Departments of Pharmacology and 4Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Received on February 2, 1999; revised on July 7, 1999; accepted on July 7, 1999.
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Abstract |
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Key words: sialic acid biosynthesis/N-acetylmannosamine/N-acetylneuraminic acid/N-glycolylneuraminic acid/siglec recognition
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Introduction |
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N-Acetylneuraminic acid (NeuAc), the predominant sialic acid in nature, is synthesized in vivo by a multistep pathway (Roseman, 1970) beginning with the conversion of N-acetylglucosamine to N-acetylmannosamine-6-phosphate by a bifunctional epimerase/kinase (Kundig et al., 1966
; Hinderlich et al., 1997
). ManNAc-6-P is converted to NeuAc-9-P by condensation with phosphoenol pyruvate, and then to CMP-NeuAc which is the activated NeuAc donor for glycolipid and glycoprotein oligosaccharide biosynthesis (Kean and Roseman, 1966
; Watson et al., 1966
). Hydroxylation of NeuAc (in the CMP-NeuAc form) by a specific hyroxylase converts NeuAc to N-glycolylneuraminic acid (NeuGc), a member of the sialic acid family which is rare (or absent) in humans, but is common in non-neural tissues of many other species (Kawano et al., 1995
; Chou et al., 1998
).
To experimentally introduce modified sialic acids on intact cells and tissues, this pathway has been short-circuited by addition of unnatural N-acylmannosamines, including N-propanoyl-, N-butanoyl-, N-pentanoyl-, and N-levulinoylmannosamine, among others (Angelino et al., 1995; Keppler et al., 1995
; Yarema et al., 1998
). These precursors are taken up, converted to the corresponding sialic acids, and expressed on cell surface glycoconjugates. Cells engineered to display modified sialic acids demonstrate altered pathogen binding, antigenicity, and chemical reactivity (Keppler et al., 1995
; Herrmann et al., 1997
; Mahal et al., 1997
; Schmidt et al., 1998
; Yarema et al., 1998
).
We and others have recently reported the carbohydrate binding specificity of a sialic acid binding lectin, myelin-associated glycoprotein (MAG), a member of the "siglec" family of immunoglobulin-like lectins (Kelm et al., 1994a; Collins et al., 1997a
,b; Crocker et al., 1998
; Kelm et al., 1998
). In the nervous system, MAG functions in the stabilization of the myelin sheath surrounding axons (Fruttiger et al., 1995
; Bartsch et al., 1997
; Lassmann et al., 1997
; Sheikh et al., 1999
), and in the control of axon cytoarchitecture (Yin et al., 1998
). In addition, MAG inhibits nerve regeneration, and has been proposed to contribute to the limited functional recovery typical of central nervous system (e.g., spinal cord) injury (McKerracher et al., 1994
; Mukhopadhyay et al., 1994
; Schnaar et al., 1998
). Presumably, MAG binds to sialoglycoconjugate targets on nerve cells to initiate its physiological and pathological effects. MAG is highly restrictive in its requirements for sialic acid substructure, in that it requires the N-acetyl group, the glycerol side chain, and the carboxylic acid group for recognition (Collins et al., 1997a
,b; Kelm et al., 1998
). Given the sensitivity of MAG binding to even minor changes in sialic acid substructure, the goal of the experiments described here was to develop a highly efficient N-acylmannosamine precursor which would result in modification of a large proportion of the nerve cell sialic acid, rendering the cells resistant to MAG binding.
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Results |
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The glycoprotein sialic acid concentration was more sensitive to the precursor pool size, with total glycoprotein sialic acid increasing 2-fold in cells treated with ManNAc, increasing
1.4-fold in cells treated with ManNPr, and decreasing by
40% in cells treated with ManNGcMA (Figure 2, bottom panel). As with the gangliosides, treatment with 20 mM ManNPr converted
70% of the glycoprotein sialic acid to the NeuPr form within 48 h, whereas treatment with 20 mM ManNGcMA resulted in
30% conversion of glycoprotein sialic acids to the NeuGc form.
Although ManNPr was efficiently incorporated into gangliosides and glycoproteins as NeuPr, it was not used to modify MAG binding, since NeuPr is reported to support MAG (Kelm et al., 1998), whereas NeuGc does not support MAG binding (Collins et al., 1997a
; Kelm et al., 1998
). Therefore, improved methods to deliver ManNGc into cells were tested.
NeuGc biosynthesis from ManNGc pentaacetate
Peracetylation has been shown to greatly increase uptake and anabolic utilization of carbohydrates (Sarkar et al., 1995, 1997). Therefore, ManNGcMA was peracetylated to give N-glycolylmannosamine pentaacetate (ManNGcPA, Figure 1, R1 = Ac) and tested as a precursor for NeuGc biosynthesis. NG10815 cells were incubated with 0.1 or 1.0 mM ManNGcPA (or 5 mM ManNGcMA for comparison) for 48 h. Peracetylated sugars were routinely dissolved in dimethylsulfoxide (DMSO), and delivered to the cells in medium containing a final concentration of 0.5% DMSO, which was non-toxic to cells (see below). Whereas none of these treatments significantly altered the sialic acid precursor pool size, treatment with 1 mM ManNGcPA resulted in 82% conversion from NeuAc to NeuGc (Figure 3, top panel). Treatment with 0.1 mM ManNGcPA resulted in
2-fold greater conversion to the NeuGc form than did treatment with 5 mM ManNGcMA, indicating an increase in potency of 100-fold due to peracetylation. Incorporation of NeuGc into gangliosides and glycoproteins was related to the precursor pool composition, resulting in
50% NeuGc within 48 h when cells were treated with 1 mM ManNGcPA (Figure 3, center and bottom panels) and much less when cells were treated with 5 mM ManNGcMA. Total ganglioside and glycoprotein sialic acid levels were not significantly altered by treatment with the peracetylated precursor.
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Discussion |
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The high selectivity of MAG for NeuAc rather than NeuGc offers an opportunity to subtly modify nerve cell sialic acids and thereby block MAG binding. Since MAG is implicated in maintaining stable myelinaxon interactions (Fruttiger et al., 1995; Bartsch et al., 1997
; Lassmann et al., 1997
; Sheikh et al., 1999
), directing axon cytoarchitecture (Yin et al., 1998
), and controlling nerve regeneration after injury (McKerracher et al., 1994
; Mukhopadhyay et al., 1994
; Schäfer et al., 1996
; Schnaar et al., 1998
), such modification has implications both for studying and possibly modulating important neural cellcell interactions.
Modification of sialic acids in live cells or animals using synthetic N-acylmannosamines was pioneered by the Reutter laboratory (Kayser et al., 1992a). The incorporation of synthetic N-acylmannosamines into sialic acids, and their effects on cell growth, morphology, antigen expression, viral susceptibility, and tumor growth have been reported (Kayser et al., 1992a
; Angelino et al., 1995
; Keppler et al., 1995
; Weiser et al., 1996
; Herrmann et al., 1997
; Hinderlich et al., 1997
; Schmidt et al., 1998
). More recently, synthetic N-levulinoylmannosamine was used to introduce novel chemical reactivity onto cell surfaces (Mahal et al., 1997
; Yarema et al., 1998
).
In the current study, we applied sialic acid mass analysis to determine, for the first time, absolute levels of incorporation of synthetic and natural N-acylmannosamines into the sialic acid precursor pool, glycolipids (gangliosides) and glycoproteins in the same cell population. The observation that the sialic acid precursor pool size is highly elastic is consistent with a prior report on the effect of ManNAc treatment on sialic acid synthesis (Thomas et al., 1985). Although our analytical conditions did not routinely distinguish between the free and nucleotide sugar forms of sialic acid, it has been reported that the precursor pool is predominantly free sialic acid (Thomas et al., 1985
), a contention supported by thin layer chromatography analysis in this study (Figure 5). Large changes in the precursor pool size did not quantitatively impact sialic acid incorporation into gangliosides, and only modestly affected incorporation into glycoproteins. However, conversion of the precursor pool from exclusively NeuAc to largely NeuPr or NeuGc resulted in incorporation of the modified sialic acids into both gangliosides and sialoglycoproteins. The delay of incorporation of modified sialic acids into glycoconjugates compared to their appearance in the precursor pool (e.g., Figure 4) may reflect the rate of turnover of gangliosides and sialoglycoproteins in these cells.
Our finding of equivalent incorporation of precursor NeuPr or NeuGc into both gangliosides and sialoglycoproteins in living cells suggests that various sialyltransferases readily use the modified sialic acid precursors. These results are consistent with previous reports demonstrating insensitivity of sialyltransferases to chemically modified CMP-sialic acid derivatives in vitro (Higa and Paulson, 1985; Kelm et al., 1998
), but do not rule out the possibility that some of the >15 known sialyltransferases (Tsuji, 1996
) may distinguish between natural and synthetic precursors, resulting in differential incorporation into different specific sialic acid glycoforms. Our data are consistent with prior reports of modified sialic acid incorporation into glycolipids (Kayser et al., 1992b
) and glycoproteins (Keppler et al., 1995
), but conflict with a recent report indicating no incorporation of NeuPr into gangliosides in ManNPr-treated neural cells (Schmidt et al., 1998
). This issue is worthy of further study to determine the basis for differences in precursor utilization.
As with other N-acylmannosamine precursors, treatment with ManNGc (as the monoacetate or pentaacetate) led NG10815 cells to synthesize and incorporate NeuGc, a major sialic acid found in non-neural tissues of many nonhuman species. Our testing of peracetylation to enhance uptake and anabolic utilization of carbohydrates was based on the work of Sarkar et al. (Sarkar et al., 1995, 1997). As in their system, peracetylation greatly increased utilization, and this simple modification is likely to be useful for other synthetic sialic acid precursors. It is assumed that the peracetylated species enter the cells more rapidly, where they are fully de-O-acetylated (perhaps by nonspecific esterases) prior to or concurrent with their conversion to the corresponding sialic acids, as shown in Figure 5.
The demonstration that ManNGcPA is an effective NeuGc precursor, and that conversion of neural NeuAc to NeuGc abrogates MAG binding (Figures 8, 9), opens the way for future animal testing. A prior study showed little incorporation of intraperitoneally injected ManNPr into sialic acids in the brains of rats, even though it was incorporated into liver and serum glycoproteins (Kayser et al., 1992a). It is hoped that peracetylation, or modification of the route of delivery, will result in higher nervous system conversion from NeuAc to NeuGc using ManNGcPA.
Animals which express NeuGc as a major sialic acid outside of the nervous system express only minor amounts within the nervous system (Chou et al., 1998; Mikami et al., 1998
). Although the mechanism of NeuGc exclusion in the brain has yet to be determined, we demonstrate that cultured rodent neural cells readily convert ManNGcPA to NeuGc, which is effectively incorporated into both gangliosides and sialoglycoproteins.
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Materials and methods |
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N-Glycolylmannosamine pentaacetate (ManNGcPA) was prepared by treating ManNGcMA (0.3 g) with acetic anhydride (2 ml) in pyridine (2 ml) for 1 h at ambient temperature. The reaction was monitored by silica gel thin layer chromatography, using toluene-ethanol (10:1) as the developing solvent and orcinol-sulfuric acid reagent to visualize sugars (product Rf = 0.32). The reaction mixture was concentrated and the product purified by silica gel column chromatography using step-wise elution with toluene, toluene-ethanol (50:1), and toluene-ethanol (20:1) as the eluants. Fractions containing the desired product were evaporated, resuspended in DMSO and the concentration determined by quantitative high pressure liquid chromatography. The structure of the peracetylated compound was confirmed by 1H NMR, which revealed full O-acetylation and an :ß ratio of 2:1.
N-Propanoylmannosamine (ManNPr) was synthesized according to the method of Keppler et al. (Keppler et al., 1995). D-MannosamineHCl (3 mmol), sodium methoxide (3.3 mmol), and propionic anhydride (3.6 mmol) in 300 ml of methanol were stirred for 2 h at 0°C. The desired product was isolated by silica gel column chromatography using ethyl acetatemethanolwater (5:2:1) as the eluant. Fractions were analyzed by thin layer chromatography using the same solvent for development and orcinol-sulfuric acid reagent for detection (Rf = 0.59). Fractions containing product were combined, evaporated to dryness, the residue dissolved in water, and stored at -20°C. N-Propanoylneuraminic acid (NeuPr) standard was prepared enzymatically (Comb and Roseman, 1960
) by reacting ManNPr with pyruvate using N-acetylneuraminic acid aldolase (Sigma).
Cell culture and treatment with sialic acid precursors
NG10815 cells were maintained in Dulbeccos modified Eagles medium (high glucose formulation) containing 5% iron-enriched calf serum, 100 µM hypoxanthine, 16 µM thymidine, and 5 µM aminopterin. Cells were cultured at 37°C in a humidified atmosphere of 90% air/10% CO2. For treatment with sialic acid precursors, mannosamine derivatives were diluted into the appropriate medium and filter sterilized. The growth medium was then replaced and cells were cultured in the presence of the precursor for 24144 h. In experiments testing ManNGcPA, all cells (control and experimental) were grown in the presence of 0.5% (v/v) DMSO, the carrier for ManNGcPA.
Sialic acid analyses
Cells were harvested using hypertonic Ca2+- and Mg2+-free phosphate-buffered saline containing 1 mM EDTA (Yang et al., 1996a), collected by centrifugation, and homogenized in ice-cold water using a Potter-Elvehjem glass/Teflon homogenizer. Methanol (2.6 volumes) was added, the suspension was mixed and brought to ambient temperature, and then chloroform (1.3 volumes, based on the original homogenate) was added and the suspension mixed vigorously. The suspension was centrifuged 30 min at 2000 x g. After collecting the supernatant, the pellet (containing precipitated proteins) was resuspended in water or concentrated ammonium hydroxide and a portion was used for protein assay (BCA, Pierce, Rockford, IL). The supernatant was partitioned by adjusting the chloroformmethanolwater ratio to 4:8:5.6 by addition of water, mixing thoroughly, and centrifuging as above. The upper phase was collected and a portion was subjected to reverse phase chromatography, using Sep-Pak C18 cartridges (Waters, Milford, MA), to isolate gangliosides (Schnaar, 1994
).
To quantify and identify sialic acids, a portion of resolublized protein, organic/aqueous soluble pool, or reverse-phase purified gangliosides was evaporated to dryness in a 500-µl polypropylene tube and 20 µl of 0.1 M HCl/0.25 M NaCl was added. One Ampliwax bead (Perkin Elmer Corp, Foster City, CA) per reaction was added to block evaporation, and the sample hydrolyzed for 3 h at 80°C. Released sialic acids were analyzed by injecting an aliquot (110 µl) onto a Dionex high pressure liquid chromatography system (Dionex Corporation, Sunnyvale, CA) using a HPIC-AS6 column and a pulsed amperometric detector (Manzi et al., 1990). Elution solutions were: A, 0.75 mM NaOH; B, 200 mM NaOH; and C, 400 mM sodium acetate in 50 mM NaOH. NeuAc and NeuPr were resolved by elution with 40% A, 50% B, 10% C for 15 min at 1 ml/min. NeuGc was resolved in separate injections using step gradient elution: 01.8 min, 18% A, 50% B, 32% C; 210 min, 40% B, 60% C. Sialic acids were identified by comparison of their elution time with those of standard NeuAc, NeuPr and NeuGc and were quantified in comparison to a standard curve of commercial NeuAc (for NeuAc and NeuPr) or NeuGc.
To confirm that the quantified peaks represented sialic acids, 10 µl of acid hydrolysates were treated with 1 µl of 1 M sodium phosphate (pH 7.2) and 10 µl of 0.1 M NaOH. Neutralized samples were incubated at 37°C for 2 h with or without 0.9 U of N-acetylneuraminic acid aldolase (Sigma), 32 µM NADH and 0.1 µg lactate dehydrogenase. Enzyme-dependent disappearance of the presumed sialic acid peak (Comb and Roseman, 1960; Manzi et al., 1990
) was taken as evidence for its identity (data not shown). Sialic acid content in the ganglioside and glycoprotein fractions is expressed per milligram of total cell protein. Precursor pool sialic acid (sialic acid plus CMP-sialic acid) was calculated by subtracting the ganglioside value from the organic/aqueous pool value, both expressed per milligram of cell protein.
MAG binding
Flow cytometry was used to test binding of a soluble MAG-Fc chimera to precursor-treated and control NG10815 cells. A chimeric protein consisting of the entire extracellular domain of MAG fused via a three amino acid bridge (TGK) to the human Fc domain was produced using the "pIgPlus" vector (Novagen, Madison, WI). A PCR fragment coding for the extracellular domain of MAG was prepared using MAG in pCDM8 as template (Yang et al., 1996a), the T7 5' primer (TAATACGACTCACTATAGG) and GATCGGATCCTTACCTGTTTTGGCCCACATCAGTCGGTGTGC as the 3' primer. The fragment was cut with BamH1 and HindIII and directionally cloned into the pIgPlus vector. The resulting construct was sequenced to confirm its identity, transfected into COS cells, and the resulting MAG-Fc chimera was purified from the culture medium using Protein G affinity chromatography.
For MAG binding studies, NG10815 cells were cultured in the presence of sialic acid precursors for the indicated times, then were released from culture dishes using hypertonic Ca2+/Mg2+-free phosphate-buffered saline (Yang et al., 1996a) and resuspended (at 2 x 105 cells/ml) in 25 mM Hepes-buffered Hanks balanced salt solution (Bashor, 1979
) containing 5 mg/ml bovine serum albumin. MAG-Fc (6 µg) was added to 100 µl of the same buffer containing 6 µg fluorescein isothiocyanate-labeled goat anti-human Fc (Jackson Immunoresearch, West Grove, PA) and incubated on ice for 1 h 45 min with frequent mixing. Cell suspension (100 µl containing 50,000 cells) was added to the premixed antibody solution and the mixture incubated on ice for 45 min. The cells were then centrifuged for 4 sec at 16,000 x g and washed three times by resuspension in 200 µl of buffer with bovine serum albumin and centrifugation. The resulting cell pellet was resuspended in 100 µl buffer with bovine serum albumin and flow cytometry was performed on an Epics Profile II cytometer (Coulter Corp, Hialeah, FL).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 Present address: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA
3 To whom correspondence should be addressed at: Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185
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