Sialylated glycoconjugates have many functions in prokaryotes and vertebrates (Rosenberg, 1995). Polysialic acid and sialylated polysaccharides are important virulence factors for bacteria providing a means of evading the host immune system (Robbins et al., 1974). Our laboratory is interested in the biosynthesis of sialylated glycoconjugates in bacteria. Most sialic acid residues are added to glycoconjugates by sialyltransferase. Cytidine-5-monophosphate-[beta]-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for all sialyltransferases and is formed by the enzyme CMP-neuNAc synthetase (Roseman, 1962; Warren and Blacklow, 1962). CMP-neuNAc synthetase catalyzes the following reaction:
CTP + neuNAc -> CMP-neuNAc + PPi
This enzyme has been reported in some pathogenic bacteria and has been detected in all vertebrate sources analyzed to date (Kean, 1991). While the bacterial and vertebrate CMP-neuNAc enzymes share many catalytic properties, several distinct differences have been reported which include localization, substrate specificity, tertiary structure, and inhibitor sensitivity. The most striking difference is the localization of the vertebrate enzyme in the nucleus (Coates et al., 1980; Kean, 1991).
The bacterial CMP-neuNAc synthetases are the best characterized. They have been cloned from several sources and their amino acid sequences deduced (Zapata et al., 1989; Ganguli et al., 1994; Haft et al., 1994; Tullius et al., 1996). These bacterial enzymes are cytosolic and appear to be homodimers with monomeric molecular weights ranging from 28,000 to 49,000 Da. The sequences of the bacterial enzymes reveal conserved amino acid residues in the amino terminal domain of the protein which play a role in catalytic activity (Stoughton et al., unpublished observations). We would like to determine the common structural features of CMP-neuNAc synthetase enzymes in both bacteria and vertebrates. While it was demonstrated that the vertebrate enzyme elutes in gel filtration with a molecular weight of 160 kDa (Schauer, 1980), the size of the monomeric protein is still undetermined (Rodriguez-Aparicio et al., 1992). Unlike the cytosolic bacterial enzyme, the vertebrate enzyme is nuclear and is therefore expected to have nuclear targeting sequences and perhaps posttranslational modifications such as phosphorylation or glycosylation (Hanover et al., 1987; Hart, 1997). Purification of the naturally occurring eukaryotic protein would help to answer some of these questions regarding molecular weight, postranslational modification, and tertiary structure.
Only partial purification of the CMP-neuNAc synthetase from several vertebrate sources has been described (Schauer, 1980; Schmelter et al., 1993; Kean and Roseman, 1966). Schmelter et al. reported a comparison of several partial purification procedures of the enzyme from trout liver (Schmelter et al., 1993). Rodriguez-Aparicio et al. reported an enzyme in rat liver with a subunit molecular weight of 58 kDa and a native molecular weight of 110 kDa which differs from previous reports for frog and bovine enzymes (Rodriguez-Aparicio et al., 1992). However, this group of researchers failed to demonstrate that the 58 kDa polypeptide observed in SDS-PAGE is indeed a CMP-neuNAc synthetase. Thus, it is unclear whether there are tissue-specific in addition to species-specific differences.
In order to facilitate a comparison of the structure and function relationships of CMP-neuNAc synthetase enzymes our laboratory purified this activity from a mammalian source. The data presented below outline the purification of this enzyme from bovine anterior pituitary glands with higher specific activity than previously described. Evidence to confirm the identity of the purified protein with CMP-neuNAc synthetase is presented.
Table I.
Purification step | Tot. Prot. (mg) | Tot. Act. (nmol/min) | Sp. Act. (nmol/min/mg) | Fold | Percent recovery |
NaCl Supernatant | 42,071 | 427,136 | 10 | 1 | 100 |
65% (NH4)SO4 ppt | 5971 | 312,419 | 52 | 5 | 73 |
Blue-Sepharose-90% (NH4)SO4 | 643 | 93,492 | 145 | 14 | 22 |
Sephacryl S-300 | 214 | 60,373 | 282 | 28 | 14 |
Q-Sepharose HR | 41 | 37,868 | 934 | 92 | 9 |
CTP-Agarose | 1.36 | 17,472 | 12,890 | 1270 | 4 |
[beta]-neuNAc-Agarose | 0.13 | 2310 | 18,252 | 1798 | 0.54 |
Purification of bovine pituitary CMP-neuNAc synthetase
We have purified the bovine anterior pituitary gland CMP-neuNAc synthetase to apparent homogeneity with a specific activity of 18,000 mU/mg (Table I). Our approach was to use dye binding chromatography, size exclusion and ion exchange to obtain a partially purified preparation. The enzyme was then purified to apparent homogeneity by affinity chromatography steps using resins based on both substrates, CTP-agarose and [beta]-sialic acid agarose (Figure
Figure 1. Affinity agarose resins used to purify CMP-neuNAc synthetase.
Figure 2. SDS-PAGE of purified CMP-neuNAc synthetase. Lanes 1 and 6, kaleidoscope MW standards; lanes 2 and 3, [beta]-neuNAc-agarose purified fraction; lane 4 and 5, CTP-agarose purified fraction; lane 7, E.coli CMP-neuNAc synthetase. Native gel electrophoresis
To demonstrate that the 52 kDa polypeptide observed in SDS-PAGE electrophoresis of the [beta]-sialic acid agarose purified fraction is indeed the CMP-neuNAc synthetase, the protein purified by this affinity column was applied to nondenaturing gel electrophoresis. Enzyme activity of the gel slices appears as two closely spaced regions of CMP-neuNAc acid synthetase activity. The two major protein bands observed in the parallel half of the gel under these nondenaturing conditions coincided with the slices having enzyme activity. The Coomassie-stained protein bands coinciding with enzyme activity were then subjected to SDS-PAGE electrophoresis and shown to exhibit a single major band in the region of the gel expected for a 52 kDa polypeptide (data not shown). Photoaffinity labeling
CMP-neuNAc synthetase is inhibited by CDP and binds to CDP-ethanolamine-agarose. Thus, we reasoned that a CMP-neuNAc synthetase enzyme would be covalently tagged by an affinity label based on this interaction. CDP-ethanolamido-azido-salicylic acid was prepared from CDP-ethanolamine and then iodinated with 125I. Purified fractions of bovine pituitary CMP-neuNAc synthetase were photoaffinity labeled with this substrate analogue in the presence and absence of CTP in order to demonstrate the specificity of the photoaffinity labeling. As shown in Figure
Figure 3. Photoaffinity labeling of bovine pituitary CMP-neuNAc synthetase. Purified and partially purified fractions of pituitary gland CMP-neuNAc synthetase were photoaffinity labeled with the substrate analog, 125I -ASA-CDP-ethanolamide. Protein fractions eluted from the affinity resins [beta]-neuNAc-agarose and CTP-agarose, or ion exchange resin were incubated with 125I -ASA-CDP-ethanolamide under a UV light, with or without the substrate CTP as inhibitor at 0°C (Materials and methods). Control reactions were incubated in the dark. Sample buffer was added directly to the reaction mixtures which were boiled, electrophoresed on SDS-PAGE, and subjected to autoradiography. [beta]-neuNAc-agarose (1 -CTP, 2 +CTP), CTP-agarose (3 -CTP, 4 +CTP), Q-Sepharose (5 -CTP, 6 +CTP), lane 7 E.coli CMP-neuNAc synthetase, and lane 8 dark reaction with [beta]-neuNAc-agarose fraction. Immunoaffinity chromatography
The enzyme fraction purified on [beta]-sialic acid agarose was used to prepare hyperimmune chicken serum. A globulin fraction of this serum was used to prepare an immunoaffinity resin. CMP-neuNAc synthetase activity was adsorbed from partially purified fractions of bovine anterior glands onto this resin and eluted with 3 M MgCl2 (Figure
Figure 4. Immunoaffinity chromatography of bovine pituitary CMP-neuNAc synthetase. An immunoadsorbent was prepared from avian antibody to the [beta]-neuNAc-agarose purified CMP-neuNAc synthetase as described in Materials and methods. Enzyme eluted from the dye binding resin, Blue-Sepharose, was further purified by immunoaffinity chromatography. The enzyme activity was absorbed onto the antibody resin in 50 mM Tris, 100 mM NaCl, pH 8.0. CMP-neuNAc synthetase activity eluted with 3 M MgCl2 (arrow). Molecular weight and isoelectric point determination
The active molecular weight of the bovine pituitary gland CMP-neuNAc synthetase was determined by gel filtration on a Superose-12 FPLC column (Figure
Figure 5. Active molecular weight of pituitary CMP-neuNAc synthetase. The active molecular weight of pituitary CMP-neuNAc synthetase was determined by FPLC gel filtration of a partially purified enzyme fraction. The absorbance at 280 nm of this fraction shown as the solid line and the molecular weight standards as the broken line. Standards are: 1, bovine thyroglobulin = 670,000; 2, IgG = 158,000; 3, ovalbumin = 44,000; 4, horse myoglobin = 17,000; 5, vitamin B12 = 1350 (at 33 min). Glycosylation and DNA binding of bovine pituitary enzyme
Vertebrate CMP-neuNAc synthetase is located in the nucleus. Nuclear proteins are often glycosylated with O-glcNAc. Thus, we tested purified pituitary gland CMP-neuNAc synthetase for the presence of carbohydrates. No carbohydrates could be detected by specific assays for O-glcNAc, or by a general method based on periodate oxidizable sugars.
Nuclear proteins such as histones, nucleotide polymerase and proteins with a regulatory role often bind DNA. Heparin agarose and calf thymus DNA-agarose are often used to purify nucleic acid binding proteins (Zhang et al., 1991; Scopes, 1993). During the course of our development of a purification strategy for bovine pituitary gland CMP-neuNAc synthetase, we tested the ability of this nuclear enzyme to bind to these resins. Enzyme fractions which had been eluted from a series of dye binding and ion-exchange chromatography were applied to either a DNA-agarose or heparin-agarose column and eluted with a NaCl gradient. Pituitary gland CMP-neuNAc synthetase despite its acidic isoelectric point was adsorbed on to both resins suggesting an ability to bind DNA. Elution of enzyme activity from the heparin resin required 0.5 M NaCl, and DNA-agarose required 0.25 M NaCl.
CMP-neuNAc synthetase is present in most sialic acid containing organisms, which includes vertebrates and some pathogenic bacteria (Kean, 1991). We describe in this report the purification of bovine pituitary CMP-neuNAc synthetase. The enzyme preparation is apparently homogeneous, as determined by gel electrophoresis, with a polypeptide molecular weight of 52,000 Da. The isolation of the naturally occurring enzyme will facilitate the detailed characterization of this enzyme in vertebrate tissues and its relationship to the family of bacterial homologues. This purification procedure unlike previous reports yields a high specific activity preparation. The proposal that the 52 kDa protein band is CMP-neuNAc synthetase is supported by several lines of evidence. The major band observed in a nondenaturing gel electrophoresis of bovine pituitary gland preparation comigrates with enzyme activity. The nucleotide photoaffinity label CDP-ethanolamido-azido-[125I] salicylic acid modifies the 52 kDa polypeptide. This modification is preferentially decreased in a partially purified preparation by the presence of the unlabeled substrate suggesting that the 52 kDa protein is binding the analog at a CTP binding site. Antibody prepared against the highest purified preparation adsorbs enzyme activity from crude preparations by immunoaffinity chromatography and binds a 52 kDa band on Western blots. These data strongly support the identity of this protein as CMP-neuNAc synthetase.
The vertebrate CMP-neuNAc synthetase like the bacterial enzymes probably has multiple subunits. The molecular weight of active CMP-neuNAc synthetase isolated from bovine, frog, and trout liver has been reported to be 160,000 (Schauer et al., 1980; Schmelter et al., 1993). The molecular weight of the active enzyme reported for rat liver was determined to be 110,000. We measured a native molecular weight by gel filtration of 158,000, which is more consistent with reports for frog liver (Schauer et al., 1980). These results may imply that the bovine pituitary and frog and trout liver enzymes exist either as homotrimers or are complexed to another protein of the same molecular weight. In any event these vertebrate enzymes appear to differ in tertiary structure from the dimeric bacterial enzymes. Furthermore, most of the bacterial CMP-neuNAc synthetases have a monomeric molecular weight of less than 35 kDa. The E.coli enzyme is largest and is the only bacterial CMP-neuNAc synthetase with a polypeptide size, ~49 kDa, similar to that described here for bovine pituitary gland. Highly purified bovine CMP-neuNAc synthetase is unstable. This instability could be due to dissociation of the multimer in dilute solution, or could also be explained by the removal of a component associated with the enzyme.
One of the more puzzling properties of this enzyme is its localization. While it has been shown that the enzyme is associated with the nucleus, many questions remain regarding the specific localization of CMP-neuNAc synthetase within this organelle. Does the enzyme play a regulatory role in the nucleus? The antibodies prepared in this report to CMP-neuNAc synthetase might be useful reagents for investigating the localization of the enzyme in the nucleus. A common feature of nuclear proteins is their ability to bind DNA. The bovine pituitary enzyme is acidic yet binds to heparin and DNA agarose. If indeed CMP-neuNAc synthetase does bind DNA, perhaps it has an additional non catalytic function which requires DNA interactions. The glycosylation of nuclear proteins has been described by several investigators (Holt and Hart, 1986; Hanover et al., 1987; Hart, 1997). Posttranslational modification of these proteins appears to play a role in their function. The enzyme described in this report, however, does not appear to be glycosylated with N-acetylglucosamine as has been reported for several other nuclear proteins. It is quite possible that an inactive form of the enzyme is glycosylated since we have necessarily purified enzyme on the basis of activity.
We have not been able to obtain amino-terminal sequence of the bovine pituitary CMP-neuNAc synthetase, perhaps due to amino terminal blockage. In the absence of a nucleotide derived sequence it is not easy to confirm an amino terminal sequence derived by Edman degradation. One might expect a vertebrate CMP-neuNAc synthetase to have an amino-terminal nuclear targeting sequence which would not be present in the homologous bacterial enzymes. Furthermore, internal sequencing of peptides by tryptic digest could also yield sequences not homologous with the bacterial family of enzymes. An amino-terminal sequence was published for the rat liver enzyme (Rodriguez-Aparicio et al., 1992); however, this sequence does not share homology with any published sequence nor with the product of the recently published rat pituitary gene (see note added in proof below).
The methods and reagents developed in this report should be more generally applicable to the purification and identification of CMP-neuNAc synthetase from other vertebrate sources. During the development of this procedure for pituitary glands we have partially purified the enzyme from bovine brain and Chinese hamster ovary cells using similar methods. The photoaffinity label CDP-ethanolamido-azido-[125I] salicylic acid was used to detect sialyltransferase in crude extracts. We have used this reagent to label CMP-neuNAc synthetase. In future experiments this reagent would be useful in defining active site residues in CMP-neuNAc synthetases.
The six known bacterial CMP-neuNAc synthetase proteins have conserved regions in the amino terminal half of the protein. It is expected that the pituitary CMP-neuNAc synthetase will share these conserved regions. The role of these residues in vertebrate CMP-neuNAc synthetase can be addressed by site directed mutagenesis of a recombinant form of this enzyme. The methodology that we have developed for the purification of this enzyme from pituitary tissue should be helpful in designing strategies for the isolation of recombinant enzyme expressed in cell culture. Materials
Bovine anterior pituitary glands obtained from Pel-Freez Biologicals (Rogers, AR) were excised and immediately stored in liquid nitrogen prior to shipping in batches of 300-400 glands. Blue-Sepharose, Q-Sepharose, cyanogen bromide activated Sepharose, and Sephacryl S-300 resins were purchased from Pharmacia. CTP-agarose for affinity chromatography was a generous gift of Carlos Hirschberg (Department of Biochemistry, University of Massachusetts, Worcester, MA). Enzyme activity
All fractions were assayed for CMP-neuNAc synthetase activity by the thiobarbituric acid assay as described previously (Warren and Blacklow, 1962; Vann, 1987). This assay measures the formation of glycosidically linked thiobarbituric acid reactive neuNAc. Purification of CMP-neuNAc synthetase
Typically, 780 gm of frozen pituitary glands were thawed overnight and washed in 50 mM Tris, 1mM DTT, pH 8.0 to remove excess blood. The glands were homogenized in a Waring Blender in 1 l cold 50 mM Tris, 1 mM DTT, and 0.5 mM Pefabloc protease inhibitor (Boehringer-Mannheim), pH 8.0 (buffer A) and strained through cheese cloth to remove large tissue fragments. The homogenate was passed through a YAMATO LH-41 Teflon tissue homogenizer (90 r.p.m.). The soluble fraction was adjusted to 1 M NaCl by addition of 1 l of cold 2 M NaCl in buffer A, homogenized to lyse the nuclei, and the homogenate centrifuged at 15,000 × g 15min. 4°C. The NaCl supernatant was adjusted to 0.5% polyethylenimine (PEI), stirred at 4°C for 1 h, then centrifuged at 15,000 × g for 15 min to remove nucleic acids. Ammonium sulfate fractionation
The PEI supernatant (1.7 l) was adjusted to 35% saturation with ammonium sulfate, stirred at 4°C for 1 h and centrifuged at 15,000 × g for 15 min at 4°C. The insoluble material was removed by centrifugation, and the supernatant adjusted to 65 % saturation with ammonium sulfate. Most of the activity precipitated in the 35-65% ammonium sulfate fraction. The 65% saturated ammonium sulfate precipitate was collected by centrifugation and dissolved in 500 ml of cold 50 mM Tris, 1 mM DTT, pH 8.0 (buffer B) and then dialyzed extensively against buffer B. The precipitate formed during dialysis was removed by centrifugation at 15,000 × g, 15 min. at 4°C. A Bio-Rad Biologic LP Chromatography System was used to purify the enzyme on all subsequent chromatography steps. Dye binding chromatography
The dialyzed 65% ammonium sulfate precipitate fraction (545 ml) was applied to a Blue Sepharose column (2.5 × 30 cm) equilibrated in buffer B. The column was washed at a flow rate of 1 ml/min with two column volumes of buffer B and eluted with 2.5 volumes of 0.4 M NaCl, 50 mM Tris, 1 mM DTT, pH 8.0. Gel filtration chromatography
The active fractions from the Blue Sepharose column were concentrated by precipitation with 90% saturation with ammonium sulfate and the protein precipitate taken up in a minimum volume (35 ml) of buffer B. The concentrated enzyme fraction was applied to a Sephacryl S-300 column (2.5 × 90 cm) in buffer B and eluted at 0.5 ml/min. Ion exchange chromatography
Active fractions from the Sephacryl S-300 column were applied to a Q-Sepharose HR column (1.6 × 20 cm) equilibrated with buffer B. The column was washed at 0.25 ml/min with one column volume of buffer B, and eluted with three column volumes of a linear gradient of 0-0.15 M NaCl followed by a 0.15-0.4 M NaCl gradient. Enzyme activity eluted between 0.08 M NaCl and 0.12 M NaCl. CTP-agarose/affinity chromatography
The active Q Sepharose fractions were pooled (38 ml) and adjusted to 10 mM MgCl2. This fraction was applied to 60 ml of CTP-agarose equilibrated with 10 mM MgCl2, 50 mM Tris, 1 mM DTT, pH 8.0. The column was washed at 0.5 ml/min with 1.5 column volumes of 10 mM MgCl2, 50 mM Tris, 1 mM DTT, pH 8.0, and eluted with 1.5 column volumes of 1 M NaCl, 10 mM MgCl2, 50 mM Tris, 1 mM DTT, pH 8.0. [beta]-N-Acetylneuraminic acid agarose/affinity chromatography
[beta]-neuNAc-agarose was prepared as described by Thiem et al. (Thiem et al., 1992). Active CTP fractions were pooled and dialyzed against buffer B. The dialysate was then applied to 6 ml of [beta]-neuNAc-agarose equilibrated with buffer B at a flow rate of 0.5 ml/min. The column was washed with 2 column volumes of buffer, and eluted with 2 column volumes of 0.4 M NaCl, 50 mM Tris, 1 mM DTT, pH 8.0. Due to instability of the enzyme at this stage in the purification, the column was used as a final purification step to obtain homogeneous enzyme for analysis as needed. Active molecular weight of pituitary CMP-neuNAc synthetase
The active molecular weight of pituitary CMP-neuNAc synthetase was determined by FPLC gel filtration of a partially purified enzyme fraction after either the CTP-agarose step or earlier in the purification. Enzyme (20 µl of 0.5-2 mg/ml) was injected onto a Superose 12 column in 0.05 M bicine, 10 mM MgCl2, 0.2 mM DTT, pH 8.0, and chromatographed at 0.5 ml/min. Bio-Rad molecular weight standards for gel filtration were used to calculate the elution molecular weight. Isoelectric Focusing of CMP-neuNAc synthetase
Partially purified pituitary CMP-neuNAc synthetase obtained from a Blue-Sepharose column was subjected to isoelectric focusing in a Bio-Rad preparative IEF cell for 75 min at 4°C using pH 4-8 Buffalyte (Pierce). Each sample contained 2.3 mg of protein, 0.1% Tween 20, and 16 mM Buffalyte in 50 ml. Isoelectric focusing was performed according to the manufacturer's instructions. Fractions were collected and assayed for enzyme activity, and the pH was measured. Native polyacrylamide gel electrophoresis
Non-denaturing polyacrylamide gel electrophoresis was performed with 7.5% running gels (pH 8.94) and 4% stacking gels (pH 6.89) in the Jovin's MZE buffer systems (Moos et al., 1988). Purified enzyme fractions were mixed with sample buffer and loaded in duplicate so as to create two identical gel halves and run for 3 h at 100 V at 4°C. Upon completion of electrophoresis one half of the gel was immediately stained with Coomassie blue. The stained bands were then excised and loaded onto an SDS-PAGE gel to determine their molecular weight. The other half of the gel was cut into 1 mm slices. Each slice was placed in a tube with 100 µl of cold 50 mM bicine, 1 mM DTT, pH 8.0, and the bands were assayed for CMP-neuNAc synthetase activity. Preparation of 125I-ASA-CDP ethanolamine
N-Hydroxylsuccinimide azido salicylic acid (55 mg) was dissolved in 5 ml of dimethylformamide (Warner et al., 1992). To this solution was added 2 ml of a solution of CDP-ethanolamine (50 mg) in 0.05 M triethylammonium bicarbonate, pH 7.5. The mixture was stirred overnight in the dark after which the solvents were removed under vacuum. The progress of the reaction was followed by thin layer chromatography in 2-propanol:H2O (7/3,v/v). The analog was purified by silica gel chromatography, concentrated to dryness and taken up in 200 µl H2O.
The nucleotide derivative was iodinated with sodium [125]iodide catalyzed by Iodogen (Pierce). Iodogen (2.5 mg/ 400 µl in chloroform) was dried in 50 µl aliquots on the walls of a test tube. ASA-CDP-ethanolamide (5 µl) and 60 µl of H2O was added to the Iodogen coated tube, followed by 10 µl (1 mCi) of Na 125I (Amersham). The reaction mixture was incubated at room temperature 10 min and loaded onto a SEP-PAK (rinsed with 1 ml acetonitrile, followed by 2 ml of H2O). After loading the sample, the SEP-PAK was washed with 15-17 ml of H2O to remove unbound 125I labeled reactants. 125I -ASA-CDP-ethanolamide was eluted with 2 ml aliquots of acetonitrile followed by 2 ml aliquots of propanol:H2O (7:3). The organic solvent fractions with highest levels of product were pooled (0.5 mCi) and concentrated under a stream of nitrogen to 300 µl. Photoaffinity labeling
Identical mixtures for light and dark reactions were prepared on a bed of ice consisting of 10 µl of 125I -ASA-CDP-ethanolamide, 5 µl of 1 M bicine, 1 mM DTT, 0.25 M MgCl2, pH 9.0, 10 µl of purified CMP-neuNAc synthetase, 2 µl of H2O, and 3 µl of 50 mM [beta]-mercaptoethanol (Warner et al., 1992). Light reactions were exposed to 365 nm light at 1 cm for 10 min. Dark reactions were not exposed to UV light. Reactions were electrophoresed on SDS-PAGE gels, dried, and autoradiographed for 16 h. For protection experiments 0.01 M CTP, pH 7.0, was added to the reaction prior to UV exposure. Preparation of chicken CMP-neuNAc synthetase antibodies
Purified enzyme from the [beta]-N-acetylneuraminic acid agarose column was used to immunize a chicken (Lofstrand). Antibody titers were determined by ELISA to be greater than 1:32,000. A hyper immune globulin fraction was prepared from serum by 30% ammonium sulfate precipitation. Antibody was purified from egg yolks of the same chicken as described by Schmidt et al. (Schmidt et al., 1993) and dialyzed against PBS. Preparation of immunoadsorbent
Cyanogen bromide activated Sepharose 4B (7.5 g) was swollen in cold 1 mM HCl and washed with 1 l of cold 1 mM HCl and then 1 l of cold H2O. Immediately before the addition of antibody, the agarose gel was washed with 100 ml of cold 0.1 M sodium bicarbonate pH 8.2. The resin (30 ml) was tumbled with 30 ml purified chicken yolk antibody in PBS, and 30 ml cold 0.1 M sodium bicarbonate pH 8.2 at 4°C overnight. The remaining active sites on the resin were blocked by tumbling with an equal volume of 1M ethanolamine pH 8.2 at 4°C for 3 h. The resin was then washed with 1 l of buffer B. Immunoaffinity chromatography of CMP-neuNAc synthetase
A small aliquot (250 µl) of the active fractions from the Blue Sepharose chromatography step was applied to 10 ml immunoadsorbent equilibrated with cold 50 mM Tris, 0.1 M NaCl, pH 8.0 at a flow rate of 0.25 ml/min. The column was washed with 50 mM Tris, 0.1 M NaCl, pH 8.0 until baseline returned to zero; then it was eluted with 3 M MgCl2, pH 7.0. Protein sequencing
Protein purified by CTP-agarose and [beta]-neuNAc-agarose affinity steps was subjected to amino terminal sequencing after electroblotting (Moos et al., 1988). The sequence of derived trypsin peptides was obtained by Edman degradation following purification by HPLC (Elliott et al., 1993). General carbohydrate method
Glycosylation of proteins in SDS-PAGE gel was detected by an immunochemical method following periodate oxidation (Chen et al., 1995). Following SDS-PAGE electrophoresis proteins were transferred onto a nylon membrane (Magnacharge, MSI). The membrane was rinsed with H2O and dried overnight. The dried membrane was then oxidized with periodate, coupled with digoxigenin-3-O-succinyl-[epsis]-aminocaproic acid-hydrazide, and stained as described previously (Chen et al., 1995). O-N-Acetylglucosamine modification
Attempts to detect O-glcNAc modification were carried out using succinylated wheat germ agglutinin binding and galactosyltransferase assays as described previously (Hanover et al., 1987). DNA and heparin-agarose chromatography
The enzyme was tested for ability to bind heparin as follows. A solution of CMP-neuNAc synthetase was applied to a 15 ml heparin-agarose (Sigma Chemical Co.) column in buffer B. The enzyme was eluted with a linear gradient of 0-0.5 M NaCl in the same buffer. Enzyme activity eluted at 0.5 M NaCl. To measure binding to DNA, a solution of CMP-neuNAc synthetase purified from the CTP-agarose step was applied to a 0.5 ml double stranded DNA-cellulose (Sigma Chemical Co.) column in buffer B. The column was washed with 5 ml of buffer B, then eluted stepwise with increasing concentrations of NaCl. Enzyme activity eluted at 0.25 M NaCl.
We thank Dr. Nga Nguyen of CBER, Facility for Biotechnology Resources for amino-terminal microsequencing. We thank the W.M. Keck Facility for Protein Sequencing at Yale University for internal amino acid sequencing.
CMP-neuNAc, cytidine 5[prime]-monophospho-[beta]-N-acetylneuraminic acid; neuNAc, N-acetylneuraminic acid; ASA-CDP-ethanolamide, cytidine 5[prime]-diphosphoethanolamide azidosalicylic acid; DTT, dithiothreitol; O-glcNAc, O-linked N-acetylglucosamine; IEF, isoelectric focusing; PEI, polyethylenimine.
During the preparation and review of this article, the cloning of a gene encoding a murine pituitary CMP-sialic acid synthetase was reported (Anja K.Munster, M.Eckhardt, B.Potvin, M.Muhlenhoff, P.Stanley, and R.Gerardy-Schahn (1998) Mammalian cytidine 5[prime]-monophosphate N-acetylneuraminic acid synthetase: a nuclear protein with evolutionarily conserved structural motifs. Proc. Natl. Acad. Sci. USA, 95, 9140-9145). This gene encodes a 48,058 Da polypeptide which migrates on SDS-polyacrylamide gels at about 50 kDa. This observation agrees with our findings and supports the identity of the 52 kDa CMP-sialic acid synthetase protein band purified in our laboratory from bovine pituitary glands.
These investigators reported that the amino terminus of the rat pituitary enzyme begins with a 45 amino acid sequence which contains a putative nuclear targeting sequence. This sequence is upstream of the homologous regions homologous with the bacterial CMP-sialic acid synthetase enzymes. Two other putative nuclear targeting sequences were identified in the rat protein in this report. It is likely that the bovine enzyme like the rat pituitary enzyme will have nuclear targeting sequences.
4To whom correspondence should be addressed at: Laboratory of Bacterial Toxins, Division of Bacterial Products, OVRR, CBER, FDA, 8800 Rockville Pike, Bethesda, MD 20892
Discussion
Materials and methods
Acknowledgments
Abbreviation
Note added in proof
References
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