Functional Relationships of the Sialyltransferases Involved in Expression of the Polysialic Acid Capsules of Escherichia coli K1 and K92 and Neisseria meningitidis Groups B or C*

Susan M. Steenbergen and Eric R. VimrDagger

From the Laboratory of Sialobiology, Department of Pathobiology, University of Illinois, Urbana, Illinois 61802

Received for publication, August 29, 2002, and in revised form, January 6, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Polysialic acid (PSA) capsules are cell-associated homopolymers of alpha 2,8-, alpha 2,9-, or alternating alpha 2,8/2,9-linked sialic acid residues that function as essential virulence factors in neuroinvasive diseases caused by certain strains of Escherichia coli and Neisseria meningitidis. PSA chains structurally identical to the bacterial alpha 2,8-linked capsular polysaccharides are also synthesized by the mammalian central nervous system, where they regulate neuronal function in association with the neural cell adhesion molecule (NCAM). Despite the structural identity between bacterial and NCAM PSAs, the respective polysialyltransferases (polySTs) responsible for polymerizing sialyl residues from donor CMP-sialic acid are not homologous glycosyltransferases. To better define the mechanism of capsule biosynthesis, we established the functional interchangeability of bacterial polySTs by complementation of a polymerase-deficient E. coli K1 mutant with the polyST genes from groups B or C N. meningitidis and the control E. coli K92 polymerase gene. The biochemical and immunochemical results demonstrated that linkage specificity is dictated solely by the source of the polymerase structural gene. To determine the molecular basis for linkage specificity, we created chimeras of the K1 and K92 polySTs by overlap extension PCR. Exchanging the first 52 N-terminal amino acids of the K1 NeuS with the C terminus of the K92 homologue did not alter specificity of the resulting chimera, whereas exchanging the first 85 or reciprocally exchanging the first 100 residues did. These results demonstrated that linkage specificity is dependent on residues located between positions 53 and 85 from the N terminus. Site-directed mutagenesis of the K92 polyST N terminus indicated that no single residue alteration was sufficient to affect specificity, consistent with the proposed function of this domain in orienting the acceptor. The combined results provide the first evidence for residues critical to acceptor binding and elongation in polysialyltransferase.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Homopolymers of N-acetylneuraminic acid (Neu5Ac,1 the most common sialic acid), also known as polysialic acids (PSAs), are essential virulence factors in neuroinvasive diseases caused by Escherichia coli K1 and K92 and certain strains of Neisseria meningitidis, Moraxella nonliquefaciens, and Mannheimia (Pasteurella) hemolytica (1). PSA also regulates cell-cell apposition when expressed in higher eukaryotes on the neural cell adhesion molecule (NCAM) (2). In E. coli K1 or N. meningitidis group B, the polymeric sialyl residues are connected by alpha 2,8 linkages, whereas group C N. meningitidis and E. coli K92 express PSA homopolymers with alpha 2,9 or alternating alpha 2,8/2,9 linkages, respectively (3, 4). Despite extensive investigation of PSA biosynthesis, including identification of the polySTs catalyzing the polymerization of sialyl residues, we do not know how PSA synthesis is initiated, how chain extension is terminated, or how polySTs interact with donor (CMP-Neu5Ac) and acceptor (nascent PSA) substrates. In contrast, all mammalian sialyltransferases investigated to date include a common primary structural motif (the L, or large sialyl motif consisting of 48 or 49 amino acid residues), which is thought to function in donor binding, and an S (small)-sialyl motif of 23 amino acid residues believed to bind both donor and acceptor substrates (5-7). Despite the structural identity between bacterial and mammalian PSA, the respective polySTs are not homologous glycosyltransferases, indicating the existence of at least two origins if not completely different mechanisms of sialyl homopolymer biosynthesis.

The biosynthesis of bacterial PSA is thought to take place on the inner surface of the cytoplasmic membrane through the polyST-catalyzed addition of Neu5Ac residues from CMP-Neu5Ac donor to the nonreducing ends of nascent (acceptor) PSA chains. The functions of most of the proteins required for biosynthesis (encoded by the neu genes) have been identified (8), including those needed for Neu5Ac synthesis, activation, and polymerization (see Fig. 1). Epistasis analysis and complementation experiments showed that polymerase activity is dependent on the structural integrity of the polysaccharide translocation and capsule assembly apparatus (9, 10), suggesting the multiprotein complex diagramed below in Fig. 1. Using an immunological approach, a similar conclusion was reached for biosynthesis of the K5 capsule (11, 12). Thus, K1 or K5 mutants with defects in conserved polypeptides encoded by the kps genes exhibit reduced rates of polymerization and accumulate intracellular (untranslocated) polysaccharides. Our working hypothesis, that polymerization is closely linked to translocation through protein-protein and protein-polysaccharide interactions, is supported by the apparent processivity of polyST and the loss or reduction of activity when polyST is expressed in the absence of other neu or kps gene products. Understanding how capsular polysaccharide production is regulated requires knowledge of polyST function.

To extend our analysis of polyST function, we carried out heterologous complementation experiments, establishing the interchangeability of polymerases for PSA expression in an E. coli K1 polyST-deficient recipient and providing direct evidence for the exclusive role of the polymerases in specifying all known PSA linkages. Then, on the basis of primary structural similarities, we created reciprocal chimeras between the K1 and K92 polySTs to identify the enzymic domains conferring linkage specificity. Site-directed alteration of selected K1 or K92 polyST residues provided additional functional information. The results extend our earlier conclusion that the K92 polyST is a bifunctional enzyme (13), demonstrate for the first time cross-species complementation of neuS, and establish the domain organization of the K1 and K92 enzymes responsible for specifying product linkage. These conclusions provide an experimental scaffold for future studies aimed at better understanding the functions of polySTs and the regulation of PSA biosynthesis in bacteria, neurons, and tumor cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains, Plasmids, and Growth Conditions-- Laboratory E. coli K-12 strain DH5alpha was used as the recipient for all molecular cloning of the various polyST structural genes. E. coli K1 hybrid strains EV136 (neuS::Tn10) and EV240 (neuS::Tn10 nanA4) are derivatives of the wild type strain EV36 (13, 14), whereas RS218 is a clinical K1 isolate kindly provided by Dr. R. Silver of the University of Rochester School of Medicine, Rochester, NY. These strains do not synthesize a PSA capsule due to insertional inactivation of the polyST structural gene, neuS, but retain all other genetic information for precursor biosynthesis, polymer assembly, and PSA translocation. The nonpolar nanA4 mutation inactivates sialate lyase (aldolase), thus the strain cannot degrade free sialic acid. EV239 is a neuB25 relative of EV240 and will not synthesize PSA even with neuS+ expressed in trans, unless provided with an exogenous source of free Neu5Ac, because of the loss of sialate synthase activity encoded by neuB (13). EV136 and EV240 synthesize PSA when neuS+ is provided in trans by transformation with either pSX92 or pSX305, encoding polySTs from E. coli K1 or K92 wild type strains (Fig. 2), respectively (13). EV138 harboring pLysS is a nanA4, neuB2 double mutant whose construction has been previously described (15). Plasmids pUE3 and pUE14 (Fig. 2) contain the synD (polyST) or synB (CMP-Neu5Ac synthetase) structural genes from group B N. meningitidis and were the kind gift of Matthias Frosch (16). The plasmid pSX306 harboring synE-encoding polyST from group C N. meningitidis was constructed from the strain Fam18 PCR product kindly provided by David Stephens (17), which was subsequently ligated into the Promega vector pGEM-T Easy following the manufacturer's suggestions for direct cloning of PCR amplicons (see Fig. 2). Unless indicated otherwise, all experiments were carried out with bacteria grown in Luria-Bertani medium with vigorous aeration at 37 °C. Minimal medium for reconstitution of PSA expression in strain EV138 was carried out as previously described (15). Plasmids were maintained by cultivating bacteria with 100 µg ml-1 ampicillin. Cells were harvested by centrifugation during the late exponential or early stationary growth phase. Immunochemical determination of capsule chemotype was accomplished by the halo test (13, 14) using H46 or alpha GpC antiserum kindly donated by Willie Vann. H46 is specific for a minimal epitope of approximately eight alpha 2,8-linked Neu5Ac residues, whereas alpha GpC recognizes the alpha 2,9 linkages in PSAs from E. coli K92 or group C meningococci. Bacterial colonies that synthesize and export capsular polysaccharides on agar plates supplemented with 10% (v/v) antiserum produce precipitin halos that are indicative of PSA linkage (chemotype). endo-N was purified from phage K1F lysates as previously described (18). All other chemicals were obtained from standard commercial sources and were of the highest available purity.

Sialyltransferase Assays-- Endogenous or exogenous polyST activity was determined in a total reaction volume of 15 µl in 10 mM Tris-HCl (pH 8.0), 5 mM magnesium acetate, and 5 mM dithiothreitol as described previously (9, 13, 14), except that CMP-[4-14C]Neu5Ac (55 mCi mmol-1) was purchased from American Radiolabeled Chemicals, Inc. Data were expressed as picomoles of Neu5Ac transferred per unit of time per milligram of protein. Transfer to exogenous acceptors was normalized per milligram of acceptor. Neu5Ac tritiated on carbon-6 was obtained from CMP-[6-3H]Neu5Ac (20 Ci mol-1) by acid hydrolysis as previously described (18).

Overlap Extension PCR-- Overlap extension PCR for the construction of genes encoding chimeric polypeptides was carried out as originally described (19). In two separate PCR amplifications, double-stranded fragments are generated and subsequently fused in PCR reaction 3. PCR reaction 1 uses a common 5'-far primer located 152 bases from the ATG start codons of either the K92 or K1 neuS genes (5'-GCCGCCAAATGTTAATGTTAGGAC-3'), and the unique reverse overlap primer (Table I) that carries information for constructing the intended chimera. In PCR reaction 2, a 3'-far primer located 175 bases from the stop codon of either the K92 or K1 neuS genes (5'-CGGTTTATTATGGGGGGAACACAA-3') and a primer that is the perfect complement of the overlap primer used in PCR reaction 1 (forward primers, Table I) are used to generate the second, or C-terminal fragment. When these two PCR products are mixed, denatured, and cooled the complementary overlaps anneal and the junction is extended by the template-dependent DNA polymerase. Finally, nested primers (5'-near and 3'-near) amplify the fragment bearing the chimeric construction and are cloned in pGEM-T Easy. The near primers are as follows: K1-5'-near, 5'-GGACTTTTGGAATTAAAAGATCTAC-3'; K92-5'-near, 5'-GGACTTTTGGAATTAAAAGATCGA-3'; K1-3'-near, 5'-CCATCCTCTTCAAAGAAAAGTAAC-3'; and K92-3'-near, 5'-GCATCCTCTTCAAAGAAA- AGTTAC-3'.


                              
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Table I
Specific primers used for chimeric enzyme synthesis and mutagenesis by overlap extension PCR

Sufficient flanking DNA sequence was available for constructions involving K1 neuS (accession number M76370), but there was insufficient 3'-flanking data to construct chimeras fusing the C terminus of K92 NeuS (accession number M88479) to the N terminus of the K1 enzyme. Therefore, we sequenced pSX305 with a primer (5'-ATTAGCATCATCTTCTTTGGT-3') identical to bp 1002-1022 of K92 neuS to yield the 529 bp of new sequence 3' to the K92 neuS stop codon. These new sequence data are deposited under accession number AF318310.

The conditions used for the first round of PCR were 35 cycles at 95 °C for 30 s interspersed at 60-65 °C for 30 s for annealing (this annealing temperature was optimized within the indicated range as determined individually for each primer set) and extension at 72 °C for 1 min. PCR reaction 3 was 35 cycles at 95 °C for 30 s, 65 °C for 30 s, and 72 °C for 90 s. All primers (synthesized and PAGE-purified by Integrated DNA Technologies, Inc.) were used at a concentration of 50 pmol reaction-1. Templates used for the first round of PCR were either the K1 (pSX92) or K92 (pSX305) neuS genes. In the second round of amplification, PCR reaction 3, 1 µl of each first round reaction was added as template. Taq polymerase was used in the PCR reactions for the initial set of chimeric polySTs. DNA sequencing of these constructs revealed unintended base changes introduced by the polymerase. Therefore, all subsequent amplifications were carried out using the PCR SuperMix High Fidelity purchased from Invitrogen. This PCR mix is a combination of the proofreading polymerase from Pyrococcus sp. GB-D and Taq DNA polymerase. After verification of product formation the PCR product was purified using the Wizard PCR Prep DNA purification system from Promega. After purification the products were A-tailed and cloned into the pGEM-T Easy Vector following the manufacturer's directions and transformed into DH5alpha for further analysis or storage. All constructs were sequenced using the pUC/M13 Forward and Reverse primers to verify the junction formed and to ensure no unplanned mutations were introduced during PCR amplifications. Any construct with an altered phenotype was completely sequenced on both strands using the previously named universal primers as well as three neuS-specific primers that allowed complete double-stranded sequencing in two to three more reactions. DNA sequencing was carried out at the University of Illinois Keck Center for Biotechnology using standard automated sequencing and data retrieval systems. The plasmids were transformed into EV240 to test for polyST activity.

Site-directed Mutagenesis-- Mutagenesis of the K1 or K92 neuS genes was performed either by overlap extension (20) or using the Altered Sites II in vitro Mutagenesis System from Promega, in which the BglII-BamHI fragments of pSX92 or pSX305 were cloned into the mutagenesis vector. The primers used for the mutagenesis reactions by overlap extension are listed in Table I. The other mutagenic primers with their respective plasmids are as follows: pSX624, 5'-TCTTGGCATTTCAGTGTTTTTTTCAGTTGCCAAGACGATAAGAAA-3'; pSX625, 5'-CCTACTAAATTTTTGTATAAATGACTCTGCTTTCTTTAATTGCCC-3'; pSX626, 5'-GACGATAAGAAAATTACTACGCTTACTAAATTTTTGTATAAAAAG-3'; where the underlined residues indicate the base(s) altered to make the desired mutant polySTs. All mutant neuS copies were sequenced as described above to ensure that only the intended alteration was introduced by the mutagenesis.

Product Analysis-- In addition to the immunological analysis of PSA chemotypes with linkage-specific antisera described above, sensitivity to K1-specific phage was used to confirm the synthesis of alpha 2,8 sialyl linkages by plaque assay. Strains synthesizing K1 or K92 capsules are sensitive to bacteriophage infection and produce a characteristic plaque phenotype (21). Where indicated, linkage assignments were confirmed by endo-N digestion and TLC analysis on silica gel G (Merck Research Laboratories) in an isopropanol, ammonium hydroxide, water (6:1:2) solvent system as previously described (18). Sialyl oligomers of defined length for use as chromatography standards were produced by partial acid hydrolysis of colominic acid as previously described (22). Oligomers were identified colorimetrically with orcinol or by autoradiography using a Kodak BioMax Transcreeen LE intensifying screen for 14C-labeled sialic acids or Dupont Cronex Quanta-III intensifying screen after autoradiographic enhancement with Fluoro-Hance (rpi Research Products, Mount Prospect, IL).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Exogenous Acceptor Activities of Heterologously Expressed PolySTs-- We previously showed that when neuS is expressed in an E. coli K-12 strain such as DH5alpha , a genetic background lacking the kps and neu genes for capsule biosynthesis (Fig. 1), polyST is unable to initiate PSA synthesis in vitro but transfers Neu5Ac from CMP-Neu5Ac to the exogenous acceptor colominic acid (13, 14). As expected from these previous results, we observed that neither K92 neuS nor synD or synE expressed a polyST that was capable of initiating PSA synthesis in the K-12 background when provided with an in vitro source of the nucleotide sugar donor, CMP-Neu5Ac (Table II). A control plasmid (pUE14) expressing the group B meningococcal CMP-Neu5Ac synthetase encoded by synB (Fig. 2), as expected, also had no polyST activity (Table II). In contrast, each of the recombinant polySTs expressed in E. coli K-12 transferred sialyl residues from CMP-Neu5Ac to both the cognate and heterologous exogenous acceptors, although with different relative efficiencies (Table II), indicating that at least for exogenous acceptors the mechanism of acceptor binding does not absolutely discriminate on the basis of sialyl linkage. Addition of monomeric Neu5Ac to the extracts as a potential exogenous acceptor did not result in polyST activities above the no-acceptor controls, indicating that none of the polySTs tested could use free sialic acid as exogenous acceptor. We concluded that all bacterial polySTs possess structurally similar acceptor binding sites. However, exogenous sialyl oligomers are poorly elongated (23-25), with most acting as acceptor for a single sialyl addition unless tethered to the membrane by a lipid anchor (26). Therefore, broadly extrapolating results derived from the use of exogenous acceptors to natural PSA elongation may be suspect to misinterpretation because of the failure to duplicate the in vivo process. To overcome the problems inherent with these in vitro systems we developed an in vivo complementation system that permits the detection of polyST activity under isogenic conditions.


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Fig. 1.   Proposed mechanism of PSA biosynthesis in E. coli K1. The inner (cytoplasmic) and outer membranes of E. coli are depicted with PSA (golden chain) in phosphodiester linkage to phospholipid in the outer leaflet of the outer membrane. The predominant outer membrane lipid A of lipopolysaccharide is indicated by the hexa-acylated GlcNAc disaccharide. CMP-Neu5Ac is synthesized in four steps from the pool of UDP-GlcNAc and the polymerized homopolymer translocated to the outer membrane through a multiprotein channel spanning the periplasmic space; the lipidation reaction is not indicated. Numbers refer to the following polypeptides in E. coli K1 with their meningococcal orthologues given in parentheses: 1, Omp, unknown outer membrane protein (CtrA); 2, KpsD (none); 3, KpsE (CtrB); 4, KpsM (CtrC); 5; KpsT (CtrD); 6, NeuE (none); 7, NeuS (SynD or E); 8, KpsF (KpsF homologue); 9, NeuA (SynB, SiaB); 10, NeuB (SynC, SiaC); 11, NeuC (SynX, SiaA); 12, NeuD (none); 13, KpsC (LipA); and 14, KpsS (LipB).


                              
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Table II
Exogenous polyST activity expressed in E. coli K-12


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Fig. 2.   Plasmids used in this study. The genetic maps of the E. coli or N. meningitidis capsular biosynthetic loci (open boxes) are not drawn to scale. A, the three regions that include the kps or neu genes of E. coli K1 and K92 are shown with transcriptional directions indicated by arrows. Region 2 is expanded to indicate the individual biosynthetic neu genes comprising this region. B, the meningococcal DNA required for groups B or C capsule production is comprised of five regions, with only the directions of transcription of Regions A and C indicated by arrows. The synXBCD (siaABCD) or E gene products are homologous with E. coli K1 NeuC, -A, -B, and -S, respectively, whereas ctr gene products are homologous with Kps polypeptides (see Fig. 1). Plasmids pSX305 and pSX92 harbor the cloned neuS genes from E. coli K92 or K1, respectively, whereas pUE3 and pSX306 harbor synD or E from groups B and C meningococci, respectively. Plasmid pUE14 harbors the meningococcal group B homologue (synB) of neuA, encoding CMP-Neu5Ac synthetase. B, BamHI; Bg, BglII; E, EcoRI restriction endonuclease sties.

Detection of PolyST Activity by Complementation Analysis-- PolyST activity can be detected by expressing an exogenous polymerase gene in trans to an E. coli K1 genetic background lacking endogenous polymerase due to transposon (insertional) inactivation of neuS (13, 14). Similarly, a double mutant with defects in neuB and nanA is dependent on an exogenous source of free Neu5Ac for PSA biosynthesis due to lack of an active synthase encoded by neuB (15). Fig. 3 shows the time course of PSA synthesis when strain EV138 (nanA4 neuB25) is continuously pulsed with exogenous radiolabeled Neu5Ac. The sialic acid internalized by the sialate permease encoded by nanT is spared from the catabolic pathway by inactivation of sialate lyase (nanA) and thus can only participate as an intermediate in PSA biosynthesis (27, 28). The internalized Neu5Ac is activated for glycosyltransfer by the synthetase encoded by neuA, which couples Neu5Ac with CTP to produce intracellular CMP-Neu5Ac detectable by 5 min after beginning the pulse (Fig. 3). PSA steadily accumulated at the origin of the chromatogram without observable intermediates. The solvent system used for the TLC analysis separates Neu5Ac monomers through oligomers of about 10 sialyl units in length. The absence of detectable oligomeric sialic acid in vivo is consistent with the processive activity of polyST inferred previously from in vitro results (23-25). The simplest interpretation of the current results is that PSA is elongated processively in vivo by a single polyST before chain termination by an unknown mechanism.


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Fig. 3.   Reconstitution of PSA biosynthesis. Strain EV138 harboring pLysS was grown in minimal medium and labeled with tritiated Neu5Ac as described under "Experimental Procedures." Samples of the culture were taken at the indicated times for analysis of the accumulation of free Neu5Ac, CMP-Neu5Ac, or PSA with reference to the authentic standards shown at right. The identity as PSA of the material that accumulated at the origin of the chromatogram was confirmed in a separate control (not shown) by depolymerization after endo-N pretreatment.

To determine if all extant bacterial PSA chemotypes were dependent on the genetic origin of their cognate polymerases, we carried out complementation experiments with the neuS homologues synD or synE from N. meningitidis groups B and C, respectively (Fig. 2). The immunoreactivities of the resulting polysaccharide products confirmed that polyST linkage specificity is retained when these enzymes catalyzed PSA synthesis in the heterologous mutant K1 background (Table III). Structural linkage assignments were confirmed by plaque assay after infection with K1F or PK1E phage. As expected, all strains expressing PSA capsules, except those complemented by synE, were sensitive to infection and lysis. Complementation with the meningococcal-derived polyST structural genes synD and synE indicates that the K1 assembly and translocation apparatus recognizes PSAs synthesized by heterologously expressed polymerases. We assume that the qualitatively greater PSA production in the EV240 background compared with that of EV136 reflects the genetically engineered absence of an active aldolase (sialic acid lyase) in strain EV240, which spares biosynthesized Neu5Ac from depletion by the catabolic pathway encoded by nanATEK (27, 28). On the basis of the complementation results shown in Table III we chose EV240 as the strain for all subsequent analyses of polyST function.


                              
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Table III
Complementation analysis of polySTs expressed in different polymerase-deficient mutant backgrounds

Structural Analysis of E. coli K92 PSA-- With the complementation system provided by strain EV240 we were able to infer the expression of the alternating alpha 2,8/2,9 PSA synthesized by K92 neuS by its immunochemical cross-reactivity against alpha GpC antiserum and susceptibility to digestion with endo-N, which is specific for PSA oligomers with at least eight alpha 2,8 linkages (18, 22). This conclusion was independently supported by proton NMR spectroscopic analysis of the heterologously synthesized K92 PSA capsular polysaccharide expressed in EV240 by complementation with cloned K92 neuS (29). However, without periodate oxidation or some other method that is capable of directly demonstrating the alternating linkages of K92-derived oligosialic acids, NMR analysis simply indicates the simultaneous presence of both alpha 2,8 and alpha 2,9 linkages, not necessarily the order in which these linkages occur in a given PSA chain. To overcome this limitation of NMR analysis, we developed a simple method for determining the structure of the K92 PSA that avoids lengthy polysaccharide isolation techniques or chemical treatments.

Fig. 4 shows the repeat structures of PSAs from E. coli K1 and group B meningococci, E. coli K92, and group C meningococci. The alpha 2,9 linkages of group C PSA (Fig. 4C) are resistant to digestion with the endosialidase from the K1-specific phage as reflected by the resistance of EV240 expressing the group C capsular PSA to phage infection. In contrast, PSA chains containing alpha 2,8 linkages (Fig. 4, A and B) are susceptible to endo-N digestion (13) and strains expressing these capsules are sensitive to K1F infection (Table II). Digestion of K1 PSA with endo-N produces sialyl oligomers primarily ranging in length from trimers to heptamers (18), whereas prolonged incubation at high endo-N to substrate ratios produces mostly monomers and dimers (30). In contrast, digestion of K92 PSA with endo-N is predicted to yield oligomers with an even number of sialyl residues because of the alternating depolymerase-resistant alpha 2,9 linkages. As shown in Fig. 5 (lane 1), TLC analysis of K92 digestion products revealed the expected product ladder consistent with dimers, tetramers, hexamers, and octamers relative to the dimeric, trimeric, tetrameric, and pentameric standards (Fig. 5, lanes 4-7). Because separation of dimers from tetramers was the most pronounced relative mobility difference in the chromatograms, we looked for the presence of timers at very high sample concentrations (Fig. 5, lanes 2 and 3). The absence of trimers in the limit K92 PSA digest shows it is composed solely of alternating alpha 2,8/2,9 linkages (Fig. 4B). This conclusion is consistent with the fractionation of K92 oligosialic acids observed after depolymerization with intact phage and size exclusion chromatography of the digestion products (31). The results of structural analysis with endo-N indicate that the immunochemical responses of structurally distinct PSAs to linkage specific antisera and sensitivity or resistance to K1-specific phage are sufficient criteria for establishing PSA linkage. Our method obviates the need to purify PSAs and the ambiguities of NMR spectroscopy noted above for the structural analysis.


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Fig. 4.   Repeat structures of PSA homopolymers. A, poly-alpha 2,8-linked sialic acid; B, poly-alpha 2,8/2,9-linked sialic acid; C, poly-alpha 2,9-linked sialic acid. Homopolymer repeat units are shown in brackets. Gentle purification of PSAs from bacterial sources generally yields polymers where n = 100-200 sialyl residues (35).


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Fig. 5.   Structural analysis of K92 PSA. Purified K92 polysaccharide was digested exhaustively for 16 h at 30 °C with endo-N purified from bacteriophage K1F. The amount of endo-N used was sufficient to degrade an equivalent sample of colominic acid to oligomers ranging in size from three to seven sialyl residues in 3 h. Samples of the K92 limit digest were spotted for thin layer chromatographic analysis and detection of sialyl oligomeric breakdown products with the orcinol reagent. Lane 1, 50 µg; lane 2, 150 µg; lane 3, 450 µg. Lanes 4-7 are oligosialic acid markers representing dimers, trimers, tetramers, and pentamers, respectively, derived from colominic acid, where numbers to the right of each band is the degree of polymerization of the oligomer. Note the absence of odd-numbered oligomers in the K92 digest indicated by numbers at the left.

Structural Basis for PolyST Linkage Specificity-- Fig. 6 shows the dendrogram derived from multiple alignment of bacterial polySTs, monosialyltransferases from Campylobacter jejuni, Haemophilus influenzae, N. meningitidis, and selected human sialyltransferases, including one polyST that sialylates NCAM. It is clear that the bacterial polySTs form a separate clade that is separate from both the other bacterial and the human sialyltransferases. Multiple alignment of the bacterial polySTs indicates the shared primary structures of family members and the very high (83%) identity between the two E. coli NeuS homologues (Fig. 7). The results shown in Figs. 6 and 7 indicate that the bacterial polySTs are derived from a common ancestor. This ancestral sialyltransferase may also exist for the bacterial monosialyltransferases. In contrast, bacterial polySTs are no more closely related to the human polyST than they are to the other human sialyltransferases shown in Fig. 6. The high similarity between K1 and K92 polySTs suggested it should be possible to determine the salient features of enzyme structure that account for linkage specificity. Although we considered using site-directed mutagenesis to sequentially alter the unconserved amino acid residues in K92 NeuS in expectation of generating a mutant with the specificity of the K1 enzyme, this approach did not seem feasible due to the lack of structural and functional information on polySTs. Instead, we decided to construct polypeptide chimeras of reciprocally exchanged K1 and K92 polyST domains and then screen these hybrid polySTs for altered specificity by complementation analysis. This approach was predicated on the assumption that the alteration of structural elements or domains of the polySTs, rather than individual amino acid residues, would be most likely to provide information relevant to linkage specificity.


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Fig. 6.   Dendrogram of selected bacterial and human sialyltransferases. The primary structures of the indicated sialyltransferases were aligned using the ClustalW program from which the unrooted dendrogram was derived using Draw Tree in the PHYLIP (Phylogeny Inference Package) suite of Biology WorkBench programs available at workbench.sdsc.edu. The following sialyltransferases were investigated, with accession numbers given in parentheses: SynE (AAB53842), SynD (S60761), K92 NeuS (AAA24215), K1 NeuS (AAA24213), C. jejuni bifunctional sialyltransferase (AAL06004), N. meningitidis lipooligosaccharide sialyltransferase (Q9JUV5), Haemophilus ducreyi lipooligosaccharide sialyltransferase (AAD28703), ST3GalVI (NP006091), ST3GalV (NM003896), human polyST (Q92187), ST3GalII (Q9H4F1), sialyltransferase 4A (NM152996), ST6GalNAcVI (NM013443), and ST6GalNAcIV (AF162789.2).


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Fig. 7.   Multiple alignment of polySTs from E. coli and meningococci. E. coli K1 NeuS (accession number M76370), E. coli K92 NeuS (accession number M88479), group B meningococcus SynD (accession number AF432602), and group C meningococcal SynE (accession number U75650) primary structures were aligned using default parameters of MacVector version 7.0 software. Numbering is from the N terminus of K1 NeuS, with shared residues in shaded boxes. The consensus primary structure is given in the fifth line where a dot indicates a presumed conservative change. Asterisks indicate residues in either the K1 or K92 NeuS altered by site-directed mutagenesis as defined in the text. Note that, due to gaps introduced by the multiple alignment program, allotted residue numbers may not conform exactly with the text.

Construction and Analysis of Chimeric PolySTs-- We considered three possible phenotypes for chimeras generated between the K1 and K92 polySTs: (i) linkage specificity remains unchanged, (ii) linkage specificity is reciprocally altered, or (iii) polyST does not tolerate the structural changes and activity is lost. Fig. 8 shows that the first two chimeras we constructed, fusing the N-terminal 100 amino acid residues of K1 NeuS to the 309 C-terminal residues of the K92 homologue, expressed polySTs that directed synthesis of PSAs with immunoreactivity to the poly-alpha 2,8-specific antibody H46, suggesting that linkage specificity depends on the N-terminal domain. However, the error-prone nature of Taq likely produced unintended changes, as shown for pSX611 and pSX612 (Fig. 8). Therefore, we could not unambiguously conclude that the observed specificity change was due to the exchanged domains. Similarly, the reciprocal Taq-generated amplicon that was intended to fuse the N terminus of K92 NeuS with the C-terminal 309 K1 NeuS residues bore a chain terminating mutation predicted to encode a truncated chimeric polyST of 293 residues (pSX613), which as expected had no activity (Fig. 8). To overcome the introduction of random mutations, probably exacerbated by the three separate PCR reactions needed for chimera synthesis, we carried out the overlap extensions with a combination of Taq for efficiency and Pyrococcus sp. GB-D polymerase for its proofreading function (see "Experimental Procedures"). All subsequent chimeras were generated with this polymerase mixture.


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Fig. 8.   Structures and phenotypes of chimeric NeuS polypeptides. Plasmids expressing the wild type K1 (open box) or K92 (shaded box) NeuS polypeptides and their immunochemical phenotypes, which are indicative of the respective alpha 2,8- or repeating alpha 2,8/2,9-linked polysialic acids, compared with pSX92 and pSX305 are the wild type constructs. The scaler at the bottom of the diagram indicates the number of amino acid residues from the N terminus. Alterations caused by spurious missence or chain-terminating mutations in three of the chimeras are indicated. A plus sign indicates a positive halo response, whereas a dash indicates no immunoreactivity was detected. All plasmids were transformed into strain EV240 prior to halo testing. Results were confirmed independently at least twice for each construct. Endogenous in vitro polyST enzyme assays and sensitivity of the products to endo-N confirmed the synthesis of PSA chains (not shown). Note that the same plasmids expressed in the neuB-deficient derivative (EV239) of EV240 did not synthesize PSA, demonstrating the expected dependence of the halo response on Neu5Ac biosynthesis.

When we constructed a fusion of the K1 NeuS N terminus to the K92 C terminus that lacked any randomly introduced mutations (pSX614), the results were similar to those obtained with pSX611 or pSX612, indicating that the unintended mutations in these chimeras were phenotypically silent, and that in three independent chimeras the critical structural information for linkage specificity resides in the N-terminal polyST domain. This conclusion was confirmed by the construction of the reciprocal chimera (pSX615) expressing a polyST with K92 specificity (Fig. 8). We concluded that residues located in the first N-terminal 100 amino acids of K1 and K92 are sufficient to confer linkage specificity. Thus our initial approach to construct chimeras with successive additions of 100-residue modules resulted in detection of a structural element responsible for linkage specificity upon the first domain swap. The truncated polyST predicted by the sequence of the chimeric neuS expressed by pSX613 provided an additional control demonstrating that capsule expression is solely dependent on the in trans expression of neuS in EV240.

To confirm the specificity change in the polyST chimera encoded by pSX614, we carried out structural analysis using endo-N digestion after first labeling endogenous PSA in vitro by addition of radiolabeled CMP-Neu5Ac to isolated membranes. As shown in Fig. 9 (lane 2), the wild type K92 polyST expressed by pSX305 produced a PSA product in EV240 that did not yield even-numbered sialyl oligomers after endo-N digestion. As expected, the autoradiograph of lane 2 (Fig. 9, lane 4) was congruent with the chromogenic oligomer pattern and thus the K92 PSA structure shown in Fig. 4B. In contrast, PSA synthesized by the K1 NeuS100/K92 NeuS309 chimeric polyST expressed by pSX614 produced both the even- and odd-numbered ladders of sialyl oligomers (Fig. 9, lanes 3 and 5) expected of the K1 capsular PSA structure shown in Fig. 4A. The radiolabeled bands migrating with the mobility of 2.5-mers shown in Fig. 9 (lanes 4 and 5) are unused CMP-Neu5Ac substrate (see Fig. 3).


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Fig. 9.   Structural analysis of PSAs synthesized by EV240 harboring pSX305 or pSX614. Membranes were prepared and labeled by exposure to CMP-[14C]Neu5Ac and treated with endo-N. Lane 1, mixture of sialyl oligomers with their degree of polymerization shown by numbers at the left; lane 2, digest of membranes from EV240/pSX305; lane 3, digest of membranes from EV240/pSX614; lane 4, autoradiogram of lane 2; autoradiogram of lane 3. Oligomers in lanes 1-3 were detected with the orcinol colorimetric reagent. Numbers at the right indicate the degree of polymerization of the endo-N digestion products, whereas the asterisk indicates the relative mobility of CMP-Neu5Ac substrate.

To more precisely delineate the structural basis for K92 NeuS linkage specificity, we constructed chimeras (pSX621 and pSX622) with N-terminal K1 NeuS domains of 85 or 52 amino acid residues fused to the appropriate C terminus of the K92 enzyme. As shown in Fig. 8, residues crucial for linkage specificity were located between positions 53 and 85, because polyST specificity was altered in pSX621 but not pSX622. This conclusion was supported by the lack of an effect on linkage specificity of the unconservative H52N change in pSX623 produced by site-directed mutagenesis (Table IV). Fig. 7 shows that the region between residues 53 and 85 includes 5/33 differences between K92 and K1 NeuS, three of which (I63V, R68K, and F72Y) appear to be conservative changes. The two remaining differences between K92 and K1 polyST in this region include a hydrophobic to polar substitution (L61S) and a change from a positive to negatively charged residue (K79E).


                              
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Table IV
Polysialic acid expression in mutants with altered K1 or K92 polySTs

Site-directed Mutagenesis of the PolyST N Terminus-- The phenotypes of the chimeric polySTs shown in Fig. 8 suggested two not necessarily mutually exclusive hypotheses. First, the N terminus of the K92 polyST includes a second CMP-Neu5Ac binding site that donates Neu5Ac to an alpha 2,8-linked acceptor to produce the alternating structure of the K92 PSA upon sequential repetition of the catalytic cycle. This dual-donor site hypothesis is supported by a model of processive glycosyltransferases in which separate sugar-nucleotide binding sites were postulated to account for synthesis of polymers with alternating linkages (32). In the alternative hypothesis the N terminus of both K1 and K92 polyST includes at least part of the acceptor binding site, in which the orientation of nascent PSA dictates linkage specificity together with a single, distally located donor site. We used site-directed mutagenesis of selected K92 NeuS residues in an attempt to distinguish between these two hypotheses.

The conservation of positively charged residues in the polyST family suggests a function in binding negatively charged substrates (Fig. 7). Although lysine and arginine are not necessarily functionally interchangeable, the R86K alteration in the mutant K92 NeuS (pSX616) retained linkage specificity (Table IV), indicating that the difference between K1 (lysine) and K92 (arginine) NeuS at this position does not influence the ability of the K92 polyST to catalyze the alpha 2,9 linkage. Similarly, the two unconservative alterations made as controls in the K92 NeuS C terminus (pSX617 and pXS618) did not affect specificity of the respective mutant polySTs (Table IV), indicating that residues 225 and 276 are not involved in catalysis or required for proper enzyme folding, because the mutant enzymes retained full catalytic activity (Fig. 10). When the lysine residue at position 94 in the K92 NeuS was changed to asparagine (pSX619) to mimic the corresponding K1 NeuS residue at this position, polyST activity was unaffected suggesting no contribution of lysine 94 to catalysis or folding of the K92 polymerase (Table IV). The reciprocal change made in pSX620 (N94K) likewise had no effect on polymerization (Table IV), confirming that a seemingly major difference between the K1 and K92 polySTs involving positively charged residues was unimportant to linkage. Thus the presence of a greater number of positively charged N-terminal amino acid residues in K92 neuS over its K1 homologue might not be crucial to the bifunctional activity of the K92 polyST. Consistent with this speculation, a change in lysine to glutamate at position 79 of the K92 polyST had no effect on phenotype (Table IV). The accuracy of alpha GpC antibody to distinguish wild type from null mutant phenotypes is demonstrated by Fig. 10, in which the halos produced by five different point mutations are indistinguishable from each other, whereas no halo was visible with the control chimera expressed by pSX627.


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Fig. 10.   Immunochemical detection of PSA. Bacteria were inoculated into nutrient agar containing either H.46 or alpha GpC antiserum. PSA synthesized by the bacteria is sloughed continually form the cell surface creating an expanding "halo" that represents the antigen-antibody precipitates, which are directly proportional to the amount of PSA synthesized by a given strain (21). A, results of PSA immunodetection with H.46 antiserum. Colony 1, EV240; colony 2, EV36 (wild type parent of EV240); and colony 3, RS218 (clinical K1 isolate). Note the expected absence of a halo by strain EV240. Neither EV36 nor RS218 produced a halo when tested against alpha GpC antiserum (not shown). B, results of PSA immunodetection using alpha GpC antiserum. Colony 1, EV240/pSX616; colony 2, EV240/pSX617; colony 3, EV240/pSX618; colony 4, EV240/pSX613; and colony 5, EV240/pSX625.

To further investigate the interchangeability of lysine and arginine residues, the R68K alteration of K92 polyST (pSX626) was constructed, again with no effect on phenotype (Table IV). The remaining unconservative difference was at position 61, where the K92 polymerase has a leucine residue in place of the serine residue at this position in K1 NeuS. The L61S alteration in K92 polyST (pSX625) also had no effect on activity (Table IV), suggesting that leucine at his position in the K92 polyST is not critical to function. Therefore, when the results of site-directed mutagenesis are considered together with the results of chimeric polyST analysis (Fig. 4), the simplest interpretation is that the N-terminal domain, especially residues 53-85, functions in positioning the PSA chain during elongation, suggestive of a functional similarity with the eukaryotic sialyltransferase S motif. The alternative hypothesis, that the N terminus includes a second donor binding site, is less likely because point mutations would have been expected to reduce catalytic efficiency.

Conclusions-- Altogether our results are consistent with a model of polyST in which the orientation of the acceptor nonreducing terminus alternates between successive nucleophilic attack by the C-8 or C-9 hydroxyl groups on bound donor CMP-Neu5Ac to yield the K92 repeat structure. If this model is correct, either the enzyme or acceptor may need to rotate during catalysis, as has been suggested for other enzymes that synthesize products with alternating linkage using a single catalytic center (33). However, other studies suggest that acceptor or donor sites may fine-tune each other through local polypeptide loop movements during each catalytic cycle (3), offering the potential flexibility to explain the dual linkage specificity of the K92 polyST without invoking any special chemical property. Although an appropriate crystallographic structure of a polymerizing glycosyltransferase should prove helpful to a better understanding of these enzymes, we suggest that the polyST family is already established for investigating the specific class of inverting CMP-sialic acid-dependent polymerases (making alpha -bonds from beta -linked donor) that figure prominently in microbial pathogenesis, nervous system development, and tumorigenesis (35).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of a polyST domain with a probable function in acceptor orientation was dependent on successful adaptation of overlap extension PCR to the bacterial polyST family of glycosyltransferases. To our knowledge, the results represent the fist successful application of this procedure to the analysis of sialyltransferase structure and function. Success using this method requires several rounds of PCR and thus robust polymerase activity typically provided by Taq. However, Taq error frequency is ~2.5 × 10-4 (36) and the length of neuS (1227 bp) suggested that one unintended mutation might occur in each chimera constructed, which is the result we observed (Fig. 8). Although two of three unintended mutations were subsequently found to be phenotypically silent (pSX611 and pSX612) and thus provided independent confirmation of linkage specificity, our results indicate that studies where just Taq is used for chimera production and only the fusion junction is verified by DNA sequencing may be compromised by extraneous mutations. We controlled for unintended mutations by complete double-stranded DNA sequencing and a mixture of Taq for efficiency and a proofreading polymerase for specificity. Gel-purified primers were also used to avoid gap artifacts resulting from deletions in primer 3' termini that are present in unpurified primers. Our results demonstrate that with suitable precautions overlap extension PCR is a useful procedure for the analysis of glycosyltransferase domain organization.

The capsular polysaccharides synthesized by the polySTs investigated in this communication are critical virulence determinants in extramucosal (invasive) diseases caused by E. coli K1 and K92 and groups B or C N. meningitidis. E. coli K1 is responsible for almost all cases of Gram-negative neonatal meningitis in the United States and is the predominant cause of cystitis in North American women (37), whereas groups B and C meningococci cause most cases of meningitis in children and young adults (38). Despite extensive research the molecular mimicry of mammalian PSA by the E. coli K1 and group B meningococcal capsules has complicated efforts to produce safe and effective anti-capsular polysaccharide vaccines and research in this area has shifted to outer membrane protein immunogens (38). Understanding how bacterial polySTs synthesize PSAs with different linkages, and how these enzymes differ with respect to their functional mammalian counterparts could suggest development of a new class of antimicrobial agents with targeted yet broad therapeutic value. The comparative aspects of the K92 polyST makes it a useful model for addressing the structure and function of the bacterial PSA polymerases, and it is this fact together with the potential health benefit indicated above that has resulted in this enzyme being the focus of several recent biochemical investigations (25, 29, 39).

Chao et al. (25) suggested that there were most likely two enzymes involved in the K92 biosynthetic process: one to produce an alpha 2,9-linked disaccharide and another linking these units at a site favoring alpha 2,8 addition. McGowen et al. (29) reinvestigated the exogenous substrate specificity of K92 polyST using the EV240 system described in this communication, providing structural (NMR) evidence that a single K92 polyST catalyzes the synthesis of both sialyl linkages. However, NMR analysis merely establishes the simultaneous presence of alpha 2,8 and alpha 2,9 linkages, not necessarily the order of these linkages in a given PSA chain. Our results (Figs. 5 and 9) provide unambiguous evidence for the synthesis of alternating linkages by K92 neuS.

Our previous genetic, immunological, and biochemical data on the K92 polyST (13) did not support the two-enzyme hypothesis (25) but could not distinguish between a single polyST with one or more catalytic centers (29). Experimental support for the two-enzyme model (25) was the reported failure of the K92 polyST to transfer sialyl units from CMP-Neu5Ac to an alpha 2,9 terminus, which is clearly not the case (Table II and see Ref. 29). Our results demonstrate that a family of homologous polySTs (Fig. 6) is both necessary and sufficient for synthesis of alpha 2,8-, alpha 2,9-, and repeating alpha 2,8/2,9-sialyl homopolymers, thus representing all extant PSA chemotypes detected in bacteria, in neurons, and in cells expressing the polysialylated oncofetal NCAM antigen (35). Therefore, the linkage specificities of the various glycosyltransferases in the bacterial polyST family are dictated solely by the source of the polymerase structural gene. As shown in Fig. 7, the molecular basis for the observed linkage specificities of these glycosyltransferases could not be readily inferred from the derived polyST primary structures. Another approach was needed to further investigate the functional relationships between family members.

In contrast to previously published work on K1 and K92 polySTs, in which trimers were found to be the minimal exogenous acceptors (23-25), Shen et al. (39) concluded that K92 NeuS preferentially recognized monomeric Neu5Ac as exogenous acceptor. We have no explanation for this discrepancy, and our current results do not support this conclusion, but in further contrast to the in vitro system used by Shen et al. (39) our complementation system synthesizes authentic polysaccharides using endogenous instead of exogenous acceptors. The in vivo system we developed is especially suited for investigating the structural basis for K92 polyST linkage specificity under isogenic conditions (Tables II and III). Other discrepancies between Shen et al. (39) and previous observations (13) have been discussed (29).

Comparison of polyST primary structures reveals 92/406 (22.4%) residues shared by all current family members (Fig. 7). A striking feature of this family of glycosyltransferases is the high proportion of positively charged lysine and arginine residues contributing to the overall basic isoelectric points predicted for the various polySTs and the lack of obvious membrane-spanning regions. Despite the lack of extended hydrophobic regions, polyST is firmly membrane-associated (13). However, it has resisted all attempts at purification in an active form, because, unlike mammalian polyST (40), the bacterial enzyme loses endogenous and exogenous activity upon detergent solubilization.2 Failure to purify active polyST makes it difficult to distinguish between the effects of site-directed mutations on catalysis as opposed to enzyme folding or stability. We overcame this experimental deficiency by focusing on altered specificity instead of reduction or loss of catalytic activity. By concentrating on altered instead of loss or reduced function, we were able to compare the biosynthetic capabilities of chimeric K1 or K92 polySTs in which the relative lengths of reciprocally exchanged N termini were varied under controlled experimental conditions (Fig. 8). The PSAs synthesized by these chimeras were readily detected using a simple immunochemical screen that was confirmed by endo-N susceptibility of the products. We found that the first 85-100 residues of the K1 or K92 polySTs were sufficient to alter specificity thus interconverting the ability of each enzyme to synthesize the alpha 2,8- or alpha 2,8/2,9-linked homopolymers. These experimental observations formally mapped the polyST domains necessary for linkage specificity to between residues 53 and 85 from the N terminus. Our general approach also should be applicable to the analysis of the group C meningococcal polyST, which synthesizes an alpha 2,9 PSA.

By analogy to a recently proposed polymerization model of processive glycosyltransferases (32), McGowen et al. (29) favored a three-site model for K92 polyST in which sites 1 and 2 bind CMP-Neu5Ac and site 3 the nascent PSA acceptor, with a strong preference for an alpha 2,8 linkage at the nonreducing end (Fig. 11A). This model is consistent with the known direction of polymerization (elongation) by addition to the nonreducing terminus, the measured acceptor preferences, and the processive nature of the PSA elongation process (14). Our results (Fig. 8) are incompatible with this model unless the linkage specificity of the first two sites is reversed (Fig. 11B), which is inconsistent with the acceptor data (25, 29).


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Fig. 11.   Models of K92 PSA biosynthesis. A and B show three-site models in which the order of glycosyltransfer differs as indicated by the arrows. C shows the favored two-site model with a single alternating acceptor site and one donor binding site.

Indirect evidence against the 3-site models depicted in Fig. 11 (A and B), is the lack of obvious repeats in the K92 polyST primary structure. If the N terminus of the K92 polymerase included a second donor site, we would expect a repeated motif indicative of conserved residues in the nucleotide sugar binding domains. This expectation follows from the known inhibition of polyST by CMP (41) but not free sialic acid (23, and this study), suggesting that the one or more donor sites primarily recognize the CMP moiety instead of Neu5Ac of the CMP-Neu5Ac donor and thus should be structurally similar, as observed in the bifunctional hyaluronan synthase with two known donor sites (42). Although the bacterial polyST family and all other bacterial sialyltransferases investigated to date lack the sialyl L motif for donor binding found in mammalian sialyltransferases (5), residues 164-167 resemble the so-called DXD motif that has been shown to orient the donor nucleotide sugar in some inverting and retaining glycosyltransferases (34). However, a dominant feature of the L and S motifs are conserved cysteine residues cross-linking these sites (43); conserved cysteine residues are lacking in the bacterial polyST family (Fig. 7). Indeed, disulfide-reducing agents inhibit the mammalian monosialyltransferases (43), whereas they either weakly stimulate or have no effect on the bacterial polySTs, indicating the possibility of distinctly different architectures for substrate binding. Interestingly, mammalian polyST is not inhibited by disulfide-reducing agents (40), suggesting a possible similarity between bacterial and mammalian PSA biosynthetic mechanisms despite their lack of obvious primary structural homology.

Additional indirect evidence against the three-site model is the crystallographic data on UDP-sugar-dependent beta -glycosyltransferases, which show the requirement for at least four conserved aspartate or glutamate residues per catalytic center. Three carboxylates are needed for UDP-sugar coordination, and a fourth acts as the catalytic base in the acceptor domain (34). As shown in Fig. 3, the polyST family includes nine conserved aspartate or glutamate residues, but none are closer than 163 residues to the N terminus of any enzyme in the family. Furthermore, because these residues are conserved in all family members, they cannot be crucial for specifying linkage. We conclude that the N terminus of K92 NeuS, which our data indicate defines at least a portion of the acceptor binding site, does not include residues that participate in donor orientation or as the catalytic base(s). We are currently altering conserved aspartate or glutamate residues with the expectation of mapping those directly involved in catalysis and donor binding.

Direct evidence against the three-site model comes from site-directed mutagenesis experiments in which no single mutation was found to alter specificity of the K92 polyST (Table IV and Fig. 10). If any of these individual mutations affected donor binding, we would have expected to have observed altered specificity or loss of activity. Instead, the retention of specificity and full catalytic activity of the mutant polySTs suggests that the K92 N terminus defines an acceptor site in which the association of this domain with the catalytic nucleophile defines the type of sialyl linkage produced during the elongation process (Fig. 11C). Our current results provide the first direct evidence for an acceptor binding site in bacterial sialyltransferases.

Although we cannot be certain that the K92 neuS evolved from its K1 homologue, this conclusion seems likely given the conservation of serine at position 61 in K1 NeuS and the meningococcal polySTs (Fig. 7). E. coli K92 is a much less prevalent cause of invasive disease than E. coli K1, indicating that virulence factors other than capsules are required for systemic infection (44). However, it is difficult to see how an increased systemic disease potential would benefit the pathogen's reproductive success, suggesting that capsule glycosyltransferases evolved under selective pressures unrelated to host physiology, with capsules only occasionally and indirectly functioning as virulence determinants. We (8) and others (45) have speculated that these evolutionary pressures may derive from avoidance of environmental factors such as bacteriophage that use surface polysaccharides and proteins as receptors. Regardless of the exact evolutionary pathways taken, our results indicate that relatively few changes are necessary for the interconversion of polyST linkage specificities, which is consistent with the apparent phylogeny of these glycosyltransferases (Fig. 6).

Finally, there are two immediately obvious applications of our complementation system. The first is to exploit the translocation machinery of strain EV240 by expressing glycosyltransferases that synthesize polysaccharides with sugars unrelated to sialic acid. Such designer E. coli with potentially novel carbohydrate-coated surfaces might be useful for new vaccine development. Indeed, EV240 synthesizing the group C meningococcal capsule by expression of the heterologous polyST structural gene synE already represents an alternative to N. meningitidis as a new source of the alpha 2,9-linked PSA, which is currently the starting point for vaccination against epidemic meningitis outbreaks caused by this serotype (37). Second, monitoring PSA biosynthesis in vivo (Fig. 3) offers a convenient method for assessing the effectiveness of low molecular weight inhibitors of polySTs. Specific inhibitors of polySTs may have wide utility as chemotherapeutic agents in treating systemic disease and, possibly, in modulating the tumorigenicity of cells expressing the oncofetal form of NCAM.

    ACKNOWLEDGEMENTS

We thank Willie Vann for the kind gifts of Bos-12 and GpC exogenous acceptors and antisera used in this study, for his encouragement of the work while it was in progress, and his helpful critique of the manuscript prior to submission. We are grateful to Richard Silver, Matthias Frosch, and David Stephens for supplying strain, plasmids, or PCR amplicon.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant RO1-AI42015 (to E. R. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF318310, M76370, M88479, AF432602, and U75650.

Dagger To whom correspondence should be addressed: Laboratory of Sialobiology, Division of Microbiology and Immunology, Dept. of Pathobiology, University of Illinois, 2001 South Lincoln Ave., Urbana, IL 61802. Tel.: 217-333-8502; Fax: 217-244-7421; E-mail: e-vimr@uiuc.edu.

Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M208837200

2 S. Steenbergen and E. Vimr, unpublished results.

    ABBREVIATIONS

The abbreviations used are: Neu5Ac, N-acetylneuraminic acid; GlcNAc, N-acetylglucosamine; PSA, polysialic acid; polyST, polysialyltransferase; H46, anti-polyalpha 2,8-linked sialic acid antiserum; alpha GpC, anti-polyalpha 2,9-linked sialic acid antiserum; endo-N, endo-N-acylneuraminidase (polyalpha 2,8-linked sialic acid depolymerase, endosialidase); Bos-12, PSA purified from E. coli K92 strain Bos-12; GpC, PSA purified from group C meningococci; NCAM, neural cell adhesion molecule.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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