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Originally published In Press as doi:10.1074/jbc.M400143200 on February 11, 2004

J. Biol. Chem., Vol. 279, Issue 17, 17738-17749, April 23, 2004
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CMP-N-Acetylneuraminic Acid Synthetase from Escherichia coli K1 Is a Bifunctional Enzyme

IDENTIFICATION OF MINIMAL CATALYTIC DOMAIN FOR SYNTHETASE ACTIVITY AND NOVEL FUNCTIONAL DOMAIN FOR PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE ACTIVITY*

Guangchao Liu{ddagger}, Chunsheng Jin{ddagger}, and Cheng Jin§

From the State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China

Received for publication, January 7, 2004 , and in revised form, January 30, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Escherichia coli CMP-NeuAc synthetase (EC 2.7.7.43 [EC] ) catalyzes the synthesis of CMP-NeuAc from CTP and NeuAc, which is essential for the formation of capsule polysialylate for strain K1. Alignment of the amino acid sequence of E. coli CMP-NeuAc synthetase with those from other bacterial species revealed that the conserved motifs were located in its N termini, whereas the C terminus appeared to be redundant. Based on this information, a series of deletions from the 3'-end of the CMPNeuAc synthetase coding region was constructed and expressed in E. coli. As a result, the catalytic domain required for CMP-NeuAc synthetase was found to be in the N-terminal half consisting of amino acids 1-229. Using the strategy of tertiary structure prediction based on the homologous search of the secondary structure, the C-terminal half was recognized as an {alpha}1-subunit of bovine brain platelet-activating factor acetylhydrolase isoform I. The biochemical analyses showed that the C-terminal half consisting of amino acids 228-418 exhibited platelet-activating factor acetylhydrolase activity. The enzyme properties and substrate specificity were similar to that of bovine brain {alpha}1-subunit. Although its physiological function is still unclear, it has been proposed that the {alpha}1-subunit-like domain of E. coli may be involved in the traversal of the blood-brain barrier.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Escherichia coli is one of the leading Gram-negative bacteria that cause neonatal meningitis. The mortality and morbidity associated with this infection are significantly high, with the case fatality rates ranging from 15 to 40% (1-4), and more than half of the survivors have neurological sequelae (2, 5). Incomplete understanding of the pathogenesis and pathophysiology of this disease has contributed to the high mortality and morbidity. It is well documented that the most cases of neonatal E. coli meningitis are caused by K1-encapsulated bacteria (6). Although several E. coli structures and/or genes have been shown to be essential for interaction with brain microvascular endothelial cells (BMEC)1 (7-10), the mechanisms by which E. coli traverses across the blood-brain barrier (BBB) are not clearly elucidated. It has been revealed that a high level of bacteremia is required for the development of E. coli meningitis by investigation of infants with meningitis and animal models (11, 12). Thus, E. coli must be able to avoid host defense mechanisms and proliferate either in the blood or in tissues to maintain a high level of bacteremia.

E. coli K1 capsular polysaccharides are polymeric sialyl residues connected by {alpha}2,8-linkages. Previous investigations have determined that the K1 capsular polysialic acids (PSAs), also known as homopolymers of N-acetylneuraminic acids (Neu5Ac), contribute to the high level of bacteremia by virtue of its serum resistance and antiphagocytic properties (12). A recent study has shown that both E. coli K1+ and K1- strains are able to traverse BMEC in vitro and enter the central nervous system in vivo; however, only infections caused by K1+ strains resulted in positive cerebrospinal fluid cultures (13). Thus, the K1 capsule has, in addition to its well recognized serum resistance and antiphagocytic properties, a role in the traversal of E. coli K1 across the BBB as live bacteria.

The biosynthesis of PSAs is thought to take place on the inner surface of the cytoplasmic membrane through the {alpha}2,8-sialyltransferase-catalyzed addition of Neu5Ac residues from CMP-Neu5Ac donor to the nonreducing ends of nascent (acceptor) PSA chains. The proteins required for biosynthesis, including those needed for Neu5Ac synthesis, activation, and polymerization, are thought to form a multiprotein complex as capsule assembly apparatus (14).

Although the biosynthesis of NeuAc follows a different pathway in mammals and in bacteria, both systems converge in the activation step of the monomer, NeuAc (15). CMP-NeuAc synthetase (CS) utilizes CTP and NeuAc as substrates and produces activated CMP-NeuAc for {alpha}2,8-sialyltransferase (16). CS-deficient mutants that do not express sialylated glycoconjugates can be complemented with functional CS in both mammalian cells (17) and bacterial systems (18).

CS has been isolated from both mammalian (18-21) and bacterial sources (22-26). Although bacterial and eukaryotic CSs share many catalytic properties, several important differences have been reported, including substrate specificity, tertiary structure, inhibitor sensitivity, and cellular localization (20). Thus, CS can be targeted by rational drug design strategies. In addition, CS is also of considerable interest in the field of biotechnology. Given the expense of chemically synthesizing CMP-NeuAc and its instability, CS together with various sialyltransferases is valuable for preparative enzymatic synthesis of biologically relevant, sialylated oligosaccharides (27).

Recently, CS from Neisseria meningitidis has been crystallized as a dimer, and the active site residues, mononucleotide-binding pocket, substrate-binding pocket, and dimerization domain have also been identified (28). The structural information obtained from N. meningitidis CS enables us to gain more insight into the structure and function relationships of its counterpart in E. coli. We have reported previously the cloning of the gene for CS from E. coli K1 strain 44277. By substitution of 6 bases in the initial region and stop codon of the wild type gene, we achieved a high level of expression of CS in E. coli. The recombinant CS protein consisted of 26% of the total bacterial proteins, and the activity was 850-fold higher than that of the wild strain (29). Alignment of the amino acid sequences of E. coli CS with its counterpart in N. meningitidis revealed that the highly conserved features, which include the phosphate binding loop, Mg2+, NeuAc, cytidine binding domain, and dimerization domain, mostly concentrate in the N-terminal half of E. coli CS. We therefore hypothesized that only the N-terminal half of E. coli CS was required for synthetase activity, whereas the C terminus appeared to be redundant. In order to verify our hypothesis, we constructed a series of enzyme mutants by truncating its C terminus. It turns out that the truncated enzymes were still active when more than 189 amino acid residues were removed from C terminus, and the instability of the mutants was also observed. Based on our observations, we concluded that C-terminal half might play a role in stabilizing of the catalytic domain. In an effort to better understand how the C-terminal half stabilizes the catalytic domain in the N-terminal half, we predicted the secondary structure of the C-terminal half and simulated the three-dimensional model by using the strategy of homologous search of the secondary structure. Strikingly, the C-terminal half was found to share a high three-dimensional structural homology with the {alpha}1-subunit of platelet-activating factor acetylhydrolase (PAF-AH) isoform I. Strong evidence has also been obtained to demonstrate that E. coli CS was a bifunctional enzyme that possessed both CS and PAF-AH activities. Here we present the first report that PAF-AH-like activity was recognized in a microorganism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Enzymes and Substrates—CTP, NeuAc, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF; C16), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, glycerol triacetate, 4-nitrophenyl butyrate, 5,5'-dithiobis-(2-nitrobenzoic acid), p-bromophenacyl bromide, and phenylmethylsulfonyl fluoride were purchased from Sigma. [acetyl-3H]PAF was obtained from PerkinElmer Life Sciences. Restriction enzymes and Pfu DNA polymerase were purchased from Promega. T4 DNA ligase was purchased from Takara. All other biochemical reagents were the highest quality commercially available.

Microorganism and Plasmids—E. coli strain 44277 was obtained from the China General Microbiological Culture Collection Center. E. coli DH5{alpha} was purchased from Invitrogen. E. coli BL21(DE3)pLysS and plasmid pET-15b were obtained from Novagen. The plasmid pET-CS2 containing wild type CMP-NeuAc synthetase gene has been described previously (29).

Culture Conditions—Unless otherwise indicated, E. coli was routinely propagated at 37 °C in LB broth contained 10 g of bactotryptone, 5 g of bactoyeast extract, and 10 g of NaCl per liter of culture medium. Growth medium was supplemented with 100 µg/ml ampicillin when required.

Cloning and DNA Manipulations—All DNA manipulations were carried out using standard techniques described by Sambrook et al. (30) or according to the manufacturers' instructions.

Construction of Truncation Mutants—The C-terminal truncation mutants of CMP-NeuAc synthetase were generated by PCR with Pfu DNA polymerase. CMP-NeuAc synthetase mutant gene was amplified by using plasmid pET-CS2 as a template and a 5'-primer (5'-GTCCATGGATCTGTATCAGAACATTCATAATAGAATC) with an introduced NcoI site and a 3'-primer with a BamHI site. A stop codon was introduced into the 3'-primer so that translation could terminate at positions 395, 339, 283, 246, 229, 227, 224, 223, 221, 213, and 210. PCR was carried out by 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1.5 min. The amplified fragments were inserted into the corresponding sites of pET-15b, and the resulting plasmids were designated as pETCS395, pET-CS339, pET-CS283, pET-CS246, pET-CS229, pET-CS227, pETCS224, pET-CS223, pET-CS221, pET-CS213, and pET-CS210, respectively.

As for N-terminal truncation mutants, the 3'-primer used was 5'-ATGGATCCTTATTTAACAATCTCCGCTAT, and a BamHI site was introduced. By introducing an NcoI site into 5'-primer, the translation of mutant construction could start at a chosen position with a start codon contained in the NcoI site. A mutant fused with a His tag was constructed by cloning the coding region for 230-418 amino acid residues into pET-15b at the sites of NdeI/BamHI. Primers used in our investigation are summarized in Table I.


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  		TABLE I
Primers employed in this study

 
Protein Expression—The recombinant plasmid was transformed into E. coli BL21 (DE3) pLysS (Novagen). The resulting transformant was grown at 37 °C in LB broth containing 100 µg/ml ampicillin. When the A600 values reach 0.6-0.8, the culture was induced with 0.4 mM isopropyl-{beta}-D-thiogalactopyranoside for 10 h at 25 °C. The cells were harvested at 5,000 rpm for 15 min and frozen at -70 °C until needed.

Purification of the C-terminal Truncated CMP-NeuAc Synthetase—The cell pellets obtained from 1 liter of induced culture of transformed E. coli BL21 (DE3) pLysS was resuspended in 80 ml of 50 mM Tris-HCl (pH 8.0), and the cells were disrupted by sonication. The cellular debris was removed by centrifugation at 14,000 x g for 15 min. The supernatant was precipitated by the addition of solid ammonium sulfate. The proteins precipitated between 35 and 60% were collected and dissolved in 50 ml of 50 mM Tris-HCl (pH 8.0). After dialysis against 3 liters of 50 mM Tris-HCl buffer (pH 7.6) containing 100 mM NaCl overnight, the enzyme solution was applied to a DEAE-cellulose column (2.5 x 30 cm) pre-equilibrated with 50 mM Tris-HCl buffer (pH 7.6) containing 100 mM NaCl. The column was washed with 1 column volume of initial buffer to remove unbound proteins. The enzyme was eluted with 2 liters of a linear gradient of NaCl from 0.10 to 0.25 M. The enzyme fractions were pooled, dialyzed against the initial buffer, and concentrated with Immersible CX-10TM (nominal molecular weight leakage (NMWL) 10,000-Da, Millipore). All purification steps were carried out at 4 °C.

Purification of the N-terminal Truncated CMP-NeuAc Synthetase—Cells obtained from culture were resuspended in 50 mM Tris-HCl (pH 7.6) and disrupted by sonication. After centrifugation at 12,000 x g for 15 min, the supernatant was collected. The supernatant was loaded onto a HiPrep 16/10-DEAE FF column (50 ml, flow rate 5 ml/min) pre-equilibrated with buffer A (20 mM Tris-HCl (pH 7.6)) and eluted with a linear gradient of 0-0.3 M NaCl in buffer A. The fractions containing the target proteins were collected, dialyzed overnight in buffer A, and then loaded onto MonoQ HR 5/5 (20 ml, flow rate 0.5 ml/min), which had been equilibrated with buffer A (20 mM Tris-HCl (pH 7.6)). The mutant enzyme was eluted with a linear gradient of 0-0.5 M NaCl in buffer A. The fractions containing the target proteins were loaded onto Superdex 200/75 10/30 (100 µl, 0.5 ml/min) pre-equilibrated with buffer A containing 0.1 M NaCl. The fractions containing the target proteins were collected. All purification steps were carried out by using fast protein liquid chromatography system (Amer-sham Biosciences) at 4 °C.

Enzyme and Protein Assays—The CMP-NeuAc synthetase activity was assayed using the thiobarbituric acid method (23). The enzyme was incubated in 250 µl of buffer containing 5.5 mM CTP, 2.8 mM NeuAc, 0.2 M Tris, 20 mM MgCl2, and 0.2 mM dithiothreitol (pH 9.0). After the mixture was incubated at 37 °C for 60 min, 50 µl of 1.6 M NaBH4 was added to destroy excess NeuAc at room temperature for 15 min. The mixture was then put on ice, and 50 µl of 20 N H3PO4 was added to decompose NaBH4. After standing at 0 °C for 5 min, the mixture was then incubated at 37 °C for 10 min to cleave the phosphoester bond of the formed CMP-NeuAc. The free NeuAc was oxidized with 50 µl of 0.2 M NaIO4 at room temperature for 10 min, and 400 µl of 4% NaAsO2 in 0.5 N HCl was added. The solution mixture was then transferred to a test tube containing 1 ml of 0.6% thiobarbituric acid in 0.5 M Na2SO4 and heated in boiled water for 15 min. After the solution was cooled, 1 ml of the solution was taken out and mixed with 1 ml of cyclohexanone. The mixture was shaken and centrifuged, and the upper layer was subjected to absorbance measurement at 549 nm ({epsilon} = 4.11 mM-1 cm-1). One 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.

The standard PAF-AH activity assay was carried out as described by Hattori et al. (31) with small modifications. The specific activity of [3H]acetyl-PAF was adjusted to 200 dpm/nmol by dilution with unlabeled PAF. The standard incubation system for assaying consisted of 50 mM Tris-HCl (pH 7.4), 20 nmol of [3H]acetyl-PAF, and the sample in a total volume of 250 µl. The reaction was terminated by addition of 2.5 ml of chloroform/methanol (4:1, v/v) and 0.25 ml of water. The radioactivity of an aliquot (0.6 ml) of each upper phase was measured to determine the amount of acetate liberated. One unit is defined as the amount of enzyme that catalyzes the formation of 1 nmol of acetate/min.

The assay for serum (plasma) PAF-AH activity was carried out using Azwell Auto PAF-AH kit (Nesco, Japan) according to the manufacturer's instructions. The substrate used in this assay was 1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine.

Esterase activity assay was carried out at 25 °C in 50 mM Tris-HCl (pH 7.0), containing 4 mM of p-nitrophenyl butyrate. An extinction coefficient for p-nitrophenol of 7.2 ml mmol-1 cm-1 at 400 nm was used. One unit is defined as the amount of enzyme catalyzing the appearance of 1 µmol of p-nitrophenol per min.

Protein concentrations were determined by using the method described by Lowry et al. Bovine serum albumin was used as a standard.

TLC Analysis of Substrate Specificity—Substrate specificity was assessed by TLC as described by Grigg et al. (32) with small modifications. After incubation of 50 µg of phospholipids/lipids with the enzyme for 3 h at 37 °C, lipids were extracted by the Bligh-Dyer method (33), dried under a stream of N2, resuspended in methanol, and then applied to silica gel plates. A solvent system of chloroform/methanol/acetic acid/water (50:25:8:4, by volume) was used to resolve the phospholipid samples and hexane/diethyl ether/acetic acid (70:30:1, v/v) to resolve the lipid samples (34). The plates were sprayed with either phosphomolybdic acid or rhodamine B, and the RF values of substrates and cleavage products were determined.

Preparation of Oxidized 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (Oxidized PC)—Oxidized PC was prepared as described by Hattori et al. (35). Briefly, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (4 µmol) in 1 ml of 90% acetate was mixed with 2 ml of an oxidation solution (24 mM KMnO4, 20 mM NaIO4), and the mixture was stirred for 1 h at room temperature. Then lipids were extracted by the Bligh-Dyer method (33), dried under a stream of N2, and resuspended in methanol.

Computer Analysis—The multiple alignment of amino acid sequences was performed using Omiga (2.0). Blast search was performed using the program BLAST. The secondary structure was predicted using the method described by Pan (36). The prediction of tertiary structure of the protein was carried out using the strategy of homologous sequence search at website of Swiss-Model (www.expasy.org/swissmod/SWISS-MODEL.html) or homologous secondary structure search in PDB Bank (www.rcsb.org/pdb/).

Electrophoresis—Analysis of recombinant cell and calculation of molecular weight was performed using 12% acrylamide gel. Gels were stained with Coomassie Brilliant Blue R-250 or silver-staining reagents. Molecular weight standards used were rabbit phosphorylase b, 97,400; bovine serum albumin, 66,200; rabbit actin, 43,000; bovine carbonic anhydrase, 31,000; trypsin inhibitor, 20,100; hen egg white lysozyme, 14,400.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Minimal Functional Domain for CMPNeuAc Synthetase Activity—Several CSs have been isolated from both mammalian and bacterial sources (18-26). According to their molecular size, these enzymes can be divided into two groups as follows: (i) those with molecular mass ranging 20-30 kDa and possessing 200-300 amino acids mostly come from bacterial species, such as 24.8 kDa from N. meningitidis; (ii) those with molecular mass ranging 40-60 kDa and possessing 400-500 amino acids mostly come from eukaryotic species. The second group has a molecular mass more than twice that of the first group. It is interesting to note, with a exception, that CSs from both E. coli and Helicobacter pylori belong to the second group. E. coli CS possesses 418 amino acid residues, which is almost twice of that of N. meningitidis (228 amino acids). Alignment of amino acid sequence of E. coli CS with that of N. meningitidis CS revealed that the highly conserved residues that are present in the active site, substrate-binding pocket, and dimerization domain could be found within the N-terminal half (1-210 amino acid residues) of E. coli CS (28). We therefore hypothesized that the remaining residues in the C-terminal half were redundant.

In order to verify our hypothesis, a series of mutant genes with deletion from 3' termini were generated and subcloned into NcoI/BamHI sites of pET-15b. When the resulting plasmid was transformed into BL21(DE3)pLysS cells, the truncated enzymes CS210, CS213, CS221, CS229, CS246, CS283, CS339, and CS395 could be induced as soluble forms, which terminated at positions 210, 213, 221, 229, 246, 283, 339, and 395, respectively. The assay for CS activity showed that CS229, CS246, CS283, CS339, and CS395 were active, whereas no activity was detected with CS210, CS213, or CS221. The wild type and active mutants were partially purified (Fig. 1). As shown in Table II, the activities of CS395, CS339, and CS283 were 65, 38, and 31% of the wild type, respectively. CS246 showed 85% loss of activity, while removal of 17 additional C-terminal amino acids to produce CS229 resulted in a 3-fold increase of activity and showed 43% of activity of the wild type.



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  		FIG. 1.
SDS-PAGE analysis of the partially purified truncated CMP-NeuAc synthetase from E. coli. The recombinant protein was induced and purified as described under "Experimental Procedures." Lane 1, the wild type CS; lane 2, CS395; lane 3, CS339; lane 4, CS283; lane 5, CS246; and lane 6, CS229.

 


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  		TABLE II
Activity and specific activity of native and truncated CMP-NeuAc synthetase from E. coli

The expression vector harboring wild-type or mutant CS gene was transformed into BL21(DE3) pLysS, and the recombinant enzyme was purified from cell extract and assayed for CMP-NeuAc synthetase activity as described under "Experimental Procedures." Each enzyme species was tested at least twice in separate experiments. One unit is defined as the amount of enzyme that synthesized 1 µmol of CMP-NeuAc/min at 37°C.

 
The highest activity for each active mutant occurred at 37 °C. CS229 even showed a slightly higher catalytic capacity than the wild type at 30 °C (data no shown). The wild type enzyme exhibited the highest activity at pH 9.0; truncation of C terminus caused a shift of optimum pH values to 7-8 and showed a broader range than the wild type, with only one exception of CS229, which was similar to the wild type. Also, CS229 was more active at pH 7-8 than the wild type. These results indicated that these mutants, especially CS229, became more flexible than the wild type enzyme.

The stability of mutant enzymes was determined to ascertain whether these deletions adversely affect the stability of the enzymes. As shown in Fig. 2, the wild type was stable at 37 °C. Incubation at 40 °C for 70 min caused about 30% of activity loss. As for mutants, only CS229 showed 10% loss of activity at 37 °C for 20 min; all others were stable. The CS246 and CS229 were less stable than the wild type at 40 °C, and their activities dropped to about 60% in 20 min, whereas the wild type and other mutants remained at 80-90% of activities.



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  		FIG. 2.
Thermostabilities of truncated enzymes. The purified wild type (+), CS395 (x), CS339 ({blacktriangleup}), CS283 (), CS246 ({Delta}), or CS229 ({circ}) was incubated at 37 (A) and 40 °C (B) for different times, respectively. Each assay was repeated at least twice in separate experiment.

 
Our results showed that the functional domain of E. coli CS was composed of residues 1-229. Removal of the C-terminal half (residues 230-418) resulted in some increase of structural flexibility and therefore contributed to a slight increase in catalytic capacity under non-optimal conditions. It is reasonable to propose that the C-terminal half (residues 230-418) played a role in stabilization by interacting with the N-terminal half of the enzyme.

Computer Modeling of CS Protein—Although CS229, CS246, CS283, CS339, and CS395 remained active, a decrease in activity was observed, and a loss in stability was also documented for either CS229 or CS246. These results, at least, indicated that the C-terminal half played a role in stability. The prediction of three-dimensional structure based on homologous sequence search revealed that CS229 shared a three-dimensional structural identity with N. meningitidis CS (PDB code 1eyr [PDB] :A) (28). N. meningitidis CS has been reported as a dimer, and the active site residues, mononucleotide-binding pocket, substrate-binding pocket, and dimerization domain have also been identified (28). As shown in Fig. 3A, the first 220 amino acids of E. coli CS are needed for assembling all secondary structural units; however, no activity was detected with CS221. We also generated mutant CS223, CS224, and CS227 with the translation terminated at position of 223, 224, and 227, respectively. However, all these three mutants were expressed mainly as inclusion bodies. Only CS223 could express a small portion of soluble form. The soluble CS223 exhibited 16% activity of the wild type. These observations suggested that the coil region formed by 222QKKNRQKI229 at the C terminus, especially the hydrophobic residue Ile228, could maintain the orientation of the C-terminal {alpha}-helix (211RMDFELAITI220) and therefore allow Mg2+-binding site (Arg207) close enough to the active center. In our experiment, a decrease in stability of mutant CS229 or CS246 has been detected, whereas CS283, CS339, and CS395 were as stable as the wild type. Also, we did not detect any dimer formed by either CS229 or CS246 on native PAGE (data not shown). Thus, the stability loss displayed by either CS229 or CS246 can be ascertained to the loss of interaction with the C-terminal half, at least, with residues 230-283. In addition, the instability of CS229 or CS246 could also be explained by exposure of hydrophobic side chain of Ile27 to the surface, in comparison with N. meningitidis CS (Fig. 3, B and C).



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  		FIG. 3.
Three-dimensional model of CS229 mutant. The three-dimensional model of CS229 (A and C) was predicted based on homologous sequence search at Swiss-Model website (www.expasy.org/swissmod/SWISS-MODEL.html). The crystal structure of N. meningitidis CS dimer (B) was retrieved from PDB (PDB 1eyr [PDB] :A). B and C, the hydrophilic side chains of amino acids are marked in blue, and hydrophobic ones are in red. The C-terminal {alpha}-helix is shown in green.

 
To understand how the C-terminal half stabilizes the N-terminal half and to explore the potential role of the C-terminal half besides its stabilizing role, we carried out homologous analysis by blasting the amino acid sequence of the C-terminal half (residues 230-418) alone in GenBankTM, but no any homologous protein was retrieved. Then the secondary structure of the C-terminal half was predicted by using the method developed by Pan (36) (Fig. 4A). When the predicted secondary structure was used for computer modeling, to our surprise, the predicted tertiary structure of the C-terminal half was shown to share identity with that of {alpha}1-subunit of PAF-AH isoform I (PDB 1fxw [PDB] :A) (Fig. 4C), a 29-kDa protein isolated from bovine brain soluble fraction (37-39). PAF-AH isoform I belongs to serine esterase family and is an intracellular heterotrimeric enzyme composed of 29-({alpha}1), 30-({alpha}2), and 45-kDa ({beta}) subunits. Among these subunits, only {alpha}1-subunit served as the catalytic subunit. Also, {alpha}1 alone is enough to catalyze the removal of the acetyl moiety at the sn-2 position of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phospholine) to produce biologically inactive lyso-PAF. Ser47 has been identified as the active center residue of {alpha}1. A large family of enzymes that have an essential serine residue possesses the consensus sequence, Gly-Xaa-Ser-Xaa-Gly. These include proteases, esterases, transacylases, and lipases (40). However, the second residue from the active Ser is Val in {alpha}1-subunit. In active center, Ser47, Asp192, and His195 have been revealed to form a catalytic triad. In most serine esterases, the Asp and His are separated by more than 20 amino acid residues (41), whereas the distance in {alpha}1 is only 4 residues (Fig. 4). As revealed by the three-dimensional modeling of the C-terminal half of CS (Fig. 4D), Ser257, Asp397, and His400 were recognized as a putative catalytic triad in catalytic site. The consensus sequence surrounding Ser257 was GHSLF (Fig. 4B). Thus, we postulated that the C-terminal half (230-418 residues) in CS was an "{alpha}1-subunit-like" functional domain.



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  		FIG. 4.
Prediction of the secondary structures and three-dimensional model of the C-terminal half of CMPNeuAc synthetase. The three-dimensional structure (D) of the C-terminal half (residues 230-418) of CS was predicted based on the homologous search of the secondary structure, which was predicted using the method described under "Experimental Procedures" (B). The secondary (A) and three-dimensional structure (C) of bovine brain PAF-AH I were retrieved from Protein Data Bank (PDB 1fxw [PDB] :A). A and B, the {alpha}-helix is indicated as H, the {beta}-sheet is shown as E, and the coil region as C. The serine esterase triad is marked in red.

 
Evidence for PAF-AH Activity Associated with E. coli CS—Based on the information obtained from three-dimensional structure prediction, we first detected the PAF-AH activity with complete CS protein using [3H]acetyl-PAF as substrate. As expected, CS could catalyze the removal of the acetyl group at the sn-2 position of PAF. Moreover, both CS and PAF-AH activities were assayed at each step during the purification procedure of complete CS protein. As shown in Table III, the increase of purification fold for PAF-AH activity was found to be almost the same as that of CS activity. Both PAF-AH and CS activity could be detected when the protein was purified to homogeneity at the final step (Fig. 5). These results clearly demonstrated that the PAF-AH activity was associated with CS protein.


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  		TABLE III
Purification of the wild-type enzyme from E. coli

The cell pellets obtained from 1 liter of induced culture of E. coli harboring pET-CS2 were resuspended in 80 ml of 50 mM Tris-HCl (pH 8.0), and the cells were disrupted by sonication. The wild-type CS protein contained in the supernatant was collected and purified as described under "Experimental Procedures." The assay for either CS or PAF-AH activity was carried out. Under the assay conditions described under "Experimental Procedures," 1 unit of CS activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol of CMP-NeuAc/min, and 1 unit of PAF-AH is defined as the amount of enzyme that catalyzes the formation of 1 nmol of acetate/min.

 



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  		FIG. 5.
Purification of the wild type enzyme from E. coli. The cell lysate of E. coli harboring pET-CS2 (lane 2) was purified with DEAE-Sepharose (lane 3), Mono Q (lane 4), and Superdex 200 (lanes 5 and 9) chromatographies as described under "Experimental Procedures." Negative control (lane 1) is cell lysate of E. coli harboring pET-15b. The SDS-PAGE was stained with Coomassie Brilliant Blue R-250 (lanes 1-7) or silver staining (lanes 8 and 9).

 
We also constructed mutant enzyme CS228-418 and CS230-418 by truncating 227 and 229 residues at the N terminus, respectively. Both mutant enzymes were expressed in E. coli as soluble forms. The activity determination revealed that both mutants were devoid of CS activity; however, only CS228-418 was capable of hydrolyzing PAF, whereas CS230-418 had no PAF-AH activity. These results demonstrated that the PAF-AH functional domain was composed of amino acids 228-418 in the C-terminal half of CS. It should be pointed out that CS228-418 was not exactly initiated with Lys228 at its N terminus. In order to truncate the N terminus of CS, CCATGGAT was introduced to the 5'-end of mutant gene so that the translation of mutant gene could start from ATG contained in the NcoI site. Thus, the N terminus of CS228-418 was Met-Gly-Lys228 instead of Lys228. Due to the same reason as in CS228-418, the CS230-418 translate was initiated with Met-Asp-Leu230 but Leu230. Somehow it appeared that this modification had no effect on the PAF-AH activity, at least for the CS228-418 mutant.

General Properties of PAF-AH Functional Domain—To define the catalytic properties of the PAF-AH domain of CS protein, the mutant CS228-418 was purified. After purification on DEAE-Sepharose, Mono Q, and Superdex 75 columns, CS228-418 was purified to 19.5-fold with a specificity of 1.69 units/mg (Table IV). The CS228-418 was purified to homogeneity, which was judged by SDS-PAGE stained with both Coomassie Brilliant Blue R-250 and silver staining (Fig. 6).


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  		TABLE IV
Purification of mutant CS228-418 from E. coli

The cell culture of E. coli harboring pET-CS228-418 was collected and purified as described under "Experimental Procedures." Under the assay condition, 1 unit of PAF-AH is defined as the amount of enzyme that catalyzes the formation of 1 nmol of acetate/min.

 



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  		FIG. 6.
Purification of mutant CS228-418 from E. coli. The CS228-418 mutant protein was expressed in E. coli harboring pET-CS228-418. The mutant protein was extracted by sonication from E. coli (lane 2) and purified by DEAE-Sepharose (lane 3), Mono Q (lane 4), and Superdex 75 (lanes 5 and 8) as described under "Experimental Procedures." The gel was stained with Coomassie Brilliant Blue R-250 (lanes 1-6) or silver staining (lanes 7 and 8). Lane 1 shows the cell lysate of E. coli harboring pET-15b.

 
With purified wild type CS and CS228-418, the effects of pH values and temperatures were determined. Both wild type and mutant were most active at 30 °C. The wild type exhibited optimum pH of 7-8, whereas the optimum pH of mutant occurred at a pH value of 8-9.

Substrate Specificities of PAF-AH Catalytic Domain—PAFAHs are unique calcium-independent PLA2s, which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. As for {alpha}1 catalytic subunit of bovine brain PAF-AH isoform I, the side chains of Thr103, Leu48, and Leu194 were identified to be involved in substrate recognition (42). These three residues were also conserved in the C-terminal half of CS as Thr308, Leu258, and Leu399 (Fig. 4), which implies that the PAF-AH domain of CS shares the same substrate specificity with the {alpha}1-subunit. A previous investigation (35) has shown that the {alpha}1-subunit of PAF-AH I does not recognize oxidized phospholipids as substrates. By using purified wild type CS and mutant CS228-418 proteins, the abilities to hydrolyze PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), PC (1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphocholine), 1-myristoyl-2-(4-nitrophenylsuccinyl)-phosphatidylcholine, oxidized PC (1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine), glycerol triacetate (triacetin), and p-nitrophenyl butyrate were investigated. As shown in Fig. 7, neither the wild type CS nor CS228-418 had activity against PC, a substrate for PLA2, or oxidized PC, a substrate for PAF-AH isoform II. Both wild type CS and CS228-418 showed activities against glycerol triacetate, a substrate for lipase. When 6.1 µg of wild type CS or 1.3 µg of mutant CS228-418 was incubated with p-nitrophenyl butyrate, a substrate for esterase, the specific activity was determined as 75.6 and 319.8 units/mg, respectively. Although no activity was detected with either the wild type or CS228-418 when a substrate for plasma PAF-AH, 1-myristoyl-2-(4-nitrophenylsuccinyl)-phosphatidylcholine was used (data not shown). These results clearly demonstrated that both wild type and mutant enzymes exhibited activity against the sn-2 ester linkage in PAF-like phospholipids with short acetyl chains and also exhibited lipase- and esterase-like activities. No activity was detected toward PAF-like phospholipids with long acyl chains or oxidized phospholipids.



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  		FIG. 7.
Substrate specificities of the wild type and CS228-418. 120 µg of wild type enzyme (WT) or 54 µgofCS228-418 (M) was incubated with 50 µg of PAF, PC, oxidized PC, or triacetin at 37 °C for 3 h, respectively. Then the lipid was extracted and analyzed as described under "Experimental Procedures." PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PC, 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphocholine; oxidized PC, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine; triacetin, glycerol triacetate. Lipase used in assay was obtained from commercial sources.

 
Effect of Various Compounds on FAF-AH Activity—Various compounds were tested for their effects on the PAF-AH activity of the wild type CS or CS228-418. As summarized in Table V, p-bromophenacyl bromide, which has been shown to block various phospholipase A2 activities by derivatizing the histidine residue at their active sites, inhibited the PAF-AH activity of the wild type CS or CS228-418 with a percentage of 70% at 4 mM. Phenylmethylsulfonyl fluoride, an inhibitor of serine esterase, inhibited the PAF-AH activity by 43-45% at 4 mM (Table V). On the other hand, 5,5'-dithiobis-(2-nitrobenzoic acid) and dithiothreitol could not inhibit the activity. These results suggested the presence of both essential histidine and serine residues at the active site, and the free sulfhydryl residue was not essential for the PAF-AH activity, which was consistent with the putative catalytic triad identified in three-dimensional models. Moreover, Ca2+, Mg2+, and EDTA had no effect on the PAF-AH activity of wild type or CS228-418 (data no shown). It was interesting to note that the PAF-AH activity of wild type or CS228-418 was increased by 1-fold in the presence of 40% glycerol.


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  		TABLE V
Effect of various compounds on the activity of the PAF-AH

22 µg of wild-type CS or 11 µg of mutant CS228-418 were preincubated at room temperature for 30 min in the presence or absence of various compounds of the assay mixture. Then the PAF-AH activity assay was determined with 30 µm [3H]acetyl-PAF as described under "Experimental Procedures." Results are expressed as means for triplicate assays. The abbreviations used are as follows: PMSF, phenylmethylsulfonyl fluoride; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); DTT, dithiothreitol; {rho}BPB, p-bromophenacyl bromide.

 
Kinetics of PAF-AH Activity—By using the purified wild type and CS228-418 mutant, the effect of PAF concentration on the rate of hydrolysis was determined. In our experiments, 22 µgof the wild type CS or 11 µg of CS228-418 was incubated with various amounts of PAF at 37 °C for 30 min. The rate of PAF hydrolysis catalyzed by the wild type enzyme increased with increasing concentrations of PAF to about 10 µM, whereas the rate of hydrolysis by mutant increased with increasing concentrations of PAF to about 20 µM. The Km was estimated to be 6.7 µM for the wild type and 4.3 µM for CS228-418, respectively (Fig. 8). Apparently, the substrate-binding affinity of CS228-418 is higher than that of the wild type.



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  		FIG. 8.
Kinetics of the wild type and CS228-418 for PAF. 22 µg of wild type CS (top) or 11 µg of CS228-418 (bottom) was incubated with PAF at 37 °C for 30 min, respectively. The hydrolysis of substrate was determined. The double-reciprocal plot of these data is shown in the insets.

 
Proteolysis of Wild Type Enzyme—The mutant analyses showed that CS activity and PAF-AH activity domains of CS protein could function separately, and we carried out proteolytic analysis of CS protein to explore other potential in vivo forms instead of full-length CS. When the CS was incubated with trypsin, after 1 h of incubation, a 22-kDa protein band became visible, whereas the density of CS protein band was found decreased on SDS-PAGE. A prolonged incubation resulted in a complete conversion of CS protein band into 22 kDa (Fig. 9). It is evident that the CS could be degraded by trypsin to produce a trypsin-resistant 22-kDa polypeptide. To define the product of trypsin treatment, the 22-kDa protein was transferred to polyvinylidene difluoride membrane and subjected to the N-terminal sequencing analysis. The N terminus was determined as ILYQN, which indicated that the proteolytic product of trypsin was the C-terminal half (designated as CS229-418, which consists of amino acid residues from 229-418) of CS protein. These results suggested that the CS protein could be hydrolyzed by protease to yield active PAF-AH-like functional domain. However, when CS229-418 was recovered from PAGE gel to remove trypsin and SDS, little or no PAF-AH activity was detected.



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  		FIG. 9.
Trypsin treatment of the wild type enzyme. Trypsin was added to the wild type CS at a ratio of 1:250 (w/w). The mixture was incubated at 4 °C for 5 min (lane 2), 20 min (lane 3), 1 h (lane 4), 3 h (lane 6), and 6 h (lane 7), respectively. The reaction was stopped by the addition of SDS-PAGE loading buffer. Lanes 1 and 5, wild type CS without addition of trypsin; lane 8, molecular mass marker.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Central nervous system infection caused by bacterial, viral, fungal, or parasitic pathogens can lead to devastating neurological disability and death. It is likely that further improvements in outcome will require new therapeutic approaches based on a better understanding of molecular and cellular mechanisms of central nervous system infection. There are many steps that are required for microorganisms to cause central nervous system infection. The least understood step in the pathogenesis of central nervous system infection by microbes is how circulating microbes penetrate the BBB and enter cerebrospinal fluid. E. coli is one of the leading Gram-negative bacteria that cause neonatal meningitidis. It has been shown that the successful traversal of the BBB by circulating E. coli K1 is a complex process, requiring several bacterial and host factors and their interactions, such as a high degree of bacteremia, binding to and invasion of BMEC, BMEC actin cytoskeleton rearrangements and related signaling pathways, and traversal of BBB as live bacteria (10). Following the initial interaction between the host and E. coli, the bacteria are progressively drawn into the host by a "zipper mechanism," which utilizes the bacterium-induced actin filaments for bacterial uptake (43). K1 capsule, OmpA, Ibe proteins, AslA, TraJ, and CNF1 have been identified to be important virulence factors using in vivo and in vitro models (10). However, it is unclear how all of these E. coli determinants contribute to BMEC invasion in vitro and crossing of BBB in vivo. Previous investigations have determined that E. coli K1 capsule contributes to the high level of bacteremia, which was required for the development of meningitidis (12). It has been suggested that PSAs can mimic the host and escape detection by the immune system (44). Moreover, recent evidence suggests that the K1 capsule is not only necessary for invading BMECs but also responsible for maintaining the viability of the bacteria inside the BMECs (13). These observations strongly suggest an important role of K1 capsule in the traversal of BMEC.

The biosynthesis of K1 capsular {alpha}2,8-linked polysialic acids is involved in the activation NeuAc and polymerization of activated NeuAc. CMP-NeuAc synthetase, the enzyme that catalyzes the activation of NeuAc, has been extensively studied (23, 45, 46). Previously, we cloned a CS gene from E. coli K1 strain 44277 (29). It has been noticed that molecular weight of E. coli CS is almost twice that of other bacterial species, and all conserved structural motifs required for NeuAc activation were concentrated in the N-terminal half of E. coli CS. Thus, we hypothesized that the C termini of E. coli CS was not required for CS activity. Evidence we obtained definitely supports our hypothesis. The investigation of truncations at the C terminus revealed that the CS mutant (CS229) was still active when up to 189 amino acids were removed from the C terminus; however, activity loss and less stability were observed. By taking advantage of structural information obtained from three-dimensional modeling, CS229 was found to be the minimal truncation that could maintain the tertiary structure required for synthetase activity (Fig. 3). Our evidence also suggested that the C-terminal half (residues 230-418) of E. coli CS could stabilize the CS catalytic domain. However, it is hard to understand why E. coli produces such a long peptide at the C terminus only for stabilization. The first idea was that the C-terminal half would interact with the putative dimerization domain in the N-terminal half and probably serve as another "subunit" as occurred in the dimer of N. meningitidis CS. It is possible that the C-terminal half is an inactive subunit derived from naturally occurring mutations of a previously functional subunit. Therefore, we carried out the three-dimensional structure prediction analysis based on the homologous sequence search of the C-terminal half (residues 230-418). However, no homologous protein was found in GenBankTM. Then we predicted the three-dimensional structure of the C-terminal half based on the homologous search of secondary structure in PDB. Most interesting, the secondary structure of the C terminus was homology with the {alpha}1-subunit of PAF-AH isoform I instead of N. meningitidis CS subunit-like domain.

PAF is one of the most potent lipid messengers involved in a variety of physiological events. The acetyl group at the sn-2 position of its glycerol backbone is well known for its biological activity, and its deacetylation induces loss of activity. The enzyme that catalyzes the deacetylation is PAF-AH, which was first reported in human sera (47) and belongs to calcium-independent phospholipase A2 family. PAF-AH has been found widely distributed in mammalian tissues and blood so far. At least two types of PAF-AH exist in mammalian tissues, namely the intracellular (cytosolic) and extracellular (plasma) type. Arai et al. (48) found two types of intracellular PAF-AH from bovine brain, PAF-AH I and II. PAF-AH I is a heterotrimer composed of 29-({alpha}1), 30-({alpha}2), and 45-kDa ({beta}) subunits (35, 38). The amino acid sequences of the three subunits show extremely high homologies among the mammalian species (31). Both {alpha}1 and {alpha}2 have a catalytic center (31). It has been shown that {alpha}1-{alpha}2 complex can express the full level of activity (49). When {alpha}1 and {alpha}2 are purified or overexpressed in E. coli, they form catalytically competent homodimers (37). Although the dissociation of the dimeric protein into monomers leads to the inactivation and destabilization of protein (50), only one subunit is active in the heterodimer or homodimer (37, 38). The biochemical differences of three possible catalytic dimers, {alpha}1/{alpha}1, {alpha}1/{alpha}2, and {alpha}2/{alpha}2, have been studied. The {alpha}2/{alpha}2 homodimer is significantly more active against PAF and 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphoric acid than {alpha}1/{alpha}1 and {alpha}1/{alpha}2, whereas both homodimers have comparable catalytic efficiency against PAF (39, 51). The {beta}-subunit binds to all three catalytic dimers. The binding of {beta}-subunit accelerates the hydrolytic rate of the {alpha}2/{alpha}2 homodimer for PAF about 4 times, slightly suppresses the hydrolytic rate of the {alpha}1/{alpha}1 homodimer, and has little effect on that of the {alpha}1/{alpha}2 heterodimer (51). It is therefore proposed that the enzyme activity of PAF-AH I is regulated in multiple ways by altering the composition of catalytic subunit and by manipulating the {beta}-subunit (48).

Ser47 has been identified as an active serine residue in {alpha}1. In the active center, Ser47, Asp192, and His195 have been revealed to form a catalytic triad (37). By comparing the three-dimensional model of PAF-AH I {alpha}1 (PDB 1fxw [PDB] :A), the putative catalytic serine was identified as Ser257, and the catalytic triad was composed of Ser257, Asp397, and His400 (Fig. 4). Thus, we tested PAF-AH activity by using [3H]acetyl-PAF as substrate. Our experimental results showed, as expected, that the wild type E. coli CS was able to release acetyl group from PAF. During the purification of CS protein, the purification fold of PAF-AH activity was almost the same as that of CS activity, which clearly demonstrated that the PAF-AH activity was associated with CS protein. In addition, the mutant analysis confirmed that the residues from 228 to 418 (CS228-418) could form a functional PAF-AH-like protein. With this evidence, we concluded that E. coli CS was a bifunctional enzyme possessing a CS activity in the N-terminal half and a PAF-AH activity in the C-terminal half.

Although we were unable to detect the dimer of CS228-418 on native PAGE (data not shown), the indirect evidence still suggested the existence of catalytic dimer formed by CS228-418. The 48.6 kDa of pure CS protein exhibited a specific activity of 1.4 units/mg for PAF-AH activity (Table III). If the 22.5 kDa of pure CS228-418 protein existed as a monomer, then its theoretical specific activity should be at least twice that of CS (Figs. 5 and 6). However, according to our results, the specific activity of CS228-418 was 1.7 units/mg (Table IV). This unexpected lower specific activity of CS228-418 was not caused by instability of the mutant, because we found that CS228-418 was more stable than CS when we incubated either CS or CS228-418 at 37 °C for 1 h. Therefore, the only plausible explanation is that CS228-418 functions as a dimer, and only one subunit exhibits activity. The slight increase of the specific activity displayed by dimeric CS228-418 can be explained by the difference in purity of CS and CS228-418. The crystal structure of bovine {alpha}1-subunit reveals that the N-terminal {alpha}-helix (H1), together with the loop that follows, is part of the dimer-forming interface, which contains a significant number of charged and polar residues (38, 42). As revealed by three-dimensional modeling of CS228-418 (Fig. 4), the putative H1 {alpha}-helix formed by the residues from 231-242 (YQNIHNRINEKR) could serve as part of dimer-forming interface. According to our results, both CS230-418 and CS229-418 were inactive. By fusing a His tag with the N terminus of inactive mutant CS230-418, we were able to restore the PAF-AH activity (data not shown). These observations suggested that only H1 was not enough for dimer forming.

PAF-AH I has a strong specificity for the acetyl group attached to the glycerol backbone of PAF, whereas phospholipids having a head group other than choline are also recognized by the enzyme (51). This specificity is ascertained to Leu48, Leu194, and Thr103, which are responsible for the substrate specificity of {alpha}1 by forming contact with the methyl group of acetate (38). On the other hand, PAF-AH II shows substrate specificity similar to plasma PAF-AH. Both enzymes hydrolyze short chain diacylglycerols, triacylglycerols, and acetylated alkanols and display phospholipase A1 activity. These two enzymes do not distinguish between an ester or an ether at the sn-1 position of PAF or PAF analogues, and both can hydrolyze phospholipids with short to medium length sn-2 acyl chains including truncated chains derived from oxidative cleavage of long chain polyunsaturated fatty acyl groups (52, 53). As for the PAF-AH domain in E. coli CS, the specificity was predicted to be similar to PAF-AH I since the amino acid residues corresponding to Leu48, Leu194, and Thr103 in {alpha}1-subunit of PAF-AH I were found to be conserved (identified as Leu399, Leu258, and Thr308). The specificity analysis with the wild type CS or CS229-418 supported our prediction. Among the phospholipids tested, only PAF, glycerol triacetate, and p-nitrophenyl butyrate could be hydrolyzed. These results indicated that the PAF-AH domain of E. coli CS was specific for PAF and exhibited lipase- and esterase-like activities.

A large family of enzymes that have an essential serine residue possesses the consensus sequence, Gly-Xaa-Ser-Xaa-Gly. By comparing the amino acid sequences between {alpha}1-subunit of PAF-AH I and E. coli CS, it is interesting to note that the second Gly is substituted by Val in {alpha}1, which has been thought to contribute to the specificity against the short chain acyl moiety (37). In the PAF-AH domain of E. coli CS, the second Gly residue from the active serine is substituted by Phe. Thus, the preference of short chain acyl moiety for the C-terminal half of E. coli CS could be explained by this substitution. Another interesting fact is that the amino acid residue between the first Gly and the active serine residue in {alpha}1 is Asp, which is the same as those of most serine proteases but not those of serine esterase, in which the residue between the first Gly and the active serine residue is His, suggesting that PAF-AH I is closer to that of proteases other than that of lipases. In the PAF-AH domain of E. coli CS, the residue between the first Gly and the active serine residue is His, suggesting that the C-terminal half of E. coli CS is closer to that of lipases. As a matter of fact, the lipase activity of the PAF-AH domain of E. coli CS has been detected with glycerol triacetate, a substrate for lipase. Moreover, it has been shown that the substitution of Tyr191 of {alpha}1 with Phe that presents in {alpha}2 gives the {alpha}1-subunit a substrate specificity similar to that of {alpha}2 (37). Tyr191 was also found to be conserved in the C-terminal half of E. coli CS. By taking the structural features and substrate specificity analysis together, it can be concluded that the specificity of C-terminal half of E. coli CS is similar to that of the {alpha}1-subunit.

The evidence we present in this report clearly demonstrates that the C-terminal half of E. coli CS functions as {alpha}1-subunit of PAF-AH I. However, the physiological role of this functional domain has not been defined yet. It has been observed that the expression of {alpha}1-subunit is restricted in early developing neurons at embryonic stages of rat (54), and the highest expression level of {alpha}1 is also detected in the human fetal brain as well (55). In addition, certain PAF-AH inhibitors, which do not bind to PAF receptors, can inhibit migration of neurons in cultures (56). Thus, the {alpha}1-subunit has been suggested to play a role in neuron migration during development. Moreover, the significant levels of {alpha}1-subunit are expressed in the kidney, thymus, and colon of the adult human, in which cell migration remains prominent under normal physiological, as well as pathological, conditions. It is therefore thought that cells expressing the {alpha}1-subunit possess the ability to migrate in adult tissue. On the other hand, a recent report that {alpha}1 is not required for embryonic and early postnatal development in mouse makes the story of {alpha}1-subunit even more complicated (57, 58). It is now recognized that the {alpha}-subunit is a member of an old family of hydrolytic proteins, identified some years ago as a family of lipolytic enzymes (59, 60). Therefore, it is possible that the natural substrate of the presumed PAF-AH I could be an as yet unidentified molecule with an acetyl moiety attached via an ester bond (39). Obviously, the elucidation of endogenous substrate is of importance not only for the physiological function of mammalian {alpha}1-subunit but also for that of the E. coli {alpha}1-subunit-like domain. Somehow, considering the correlation of mammalian {alpha}1-subunit to cell migration, it is possible that the E. coli {alpha}1-subunit-like domain may play a role in E. coli motility during the infection of BMEC.

In our investigation, we found that the CS activity of the wild type enzyme had a pH optimum of 9.0 and that the truncated CS229 mutant was more active at pH 7-8. In contrast, we found that the PAF-AH activity of the wild type was optimal at 7-8, whereas the truncated mutant was most active at pH 9.0. Apparently, it appears that the wild type enzyme mainly functions as CS at pH 9.0 and serves PAF-AH at pH 7-8. However, it is now hard to understand why the wild type enzyme exhibits its highest CS activity at pH 9.0 because it catalyzes the CMPNeuAc synthesis in cytosol, which is a neutral environment. Although we did not detect PAF-AH domain alone in vivo, the proteolytic analysis showed that the PAF-AH could exist in vivo under some unknown conditions. Probably, in an environment of pH 9.0, the wild type enzyme or PAF-AH domain derived from proteolysis mainly functions as PAF-AH, which may be a clue to explore the physiological function.

In summary, we have demonstrated that the E. coli CS is a bifunctional enzyme with a CS activity in the N-terminal half and a PAF-AH-like activity in the C-terminal half. The tertiary structure of PAF-AH-like domain is significant homology with that of {alpha}1-subunit of bovine brain PAF-AH I. The biochemical properties of PAF-AH-like domain in E. coli CS are essentially similar to that of bovine brain {alpha}1-subunit. This protein is the first {alpha}1-subunit-like protein recognized in microorganisms. At present the in vivo substrate and the physiological function of this novel functional domain are still unclear. Our next challenge will be to identify the in vivo substrate for this domain and explore its physiological function.


   FOOTNOTES
 
* This work was supported by the Chinese Academy of Sciences Grant KSCX2-3-02-01 and the National Science and Technology Ministry, China, Grant 2001CCA00100. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Both authors contributed equally to this work. Back

§ To whom correspondence should be addressed. Tel.: 86-10-62587206; Fax: 86-10-62653468; E-mail: jinc{at}sun.im.ac.cn.

1 The abbreviations used are: BMEC, brain microvascular endothelial cells; BBB, blood-brain barrier; PSAs, polysialic acids; CS, CMP-NeuAc synthetase; PAF-AH, platelet-activating factor acetylhydrolase; Neu5Ac, N-acetylneuraminic acids; PDB, Protein Data Bank; PC, 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphocholine. Back


   ACKNOWLEDGMENTS
 
We thank Guoping Zhao and Yixuan Zhang of Chinese National Human Genome Center, Shanghai, China, for help in the attempt to assay the enzymatic activity for plasma PAF-AH; Xian-Ming Pan of the Institute of Biophysics, Chinese Academy of Sciences, for help in prediction of three-dimensional structure of PAF-AH.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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