Approximately 7.5 µg (15 µl) of PSA-NCAM were loaded onto the gel, which was run using a Mini PROTEAN II cell (Bio-Rad) at 180 V constant voltage in 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3. Spots were visualized with Coomassie blue, the areas of the gel containing NCAM were removed, and the protein was reduced and alkylated within the gel prior to removal of the N-linked glycans with PNGase F as described earlier (Küster et al., 1997Go).

Purification of glycans
Neutral glycans were purified with a microcolumn packed with 5 µl each of AG3-X4 (free base form), AG50-X12 (hydrogen form), and C18 resins in a GELoader pipette tip. The column was washed with 3 x 50 µl water prior to addition of the sample in 40 µl water. Glycans were eluted with 3 x 50 µl water, collected, and dried in vacuo.

Sialylated glycans were converted into their methyl esters with methyl iodide using the method described by Powell and Harvey (1996)Go. Removal of salts before MALDI-MS analysis was by a microcolumn packed with 5 µl each of AG3-X4 (free base form) and AG50-X12 (hydrogen form). The column was packed and washed, and glycans were recovered as detailed in the preceding paragraph. Samples were freeze-dried down to 2–5 µl and completely dried in vacuo.

Sulfated glycans were purified with a microcolumn packed with 2 µl C18 resin and 10 µl of PGC (Packer et al., 1998Go) (from 100 mg of resin that had been washed with 3 x 4 ml of each of the following solutions; (1) 1 M sodium hydroxide, (2) water, (3) 4:1 acetonitrile:water/0.1% TFA, (4) 1:3 acetonitrile:water/0.05% TFA, (5) 1:3 acetonitrile:water, and (6) water. After loading, salts were removed by washing with 3 x 40 µl water, followed by elution of uncharged glycans with water:acetonitrile (3:1, v/v, 3 x 40 µl). Sulfated and sialylated glycans were eluted from the microcolumn with water:acetonitrile (3:1, v/v)/0.05% trifluoroacetic acid and water:acetonitrile (3:2, v/v)/0.05% TFA, respectively. All fractions were freeze-dried and reconstituted in water (5 µl), prior to analysis by MS.

Solution PNGase F digestion of PSA-NCAM
Approximately 7 µg (15 µl) of endo N-digested PSA-NCAM were dissolved in 50 µl of 20 mM phosphate buffer, pH 7.2, containing 5 mM mercaptoethanol. Denaturation of the glycoprotein was achieved by heating at 100°C for 10 min. After cooling, a solution of 200 U/ml PNGase F (10 µl) was added to the denatured sample and incubated at 37°C for 18–24 h. Complete digestion was confirmed by SDS–PAGE (Laemmli gel; 8% T, 2.5% C) from an aliquot (10 µl) of the digest mixture. The glycans were recovered by pelleting the protein with ice-cold 2:1 ethanol:water (0.9 ml) from an in-solution digestion of 150 µg (300 µl) endo N–treated PSA-NCAM. Further precipitation of the protein was achieved after 2 days at –20°C, followed by spinning at 300 rpm for 5 min. The supernatant, containing the oligosaccharides, was removed, and the protein pellet washed with a further 2 x 300 µl of 2:1 ethanol:water and respun. The combined supernatants were dried down to a volume of 150 µl under a stream of nitrogen. Further removal of Triton X-100 and protein was carried out using an Extracti-gel D column (200 µl) equilibrated in 100 mM phosphate buffer, pH 7.1. The sample was applied to the column, and the glycans were eluted with five-column volumes of phosphate buffer. The total volume was reduced to 300 µl by drying in vacuo, and the solution was stored at –20°C.

Fluorescent labeling of glycans
The total glycan pool of released glycans was fluorescently labeled by reductive amination with 2-AB (Bigge et al., 1995Go).

Exoglycosidase digestions
The glycans obtained from in-gel PNGase F digestion were first desialylated with a mixture of NDV sialidase (0.5 U/ml) and A. ureafaciens sialidase (2 U/ml) at 37°C for 18 h. The resulting solution was then split into eight aliquots (1 µl) and each was mixed with 1 µl of each of the following exoglycosidases; (1) S. pneumoniae ß-galactosidase (1 U/ml); (2) bovine testes ß-galactosidase (4 U/ml); (3) bovine testes ß-galactosidase and bovine epididymis {alpha}-fucosidase (1 U/ml); (4) bovine testes ß-galactosidase, bovine epididymis {alpha}-fucosidase, and almond meal {alpha}-fucosidase (1 mU/ml); (5) S. pneumoniae ß-galactosidase, bovine epididymis {alpha}-fucosidase, and almond meal {alpha}-fucosidase; (6) bovine testes ß-galactosidase, bovine epididymis {alpha}-fucosidase, almond meal {alpha}-fucosidase, and S. pneumoniae N-acetyl-ß-D-glucosaminidase (6 mU/ml); (7) bovine testes ß-galactosidase, bovine epididymis {alpha}-fucosidase, almond meal {alpha}-fucosidase, and jackbean ß-N-acetylhexosaminidase (6 U/ml); and (8) bovine testes ß-galactosidase, bovine epididymis {alpha}-fucosidase, almond meal {alpha}-fucosidase, jackbean ß-N-acetylhexosaminidase, and jackbean {alpha}-mannosidase (100 U/ml). All enzyme digests were made-up to a total volume of 10 µl with an appropriate buffer as described earlier (Chen et al., 1998Go) and incubated overnight at 37°C in parallel. After incubation, the enzyme activity was terminated by heating at 100°C for 1–2 min. The samples were then deproteinated by application onto 0.45 µm cellulose nitrate Pro-Spin MicroTM centrifugal filters and left at room temperature for 20 min. Glycans were then recovered by centrifugation three times with 40 µl water:acetonitrile (19:1, v/v) and prepared for MALDI-MS by using the neutral glycan microcolumn clean-up procedure described in Purification of glycans.

WAX-HPLC
2-AB-labeled glycans were separated on a 7.5 x 50 mm Vydac 301VHP575 WAX column (Hichrom) using a Waters 2690 Alliance separation module with a column heater at 30°C and a Waters 474 scanning fluorescence detector. The gradient used was as described by Guile et al. (1994)Go: solvent A, 500 mM ammonium formate, pH 9.0; solvent B, water. Initial conditions were 0% A at a flow rate of 1 ml/min followed by a linear gradient of 0–5% A over 12 min, followed by increases to 21% A over 13 min, 80% over 25 min, and 100% over the last 5 min. The column was washed with 100% A for 5 min at a flow rate of 1 ml/min before being reequilibrated in 0% A for the next sample. The elution of the labeled glycans was monitored by fluorescence detection and calibrated with 2-AB-labeled fetuin oligosaccharides. For preparative separations, 2-AB-labeled glycans from one gel band (7.5 µg of protein) were injected onto the WAX column and collected at 1-min intervals. The resulting fractions were repeatedly dried under vacuum in a Speedvac until all of the ammonium formate buffer was removed.

NP-HPLC
2-AB-labeled glycans were separated on a 4.6 x 250 mm GlycoSep N column (Oxford GlycoSciences) using a Waters 2690 Alliance Separation Module with a column heater at 30°C and a Waters 474 scanning fluorescence detector. The gradient used was as described by Guile et al. (1996)Go: solvent A, 50 mM ammonium formate, pH 4.4; solvent B, acetonitrile. Initial conditions were 20% A at a flow rate of 0.4 ml/min followed by a linear gradient of 20–58% A over 152 min followed by 58–100% A in the next 3 min. The column was washed with 100% A for 5 min at a flow rate of 1 ml/min before being reequilibrated in 20% A for the next sample. Retention times in GUs are reported relative to a standard dextran ladder.

MALDI-MS
Oligosaccharide samples were loaded onto the mass spectrometer target in 1 µl of water, mixed with 0.5 µl of a fresh solution of D-arabinosazone (Chen et al., 1997Go) (3 mg/ml in 3:1 [v/v] ethanol:water) and allowed to air-dry. A further 0.5 µl of matrix was added and dried quickly to produce a microcrystalline surface of matrix and glycan. Alternatively, the glycans were mixed with 2,5-DHB (0.5 µl of a saturated solution in acetonitrile), allowed to dry, and then were recrystallized from ethanol (Harvey, 1993Go). All mass spectra were acquired on a Micromass TofSpec-2E reflectron TOF mass spectrometer equipped with a time-lag focusing MALDI ion source. The pulse delay time and accelerating voltage were 500 ns and 20 kV, respectively. Between 150 and 230 laser shots were averaged for each spectrum. The mass spectrometer was externally calibrated with oligothymidylic acid and 2-AB-labeled dextran and spectra were acquired and processed with a Micromass MassLynx data system. All molecular weights are quoted as monoisotopic masses except where indicated in Table II. Sulfate-containing oligosaccharides were observed as [M–H] or [M+Na–2H] ions in the negative ion spectra; all other spectra were acquired in positive ion mode where the glycans gave [M+Na]+ ions.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. D. Linder, Institute of Biochemistry, Giessen, for amino acid sequencing. This project was supported by the Deutsche Forschungsgemeinschaft (Ge386/2-1/2, Sonderforschungsbereich 535 and Scha/85/28, 1-2), and the Biotechnology and Biological Sciences Research Council and Oxford GlycoSciences.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
2-AB, 2-aminobenzamide; DHB, dihydroxybenzoic acid; DTT, dithiothreitol, EDTA, ethylenediamine tetra-acetic acid; endo N, endoneuraminidase N; GlcNAc, N-acetylglucosamine; GU, glucose unit; HPAEC, high-pH anion exchange chromatography; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; Ig, immunoglobulin; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; NCAM, neural cell adhesion molecule; NDV, Newcastle disease virus; NeuNAc, N-acetylneuraminic acid (sialic acid); NP, normal phase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PGC, porous graphitized carbon; PNGase F, peptide-N4-(N-acetyl-ß-glucosaminyl) asparagine amidase F; PSA, polysialic acid; Q, quadrupole; RP, reversed-phase; SDS, sodium dodecyl sulfate; TFA, trifluoroacetic acid; TOF, time-of-flight; WAX, weak anionic exchange.


    Footnotes
 
1 These authors contributed equally to this paper. Back

2 Present address: Mermaid Pharmaceuticals, Falkenreid 88, D-20251 Hamburg, Germany Back

3 Present address: Neuroscience Research Centre, Merck, Sharp and Dohme, Terlings Park, Harlow, Essex CM20 2QR, UK Back

4 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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Angata, K., Suzuki, M., McAuliffe, J., Ding, Y., Hindsgaul, O., and Fukuda, M. (2000) Differential biosynthesis of polysialic acid on neural cell adhesion molecule (NCAM) and oligosaccharide acceptors by three distinct alpha 2, 8-sialyltransferases, ST8Sia IV (PST), ST8Sia II (STX), and ST8Sia III. J. Biol. Chem., 275, 18594–18601.[Abstract/Free Full Text]

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Chen, Y.-J., Wing, D.R., Guile, G.R., Dwek, R.A., Harvey, D.J., and Zamze, S. (1998) Neutral N-glycans in adult rat brain tissue: complete characterization reveals fucosylated hybrid and complex structures. Eur. J. Biochem., 251, 691–703.[Abstract]

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