3Zentrum für Molekulare Neurobiologie, Universität Hamburg, D-20246 Hamburg, Germany, 4Institute of Biochemistry, University of Giessen, D-35392 Giessen, Germany, 5Institute for Molecular Biology and Biophysics, ETH Hönggerberg, CH-8093 Zürich, Switzerland, 6Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany, and 7Division of Immunochemistry, Research Center Borstel, D-23845 Borstel, Germany
Received on October 11, 2000; revised on January 3, 2001; accepted on January 4, 2001.
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Abstract |
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Key words: HNK1-epitope/NCAM/N-glycans/polysialic acid
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Introduction |
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In addition to polysialylated glycans, NCAM is known to carry the L2/HNK1-carbohydrate epitope (Kruse et al., 1984) consisting of a 3'-sulfated glucuronic acid attached to C3 of a N-acetyllactosamine unit (Chou et al., 1986
; Ariga et al., 1987
; Voshol et al., 1996
). The HNK1-carbohydrate has been recognized as an important mediator of molecular recognition in normal development of the nervous system. It is expressed by recognition molecules of the immunoglobulin (Ig) superfamily, members of the tenascin family, integrins, proteoglycans (Schachner and Martini, 1995
), as well as acidic glycolipids of the peripheral nervous system (Chou et al., 1986
; Ariga et al., 1987
). It binds to certain isoforms of laminin and to P and L selectins (Hall et al., 1993
; Needham and Schnaar, 1993
). The HNK1-epitope displays a high degree of phylogenetic conservation because it occurs not only in vertebrates but also in glycoproteins and glycolipids of insects (Dennis et al., 1988
). Its expression appears to be associated with developmental events involving the movement of cells and extensions of their processes. For example, there is a correlation of the presence of the HNK1-carbohydrate epitope with the migration of chick embryo cells, such as epiblasts (Canning and Stern, 1988
) and neural crest cells (Vincent et al., 1983
; Rickmann et al., 1985
; Bonner-Fraser, 1987
; Loring and Erickson, 1987
). Expression of HNK1 is also spatially and temporally regulated during other aspects of neural development (Schwarting et al., 1987
; Yoshihara et al., 1991
) as well as during outgrowth of astrocytic and neuronal processes and glial cell migration (Künemund et al., 1988
; Martini et al., 1992
). Furthermore, there is evidence that this carbohydrate is involved in neuron to glial cell adhesion (Keilhauer et al., 1985
).
Because of the functional importance of NCAM carbohydrates, a precise knowledge of the structures, location on the protein backbone, and the regulation of their biosynthesis is clearly mandatory. Studies performed by Finne (1982) provided early evidence for the presence of fucosylated tri- and tetraantennary core structures of the PSA glycan chains from NCAM of fetal rat brain. Later, additional sulfate groups, type 1 (Galß3GlcNAc) and type 2 (Galß4GlcNAc) antennae and lactosamine repeating units were demonstrated to occur in the polysialylated oligosaccharides from NCAM of embryonic chick brains (Kudo et al., 1996
). In the case of chicken NCAM, it could be further verified that polysialylation occurs exclusively on N-glycan chains in the fifth Ig-like domain, where two of the three potential N-glycosylation sites have been shown to be polysialylated (Nelson et al., 1995
). In the present study, we have initiated the characterization of the entire pattern of N-glycans obtained from newborn mouse NCAM. In this context, particular emphasis is laid on the specific location and the characteristic structural features of the different classes of carbohydrate chains present to achieve more detailed information on the carbohydrate structure of this glycoprotein.
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Results |
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Characterization of PSA glycans
The different glycan fractions from murine NCAM were characterized by carbohydrate constituent and linkage analyses as well as MALDI-TOF mass measurement. For all glycan fractions obtained, neutral monosaccharide constituent analysis revealed the exclusive presence of Fuc, Man, Gal, and GlcNAc, whereas GalNAc could not be detected in any of these oligosaccharide pools. Hence, the presence of N-glycans with GalNAcß4GlcNAc (LacdiNAc) units could be excluded. For the characterization of the PSA chains, aliquots of the PSA glycopeptides were subjected to methylation analysis in combination with esterification and reduction of the carboxyl group of Neu5Ac, resulting in the corresponding partially methylated D-erythro-L-manno/gluco-nonitol acetates. Due to incomplete methylationprobably as a consequence of the bulk of neuraminic acid residues presentas well as the small amounts of material available, resulting partially methylated monosaccharide derivatives were only qualitatively characterized (Table II). The presence or absence of the respective alditol acetates, however, was clearly significant in all cases. The analysis of the PSA glycopeptides revealed the presence of terminal and 8-substituted Neu5Ac, terminal fucose, 3-substituted and small amounts of terminal galactose, 2-substituted as well as 2,4-, 2,6- and 3,6-disubstituted mannose residues in addition to 4-substituted N-acetylglucosamine. From the relative amounts of 2-substituted, 2,4-disubstituted, and 2,6-disubstituted mannosyl residues found, it is proposed that the respective oligosaccharides comprise fucosylated diantennary, triantennary, and tetraantennary complex-type core structures in a ratio approaching 10:45:45, respectively, the majority of which (about 90%) is substituted at C3 of the terminal Gal. The molar ratio of 2,4-disubstituted versus 2,6-disubstituted mannosyl residues was found to be 1:2. Therefore, it can be assumed that the core structures of triantennary PSA glycans represent predominantly the 2,6-branched type of isomers.
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Characterization of non-PSA/HNK1-glycans
Non-PSA/HNK1-(glyco)peptides obtained with the flow-through of both immunoaffinity columns (see Figure 2) were preparatively desialylated by mild acid treatment. Oligosaccharides were released by incubation with PNGase F, freed from remaining peptides by RP-HPLC, and characterized by MALDI-TOF-MS in the positive-ion reflectron mode (Figure 8). To increase sensitivity (Geyer et al., 1999), remaining glycans were converted into 2-aminopyridine(PA)-derivatives by reductive amination and, subsequently, subjected to MALDI-TOF-MS in the negative-ion reflectron mode. In parallel, resulting PA-glycans were examined by linkage analysis (Table II). The results demonstrated that non-PSA/HNK1-oligosaccharides represented mainly core-fucosylated diantennary species, the majority of which comprised truncated N-acetyllactosamine chains as well as additional bisecting GlcNAc and/or one to two sulfate residues (Table III). In addition, oligosaccharides with the compositions Hex5HexNAc4dHex2, Hex4HexNAc5dHex2, Hex4HexNAc5dHex2SO4, Hex4HexNAc5dHex3, Hex5HexNAc5dHex2, Hex4HexNAc6dHex1SO4, Hex5HexNAc6dHex1, Hex5HexNAc6dHex1SO4, and Hex5HexNAc6dHex2 were detected, reflecting partially sulfated structural variants with more than one fucosyl residue and/or minor amounts of triantennary carbohydrate chains (see Table III). Due to the presence of trace amounts of 2,4- and 2,6-disubstituted mannosyl residues (cf. Table II), the latter species are again assumed to represent both 2,4- and 2,6-branched isomers. Because all glycans were released by PNGase F treatment, difucosylation of the innermost GlcNAc residue can be excluded (Tretter et al., 1991
). Instead, the additional fucose residue(s) may be assumed to reside on either of the lactosamine antennae. From the presence of 3,4-disubstituted GlcNAc residues (see Table II), it is possible that this fucose is linked to C3 of GlcNAc, thus forming a Lewis X determinant. Similar to desialylated PSA-glycans, MALDI-TOF-MS of non-PSA/HNK1-PA-glycans in the negative-ion mode provided evidence for the presence of sulfated compounds (Table III). The composition of these prevalent structures suggests the presence of incomplete, mostly diantennary species, as detected in the positive-ion mode, which carry an additional sulfate group in an as yet undefined position. Trace amounts of 3-substituted Gal, however, suggests that some residues may be linked to the C3 of terminal galactose units.
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Discussion |
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Although the ratio of terminal and 8-substituted sialic acid derivatives observed after linkage analysis did not allow any conclusions on the length of the PSA chains, the results obtained clearly verified the polymeric nature of these substituents. Respective oligosaccharide core structures comprised predominantly 2,6-branched isomers of triantennary as well as tetraantennary complex-type N-glycans. Similar to NCAM from embryonic chick brains (Kudo et al., 1996), the carbohydrate chains were found to carry a 6-linked fucosyl residue attached to the innermost GlcNAc residue and, in part, an additional sulfate group in as yet undefined position(s). Due to the experimental strategy used, the presence of small amounts of glycans with N-acetyllactosamine repeats could not be ruled out. In contrast to the above study, however, roughly 10% of the formerly polysialylated glycans were found to be of the diantennary type, indicating that, at least in murine NCAM, 2,6-branching of the (
1-6)-linked mannosyl residues is obviously not an absolute requirement for polysialylation, which is in agreement with data reported by Angata and coworkers (Angata et al., 1998
). In addition, there is evidence that a minor proportion of these glycans carried bisecting GlcNAc residues. Carbohydrate substituents carrying an HNK1 epitope could be assigned to the N-glycosylation sites 5 and 6, similar to polysialylated glycans, as well as to site 2 and, in minor proportion, also to site 3 of the molecule. This result is in partial agreement with an earlier study on chicken NCAM in which the HNK1 epitope has been assigned to glycosylation site 1 or 2 (Cole and Schachner, 1987
). The present study on murine NCAM extends this information insofar as glycans bearing this epitope could be also allocated to sites 5 and 6. HNK1 glycopeptides with amino acid sequences comprising the first and the fourth potential N-glycosylation of NCAM could not be detected in the present study. Hence, although displaying a higher degree of spreading, the presence of glycans with a HNK1 epitope is again restricted to distinct sites of the molecule. From these results, it might be concluded that the enzyme activities involved in polysialylation and HNK1 biosynthesis depend on different structural parameters of the glycoprotein.
HNK1 glycans represented predominantly fucosylated, partially truncated diantennary species carrying, in part, a bisecting GlcNAc residue. In this context, it is interesting to note that one of the two major compounds (Hex4HexNAc5dHex1HexASO4) possessed the same molecular composition as well as similar structural features, that is, core fucosylation, bisecting GlcNAc, and one truncated antenna, as a predominant HNK1-bearing glycan from bovine peripheral myelin glycoprotein P0 (Voshol et al., 1996). Although the precise assignment of the HNK1 epitope to a distinct antenna has not been possible in this study, the structural analogy of the glycan found in murine NCAM with the published carbohydrate is clearly evident. A corresponding set of variants of triantennary species as well as hybrid-type glycans were only found as minor components. In contrast to PSA glycans, triantennary oligosaccharides were found to comprise both 2,4- and 2,6-branched isomers. Furthermore, tetraantennary species representing major compounds in the case of polysialylated sugar chains could not be detected in the HNK1glycan fraction, thus displaying a separate, highly characteristic structural profile.
Due to limited amounts of material, residual non-PSA/HNK1-glycans could not be assigned to distinct N-glycosylation sites. Therefore, the question remains open as to whether the first or the fourth potential N-glycosylation sites of murine NCAM are actually used for carbohydrate substitution. Structurally, these glycans represented a highly heterogeneous population of mostly diantennary species that were individually modified by partial truncation and, in particular, by the presence of additional bisecting GlcNAc as well as deoxyhexose residues and/or one to two sulfate groups. Similar to oligosaccharides comprising the HNK1 epitope, triantennary or tetraantennary species were only present in small amounts or not detectable, respectively, underlining again the different structural properties of the three classes of N-glycans present in NCAM from newborn mouse brain.
The present study was designed to characterize and to assign the entire panel of N-glycans to individual glycosylation sites of NCAM. Carbohydrate constituent analyses (data not shown) of the peptides obtained from the non-PSA/HNK1-glycopeptide fraction after PNGase F treatment also revealed the presence of GalNAc, which might be indicative for the simultaneous occurrence of O-linked glycans in murine NCAM. The question as to the precise structure(s) and localization of the respective O-glycan(s) was, however, not addressed in this study.
NCAM plays a fundamental role during the development of the nervous system by mediating intercellular recognition and adhesion (see Introduction). The modulation of its biological activities is usually discussed in conjunction with its degree of polysialylation. In agreement with studies on the polysialylated N-glycans of NCAM from chicken embryos (Kudo et al., 1996) our study demonstrates that NCAM glycans contribute negative charges not only due to their polysialic acid substituents but also by sulfate groups, which have been demonstrated to occur in all classes of N-glycans characterized. Hence, the degree in sulfation may further subtly modulate NCAM bioactivity and may also be subject to developmental regulation. To our knowledge, however, systematic investigations concerning the degree of expression of the HNK1 epitope and/or the sulfation of NCAM N-glycans during development have not yet been performed.
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Materials and methods |
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Isolation of NCAM
Brains of 2- to 3-day-old mice (60 g; stored at 80°C) were thawed and immediately homogenized (20 min; Ultra-Turrax, Janke & Kunkel, Staufen, Germany) on ice in 60 ml buffer containing 30 mM sodium phosphate (pH 7.4), 0.5 mM PMSF, 0.1 mM DTE, and 1 mM EDTA. Soluble material was removed by centrifugation at 17,000 x g and 4°C for 90 min. The pellet was resuspended for solubilization in 300 ml buffer containing 20 mM sodium phosphate (pH 7.4), 150 mM NaCl, 0.5 mM PMSF, 0.1 mM DTE, 1 mM EDTA, and 1% (v/v) Triton X-100. The mixture was shaken overnight at 4°C and finally centrifugated at 100,000 x g and 4°C for 1 h. The supernatant was diluted six times with phosphate-buffered saline (PBS; 10 mM sodium phosphate, 150 mM NaCl, pH 7.4) and added in 300-ml aliquots to a column (1 x 20 cm) containing immobilized mAb H28. The column was washed with 30 column volumes of PBS with 1% Triton X-100, 10 column volumes of PBS containing 300 mM NaCl, and again with 10 column volumes of PBS. Bound NCAM was eluted with buffer containing 100 mM diethylamine, pH 11.5, 150 mM NaCl, 2 mM EDTA, and 0.1% (v/v) sodium deoxycholate. The eluted material was quickly neutralized by addition of 500 mM TrisHCl buffer, pH 6.8, concentrated by ultrafiltration (YM100 membrane, Millipore, Eschborn, Germany) and desalted on a PD-10 column (1 x 20 cm; BioRad, Munich, Germany).
SDSPAGE
Analytical SDSPAGE of NCAM was carried out according to Laemmli (1970) in slab gels containing 7.5% polyacrylamide. Proteins were detected by silver staining. For immunostaining, proteins were blotted onto nitrocellulose. NCAM, PSA, and the HNK1 epitope were detected with mAb H28, mAb 735, and mAb 412, respectively.
Treatment of NCAM with PNGase F and endo N
Deglycosylation of NCAM using PNGase F from Flavobacterium meningosepticum (Oxford GlycoSciences, Abingdon Oxfordshire, UK) was performed in 20 mM sodium phosphate buffer, pH 7.2 at 37°C for 24 h (2 mU PNGase F per 10 µg of NCAM). The same conditions were employed for treatment of NCAM with endo-N (0.2 µg endo-N per 10 µg of NCAM) as well as in control experiments in which protein was similarly incubated without enzyme.
Isolation of tryptic glycopeptides
NCAM was carboxymethylated and digested with trypsin (25 µg of trypsin per mg of NCAM; sequencing grade; Sigma, Deisenhofen, Germany) at 37°C for 24 h. For fractionation, the sample was first applied to an immunoaffinity column (0.5 x 10 cm) containing mAb 735 and, subsequently, the flow-through to a column (1 x 10 cm) containing mAb 412. Both columns were washed with five column volumes of PBS containing 0.5% (v/v) Triton X-100 and five column volumes of PBS. Bound material was eluted with buffer containing 100 mM diethylamine, pH 11.5, 150 mM NaCl, and 2 mM EDTA. Eluated glycopeptides were neutralized by addition of 500 mM TrisHCl buffer, pH 6.8, and desalted on a Bio-Gel P30-column (1.5 x 20 cm; BioRad) with 50 mM NH4HCO3. Fractions were monitored by absorption at 206 nm and tested for carbohydrates by orcinol/H2SO4 staining. Positive fractions were pooled and lyophilized.
Isolation of oligosaccharides and peptide moieties
Glycans were released from the different glycopeptide fractions by treatment with PNGase F (20 mU of enzyme at 37°C for 24 h; Roche, Mannheim, Germany) and separated from residual peptides by RP-HPLC using a column filled with ODS-Hypersil C18 (2.1 x 250 mm; 3 µm; Shandon, UK). Elution was carried out at 120 µl/min with a linear gradient from 0.1% trifluoroacetic acid to 60% acetonitrile containing 0.1% trifluoroacetic acid in 45 min at 30°C. Peptides were monitored by absorption at 220 nm; oligosaccharides were detected by orcinol/H2SO4 staining. Fractions of 60120 µl were collected, pooled, and immediately evaporated to dryness. Pooled glycan fractions were desalted using Bio-Gel P2 (BioRad) as described earlier (Geyer and Geyer, 1993).
Desialylation of glycopeptides and oligosaccharides
Sialic acid residues were removed by mild acid hydrolysis in 500 µl of 1 M trifluoroacetic acid for 30 min at 80°C. After drying the sample in a SpeedVac concentrator, residual acid was removed by repeated addition of methanol and evaporation under vacuum (Geyer et al., 1992).
Derivatization of oligosaccharides
Oligosaccharides were pyridylaminated according to Kondo et al. (1990). Excess 2-aminopyridine and reaction byproducts were removed by gel filtration using a column of Fractogel HW-40F (Merck, Darmstadt, Germany) as decribed earlier (Lochnit and Geyer, 1995
). Reductive amination with 2-aminobenzamide was performed as described by Bigge et al. (1995)
using the SignalTM Labelling Kit (Oxford GlycoSciences).
Anion-exchange HPLC
Anion-exchange HPLC was performed at 25°C at a flow rate of 1 ml/min using a Mikropak AX-10 column (0.46 x 25 cm; Varian, Walnut Creek, CA). For elution, a linear gradient of 0300 mM potassium phosphate buffer, pH 4.4, was applied within 60 min (Liedtke et al., 1994).
Size-fractionation HPLC
Non-PSA/HNK1-PA-oligosaccharides were subjected to size-fractionation HPLC (Ohara et al., 1991) using a MN-Carbohydrate column (0.46 x 25 cm; Macherey & Nagel, Düren, Germany) at a flow rate of 1 ml/min. A linear gradient of 2550% (v/v) 200 mM acetic acid-triethylamine buffer pH 7.3 in acetonitrile in 60 min was used. PA-oligosaccharide samples were dissolved in 75% aqueous acetonitrile prior to injection and monitored using a fluorescence spectrophotometer (Lochnit and Geyer, 1995
).
Peptide sequencing
(Glyco)peptides were amino-terminally sequenced by automated Edman degradation using a Modular Sequencer (Knauer, Berlin, Germany) that had been modified to allow isocratic identification of the phenylhydantoin amino acid derivatives as described earlier (Frank, 1989; Strobl et al., 1997
).
Carbohydrate constituent and methylation analysis
Carbohydrate constituents were identified as alditol acetates as detailed elsewhere (Geyer et al., 1982). For linkage analysis, glycopeptides or oligosaccharides were permethylated (PazParente et al., 1985
) and hydrolyzed. Partially methylated alditol acetates obtained after reduction and acetylation were analyzed by capillary combined gas liquid chromatography/mass spectrometry (GLC-MS) using the instrumentation and microtechniques described earlier (Geyer and Geyer, 1994
). For determination of sialic acid linkages, PSA glycopeptides were first treated with 250 µl of 0.05 M HCl in methanol for 1 min at room temperature. Then 500 µl of diazomethane were added. After 12 h at ambient temperature, the sample was dried in a stream of nitrogen, permethylated (Kvernheim, 1987
), hydrolyzed, and reduced as above. Thereafter, the sample was again treated with 1 M HCl in methanol (5 min) and dried, and the carboxylester was reduced with sodium borohydride (see above). Following peracetylation, the sample was analysed by GLC-MS using a Hewlett-Packard Model 5989 instrument equipped with a HP-5MS capillary column (Hewlett-Packard, Waldbronn, Germany) using a temperature gradient of 150320°C at 5°C/min. Electron impact spectra were recorded at 70 keV, and chemical ionization spectra were obtained with ammonia as reactant gas.
MALDI-TOF-MS
Data were obtained 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- or negative-ion reflectron mode. Typically, obtained spectra result from the accumulation of 525 laser shots. Either 2,5-dihydroxybenzoic acid (10 mg/ml in 0.1% [v/v] aqueous trifluoroacetic acid, 30% [v/v] acetonitrile) or 6-aza-2-thiothymine (5 mg/ml in twice distilled water; Geyer et al., 1999
) were used as matrix. One microliter of analyte solution was mixed on the stainless steel target with 1 µl of matrix solution and allowed to air-dry. In the positive-ion mode, the instrument was calibrated with an external mixture of isomaltosyl oligosaccharides containing 515 glucose units (measurement of oligosaccharides) or, in the case of peptides, with human angiotensin and bovine insulin (both from Sigma). For calibration in the negative-ion mode, isomaltosyl oligosaccharide derivatives with anthranilic acid tags were prepared (Anumula, 1994
). Given mass values represent average masses. Overall mass accuracy was about 0.025%.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed at: Biochemisches Institut am Klinikum der Universität, Friedrichstrasse 24, D-35392 Giessen, Germany.
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References |
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