2Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan, 3Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany, and 4Division of Organogenesis, Nagoya University Bioscience Center, Nagoya University, Nagoya 464-8601, Japan
Received on February 5, 2001; revised on March 23, 2001; accepted on March 27, 2001.
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Abstract |
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Key words: sialic acid/deaminoneuraminic acid/CMP-KDN synthetase/CMP-Neu5Ac synthetase/CMPsialic acid synthetase
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Introduction |
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The biosynthetic pathway of KDN and KDN ketosides in glycan chains was shown to consist of the following sequential reactions (Angata et al., 1999a,b): (i) Mannose (Man) + ATP
Man 6-phosphate + ADP; (ii) Man 6-phosphate + phosphoenolpyruvate
KDN 9-phosphate (KDN-9-P) + Pi; (iii) KDN-9-P
KDN + Pi; (iv) KDN + CTP
CMP-KDN + PPi; (v) CMP-KDN + R-OH
R-O-KDN + CMP (R, acceptor glycan). Of enzymes involved in these reactions, KDN 9-phosphate synthase catalyzing reaction (ii) (Angata et al., 1999a
), CMP-Sia synthetase catalyzing reaction (iv) (Terada et al., 1993
), and KDN transferase catalyzing reaction (v) (Angata et al., 1994
) were characterized using partially purified enzymes from rainbow trout (Kanamori et al., 1994
; Yu et al., 1991
, 1995). However, none of these enzymes have been purified or cloned so far.
The partially purified rainbow trout CMP-Sia synthetase was shown to convert both KDN and Neu5Ac to CMP-KDN and CMP-Neu5Ac, respectively (Terada et al., 1993). This property is unique because fetal calf brain CMP-Neu5Ac synthetase had only a weak activity toward KDN (Terada et al., 1993
). To determine if the synthesis of CMP-KDN and CMP-Neu5Ac in the trout enzyme preparation was catalyzed by the same enzyme or by separate enzymes, the substrate competition experiments were carried out and suggested that a single enzyme catalyzed the synthesis of both CMP-Sia compounds (Terada et al., 1993
). However, structural requirements of the trout enzyme for the recognition of both KDN and Neu5Ac still remained to be elucidated. Recently, murine CMP-Neu5Ac synthetase cDNA has been cloned as a first example of this class of enzyme of vertebrate origin (Münster et al., 1998
). The murine enzyme was shown to share four evolutionarily conserved amino-acid sequence motifs with bacterial CMP-3-deoxy-D-manno-octulosonic acid and CMP-Neu5Ac synthetases. To precisely understand the substrate specificity of the trout CMP-Sia synthetase, a homology cloning was applied to clone the trout enzyme.
Here we describe the isolation of a cDNA encoding the rainbow trout testis CMP-Sia synthetase. Cloning succeeded by polymerase chain reaction (PCR) using degenerate oligonucleotide primers corresponding to the conserved motifs in bacterial and murine enzymes. Northern blot analysis revealed developmentally regulated and tissue-dependent expression of the mRNAs in trout liver, testis, and ovary. Kinetic analyses showed that the recombinant trout enzyme expressed in Escherichia coli had high activity to synthesize both CMP-KDN and CMP-Neu5Ac, whereas the recombinant murine enzyme had little activity to synthesize CMP-KDN. These results unambiguously demonstrate that trout testis contains an enzyme that exhibits both CMP-Neu5Ac and CMP-KDN synthetase activities, as suggested by the substrate competition experiment using crude enzyme preparations (Terada et al., 1993).
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Results |
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Expression and purification of the functional CMP-Sia synthetase in E. coli
The recombinant CMP-Sia synthetases fused with thioredoxin were expressed in E. coli and assayed for the enzyme activity as described under Materials and methods. The recombinant enzymes were inducibly expressed in E. coli. About 90% of the recombinant proteins for both trout and murine enzymes were recovered in the inclusion bodies, but the salt-soluble fractions were also shown to contain the enzyme activities (Figure 2). The trout enzyme was shown to have the ability to synthesize both CMP-KDN and CMP-Neu5Ac, respectively, from KDN and Neu5Ac together with CTP (Figure 2A, B). The murine enzyme was also capable of synthesizing CMP-Neu5Ac from Neu5Ac and CTP (Figure 2D). However, CMP-KDN was not efficiently synthesized from KDN and CTP by the same murine enzyme fraction (Figure 2C). No CMP-KDN or CMP-Neu5Ac was synthesized by the enzyme fraction from transformants of the empty plasmid (Figure 2E,F), thus indicating that no endogenous CMP-Sia synthetase activity of the host E. coli strain was present in the enzyme fraction.
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Expression of mRNAs coding for the CMP-Sia synthetase in rainbow trout liver, testis, and ovary
Northern blot analysis showed that the expression pattern of mRNAs were different among the three tissues examined (Figure 5). A single 3.1-kb mRNA was detected in liver (Figure 5, lane 1), and at least five distinct mRNAs with different sizes (2.3 kb, 3.1 kb, 4.0 kb, 6.0 kb, and 7.8 kb) were detected in immature testis from rainbow trout harvested in May (Figure 5, lane 3). In mature testis, harvested in August, a month before spermiation, four different mRNAs (2.3 kb, 3.1 kb, 4.3 kb, and 7.8 kb) were detected (Figure 5, lane 4). The mRNAs of 2.3 kb, 3.1 kb, and 7.8 kb appear to be constitutively expressed during spermatogenesis. The 4.3-kb transcript was found only in mature testis, and the 4.0 kb and 6.0 kb transcripts were only in immature testis. Thus, the expression of these CMP-Sia synthetase mRNAs are suggested to be developmentally regulated. In immature ovary, three mRNAs of 2.3 kb, 3.1 kb, and 4.0 kb were observed (Figure 5, lane 2), among which a 3.1-kb mRNA was most prominent. It remains unknown why multiple mRNAs are expressed in testis and ovary, while a single 3.1-kb mRNA is expressed in liver. The 3.1-kb transcript was commonly detected in all the tissues tested, although the expression level was different from tissue to tissue. These results were highly reproducible. Sequencing of a cDNA clone for the liver CMP-Sia synthetase showed that a coding region of cDNA for the liver enzyme was identical to that of the mature testis enzyme (data not shown).
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Discussion |
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It has been recognized that KDN is a poor substrate for CMP-Neu5Ac synthetases isolated from various animal origins (Terada et al., 1993). However, the CMP-Sia synthetase from rainbow trout testis and ovary was shown to be able to convert both KDN and Neu5Ac to the corresponding CMP-Sia compounds efficiently (Terada et al., 1993
). Based on the substrate competition experiments using crude enzyme preparation, both activities to synthesize CMP-KDN and CMP-Neu5Ac were suggested to be on a single enzyme molecule. Consistent with these previous reports (Terada et al., 1993
), the recombinant trout CMP-Sia synthetase can utilize KDN and Neu5Ac as substrates to synthesize both CMP-KDN and CMP-Neu5Ac (Figure 2). Based on the Vmax/Km values (Table I), the recombinant trout enzyme exhibited a 1.6-fold higher activity with KDN than with Neu5Ac. In contrast, the recombinant murine enzyme exhibited a 15-fold lower activity with KDN than with Neu5Ac (Figure 4, Table I). It is thus concluded that the trout testis CMP-Sia synthetase has novel substrate specificity different from the murine CMP-Neu5Ac synthetase.
Several forms of mRNA for the trout CMP-Sia synthetase were detected in immature and mature trout testes (Figure 5). These mRNAs can be categorized into two groups, a constitutively expressed group and a developmentally expressed group. Further study would be necessary to elucidate how and why these multiple mRNA species are expressed in trout testis, but their occurrence may be related to the developmental changes in expression of KDN in trout testis during spermatogenesis (Yu et al., 1995; Terada et al., 1993
).
In BALB/c mouse tissues, a single form of mRNA for the murine CMP-Sia synthetase (2.0 kb) was detected (Nakata and Münster, unpublished data). Northern hybridization of mRNAs from various normal murine tissues with cRNA for the trout enzyme under less stringent conditions as well as the PCR amplification experiment showed that the mRNA for the murine CMP-Sia synthetase appears to be the only component that is homologous to the trout testis enzyme mRNAs in normal murine tissues (data not shown). Importantly, kinetic studies (Figure 4) indicate that the murine enzyme can produce CMP-KDN with low efficiency. In mammalian cells, the expression level of free KDN is very low (a few µM) (Angata et al., 1999b). Considering that this value of free KDN concentration is much lower than the Km value (0.56 mM) for KDN of the murine enzyme, CMP-KDN can not be synthesized in murine tissues unless the intracellular KDN concentration is elevated. Recently, we have revealed that Man plays an important role as a precursor in the synthesis of KDN in mammalian cells (Angata et al., 1999a
,b). In murine melanoma B16 cells, the level of intracellular free KDN is at about 1.2 µM in basal media; interestingly, it increases up to about 79 µM in parallel with an increase in intracellular Man concentration from 3 to 25 µM (Angata et al., 1999b
). If such elevation of intracellular free KDN level occurs, the murine enzyme can start converting KDN to CMP-KDN. However, it remains unknown if the formation of CMP-KDN does not directly lead to the expression of KDN residues, because the synthesis of KDN ketosides in glycoconjugates consists of still more steps after the formation of CMP-KDN, including translocation of CMP-KDN into lumen of the Golgi apparatus by a CMP-KDN transporter and transfer of KDN into acceptor glycans by KDN-transferases. Current efforts are under way in our laboratory not only to seek effect of expression of the trout CMP-Sia synthetase in mammalian cells on metabolism of KDN but also to characterize the CMP-KDN transporter and KDN-transferases through biochemical and molecular biological approaches.
With respect to the synthesis of free KDN in mammalian cells, we have recently succeeded in cloning the murine Neu5Ac 9-phosphate (Neu5Ac-9-P) synthase that catalyzes the synthesis of Neu5Ac-9-P through the condensation of ManNAc-6-P and phosphoenolpyruvate, and shown that this enzyme has very low or no activity to synthesize KDN 9-phosphate (KDN-9-P) from Man and phosphoenolpyruvate (Nakata et al., 2000). The corresponding human enzyme that has recently been cloned by Lawrence et al. (2000)
is reported to have the ability to synthesize KDN-9-P; however, again, only a very low activity to synthesize KDN-9-P compared with Neu5Ac-9-P was found. Although the mammalian Neu5Ac-9-P synthetase may possibly catalyze the synthesis of KDN-9-P under some regulated conditions, it is plausible that a Sia-9-P synthase that can preferentially catalyze the synthesis of KDN-9-P rather than Neu5Ac-9-P is expressed in mammalian cells to synthesize free KDN at the high level. Thus, further studies are necessary to gain insight into mechanisms by which mammalian cells can express free KDN.
Several enzymes have been reported that can utilize KDN as a good substrate, including the trout testis CMP-Sia synthetase, KDN-transferase from rainbow trout ovary (Angata et al., 1994), KDN-9-P synthase from rainbow trout testis (Angata et al., 1999a
), and KDN-ketosides hydrolyzing enzyme (KDNase Sm) from Sphingobacterium multivorum (Kitajima et al., 1994
). These enzymes are unique because most enzymes involved in synthesis and catalysis of N-acylneuraminic acid residues often fail to use KDN. However, none of these enzymes have been sequenced or cloned thus far, and the rainbow trout CMP-Sia synthetase is the first case of a KDN-preferential enzyme whose cDNA has been cloned. Thus, this study would open up new research areas not only to reveal structural requirements of this class of enzyme for the differential recognition of KDN from Neu5Ac but also to elucidate their biological roles in normal development and differentiation as well as in malignancy through molecular biological approaches.
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Materials and methods |
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PCR cloning
The following degenerate oligonucleotide primers based on the murine CMP-Neu5Ac synthetase cDNA sequence (Münster et al., 1998) were used for the PCR cloning: 5'-AAGGCATCCC(A/C) CTGAAGAA-3' (nucleotides 160179 in motif I) and 5'-AT(A/G)TCCAC(A/G)TC(A/G/T) ATATCCAC-3' (nucleotides 730749 in motif V). Random-primed cDNA (1 µg) from rainbow trout testis or ovary total RNA was used as a template for PCR. Both sense and antisense primers (25 pmol each) were added to a 50-µl reaction mixture containing 10 mM TrisHCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, all for dNTPs (each 200 µM), 1 unit of rTaq polymerase (TaKaRa), and template cDNA. Thirty cycles were carried out on a thermal cycler, each cycle involved incubation at 94°C for 30 s, 48°C for 30 s, and 72°C for 1 min. The PCR products were separated on a 1.0% agarose gel. Fragments of 600 bp were excised and subcloned into pGEM-T Easy vector (Promega, Madison, WI). Amplification of 5'- and 3'-ends of the cDNA fragment for the trout CMP-Sia synthetase was carried out as described previously (Ausubel et al., 1997
). Nucleotide sequences of the cloned cDNAs were determined by the dideoxy chain termination method (Sanger et al., 1977
) on DNA sequencer Model 373 (PE Applied Biosystems, Foster City, CA).
Computational analysis for DNA and protein sequences
Homology searches were carried out at the National Center for Biotechnology Information by using the BLAST 2.0 program. For multiple alignments DNASIS-Mac v3.0 (Hitachi Software, Yokohama, Japan) was used. Nuclear localization signals were searched for by using the PSORT II program.
Construction of prokaryotic expression plasmids
A DNA fragment coding for a truncated form of the trout CMP-Sia synthetase, lacking the C-terminal 12 amino acids of the open reading frame, was amplified by PCR using the following primers: 5'-GGAAGCTTCTTTCAATGGCGCT GCAA-3' and 5'-GAGACTTGGCCTTCTTCTTGAGCAG-3'. The amplified fragment was subcloned into pBluescript II KS(+) through EcoR V site. The inserted fragment was removed by digestion with Hind III and Not I, and then ligated into pET32b(+) (Novagen, Madison, WI) through the same site (pCSSrt). An expression plasmid for murine CMP-Neu5Ac synthetase was constructed as follows. The plasmid pAM16 encoding the entire open reading frame of the murine enzyme (Münster et al., 1998) was digested with BstX I and subsequently treated with T4 DNA polymerase to remove the upstream termination codons (nucleotides 36 to 34, and 33 to 31) of the first Met residue. The plasmid was self-ligated (pAM16 BstX I(-)) and then digested with BamH I and Xho I. The insert was then subcloned into the BamH I/Xho Idigested pET32b(+) (pCSSm).
Preparation and enzyme assay of the various recombinant enzymes
E. coli strain BL21(DE3)pLysS was transformed with the plasmid pCSSrt, pCSSm, or the empty pET32b(+). A single colony of transformed cells was isolated and cultured in Luria broth supplemented with ampicillin (LA). Five milliliters of LA were inoculated with 50 µl of overnight culture of the transformed cells and incubated at 37°C for 2.5 h, followed by incubation at 15°C for 2.5 h in the presence of 0.4 mM isopropyl-1-thio-ß-D-galactopyranoside. Bacterial cells were harvested and suspended in 500 µl of 50 mM TrisHCl (pH 8.0) containing 2 mM EDTA and 1 µg/ml each of aprotinin, leupeptin, and pepstatin A. The suspension was incubated at 30°C for 30 min, sonically disrupted, and centrifuged at 16,000 x g for 10 min. The resultant supernatant was used for the enzyme assay.
For purification of the recombinant enzymes, 5 ml of overnight culture of transformant cells with pCSSrt or pCSSm were incubated in 200 ml of LA at 37°C for 2.5 h and followed by the induction as described above. The supernatant obtained from the sonically disrupted cells was mixed with an equal volume of 10 mM imidazole, 1.0 M NaCl, and 40 mM TrisHCl (pH 7.9) and applied to Ni2+ chelating column (1.4 x 1.3 cm) that had been equilibrated with 5 mM imidazole, 0.5 M NaCl, and 20 mM TrisHCl (pH 7.9). After washing with 30 mM imidazole, 0.5 M NaCl, and 20 mM TrisHCl (pH 7.9), the column was eluted with 100 mM imidazole. The fractions that contained the enzyme activity were collected. SDSPAGE, Coomassie brilliant blue staining, and western blotting using His-probe (H-15) were carried out as described previously (Ziak et al., 1996; Kitajima et al., 1994
).
Assay for CMP-Sia synthetase activity
The reaction mixture (50 µl) contained 5.0 µl of enzyme fraction in 100 mM TrisHCl buffer (pH 9.0), 1.2 mM KDN or 1.0 mM Neu5Ac, 5.0 mM CTP, 20 mM MgCl2, and 0.1 mM Na3VO4 (preventing CTP dephosphorylation). After incubation at 25°C (for the trout enzyme) or 37°C (for the murine enzyme) for 1 h, the reaction mixture received 10 µl of 1 M NaOH-glycine (pH 10.0), 2 µl (30 units) of alkaline phosphatase from calf intestine (Böhringer Mannheim, Mannheim, Germany), and 38 µl of water and was further incubated at 37°C for 3 h to destroy excess CTP. Cold ethanol (300 µl) was added and centrifuged at 16,000 x g for 10 min. The supernatant was dried under reduced pressure, dissolved in 100 µl water, and applied to the high-performance liquid chromatography (HPLC) analysis. One unit of the CMP-Sia synthetase activity was defined as the amount of enzyme that produces 1 µmol of CMP-KDN per 1 min under these conditions.
HPLC was carried out on a JASCO HPLC system using a resource Q column (6.5 x 30 mm). The column was eluted at 1.0 ml/min with a linear gradient of 0 to 0.2 M NaCl in 20 mM TrisHCl buffer (pH 8.0) in 10 min. The elution profile was monitored by absorbance at 271 nm, and the peak area was integrated for quantitation. Authentic compounds of CMP-KDN and CMP-Neu5Ac were also used for quantitative analysis. Each peak was pooled and analyzed by the DMB derivatization method (Angata et al., 1999a) for the incorporation of KDN or Neu5Ac in the compound under the peak.
Kinetic analysis
For kinetic analysis, the enzyme concentration, incubation period, and CTP concentration were optimized. The enzyme concentration was determined by the BCA protein assay kit (Pierce, Rockford, IL) using bovine serum albumin as a standard. KDN at 0.3, 0.6, 1.2, or 2.4 mM or Neu5Ac at 0.25, 0.5, 1.0, or 2.0 mM was incubated with the purified recombinant trout enzyme (0.05 µg) or the purified recombinant murine enzyme (0.6 µg) in 50 µl of 0.1 M TrisHCl (pH 9.0) containing 5.0 mM CTP, 20 mM MgCl2, and 0.1 mM sodium vanadate at 25°C (for the trout enzyme) or 37°C (for the murine enzyme) for 1 h. The product was quantitated by the HPLC method as described above. The kinetic parameters were determined by double-reciprocal Lineweaver-Burk plots.
Northern blot analysis
Total RNA was extracted from rainbow trout liver, testis, or ovary with TRIZOL (Gibco BRL, Rockville, MD) according to the manufacturers instructions. Poly(A)+RNA was isolated with Oligotex-dT 30 (TaKaRa). One microgram of poly(A)+RNA was separated on 1.0% agarose/2.2 M formaldehyde gel, blotted to a Hybond N+ membrane (Amersham Pharmacia Biotech., Uppsala, Sweden), and hybridized to digoxigenin-labeled rainbow trout CMP-Sia synthetase cRNA (nucleotides 127713). Hybridization was carried out at 65°C for 12 h in 50% formamide, 5x SSC, 1.5% SDS, 0.1 mg/ml bakers yeast tRNA, and 2% blocking reagent (Böhringer Mannheim). The membrane was washed with 0.2x SSC and 0.1% SDS at 65°C for 30 min. The bound RNA probes were visualized by incubation with alkaline phosphataseconjugated sheep anti-digoxigenin-Fab (Böhringer Mannheim) and subsequently with the CDP-Star chemiluminescent substrate (New England BioLabs, Beverly, MA), as described previously (Trayhurn et al., 1995).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Angata, T., Matsuda, T., and Kitajima, K. (1998) Synthesis of neoglycoconjugates containing deaminated neuraminic acid (KDN) using rat liver 2, 6-sialyltransferase. Glycobiology, 8, 277284.
Angata, T., Nakata, D., Matsuda, T., Kitajima, K., and Troy, F.A. II (1999a) Biosynthesis of KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). Identification and characterization of a KDN-9-phosphate synthetase activity from trout testis. J. Biol. Chem., 274, 2294922956.
Angata, T., Nakata, D., Matsuda, T., and Kitajima, K. (1999b) Elevated expression of free deaminoneuraminic acid in mammalian cells cultured in mannose-rich media. Biochem. Biophys. Res. Commun., 261, 326331.[ISI][Medline]
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J, G., Smith, J.A., and Struhl, K. (1997) Current Protocols in Molecular Biology Vol. 2. John Wiley & Sons, New York, pp. 15.0.115.8.8.
Campanero-Rhodes, M.A., Solís, D., Carrera, E., de la Cruz, M.J., and Díaz-Mauriño, T. (1999) Rat liver contains age-regulated cytosolic 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid (Kdn). Glycobiology, 9, 527532.
Edwards, U., Muller, A., Hammerschmidt, S., Gerardy-Schahn, R., and Frosch, M. (1994) Molecular analysis of the biosynthesis pathway of the 2, 8 polysialic acid capsule by Neisseria meningitidis serogroup B. Mol. Microbiol., 14, 141149.[ISI][Medline]
Gil-Serrano, A.M., Rodriguez-Carvajal, M.A., Tejero-Mateo, P., Espartero, J.L., Thomas-Oates, J., Ruiz-Sainz, J.E., and Buendia-Claveria, A.M. (1998) Structural determination of a 5-O-methyl-deaminated neuraminic acid (Kdn)-containing polysaccharide isolated from Sinorhizobium fredii. Biochem. J., 334, 585594.[ISI][Medline]
Inoue, S., Kitajima, K., and Inoue, Y. (1996) Identification of 2-keto-3-deoxy-D-glycero-D-galactonononic acid (KDN, deaminoneuraminic acid) residues in mammalian tissues and human lung carcinoma cells. Chemical evidence of the occurrence of KDN glycoconjugates in mammals. J. Biol. Chem., 271, 2434124344.
Inoue, S., Lin, S.-L., Chang, T., Wu, S.-H., Yao, C.-W., Chu, T.-Y., Troy, F.A. II, and Inoue, Y. (1998) Identification of free deaminated sialic acid (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) in human red blood cells and its elevated expression in fetal cord red blood cells and ovarian cancer cells. J. Biol. Chem., 273, 2719927204.
Kanamori, A., Inoue, S., Xulei, Z., Zuber, C., Roth, J., Kitajima, K., Ye, J., Troy, F.A. II, and Inoue, Y. (1994) Monoclonal antibody specific for 2
8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry. Histochemistry, 101, 333340.[ISI][Medline]
Kitajima, K., Kuroyanagi, H., Inoue, S., Ye, J., Troy, F.A. II, and Inoue, Y. (1994) Discovery of a new type of sialidase, "KDNase, " which specifically hydrolyzes deaminoneuraminyl (3-deoxy-D-glycero-D-galacto-2-nonulosonic acid) but not N-acylneuraminyl linkages. J. Biol. Chem., 269, 2141521419.
Knirel, Y.A., Kocharova, N.A., Shashkov, A.S., Kochetkov, N.K., Mamontova, V.A., and Soloveva, T.F. (1989) Structure of the capsular polysaccharide of Klebsiella ozaenae serotype K4 containing 3-deoxy-D-glycero-D-galacto-nonulosonic acid. Carbohydr. Res., 188, 145155.[ISI][Medline]
Lawrence, S.M., Huddleston, K.A., Pitts, L.R., Nguyen, N., Lee, Y.C., Vann, W.F., Colemen, T.A., and Betenbaugh, M.J. (2000) Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid biosynthetic ability. J. Biol. Chem., 275, 1786917877.
Münster, A.K., Eckhardt, M., Potvin, B., Mühlenhoff, M, Stanley, P., and Gerardy-Schahn, R. (1998) Mammalian cytidine 5'-monophosphate N-acetylneuraminic acid synthetase: a nuclear protein with evolutionarily conserved structural motifs. Proc. Natl Acad. Sci. USA, 95, 91409145.
Nadano, D., Iwasaki, M., Endo, S., Kitajima, K., Inoue, S., and Inoue, Y. (1986) A naturally occurring deaminated neuraminic acid, 3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN). Its unique occurrence at the nonreducing ends of oligosialyl chains in polysialoglycoprotein of rainbow trout eggs. J. Biol. Chem., 261, 1155011557.
Nakai, K., and Horton, P. (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem. Sci. Sci., 24, 3436.[ISI][Medline]
Nakata, D., Close, B.E., Colley, K.J., Matsuda, T., and Kitajima, K. (2000) Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which does not have deaminoneuraminic acid (KDN) 9-phosphate synthase activity. Biochem. Biophys. Res. Commun., 273, 642648.[ISI][Medline]
Sanger, F., Nicklen, S., and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA, 74, 54635467.[Abstract]
Stoughton, D.M., Zapata, G., Picone, R., and Vann, F.W. (1999) Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase. Biochem. J., 343, 397402.[ISI][Medline]
Terada, T., Kitazume, S., Kitajima, K., Inoue, S., Ito, F., Troy, F.A. II, and Inoue, Y. (1993) Synthesis of CMP-deaminoneuraminic acid (CMP-KDN) using the CTP:CMP-3-deoxynonulosonate cytidylyltransferase from rainbow trout testis. Identification and characterization of a CMP-KDN synthetase. J. Biol. Chem., 268, 26402648.
Trayhurn, P., Thomas, M.E., Duncan, J.S., Black, D., Beattie, J.H., and Rayner, D.V. (1995) Ultra-rapid detection of mRNAs on northern blots with digoxigenin-labeled oligonucleotides and CDP-Star, a new chemiluminescence substrate. Biochem. Soc. Trans, 23, 494S.[Medline]
Tullius, M.V., Munson, R.S. Jr., Wang, J., and Gibson, B.W. (1996) Purification, cloning and expression of a cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi. J. Biol. Chem., 271, 1537315380.
Vionnet, J., Concepcion, N., Warner, T., Zapata, G., Hanover, J., and Vann, W.F. (1999) Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands. Glycobiology, 9, 481487.
Yu. S., Y., Kitajima, K., Inoue, S., and Inoue, Y. (1991) Isolation and structural elucidation of a novel type of ganglioside, deaminated neuraminic acid (KDN)-containing glycosphingolipid, from rainbow trout sperm. The first example of the natural occurrence of KDN-ganglioside, (KDN)GM3. J. Biol. Chem., 266, 2192921935.
Yu, S., Kitajima, K., Inoue, S., Khoo, K. -H., Morris, H.R., Dell, A., and Inoue, Y. (1995) Expression of new KDN-gangliosides in rainbow trout testis during spermatogenesis and their structural identification. Glycobiology, 5, 207218.[Abstract]
Ziak, M., Qu, B., Zuo, X., Zuber, C., Kanamori, A., Kitajima, K., Inoue, S., Inoue, Y., and Roth, J. (1996) Occurrence of poly(2, 8-deaminoneuraminic acid) in mammalian tissues: widespread and developmentally regulated but highly selective expression on glycoproteins. Proc. Natl Acad. Sci. USA, 93, 27592763.
Ziak, M., Meier, M., and Roth, J. (1999a) Megalin in normal tissues and carcinoma cells carries oligo/poly 2, 8 deaminoneuraminic acid as a unique posttranslational modification. Glycoconj. J., 16, 185188.[ISI][Medline]
Ziak, M., Kerjaschki, D., Farquhar, M.G., and Roth, J. (1999b) Identification of megalin as the sole rat kidney sialoglycoprotein containing poly 2, 8 deaminoneuraminic acid. J. Am. Soc. Nephrol., 10, 203209.