Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada, 2Pest Animal Control CRC, CSIRO Wildlife and Ecology, Canberra ACT 2601, Australia, and 3Laboratory for Molecular Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako, Saitama 3510198, Japan
Received on July 30, 1999; revised on October 1, 1999; accepted on October 1, 1999.
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Abstract |
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Key words: myxoma virus/2,3-sialyltransferase/substrate specificity
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Introduction |
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Mammalian sialyltransferase enzymes can be classified into three categories, 2,3 (ST3),
2,6 (ST6), and
2,8-sialyltransferases (ST8), according to the regiochemistry of the resulting sialylated reaction products (Tsuji et al., 1996
).
2,3-Sialyltransferases (ST3Gals) have been further categorized into six subfamilies, ST3Gal I to ST3Gal VI, by the chronological order of cDNA publication (Tsuji et al., 1996
; Tsuji, 1998
). Each subfamily possesses a characteristic acceptor specificity. ST3Gal I is highly active toward Type III disaccharide acceptors (Galß1-3GalNAc
) (Gillespie et al., 1992
; Lee et al., 1993
; Kitagawa and Paulson, 1994b
; Kurosawa et al., 1995
). STGal II also utilizes Type III acceptors with a preference for glycolipid aglycons (Lee et al., 1994
). ST3Gal III and ST3Gal IV work mainly on Type I (Galß1-3GlcNAcß) and Type II (Galß1-4GlcNAcß) acceptors, respectively (Wen et al., 1992
; Kitagawa and Paulson, 1993
; Sasaki et al., 1993
; Kitagawa and Paulson, 1994a
). Neither acts on glycolipids. ST3Gal V (Ishii et al., 1998
; Kono et al., 1998
) exclusively produces sialylated lactosylceramides with no activity observed towards Type I, II, or III disaccharide acceptors. ST3Gal VI (Okajima et al., 1999
) is believed to be responsible for the sialylation of glycolipid substrates with Type II disaccharide acceptors at their nonreducing ends.
To date 16 distinct mammalian sialyltransferases have been cloned including the six kinds of ST3Gals. The amino acid sequences of all cloned mammalian sialyltransferases have been analyzed and all of them contain L-, S-, and VS-sialylmotif sequences (Drickamer, 1993; Kurosawa et al., 1994
; Geremia et al., 1997
). These motifs are highly conserved regions comprising about 20% of the total protein sequence. The L-sialylmotif has been shown to bind to the donor CMP-NeuAc (Datta and Paulson, 1995
), while the S-sialylmotif participates in the binding of both donor and acceptor saccharide substrates (Datta et al., 1998
). The exact role for VS-sialylmotif in the catalytic process has not been identified.
Recently, three bacterial sialyltransferases have been cloned and characterized (Gilbert et al., 1996 (ST3); Yamamoto et al., 1998
(ST6); Bozue et al., 1999
(ST3)). Interestingly, the sialylmotif has not been found in the amino acid sequences of these bacterial sialyltransferases even though they transfer NeuAc from CMP-NeuAc to oligosaccharides in analogous to the mammalian enzymes. Characterization of recombinant Neisseria meningitidis ST3Gal revealed that this enzyme can utilize both
-and ß-linked terminal galactoses as acceptors, suggesting that bacterial enzymes have broader acceptor tolerances compared to mammalian enzymes (Gilbert et al., 1997
).
We report here the acceptor specificity of an 2,3-sialyltransferase that is encoded by myxoma virus. Myxoma virus is a member of the poxvirus family of double stranded DNA viruses, and it is known to infect Old World or European rabbits causing highly lethal myxomatosis (Fenner, 1994
). It has been reported that a novel myxoma virus early gene, MST3N, is a member of the eukaryotic sialyltransferase gene family (Jackson et al., 1999
). Myxoma viral infection of mammalian cells produces viral
2,3-sialyltransferase in the cells. In order to characterize this enzyme kinetically, partially purified extracts of cells infected with myxoma virus were prepared for substrate specificity evaluations. Comparisons with mammalian and bacterial enzymes revealed that the myxoma sialyltransferase has a unique ability to sialylate fucosylated acceptors.
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Results |
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Preliminary characterizations of the reaction products from the incubations with Lewisx and Lewisa were carried out employing capillary electrophoresis with laser induced fluorescence detection (CE-LIF; Zhang et al., 1995). Figure 1a illustrates the electropherogram obtained from an incubation of the myxoma viral sialyltransferase with CMP-NeuAc and Lewisx-TMR (10) (Le et al., 1997
), an acceptor with a highly fluorescent aglycone (Scheme 01). The two peaks were identified by comigration with authentic standards as Lewisx-TMR (10) and 2,3-sialylated Lewisx-TMR (11). Figure 1b illustrates the electropherogram obtained following enzyme incubation with Lewisa-TMR (12) and CMP-NeuAc. The two peaks were also identified by comigration with authentic standards, as Lewisa-TMR (12) and 2,3-sialylated Lewisa-TMR (13). As shown in the radiochemical assay results, the CE data also revealed that more 2,3-sialylated Lewisa-TMR was formed than 2,3-sialylated Lewisx-TMR. There were no other products formed under the conditions described. The reaction mixtures were also treated with neuraminidase. Sialic acids were completely removed in both cases to give starting Lewisx-TMR and Lewisa-TMR (data not shown).
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Discussion |
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In 1996, a systematic nomenclature for sialyltransferases was proposed by Tsuij and Paulson (Tsuji et al., 1996). This nomenclature focused on vertebrate sialyltransferases that possess sialylmotifs in their amino acid sequence. Since the viral enzyme contains sialylmotifs, the vertebrate nomenclature system was employed to name the viral enzyme. The
2,3-sialyltransferase encoded by myxoma virus is named v-ST3Gal I with a v- to denote it as a viral enzyme. The viral enzyme gene is distinct from vertebrate sialyltransferase gene families, and a roman numeral I corresponds to the first subfamily found in v-ST3Gals.
A dendrogram constructed by analyzing the deduced amino acid sequences of cloned sialyltransferases according to Tyler et al. (1991) suggested interesting relationships between v-ST3Gal I and vertebrate sialyltransferases. Figure 2 was constructed with amino acid sequences of sialyltransferases from mouse. ST6Gal I from four different animals were also included to show homologues of one enzyme subfamily among different species. The v-ST3Gal I is most closely related to mST3Gal IV. However, because of the completely different substrate specificities of these enzymes, they should be considered as different enzyme subfamily. The branch length in the dendrogram also supports this assignment.
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Examples of complete genome sequences of viruses belonging to different poxvirus genera have been determined in recent years and it has been observed that they encode homologues of known cellular genes (Goebel et al., 1990; Senkevich et al., 1996
; and Afonso et al., 1999
). It is thought that poxviruses have acquired these cellular genes from their hosts throughout evolutionary development, conferring enhanced virulence and favoring virus survival. Like all poxvirus genes, these integrated "cellular genes" do not contain introns. This fact suggests that they are cDNA copies of mRNA. At the amino acid level the v-STGal I is most similar to the mSTGal IV. This may suggests that an ancestral ST3Gal IV gene may have been acquired, which then diverged with the viral encoded sialyltransferase gaining broader acceptor specificity. It would be interesting to compare the myxoma v-ST3Gal I to ST3 genes encoded by Lagomorphs (the natural hosts of leporipoxviruses) to determine their evolutionary relationship.
Sialic acids residues on the termini of oligosaccharides are known to play significant roles in a variety of biological functions including; maintenance of soluble glycoproteins in circulation, masking of microbial antigens, and receptorligand interactions between cells involved in immunological and inflammatory responses (Varki, 1993; Kelm and Schauer, 1997
). Therefore, sialylation of myxoma virus or host encoded glycoproteins could influence multiple "viral stealth" strategies involved in subverting the rabbits responses to infection. Inactivation of the MST3N gene in a recombinant myxoma virus has demonstrated that the sialyltransferase activity is not essential for viral infection or replication in cell culture, yet the viral directed sialylation is required for the full virulence phenotype when laboratory rabbits are infected (Jackson et al., 1999
). The observed decrease in virulence may simply result from the partial unmasking and enhanced antigenic profile of secreted or cell-associated virus. Alternatively, it may result from a decreased half-life of secreted virulence factors within the infected tissues. However, the potential products of the myxoma virus sialyltransferase are known ligands for the cellular receptors involved in inflammation and immune responses. Two members of the sialoadhesin family, macrophage/myeloid marker CD33, and sialoadhesin (Sn) expressed on activated macrophages in lymphoid tissues and sites of inflammation, bind terminal 2,3-sialylated type I, II, and III acceptors of glycoproteins (Kelm et al., 1996
; Crocker et al., 1997
). While L-selectin (expressed on lymphocytes), E-selectin (expressed on endothelial cells) and P-selectin (expressed on platelets and endothelial cells) are "C type" mammalian lectins involved with leukocyte trafficking. These selectins bind cell surface sialylated and fucosylated lactosamines of which 2,3-sialylated Lewisa and 2,3-sialylated Lewisx are the simplest examples (Lasky, 1995
; Tedder et al., 1995
).
It has recently been shown that myxoma virus infected cells secrete an important anti-inflammatory factor, SERP-1, which is preferentially sialylated by the viral encoded sialyltransferase (Nash et al., 2000). Although differing levels of sialylation resulting from alternative viral and cellular sialyltransferase activities had no observable effect upon the in vitro serine proteinase activity of SERP-1 (Nash et al., 2000
), it may have consequences on the tissue tropism of naturally secreted SERP-1. SERP-1 is a potent anti-inflammatory agent, and thus, the natural site of action is within viral infected tissues where activated macrophages express Sn and vascular endothelium expresses high levels of E- and P-selectin. In speculation, the presence of the selectin or Sn ligands on the secreted SERP-1 may target the glycoprotein to its site of action where its anti-inflammatory effects could be 2-fold. First, SERP-1 could downregulate cellcell interactions by blocking sialic acid dependent receptors. For example, it could block endothelial selectin receptors resulting in a reduction of neutrophil (Lasky, 1995
; Tedder et al., 1995
) or CD4+ Th1 cell (Austrup et al., 1997
) adhesion and migration into sites of inflammation. Second, localization of the SERP-1 glycoprotein to its site of action where low concentrations serine proteinase activity are sufficient to block a proteinase-mediated pro-inflammatory cascade (Lucas et al., 1996
). Myxoma virus is known to encode a number of other anti-inflammatory and immunomodulatory glycoproteins (McFadden et al., 1998
; Nash et al., 1999
) and probably additional undiscovered "virokines" and "viroreceptors." Many of these virulence factors are either cell membrane associated or secreted glycoproteins, and therefore, they are potential substrates for the viral encoded sialyltransferase. The activity of the myxoma virus
2,3-sialyltransferase upon the biological activity of viral virulence factors has the potential to be highly complex and will undoubtedly be the subject of intensive research over the coming years.
v-ST3Gal I is also an attractive enzyme for the synthesis of sialylated oligosaccharides because of its broad acceptor specificity and stability in lengthy incubations. The large-scale production of this enzyme for utilization in oligosaccharide synthesis is ongoing.
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Materials and methods |
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Source of sialyltransferase
Ten T180 flasks of confluent monolayers of European rabbit kidney cells (Oryctolagus cuniculus RK13) were infected with Brazilian myxoma virus strain, Lausanne (ATCC VR-115). The cells were kept at 37°C for 24 h and then detached with a cell scraper and washed twice with PBS at 4°C. Cell lysates were prepared by suspension of the cells (1.83 g wet weight) in 20 ml of extraction buffer (50 mM MES, pH 6.1, 0.5% Triton X-100, 100 mM NaCl, 1.5 mM MgCl2, 0.1 mM PMSF, 10 µg/ml aprotinin) at 4°C, for 45 min. The lysate was clarified by centrifugation at 2000 x g at 4°C for 15 min. The negative controls were RK13 cells that had not been infected with the virus and RK13 cells infected with the virus in which the 2,3-sialyltransferase gene-encoding regions had been disrupted by the insertion of the E.coli lacZ gene. Cell lysate from CV1 cells were also prepared in a similar way.
Partial purification of cell lysate
The supernatant was applied to 5 ml HiTrap Blue Affinity chromatography column equilibrated with 50 mM MES, pH 6.1, 0.1% Triton CF54, 100 mM NaCl and 25% glycerol. The column was washed with 25 ml of column equilibration buffer and then sialyltransferase was eluted stepwise with NaCl (0.5 M, 1.0 M, 1.5 M, and 2.0 M NaCl) in equilibration buffer. The majority of the enzyme was eluted in the 1.0 M NaCl step. The 2,3-sialytransferase was desalted by passing the eluate through a PD-10 column in column buffer, 50 mM MES, pH 6.1, 0.1% Triton CF54, 25% glycerol. The total protein concentration of the desalted extract was 0.58 mg/ml.
Radiochemical assays
For relative rates of transfer, acceptor (54 nmol), CMP-NeuAc (20 mM solution in H2O, 2 µl, 40 nmol), CMP-3H-NeuAc (150,000180,000 dpm, in 1 µl of H2O), and assay buffer (250 mM MES, 0.5% Triton CF54, pH 7.0, 1 µl) were added to desalted enzyme (16 µl, 9 µg protein) in a final volume of 20 µl. Reaction mixtures were incubated at 37°C for 90 min, diluted with water to 200 µl and loaded onto Sep-Pak Plus C18 reverse phase cartridges. After application of the reaction mixture, the cartridge was washed with 50 ml water, and then the product was eluted with 4 ml of MeOH into a scintillation vial. The radioactivity of the MeOH eluate was quantitated in a liquid scintillation counter after adding 10 ml of EcoLite (+) scintillation cocktail. A mU of enzyme activity is the amount enzyme that catalyzes the conversion of 1 nmol of acceptor to product per minute under standard screening conditions. When asialofetuin (Type I, 500 µg for one assay) was used as an acceptor, Sephadex G-50 columns (16 cm x 0.8 cm i.d.) were used for the separation of radioactive glycoprotein from CMP-3H-NeuAc employing 0.2 M NaCl as a solvent. Complete substrate kinetics were carried out in an analogous method by varying the acceptor concentration from 0.2 x Km to 3 x Km.
Capillary electrophoresis assays
Lewisa- or Lewisx-TMR (10, 12) (35 nmol) and CMP-NeuAc (200 nmol) were incubated with 4.9 µl viral sialyltransferase and 0.1 µl alkaline phosphatase solution (5 µl of alkaline phosphatase at 1000 U/ml and 1 µl BSA solution at 100 mg/ml). After gentle rotation at room temperature (25°C) for 42 h, additional alkaline phosphatase solution (0.2 µl) and 100 mM CMP-NeuAc solution (0.2 µl) were added to the mixture, which was incubated for another 48 h at room temperature. The reaction temperature was then elevated and maintained at 37°C for another 48 h. Reaction mixtures were then loaded onto Sep-Pak Plus C18 reverse phase cartridges. The cartridges were washed with 15 ml of water, and products were eluted with 4 ml of HPLC grade methanol. These MeOH eluates were concentrated to dryness and the resulting residues were dissolved in water (10 ml). These solutions were passed through Millex-GV filters (0.22 µm), and the filtrates were lyophilized. The residues were dissolved in water to a concentration of 100 µM TMR. This solution (0.5 µl) was mixed with CE running buffer (499.5 µl) to give a 100 nM solution in CE running buffer. This was used for separation and analysis by capillary electrophoresis with laser induced fluorescence detection (CE-LIF; Zhang et al., 1995; Le et al., 1997
). New products formed in the enzymatic reactions had migration times the same as that of authentic 2,3-sialylated Lewisx-TMR (11) and 2,3-sialylated Lewisa-TMR (13).
A 10 µl sample of the incubation mixture (100 µM TMR solution in H20) containing 1 nmol total TMR-labeled compounds was mixed with 1 µl Arthrobacter ureafaciens neuraminidase (8.4 mU in 10 mM sodium phosphate, 0.025% (w/v) BSA, 0.05% (w/v) NaN3, pH 7.0), and the mixture was incubated with gentle rotation at 37°C. The mixture was then loaded onto a Sep-Pak Plus C18 reverse phase cartridge. A solution containing 100 nM of sample in CE running buffer was prepared in the same manner as described for separation and detection with CE-LIF. The new product peaks were converted back to the starting acceptors by the treatment with neuraminidase.
Capillary electrophoresis with laser-induced fluorescence detection
A capillary electrophoresis system equipped with post-column laser-induced fluorescence detection was used for separation and analysis of oligosaccharides. Separation was done in a fused silica capillary (60 cm x 10 µm i.d.) with an electric field of 400 V/cm. A 5 mW He-Ne laser with a wavelength of 543.5 nm was used to excite the TMR molecules. Sample was introduced into the capillary electrokinetically by applying a potential of 1 kV for 5 s; each injection volume was 13 pl (determined by Knolls method; Knoll et al., 1985). All separations were performed at ambient temperature without temperature control.
Representative preparative synthesis: synthesis of 2,3-sialylated Lewisx from Lewisx
BSA (5 µl, 100 mg/ml) was added to 14 ml of desalted enzyme and the mixture was concentrated to 1.8 ml in a Slide-A-lyzer. Lewisx-O(CH2)8CO2CH3 (5) (4.7 mg, 6.7 µmol), CMP-NeuAc (6.8 mg, 10.3 µmol) and alkaline phosphatase (10 µl, 10 U) were added to 200 µl of concentrated enzyme. The mixture was gently rotated end-over-end for 24 days at room temperature. CMP-NeuAc was added during this incubation, 3 mg after 3, 7, and 11 days then 2 mg after 15, 19, and 21 days. The mixture was loaded onto a Sep-Pak Plus C18 reverse phase cartridge. The cartridge was washed with water (40 ml), then the product was eluted with 50 ml of a 10% aqueous solution of methanol. The eluate was dried under vacuum and again loaded onto a Sep-Pak C18 reverse phase cartridge. After washing with water (10 ml), 1% MeOH (10 ml), and 5% MeOH (10 ml), the desired product was eluted with 10% methanol (63 ml). This eluate was concentrated to dryness and the resulting residues were dissolved in water (10 ml). This solution was then passed through a Millex-GV filter (0.22 µm), and the filtrate was lyophilized to give 2,3-sialylated Lewisx-O(CH2)8CO2CH3 (2.2 mg, 33% yield). 1H-NMR spectroscopy and mass spectrometry were used to characterize the reaction product. A 1H-NMR spectrum of the product was identical to the chemically synthesized authentic sample.
NMR (300 MHz, only characteristic peaks are given, D2O) 5.10 (d, H, J = 3.9 Hz, H-1 (Fuc)), 4.51 (d, 2H, J = 8.0, H-1 (Gal, GlcNAc)), 3.69 (s, 3H, CO2Me), 2.76 (dd, H, J = 4.7, 12.6 Hz, H-3 (NeuAc)), 2.38 (t, 2H, J = 11.4 Hz, CH2CO2Me), 2.04 (s, 3H, Ac), 2.02 (s, 3H, Ac), 1.79 (t, H, J = 12.2, 1.8 Hz, H-3 (NeuAc)), 1.17 (d, 3H, J = 6.6 Hz, H-6 (Fuc)). Mass (negative) calculated for C41H69N2O25 989.4, found 989.0.
Preparative synthesis of 2,3-sialylated Lewisa from Lewisa
2,3-Sialylated Lewisa-O(CH2)8CO2CH3 was also synthesized from Lewisa-O(CH2)8CO2CH3 (6) in a similar manner (35% yield). Both NMR spectroscopy and mass spectrometry confirmed its structure. A 1H-NMR spectrum of the product was identical to the chemically synthesized authentic sample.
NMR (300 MHz, only typical peaks are shown, D2O) 5.00 (d, H, J = 3.9 Hz, H-1 (Fuc)), 4.52 (d, 2H, J = 7.7, H-1 (Gal, GlcNAc)), 3.69 (s, 3H, CO2Me), 2.76 (dd, H, J = 4.7, 12.5 Hz, H-3 (NeuAc)), 2.39 (t, 2H, J = 7.3 Hz, CH2CO2Me), 2.02 (s, 6H, Ac), 1.76 (t, H, J = 12.3, H-3 (NeuAc)), 1.16 (d, 3H, J = 6.4 Hz, H-6 (Fuc)). Mass (negative) calculated for C41H69N2O25 989.4, found 989.0.
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Acknowledgments |
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Footnotes |
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References |
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