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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Key words: polysialyltransferase/NCAM/sialic acid/biosynthesis
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Introduction |
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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 -2
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 Wilms 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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Results |
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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 2030 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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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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Discussion |
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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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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 Dulbeccos modified Eagles 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 TrisHCl, 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% SDSPAGE 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 Eagles 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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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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