Department of Neurology and 2Department of Pathology, Medical University of South Carolina, Charleston SC, USA and 3School of Medicine, University of California San Diego, La Jolla CA, USA
Received on June 3, 1999; revised on July 19, 1999; accepted on July 21, 1999.
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Abstract |
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Key words: bovine brain/glycosphingolipid/immunohistochemistry/NMR spectroscopy/neurons
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Introduction |
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At present, the role of glycoconjugates in CNS development has not been fully delineated and this is particularly true of neutral glycosphingolipids (Ngsls). In recent years, we have been examining their potential role in CNS development. Several minor long chain (i.e., containing more than three glycosyl residues) Ngsls have been identified in brain by digoxigenin immunostaining (DIG-IS) after removal of galactocerebroside (Dasgupta et al., 1992). Three of them have been characterized as GA1 (Dasgupta et al., 1992
; Dasgupta and Hogan, 1993
), GalGbOse4Cer (Dasgupta et al., 1995b
), and FucnLcOse4Cer (Dasgupta et al., 1996
). The topography of GA1 and FucnLcOse4Cer has been examined immunohistochemically in rodent brain (Dasgupta et al., 1996
).
We now report on identification, purification and chemical characterization of another pair of minor brain Ngsls and their subsequent histochemical localization in adult rat brain. This report confirms and extends our preliminary data (Dasgupta et al., 1995a).
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Results |
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GalNAcß14Galß13GalNAcß14Galß14Glc.
It is worth mentioning that the oligosaccharide GgOse5 showed some peculiar 1H NMR features. The presence of NAc methyl signals with total intensity equivalent to six protons (see Figure 6) confirmed the presence of two GalNAc residues in the structure. One of the NAc signals (the singlet at 2.056 p.p.m., with intensity equivalent to three protons) was assigned to the GalNAc-V residue that is relatively remote from the anomeric center of the reducing Glc-I residue. Thus, the remaining two NAc signals (at 2.039 and 2.037 p.p.m.; combined intensity equivalent to three protons) both belong to the second GalNAc residue (GalNAc-III). This pair of singlets was observed in the anomeric intensity ratio (:ß
1:2), reflecting the position of the GalNAc-III residue relatively close in space to the reducing end of the oligosaccharide GgOse5. The H1 doublet of the internal GalNAc-III residue was observed doubled by the anomerization effect as well (
4.697 and 4.690, in ratio ~ 1:2). The aforementioned observations may prove useful in future conformational studies of GgOse5.
Immunohistochemical localization of GalNAc-GA1 using anti-GalNAc-GA1 antibody
Monospecific polyclonal anti-GalNAc-GA1 antibody (anti-rabbit IgG) was used to localize the novel Ngsl in adult rat brain parasagittal sections. The antibody stained neurons in cerebral cortex and Purkinje cells in cerebellum and spared the myelin (Figure 7a,b).
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Discussion |
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In order to exclude the possibility that the Ngsls A and B were generated by the removal of sialic acid from relevant gangliosides (such as GalNAc-GM1, GalNAc-GM1b, etc.) induced by the silicic acid column during the initial purification step, the brain Ngsls were purified from the total brain lipid extract by a DEAE-Sephadex column. The long-chain Ngsls fraction was then purified from other neutral lipids, phospholipids, and monoglycosylceramides by a silicic acid column. The purified Ngsl fraction was examined by ELISA using anti-GalNAc-GA1 antibody along with the purified Ngsl A/B and a Ngsl fraction prepared as described above (silicic acid chromatography followed by DEAE-Sephadex). An identical antibody titer (1/160) in both Ngsl preparations suggests that both Ngsls A and B are naturally occurring components of bovine brain.
Previously, gangliosides containing this GalNAc1-4Gal repeating unit, namely GalNAc-GM1, GalNAc-GM1b, GalNAc-GD1a, and GalNAc-GD1b, have been characterized as minor brain components. But, so far, their tissue and cell localization have not been determined because specific antibodies are unavailable. This stems from the difficulties of producing monoclonal antibodies against purified gangliosides (Ozawa et al., 1992; Kotani et al., 1994
) and the low immunogenicity of gangliosides in rodents (Kotani et al., 1994
). Ozawa et al. developed a method to prepare anti-ganglioside monoclonal antibodies in C3/HeN mice and studied ganglioside localization in adult rat brain (Kotani et al., 1994
), but no ganglioside containing terminal GalNAc was examined.
We prepared a monospecific polyclonal antibody (anti-IgG) to GalNAc-GA1 in rabbit. The substrate affinity purified anti-IgG was specific for GalNAc-GA1 when determined by enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography immunostaining (TLC-IS); our antibody did not react with a series of Gsls having close structural similarity to GalNAc-GA1 used as comparison (namely GA1, GM1, GalNAc-GM1, etc.).
In adult rat CNS, the anti-GalNAc-GA1 antibody stained neurons in cerebral cortex and Purkinje cells, as well as other neuronal perikarya in cerebellum, suggesting that GalNAc-GA1 is neuronal cell specific. This is in marked contrast to GA1 which has been localized specifically in myelin (Dasgupta et al., 1996). Our recent DIG-IS examination of Ngsls in whole brain, spinal cord, white matter and gray matter identifies GalNAc-GA1 in whole brain and gray matter but not in spinal cord or white matter (Dasgupta et al., unpublished observations). Its higher concentration in gray matter than in whole brain supports our observation and interpretation that GalNAc-GA1 is a neuron-associated Ngsl.
Several N-acetylgalactosaminyltransferases with specificity for glycoprotein and glycolipid substrates have been described previously (Basu et al., 1987), but none of them catalyzes transfer of GalNAc to GA1. The characterization of GalNAc-GA1 in brain suggests that a novel galactosaminyltransferase may exist exclusively in CNS. We are actively pursuing the characterization of this enzyme and are investigating the possibility of its developmental regulation associated with neuronal cell metabolism. It is noteworthy that brains of mice, engineered to lack the ß14 galactosaminyl transferases responsible for synthesis of GM2/GD2, are enriched in GM3/GD3 but deficient in complex gangliosides. The brain appeared to develop normally though examined by only simple criteria of histogenesis, nerve conduction velocity, and behavioral study suggesting a compensatory role for GM3/GD3 for complex gangliosides (Takamiya et al., 1996
). However, since the GalNAcT specific to the synthesis of GalNAc-GA1 appears to be a different enzyme (Hashimoto et al., 1993
; Kanzuya et al., 1994
), this interesting observation would not settle the question. At this point, we have shown that GalNAc-GA1, a minor brain Ngsl with a novel carbohydrate sequence, is localized in neuronal cells and may well be a mediator of neuronal development.
An antibody to GalNAc-GD1a has been reported to occur in the serum of patients with neuropathy associated with gammopathy (Ilyas et al., 1988) and in acute demyelinating inflammatory polyradiculopathy (Guillain-Barré disease) (Kusunoki et al., 1994
). Bovine brain gangliosides containing terminal GalNAc residue have been reported to be T-cell markers (Muthing et al., 1989
), antigens in human neuronal diseases (Kusunoki et al., 1994
), and putative receptors for human pathogenic bacteria (Krivan et al., 1988a
) which can be identified by TLC-IS using the mouse monoclonal antibody 2D4 (Muthing and Ziehr, 1990
; Krivan et al., 1988a
). Glycosphingolipids containing GalNAcß14Gal either as a terminal or internal epitope are binding receptors for P.aeruginosa and P.cepacia isolated from sputum and lung, respectively, of patients with cystic fibrosis (Krivan et al., 1988a
). The pulmonary pathogens Streptococcus pneumoniae and Klebsiella pneumoniae, and certain strains of Escherichia coli (VJI and 6883) specifically bind fucosylated-GM1, asialo-GM1 (GA1) and asialo-GM2 (GA2); all containing the GalNAcß14Gal moiety (Krivan et al., 1988a
), and with evidence that the minimal binding requirement of these bacteria is a terminal or internal GalNAcß14Gal sequence unsubstituted with a sialyl residue (Krivan et al., 1988b
). In this context, GalNAc-GA1 with a terminal and an internal consecutive repeating GalNAcß14Gal epitope might be a carbohydrate receptor for yet unidentified neuropathogens (microbial, viral, etc.) and is also a candidate antigen for inducing autoimmune pathogenic events and neurological disease. The specificity of the GalNAc-GA1 in neuronal development and disorder is now being examined in our laboratory.
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Materials and methods |
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Purification of neutral glycosphingolipids
Total Ngsl was purified from bovine brain as described previously (Dasgupta et al., 1996). In brief, the lipid extracted from acetone-dried powder was applied to a silicic acid column (1.2 cm x 30 cm), washed with chloroform:methanol 85:15 (v/v) to remove cerebroside and eluted with tetrahydrofuran:water 7:1 (v/v) (Dasgupta et al., 1994
). Ngsls and gangliosides were separated from the dialyzed eluate using a DEAE-Sephadex A50 (acetate form) column. Individual Ngsls were further fractionated on a silicic acid (1 cm x 40 cm) column using chloroform:methanol:water as the solvent system. Twenty microliter aliquots from alternate fractions (4 ml) were examined by TLC. The fractions containing novel Ngsls were further purified through another silicic acid column (0.8 cm x 30 cm) using chloroform:methanol: 2.5N ammonia as the eluting solvent. Fractions containing purified Ngsls were pooled, and the purified compounds were found homogeneous when reexamined by TLC using neutral and alkaline solvent systems.
Chemical composition analysis
The carbohydrate composition of Ngsl A and B was determined as alditol acetates (Bjorndal et al., 1967). Carbohydrate linkage positions were determined by methylation analysis by GLC-MS of permethylated alditol acetates (PMAAs). Approximately 50 µg of each Ngsl was permethylated (Gunnarson, 1987
) and acetylated and the resulting PMAAs were separated and analyzed on a DB-1 column by a Hewlett Packard 5890 series gas chromatograph attached to a 5972 mass spectrometer (Dasgupta et al., 1996
). The fatty acids and bases of Ngsl A and B were analyzed by GLC as methyl esters and trimethylsilyl derivatives, respectively (Dasgupta et al., 1994
).
Stepwise exoglycosidase digestion
Approximately 20 µg of each Ngsl was treated with ß-hexosaminidase in 50mM citrate buffer (pH 4.2) containing 0.05% sodium taurodeoxycholate (Dasgupta et al., 1996). The reaction was terminated with 4 volumes of chloroform:methanol (2:1, v/v) and the lower phase was examined by TLC. The product was further treated with ß13 galactosidase, ß-hexosaminidase and ß14 galactosidase, respectively (Dasgupta et al., 1994
).
Immunochemical and chemical composition analysis of the ß-hexosaminidase digested Ngsls
The products obtained from Ngsl A and B after enzymatic removal of the terminal hexosamine residue and the parent compounds were resolved on a TLC plate and overlaid with anti-GA1 monoclonal antibody (a generous gift from Dr. P.Fredman, University of Goteborg, Sweden). Excess antibody was removed by thorough washing; the plate was treated with an anti-mouse IgM antibody coupled to peroxidase and the reaction was visualized with 3,3'-diaminobenzidine (Dasgupta et al., 1996). Approximately 20 µg of the hexosaminidase hydrolyzed product was permethylated and the PMAAs were analyzed by GLC-MS as described above.
NMR spectroscopy of the oligosaccharide released from the Ngsl
Ngsl B (100µg) was hydrolyzed with ceramide-glycanase (Zhou et al., 1989) in acetate buffer containing sodium taurodeoxycholate (0.05%). The oligosaccharide, purified by Biogel P2 chromatography (0.8 cm x 40 cm; water as eluent), was repeatedly dissolved in D2O [Cambridge Isotope Laboratories (Andover, MA); 99.96 atom % D] at room temperature and pD 6.7, with intermediate lyophilization. Prior to NMR spectroscopic analysis the sample was redissolved in 0.5 ml of D2O and transferred into a 5 mm NMR tube (Wilmad; 535-PP). 1H NMR spectroscopy was performed on a Bruker AMX-600 spectrometer interfaced with an Aspect-X32 computer. The probe temperature was maintained at 23°C, with experimental details as described previously (Van Halbeek, 1994
). One-dimensional (1D) data were processed on an IBM-compatible PC using Felix for Windows version 1.01 (Biosym/MSI, San Diego, CA). Chemical shifts (
) for the oligosaccharide are expressed in "p.p.m." (parts per million) downfield from internal sodium 4,4-dimethyl-4-silapentane-1-sulfonate, measured by reference to internal acetate (
1.908 at pD 6.7) with an accuracy of 0.002 p.p.m..
Two-dimensional 1H TOCSY (Braunschweiler and Ernst, 1983; Bax and Davis, 1985
) and ROESY (Bothner-By et al., 1984
) data sets were collected in phase-sensitive mode. In each of the 2D experiments, 200 FIDs of 2048 data points were acquired. For the TOCSY experiment, 256 scans per t1 increment were collected. The TOCSY pulse program contained a 200 ms MLEV-17 spin-lock pulse (Bax and Davis, 1985
). The ROESY experiment used a 189 ms 2 kHz CW spin-lock pulse flanked by two 90° pulses for offset compensation (Griesinger and Ernst, 1987
); 512 scans per t1 increment were collected. The 2D data were processed with a Lorentzian-to-Gaussian function applied in the t2 dimension and a shifted squared sine bell function and zero-filling applied in the t1 dimension. Data were processed in Felix 2.1 on a Silicon Graphics Indy workstation.
Preparation of antibody for immunohistochemical localization
Monospecific polyclonal anti-GalNAc-GA1 antibody was prepared by subcutaneous injection of the emulsified GalNAc-GA1 into New Zealand rabbits using Freunds adjuvant and keyhole limpet hemocyanin (Dasgupta et al., 1996) and purified by substrate affinity chromatography (Nair et al., 1993
; Dasgupta et al., 1995b
). The purified antibody was highly specific for GalNAc-GA1 by ELISA and TLC-IS when assayed with the substrates GM1, GA1, GalNAc-GA1, and GalNAc-GM1 (the latter compound was received as a gift from Professor S. Sonnino, University of Milan, Italy).
In situ localization in adult rat brain
The immunohistochemical localization of GalNAc-GA1 in rat brain tissue was carried out according to the methods previously utilized to localize glycoconjugates in rodent brain (Nair et al., 1993; Dasgupta et al., 1995b
). Briefly, anesthetized rats were perfused intraortically with 2000 U of heparin in 10 ml of phosphate-buffered saline (PBS) followed by 0.1 M phosphate buffer (pH 7.4) containing 2% glutaraldehyde and 2% paraformaldehyde. Parasagittal sections (10 µm) were postfixed with cold acetone (-20°C) for 10 min and stained as described (Nair et al., 1993
) using 0.2% Triton X-100 during staining. The fixed sections were washed with PBS, blocked with 1% normal goat serum (NGS) and incubated with the primary antibody (neat) for 2448 h at 4°C and the antibody binding was visualized by diaminobenzidine staining.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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