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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: NCAM/N-glycans/polysialic acid
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1992, 1994). NCAM also mediates heterophilic interactions with other cell adhesion molecules, such as L1 (Kadmon et al., 1990a
,b) and components of the extracellular matrix (Reyes et al., 1990
; Storms et al., 1996
; Kiselyov et al., 1997
). 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, 1993
; Rousselot et al., 1995
). Under conditions of regeneration and plasticity, PSA upregulation has been observed (Muller et al., 1994
, 1996; Becker et al., 1996
). 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., 1991
; Garcia-Segura et al., 1995
; Storms and Rutishauser, 1998
; Muller et al., 2000
; Vutskits et al., 2001
). The PSA-mediated modulation of these interactions may strongly influence neurite outgrowth (Doherty et al., 1990
), synaptic plasticity (Becker et al., 1996
; Muller et al., 1996
; Eckhardt et al., 2000
), and neural regeneration in the adult (Muller et al., 1994
), 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, 1982), 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., 1996
). In a recent study, NCAM glycosylation from newborn mice was analyzed (Liedtke et al., 2001
) 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., 1995
).
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). In addition, the chain length of polysialic acid was analyzed by partial hydrolysis.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
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, 1996). 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., 1984
) to cleave PSA chains attached to N-linked glycans providing that at least five consecutive
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., 1984
). 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.
|
|
|
Further desialylation of the glycans from the in-gel release with Arthrobacter ureafaciens sialidase additionally removed all 26-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
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 -fucosidase and S. pneumoniae ß-galactosidase (and, in each case, removal of a fucose from the reducing terminal by bovine epidydimis
-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., 1999
). 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., 1998
).
|
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 -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(
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
-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
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 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) (
2-6-linked) as well as the galactose residue itself (Zamze et al., 1998
).
|
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 -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 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
-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 22was found to exist in a ratio of 1:3 as sialylated and naturally neutral forms, again with the proximal sialic acid in 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
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
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 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
2-6-linked sialic acid, but the remaining 10% had only
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 4854 having isobaric monosaccharide compositions in the series (Gal)4(GlcNAc)6(Man)3(Fuc)04 and compounds 5558 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, 2001). 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 39were 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).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1995) 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., 2001
). 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., 1992
). 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, 1982; Finne et al., 1983
; Livingston et al., 1988
). The question as to whether higher degrees of polymerization would have been detectable when employing different hydrolysis and chromatographic conditions (Zhang et al., 1997
; Inoue et al., 2000
) 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 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., 1996
). This fucose substitution was not observed for PSA-carrying N-glycans of NCAM from the brains of young mice (Liedtke et al., 2001
). The terminal sialic acid monomers bound to the core glycans showed a slight preference for an
2-6-linkage and, to a smaller degree, an
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
2-3-linkage in glycans from young chicken and young mouse brain (Kudo et al., 1996
; Liedtke et al., 2001
). 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)
and Liedtke et al. (2001)
. 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., 1996
).
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., 1992; Horstkorte et al., 1993
; Schmitz et al., 1993
; Heiland et al., 1998
). 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., 1990b
; Horstkorte et al., 1993
; Schmitz et al., 1993
; Porwoll et al., 1998
; Helenius and Aebi, 2001
). 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., 1996; Liedtke et al., 2001
) 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., 1983; Rutishauser et al., 1985
; Doherty et al., 1990
). 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., 1984
; Schachner and Martini, 1995
; Voshol et al., 1996
). 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., 1999
), 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, 1991
). Three different enzymes can attach PSA to NCAM, the polysialyltransferases ST8SiaII/STX (Kitagawa and Paulson, 1993
; Kojima et al., 1996
), ST8SiaIV/PST (Eckhardt et al., 1995
; Nakayama et al., 1995
)which both show a different spatial and temporal expression (Angata et al., 1998
; Seidenfaden et al., 2000
; Kitazume-Kawaguchi et al., 2001
)and ST8SiaIII (Angata et al., 2000
). Although different studies were done looking for an acceptor for the polysialyltransferases ST8SiaII, ST8SiaIV and ST8SiaIII (Angata et al., 2000
) 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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), dried in vacuo, and stored at 20°C.
Endo N was purified from E. coli bacteriophage PK1E (Gerardy-Schahn et al., 1995). Recombinant (E. coli) PNGase F from Flavobacterium meningosepticum and recombinant NDV sialidase were purchased from Boehringer Mannheim (Mannheim, Germany). Almond meal
-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 TrisHCl (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 310 (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 TrisHCl, 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% SDSPAGE. Proteins were detected by silver staining according to the method described by Heukeshoven and Dernick (1988).
Gradient gel electrophoresis was carried out in a 48% 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 acidfree) 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 peroxidaseconjugated 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 TrisHCl 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., 1990). A linear gradient (060 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 ( = 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 525 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
SDSPAGE gels (80 x 80 x 0.75 mm) were prepared as described previously (Küster et al., 1997). 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., 1997
).
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). 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 25 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., 1998) (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 1824 h. Complete digestion was confirmed by SDSPAGE (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 Ntreated 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., 1995).
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 -fucosidase (1 U/ml); (4) bovine testes ß-galactosidase, bovine epididymis
-fucosidase, and almond meal
-fucosidase (1 mU/ml); (5) S. pneumoniae ß-galactosidase, bovine epididymis
-fucosidase, and almond meal
-fucosidase; (6) bovine testes ß-galactosidase, bovine epididymis
-fucosidase, almond meal
-fucosidase, and S. pneumoniae N-acetyl-ß-D-glucosaminidase (6 mU/ml); (7) bovine testes ß-galactosidase, bovine epididymis
-fucosidase, almond meal
-fucosidase, and jackbean ß-N-acetylhexosaminidase (6 U/ml); and (8) bovine testes ß-galactosidase, bovine epididymis
-fucosidase, almond meal
-fucosidase, jackbean ß-N-acetylhexosaminidase, and jackbean
-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., 1998
) and incubated overnight at 37°C in parallel. After incubation, the enzyme activity was terminated by heating at 100°C for 12 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): 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 05% 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): 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 2058% A over 152 min followed by 58100% 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., 1997) (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, 1993
). 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 [MH] or [M+Na2H] ions in the negative ion spectra; all other spectra were acquired in positive ion mode where the glycans gave [M+Na]+ ions.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
2 Present address: Mermaid Pharmaceuticals, Falkenreid 88, D-20251 Hamburg, Germany
3 Present address: Neuroscience Research Centre, Merck, Sharp and Dohme, Terlings Park, Harlow, Essex CM20 2QR, UK
4 To whom correspondence should be addressed
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Angata, K., Suzuki, M., and Fukuda, M. (1998) Differential and cooperative polysialylation of the neural cell adhesion molecule by two polysialyltransferases, PST and STX. J. Biol. Chem., 273, 2852428532.
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, 1859418601.
Becker, C.G., Artola, A., Gerardy-Schahn, R., Becker, T., Welzl, H., and Schachner, M. (1996) The polysialic acid modification of the neural cell adhesion molecule is involved in spatial learning and hippocampal long-term potentiation. J. Neurosci. Res., 45, 143152.[CrossRef][ISI][Medline]
Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., and Parekh, R.B. (1995) Nonselective and efficient fluorescent labeling of glycans using 2-aminobenzamide and anthranilic acid. Anal. Biochem., 230, 229238.[CrossRef][ISI][Medline]
Bitter-Suermann, D. and Roth, J. (1987) Monoclonal antibodies to polysialic acid reveal epitope sharing between invasive pathogenic bacteria, differentiating cells and tumor cells. Immunol. Res., 6, 225237.[ISI][Medline]
Chen, P., Baker, AG., and Novotny, M.V. (1997) The use of osazones as matrices for the matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates. Anal. Biochem., 244, 144151.[CrossRef][ISI][Medline]
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, 691703.[Abstract]
Doherty, P., Cohen, J., and Walsh, F.S. (1990) Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid. Neuron, 5, 209219.[ISI][Medline]
Eckhardt, M., Muhlenhoff, M., Bethe, A., Koopman, J., Frosch, M., and Gerardy-Schahn R. (1995) Molecular characterization of eukaryotic polysialyltransferase-1. Nature, 373, 715718.[CrossRef][ISI][Medline]
Eckhardt, M., Bukalo, O., Chazal, G., Wang, L., Goridis, C., Schachner, M., Gerardy-Schahn, R., Cremer, H., and Dityatev, A. (2000) Mice deficient in the polysialyltransferase ST8SiaIV/PST-1 allow discrimination of the roles of neural cell adhesion molecule protein and polysialic acid in neural development and synaptic plasticity. J. Neurosci., 20, 52345244.
Edelman, G.M. and Crossin, K.L. (1991) Cell adhesion molecules: implications for a molecular histology. Annu. Rev. Biochem., 60, 155190.[CrossRef][ISI][Medline]
Finne, J. (1982) Occurrence of unique polysialosyl carbohydrate units in glycoproteins of developing brain. J. Biol. Chem., 257, 1196611970.
Finne, J. and Mäkelä, P.H. (1985) Cleavage of the polysialosyl units of brain glycoproteins by a bacteriophage endosialidase. Involvement of a long oligosaccharide segment in molecular interactions of polysialic acid. J. Biol. Chem., 260, 12651270.
Finne, J., Finne, U., Deagostini-Bazin, H., and Goridis, C. (1983) Occurrence of alpha 2-8 linked polysialosyl units in a neural cell adhesion molecule. Biochem. Biophys. Res. Commun., 112, 482487.[ISI][Medline]
Frosch, M., Gorgen, I., Boulnois, G.J., Timmis, K.N., and Bitter-Suermann, D. (1985) NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an IgG antibody to the polysaccharide capsule of E. coli K1 and group B meningiococci. Proc. Natl. Acad. Sci. USA, 1982, 11941198.
Garcia-Segura, L.M., Canas, B., Parducz, A., Rougon, G., Theodosis, D., Naftolin, F., and Torres-Aleman, I. (1995) Estradiol promotion of changes in the morphology of astroglia growing in culture depends on the expression of polysialic acid of neural membranes. Glia, 13, 209216.[ISI][Medline]
Gerardy-Schahn, R. and Eckhardt, M. (1994) Hot spots of antigenicity in the neural cell adhesion molecule NCAM. Int. J. Cancer Suppl., 8, 3842.
Gerardy-Schahn, R., Bethe, A., Brennecke, T., Muhlenhoff, M., Eckhardt, M., Ziesing, S., Lottspeich, F., and Frosch, M. (1995) Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE. Mol. Microbiol., 16, 441450.[ISI][Medline]
Griffith, L.S., Schmitz, B., and Schachner, M. (1992) L2/HNK-1 carbohydrate and proteinprotein interactions mediate the homophilic binding of the neural adhesion molecule P0. J. Neurosci. Res., 33, 639648.[ISI][Medline]
Guile, G.R., Wong, S.Y.C., and Dwek, R.A. (1994) Analytical and preparative separation of anionic oligosaccharides by weak anion exchange high performance liquid chromatography on an inert polymer column. Anal. Biochem., 222, 231235.[CrossRef][ISI][Medline]
Guile, G.R., Rudd, P.M., Wing, D.R., Prime, S.B., and Dwek, R.A. (1996) A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem., 240, 210226.[CrossRef][ISI][Medline]
Hallenbeck, P.C., Vimr, E.R., Yu, F., Bassler, B. and Troy, F.A. (1987) Purification and properties of a bacteriophage-induced endo-N-acetylneuraminidase specific for poly-alpha-2,8-sialosyl carbohydrate units. J. Biol. Chem., 262, 35533561.
Harvey, D.J. (1993) Quantitative aspects of the matrix-assisted laser desorption mass spectrometry of complex oligosaccharides. Rapid Commun. Mass Spectrom., 7, 614619.[ISI][Medline]
Heiland, P.C., Griffith, L.S., Lange, R., Schachner, M., Hertlein, B., Traub, O., and Schmitz, B. (1998) Tyrosine and serine phosphorylation of the neural cell adhesion molecule L1 is implicated in its oligomannosidic glycan dependent association with NCAM and neurite outgrowth. Eur. J. Cell Biol., 75, 97106.[ISI][Medline]
Helenius, A. and Aebi, M. (2001) Intracellular functions of N-linked glycans. Science, 291, 23642369.
Hellerqvist, C.G. (1990) Linkage analysis using Lindberg method. Methods Enzymol., 193, 554573.[Medline]
Heukeshoven, J. and Dernick, R. (1988) Improved silver staining procedure for fast staining in Plast System Development Unit. Staining for sodium dodecyl sulfate gels. Electrophoresis, 9, 2832.[ISI][Medline]
Horstkorte, R., Schachner, M., Magyar, J.P., Vorherr, T., and Schmitz, B. (1993) The fourth immunoglobulin-like domain of NCAM contains a carbohydrate recognition domain for oligomannosidic glycans implicated in association with L1 and neurite outgrowth. J. Cell Biol., 121, 14091421.[Abstract]
Inoue, S. and Iwasaki, M. (1978) Isolation of a novel glycoprotein from the eggs of rainbow trout: occurrence of disialosyl groups on all carbohydrate chains. Biochem. Biophys. Res. Commun., 83, 10181023.[ISI][Medline]
Inoue, S. and Iwasaki, M. (1980) Characterization of a new type of glycoprotein saccharides containing polysialosyl sequence. Biochem. Biophys. Res. Commun., 93, 162165.[ISI][Medline]
Inoue, S., Lin, S.L., and Inoue, Y. (2000) Chemical analysis of the developmental pattern of polysialylation in chicken brain. Expression of only an extended form of polysialyl chains during embryogenesis and the presence of disialyl residues in both embryonic and adult chicken brains. J. Biol. Chem., 275, 2996829979.
Iwasaki, M., Inoue, S., and Troy, F.A. (1990) A new sialic acid analogue, 9-O-acetyl-deaminated neuraminic acid, and alpha-2,8 linked O-acetylated poly(N-glycolylneuraminyl) chains in a novel polysialoglycoprotein from salmon eggs. J. Biol. Chem., 265, 25962602.
James, W.M. and Agnew, W.S. (1989) Alpha-(28)-polysialic acid immunoreactivity in voltage sensitive sodium channel of eel electric organ. Proc. R. Soc. Lond. B Biol. Sci., 237, 233245.
Joliot, A.H., Triller, A., Volovitch, M., Pernelle, C., and Prochiantz, A. (1991) Alpha-2,8-polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide. New Biol., 3, 11211134.[ISI][Medline]
Kabat, E.A., Liao, J., Osserman, E.F., Gamian, A., Michon, F., and Jennings, H.J. (1988) The epitope associated with the binding of the capsular polysaccharide of the group B meningococcus and of Escherichia coli K1 to a human monoclonal macroglobulin, IgMNOV. J. Exp. Med., 168, 699711.[Abstract]
Kadmon, G., Kowitz, A., Altevogt, P., and Schachner, M. (1990a) The neural cell adhesion molecule N-CAM enhances L1-dependent cellcell interactions. J. Cell Biol., 110, 193208.[Abstract]
Kadmon, G., Kowitz, A., Altevogt, P., and Schachner, M. (1990b) Functional cooperation between the neural adhesion molecules L1 and N-CAM is carbohydrate dependent. J. Cell Biol., 110, 209218.[Abstract]
Kiselyov, V.V., Berezin, V., Maar, T.E., Soroka, V., Edvardsen, K., Schousboe, A., and Bock, E. (1997) The first immunoglobulin-like neural cell adhesion molecule (NCAM) domain is involved in double-reciprocal interaction with the second immunoglobulin-like NCAM domain and in heparin binding. J. Biol. Chem., 272, 1012510134.
Kitagawa, H. and Paulson, J.C. (1993) Cloning and expression of human Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase. Biochem. Biophys. Res. Commun., 194, 375382.[CrossRef][ISI][Medline]
Kitazume-Kawaguchi, S., Kabata, S., and Arita, M. (2001) Differential biosynthesis of polysialic or disialic acid structure by ST8Sia II and ST8Sia IV. J. Biol. Chem., 276, 1569615703.
Kojima, N., Tachida, Y., Yoshida, Y., and Tsuji, S. (1996) Characterization of mouse ST8Sia II (STX) as a neural cell adhesion molecule-specific polysialic acid synthase. Requirement of core alpha1,6-linked fucose and a polypeptide chain for polysialylation. J. Biol. Chem., 271, 1945719463.
Kruse, J., Mailhammer, R., Wernecke, H., Faissner, A., Sommer, I., Goridis, C., and Schachner, M. (1984) Neural cell adhesion molecules and myelin-associated glycoprotein share a common carbohydrate moiety recognized by monoclonal antibodies L2 and HNK-1. Nature, 311, 153155.[ISI][Medline]
Kudo, M., Kitajima, K., Inoue, S., Shiokawa, K., Morris, H.R., Dell, A., and Inoue, Y. (1996) Characterization of the major core structures of the alpha 28-linked polysialic acid-containing glycan chains present in neural cell adhesion molecule in embryonic chick brains. J. Biol. Chem., 271, 3266732677.
Küster, B., Wheeler, S.F., Hunter, A.P., Dwek, R.A., and Harvey, D.J. (1997) Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high performance liquid chromatography. Anal. Biochem., 250, 82101.[CrossRef][ISI][Medline]
Liedtke, S., Geyer, H., Wuhrer, M., Geyer, R., Frank, G., Gerardy-Schahn, R., Zähringer, U., and Schachner, M. (2001) Characterization of N-glycans from mouse brain neural cell adhesion molecule. Glycobiology, 11, 373384.
Livingston, B.D., Jacobs, J.L., Glick, M.C., and Troy, F.A. (1988) Extended polysialic acid chains (n greater than 55) in glycoproteins from human neuroblastoma cells. J. Biol. Chem., 263, 94439448.
Michon, F., Brisson, J.R., and Jennings, H.J. (1987) Conformational differences between linear alpha (2-8)-linked homosialooligosaccharides and the epitope of the group B meningococcal polysaccharide. Biochemistry, 26, 83998405.[ISI][Medline]
Muller, D., Stoppini, L., Wang, C., and Kiss, J.Z. (1994) A role for polysialylated neural cell adhesion molecule in lesion-induced sprouting in hippocampal organotypic cultures. Neuroscience, 61, 441445.[CrossRef][ISI][Medline]
Muller, D., Wang, C., Skibo, G., Toni, N., Cremer, H., Calaora, V., Rougon, G., and Kiss, J. Z. (1996) PSA-NCAM is required for activity-induced synaptic plasticity. Neuron, 17, 413422.[ISI][Medline]
Muller, D., Djebbara-Hannas, Z., Jourdain, P., Vutskits, L., Durbec, P., Rougon, G., and Kiss, J.Z. (2000) Brain-derived neurotrophic factor restores long-term potentiation in polysialic acid-neural cell adhesion molecule-deficient hippocampus. Proc. Natl. Acad. Sci. USA, 97, 43154320.
Nakayama, J., Fukuda, M.N., Fredette, B., Ranscht, B., and Fukuda, M. (1995) Expression cloning of a human polysialyltransferase that forms the polysialylated neural cell adhesion molecule present in embryonic brain. Proc. Natl. Acad. Sci. USA, 92, 70317035.[Abstract]
Nelson, R.W., Bates, P.A., and Rutishauser, U. (1995) Protein determinants for specific polysialylation of the neural cell adhesion molecule. J. Biol. Chem., 270, 1717117179.
Nomoto, H., Iwasaki, M., Endo, T., Inoue, S., Inoue, Y., and Matsumura, G. (1982) Structures of carbohydrate units isolated from trout egg polysialoglycoproteins: short-cored units with oligosialosyl groups. Arch. Biochem. Biophys., 218, 335341.[ISI][Medline]
Packer, N.H., Lawson, M.A., Jardine, D.R., and Redmond, J.W. (1998) A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj. J., 15, 737747.[CrossRef][ISI][Medline]
Pfeiffer, G., Geyer, H., Geyer, R., Kalsner, I., and Wendorf, P. (1990) Separation of glycoprotein-N-glycans by high-pH anion-exchange chromatography. Biomed. Chromatogr., 4, 193199.[ISI][Medline]
Porwoll, S., Loch, N., Kannicht, C., Nuck, R., Grunow, D., Reutter, W., and Tauber, R. (1998) Cell surface glycoproteins undergo postbiosynthetic modification of their N-glycans by stepwise demannosylation. J. Biol. Chem., 273, 10751085.
Powell, A.K. and Harvey, D.J. (1996) Stabilization of sialic acids in N-linked oligosaccharides and gangliosides for analysis by positive ion matrix-assisted laser desorption-ionization mass spectrometry. Rapid Commun. Mass Spectrom., 10, 10271032.[CrossRef][ISI][Medline]
Rao, Y., Wu, X.F., Gariepy, J., Rutishauser, U., and Siu, C.H. (1992) Identification of a peptide sequence involved in homophilic binding in the neural cell adhesion molecule NCAM. J. Cell Biol., 118, 937949.[Abstract]
Rao, Y., Zhao, X., and Siu, C.H. (1994) Mechanism of homophilic binding mediated by the neural cell adhesion molecule NCAM. Evidence for isologous interaction. J. Biol. Chem., 269, 2754027548.
Reyes, A.A., Akeson, R., Brezina, L., and Cole, G.J. (1990) Structural requirements for neural cell adhesion molecule-heparin interaction. Cell Regul., 1, 567576.[ISI][Medline]
Rousselot, P., Lois, C., and Alvarez-Buylla, A. (1995) Embryonic (PSA) N-CAM reveals chains of migrating neuroblasts between the lateral ventricle and the olfactory bulb of adult mice. J. Comp. Neurol., 351, 5161.[ISI][Medline]
Rudd, P.M., Endo, T., Colominas, C., Groth, D., Wheeler, S.F., Harvey, D.J., Wormald, M. R., Serban, H., Prusiner, S.B., Kobata, A., and Dwek, R.A. (1999) Glycosylation differences between the normal and pathogenic prion protein isoforms. Proc. Natl. Acad. Sci. USA, 96, 1304413049.
Rutishauser, U., Watanabe, M., Silver, J., Troy, F.A., and Vimr, E.R. (1985) Specific alteration of NCAM-mediated cell adhesion by an endoneuraminidase. J. Cell Biol., 101, 18421849.[Abstract]
Sadoul, R., Hirn, M., Deagostini-Bazin, H., Rougon, G., and Goridis, C. (1983) Adult and embryonic mouse neural cell adhesion molecules have different binding properties. Nature, 304, 347349.[ISI][Medline]
Schachner, M. and Martini, R. (1995) Glycans and the modulation of neural-recognition molecule function. Trends Neurosci., 18, 183191.[CrossRef][ISI][Medline]
Schmitz, B., Peter-Katalinic, J., Egge, H., and Schachner, M. (1993) Monoclonal antibodies raised against membrane glycoproteins from mouse brain recognize N-linked oligomannosidic glycans. Glycobiology, 3, 609617.[Abstract]
Seidenfaden, R., Gerardy-Schahn, R., and Hildebrandt, H. (2000) Control of NCAM polysialylation by the differential expression of polysialyltransferases ST8SiaII and ST8SiaIV. Eur. J. Cell Biol., 79, 680688.[ISI][Medline]
Seki, T. and Arai, Y. (1993) Distribution and possible roles of the highly polysialylated neural cell adhesion molecule (NCAM-H) in the developing and adult central nervous system. Neurosci. Res., 17, 265290.[CrossRef][ISI][Medline]
Storms, S.D. and Rutishauser, U. (1998) A role for polysialic acid in neural cell adhesion molecule heterophilic binding to proteoglycans. J. Biol. Chem., 273, 2712427129.
Storms, S.D., Kim, A.C., Tran, B.H., Cole, G.J., and Murray, B.A. (1996) NCAM-mediated adhesion of transfected cells to agrin. Cell Adhes. Commun., 3, 497509.[ISI][Medline]
Streit, A., Yuen, C.T., Loveless, R.W., Lawson, A.M., Finne, J., Schmitz, B., Feizi, T., and Stern, C.D. (1996) The Le(x) carbohydrate sequence is recognized by antibody to L5, a functional antigen in early neural development. J. Neurochem., 66, 834844.[ISI][Medline]
Troy, F.A. and McCloskey, M.A. (1979) Role of a membranous sialyltransferase complex in the synthesis of surface polymers containing polysialic acid in Escherichia coli. Temperature-induced alteration in the assembly process. J. Biol. Chem., 254, 73777387.[Abstract]
Vimr, E.R., McCoy, R.D., Vollger, H.F., Wilkison, N.C., and Troy, F.A. (1984) Use of prokaryotic-derived probes to identify poly(sialic acid) in neonatal neuronal membranes. Proc. Natl. Acad. Sci. USA, 81, 19711975.[Abstract]
Voshol, H., van Zuylen, C.W., Orberger, G., Vliegenthart, J.F., and Schachner M. (1996) Structure of the HNK-1 carbohydrate epitope on bovine peripheral myelin glycoprotein P0. J. Biol. Chem., 271, 2295722960.
Vutskits, L., Djebbara-Hannas, Z., Zhang, H., Paccaud, J.P., Durbec, P., Rougon, G., Muller, D., and Kiss, J.Z. (2001) PSA-NCAM modulates BDNF-dependent survival and differentiation of cortical neurons. Eur. J. Neurosci., 13, 13911402.[CrossRef][ISI][Medline]
Wheeler, S.F. and Harvey, D.J. (2001) Extension of the in-gel release method for structural analysis of neutral and sialylated N-linked glycans to the analysis of sulfated glycans. Anal. Biochem., 296, 92100.[CrossRef][ISI][Medline]
Zamze, S., Harvey, D.J., Chen, Y.-J., Guile, G.R., Dwek, R.A., and Wing, D.R. (1998) Sialylated N-glycans in adult rat brain tissue: a widespread distribution of disialylated antennae in complex and hybrid structures. Eur. J. Biochem., 258, 243270.[Abstract]
Zamze, S., Harvey, D.J., Pesheva, P., Mattu, T.S., Schachner, M., Dwek, R.A., and Wing, D.R. (1999) Glycosylation of a CNS-specific extracellular matrix glycoprotein, tenascin-R, is dominated by O-linked sialylated glycans and "brain-type" neutral N-glycans. Glycobiology, 9, 823831.
Zhang, Y., Inoue, Y., Inoue, S., and Lee, Y.C. (1997) Separation of oligo/polymers of 5-N-acetylneuraminic acid, 5-N-glycolylneuraminic acid, and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid by high-performance anion-exchange chromatography with pulsed amperometric detector. Anal. Biochem., 250, 245251.[CrossRef][ISI][Medline]
Zuber, C., Lackie, P.M., Catterall, W.A., and Roth, J. (1992) Polysialic acid is associated with sodium channels and the neural cell adhesion molecule N-CAM in adult rat brain. J. Biol. Chem., 267, 99659971.