Molecular cloning of a unique CMP–sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates

Daisuke Nakata2, Anja-K. Münster3, Rita Gerardy-Schahn3, Naohito Aoki2, Tsukasa Matsuda2 and Ken Kitajima1,2,4

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a sialic acid (Sia) that is ubiquitously expressed in vertebrates during normal development and tumorigenesis. Its expression is thought to be regulated by multiple biosynthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synthetases had very low activity to synthesize CMP-KDN from KDN and CTP, and the corresponding enzyme from rainbow trout testis had high activity to synthesize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al. [1993] J. Biol. Chem., 268, 2640–2648). To demonstrate if the unique substrate specificity found in the crude trout enzyme is conveyed by a single enzyme, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based strategy. The trout enzyme was shown to consist of 432 amino acids with two potential nuclear localization signals, and the cDNA sequence displayed 53.8% identity to that of the murine enzyme. Based on the Vmax/Km values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min–1). In contrast, the recombinant murine enzyme had 15 times lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min–1). Northern blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicate that at least one cloned enzyme has the ability to utilize both KDN and Neu5Ac as substrates efficiently and is useful for the production of CMP-KDN.

Key words: sialic acid/deaminoneuraminic acid/CMP-KDN synthetase/CMP-Neu5Ac synthetase/CMP–sialic acid synthetase


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is a sialic acid in which an acylamino group at C-5 position of N-acylneuraminic acid is replaced by a hydroxyl group. KDN is now acknowledged to be a ubiquitous component of vertebrate glycoconjugates (Nadano et al., 1986Go; Yu et al., 1991Go, 1995; Kanamori et al., 1994Go; Ziak et al., 1996Go; Inoue et al., 1996Go, 1998; Angata et al., 1998Go, 1999a and references therein) and bacterial capsular polysaccharides (Knirel et al., 1989Go; Gil-Serrano et al., 1998Go), although it was first identified in rainbow trout egg polysialoglycoprotein in 1986 (Nadano et al., 1986Go). In rainbow trout, KDN is highly expressed in ovary and testis, but not in liver and other somatic tissues tested (Angata et al., 1999aGo; Yu et al., 1991Go). In mammals, KDN is expressed in some lung carcinoma cells (Inoue et al., 1996Go), and in rat fetal lung but not in adult lung (Ziak et al., 1996Go). It has also been shown that KDN exists in human fetal cord red blood cells and ovarian cancer cells (Inoue et al., 1998Go). Recently, in normal rat kidney, megalin has been identified as the sole sialoglycoprotein containing an oligopolymeric form of KDN (Ziak et al., 1999aGo,b). Furthermore, KDN is reported to be expressed in rat tissues in an age-dependent manner (Campanero-Rhodes et al., 1999Go). Thus, expression of KDN in vertebrates is regulated during normal development and tumorigenesis.

The biosynthetic pathway of KDN and KDN ketosides in glycan chains was shown to consist of the following sequential reactions (Angata et al., 1999aGo,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., 1999aGo), CMP-Sia synthetase catalyzing reaction (iv) (Terada et al., 1993Go), and KDN transferase catalyzing reaction (v) (Angata et al., 1994Go) were characterized using partially purified enzymes from rainbow trout (Kanamori et al., 1994Go; Yu et al., 1991Go, 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., 1993Go). This property is unique because fetal calf brain CMP-Neu5Ac synthetase had only a weak activity toward KDN (Terada et al., 1993Go). 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., 1993Go). 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., 1998Go). 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., 1993Go).


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Five highly conserved motifs in the CMP-Neu5Ac synthetases from mouse, human, and bacteria
A murine CMP-Neu5Ac synthetase cDNA has recently been cloned and four deduced amino acid sequences (motifs I–IV in Figure 1B) conserved between murine and bacterial CMP-Neu5Ac synthetases have been reported (Münster et al., 1998Go). We screened the expression sequence tags (EST) database for homologous sequences to the murine CMP-Neu5Ac synthetase and found sequences with the highest homology from the human EST database. Comparison of the deduced amino acid sequences of the murine, human, and bacterial CMP-Neu5Ac synthetase cDNAs revealed five conserved sequence motifs, including four known motifs, I–IV, and a newly found motif, V (Figure 1B).




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Fig. 1. Nucleotide and deduced amino acid sequences of the cDNA coding for the trout CMP-Sia synthetase. (A) The nucleotide and amino acid sequences are numbered from the presumed start codon and initiation Met, respectively. Two predicted nuclear localization signals are underlined. Five homologous amino acid sequence motifs are shown as shaded boxes. Three potential N-glycosylation sites are located at Asn-201, -267, and -324. (B) Alignment of the five amino acid sequence motifs (shown in panel A), which are conserved in CMP-Sia synthetase of vertebrates and bacteria. Accession numbers are AB027414 for rainbow trout; AJ006215 for mouse; AF271388 for human; U19899 for Streptococcus agalactiae; P13266 for Escherichia coli; and X78068 for Neisseria meningitidis. (C) Alignment of the two nuclear localization signals underlined in panel A with the corresponding signals reported in the murine enzyme (Münster et al., 1998Go). The amino acid residues identical to those of the trout enzyme are shadowed.

 
Molecular cloning and sequencing of the rainbow trout CMP-Sia synthetase cDNA
By reverse transcription (RT)-PCR amplification using rainbow trout testis mRNA (harvested in August) and degenerate primers designed for conserved motifs I and V, a cDNA fragment containing a similar sequence to murine CMP-Neu5Ac synthetase cDNA was obtained, and the 5'- and 3'-ends of the fragment were extended by the 5'– and 3'–rapid amplification of cDNA end (RACE) procedures. As shown in Figure 1A, we determined a 1468-bp sequence of overlapping cDNA clones for rainbow trout CMP-Sia synthetase, which contained an open reading frame of 1296 bp preceded by an in-frame termination codon TAG at –102 to –100. The open reading frame encoded a protein of 432 amino acids with a predicted molecular mass of approximately 48 kDa. The deduced amino acid sequence showed 53.8% identity to the murine CMP-Neu5Ac synthetase. All the conserved motifs I–V were also found and a PSORT II program search (Nakai and Horton, 1999Go) revealed two predicted nuclear localization signals in the sequence (Figure 1A,C).

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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Fig. 2. Synthesis of CMP-KDN or CMP-Neu5Ac from KDN or Neu5Ac and CTP by the recombinant CMP-Sia synthetases. The substrate mixture containing 1.2 mM KDN (A, C, E) or 1.0 mM Neu5Ac (B, D, F) and 5.0 mM CTP was incubated with the recombinant enzymes prepared from the bacterial cells transformed with pCSSrt (A, B), pCSSm (C, D), or the empty plasmid (E, F). The products were analyzed with HPLC as described under Materials and methods. The protein amounts of the enzyme fractions used were 99 µg for A and B, 78 µg for C and D, and 82 µg for E and F. Peaks 1 and 2 were assigned as CMP-KDN and CMP-Neu5Ac, respectively, according to the retention times for authentic CMP-KDN (9.56 min) and CMP-Neu5Ac (8.99 min). Note that no CMP-KDN or CMP-Neu5Ac was synthesized by the enzyme fraction prepared from the empty plasmid-transformed cells.

 
As shown in Figure 3, these enzyme fractions gave a single major band with a few other weak bands on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE)/Coomassie brilliant blue staining. The major bands were only detected on western blot analyses using His-probe (H-15) antibody, showing that they are the recombinant enzymes. The molecular masses of them were 69 kDa and 66 kDa for trout and murine enzymes, respectively (Figure 3). This was consistent with the expected molecular masses (66 kDa) of both of the thioredoxin-CMP-Sia synthetase fusion proteins.



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Fig. 3. SDS–PAGE/Coomassie brilliant blue staining and western blot analysis of the recombinant CMP-Sia synthetases. The recombinant enzymes were purified from the transformant cells with pCSSrt and pCSSm and subjected to SDS–PAGE/western blot analysis using His-probe (H-15) as described under Materials and methods. (A) SDS–PAGE/Coommassie brilliant blue staining for the crude (lane 1) and purified (lane 2) recombinant trout enzyme; (B) SDS–PAGE/ Coommassie brilliant blue staining for the crude (lane 1) and purified (lane 2) recombinant murine enzyme; (C) SDS–PAGE/western blotting for the purified trout (lane 1) and murine (lane 2) enzymes.

 
Kinetic analyses
For kinetic analysis, the purified recombinant enzymes (Figure 3) were used. The amount of trout enzyme and the incubation period used in the kinetic assay were set at 0.05 µg and 1 h, respectively, because incubation at 25°C with up to 0.2 µg for up to 3 h resulted in linearly increasing formation of CMP-KDN or CMP-Neu5Ac. At 5.0 mM CTP, which was a saturating concentration, the production of the CMP-KDN or CMP-Neu5Ac reach a maximum level when 0.6 mM KDN or Neu5Ac was used. Lineweaver-Burk plots for KDN and Neu5Ac are shown in Figure 4A. The Km and Vmax values of the trout enzyme were 3.0 mM and 450 min–1 for KDN, and 2.8 mM and 270 min–1 for Neu5Ac, respectively (Table I). The trout enzyme was shown to convert both KDN and Neu5Ac to their CMP-Sia compounds with twice as high efficiency toward KDN as Neu5Ac, based on the Vmax/Km values: 1.1 min–1 for KDN, 0.68 min–1 for Neu5Ac (Table I). The relatively higher values of standard deviations of the kinetic parameters were obtained for the trout enzyme. This is possibly because the trout enzyme is unstable even at 25°C in comparison with the murine enzyme. The instability of the trout enzyme at 25 and 37°C was reported previously (Terada et al., 1993Go).



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Fig. 4. Lineweaver-Burk plots for the synthesis of CMP-KDN and CMP-Neu5Ac catalyzed by the purified recombinant CMP-Sia synthetase from rainbow trout and mouse. The substrate mixture containing 5.0 mM CTP and varying concentrations of KDN (open square) or Neu5Ac (open circle) was incubated with (A) the recombinant trout enzyme (0.05 µg) at 25°C for 1 h and (B) the recombinant murine enzyme (0.6 µg) at 37°C for 1h. The initial velocities (V) were measured by quantitating the products on HPLC as described under Materials and methods. [S] represents a substrate concentration for KDN or Neu5Ac.

 

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Table I. Kinetic parameters for the recombinant trout and murine CMP-Sia synthetases
 
The same kinetic experiments were carried out for the murine enzyme. The amount of enzyme and the incubation period were set at 0.6 µg and 1 h, respectively, because incubation at 37°C with up to 1.0 µg for up to 3 h gave the linear formation of the products. At 5.0 mM CTP, the production of CMP-KDN or CMP-Neu5Ac reach a maximum level when 0.6 mM KDN or Neu5Ac was used. Based on the results shown in Figure 4B, the Km and Vmax values of the murine enzyme were 0.56 mM and 3.3 mM min–1 for KDN, and 0.26 mM and 0.89 mM min–1 for Neu5Ac, respectively (Table I). The murine enzyme could convert both KDN and Neu5Ac to the respective CMP derivatives but showed 15 times lower activity toward KDN compared with Neu5Ac (the Vmax/Km values: 0.23 min–1 and 3.5 min–1, respectively).

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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Fig. 5. Northern blot analysis of the rainbow trout CMP-Sia synthetase mRNA in liver, ovary and testis. One microgram of poly(A)+RNA was resolved on a 1% agarose/2.2 M formaldehyde gel and, after transfer to a nylon membrane, was probed with a digoxigenin-labeled cRNA as described under Materials and methods. Lane 1, poly(A)+ RNA from liver; lane 2, that from immature ovary; lane 3, that from immature testis; lane 4, that from mature testis. The rainbow trout GAPDH (accession number AF027130) probe was used as a control. kb, kilobases.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Sequence analysis of the cDNA for the trout CMP-Sia synthetase indicates that the enzyme is a 48-kDa protein with 432 amino acid residues. On gel filtration, the CMP-Sia synthetase from rainbow trout testis was reported to be eluted at 260 kDa as a single peak (Terada et al., 1993Go). Thus, the CMP-Sia synthetase is suggested to exist as an oligomer in a native form. It was also reported that the bovine anterior pituitary gland enzyme existed as a 158-kDa oligomeric form of 52-kDa monomers (Vionnet et al., 1999Go). Interestingly, the murine CMP-Neu5Ac synthetase contains the same number (432) of amino acid residues (Münster et al., 1998Go), and the overall identities of the deduced amino acid sequences between the murine and trout enzymes are 54%. Based on the identities between the two polypeptide sequences, this class of enzymes can be tentatively separated into three domains: the first 30 amino acids (20% identities), the N-terminal half domain of amino acid residues 31–254 (76% identities), and the C-terminal half domain of amino acid residues 255–432 (37% identities). The N-terminal half domain is more highly conserved than the other domains. Five evolutionarily conserved structural motifs (motifs I–V) are also conserved in the rainbow trout enzyme (Figure 1B). Interestingly, all these motifs exist in the N-terminal half domain. Motif I was required for the catalytic activity of the enzyme (Stoughton et al., 1999Go). Furthermore, Haemophilus influenzae, Haemophilus ducreyi, and Neisseria meningitidis CMP-Neu5Ac synthetases were shown to consist of a shorter polypeptide chain corresponding to the N-terminal half domain with all the conserved motifs I–V and were devoid of the sequence corresponding to the C-terminal half domain (Tullius et al., 1996Go; Edwards et al., 1994Go). Therefore, the N-terminal half domain appears to be a catalytic domain of this class of enzyme. On the other hand, the less conserved C-terminal half domain may possibly be a regulatory domain that is involved in some cell type–specific functions of the enzyme.

It has been recognized that KDN is a poor substrate for CMP-Neu5Ac synthetases isolated from various animal origins (Terada et al., 1993Go). 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., 1993Go). 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., 1993Go), 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., 1995Go; Terada et al., 1993Go).

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., 1999bGo). 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., 1999aGo,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., 1999bGo). 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., 2000Go). The corresponding human enzyme that has recently been cloned by Lawrence et al. (2000)Go 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., 1994Go), KDN-9-P synthase from rainbow trout testis (Angata et al., 1999aGo), and KDN-ketosides hydrolyzing enzyme (KDNase Sm) from Sphingobacterium multivorum (Kitajima et al., 1994Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
KDN and CMP-KDN were prepared as described previously (Angata et al., 1998Go). Neu5Ac, CMP-Neu5Ac, aprotinin, and leupeptin were from Sigma (St. Louis, MO). Pepstatin A was from Peptide Institute Inc. (Minoh, Japan). The restriction endonucleases and T4 DNA polymerase were from TaKaRa (Kyoto, Japan). Synthetic oligonucleotide primers were obtained from Rikaken (Nagoya, Japan). His-probe (H-15) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Male and female fish of rainbow trout were kindly provided by the Shiga Prefectural Samegai Trout Farm. Immature trout ovaries were excised from females harvested in May (5 months before ovulation). Immature and mature trout testes were excised from males harvested in May (5 months before spermiation) and in August (immediately before spermiation), respectively. Various tissues of male and female BALB/c mice (8 weeks old, SCL, Japan) were excised, frozen immediately using liquid nitrogen and kept at –80°C until use.

PCR cloning
The following degenerate oligonucleotide primers based on the murine CMP-Neu5Ac synthetase cDNA sequence (Münster et al., 1998Go) were used for the PCR cloning: 5'-AAGGCATCCC(A/C) CTGAAGAA-3' (nucleotides 160–179 in motif I) and 5'-AT(A/G)TCCAC(A/G)TC(A/G/T) ATATCCAC-3' (nucleotides 730–749 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 Tris–HCl (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., 1997Go). Nucleotide sequences of the cloned cDNAs were determined by the dideoxy chain termination method (Sanger et al., 1977Go) 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., 1998Go) 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 I–digested 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 Tris–HCl (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 Tris–HCl (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 Tris–HCl (pH 7.9). After washing with 30 mM imidazole, 0.5 M NaCl, and 20 mM Tris–HCl (pH 7.9), the column was eluted with 100 mM imidazole. The fractions that contained the enzyme activity were collected. SDS–PAGE, Coomassie brilliant blue staining, and western blotting using His-probe (H-15) were carried out as described previously (Ziak et al., 1996Go; Kitajima et al., 1994Go).

Assay for CMP-Sia synthetase activity
The reaction mixture (50 µl) contained 5.0 µl of enzyme fraction in 100 mM Tris–HCl 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 Tris–HCl 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., 1999aGo) 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 Tris–HCl (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 manufacturer’s 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 127–713). Hybridization was carried out at 65°C for 12 h in 50% formamide, 5x SSC, 1.5% SDS, 0.1 mg/ml baker’s 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 phosphatase–conjugated 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., 1995Go).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Drs. Karen Colley and Brett Close for a critical reading and an English editing of this manuscript. This research was supported in part by Grants-in-Aid for International Scientific Research, Joint Research, for Scientific Research on Priority Areas, and for Scientific Research (to K.K.) from the Ministry of Education, Science, Sports and Culture, and by the Asahi Glass Foundation and by the Deutsche Forschungsgemeinschaft (GE801/5-1, to R.G.S. and A.M.).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
9-P, 9-phosphate; EST, expression sequence tags; HPLC, high-performance liquid chromatography; KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid; LA, Luria broth supplemented with ampicillin; Neu5Ac, N-acetylneuraminic acid; Man, mannose; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcription polymerase chain reaction; SDS, sodium dodecyl sulfate; Sia, sialic acid; SSC, 0.3M sodium chloride–0.03M sodium citrate buffer (pH 7.0).


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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