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Originally published In Press as doi:10.1074/jbc.M111619200 on January 10, 2002

J. Biol. Chem., Vol. 277, Issue 11, 9255-9261, March 15, 2002
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Differential Effects of Unnatural Sialic Acids on the Polysialylation of the Neural Cell Adhesion Molecule and Neuronal Behavior*

Neil W. CharterDagger §, Lara K. Mahal||, Daniel E. Koshland Jr.Dagger , and Carolyn R. BertozziDagger **

From the Departments of  Chemistry and Dagger  Molecular and Cell Biology and the ** Howard Hughes Medical Institute, University of California, Berkeley, California 94720

Received for publication, December 5, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCODURES
RESULTS
DISCUSSION
REFERENCES

In this study we have examined how unnatural sialic acids can alter polysialic acid expression and influence the adhesive properties of the neural cell adhesion molecule (NCAM). Unnatural sialic acids are generated by metabolic conversion of synthetic N-acyl mannosamines and are typically incorporated into cell-surface glycoconjugates. However, N-butanoylmannosamine and N-pentanoylmannosamine are effective inhibitors of polysialic acid (PSA) synthesis in stably transfected HeLa cells expressing NCAM and the polysialyltransferase STX. These cells were used as substrates to examine the effect of inhibiting PSA synthesis on the development of neurons derived from the chick dorsal root ganglion. N-butanoylmannosamine blocked polysialylation of NCAM and significantly reduced neurite outgrowth comparable with enzymatic removal of PSA by endoneuraminidases. As a result, neurite outgrowth was similar to that observed for non-polysialylated NCAM. In contrast, previous studies have shown that N-propanoyl sialic acid (SiaProp), generated from N-propanoylmannosamine, is readily accepted by polysialyltransferases and permits the extension of poly(SiaProp) on NCAM. Despite being immunologically distinct, poly(SiaProp) can promote neurite outgrowth similarly to natural polysialic acid. Thus, subtle structural differences in PSA resulting from the incorporation of SiaProp residues do not alter the antiadhesive properties of polysialylated NCAM.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCODURES
RESULTS
DISCUSSION
REFERENCES

Polysialic acid (PSA)1 is a unique carbohydrate because of its unusual structure and importance during neuronal development, synaptic plasticity, and tumor metastasis. PSA is a linear polysaccharide composed entirely of alpha 2,8-linked sialic acid that can reach up to 200 residues in length in mammalian cells (1-3). There are two major polysialyltransferases, STX and PST, which are responsible for polysialylation (4). STX (ST8SiaII) is predominately expressed during development, whereas PST (ST8SiaIV) is the major polysialyltransferase expressed in the adult central nervous system (5-11). The neural cell adhesion molecule (NCAM) appears to be, by far, the primary substrate for polysialylation because the absence of NCAM expression results in the almost complete loss of PSA immunoreactivity (12). NCAM is a major cell adhesion molecule in the central nervous system and is thought to operate by promoting cell-cell interactions via homophilic binding of NCAM molecules on apposing membranes (13). Glycosylation of NCAM by PSA significantly reduces the ability of NCAM molecules to interact and as a result enhances cell migration (14, 15).

During central nervous system development, the majority of NCAM is highly polysialylated and has a major role in axon pathfinding, target innervation, and fasciculation. In the adult, polysialylation is restricted to only those regions of the central nervous system that remain plastic and is involved in processes underlying memory formation (12, 16-19). In addition to its normal occurrence, PSA-NCAM has been associated with an increasing number of cancers, including the small cell lung carcinoma, neuroblastomas, and Wilm's tumor (5, 20), and its expression is associated with metastasis and poor patient prognosis (21).

The role that polysialylation of NCAM has in altering neuronal behavior is not fully understood. One approach to studying the effect of glycosylation on protein function and cell behavior involves the use of unnatural saccharide derivatives that can substitute for the native sugar in glycosides (22). The sialic acid biosynthesis pathway is particularly amenable because it is permissive for replacement of the natural precursor N-acetylmannosamine with synthetic derivatives that contain modifications at the N-acyl side chain (23-26). This approach has been used to study the effects of unnatural sialic acid derivatives on viral binding, glial cell proliferation, and myelin-associated glycoprotein binding to neural cells (27-29).

Polysialyltransferases catalyze a significantly different reaction from other types of sialyltransferases, namely the formation of polymeric alpha 2,8-linked sialic acids. Because sialic acids serve as both glycosyl donor and acceptor for these enzymes, we considered that unnatural sialic acids might dramatically affect their efficiency. We previously demonstrated that cultured human neurons were capable of incorporating low levels of N-levulinoyl sialic acid into PSA-NCAM, which indicated that polysialyltransferases were able to accept unnatural sialic acids in the synthesis of PSA (30). Mammalian cells appear to readily utilize N-propanoyl sialic acid in PSA synthesis, resulting in the incorporation of SiaProp sialosides in PSA on NCAM (31). The expression of poly(SiaProp) on NCAM results in altered antigenicity compared with natural polysialic acid. However, our studies also demonstrated that N-butanoylmannosamine, which possesses an additional methylene group at the N-acyl domain, is an effective inhibitor of PSA synthesis in mammalian cells (32).

Given the wide range of roles that PSA-NCAM plays in both the central nervous system and disease, we sought to examine the effect that unnatural sialic acid analogs can have on cell behavior that is regulated by PSA expression. Here we compare how unnatural poly(SiaProp) (generated by N-propanoylmannosamine treatment) and PSA synthesis inhibition (as a result of N-butanoylmannosamine treatment) affect the ability of neurons to project neurites.

    EXPERIMENTAL PROCODURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCODURES
RESULTS
DISCUSSION
REFERENCES

Materials and Antibodies-- The anti-NCAM mouse monoclonal antibody (mAb) NCAM-OB11 was obtained from Sigma. The rat anti-PSA mAb 12F8 was obtained from BD PharMingen. The mouse anti-NF160 monoclonal antibody RM0270 was obtained from Zymed Laboratories Inc. The mouse anti-PSA mAb 735 was a generous gift from Dr. Rita Gerady-Schahn (Institut für Medizinishe, Mikrobiologie, Hanover, Germany). Conjugated secondary antibodies and normal goat and normal rabbit sera were obtained from Jackson Immunoresearch. Poly-D-lysine was obtained from Sigma. Matrigel was obtained from Collaborative Biosciences. Nerve growth factor was obtained from Calbiochem. All cell culture media were obtained from Invitrogen, unless specified otherwise.

Synthesis of Unnatural Mannosamine Derivatives-- Synthesis of N-propanoylmannosamine, N-butanoylmannosamine, and N-pentanoylmannosamine and their peracetylated derivatives was performed as previously described (23, 32).

Cell Lines and Culture-- Clonal HeLa cell lines that stably expressed NCAM-140 with or without concomitant expression of either polysialyltransferases STX or PST were a generous gift from Dr. Minoru Fukuda (The Burnham Institute) (7, 8). The cells were maintained in Dulbecco's modified Eagle's medium/10% heat-inactivated fetal bovine serum supplemented with 400 µg/ml G418 and 400 µg/ml hygromycin (HeLa-NCAM/STX and HeLa-NCAM/PST only).

Detailed methods for the culture of NT2 cells and differentiation into terminally differentiated neurons have been described previously (30, 33-35). Purified NT2 neurons were seeded onto poly-D-lysine- and Matrigel-coated 12-well cultures plates.

Quantification of PSA Expression by Whole Cell ELISA-- PSA expression was quantified using modified whole cell ELISA (36). HeLa-NCAM/STX and HeLa-NCAM/PST cells, seeded on 96-well culture plates (Corning), were incubated for 3 days with various concentrations of mannosamine derivatives. The cells were washed and fixed for 20 min with MeOH at -20 °C. Following fixation, the cells were rehydrated for 10 min with PBS/0.1% Triton X-100 (PBS-X) and blocked for 30 min with blocking buffer containing 2.5% normal rabbit serum in PBS-X. Cells were incubated with a 1:1000 dilution of rat anti-PSA monoclonal antibody (12F8, BD PharMingen) in blocking buffer for 1 h. The plates were washed twice with PBS-X, treated with ImmunoPure peroxidase suppressor (Pierce) to suppress endogenous peroxidase activity, and washed another two times with PBS-X. The cells were incubated for 1 h with a 1:1000 dilution of rabbit anti-rat IgM-horseradish peroxidase conjugate in blocking buffer. After washing with PBS-X, the cells were incubated with Turbo-TMB substrate buffer (Pierce) for 15 min, and the reaction was quenched with 1 N H2SO4. Anti-PSA labeling was measured using a SpectroMax 340 plate reader (Molecular Devices, Sunnyvale, CA) at an absorbance of 450 nm.

Concentration-response curves were fitted to the data with SoftMax Pro (Molecular Devices) using the equation E = ((E0 - Emax)/(1 + (L/IC50)n)) + Emax, where E0 is the level of PSA expression in the absence of sugar, Emax is the level of PSA expression in the presence of maximally effective concentration of sugar, L is the concentration of the sugar, IC50 is the concentration at which a half-maximal effect is produced, and n is the Hill coefficient.

Immunological Analysis of PSA-NCAM Expression-- Detailed methods for the detection of PSA and NCAM by Western analysis using the anti-PSA mAbs 735 and the anti-NCAM mAb OB11 (Sigma) have been described previously (30). Whole cell lysates were generated from HeLa-NCAM/STX and HeLa-NCAM/PST cells following incubation for 3 days with various concentrations of mannosamine derivatives. Purified NT2 neurons were seeded onto poly-D-lysine- and Matrigel-coated culture plates and incubated for 5 days with varying concentrations of mannosamine derivatives prior to lysis for Western analysis.

Assay for Neurite Outgrowth on HeLa Cells Stably Expressing NCAM and PSA-- PSA expression was found to be highly unstable in NCAM- and PST-transfected HeLa cells resulting in heterogeneous cultures that expressed either NCAM only or PSA-NCAM. Therefore, the effect of PSA expression on neurite growth was only examined using HeLa substratum cells that stably expressed NCAM and STX. Sensory neurons from the dorsal root ganglia were isolated from 10-day-old chick embryos, dissociated with 0.5% trypsin, and seeded onto confluent cultures of HeLa monolayer in minimal essential medium containing 10% fetal bovine serum and 10 ng/ml nerve growth factor (7, 8). The neuron/HeLa co-cultures were grown for 15 h, fixed using 4% formaldehyde in PBS, stained with antineurofilament mAb RM0270 (37), and visualized by immunofluorescence. Neurite length analysis was performed on a Macintosh computer using the public domain NIH Image program (developed at the National Institutes of Health and available on the Internet at rsb.info.nih.gov/nih-image). The mean neurite lengths per neuron were compared among the different substrate conditions using one-way ANOVA analysis. Comparisons between individual assays were made after normalizing neurite outgrowth to that on HeLa-NCAM cells.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCODURES
RESULTS
DISCUSSION
REFERENCES

The Effect of N-Acyl Mannosamine Derivatives on PSA Expression in NCAM- and STX- or PST-transfected HeLa Cells-- To compare the effect of unnatural N-acyl mannosamine derivatives on the biosynthesis of PSA mediated by the polysialyltransferases PST and STX separately, HeLa cells expressing NCAM and either STX or PST were incubated with N-propanoylmannosamine (ManProp), N-butanoylmannosamine (ManBut), or N-pentanoylmannosamine (ManPent). These derivatives possess an additional one, two, or three methylene groups, respectively, at the N-acyl domain when compared with the natural precursor in sialic acid biosynthesis N-acetylmannosamine (ManNAc). PSA expression was examined with a whole cell ELISA using the PSA-specific antibody 12F8 (Fig. 1). Both ManBut and ManPent treatment resulted in a dose-dependent loss of anti-PSA staining that was complete at 10 mM for ManBut and 30 mM for ManPent. Therefore, ManBut appeared to be the more potent of the two unnatural derivatives for blocking PSA staining, with an IC50 of 2.7 mM for HeLa-NCAM/STX and 2.0 mM for HeLa-NCAM/PST cells. ManPent inhibited anti-PSA labeling with slightly higher IC50 of 4.1 mM for HeLa-NCAM/STX and 5.9 mM for HeLa-NCAM/PST cells. In contrast, ManProp gave complex results, which will be discussed in greater detail later in this section.


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  		Fig. 1.   Effects of ManProp, ManBut, and ManPent on PSA expression in HeLa-NCAM/STX and HeLa-NCAM/PST cells. HeLa-NCAM/STX and HeLa-NCAM/PST cells were cultured in 96-well plates for 3 days in the presence of various concentrations of ManPent, ManBut, ManProp, or ManNAc. PSA immunoreactivity was determined by ELISA using anti-PSA mAb 12F8 and quantified by colorimetry. Each data point is the mean percent anti-PSA labeling ± S.E. of four determinations from a representative experiment.

Because the whole cell ELISA measures anti-PSA labeling, the loss of labeling observed may be due to the inability of the anti-PSA antibody 12F8 to recognize PSA containing unnatural sialosides. To determine whether the loss of PSA labeling was due to the inhibition of PSA synthesis or to the incorporation of SiaBut residues in PSA, total protein from ManBut-treated HeLa-NCAM/STX was analyzed by immunoblot using antibodies specific to PSA (mAb 735) or NCAM (mAb OB11) (Fig. 2). Loss of PSA labeling and a concomitant reduction in the apparent molecular mass of NCAM consistent with a lack of polysialylation were observed at 3 mM ManBut. Therefore the primary effect of ManBut treatment appears to be the inhibition of PSA synthesis, and the measured IC50 value of 2.7 mM appears to be accurate. Western analysis of PSA synthesis by PST was complicated by the fact that its expression or activity was highly unstable in HeLa-NCAM/PST cells. As a result, two populations of cells were present, one expressing NCAM-PSA and the second expressing only NCAM (Fig. 2). A number of attempts were made to generate a single population of NCAM-PSA-expressing cells, but in each case the cultures quickly reverted to a mixed population of PSA- and non-PSA-expressing cells. Because ManBut readily inhibited PSA synthesis in HeLa-NCAM/STX cells, these cells were used as a substrate for neuronal cell growth to determine the effect of ManBut treatment on neurite outgrowth.


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  		Fig. 2.   Comparison of the effects of ManProp and ManBut on polysialylation of NCAM in HeLa-NCAM/STX cells. HeLa-NCAM/STX cells were incubated with various concentrations of ManProp or ManBut for 3 days. Western analysis was performed on whole cell lysates from treated cells using the anti-NCAM mAb OB11 (alpha NCAM) and the anti-PSA antibody 735 (alpha PSA). Note that PSA expression in HeLa-NCAM/PST cells (P) was very poor, and the majority of NCAM appeared not to be polysialylated. Immunoreactivity was detected using an HRP-conjugated secondary antibody and visualized by chemiluminescence.

ManProp treatment of either HeLa-NCAM/STX or HeLa-NCAM/PST cells resulted in a dose-dependent reduction but not a complete loss of anti-PSA labeling by ELISA (Fig. 1). At concentrations up to 30 mM ManProp, anti-PSA labeling using the mAb 12F8 could still be detected in HeLa-NCAM/STX or HeLa-NCAM/PST cells, whereas comparable doses of ManBut and ManPent had resulted in a complete loss of anti-PSA staining by ELISA. Analysis of whole cell lysates from ManProp-treated HeLa-NCAM/STX cells revealed a dose-dependent loss in PSA staining when the anti-PSA-specific antibody 735 was used (Fig. 2). In contrast to the effect observed with ManBut, the loss of anti-PSA labeling as a result of ManProp treatment was not associated with a concomitant reduction in molecular mass of NCAM to non-polysialylated levels. Rather, NCAM migrated with an apparent molecular mass in excess of 200 kDa, which was indicative of a high degree of polysialylation.

Incubation with ManProp treatment has previously been shown to generate SiaProp-containing PSA in tumor and neuronal cells and to result in the loss of mAb 735 immunoreactivity (31, 32). In contrast, the anti-PSA antibody 12F8 appears to tolerate the inclusion of unnatural SiaProp sialosides in PSA. Although a decrease in labeling of PSA by 12F8 was observed in ManProp-treated cells (Fig. 1), there was a corresponding dose-dependent reduction in the molecular mass of NCAM by Western analysis (Fig. 2). Taken together, these data indicate that in HeLa-NCAM/STX cells, ManProp treatment results in a reduction of the degree of polymerization of PSA. Hence, 12F8 immunoreactivity does not appear to be affected by the inclusion of SiaProp sialosides in PSA. Despite the observation that ManProp reduces polymer length of PSA in HeLa-NCAM/STX cells, the data presented here demonstrate that treatment of these cells with ManProp results in the expression of SiaProp-containing PSA. Therefore, these cells can be used as substrate on which to examine the effect of poly(SiaProp) on PSA-mediated neurite outgrowth.

Acetylation Increases the Potency of Mannosamine Derivatives in NT2 Neurons-- All of the mannosamine derivatives used in this and previous studies have poor membrane permeability, demanding millimolar concentrations to observe metabolic effects. Therefore, we synthesized more lipophilic, acetylated derivatives that can diffuse passively through the plasma membrane. Once inside the cell, these acetyl groups are hydrolyzed by nonspecific esterases (38). We examined whether the acetylated forms of ManProp (Ac4ManProp), ManBut (Ac4ManBut), and ManPent (Ac4ManPent) derivatives were able to inhibit PSA synthesis at lower concentrations than the non-acetylated counterparts without having any detrimental side effects on cell survival.

Human NT2 neurons were treated for 5 days with varying concentrations of acetylated N-acyl mannosamine derivatives, and whole cell lysates were examined by Western analysis for PSA and NCAM expression. Ac4ManBut or Ac4ManPent treatment resulted in a dose-dependent loss in anti-PSA labeling that was apparent at 10 µM and complete at 30 µM (Fig. 3). PSA inhibition in NT2 neurons with 30 µM of the acetylated derivatives was comparable with 1 mM of the non-acetylated counterparts and indicated that acetylation increased inhibitory potency by at least 30-fold (32). The loss of PSA immunoreactivity was associated with a concomitant reduction in the molecular mass of NCAM to less than 200 kDa, indicative of the inhibition of PSA synthesis (Fig. 2C).


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  		Fig. 3.   The effects of acetylated ManProp, ManBut, and ManPent on polysialylation of NCAM in HeLa-NCAM/STX cells. HeLa-NCAM/STX cells were incubated with various concentrations of the acetylated forms of ManNAc (Ac4ManNAc), ManProp (Ac4ManProp), ManBut (Ac4ManBut), or ManPent (Ac4ManPent) for 3 days. Western analysis was performed on whole cell lysates from treated cells using the anti-NCAM mAb OB11 (alpha NCAM) and the anti-PSA antibody 735 (alpha PSA). Immunoreactivity was detected using a horseradish peroxidase-conjugated secondary antibody and visualized by chemiluminescence.

Treatment of NT2 neurons with Ac4ManProp had a similar effect as unprotected ManProp in reducing PSA immunoreactivity without affecting the polysialylation of NCAM. Loss of PSA immunoreactivity was apparent at 30 µM Ac4ManProp, similar to the effect of 3 mM ManProp on NT2 neurons in earlier studies (32). Therefore, as with the acetylated forms of ManBut and ManPent, Ac4ManProp possessed similar properties to ManProp in altering the composition of polysialic acid but at significantly lower doses.

In performing these experiments, we observed that incubation of NT2 neurons with higher concentrations of the acetylated N-acyl mannosamine derivatives (100 µM or above) appeared to be toxic to the cells. The toxicity did not appear to be due to the modified N-acyl domain itself because acetylated ManNAc had a similar effect. We were concerned that because HeLa-NCAM/STX cells typically require a 3-fold higher dose of N-acyl mannosamine derivatives to produce similar alterations in PSA synthesis compared with NT2 neurons, it was likely such doses may have a significant effect on cell viability. Therefore, despite the observation that acetylated N-acyl mannosamine derivatives are effective at significantly lower dosages than the free sugars, the possible effect of acetylated mannosamine derivatives on cell viability led us to conduct our studies on modifying neurite outgrowth with the non-acetylated forms that exhibit no apparent toxicity.

PSA Inhibition by ManBut Reduces Neurite Outgrowth in Chick Dorsal Root Ganglion Neurons-- The expression of polysialic acid on neural cells has a significant effect on cell migration and axon outgrowth (8, 14, 39). We examined the effects of ManBut, a PSA synthesis inhibitor, on the neurite outgrowth of chick dorsal root ganglia neurons when cultured on a substratum of HeLa-NCAM/STX cells. Consistent with earlier studies, we found that neurite outgrowth was promoted by the polysialylation of NCAM on HeLa cell substrata (7, 8, 40). In one such experiment, summarized in Fig. 4, neurons cultured on HeLa cells expressing NCAM alone exhibited modest outgrowth (137.8 ± 7.5 µm, n = 58), whereas those grown on HeLa cells expressing NCAM and the polysialyltransferase STX produced significantly longer neurites (182.2 ± 7.4 µm, n = 70; p < 0.001). Similar behavior was observed in other experiments, and a high degree of correlation was observed between experiments after normalizing neurite outgrowth to that observed on NCAM (Fig. 5B). Typically, polysialylation of NCAM on substrate cells was found to promote neurite outgrowth by over 35% (STX: 138.2 ± 2.5%, compared with normalized outgrowth on NCAM of 100%, in five independent experiments).


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  		Fig. 4.   Inhibition of PSA expression in substratum cells by ManBut reduces neurite outgrowth of chick dorsal root ganglia neurons. HeLa cells expressing either NCAM-140 alone or NCAM and STX were cultured in the presence or absence of 10 mM ManBut. Sensory neurons from embryonic chick dorsal root ganglia were seeded onto the resulting monolayers. A, after 15 h incubation, the co-cultures were fixed, stained for neurofilament, and visualized by fluorescence microscopy (magnification ×200). B, the length of the neurites was measured using NIH Image. For each condition, the mean neurite length per neuron was determined. The data presented here are from a representative experiment and show the average neurite length ± S.E. of over 40 neurons per condition (see "Results"). Significant differences shown between data points were calculated by one-way ANOVA.


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  		Fig. 5.   Comparison of the effects of ManBut on neurite outgrowth between individual experiments and on the distribution of neurite lengths. HeLa cells expressing either NCAM-140 alone or NCAM and STX were cultured in the presence or absence of 10 mM ManBut. Sensory neurons from embryonic chick dorsal root ganglia were seeded onto the resulting monolayers. A, the correlation between individual assays was determined by normalizing average neurite length (per neuron) to that on HeLa-NCAM substrate cells. Each data point represents the average neurite length from five independent assays ± S.E. Significant differences shown between data points were calculated by one-way ANOVA. B, distribution of neurite lengths of different substrata. The percentage of neurites that were identical to or longer than a given size for each substrate was determined.

As illustrated in Fig. 3, a 3-day treatment with 10 mM ManBut is sufficient to completely inhibit PSA synthesis in HeLa-NCAM/STX cells. Therefore we compared neurite outgrowth on ManBut-treated HeLa-NCAM/STX cells and observed a significant reduction in the length of neurites compared with untreated HeLa-NCAM/STX cells (STX+ManBut: 142.4 ± 6.9 µm, n = 68 versus STX: 182.2 ± 7.4 µm, p < 0.001) (Fig. 4). Neurite outgrowth on HeLa-NCAM/STX cells was reduced by ManBut treatment to the extent that there was no significant difference with outgrowth observed on HeLa-NCAM substrate cells that lack STX. Overall, in five independent experiments, the inhibition of PSA synthesis by ManBut resulted in a 38% decrease in neurite outgrowth (STX+ManBut: 100.9 ± 4.0% versus STX: 138.2 ± 2.5%) (Fig. 5A).

This suggested that the loss of enhanced neurite outgrowth as a result of ManBut treatment was because of the loss of PSA expression. However, in addition to inhibiting PSA synthesis, ManBut treatment will also result in the incorporation of SiaBut residues into other cell-surface sialoglycoconjugates (26) and could possibly affect the ability of the HeLa cells to act as a suitable substrate for neurite outgrowth. To address this, we treated HeLa-NCAM cells with ManBut and did not observe any effect on neurite length compared with untreated HeLa-NCAM cells, indicating that the presence of SiaBut-containing glycoconjugates did not alter neurite outgrowth (NCAM+ManBut: 143.6 ± 6.9 µm, n = 41 versus NCAM: 137.8 ± 7.5 µm, n = 58, not significant). Therefore, ManBut treatment exerted its influence on neurite outgrowth solely through the inhibition of PSA synthesis.

As shown in Fig. 5B, polysialylation of NCAM has a profound effect in promoting outgrowth throughout the population of neurites (40). Inhibition of PSA synthesis by ManBut resulted in a distribution of neurite lengths that was comparable with that observed on non-polysialylated NCAM. The inverse experiment, namely inhibiting PSA synthesis in chick neurons rather than their substratum, was not attempted because of the extreme difficulty in blocking PSA synthesis prior to seeding onto the substratum cells. In addition, the effectiveness of ManBut treatment on the inhibition of PSA synthesis by avian polysialyltransferases has not yet been determined.

Effect of Incorporation of SiaProp Residues into PSA on Neurite Outgrowth-- In contrast to the inhibitory effect of ManBut on PSA synthesis, the derivative ManProp, which possesses one less methylene group in the N-acyl group, is readily utilized by mammalian cells in the synthesis of SiaProp-containing polysialic acid. A consequence of the expression of poly(SiaProp) in PSA is altered immunoreactivity (31, 41). Changes in epitope structure of PSA as a result of the inclusion of SiaProp residues clearly can have a profound effect on antibody recognition. We were curious whether the subtle alterations in the structure of PSA as a consequence of the additional methylene group at the N-acyl domain of SiaProp was significant enough to alter the antiadhesive properties of PSA. Therefore, we compared the ability of neurons to project neurites on substrate cells expressing poly(SiaProp) with outgrowth on naturally occurring PSA.

The results summarized in Fig. 6 clearly demonstrate that treatment of HeLa-NCAM/STX cells with 10 mM ManProp did not have any apparent effect on the neurite outgrowth. In one such experiment (Fig. 6A), neurite outgrowth on ManProp-treated HeLa-NCAM/STX cells was not significantly different from outgrowth on untreated HeLa-NCAM/STX cells (STX+ManProp: 171.6 ± 6.2 µm, n = 105 versus STX: 171.5 ± 6.3 µm, n = 97). In contrast, outgrowth on substrate cells expressing non-polysialylated NCAM was significantly lower than on HeLa-NCAM/STX substrates, irrespective of whether the cells were treated with ManProp (NCAM: 132.5 ± 7.0 µm, n = 89, p < 0.001).


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  		Fig. 6.   Inhibition of PSA expression in substratum cells by ManBut reduces neurite outgrowth of chick dorsal root ganglia neurons. HeLa cells expressing either NCAM-140 alone or NCAM and STX were cultured in the presence or absence of 10 mM ManProp. Sensory neurons from embryonic chick dorsal root ganglia were seeded onto the resulting monolayers. A, the length of the neurites was measured using NIH Image. For each condition, the mean neurite length per neuron was determined. The data presented here are from a representative experiment and show the average neurite length ± S.E. of over 70 neurons per condition (see "Results"). Significant differences shown between data points were calculated by one-way ANOVA. B, the correlation between individual assays was determined by normalizing average neurite length (per neuron) to that on HeLa-NCAM substrate cells. Each data point represents the average neurite length from three independent assays ± S.E. Significant differences shown between data were calculated by one-way ANOVA. C, distribution of neurite lengths of different substrata. The percentage of neurites which were identical to or longer than a given size for each substrate was determined.

Incubation of HeLa-NCAM/STX cells with ManProp will also result in the incorporation of SiaProp residues into other cell surface sialoglycoconjugates, which conceivably could affect neurite outgrowth independently of NCAM and PSA expression. However, no significant difference was observed in the neurite lengths on HeLa-NCAM substrate cells in the presence or absence of ManProp treatment (NCAM+ManProp: 133.4 ± 8.1 µm, n = 71). Similar data obtained from a total of three independent experiments showed a high degree of correlation after normalizing the results to outgrowth on HeLa-NCAM cells (Fig. 6B). Furthermore, incorporation of SiaProp residues had no apparent effect on the distribution of neurite lengths on substrates expressing polysialylated NCAM (Fig. 6C). Neurite outgrowth on ManProp-treated HeLa-NCAM/STX cells was promoted to a similar degree to those on a substrate of natural polysialic acid. In conclusion, these results demonstrate that ManProp can be used to readily incorporate unnatural SiaProp into polysialic acid without altering the antiadhesive properties of PSA.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCODURES
RESULTS
DISCUSSION
REFERENCES

Several studies have illustrated that the sialic acid biosynthesis machinery is permissive for a variety of N-acyl mannosamine precursors (26) and that the resulting N-acyl sialic acids are accepted by the majority of sialyltransferases (27). However, we have demonstrated that, although it has no effect on sialylation in general, ManBut is a metabolic inhibitor of PSA synthesis in vivo (32). Here we show that ManPent is also effective in blocking PSA synthesis. Therefore, these observations indicate that ManBut and ManPent are converted to downstream metabolites that interfere with polysialyltransferase activity. We have previously demonstrated that CMP-SiaBut can act as a substrate for polysialyltransferases in the synthesis of PSA in vitro, albeit at reduced efficiency compared with CMP-sialic acid (32). Moreover, NT2 neurons are able to incorporate low levels of N-levulinoyl sialic acid into PSA-NCAM (30). The ability of N-acyl mannosamine derivatives to act as inhibitors of PSA biosynthesis may be a consequence of poor substrate activity of the corresponding CMP-sialic acids and poor recognition of the growing PSA chain once a few residues of unnatural sialic acid have been installed. Given that polysialylation in vivo occurs during the passage of NCAM through the Golgi apparatus, a reduction in polysialyltransferase activity in the presence of CMP-SiaBut could result in little or no PSA biosynthesis on NCAM prior to the export of the glycoprotein to the cell surface.

Polysialylation has a significant impact on the ability of NCAM to promote cell-cell interactions and is important in such processes as cell migration, axon outgrowth, and synaptic plasticity. Previous studies have demonstrated that polysialylation of NCAM in substratum cells promotes the growth of neurites of chick sensory neurons (7, 8, 40). Here, we demonstrate that ManBut can block PSA synthesis in substratum cells expressing NCAM and the polysialyltransferase STX and significantly reduce the ability of neurons to project neurites. This effect was comparable with the effect observed with the enzymatic removal of PSA using the PSA-specific endoneuraminadase Endo NE (8). As a result, neurite outgrowth was substantially reduced to such an extent that it was similar to outgrowth on a substrate of non-polysialylated NCAM. Because ManBut treatment will result not only in the inhibition of PSA synthesis but also in the expression of SiaBut residues on other cell surface glycoconjugates, it is possible that the presence of unnatural sialic acids impeded neurite outgrowth, irrespective of PSA expression. However, ManBut treatment of substratum cells expressing NCAM alone had no significant effect on neurite outgrowth, indicating that the presence of SiaBut residues on sialoglycoconjugates was not inhibitory to neurite development. Therefore, we conclude that ManBut, as a specific inhibitor of PSA biosynthesis, can be used to modulate PSA-mediated cell behavior.

The ability to inhibit PSA synthesis is restricted to mannosamine derivatives that possess at least two additional methylene groups at the N-acyl domain, compared with the natural saccharide. ManProp, which possesses one less methylene unit than the PSA biosynthesis inhibitor ManBut, does not block synthesis of PSA. Instead, SiaProp readily replaces sialic acid in the synthesis of PSA. As a consequence of the inclusion of SiaProp sialosides, the resulting polysaccharide exhibits altered antigenic properties (31). Evidence that CMP-SiaProp is not as good a substrate for polysialyltransferases as CMP-sialic acid is provided by the reduced activity in vitro and a lower degree of polymerization in vivo with HeLa-NCAM/STX (32) (Fig. 2). Despite this, sufficient SiaProp-containing polysialic acid is generated on NCAM to promote neurite outgrowth of chick dorsal root ganglia neurons. Furthermore, the presence of an additional methylene group in the N-acyl group did not affect the ability of PSA to modulate NCAM-dependent neurite extension. This evidence indicates that replacement of sialic acid with SiaProp in PSA does not hinder the ability of the polymer to perform its antiadhesive function.

In summary, ManBut, as a PSA biosynthesis inhibitor, can modulate cell-cell interactions that are normally affected by polysialylation of NCAM. ManBut is the first example of a small molecule inhibitor of PSA biosynthesis, and here we have illustrated its potential for use in studying the role of PSA in modifying neuronal behavior important in development and plasticity. Because PSA-NCAM expression in tumor cells has been associated with metastasis and poor patient prognosis (5), the ability to block PSA synthesis using a small molecule inhibitor such as ManBut could significantly reduce the probability of cell migration and growth of PSA-expressing tumors (20). In contrast, SiaProp is readily incorporated into polysialic acid without altering its antiadhesive properties. The combination of incorporation of SiaProp in PSA without loss of function and the altered immunoreactivity of poly(SiaProp) provides a means to readily label and identify de novo expression of PSA.

    ACKNOWLEDGEMENTS

We thank Kiyohiko Angata and Minoru Fukuda for the kind donation of STX- and PST-transfected HeLa cell lines. N. W. C thanks Sofia Ryzhikov and Wendy Yan for excellent technical assistance in generating and isolating NT2 neurons and Danielle Dube for providing peracetylated-N-acetylmannosamine.

    FOOTNOTES

* This work was supported by the Director, Office of Energy Research, Office of Basic Energy Sciences, Div. of Materials Sciences, United States Dept. of Energy under Contract No. DE-AC03-76SF00098, by Office of Naval Research Grant N00014-98-1-0605, and by National Institutes of Health Grants GM58867-01 and DK09765. This research was also supported by the W. M. Keck Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 510-642-0529; Fax: 510- 643-6386; E-mail: ncharter@uclink4.berkeley.edu.

|| Supported by a graduate fellowship from the American Chemical Society Division of Medicinal Chemistry.

Published, JBC Papers in Press, January 10, 2002, DOI 10.1074/jbc.M111619200

    ABBREVIATIONS

The abbreviations used are: PSA, polysialic acid; ManNAc, N- acetyl-mannosamine; ManProp, N-propanoylmannosamine; ManBut, N-butanoylmannosamine; ManPent, N-pentanoylmannosamine; Ac4ManNAc, 1,3,4,6-tetra-O-acetyl-N-acetylmannosamine; Ac4ManProp, 1,3,4,6-tetra-O-acetyl-N-propanoylmannosamine; Ac4ManBut, 1,3,4,6-tetra-O-acetyl-N-butanoylmannosamine; Ac4ManPent, 1,3,4,6-tetra-O-acetyl-N-pentanoylmannosamine; ELISA, enzyme-linked immunosorbant assay; SiaProp, N-propanoyl sialic acid; SiaBut, N-butanoyl sialic acid; mAb, monoclonal antibody; NCAM, neural cell adhesion molecule; ANOVA, analysis of variance; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCODURES
RESULTS
DISCUSSION
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