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-Monophosphate N-Acetylneuraminic Acid Synthetase
from Haemophilus ducreyi*
(Received for publication, February 14, 1996, and in revised form, April 10, 1996)
§,
,
''
From the
Department of Pharmaceutical Chemistry,
University of California, San Francisco, California 94143-0446, the
¶ Children's Hospital Research Foundation, and the
Departments of Pediatrics and Medical Microbiology and
Immunology, Ohio State University, Columbus, Ohio 43205-2696
An N-acetylneuraminic acid cytidylyltransferase (EC) (CMP-NeuAc synthetase) was isolated from a Haemophilus ducreyi strain 35000 cell lysate and partially characterized. The enzyme catalyzes the reaction of CTP and NeuAc to form CMP-NeuAc, which is the nucleotide sugar donor used by sialyltransferases. Previous studies have shown that the outer membrane lipooligosaccharides of H. ducreyi contain terminal sialic acid attached to N-acetyllactosamine and that this modification is likely important to its pathogenesis. Therefore, to investigate the role of sialic acid in H. ducreyi pathogenesis, the gene encoding the CMP-NeuAc synthetase was cloned using degenerate oligonucleotide probes derived from NH2-terminal sequence data, and the nucleotide sequence was determined. The derived amino acid sequence of the CMP-NeuAc synthetase gene has homology to other CMP-NeuAc synthetases and to a lesser extent to CMP-2-keto-3-deoxy-D-manno-octulosonic acid synthetases. The gene was cloned into a T7 expression vector, the protein expressed in Escherichia coli, and purified to apparent homogeneity by anion exchange, Green 19 dye, and hydrophobic interaction chromatography. The final step yielded 20 mg of pure protein/liter of culture. The protein has a predicted molecular mass of 25440.6 Da, which was confirmed by electrospray mass spectrometry (Mexpt = 25439.9 ± 1.4 Da). The enzyme appears to exist as a dimer by size exclusion chromatography. In contrast to other bacterial CMP-NeuAc synthetases, the H. ducreyi enzyme exhibited a different substrate specificity, being capable of also using N-glycolylneuraminic acid as a substrate.
Haemophilus ducreyi is the Gram-negative bacterium that causes the sexually transmitted disease chancroid. Chancroid is common in developing countries and has been shown to be an independent risk factor in the transmission of the human immunodeficiency virus (1). Although there are relatively few reported cases of chancroid each year in the United States, there have been several outbreaks in urban areas, and recent studies suggest that chancroid may be greatly underreported due to inadequate methods of detection (2, 3, 4). Although chancroid is still treatable with antibiotic regimens, resistant strains of H. ducreyi have been increasing (5, 6, 7).
Lipooligosaccharides (LOS)1 present in the outer membrane of H. ducreyi have been shown to be a virulence factor (8, 9, 10, 11). Recently, we have shown that many H. ducreyi strains synthesize LOS glycoforms that contain a terminal N-acetyllactosamine that further undergoes the addition of sialic acid (12, 13). The LOS from H. ducreyi and other Gram-negative mucosal pathogens, such as Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae, are structurally similar, and some of these LOS glycoforms have been shown to mimic human antigens (14, 15). This mimicry may contribute to the organism's pathogenicity by allowing the bacteria to evade the host's immune system or establish infection by adhering to host cells (16). Incorporation of sialic acid in the LOS of H. influenzae, N. meningitidis, and N. gonorrhoeae has also been described (15). In addition to the ability of sialylated molecules to downregulate complement as part of a polyanionic surface, sialic acid plays important roles both in cellular recognition and the masking of epitopes, especially terminal galactose residues (17, 18). Sialylation of N. gonorrhoeae LOS has been shown to block neutrophil phagocytosis, block bactericidal antibody binding, and increase serum resistance (19, 20, 21). Also, the sialic acid containing capsules of N. meningitidis, Escherichia coli K1, and group B streptococci all have been identified as virulence factors (17, 22, 23, 24). To date, there are no conclusive results demonstrating that H. ducreyi produces a capsule (25, 26).
The following reactions (I-III) are involved in the biosynthesis of sialic acid containing molecules and are catalyzed by the enzymes shown in parenthesis to the right. In the final step, the hydroxyl group of a carbohydrate serves as the acceptor (HO acceptor) for the nucleotide-sialic acid donor, CMP-NeuAc:
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Materials
Reactive Green 19-Agarose, Cibacron Blue 3GA-Agarose, CTP,
N-acetylneuraminic acid, N-glycolylneuraminic
acid, 2-keto-3-deoxy-D-manno-octulosonic acid, ampicillin,
kanamycin, and SDS-PAGE molecular weight standards were from Sigma.
Size exclusion chromatography standards were from Sigma and
Bio-Rad. Isopropyl
-D-thiogalactopyranoside was from
Life Technologies, Inc. H. ducreyi strain 35000 is a typed
CDC strain and was a generous gift of Dr. A. Campagnari (SUNY at
Buffalo).
Methods
Growth of Bacteria H. ducreyi strain 35000 was
grown on chocolate agar plates (GC Medium Base (Difco), 1% (w/v)
hemoglobin (Difco), and 1% (v/v) IsoVitaleX (Becton Dickinson) or
0.1% (w/v) glucose, 0.01% (w/v) L-glutamine, 0.026%
(w/v) L-cysteine hydrochloride) incubated in candle jars at
33-35 °C for 1-2 days. The bacteria were suspended in 50 mM Bis-Tris-HCl, pH 6.5 (buffer A), and used immediately
for purification of the CMP-NeuAc synthetase or frozen and stored at
80 °C. E. coli strains were grown on Luria-Bertani (LB)
agar plates and in LB broth along with the appropriate antibiotic (50 µg/ml ampicillin or kanamycin).
The bacterial suspension (3 g of wet weight cells in 20 ml of buffer A) was adjusted to 2 mM EDTA and 0.2 mg/ml lysozyme and was stirred for 20 min at room temperature. Unlysed cells and cellular debris were removed by centrifugation at 13,000 × g for 10 min. The supernatant was applied to two 5-ml EconoPac Q cartridges (Bio-Rad) connected in series and equilibrated in buffer A. The enzyme was eluted with 0.2 M NaCl in buffer A. The active fraction (11 ml) was applied to a 5-ml Cibacron Blue 3GA-Agarose dye column also equilibrated with buffer A. The column breakthrough (20 ml), which contained most of the enzyme activity, was concentrated to 0.5 ml with two Centricon-30 concentrators (Amicon) and applied to two Bio-Sil TSK-125 (Bio-Rad; 7.5 × 600 mm each) size exclusion columns connected in series and equilibrated with 20 mM KH2PO4, 0.5 mM EDTA, 20% (v/v) glycerol, pH 7.0. The active fractions (4.5 ml) were pooled, 0.5 ml of 0.5 M Tris-HCl, pH 8.5, was added to increase the pH, and the sample applied to a MonoQ HR 5/5 column (Pharmacia Biotech Inc.; 5 × 50 mm) equilibrated with 50 mM Tris-HCl, 20% (v/v) glycerol, pH 8.0. The enzyme was eluted with a KCl gradient (0.5 ml/min, 50-200 mM KCl in 40 min) in the same buffer. The active fractions (3 ml) were pooled, concentrated to 0.4 ml with a Centricon-30 concentrator, and adjusted to 1.5 M (NH4)2SO4. The sample was applied to a Hydropore-5-HIC column (Rainin; 4.6 × 100 mm), equilibrated with 0.1 M KH2PO4, 0.5 mM EDTA, 1.2 M (NH4)2SO4, pH 7.0 (buffer B), and eluted with a gradient of 0.1 M KH2PO4, 0.5 mM EDTA, pH 7.0 (buffer C) (0.5 ml/min, 1.2-0 M (NH4)2SO4 in 40 min). The active fractions (3 ml) were readjusted to 1.2 M (NH4)2SO4 and rechromatographed on the HIC column using a shallower gradient (0.4 ml/min, 1.125-0.6 M (NH4)2SO4 in 52.5 min). The active fractions (2.4 ml) were pooled, concentrated, and desalted with a Centricon-30 concentrator to a volume of 0.5 ml and applied to a C4 reverse phase HPLC column (Vydac; 2.1 × 150 mm). The C4 column was equilibrated with 0.1% trifluoroacetic acid, and the proteins were eluted with a gradient of 0.08% trifluoroacetic acid, 70% CH3CN over 70 min at a flow rate of 0.2 ml/min.
Amino Acid SequencingThe three major peaks eluted from the HPLC C4 column were each subjected to NH2-terminal sequence analysis using an Applied Biosystems 470A gas-phase sequencer with an on-line ABI Model 130A phenylthiohydantoin analyzer. For the fraction identified as containing the CMP-NeuAc synthetase, 43 cycles were obtained in which the amino acid residue could be assigned with confidence. The first cycle produced 4 pmol of methionine with the yield dropping to 0.1 pmol phenylalanine by the 43rd cycle. Based on typical conversion yields (30-40%), approximately 10-13 pmol (0.25-0.33 µg) of CMP-NeuAc synthetase was loaded on the sequencer.
Screening of the H. ducreyi Genomic DNA LibraryThe
NH2-terminal amino acid sequence was reverse-translated
employing the CompuGene program (38). Two degenerate oligonucleotides
were constructed (see Table I). Oligonucleotide 1, a 23-mer containing
a single inosine at position 15 and a degeneracy of 108, corresponds to
the reverse translation of amino acids 1-8. Oligonucleotide 2 corresponds to the reverse translation of amino acids 14-20 and is a
20-mer with a single inosine and a degeneracy of 48. Both
oligonucleotides were prepared by the Protein Chemistry Facility at
Washington University School of Medicine. A
DASHII library of
H. ducreyi 35000 DNA (39) was plated, plaques were
transferred to Hybond-N, and DNA was immobilized by UV cross-linking
(Stratalinker) employing standard methodologies. Oligonucleotide 1 was
end-labeled with
-32P using T4 polynucleotide kinase,
and hybridization was performed overnight in 5 × SSPE (1 × SSPE = 0.18 M NaCl, 0.01 M sodium phosphate, and 1 mM EDTA, pH 7.7), 5 × Denhardt's solution, 0.5% SDS, and
20 µg/ml Salmon testes DNA at 30 °C. Following hybridization, the
membrane was washed three times at 35 °C in 2 × SSPE-0.1% SDS. The
membrane was then wrapped in Saran Wrap and autoradiographed. Phage
plaques, which gave a positive hybridization signal, were picked and
rescreened. Phage DNA was isolated from 10 ml of liquid lysates and
characterized by restriction analysis.
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The low copy number vector pWSK30 was employed for the initial subcloning (40). For subsequent studies, the DNA fragment containing CMP-NeuAc synthetase was amplified by 20 rounds of PCR using primers 3 and 4 (see Table I). The PCR product was cloned with the TA cloning kit into pCRII according to the manufacturer's instructions (Invitrogen). Plasmid vectors pT7-7 (41) and pET24b (Novagen) were employed for the preparation of the T7 expression constructs. Plasmid DNA was purified by using the Qiawell-8 plasmid kit (Qiagen, Chatsworth, CA).
DNA sequence was determined by the dideoxychain termination method employing Sequenase according to the manufacturer's directions (U. S. Biochemicals Corp.). Lasergene software (DNASTAR, Madison, WI) was employed for contig assembly and sequence analysis.
Protein Sequence AlignmentProtein sequence data bases (GenBank, Release 92.0, Dec. 1995; SWISS-PROT, Release 32.0, Dec. 1995; PIR, Release 45.0, June 1995) were searched using the search program BLAST (42), and protein sequences were aligned with the PILEUP program from the Wisconsin Sequence Analysis Package, version 8.0 (Genetics Computer Group, Inc., Madison, WI). The similarity matrix was calculated using the scoring method in Ref. 43. In pairwise comparisons, the smaller of the two sequences was taken as the denominator.
Protein Expression E. coli BL21(DE3) harboring
the pET24 expression construct containing the CMP-NeuAc synthetase
(designated pMVT1) was grown at 30 °C in LB broth containing 50 µg/ml kanamycin. After reaching an A600
between 0.3 and 0.8, the culture was induced with 0.1 mM
isopropyl
-D-thiogalactopyranoside for 10 h at
30 °C. The cells were harvested and frozen at
80 °C until
needed.
The cell paste (11 g) from a 3-liter culture was suspended in 40 ml of 50 mM Tris-HCl, 0.1 M NaCl, pH 7.5 (buffer D), and the cells were lysed by two passages through a French press (approximately 20,000 psi). The lysate was sonicated (four 30-s pulses at 25 watts with 2-min pauses) to shear nucleic acids and reduce its viscosity. The lysate was then centrifuged at 15,000 × g for 90 min to remove cellular debris. The supernatant (39 ml) was applied to a DEAE-cellulose column (2.5 × 13.5 cm) equilibrated with buffer D. After washing the column with 120 ml of buffer D, the enzyme was eluted with a gradient of 0.1-1.0 M NaCl of 270 ml. The active fractions were pooled (120 ml) and adjusted to 50% (NH4)2SO4. The precipitate was pelleted, redissolved to 10 ml, and desalted into buffer D using a 15-ml Swift desalting column (Pierce). The sample was applied to a Reactive Green 19-Agarose dye column (2.5 × 14 cm) equilibrated with buffer D. The column was washed with 120 ml of buffer D, and most of the enzyme was then eluted with a step gradient of 50 mM Tris-HCl, 0.5 M NaCl, pH 7.5. The sample (25 ml) was adjusted to 50% (NH4)2SO4, and the precipitate was pelleted. The pellet was redissolved to 4.8 ml with buffer C, and then saturated (NH4)2SO4 (1.2 ml) was slowly added. The sample was centrifuged at 12,000 × g for 30 min to remove particulates before loading on the HIC column equilibrated with buffer B. The enzyme was eluted with a gradient of buffer C (1 ml/min, 1.2-0.6 M (NH4)2SO4 in 40 min). Four separate runs were required to purify the entire sample. Fractions judged to be pure by SDS-PAGE were pooled (13.5 ml) and adjusted to 65% (NH4)2SO4 for storage as a suspension at 4 °C.
CMP-NeuAc Synthetase AssayCMP-NeuAc synthetase activity
was assayed by the following procedure: 10 µl of enzyme diluted in
0.1 mg/ml bovine serum albumin (BSA) was added to 90 µl of assay mix
and incubated at 37 °C for 10 min. The reaction was stopped by
adding 400 µl of cold water and freezing the sample in dry ice. The
final concentrations of components in the assay mixture were 1 mM CTP, 2 mM NeuAc, 20 mM
MgCl2, 0.2 M MOPS, pH 7.1, 10 µg/ml BSA, and
4-10 milliunits/ml (nmol/min/ml) CMP-NeuAc synthetase. The assay
solutions were thawed directly before analysis. A 20-µl loop was
completely filled by a 100-µl injection on a Rainin (Woburn, MA) HPLC
system. A nucleotide analysis column (Vydac; 4.6 × 50 mm) was used to
resolve CMP-NeuAc from the substrate CTP and any small amounts of CMP
or CDP present. Solvent A was water and solvent B was 0.5 M
ammonium formate (adjusted to pH 3.5 with formic acid). The column was
operated at a flow rate of 2 ml/min, and the separation was complete in
2 min followed by a 1.5-min reequilibration cycle (Fig.
1). Quantitation of the CMP-NeuAc peak area was done
using a standard curve of CMP (44). Detection was at 280 nm
(
max of CMP and CMP-NeuAc at pH 3.5) using an Applied
Biosystems 1000S diode array detector and was linear from 10 to 10,000 pmol. A unit is defined as the amount of enzyme that catalyzes the
formation of 1 µmol of CMP-NeuAc/min under the conditions of the
assay.
For calculation of the Km for CTP and NeuAc, the enzyme was diluted with 0.2 M NaCl, 1 mM MgCl2, 1 mg/ml BSA to 1 and 4 µg/ml, respectively. The diluted enzyme (35 µl) was added to 315 µl of assay mix preheated to 37 °C and incubated at 37 °C for up to 10.5 min. Each substrate was measured with the other held constant at a saturating concentration. NeuAc was varied from 0.25-8 mM with CTP kept at 1 mM, and CTP was varied from 0.025-0.8 mM with NeuAc at 10 mM. The final concentrations of the other components of the assay mixture were 20 mM MgCl2, 20 mM NaCl, 0.2 M MOPS, pH 7.1, 0.1 mg/ml BSA, and 0.1 or 0.4 µg/ml CMP-NeuAc synthetase. Aliquots (100 µl) were removed from the assays at 3.5 and 7 min and frozen in dry ice along with the final time point at 10.5 min. CMP-NeuAc product for each time point was quantitated as above, except that a 50-µl loop was filled with a 100-µl injection. A straight line was fit to the three time points, and the slope was taken as the initial velocity. The Km for CTP and NeuAc were determined by fitting the data directly to the equation v = Vmax[S]/(Km + [S]) using the computer program KinetAsyst (IntelliKinetics, State College, PA).
Protein DeterminationProtein was determined using the Bio-Rad DC Protein Assay, which is a modification of the Lowry method (45), according to the manufacturer's instructions. BSA was used as a standard.
SDS-PAGEAnalysis of chromatography fractions and
calculation of molecular weight was performed using 15% acrylamide
gels (46). Gels were stained with Coomassie Brilliant Blue R-250.
Molecular weight standards used were albumin, 66000; ovalbumin, 45000;
glyceraldehyde-3-phosphate dehydrogenase, 36000; carbonic anhydrase,
29000; trypsinogen, 24000; trypsin inhibitor, 20000;
-lactalbumin,
14200; and aprotinin, 6500.
To estimate the native molecular weight, purified enzyme (25-200 µg) was applied to two Bio-Silect SEC 250-5 columns (Bio-Rad; 7.8 × 300 mm each) connected in series and equilibrated with 0.1 M KH2PO4, 0.15 M NaCl, pH 7.0. Elution was at 1 ml/min. The column was calibrated with molecular weight standards from Bio-Rad (thyroglobulin, 670000; IgG, 158000; ovalbumin, 44000; and myoglobin, 17500) and Sigma (albumin, 66000; carbonic anhydrase, 29000; cytochrome c, 12400; and aprotinin, 6500), both run separately from each other and the CMP-NeuAc synthetase. The CMP-NeuAc synthetase was exchanged into the column buffer with a 15-ml Swift desalting column, desalted with water using a Microcon-10 microconcentrator (Amicon), or resuspended from an ammonium sulfate pellet with the column buffer prior to chromatography.
Electrospray Mass SpectrometryThe CMP-NeuAc synthetase was
desalted by gradient elution from a Vydac C4 reverse phase HPLC column
or by repeated concentration and dilution with water using a
Microcon-10 microconcentrator. A 5 µl aliquot (
5 µg) of this
solution was injected into a stream of
H2O/CH3CN (1:1) (with or without 1% acetic
acid) at a flow rate of 20 µl/min, which was coupled to the
electrospray ionization source of a Fison Platform quadrupole mass
spectrometer (Fison, Manchester UK). The mass spectrometer was run in
the positive-ion mode and scanned from m/z
600-2000 in 8.1-s intervals. Horse heart myoglobin (average
Mr = 16951.5) was used as a reference compound
and external calibrant, and a typical mass accuracy of ± 0.01%
was obtained.
The CMP-NeuAc synthetase from the H. ducreyi cell
lysate was purified to homogeneity using a procedure consisting of
anion exchange, dye, size exclusion, hydrophobic interaction, and
reverse phase chromatography (Fig. 2). The final step
using reverse phase chromatography resolved three major peaks, which
were sequenced by Edman degradation. The CMP-NeuAc synthetase was
identified by homology to other known CMP-NeuAc synthetases.
The relatively crude preparation of enzyme eluted from the first anion
exchange column was stable to storage as a 65% ammonium sulfate
suspension or 20% glycerol solution at 4 °C or as a 50% glycerol
solution at
20 and
80 °C for at least several months. This
material was used for initial characterization of the enzyme's
properties. The enzyme has a rather broad pH optimum from 8 to 9.5, which is similar to other CMP-NeuAc synthetases (30, 31). The enzyme
has a requirement for Mg2+, with 20 mM giving
the most activity under the assay conditions used. Mn2+ is
only 40% as effective at 20 mM, whereas Ca2+
gave no reaction. The divalent cation requirements of other bacterial
CMP-NeuAc synthetases are similar, whereas enzymes from animal sources
are capable of using Ca2+ and other divalent cations in
addition to Mg2+ and Mn2+ (30, 32). Sulfhydryl
reagents are necessary for or greatly stimulate the enzyme activity of
some other CMP-NeuAc synthetases (30, 31). However, dithiothreitol
was found to have little effect on enzyme activity and so was not
included in purification buffers or enzyme assays. Interestingly,
N-glycolylneuraminic acid was also a substrate for the
H. ducreyi CMP-NeuAc synthetase.
N-Glycolylneuraminic acid can be used as a substrate by
CMP-NeuAc synthetases from animal tissues but not by the enzymes from
E. coli or N. meningitidis (31). The
monosaccharide, KDO, was not a substrate.
In order to clone the gene for the H. ducreyi CMP-NeuAc
synthetase, the amino-terminal sequence was reverse translated, and two
degenerate oligonucleotides were constructed (Table I).
Approximately 1200 clones from a
DASHII library of strain 35000 DNA
were screened with oligonucleotide 1, and seven hybridization-positive
clones were identified. After plaque purification and rescreening, DNA
was prepared from each clone and characterized by restriction analysis
and by Southern hybridization. Two of the clones were identical, and
the additional five clones had overlapping restriction maps. One clone
was chosen for further characterization. H. ducreyi DNA
fragments of 5.5 and 8 kb were identified in a NotI digest
and cloned into NotI-digested pWSK30, and the mixture was
transformed into E. coli DH5
. Clones containing either
the 5.5- or the 8-kb fragment were identified. Southern analysis using
oligonucleotides 1 and 2 indicated that the H. ducreyi
CMP-NeuAc synthetase gene was localized to the 8-kb fragment. A clone
containing the 8-kb NotI fragment, designated pRSM1627, was
saved. Oligonucleotide 1 was used as a sequencing primer and sequence
was determined with Sequenase. Additional primers were constructed, and
the complete DNA sequence of the H. ducreyi CMP-NeuAc
synthetase gene (neuA) was determined in both directions.
The position and direction of transcription of the H. ducreyi CMP-NeuAc synthetase gene in pRSM1627 was determined
by PCR analysis (Fig. 3).
An open reading frame of 684 base pairs encoding a 228-residue protein
was identified (Fig. 4). An inverted repeat
characteristic of a rho-independent transcriptional terminator was
identified 63 nucleotides downstream of the termination codon. Putative
promoter sequences were not identified.
A T7 expression system was employed to generate large quantities of the
H. ducreyi CMP-NeuAc synthetase. The gene was first
amplified by PCR and recloned by TA cloning. The 5
PCR primer
contained an NdeI site (Table I, oligonucleotide 3), and the
3
primer contained a BamHI site (Table I, oligonucleotide
4). The sequence of the clone in the TA vector was again determined to
verify that there were no PCR errors. The CMP-NeuAc synthetase gene was
initially cloned as a NdeI-BamHI fragment into
pT7-7, which had been digested with the same enzymes. The construct
was readily transformed into E. coli strains lacking a
source of T7 polymerase; however, no transformants were obtained with
competent BL21(DE3) cells, suggesting that the synthetase gene product
was being expressed and was toxic to the cells. Transformants were
obtained after cloning the gene as a NdeI-BamHI
fragment into the more tightly regulated T7lac pET24 vector. Initial
expression experiments at 37 °C and inducing with 1 mM
isopropyl
-D-thiogalactopyranoside showed the synthetase
to be the most abundant protein in cell lysates and enzyme activity
levels in a crude lysate to be several hundred times higher than from
H. ducreyi lysates. However, a large portion of the enzyme
(approximately 75%) was found in the insoluble cell pellet. Lower
temperatures (20-30 °C) and isopropyl
-D-thiogalactopyranoside concentration (0.1 mM) favored more soluble enzyme, although insoluble
CMP-NeuAc synthetase was still found in the lysate pellet under the
best conditions identified.
The enzyme was the major component of the soluble cell lysate (Fig.
5). This greatly simplified the purification and allowed
a homogeneous preparation of enzyme to be obtained in only three steps.
For this, successive anion exchange, Green 19 dye, and hydrophobic
interaction chromatography steps were used, and 20 mg of pure protein
per liter of culture was obtained. In the final purification step a
single, large peak was eluted from an HIC column using a shallow
gradient (23.5 column volumes over 40 min) (Fig. 6). A
summary of the purification of the recombinant enzyme is given in Table
II. The enzyme ran as a single band by SDS-PAGE with a
molecular mass of 25.6 kDa. The enzyme also appeared homogeneous by
reverse phase chromatography. The calculated average mass of 25440.6 Da
was confirmed by electrospray mass spectrometry with an accuracy to
within 1.4 Da (Fig. 7). By size exclusion chromatography
the enzyme's molecular mass was calculated as 47-74 kDa in 0.1 M KH2PO4, 0.15 M NaCl,
pH 7.0 buffer and thus appears to exist as a dimer or possibly larger
species. The concentration of enzyme affected the retention time, with
higher concentrations shifting the peak to earlier retention times
(higher molecular mass). Whether the enzyme was resuspended from an
ammonium sulfate suspension or desalted with the column buffer or
water, most of the enzyme eluted as a higher molecular weight species
with little evidence for the presence of a monomeric form. However, if
the enzyme was left at 4 °C overnight in water or column buffer,
both dimer and monomer (26-28 kDa) were detected by size exclusion
chromatography. Incubation overnight at 4 °C in 0.2 M
NaCl (no phosphate buffer) resulted in very little of the monomeric
species. The retention time of the monomer showed little dependence on
enzyme concentration. The rat liver enzyme was identified as a dimer by
size exclusion chromatography (32). The E. coli enzyme is
thought to be active as a monomer but did form dimers and larger
aggregates in some buffers (47). The N. meningitidis enzyme
also possibly exists as a dimer based on size exclusion chromatography
results (29). The apparent Km for CTP and NeuAc were
found to be 0.035 mM and 0.26 mM (mean of three
experiments), respectively, at pH 7.1. The Km for
NeuAc is similar to that measured for the N. meningitidis
enzyme, but the Km for CTP is much lower than values
obtained for previously described activities (see Ref. 30 for review).
A possible reason for such a large difference is that most other
CMP-NeuAc synthetases have been assayed at a higher pH where activity
is greater, whereas the H. ducreyi enzyme was assayed at a
more physiological pH.
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There are now five sequences in the sequence data bases identified as
CMP-NeuAc synthetases. A sixth sequence from Campylobacter
coli is not identified as such but is highly similar (ptmB,
U25992; identified as being involved in the posttranslational
modification of flagellin). All six sequences are from bacteria. Twenty
residues from the NH2 terminus of the rat liver enzyme are
known, but the sequence is not similar to any of the bacterial
sequences. The H. ducreyi CMP-NeuAc synthetase is most
similar to the H. influenzae and N. meningitidis
sequences being 65 and 45% identical (79 and 62% similar),
respectively (Table III). These three enzymes all have
nearly the same molecular weight, whereas the E. coli and
Streptococcus agalactiae enzymes are nearly twice as large.
The H. ducreyi CMP-NeuAc synthetase is 32% identical with
the E. coli and S. agalactiae enzymes. Taken as a
whole, the CMP-NeuAc synthetases have 32-65% identity and 50-79%
similarity with each other. The CMP-NeuAc synthetases also have
homology to the four known CMP-KDO synthetase sequences (11-18%
identity, 32-39% similarity). Although the specificity of these
enzymes for their sugar substrate is high, they catalyze a very similar
reaction in which CMP is transferred from CTP to either NeuAc or KDO;
both of which are
-keto acid sugars (48, 49, 50). These proteins do not
contain the HiGH motif that is involved in nucleotide binding in the
recently elucidated cytidylyltransferase superfamily (51). The highest
homology is found in the NH2-terminal region. The two
previously noted regions of highest similarity between the E. coli NeuA and KdsB proteins largely holds true for the larger set
of sequences as well (residues 6-10 and 46-51 in H. ducreyi NeuA) (47). There are only a few residues conserved
throughout in these two enzyme classes, and these are likely to be
involved in catalysis and binding of the common substrate MgCTP (Fig.
8). A conserved arginine and lysine are found nine
residues apart in the NH2 terminus along with a third
arginine or lysine in between. Chemical modification and site-directed
mutagenesis of the E. coli CMP-NeuAc synthetase suggests
that arginine and lysine are important for enzyme activity (52, 53). In
addition, there is almost a complete lack of negatively charged amino
acids in this NH2-terminal region. There is also a third
region of high similarity with a conserved glutamine, proceeded by
several hydrophobic residues, and a conserved proline four residues
apart (residues 98-107 in H. ducreyi NeuA). The residues in
between are conserved within each class but differ between the two
classes ((P/V)TS and GDE for CMP-NeuAc and CMP-KDO synthetases,
respectively).
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) signs above the
residues. In two cases the conserved charged residues differ by one
position between the two classes, so the sign is placed between the
residues. Boxes indicate areas of a high percentage of
identity among each class of enzymes. The large bars
underline residues that are conserved within each class but differ
between the two. The abbreviation used are: HD, H. ducreyi; HI, H. influenzae; NM,
N. meningitidis; EC, E. coli;
SA, S. agalactiae; and CT,
Chlamydia trachomatis.
The NH2-terminal regions of CMP-NeuAc and CMP-KDO synthetases also share some homology with other nucleotidyltransferases that catalyze the general reaction:
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Regarding the role of CMP-NeuAc synthetase in H. ducreyi, it has been recently reported that many strains contain terminal lactosamine and sialylated lactosamine as part of their LOS (12, 13). These epitopes are also a component of human blood group antigens and have important roles in molecular and cellular recognition (16, 18). Understanding the function of these sugars in the interaction of H. ducreyi with host cells should lead to a better understanding of the pathogenesis of this organism. Recent studies have shown that H. ducreyi can adhere to and invade various human cells (56, 57, 58, 59), and LOS appears to play an important role in this process. Sialic acid may offer the bacteria more protection from the host immune defenses, but exposed terminal galactose may be necessary for the initial infection as has been suggested for N. gonorrhoeae LOS (60). An isogenic mutant of H. ducreyi incapable of producing sialylated LOS will enable a better understanding of the role of sialic acid and the terminal galactose in adhesion to and invasion of host cells and susceptibility of the organism to host defenses. Such a mutant is currently under construction.
The importance of sialylated molecules in general and as virulence factors in pathogenic bacteria in particular make understanding the biosynthesis of sialic acid containing molecules an area of great interest. Further study will hopefully better define the role of sialic acid and LOS in the pathogenesis of H. ducreyi. With large quantities of recombinant CMP-NeuAc synthetase now available, more detailed characterization of this enzyme can be accomplished, including screening conditions for crystallographic analysis. Along with the information gained from conserved residues and domains in the nine sequences of CMP-NeuAc and CMP-KDO synthetases now available, site-directed mutagenesis and active site labeling with reactive substrate analogs will hopefully lead to a better understanding of the residues necessary for substrate binding and catalysis in CMP-NeuAc synthetases.
We thank Lori Andrews for efforts in supplying the Edman sequence data and Prof. A. Campagnari (SUNY at Buffalo, NY) for supplying several preparations of H. ducreyi strain 35000. We also thank Dr. Patricia Babbitt for helpful discussions and Lisa Kim-Shapiro for the computer program to calculate protein similarity.