Glycobiology, 2000, Vol. 10, No. 10 1049-1056
© 2000 Oxford University Press
Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells
3Departments of Chemistry and 4Molecular and Cell Biology, University of California, and 5Center for Advanced Materials, Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
Received on February 3, 2000; revised on May 24, 2000; accepted on May 30, 2000.
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Abstract |
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In this study we demonstrate that polysialyltransferases are capable of accepting unnatural substrates in terminally differentiated human neurons. Polysialyltransferases catalyze the glycosylation of the neural cell adhesion molecule (NCAM) with polysialic acid (PSA). The unnatural sialic acid analog, N-levulinoyl sialic acid (SiaLev), was incorporated into cell surface glycoconjugates including PSA by the incubation of cultured neurons with the metabolic precursor N-levulinoylmannosamine (ManLev). The ketone group within the levulinoyl side chain of SiaLev was then used as a chemical handle for detection using a biotin probe. The incorporation of SiaLev residues into PSA was demonstrated by protection from sialidases that can cleave natural sialic acids but not those bearing unnatural N-acyl groups. The presence of SiaLev groups on the neuronal cell surface did not impede neurite outgrowth or significantly affect the distribution of PSA on neuronal compartments. Since PSA is important in neural plasticity and development, this mechanism for modulating PSA structure might be useful for functional studies.
Key words: polysialyltransferase/NCAM/sialic acid/biosynthesis
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Introduction |
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The ability to alter cell surface oligosaccharide structures using metabolic pathways has provided a novel avenue for biological studies of cell–cell recognition. The sialic acid biosynthetic pathway is notably amenable to this approach. Unnatural sialosides can be introduced into glycoconjugates by feeding cells their precursors, unnatural N-acyl mannosamines (Kayser, 1992; Keppler et al., 1995
). This method has been used to alter viral binding efficiency, stimulate proliferation of neural cell lines and alter myelin associated glycoprotein (MAG) binding to neural cells (Keppler et al., 1995; Schmidt et al., 1998; Collins et al., 2000).
We have extended this approach by replacing the N-acyl group of the precursor mannosamine with a levulinoyl moiety that contains a reactive ketone group (Mahal et al., 1997; Yarema et al., 1998). Cultured cells treated with N-levulinoylmannosamine (ManLev) incorporated the corresponding ketone-containing N-levulinoyl sialic acid (SiaLev) into cell surface oligosaccharides. The ketone group on the cell surface was then covalently ligated with biotin hydrazide permitting detection of SiaLev on cultured cells. The presence of SiaLev within cell surface glycoconjugates was confirmed by inhibiting ketone expression with N-linked and O-linked glycosylation inhibitors, and by the failure of sialidases to hydrolyze SiaLev (Yarema et al., 1998). Generally, it appears that sialyltransferases are capable of incorporating unnatural sialic acids into cell surface glycoconjugates. However, the individual substrate specificities of the numerous cloned sialyltransferases in vivo have not yet been directly studied.
Polysialic acid (PSA) is a linear homopolymer of 
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8 linked sialic acid that can be over 50 residues in length (Finne, 1982; Troy, 1992; Rutishauser, 1998). The neural cell adhesion molecule (NCAM) is likely to be the primary carrier of PSA since NCAM-deficient mice are almost completely devoid of PSA expression (Cremer et al., 1994; Ono et al., 1994). Polysialylation of NCAM disrupts the homophilic association of NCAM molecules by impeding the molecular contacts between aposing membranes (Rutishauser et al., 1988) and thus promotes cell migration and axon outgrowth (Szele et al., 1994; Hu et al., 1996).
During development, the majority of NCAM in the central nervous system is highly polysialylated (Probstmeier et al., 1994). In contrast, in adults only specific regions of the brain associated with synaptic plasticity are found to express polysialylated NCAM (PSA-NCAM; Szele et al., 1994) which is known to be critical for long-term potentiation in the hippocampus (Becker et al., 1996; Muller et al., 1996; Cremer et al., 1998). Abnormal expression of PSA-NCAM has been described in a number of human cancers including small cell lung carcinomas (SCLC) and Wilm’s tumors (Troy, 1992; Fukuda, 1996; Martersteck et al., 1996) and is thought to promote tumor cell metastasis (Michalides et al., 1994; Scheidegger et al., 1994).
Two polysialyltransferases, PST (ST8Sia IV) and STX (ST8Sia II), have been identified that catalyze the formation of the PSA polymer (Nakayama and Fukuda, 1996; Angata et al., 1998). Each enzyme has been shown to be capable of catalyzing all steps involved in PSA synthesis (initiation, polymerization and termination) using CMP-sialic acid as the sialic acid donor (Kojima et al., 1995a,b, 1996; Mühlenhoff et al., 1996; Angata et al., 1998). The donor substrate specificities of the enzymes have not been investigated. If these polysialyltransferases accept modified substrates then the composition of PSA could be altered in living cells, providing a novel approach to the study of processes that involve PSA-NCAM.
Herein we report that polysialyltransferases are capable of accepting an unnatural CMP-sialic acid analog generated by cellular metabolism. Specifically, we show that differentiated human NT2 neurons fed the unnatural sugar precursor N-levulinoylmannosamine install N-levulinoyl sialic acid in PSA chains. The metabolic incorporation of this chemically modified saccharide into PSA provides a novel means to alter the composition of this biologically important polysaccharide.
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Terminally differentiated NT2 neurons incorporate unnatural sialic acids into cell surface glycoconjugates
To establish whether terminally differentiated neurons are able to metabolize ManLev and incorporate SiaLev residues into cell-surface glycoconjugates, NT2 neurons were cultured in the presence of ManLev and monitored for cell surface ketone expression. NT2 neurons closely resemble embryonic hippocampal neurons in that they form axons and dendrites, segregate proteins into the correct compartments and readily form functional synapses (Pleasure et al., 1992
; Younkin et al., 1993; Munir et al., 1995, 1996). Primary mouse astrocytes served as a support for purified neurons in order to reduce the damage caused by washing steps during the labeling procedure. After incubation with ManLev for 5 days, NT2 neurons were examined for ketone expression using the ketone-specific probe biotin-X-hydrazide. Biotinylation of the neuronal cell surface was visualized by immunofluorescence using specific anti-biotin antibodies under conditions that minimized intracellular labeling. Fluorescence micrographs of NT2 neurons treated with varying concentrations of ManLev or the native substrate ManNAc are shown in Figure 1.
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The appearance of ketones on the surface of NT2 neurons was first detected after 2 days of ManLev treatment (data not shown) and reached a steady-state level within 5 days as determined by immunofluorescence. These data suggest that NT2 neurons utilize the unnatural precursor in sialic acid biosynthesis and incorporate ketone-containing sialic acids into cell surface glycoconjugates. In contrast, control cultures incubated with ManNAc produced little or no immunofluorescence after biotinylation, confirming that staining of ManLev-treated cells is a ketone-specific process. Ketone incorporation appeared to be evenly distributed along the surfaces of soma and projecting neurites, indicating that SiaLev glycoconjugates are incorporated into multiple compartments of the neurons.
The incorporation of SiaLev residues into NT2 cell surface glycoconjugates was dependent on the concentration of ManLev in the media. Significant levels of cell surface ketones were detected after 5 days of incubation with ManLev at concentrations as low as 0.1 mM (Figure 1). Robust staining of both the soma and neurites of NT2 neurons was observed using 1 mM ManLev. Incubation of NT2 neurons with concentrations up to 30 mM ManLev did not significantly increase the level of cell surface ketones above that observed with 3 mM ManLev (data not shown). In previous work, expression of SiaLev in a variety of human cell lines reached a maximum at 20–30 mM ManLev (Yarema et al., 1998); in these cell lines, little or no staining was observed using 0.1 mM ManLev as detected by flow cytometry. In contrast, NT2 neurons appear to incorporate SiaLev residues and reach maximal incorporation when incubated with significantly lower concentrations of ManLev.
Ketone expression was not detected on the underlying primary mouse astrocytes, even with 10 mM ManLev in the media. Since these cells are quiescent, the lack of ketone incorporation may reflect a relatively low turnover rate of the plasma membrane. Post-mitotic NT2 neurons, on the other hand, are still developing through neurite extension. As a result, the relative rate of de novo glycoprotein and glycolipid biosynthesis and the concomitant expression of SiaLev on the neuronal cell surface should be high. Another possibility is that the murine origin of the primary astrocytes may influence their ability to utilize ManLev as a substrate for sialic acid biosynthesis. It has been observed previously that murine cell lines metabolize ManLev less efficiently than their human counterparts (Yarema et al., 1998; Lee et al., 1999).
Glycosylation of NCAM with PSA occurs in the presence of ManLev
To address the effects of ManLev treatment on polysialylation, NT2 neurons were incubated with ManLev for five days at concentrations ranging from 0.3 mM to 3 mM. The cultures were then stained for PSA using the anti-PSA antibody 12F8 and visualized by immunofluorescence (Figure 2). The anti-PSA antibody readily labeled both the soma and projecting neurites of NT2 neurons. Labeling was also observed in growth cones emanating from developing axons. Interestingly, we observed significant PSA expression in the mouse primary astrocytes on which NT2 neurons were cultured (Figure 2, background). This is in agreement with other studies which have shown that primary astrocyte cultures express polysialylated NCAM (Kiss, 1998; Miñana et al., 1998). At 3 mM ManLev, a concentration at which ketone incorporation appears to reach a maximum, the level of PSA staining was comparable to the ManNAc control. These results indicate that incubation with ManLev in this concentration range has no detectable effect on PSA as seen by immunofluorescence.
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Western blot analysis of NCAM from ManLev-treated NT2 neurons was performed to directly examine any effects unnatural sialosides might have on PSA biosynthesis. First we verified the expression of NCAM and its associated polysialylation in NT2 neurons by analysis of total cell lysates. Treatment of NT2 neurons with the PSA-specific endoneuraminidase, endo NE, followed by Western blot analysis confirmed the polysialylation of NCAM and revealed that the 180 kDa isoform of NCAM is the major isoform observed in these cells (vida infra). To determine the effects of ManLev on polysialylation, we cultured NT2 neurons in the absence of primary astrocytes with 0.3–3 mM ManLev or ManNAc and analyzed whole cell lysates by Western blot (Figure 3). Incubation of NT2 neurons with ManLev at these concentrations did not block the expression of PSA on NCAM; the anti-PSA monoclonal antibody, 735, revealed a diffuse band of protein of molecular weight >200 kDa, which is indicative of highly polysialylated NCAM. No alteration in the molecular weight range was detected for PSA-NCAM in cells treated with ManLev at 0.1–1 mM. However, we did observe lower amounts of PSA immunoreactivity and a slight shift in molecular weight for cells treated with 3 mM ManLev compared to controls. This result may reflect a partial inhibition of PSA biosynthesis in the presence of high levels of unnatural sialic acids. Alternatively the presence of SiaLev residues in PSA may inhibit recognition by the anti-PSA antibody. The molecular basis of this possible inhibitory effect is currently under investigation.
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PSA bearing SiaLev residues resists sialidase digestion
To demonstrate the incorporation of SiaLev into PSA on NCAM, we took advantage of the inability of sialidases to cleave SiaLev glycosides (Yarema et al., 1998
). Sialidases hydrolyze terminal sialic acid residues and digest the polymer from the terminus of the PSA chain. If the unnatural analog SiaLev is incorporated into PSA, sialidases would digest the polymer until a SiaLev residue was encountered, at which point the PSA chain would resist further degradation. We incubated NT2 neurons with ManLev or ManNAc at concentrations ranging from 0.3–3 mM and then subjected them to treatment with sialidase from Clostridium perfringens (C.P. sialidase) prior to staining for PSA immunoreactivity. At all concentrations of ManNAc, PSA immunoreactivity was nearly abolished after treatment with the sialidase (Figure 4). By contrast, PSA immunoreactivity, although diminished after sialidase treatment, was readily apparent in ManLev-treated cultures. This finding is consistent with the replacement of some sialic acid residues in PSA with sialidase-resistant SiaLev residues. Significant PSA immunoreactivity was observed at all three concentrations of ManLev examined, indicating that even at 0.3 mM, some SiaLev residues were incorporated into PSA.
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To better quantitate the effect of SiaLev on digestion of PSA by C.P. sialidase, PSA-NCAM was analyzed by Western blot after sialidase treatment (Figure 5). NT2 neurons were treated with 0.3–3 mM ManLev or ManNAc for 5 days then subjected to sialidase treatment prior to isolation of total protein. Sialidase treatment of ManNAc-treated NT2 neurons resulted in the removal of the majority of PSA from NCAM, shifting the apparent molecular weight of NCAM from >200 kDa to
200 kDa. This is consistent with the removal of PSA, as treatment of NT2 neurons with endo NE results in a band at 200 kDa (vida infra). Densitometry analysis of Western blots from ManNAc-treated NT2 neurons showed that sialidase reduces PSA immunoreactivity to 15% of controls (Table I). In contrast, after sialidase treatment, 50% of PSA remained intact in whole cell lysates of NT2 neurons cultured with 1 mM ManLev. This supports the evidence from immunofluorescence staining indicating that treatment of NT2 neurons with 1 mM ManLev provides sialidase protection. Curiously, densitometry analysis of the anti-PSA Western blots did not show any evidence of sialidase protection in cultures treated with 0.3 and 3 mM ManLev (Figure 5). However, the anti-NCAM Western blots clearly indicate that PSA-NCAM on cells treated with 0.3 and 3 mM ManLev are partially protected from sialidase digestion as indicated by the apparent molecular weight (Figure 5). The difference in these results may be due to reduced immunoreactivity of anti-PSA antibody 735 with unnatural polysialic acids.
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Overall, the results are consistent with the presence of the unnatural sialic acid in PSA on NCAM, and imply that the
-28 polysialyltransferases tolerate the unnatural substrate CMP-SiaLev. The presence of SiaLev residues in the inner segment of PSA cannot be established from these experiments since it is possible that incorporation of SiaLev prevents extension of the polysaccharide chain. Unfortunately, the biochemical analysis of SiaLev-bearing PSA chains in order to identify such internal residues was frustrated by the amounts of polysialic acid necessary for analysis which cannot be obtained from NT2 neurons (Hallenbeck et al., 1987; Pelkonen et al., 1988; Zhang et al., 1997). The sensitivity of SiaLev to acid mediated decomposition (unpublished observations) complicates analyses that require acid hydrolysis (Sato et al., 1998; Zhang et al., 1997), further impeding detailed chemical studies.
Persistence of SiaLev on NT2 neurons after C.P. sialidase treatment
To confirm that SiaLev persists on the cell surface of NT2 neurons after sialidase treatment, the cells were cultured in the presence of ManLev or ManNAc for 5 days, then subjected to sialidase treatment and assayed for the presence of ketones (Figure 6). After sialidase treatment, significant ketone-specific fluorescence was observed on the surface of ManLev-treated NT2 neurons. The level of ketones was partially reduced by sialidase treatment, but this might reflect the activity of proteases that contaminate the commercial C.P. sialidase preparation.
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The minimal degree of polymerization of PSA containing SiaLev is eight residues
To determine a minimal degree of polymerization for polysialic acid chains containing the unnatural sialic acid SiaLev, we examined the ability of endo NE to cleave polysialic acid chains on ManLev-treated NT2 neurons. Endo NE, which derives from the Escherichia coli K1 bacteriophage PK1E, requires a minimum of eight sialic acid residues in a chain for cleavage (Finne and Mäkelä, 1985
). The cleavage pattern of endo NE leaves five sialic acid residues attached to an oligosaccharide chain on NCAM and releases three or more residues (Pelkonen et al., 1989). We incubated NT2 neurons with ManLev at concentrations ranging from 0–3 mM and treated the cells with either C.P. sialidase, endo NE, or sequentially with C.P. sialidase treatment followed by endo NE. Total protein lysates were obtained and visualized by Western blot (Figure 7). NCAM from untreated NT2 neurons demonstrated a loss of polysialic acid with both C.P. sialidase and endo NE treatment as shown by anti-NCAM (Figure 7) and anti-PSA (data not shown) staining. In contrast, polysialic acid on NCAM from NT2 neurons treated with 1 or 3 mM ManLev was resistant to C.P. sialidase but not to endo NE. We conclude that PSA on NT2 neurons treated with 1 and 3 mM ManLev contain unnatural sialic acids and must comprise a minimum of eight residues. The difference in molecular weight (Figure 7) between endo NE-digested NCAM, bearing five sialic acid residues, and C.P. sialidase resistant PSA-NCAM, bearing SiaLev, suggests that the degree of polymerization of unnatural PSA can significantly exceed eight residues.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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In this study, we have investigated the possibility of incorporating unnatural sialic acids into PSA. Our results show that the unnatural sialic acid precursor, ManLev, is metabolized by terminally differentiated NT2 neurons to the corresponding ketone-containing sialic acid, SiaLev, within cell surface glycoconjugates. Although this paper examines ManLev concentrations in the millimolar range, it should be noted that concentrations of peracetylated ManLev in the micromolar range have been shown to produce similar amounts of ketones on other types of human cells (Lemieux et al., 1999
). In examining the distribution of SiaLev-containing glycoconjugates, there was little evidence of any restriction in their expression among the structurally and functionally distinct compartments of NT2 neurons. Detailed examination revealed that ketone expression occurred at the leading edge of axon outgrowth, the growth cone, indicating SiaLev-labeled glycoconjugates are effectively transported to the periphery of NT2 neurons. In addition, there appeared to be no negative impact of ManLev treatment on the development of NT2 neurons. Axonal and dendritic processes developed at a similar rate as controls and no apparent toxicity was observed. After seeding, purified NT2 neurons rapidly extended neurites, resulting in a large increase in plasma membrane surface area with the concomitant appearance of de novo glycoconjugates and SiaLev on the cell surface. This implies that expression of SiaLev-containing glycoconjugates on the cell membrane might be a useful method for monitoring membrane synthesis and glycoconjugate turnover.
The ability to incorporate SiaLev residues into PSA-NCAM was demonstrated by resistance to C.P. sialidase, an exoglycosidase that is unable to cleave unnatural sialic acids modified at the N-acyl group (Yarema et al., 1998). The polysialylation of NCAM is important in both the developing and adult central nervous system; it is thought to mediate cell migration, axon outgrowth and synaptic remodeling. However, most conclusions regarding the activity of PSA have been derived from studies in which PSA was either absent or removed. The ability to metabolically label PSA on NCAM could provide significant information regarding its temporal and spatial expression and its relation to neuronal behavior. Further work is necessary to establish that polysialyltransferases can incorporate SiaLev into the inner segment of PSA before it can be used for such applications.
Metabolic labeling of PSA-NCAM and other neuronal glycoconjugates with SiaLev offers the potential to study activity-induced changes in neuronal networks. Recent studies have shown that plasticity in the CNS involves the formation of new synaptic structures (Toni et al., 1999). Incorporation of SiaLev into cell surface glycoconjugates appears to be dependent on de novo membrane and glycoconjugate biosynthesis. Since the biotinylation of SiaLev in cell surface glycoconjugates can be readily performed under physiological conditions, ManLev could potentially be a useful tool for visualizing physical changes in neuronal networks. By the nondestructive labeling of structures that have undergone remodeling it may be possible to identify and map neuronal networks involved in synaptic plasticity.
Most importantly, the results herein indicate that polysialyltransferases recognize unnatural analogs of CMP-sialic acid and can biosynthesize modified PSA in living cells. This creates a new avenue for biological investigation, as a correlation can be made between modified PSA structure and function. Other modifications to sialic acid might also be tolerated by the polysialyltransferases, and the extent of this is a subject worthy of further study.
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Materials and methods |
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Materials and antibodies
The anti-NCAM mouse monoclonal antibody (mAb) NCAM-OB11, goat polyclonal anti-biotin antibody, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody were obtained from Sigma. Fluorescein- and rhodamine-conjugated secondary antibodies and normal donkey and rabbit sera were obtained from Jackson Immunoresearch. The rat anti-PSA mAb 12F8 was obtained from Pharmingen. The mouse anti-PSA mAb 735 and the PSA-specific endoneuraminidase endo NE were the generous gifts of Dr. Rita Gerady-Schahn (Institut für Medizinishe, Mikrobiologie, Hanover, Germany). Neuraminidase from Clostridium perfringens (C.P. sialidase) was purchased from Boehringer Mannheim. Matrigel was obtained from Collaborative Biosciences. Epidermal growth factor (EGF) and biotinamidocaproyl hydrazide (biotin-X-hydrazide) were obtained from Calbiochem. All cell culture media were obtained from Irvine Scientific, unless specified otherwise. N-Levulinoylmannosamine (ManLev) was synthesized as described previously (Yarema et al., 1998
).
Cell culture
NT2 cells are a human embryonic carcinoma cell line that differentiates into neurons when treated with retinoic acid (Andrews, 1984; Lee and Andrews, 1986). Pure cultures of post-mitotic neurons can be obtained by a combination of multiple platings and treatment with inhibitors to mitotic growth (Pleasure et al., 1992). Briefly, NT2 cells were maintained in Opti-MEM-I (Gibco) supplemented with 5% heat-inactivated fetal bovine serum (HI-FBS), 1% penicillin/streptomycin (P/S). For differentiation, 7 x 106 NT2 cells were seeded into a T225 tissue culture flask in Dulbecco’s modified Eagle’s medium with high glucose (DMEM-HG) containing 10% HI-FBS, 1% P/S and 10 µM retinoic acid. The cultures were fed two times/week for 5 weeks. After differentiation, the cells were dissociated with trypsin/EDTA and split 1:6 in DMEM-HG containing 10% HI-FBS and 1% P/S. Two days later, NT2 neurons were mechanically dislodged following a 2 min incubation with 0.05% trypsin (Sigma, 1:250) in PBS. The purified neurons were then plated onto either mouse primary astrocytes or Matrigel in DMEM-HG supplemented with 5% HI-FBS, 1% P/S, 1 µM 1-ß-D-arabinofuranosylcytosine (Ara C), 10 µM fluorodeoxyuridine and 10 µM uridine.
Mouse primary astrocyte cultures were generated from cortices of neonatal pups essentially as described (Banker and Goslin, 1991) with the exception that the cells were seeded into DMEM-HG, 5% HI-FBS, 5% heat-inactivated horse serum, 1% P/S and 10 ng/ml EGF at 1.5 x 105 cells per ml (Turetsky et al., 1993). The cultures were allowed to reach confluency without feeding for 2 weeks before plating NT2 neurons. For immunofluorescence studies, the astrocytes were cultured on poly-L-lysine (Sigma)-coated German coverglass (Assistant, Carolina Biological) as described previously (Banker and Goslin, 1991).
Detection of ketones by fluorescence microscopy
NT2 neurons were treated with varying concentrations of ManLev or ManNAc at the time of plating onto primary mouse astrocytes grown on coverslips. The cultures were incubated with either sugar for 5 days without feeding. NT2 neuron/astrocyte cultures were then washed three times with biotin-staining buffer [BSB: 1% HI-FBS in calcium and magnesium containing phosphate-buffered saline (CMPBS, Gibco), pH 6.5]. The cultures were incubated with 2.5 mM biotin-X-hydrazide in CMPBS with 0.5% HI-FBS at pH 6.5 for 1.5 hours at room temperature. After biotinylation, the cultures were washed three times with labeling buffer (CMPBS, 0.5% HI-FBS, 15 mM sodium azide) at 4°C and incubated with 1 µg/ml goat anti-biotin antibody in labeling buffer for 15 min at 4°C. Following three washes with labeling buffer to remove unbound excess antibody, the cultures were rinsed in CMPBS and fixed with methanol for 20 min at –20°C. The fixed cells were then rehydrated with PBS, blocked with 5% normal donkey serum, and washed with PBS. The samples were incubated with 1:100 dilution of fluorescein-conjugated donkey anti-goat IgG, washed with PBS and mounted with Vectashield mounting medium. Cells were visualized by fluorescence microscopy.
PSA detection on NT2 neurons by fluorescence microscopy
NT2 neurons were seeded onto primary mouse astrocytes grown on coverslips and treated with varying concentrations of ManLev or ManNAc for 5 days. The cultures were fixed and permeabilized with methanol for 20 min at –20°C. The fixed cells were then rehydrated with PBS, blocked with 2.5% normal rabbit serum, and washed with PBS. The samples were incubated with 1:200 dilution of mouse anti-PSA mAb 12F8 for 1 h and washed with PBS. The cells were stained with a 1:100 dilution of rhodamine-conjugated rabbit anti-mouse IgM, washed with PBS, mounted with Vectashield mounting medium and visualized by fluorescence microscopy.
Western blot analysis of PSA-NCAM
NT2 neurons were seeded onto Matrigel and treated with varying concentrations of ManLev or ManNAc for 5 days. The cells were then lysed in pH modified Laemmli buffer (60 mM Tris–HCl, pH 8.0, 10% glycerol, 2% SDS, 0.5 M dithiothreitol) for 5 min at 4°C, boiled for 10 min and centrifuged at 12,000 x g for 10 min. Samples were then separated on a 7% SDS–PAGE gel and transferred onto a nitrocellulose filter (Bio-Rad). The filter was blocked for 2 h at room temperature with block buffer (PBS, 3% BSA, 0.01% Tween 20), and incubated for 1 h at room temperature with 1:10,000 mAb 735, or 1:1000 NCAM OB11. Filters were then washed with PBS-T (PBS, 0.01% Tween-20), and incubated with horseradish peroxidase-conjugated anti-mouse IgG. After washing with PBS-T, filters were visualized using Supersignal West Dura Extended Duration substrate (Pierce).
Enzymatic digestion of sialic acids on NT2 neuron glycoconjugates
NT2 neurons were seeded onto either primary mouse astrocytes grown on coverslips or onto Matrigel-coated plates. The cultures were incubated for 5 days in the presence of varying concentrations of ManNAc or ManLev. Digestion of sialic acids by the neuraminidase from Clostridium perfringens was performed in bicarbonate-free modified Eagle’s medium (Gibco), supplemented with 20 mM glucose and adjusted to pH 5.75 at 37°C. The cultures were washed several times, and then incubated with 100 mU of enzyme for 1 h at 37°C in a 5% CO2 atmosphere. Endoneuraminidase (endo NE) digestion was performed using 56 ng of enzyme in 1 ml serum-free DMEM-HG media for 1 h at 37°C. For analysis by immunofluorescence, NT2 neuron/astrocyte cultures on coverslips were fixed and stained for PSA immunoreactivity or ketone incorporation as described above. For analysis by immunoblot, NT2 neurons cultured on Matrigel were lysed in pH modified Laemmli buffer as described above.
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N.W.C. thanks Sophia Ryzhikov for her excellent technical assistance. L.K.M. was supported by a graduate fellowship from the American Chemical Society Division of Medicinal Chemistry. Support from the W.M.Keck Foundation is also gratefully acknowledged. This research was supported by the Director, Office of Energy Research, Office of Basic Energy Sciences, Division of Materials Sciences, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098, the Office of Naval Research (Grant No. N00014–98–1–0605), and the National Institutes of Health (GM58867–01 and DK09765).
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Biotin-X-hydrazide, biotinamidocaproyl hydrazide; C.P. sialidase, neuraminidase (exoglycosidase) from Clostridium perfringens; endo NE, endoneuraminidase from bacteriophage PK1E; mAb, monoclonal antibody; ManLev, N-levulinoylmannosamine; ManNAc, N-acetylmannosamine; NCAM, neural cell adhesion molecule; PSA, polysialic acid; SiaLev, N-levulinoyl sialic acid.
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Footnotes |
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1 Authors contributed equally
2 To whom correspondence should be addressed
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References |
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