Glycobiology Advance Access originally published online on December 12, 2007
Glycobiology 2008 18(2):187-194; doi:10.1093/glycob/cwm132
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CMP substitutions preferentially inhibit polysialic acid synthesis
4 Tumor Microenvironment Program, Glycobiology Unit, Cancer Research Center, Burnham Institute for Medical Research, La Jolla, CA 92037, USA
1 To whom correspondence should be addressed: Tel: +858-646-3144; Fax: +858-646-3193; e-mail: minoru{at}burnham.org
Received on August 15, 2007; revised on November 9, 2007; accepted on December 3, 2007
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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It is widely reported that derivatives of sugar moieties can be used to metabolically label cell surface carbohydrates or inhibit a particular glycosylation. However, few studies address the effect of substitution of the cytidylmonophosphate (CMP) portion on sialyltransferase activities. Here we first synthesized 2'-O-methyl CMP and 5-methyl CMP and then asked if these CMP derivatives are recognized by
2,3-sialyltransferases (ST3Gal-III and ST3Gal-IV), 2,6-sialyltransferase (ST6Gal-I), and 2,8-sialyltransferase (ST8Sia-II, ST8Sia-III, and ST8Sia-IV). We found that ST3Gal-III and ST3Gal-IV but not ST6Gal-I was inhibited by 2'-O-methyl CMP as potently as by CMP, while ST3Gal-III, ST3Gal-IV, and ST6Gal-I were moderately inhibited by 5-methyl CMP. Previously, it was reported that polysialyltransferase ST8Sia-II but not ST8Sia-IV was inhibited by CMP N-butylneuraminic acid. We found that ST8Sia-IV as well as ST8Sia-II and ST8Sia-III are inhibited by 2'-O-methyl CMP as robustly as by CMP and moderately by 5-methyl CMP. Moreover, the addition of CMP, 2'-O-methyl CMP, and 5-methyl CMP to the culture medium resulted in the decrease of polysialic acid expression on the cell surface and NCAM of Chinese hamster ovary cells. These results suggest that 2'-O-methyl CMP and 5-methyl CMP can be used to preferentially inhibit sialyltransferases, in particular, polysialyltransferases in vitro and in vivo. Such inhibition may be useful to determine the function of a carbohydrate synthesized by a specific sialyltransferase such as polysialyltransferase.
Key words:
2,8-sialyltransferase
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2'-O-methyl CMP
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5-methyl CMP
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polysialic acid
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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Glycosylation requires several enzyme-catalyzed steps to synthesize an activated form of a monosaccharide. For sialic acid synthesis, N-acetylglucosamine is first converted to N-acetylmannosamine by N-acetylglucosamine 2-epimerase. N-Acetylmannosamine is then phosphorylated to N-acetylmannosamine 6-phosphate by N-acetylmannosamine-kinase. The resulting N-acetylmannosamine 6-phosphate is conjugated to phosphoenolpyruvate by N-acetylneuraminic 9-phosphate synthase, through aldol condensation, forming N-acetylneuraminic 9-phosphate. The product in turn yields N-acetylneuraminic acid (NeuAc) by N-acetylneuraminic acid 9-phosphatase. N-Acetylneuraminic acid is then activated by conjugation to CTP by N-acetylneuraminyl cytidyltransferase, forming cytidyl N-acetylneuraminic acid 5'-phosphate, cytidylmonophosphate (CMP)-NeuAc. These reactions occur in the nucleus. In animal and plant cells, the transfer of sialic acid to acceptor carbohydrates takes place in the Golgi apparatus; thus, CMP-NeuAc needs to be transported to the lumen of the Golgi by the CMP-NeuAc transporter. Once there, CMP-NeuAc is transferred to acceptor glycans by various sialyltransferases (Schauer 1982
; Varki 1992). Mutation in various enzymes in this multistep process results in genetic conditions known as congenital disorders of glycosylation (CDGs) (Freeze 2006). Indeed, in one CDG, the CMP-NeuAc transporter is mutated, leading to thrombocytopenia and neutropenia (Willig et al. 2001; Martinez-Duncker et al. 2005).
Polysialic acid is a homopolymer of 2,8-linked sialic acid attached to the neural cell adhesion molecule (NCAM) and is highly expressed in embryonic brain (Angata and Fukuda 2005). Recently, polysialic acid-deficient mutant mice were generated by inactivating two polysialyltransferase genes (ST8Sia-II and ST8Sia-IV). Polysialic acid-deficient mice die as neonates (Weinhold et al. 2005) and exhibit impaired migration of neural cells, resulting in apoptosis of those cells (Angata et al. 2007). In certain tumors such as neuroblastoma and glioma, polysialic acid is ectopically expressed even in the adult and is thought to play a role in tumor invasion (Scheidegger et al. 1994; Suzuki et al. 2005). Inhibiting polysialic acid synthesis in those tumors could thus have an antitumorigenic effect. Indeed Bertozzi's group showed that feeding cells N-butylmannosamine can inhibit polysialyltransferase activity (Mahal et al. 2001). In their study, it was critical that all of the enzymes required for CMP-NeuAc synthesis noted above tolerate the increase in size of the N-acyl group from an acetyl (O=C–CH3) to a butyl (O=C–CH2–CH2–CH3) group. By contrast, polysialyltransferases, in particular ST8Sia-II (STX), utilize CMP-N-butylmannosamine much less efficiently than CMP-N-acetylneuraminic acid or CMP-N-propanyl neuraminic acid. This inhibition leads to a decreased synthesis of polysialic acid by ST8Sia-II (Mahal et al. 2001). These observations suggest that glycosylation efficiency may decrease substantially when the structure of a precursor carbohydrate residue is modified.
Monosaccharide derivatives have also been used to tag glycans. A well-characterized example is the use of N-azido-acetyl mannosamine containing O=C–CH2–N3 instead of O=C–CH3 (Prescher et al. 2004). The azido group is intact during metabolic conversion and the sialic acid-containing azido group can be expressed on the cell surface. Cell surface glycoproteins expressing sialic acid can be tagged using the Staudinger ligation of FLAG peptide to the azido group, which can be detected by fluorescent anti-FLAG peptide antibody. In this approach, it is essential that each metabolic step required to form sialylated glycoproteins, including sialylation by sialyltransferases, tolerate the N-azido acetyl group (Prescher et al. 2004).
Monosaccharides with modified groups have been used in other studies (Rabuka et al. 2006; Sawa et al. 2006), but only a few address how modification of the nucleotide phosphate portion influences enzymatic activity. Since the nucleotide portion is far from the activated monosaccharide, which is attached to the nucleotide through a phosphate or diphosphate, glycosyltransferases may tolerate nucleotide modification differently than the modification of an activated monosaccharide.
In the present study, we determine how different modifications of a sugar nucleotide differentially affect glycosyltransferase activity. Specifically, we asked whether methylation of the C-5 or the 2'-OH of cytidine 5'-monophosphate (CMP) affects 2,3-sialyltransferase (ST3Gal-III) and 2,6-sialyltransferase (ST6Gal-I) activity. We found that ST3Gal-III and ST3Gal-IV but not ST6Gal-I was inhibited by 2'-O-methyl CMP as much as by unmodified CMP, while ST6Gal-I, ST3Gal-III, and ST3Gal-IV were moderately inhibited by 5-methyl CMP. We also found that ST8Sia-IV (Eckhardt et al. 1995; Nakayama et al. 1995), which is barely inhibited by N-butyl neuraminic acid, was inhibited by 5-methyl CMP and substantially by 2'-O-methyl CMP. Moreover, the addition of CMP, 2'-O-methyl CMP, and 5-methyl CMP to a culture medium resulted in the inhibition of polysialic acid on the cell surface and NCAM in Chinese hamster ovary (CHO) cells. These results suggest that it is possible to decrease the amount of a particular sialic acid linkage using modified CMP.
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Results |
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2'-O-Methyl and 5-methyl derivatives of cytidyl 5'-monophosphate
Since a methyl group represents a small substitution group without a charge, the effect of this substitution should implicate whether a sialyltransferase tolerates a small change in CMP. We first synthesized 2'-O-methyl CMP and 5-methyl CMP (Figures 1). For this, we phosphorylated 2'-O-methyl cytidine by reacting it with phosphoryl chloride in triethanol phosphate. After stopping the reaction by the addition of water, the 2'-O-methyl CMP product was isolated using a DEAE-Sephacel column as described (Beres et al. 1989
). Using the same method, we synthesized 5-methyl CMP. NMR analysis confirmed the products as 2'-O-methyl CMP and 5-methyl CMP (Figures 2 and 3).
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Inhibition of ST3Gal-III and ST6Gal-I by 2'-O-methyl CMP and 5-methyl CMP
To determine first whether ST3Gal-III and ST6Gal-I recognize 2'-O-methyl or 5-methyl CMP derivatives, we undertook an enzyme assay using an acceptor substrate (N-acetyllactosamine) and donor CMP-[14C]-NeuAc in the presence of the CMP derivatives. The rationale was that if the enzyme recognized the derivative, transfer of [14C]-labeled NeuAc would be inhibited. CMP served as a positive control, and as predicted, ST3Gal-III and ST6Gal-I activity was inhibited by CMP (Table I). 2'-O-Methyl CMP (0.25 mM) inhibited ST3Gal-III more efficiently than CMP itself (0.25 mM), while it did not inhibit ST6Gal-I under the same conditions. On the other hand, 5-methyl CMP (0.25 mM) moderately inhibited both ST3Gal-III and ST6Gal-I. These results indicate that ST3Gal-III is preferentially inhibited by 2'-O-methyl CMP.
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These analyses were extended to other sialyltransferases including ST3Gal-IV and ST8Sia-III using asialo fetuin and fetuin as an acceptor as described previously (Angata et al. 2000
). ST3Gal-IV and ST8Sia-III were produced as a recombinant protein consisting of an IgG-binding domain of protein A fused with a catalytic domain of the transferases. These chimeric proteins were adsorbed to IgG-Sepharose and then incubated with acceptor glycoproteins and CMP-[14C]-NeuAc. After the incubation for 24 h, the reaction mixture was centrifuged and the supernatant obtained was subjected to SDS–polyacrylamide gel electrophoresis (PAGE) followed by fluorography as described previously (Angata et al. 2000). The results showed that both ST3Gal-IV and ST8Sia-III can be inhibited by 2'-O-methyl CMP slightly more than 5-methyl CMP (Table II).
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Inhibition of ST8Sia-IV by 2'-O-methyl CMP but moderately by 5-methyl CMP
Previously, we showed that ST8Sia-II is inhibited by CMP-N-butyl neuraminic acid, while ST8Sia-IV is only moderately inhibited by the same compound (Mahal et al. 2001
). We thus asked if either 2'-O-methyl CMP or 5-methyl CMP inhibits ST8Sia-IV activity. To do so, transfer of [14C]-NeuAc from CMP-[14C]-NeuAc to NCAM was measured in the presence or absence of 2'-O-methyl CMP or 5-methyl CMP, and the product was analyzed by SDS–PAGE followed by fluorography. As shown in Figure 4 (lane 1), in the absence of inhibitor, ST8Sia-IV formed large and heterogeneous [14C]-NeuAc-labeled NCAM. That reaction was strongly inhibited by the addition of CMP in a dose-dependent manner, and in the presence of 0.25 mM CMP, only a small amount of [14C]-NeuAc was incorporated (Figure 4, lane 4). Similarly, ST8Sia-IV activity was inhibited by 2'-O-methyl CMP dose dependently (Figure 4 and Table II). In the presence of 0.25 mM 2'-O-methyl CMP, almost no high molecular weight polysialylated NCAM was produced (Figure 4, lane 7). 5-Methyl CMP had less effect than 2'-O-methyl CMP (Figure 4, lanes 8–10 and Table II). These results indicate that 2'-O-methyl CMP can inhibit ST8Sia-IV activity, while 5-methyl CMP moderately inhibits ST8Sia-IV activity in vitro. Similar experiments were carried out on ST8Sia-II. As shown in Table II, ST8Sia-II was also inhibited more efficiently by 2'-O-methyl CMP than 5-methyl CMP. CMP and 2'-O-methyl CMP almost equally inhibited ST8Sia-II.
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Inhibition of polysialic acid synthesis in CHO cells by 2'-O-methyl CMP and 5-methyl CMP
It has been shown that CHO cells express ST8Sia-IV and cell surface polysialic acid (Windfuhr et al. 2000
). It has also been reported that CMP added in the culture medium can be incorporated into intestinal epithelial cells (Tanaka et al. 1996). We thus tested if an exogenous addition of CMP, 2'-O-methyl CMP, and 5'-methyl CMP has inhibiting effect on polysialic acid synthesis of CHO cells. Apparently, the treatment by these compounds does not have cytotoxic effect since the change of the cell shape was not observed under light microscopy after treatment with these compounds (Figure 5).
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CHO cells were cultured in the presence of 0.5 mM CMP, 2'-O-methyl CMP, or 5-methyl CMP for 24 h since 0.2 mM of CMP was used in the previous study (Tanaka et al. 1996). After dissociating cells, monodispersed cells were incubated with antipolysialic acid antibody or anti-NCAM antibody followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibody. In parallel, dissociated CHO cells were incubated with FITC-conjugated Maackia amurensis agglutinin (MAA), which reacts with
2,3-linked sialic acid (Wang and Cummings 1988), for flow cytometry. As shown in Figure 6, the expression levels of polysialic acid was decreased by CMP, 2'-O-methyl CMP, and 5-methyl CMP while those of NCAM and MAA were barely changed with the same treatment. These results showed that exogenously added CMP, 2'-O-methyl CMP, and 5-methyl CMP inhibit polysialic acid synthesis. Since CHO cells lack 2,6-linked sialic acid in N-glycans (Sasaki et al. 1987), the expression level of 2,6-linked sialic acid was not examined.
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To determine if the polysialylation of NCAM is affected by CMP, 2'-O-methyl CMP, and 5'-methyl CMP, NCAMs from these CHO cells were examined. After 24 h of incubation, the cells were collected and proteins in the cell lysates were separated by SDS–PAGE, and Western blot analysis was carried out using anti-NCAM antibody. As shown in Figure 7 (lane 1), NCAM, most likely NCAM-140, displays a polydisperse band with high molecular weight. This was converted to a relatively homogenous band with lower molecular weight after endoneuraminidase treatment (Figure 7, lane 5). In the presence of CMP, 2'-O-methyl CMP, or 5-methyl CMP, the polydisperse band and high molecular weight NCAM were converted to a relatively homogeneous band with a lower molecular weight (lanes 2–4 of Figure 7). These results indicate that 2'-O-methyl CMP and 5-methyl CMP in the culture medium can be incorporated into cells and inhibit polysialyltransferase ST8Sia-IV.
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Discussion |
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The present study showed that the derivatization of 2'-OH or 5-C of CMP has differential effects on inhibitory activity toward
2,3-, 2,6-, and 2,8-sialyltransferases. 2'-O-methyl CMP at 0.25 mM significantly inhibited 2,3-sialyltransferase (ST3Gal-III and ST3Gal-IV) and 2,8-sialyltransferase (ST8Sia-II, ST8Sia-III, and ST8Sia-IV) but showed a small effect on 2,6-sialyltransferase (ST6Gal-I). These results indicate that ST3Gal-III, ST3Gal-IV, ST8Sia-II, ST8Sia-III, and ST8Sia-IV tolerate substitution at the CMP 2'-OH group while ST6Gal-I does not. This differential effect likely reflects steric differences in the CMP-sialic acid-binding site of the three sialyltransferases.
Previously, Kleineidam et al. (1997) reported that 5-methyl CMP inhibits 2,6-sialyltransferase; however, they did not report if 5-methyl CMP inhibits 2,3-sialyltransferase. In their study, 2'-O-methyl CMP was not tested. We showed here that 5-methyl CMP inhibits the activity of ST3Gal-III, ST3Gal-IV, ST6Gal-I, ST8Sia-II, ST8Sia-III, and ST8Sia-IV, although less efficiently than unmodified CMP. These results suggest that all six enzymes recognize the cytidyl portion of CMP in a similar manner and that substitution with methyl at the 5-position of CMP is tolerated by all of them.
It has been shown that amino acid sequences of sialyltransferases in the sialyl motif L and the sialyl motif S are essential for CMP-sialic acid recognition and recognition of CMP-sialic acid and acceptor, respectively (Datta and Paulson 1995; Datta et al. 1998), and that these two domains are connected by disulfide bonds (Angata et al. 2001). Comparison of the amino acid sequence in the sialyl motif L shows that ST3Gal-III, ST8Sia-IV, and ST6Gal-I are highly homologous to each other (Figure 8). ST3Gal-III, ST3Gal-IV, ST8Sia-II, ST8Sia-III, and ST8Sia-IV are also highly homologous in the sialyl motif S, while homology between ST6Gal-I and the rest of sialyltransferases tested is slightly lower in this domain (Figure 8). The present study suggests that small differences in the sialyl motif S among these sialyltransferases may result in the differential recognition of 2'-O-methyl CMP. In particular, the G
IIIM sequence in ST6Gal-I is M/AY/ITL/MA in other sialyltransferases (Figure 8) and this difference may make ST6Gal-I unrecognize 2'-O-methyl CMP.
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It has also been reported that a methyl ester of fully acetylated sialic acid attached to 5-fluoro-2',3'-isopropylidate CMP inhibits pulmonary metastasis of mouse colon carcinoma (Kijima-Suda et al. 1986
, 1988; Harvey and Thomas 1993). This compound was shown to inhibit the CMP-sialic acid transporter, thereby decreasing the amount of CMP-sialic acid in the Golgi (Harvey and Thomas 1993). On the other hand, Gross et al. (1996) showed that cell surface sialyltransferase-catalyzed sialylation of the human B lymphocyte cell line is stimulated by exogenous CMP-NeuAc. A similar approach using substituted fucose was utilized for the cell surface modification by a specific carbohydrate. Substitution of C-6 of fucose in GDP-fucose does not inhibit 1,3-fucosyltransferase. Using this tolerant property, even 6-sulfosialyl Lewis X, NeuAc 2
3Galβ14 [Fuc13 (SO36)]GlcNAc attached to C-6 of fucose, can be transferred to cell surface carbohydrates, thus generating 6-sulfo sialyl Lewis X-expressing CHO cells (Tsuboi et al. 1996).
In the present study, we showed that CMP, 2'-O-methyl CMP, and 5-methyl CMP almost equally inhibit ST8Sia-IV expressed in CHO cells when these compounds are added to the culture medium. The expression of cell surface polysialic acid was decreased 30–40% after 24 h treatment of 0.5 mM CMP, 2'-O-methyl CMP or 5-methyl CMP as estimated by FACS analysis. Western blot analysis of NCAM expressed in CHO cells showed significant loss of polysialylated NCAM after treatment of these compounds. It is noteworthy that the effect on polysialylation is more apparent than the effect on 2,3-sialylation. Since multiple continuous reactions of polysialyltransferase ST8Sia-II or ST8Sia-IV are necessary for polysialic acid synthesis, a small decrease of each reaction culminates in a significant decrease of polysialic acid synthesis, in contrast to a small decrease of other sialic acid structures, which require only one reaction.
CMP, 2'-O-methyl CMP, and 5-methyl CMP almost equally inhibit in vivo polysialylation while CMP is more effective inhibitor in vitro than the others. It has been reported that polysialyltransferases ST8Sia-II and ST8Sia-IV are mostly present in the Golgi apparatus; thus, polysialylation takes place mostly in the Golgi apparatus (Close and Colley 1998). Thus it is reasonable to predict that CMP, 2'-O-methyl CMP, and 5-methyl CMP are transported into the Golgi apparatus to compete with CMP-NeuAc. It is possible that the transport of these compounds may be a rate-limiting step for the inhibitory activity of these compounds. The effect by these compounds is almost equal probably because transport efficiency may not be greatly different for CMP, 2'-O-methyl CMP, and 5-methyl CMP. These results indicate that 2'-O-methyl CMP and 5-methyl CMP may be used to inhibit polysialic acid synthesis in vivo. Since CMP may be converted and used for many natural synthetic reactions, the use of CMP may have an unwanted side effect. Glioma cells express increased amount of polysialic acid, which is thought to increase glioma invasion (Suzuki et al. 2005). Further studies are needed in order to determine if polysialic acid-mediated tumor invasion can be attenuated by the treatment of 2'-O-methyl CMP and 5-methyl CMP.
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Materials and methods |
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General methods
NMR spectra were recorded with Varian UNITYINOVA 300 instruments at 303 K. The chemical shifts of 1H NMR are presented in ppm and referenced to HOD (
= 4.81 ppm) in D2O as an internal standard. The chemical shifts of 31P NMR spectra are expressed in ppm and referenced to phosphoric acid ( = 0.00 ppm) in D2O as an external standard. For thin-layer chromatography we used DC-Platten Kieselgel 60 F254 (EMD Chemicals Inc., Gibbstown, NJ). ESI-MS was carried out using a ZQ2000 mass spectrometer (Waters, Milford, MA).
Materials
Solvents and reagents were purified according to standard procedures. -2,3-(N)-Sialyltransferase ST3Gal-III (EC 2.4.99.5; rat recombinant) (Wen et al. 1992) and -2,6-(N)-sialyltransferase ST6Gal-I (EC 2.4.99.1; rat recombinant) (Weinstein et al. 1987) were obtained from EMD Chemicals Inc. Cytidine 5'-monophosphate-β-D-[4,5,6,7,8,9-14C]-sialic acid (325 mCi/mmol, CMP-[14C]-NeuAc) was obtained from Perkin Elmer Life and Analytical Science (Waltham, MA). Calf intestinal alkaline phosphatase (CIAP, EC 3.1.3.1) was purchased from Roche (Indianapolis, IN). 2'-O-methylcytidine and 5-methylcytidine were purchased from Sigma, and liquid scintillation cocktail (EcoLume) was obtained from Mr Biomedicals (Solon, OH). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless indicated.
Phosphorylation of cytidine derivatives
Cytidine-5'-monophosphate derivatives were synthesized according to reported methods (Beres et al. 1989), except for the purification methods described below.
2'-O-Methylcytidine 5'-Monophosphate (1)
To a stirred solution of 2'-O-methyl-cytidine (22.1 mg, 86 µmol) in triethyl phosphate (500 µL) was added phosphoryl chloride (16 mL, 175 µmol) at 0°C. After 2 h, the reaction mixture was quenched with H2O (2 mL), and the pH adjusted to 7 with 2 N NaOH. The solution was applied to a DEAE-Sephacel column (1.5 x 40 cm, HCO3– form), and the column was washed with H2O (100 mL) to remove impurities. The product was eluted with 200 mM NH4HCO3. Activated charcoal (1.2 g), which was washed with H2O and ethanol, was added to the solution containing the product. After the supernatant was removed, the product was eluted with ethanol: H2O: 28% NH4 at 5:4:1 (10 mL). Lyophilization of eluate afforded compound 1 (12.3 mg, yield = 38%) as an amorphous mass; 1H NMR (300 MHz, D2O) 8.12 (d, 1H, J5,6 7.6 Hz, H-6), 6.19 (d, 1H, J5,6 7.6 Hz, H-5), 6.12 (d, 1H, J1',2' 3.8 Hz, H-1'), 4.50 (dd, 1H, J2',3' 4.9 Hz, J3',4' 5.7 Hz, H-3'), 4.29 (ddd, 1H, J3',4' 5.7 Hz, J4',5a' 2.5 Hz, J4',5b' 5.1 Hz, H-4'), 4.23 (ddd, 1H, J4',5a' 2.5 Hz Jgem 11.8 Hz, J5a',P 4.1 Hz, H-5a'), 4.12 (ddd, 1H, J4',5b' 5.1 Hz Jgem 11.8 Hz J5b',P 2.8 Hz, H-5b'), 4.10 (dd, 1H, J1',2' 3.8 Hz, J2',3' 4.9 Hz, H-2'), 3.60 (s, 3H, 2'-O-Me) (Figure 2); 31P NMR (121 MHz, D2O) 1.84; calculated for C10H17N3O8 P (M+H+) 338.07, and ESI-MS experiment showed 338.12. The physical data were identical to the reported data (Beres et al. 1989).
5-Methylcytidine 5'-Monophosphate (2)
5-Methylcytidine (18.5 mg, 72 µmol) was phosphorylated as described for 1 to give compound 2 (10.2 mg, yield = 38%) as an amorphous mass; 1H NMR (300 MHz, D2O) 7.88 (bs, 1H, H-6), 6.05 (d, 1H, J1',2' 4.1 Hz, H-1'), 4.42 
4.34 (m, 2H, H-2', H-3'), 4.28 (m, 1H, H-4'), 4.18 3.98 (m, 2H, H-5a', H-5b'), 2.05 (s, 3H, 5-Me); 31P NMR (121 MHz, D2O) 2.94; ESI-MS showed 338.05 (calculated for C10H17N3O8 P (M+H+) is 338.07). The physical data were identical to the reported data (Beres et al. 1989).
2,3- and 2,6-sialyltransferaseinhibition assay in the presence of CMP, 5-methyl CMP, and 2'-O-methyl CMP
The substrate solution contained 6 nmol of 8-methoxycarbonyloctyl LacNAc (Field et al. 1995), 2.5 mM MnCl2, 2.5 mM MgCl2, 2.5 mM CaCl2, 0.1% Triton CF-54, 1.0 nmol (0.15 µCi) of CMP-[14C]-NeuAc, and various amounts of CMP derivatives in 40 µL of 100 mM sodium cacodylate buffer, pH 6.0 (Palcic et al. 1988). 0.9 mU of 2,3-(N)-sialyltransferase or -2,6(N)-sialyltransferase was added to this substrate solution, and the reaction mixture was incubated at 37°C for 1 h. Controls were run simultaneously without CMP derivative. The negative control was incubated without sialyltransferase. All assays were performed in duplicate. After the incubation, the reaction mixture was diluted to 1.2 mL with water and applied to a conditioned SepPak cartridge. The cartridge was washed with 10 mL water to remove CMP-[14C] NeuAc. Radiolabeled product was then eluted with 3.5 mL of methanol and counted in 12 mL scintillation cocktail (EcoLume).
Inhibition assay of ST8Sia-IV using CMP, 5-Me-CMP and 2'-O-Me CMP
NCAMIgG was purified from the cultured medium of COS-1 cells that were transfected with pIG-NCAM as described previously (Angata et al. 2001). The substrate solution contained 10 pmol of NCAMIgG (containing 60 pmol of N-glycans), 2.5 mM MnCl2, 2.5 mM MgCl2, 2.5 mM CaCl2, 0.1% Triton CF-54, 2.0 nmol (0.3 µCi) of CMP-[14C]-NeuAc, and the CMP derivative (2, 5, or 10 nmol) in 40 µL of 100 mM sodium cacodylate buffer, pH 6.0. 10 µL of ST8Sia-IV protein produced in insect cells infected with baculovirus (Angata et al. 2001) was added to the substrate solution, and the reaction mixture was incubated at 37°C for 2 h. After incubation, the reaction mixture was analyzed by SDS–PAGE followed by fluorography as described (Nakayama and Fukuda 1996). Fluorographic images were captured and analyzed densitometrically using the AlphaImager system (Alpha Innotech Corporation, San Leandro, CA).
ST8Sia-II was produced as a chimeric enzyme consisting of IgG-binding domain of protein A fused with a catalytic domain of human ST8Sia-II as described previously (Angata et al. 2000). Briefly, pcDNAI-harboring cDNA encoding the chimeric ST8Sia-II protein was transfected into COS-1 cells. The chimeric enzyme released into the cultured medium was absorbed to IgG-Sepharose as described and the enzyme on the beads was incubated with purified NCAMIgG and CMP-[14C]-NeuAc for 24 h. After a brief centrifugation, the supernatant was analyzed as described above.
Similarly, ST8Sia-III and ST3Gal-IV were produced as chimeric enzyme consisting of IgG-binding domain of protein A and catalytic domain of the enzymes. ProA ST3Gal-IV (Sasaki et al. 1993) was subcloned from the pAMo vector into pcDNAI while ST8Sia-III chimeric enzyme was cloned into pcDNAI as described previously (Angata et al. 2000). The substrate (fetuin and asialo fetuin for enzyme reaction) was at the concentration of 0.1 mg/mL.
Effect of 2'-O-methyl CMP and 5-methyl CMP on polysialic acid synthesis in vivo
The cells were cultured in an -MEM medium containing 10% dialyzed fetal bovine serum (Tissue Culture Biologicals). After seeding CHO cells, 0.5 mM (final concentration) of CMP, 2'-O-methyl CMP, and 5-methyl CMP was added to the culture medium. After 24 h incubation, cells were harvested and dissociated with enzyme-free dissociation solution. The Hanks-based saline solution was purchased from Cell and Molecular Technologies. Monodispersed cells were incubated with antipolysialic acid antibody 12F8 (BD Biosciences, San Jose, CA) or polyclonal rabbit anti-NCAM antibody (Millipore, Billerica, MA) followed by FITC-conjugated secondary antibody and subjected to FACS analysis as described previously (Angata et al. 1998). The monodispersed cells were also incubated with FITC-conjugated MAA (EY Laboratories, San Moteo, CA) and subjected to FACS analysis under the same conditions.
Separately, CHO cells treated with CMP, 2'-O-methyl CMP, or 5-methyl CMP were lysed using RIPA, which contains 150 mM NaCl, 50 mM Tris–HCl (pH 7.4), and 5 mM EDTA, 1% NP-40, 0.1% SDS and protease inhibitor cocktail (Roche), at 4°C for 2 h. After centrifugation, supernatant was kept for further analysis. Aliquots of the lysates were treated by endoneuraminidase-N at 37°C for 1 h before analysis. Samples were separated by SDS–PAGE (5%). Separated proteins were blotted to a PVDF membrane and incubated with polyclonal anti-NCAM antibody. HRP-conjugated secondary antibody was used to detect the binding of NCAM antibody and ECL reagents (GE Healthcare, Piscataway, NJ) were used to detect signals.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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National Institutes of Health (CA33895 to M.F.); (PO1 CA 71932 to P.H.S., O.H., and M.F.).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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We declare no conflict of interest.
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Acknowledgements |
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The authors thank Dr. Elise Lamar for critical reading of the manuscript and Aleli Morse for organizing the manuscript.
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Footnotes |
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2 Current address: Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, 956-8603, Japan.
3 These authors contributed equally to this work.
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Abbreviations |
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CDGs, congenital disorders of glycosylation; CHO, Chinese hamster ovary; CMP, cytidylmonophosphate; FITC, fluorescein isothiocyanate; MAA, Maackia amurensis agglutinin; NCAM, the neural cell adhesion molecular; NeuAc, N-acetylneuraminic acid; PST, polysialyltransferase (ST8Sia-IV); SDS-PAGE, SDS-polyacrylamide gel electrophoresis; STX, ST8sia-II
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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