Glycobiology Advance Access originally published online on August 17, 2007
Glycobiology 2007 17(11):1167-1174; doi:10.1093/glycob/cwm086
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A β-galactoside 
2,6-sialyltransferase produced by a marine bacterium, Photobacterium leiognathi JT-SHIZ-145, is active at pH 8
2 Glycotechnology Business Unit, Japan Tobacco Inc, 700 Higashibara, Iwata, Shizuoka 438-0802, Japan
1 To whom correspondence should be addressed: Tel: +81-538-32-7389; Fax: +81-538-33-6046; e-mail: takeshi.yamamoto{at}ims.jti.co.jp
Received on June 9, 2007; revised on July 24, 2007; accepted on August 6, 2007
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Abstract |
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A gene encoding a sialyltransferase produced by Photobacterium leiognathi JT-SHIZ-145 was cloned, sequenced, and expressed in Escherichia coli. The sialyltransferase gene contained an open reading frame of 1494 base pairs (bp) encoding a predicted protein of 497 amino acid residues. The deduced amino acid sequence of the sialyltransferase had no significant similarity to mammalian sialyltransferases and did not contain sialyl motifs, but did show high homology to another marine bacterial sialyltransferase, a β-galactoside
2,6-sialyltransferase produced by P. damselae JT0160. The acceptor substrate specificity of the new enzyme was similar to that of the 2,6-sialyltransferase from P. damselae JT0160, but its activity was maximal at pH 8. This property is quite different from the properties of all mammalian and bacterial sialyltransferases reported previously, which have maximal activity at acidic pH. In general, both sialosides and cytidine-5'-monophospho-N-acetylneuraminic acid, the common donor substrate of sialyltransferases, are more stable under basic conditions. Therefore, a sialyltransferase with an optimum pH in the basic range should be useful for the preparation of sialosides and the modification of glycoconjugates, such as asialo-glycoproteins and asialo-glycolipids. Thus, the sialyltransferase obtained from P. leiognathi JT-SHIZ-145 is a promising tool for the efficient production of sialosides. Key words: genus Photobacterium / marine bacterium / sialyltransferase
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementReferences |
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All mammalian cells have various carbohydrates on their surfaces in the form of sugar chains covalently bound to membrane proteins and lipids. The carbohydrate moieties of these glycoproteins and glycolipids play important roles in a variety of biochemical phenomena, such as cell–cell recognition and cell differentiation (Varki 1993
; Gagneux and Varki 1999). The sugar chain moieties of glycoconjugates are synthesized in the cells by a series of glycosyltransferases (Stults et al. 1988; Burda and Aebi 1999).
Lipopolysaccharides are the major outer surface membrane components expressed by majority of Gram-negative bacteria species and composed of specific carbohydrate lipid moiety and a covalently linked hydrophilic polysaccharide portion (Holst et al. 1996; Wakarchuk et al. 1998; Muldon et al. 2002). The sugar chain structures of lipopolysaccharides in human pathogens have been especially well studied. Bacterial pathogens have evolved to escape the immune systems of hosts by mimicking the surface carbohydrates of the host cells, which are crucial for self/nonself recognition. Thus, the glycoconjugates of the pathogens contribute to their virulence and adhesion to host cells (Guerry et al. 2002).
Unlike lipopolysaccharides, the glycosylation of proteins was once generally thought to be restricted to eukaryotes. However, recent studies have revealed that glycoproteins are also present in prokaryotic organisms (Erickson and Herzberg 1993; Young et al. 2002). Although bacterial N-linked sugar chains differ structurally from their eukaryotic counterparts, protein glycosylation pathways similar to those in eukaryotes exist in Gram-negative bacteria. A functional N-linked glycosylation pathway of a Gram-negative bacterium has been transferred into E. coli (Wacker et al. 2002). Therefore, the bacterial glycosylation capability will likely be modified to express human-type glycosylated proteins in E. coli in the near future. Indeed, research on glycosyltransferases from diverse bacteria has been quite active lately (Koizumi 2003), and various types of carbohydrate chains have been synthesized using bacterial enzymes (Johnson 1999; Izumi et al. 2001). In general, bacterial enzymes are more stable and productive in E. coli expression systems than are mammalian enzymes, and bacterial glycosyltransferases have a broader acceptor substrate specificity than mammalian glycosyltransferases. Therefore, bacterial enzymes are expected to serve as good tools for the synthesis and modification of several glycans that thought to be drug candidates (Izumi and Wong 2001).
During the course of our research, we have identified over 20 bacteria that produce sialyltransferase, many of which are classified in the genus Photobacterium or the closely related genus Vibrio. For instance, P. phosphoreum JT-ISH-467, Photobacterium sp. JT-ISH-224, and Vibrio sp. JT-FAJ-16 all show 2,3-sialyltransferase activity, and P. damselae JT0160 expresses 2,6-sialyltransferase activity (Yamamoto et al. 2006).
In this paper, we report on the cloning of a β-galactoside 2,6-sialyltransferase gene from P. leiognathi JT-SHIZ-145 and the expression and the characterization of the recombinant enzyme. Because this enzyme is most active at basic pH, we believe it is a promising tool for the effective synthesis of sialosides and the modification of glycoconjugates.
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Results |
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Isolation of a marine bacterium showing sialyltransferase activity
We examined more than 3000 isolates of bacteria for sialyltransferase activity. From these, we isolated the JT-SHIZ-145 strain that was showing sialyltransferase activity from the surface of a Japanese common squid. Using pyridylaminated lactose as an acceptor substrate, we performed an enzymatic reaction with crude extract prepared from JT-SHIZ-145 and found pyridylaminated 6'-sialyllactose in the reaction mixture, indicating that JT-SHIZ-145 produces an
2,6-sialyltransferase. A partial DNA sequence of the 16S ribosomal RNA gene of JT-SHIZ-145 classified it into the cluster of the genus Photobacterium, and the sequence showed 99.8% homology to Photobacterium leiognathi (ATCC 25521). We therefore named this strain P. leiognathi JT-SHIZ-145.
The 2,6-sialyltransferase gene is a homologue of the P. damselae JT0160 2,6-sialyltransferase gene
We previously cloned an 2,6-sialyltransferase gene from P. damselae JT0160 (Yamamoto, Nakashizuka, et al. 1998). To investigate whether there is a homologue of this 2,6-sialyltransferase gene in the P. leiognathi JT-SHIZ-145 genome, we performed a Southern blot analysis. We detected a 5.5-kb EcoRI fragment, a 4.8-kb HindIII fragment, a 4.8-kb BamHI fragment, and a 1.6-kb Pst1 fragment that hybridized to a 1200-bp EcoRI and HindIII fragment of the 2,6-sialyltransferase gene from P. damselae JT0160. We therefore concluded that the 2,6-sialyltransferase gene in the P. leiognathi JT-SHIZ-145 genome is a homologue of the P. damselae JT0160 2,6-sialyltransferase gene.
Cloning of the 2,6-sialyltransferase gene homologue
The genomic DNA of JT-SHIZ-145 was digested with Pst1 and the DNA fragments were separated in an agarose gel. The DNA fraction containing the 1.6-kb Pst1 fragment was recovered and purified from the gel and then subcloned into the Pst1 site of pUC18 and transformed into E. coli TB1 cells. To obtain positive clones, we performed colony hybridization using the 1200-bp EcoRI and HindIII fragment from the P. damselae JT0160 2,6-sialyltransferase gene as a hybridization probe. We recovered a plasmid DNA containing a 1.6-kb Pst1 fragment from the positive clones. Both strands of the inserted DNA fragment were sequenced. The amino acid sequence deduced from the sequenced DNA fragment showed significant homology with the sialyltransferase of P. damselae JT0160. Next, using the sequence information of the 1.6-kb Pst1 fragment described above, we designed two sequence primers, named SHIZ145/26/N1 and SHIZ145/26 N2 (Table I), to determine the nucleotide sequence of the 1.6-kb Pst1 DNA fragment. The amplified DNA fragments were sequenced completely. Figure 1 shows the nucleotide sequence of the 1.6-kb Pst1 DNA fragment and the predicted amino acid sequence of the sialyltransferase of P. leiognathi JT-SHIZ-145. An open reading frame of 1494 nucleotides was found; the amino acid sequence deduced from the nucleotide sequence of the sialyltransferase had 497 amino acid residues with a calculated molecular weight of 56.6 kDa. The first 15 amino acid residues contained a hydrophobic region predicted to be a signal peptide by Genetyx version 7.0 (Genetyx Co, Tokyo, Japan). The DNA sequence of the P. leiognathi JT-SHIZ-145 2,6-sialyltransferase gene had 68.2% homology to that of the 2,6-sialyltrasferase in P. damselae JT0160. The amino acid sequence of P. leiognathi JT-SHIZ-145 2,6-sialyltransferase had 66.3% homology to that of the 2,6-sialyltransferase in P. damselae JT0160.
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Expression of the
2,6-sialyltransferase in E. coli and purification of the recombinant 2,6-sialyltransferaseExpression vectors expressing the full-length gene for P. leiognathi JT-SHIZ-145
2,6-sialyltransferase (N0C0/pTrc99A) and the truncated form (N1C0/pTrc99A), which lacks the 15 amino acids of the N-terminal hydrophobic region were expressed in E. coli. The crude extracts from both isopropyl-1-thio-β-D-galactopyranoside (IPTG)-treated E. coli strains showed sialyltransferase activity. The sialyltransferase activity of the extract from E. coli with N1C0/pTrc99A was much more productive than that of N0C0/pTrc99A (data not shown). The truncated enzyme produced from E. coli harboring N1C0/pTrc99A was purified through five steps of chromatography in the presence of a detergent, with a yield of 6.5% (Table II). The enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 50 kDa (Figure 2). The specific activity of the purified enzyme was 3.8 U/mg. The crude extract of the truncated form was reacted with pyridylaminated lactose, and its products were analyzed by high-performance liquid chromatography (HPLC). The retention time of the products was consistent with that of an authentic pyridylaminated 6'-sialyllactose (Yamamoto et al. 1996).
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Identification of the enzymatic reaction product by 1H-NMR (nuclear magnetic resonance) spectroscopy
We performed an enzymatic reaction using the purified recombinant truncated enzyme. The product was isolated and analyzed by 1H-NMR spectroscopy in D2O at 298 K. The product formed with lactose as an acceptor substrate was N-acetylneuraminic acid (NeuAc)
2,6-Galβ1-4Glc and 22.5 mg of this product was obtained. The structural reporter group signals were as follows: 
5.21 (d, 0.4 H, J = 3.5 Hz, Glc H-1), 4.66 (d, 0.6 H, J = 8.0 Hz, Glc β H-1), 4.42 (d, 1 H, J = 7.8 Hz, Gal β H-1), 3.30 (dd, 0.6 H, J = 8.4 Hz, 8.4 Hz, Glc β H-2), 2.70 (dd, 1 H, J = 4.5 Hz, 12.35 Hz, NeuAc H-3 eq), 2.02 (s, 3 H, Ac), 1.73 (dd, 1 H, J = 12.3 Hz, 12.3 Hz, NeuAc H-3 ax). The 1H-NMR spectrum of the product was in good agreement with that reported for NeuAc 2,6-Galβ1-4Glc (Sabesan and Paulson 1986). These results confirm that the isolated sialyltransferase is an 2,6-sialyltransferase.
Functional properties of the truncated 2,6-sialyltransferase
The optimum temperature and pH for the activity of the sialyltransferase were found to be 30°C (data not shown) and pH 8 (Figure 3), respectively. We tested a variety of substrates for their ability to act as acceptors for the sialyltransferase. Methyl-β-D-galactopyranoside was the best acceptor substrate among the monosaccharides, although N-acetyl-D-galactosamine was also a good acceptor substrate. N-Acetyllactosamine was the best acceptor substrate among the disaccharides tested (Table III). The apparent Km value for cytidine monophosfate (CMP)-NeuAc was 0.2 mM (Table IV), which is almost the same as the value for the 2,6-sialyltransferase obtained from P. damselae JT0160 (Yamamoto et al. 1996). The apparent Km values for N-acetyllactosamine and lactose were 20 mM and 13 mM, respectively (Table IV). The enzyme transferred NeuAc to the all asialo-glycoproteins used in our assay (Table V).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementReferences |
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In this study, we identified a novel sialyltransferase from a bacterium that we named P. leiognathi JT-SHIZ-145. The sialyltransferase had the unusual characteristic of being most active at basic pH. This feature of the enzyme is likely to be advantageous for the production of several sialosides.
One of the major problems in the practical-scale production of sialosides using sialyltransferases has been to provide sufficient quantities of the unstable donor, CMP-NeuAc. This problem was almost solved by introducing a sugar–nucleotide recycling system (Ichikawa et al. 1991), and highly efficient one-pot synthesis of sialyl-oligosaccharides has been carried out with NeuAc, oligosaccharide, phosphoenolpyruvate, and catalytic amounts of adenosine triphosfate (ATP) and CMP as synthetic materials using nucleoside monophosfate kinase, pyruvate kinase, CMP-NeuAc synthetase, pyrophosfatase, and sialyltransferase as catalysts with in situ regeneration of CMP-NeuAc (Ichikawa et al. 1991). However, this method requires the handling of several enzymes, and in vitro synthesis offers significant advantages over other methods from the viewpoint of sialoside purification and reaction efficiency. One-pot sialylation using sialyltransferase and CMP-NeuAc synthetase simultaneously is thought to be the best method, because it is not strongly affected by the decomposition of CMP-NeuAc. However, most of the reported sialyltransferases, of both mammalian and bacterial origin, have their maximal activity under acidic conditions, around pH 6.0, whereas most of the reported CMP-NeuAc synthetases have their maximal activity under basic conditions, around pH 8.0 (Warren and Blacklow 1962; Kean and Roseman 1966; Tullius et al. 1996), and quickly decompose to CMP and NeuAc under acidic conditions. Therefore, it has been difficult to synthesize sialosides efficiently using sialyltransferase and CMP-NeuAc simultaneously in the same reaction mixture. However, the β-galactoside 2,6-sialyltransferase cloned from P. leiognathi JT-SHIZ-145 has its maximal activity at pH 8, a condition where CMP-NeuAc is stable. Most sialic acids attached to acceptor substrates are also more stable under basic conditions. Therefore, the sialyltransferase cloned from P. leiognathi JT-SHIZ-145 is likely to be a useful tool for practical-scale sialylation.
The expression of the recombinant gene has not yet been optimized for productivity, but we have produced approximately 340 units of the truncated enzyme per liter of culture of the E. coli TB1 containing the N1C0/pTrc99A expression vector. Furthermore, this sialyltransferase can transfer NeuAc to all of the asialo-glycoproteins we used (Table V). From these results, this sialyltransferase can transfer NeuAc to both N-linked and O-linked asialo-glycoproteins. This high-level expression of the truncated enzyme and broad acceptor substrate specificity are also thought to be advantageous for practical-scale production of several sialosides.
In general, β-galactoside 2,6-sialyltransferases transfer NeuAc from CMP-NeuAc to the galactose residue of the carbohydrate chain at position 6. The acceptor substrate specificity of mammalian sialyltransferases is generally very strict. In contrast, the acceptor specificity of bacterial sialyltransferases is broad, and this property is considered to be useful when various sialylated glycans are to be prepared. The acceptor substrate specificity of our enzyme was similar to that of the β-galactoside 2,6-sialyltransferase produced by P. damselae JT0160 (Yamamoto et al. 1996). Preference for the β-anomer was observed for monosaccharide acceptor substrates. The transfer assay clearly showed that methyl-β-D-galactopyranoside is the best acceptor substrate among the monosaccharides, although N-acetyl-D-galactosamine was also a good acceptor substrate (Table III). In the case of the 2,6-sialyltransferase produced by P. damselae JT0160, the Km values for lactose and N-acetyllactosamine are lower than the value for methyl-β-D-galactopyranoside (Yamamoto et al. 1996). From this result, we summarize that the 2,6-sialyltransferase produced by P. damselae JT0160 recognizes not only the nonreducing terminal galactose unit, but also the reducing terminal unit to which the galactose is attached. However, the recognition of the nonreducing terminus by the 2,6-sialyltransferase of P. leiognathi JT-SHIZ-145 seemed to be different from that of the sialyltransferase obtained from P. damselae JT0160, because the Km values for lactose, N-acetyllactosamine, and methyl-β-D-galactopyranoside were almost the same (Table IV). This result strongly indicates that the sialyltransferase cloned from P. leiognathi JT-SHIZ-145 does not recognize the reducing terminal unit to which the galactose is attached. An X-ray crystallographic analysis may give important additional information about this point.
The cloning of genes of mammalian sialyltransferases revealed three conserved regions, named "sialyl motif L", "sialyl motif S" and "sialyl motif VS" (Wen et al. 1992; Sasaki 1996). However, the genes encoding bacterial sialyltransferases show no similarity to those of mammalian sialyltransferases. As with other bacterial sialyltransferases, the 2,6-sialyltransferase of P. leiognathi JT-SHIZ-145 has no regions homologous to the cloned mammalian sialyltransferases. However, the amino acid sequence of the P. leiognathi JT-SHIZ-145 2,6-sialyltransferase shows 66.3% similarity to that of the 2,6-sialyltransferase from P. damselae JT0160. This similarity suggests that the three-dimensional structures of these two enzymes are likely to resemble each other, but differences of the acceptor substrate specificity and optimum pH might imply that there are structural differences in the substrate-binding site and catalytic acid and/or catalytic base between the two enzymes.
P. leiognathi JT-SHIZ-145 is a Gram-negative marine bacterium, and there is a candidate signal peptide in the NH2-terminal region of the deduced amino acid sequence of the enzyme. Thus, the enzyme may be translocated across the cytoplasmic membrane to the periplasm. The conditions in the periplasm are thought to be similar to those of the environment in which the bacterium grows. The average pH value and sodium chloride (NaCl) concentration of seawater are about 8.0 and 500 mM, respectively. The enzyme has its optimum pH under basic conditions (Figure 3), and increasing the concentration of NaCl increases the enzyme activity (data not shown). When we consider all of our results together, we conclude that this sialyltransferase may be involved in the biosynthesis of glycans which contain sialic acid with the 2-6 linkage, and we have confirmed using Salvia sclarea agglutinin (SSA) lectin staining that an 2,6-linked sialic acid is present on the surface of the bacterium (data not shown). We are currently studying the glycans containing 2,6-linked sialic acid that exist on the surface of this bacterium.
In this study, we isolated and described the fundamental characteristics of a β-galactoside 2,6-sialyltransferase from P. leiognathi JT-SHIZ-145. This is the first example of a sialyltransferase having its greatest activity at basic pH. This property would make this enzyme a powerful tool for the industrial production of glycans and the modification of glycoconjugates, such as glycoproteins and glycolipids.
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Materials and methods |
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Screening, cultivation, and identification of the marine bacterium
The bacteria were screened for sialyltransferase activity using the methods described in a previous paper (Yamamoto et al. 2006
). Marine bacteria were cultured in marine broth 2216. Bacteria were identified by sequence analyses of the 16S rRNA gene. The 16S rRNA gene was amplified from the genomic DNA by polymerase chain reaction (PCR) using primers 9F (GAGTTTGATCCTGGCTCAG) and 1510R (GGCTACCTTGTTACGA) under the same condition, except for annealing at 55°C, elongation for 1 min, and a cycle number of 30 (Weisburg et al. 1991).
Sialyltransferase assay
Sialyltransferase activity was assayed using a standard method (Yamamoto et al. 1996) by measuring [4,5,6,7,8,9-14C]-NeuAc transferred from CMP-[4,5,6,7,8,9-14C]-NeuAc (Du Pont, Boston, MA) as a donor substrate to the following acceptor substrates: lactose (Wako Pure Chemicals, Osaka, Japan); methyl--D-galactopyranoside, methyl-β-D-galactopyranoside, methyl--D-glucopyranoside, methyl-β-D-glucopyranoside, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine (all purchased from Sigma, St. Louis, MO); methyl--D-manno- pyranoside (Fluka, St. Louis, MO); and N-acetyllactosamine (Seikagaku Kogyo, Tokyo, Japan). Sialyltransferase activity for glycoproteins was assayed using asialo-glycoproteins (asialo-fetuin, asialo-bovine submaxillary mucin, and asialo 1 acid glycoprotein) as acceptor substrates. Removal of sialic acid from 1 acid glycoprotein (Sigma) and bovine submaxillary mucin (Sigma) were carried out according the method described previously (Yamamoto, Nagae, et al. 1998). The reaction mixture, the reaction conditions, and the assay were as described in a previous paper (Yamamoto, Nagae, et al. 1998). One unit (U) is defined as the amount of enzyme that transferred 1 µmol of sialic acid from CMP-NeuAc (Marukin Bio, Kyoto, Japan) to lactose per minute. Radioactivity was measured using a Packard model TR 1900 liquid scintillation counter.
Southern blotting and colony hybridization
Genomic DNA was isolated from a bacterial pellet using Genomic-tip 500/G (Qiagen, Chatsworth, CA). DNA was digested with restriction endonucleases, separated with 0.8% agarose gel electrophoresis, and blotted onto a Hybond N+ nylon membrane filter (Amersham, Uppsala, Sweden) by alkaline blotting with 0.4 M sodium hydroxide (NaOH). An approximately 1200-bp EcoRI and HindIII fragment from the P. damselae JT0160 2,6-sialyltransferase gene was labeled with 32P and used as a hybridization probe. Hybridization was performed using an ECL direct labeling and detection system (GE Healthcare Science, Tokyo, Japan), according to the manufacturer's instructions.
DNA sequencing
DNA sequences were determined by the dideoxy chain termination method with an ABI PRISM fluorometric autocycle sequencer (Model 310 Genetic Analyzer, Applied Biosystems, Foster City, CA). The sequence was determined on both strands. DNA and amino acid sequences were analyzed by Genetyx version 9.0, and a database search was performed using the BLAST program in DDBJ and GenBank.
Construction of expression cassettes
The PCR reaction mixture of 50 µL consisted of 500 ng of the template DNA (the 1.6-kb Pst1 fragment), 50 pmol of primers, 4 µL of each dNTP (2.5 mM), 2.5 units of PyroBest DNA Polymerase (0.5 µL), and 5 µL of 10 x PyroBest buffer II, in accordance with the manufacturer's instructions (Takara Bio, Shiga, Japan). The reaction was hot-started at 96°C for 3 min; incubated at 96°C for 1 min, 55°C for 1 min, and 72°C for 2 min for five cycles; and further incubated at 72°C for 6 min in Program Temp Control System (ASTEK, Fukuoka, Japan). The PCR product was cloned into a pCR4TOPO vector (Invitrogen, Carlsbad, CA), using the protocol provided by the manufacturer. The pCR4TOPO vector containing the PCR products was introduced into E. coli TB1. Concretely, to amplify the 2,6-sialyltransferase gene homologue, we designed three primers, N0BspH1, C0Hind, and N1Pci (Table I) to yield two PCR products: N0C0 (using N0BspH1 and C0Hind to amplify the full-length form) and N1C0 (using N1Pci and C0Hind to amplify the putative mature form). Both plasmid DNAs were digested with BspHI and BamHI for the full-length form and with PciI and HindIII for the putative mature form, respectively. The digested fragments were ligated into the multiple cloning site of an expression vector, pTrc99A (Pharmacia, Uppsala, Sweden), to produce the plasmids N0C0/pTrc99A and N1C0/pTrc99A, respectively. The expression vectors were introduced into E. coli strain TB1.
Expression of the recombinant protein in E. coli
A single colony from E. coli harboring an expression vector was inoculated into LB broth (Type Miller, Becton–Dickinson, Sparks, MD) containing 100 µg/mL of ampicillin and was grown at 30°C until the A600 reached 0.5. Then, 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG, Wako) was added to the culture, which was further incubated at 30°C for 4 h. The bacterial pellet was collected by centrifugation, dissolved in 20 mM bis-Tris buffer (pH 7.0) containing 0.5 M NaCl and 0.3% Triton X-100, and sonicated on ice. The supernatant was the crude enzyme solution used for further analyses.
Purification of recombinant 2,6-sialyltransferase
Preparation of the Crude Extract:
E. coli TB1 that harbored N1C0/pTrc99A were shaken at 30°C for 8 h in 6 mL LB broth containing 100 µg/mL of ampicillin. Then, 300 mL of the same medium containing 1 mM IPTG was inoculated with the seed culture and incubated for 16 h. Bacteria were harvested by centrifugation from the culture to yield a pellet. The pellet was then suspended in 16 mL of 20 mM potassium phosfate buffer (pH 8.0) containing 0.3% Triton X-100 per gram of pellet and lysed by sonication on ice. Cellular debris was removed by centrifugation at 100,000 x g for 60 min, and the supernatant was filtered through a 0.45-µm cellulose acetate membrane. This solution was used as the crude extract.
Column Chromatography:
The filtrate was applied to a HiLoad 26/10 Q Sepharose HP column (Pharmacia) equilibrated with 20 mM potassium phosfate buffer (pH 8.0) containing 0.3% Triton X-100. The enzyme was eluted with a linear gradient of 0 to 1 M NaCl in the same buffer. The fractions containing sialyltransferase were pooled, diluted with 20 mM potassium phosfate buffer (pH 8.0) containing 0.3% Triton X-100, and applied to a Bio-Scale ceramic Hydroxyapatite Type I column (CHT20-I; Bio-Rad, Hercules, CA) equilibrated with 20 mM potassium phosfate buffer (pH 8.0). The enzyme was eluted with a linear gradient of potassium phosfate from 20 to 500 mM. The active fractions were collected, pooled, and loaded onto a Hiload 26/60 Superdex HR column (Pharmacia) equilibrated with 20 mM potassium phosfate buffer (pH 8.0) containing 0.3% Triton X-100 and 0.2 M NaCl. All of the chromatographic purification steps were performed at 4°C.
Analysis of the enzymatic reaction product by HPLC
To identify the structure of the sialylated glycan, the enzymatic reaction was carried out using pyridylaminated carbohydrate chains (Takara) as acceptor substrates. The reaction products were analyzed by HPLC using a PALPAK type R analytical column (Takara). The reaction and analyses were performed according to the procedure described in a previous report (Yamamoto et al. 1996).
Identification of the enzymatic reaction product by 1H-NMR
To clarify the glycosidic linkage formed by the sialyltransferase, enzymatic synthesis was performed using lactose as an acceptor substrate. The reaction mixture was composed of acceptor substrate (55 µmol), CMP-NeuAc (66 µmol), and 0.6 U of the purified sialyltransferase in 0.3 mL of 20 mM potassium phosfate buffer (pH 8.0) containing 0.3% Triton X-100. The reaction was allowed to proceed at 30°C for 2 h. The enzymatic reaction product was purified as follows: After the reaction, the reaction mixture was diluted with 10 mL of distilled water and loaded on a column (1 x 10 cm) containing AG 1 X8 (phosfate form; Bio-Rad). The column was washed with distilled water, and then the product was eluted with 5 mM of phosfate buffer (pH 6.8). The fractions containing glycosidic NeuAc were pooled and evaporated to dryness. The dried residues were dissolved in 5 mL of distilled water and then loaded on a Sephadex G-15 column (3.0 x 75 cm). The product was eluted with distilled water. The fractions found to contain glycosidic NeuAc were pooled and evaporated to dryness. The purified product was analyzed by 1H-NMR spectroscopy using a Brucker AM500 spectrometer.
Kinetic study
Apparent kinetic parameters of the enzyme for the donor substrate were determined under the following conditions using lactose as an acceptor substrate. The reaction mixture was composed of lactose (120 mM), various concentrations of CMP-NeuAc, and purified sialyltransferase (3.5 mU) in 5 µL of 20 mM sodium phosfate buffer (pH 8.0) containing 0.3% Triton X-100. The enzyme reaction was carried out at 30°C for 1 min. Assays were performed in duplicate. Apparent kinetic parameters for several acceptor substrates were determined with a saturating concentration of CMP-NeuAc. The reaction mixture was composed of CMP-NeuAc (2.7 mM), various concentrations of acceptor substrate, and purified sialyltransferase (3.5 mU) in 30 µL of 20 mM sodium phosfate buffer (pH 8.0) containing 0.3% Triton X-100. The enzyme reaction was carried out at 30°C for 1 min. Assays were performed in duplicate. After the reaction, the radioactive product was isolated as described in the "Sialyltransferase assay" section, the enzyme activity was calculated, and the kinetic parameters were determined.
pH profile, temperature profile, protein determinations, and SDS-PAGE
Assays for determining the pH profile of the enzyme were performed using the following buffers: 100 mM phosfate buffer (pH 6.0 to 8.0), 100 mM Tris–HCl buffer (pH 8.0 to 9.0), and 100 mM 2-N-cyclohexylamino ethanesulfonic (CHES) acid buffer (pH 9.0 to 10.0). Assays were performed in triplicate. To analyze the temperature profile of the enzyme activity, the enzyme reaction was carried out at 20, 25, 30, 35, and 40°C, according to the sialyltransferase assay. Protein determination was performed using a Coomassie Protein Assay Reagent (Pierce, Rockford, IL) according to the manufacturer's instructions. Bovine serum albumin was used as a standard. SDS-PAGE was performed using precast polyacrylamide gels (Atto, Tokyo, Japan) and molecular mass standard samples (Bio-Rad), and the gels were stained with Coomassie Brilliant Blue R-250.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We thank Mr. Masashi Mizutani, research scientist of Japan Tobacco Inc., for performing the 1H-NMR spectroscopy. We also thank Professor Sanji Matsushima of Nagahama Institute of Bio-Science and Technology, Ms. Hiroshi Yoinara of Japan Tobacco Inc. for their support.
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Abbreviations |
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ATP, adenosine triphosfate; bp, base pairs; CHES, 2-N-cyclohexylamino ethanesulfonic acid; CMP, cytidine monophosfate; HPLC, high-performance liquid chromatography; IPTG, isopropyl-1-thio-β-D-galactopyranoside; NaCl, sodium chloride; NaOH, sodium hydroxide; NeuAc, N-acetylneuraminic acid; NMR, nuclear magnetic resonance; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SSA, Salvia sclarea agglutinin
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References |
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