Elongation of alternating
2,8/2,9 polysialic acid by the Escherichia coli K92 polysialyltransferase
Margaret M. McGowen,
Justine Vionnet and
Willie F. Vann1
Laboratory of Bacterial Toxins, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, 8800 Rockville Pike, Bethesda, MD 20892, USA
Received on October 16, 2000; revised on February 6, 2001; accepted on February 12, 2001.
 |
Abstract
|
---|
We have chosen E. coli K92, which produces the alternating structure
(2-8)neuNAc
(2-9)neuNAc as a model system for studying bacterial polysaccharide biosynthesis. We have shown that the polysialyltransferase encoded by the K92 neuS gene can synthesize both
(2-8) and
(2-9) neuNAc linkages in vivo by 13C-nuclear magnetic resonance analysis of polysaccharide isolated from a heterologous strain containing the K92 neuS gene. The K92 polysialyltransferase is associated with the membrane in lysates of cells harboring the neuS gene in expression vectors. Although the enzyme can transfer sialic acid to the nonreducing end of oligosaccharides with either linkage, it is unable to initiate chain synthesis without exogenously added polysialic acid. Thus, the polysialyltransferase encoded by neuS is not sufficient for de novo synthesis of polysaccharide but requires another membrane component for initiation. The acceptor specificity of this polysialyltransferase was studied using sialic acid oligosaccharides of various structures as exogenous acceptors. The enzyme can transfer to the nonreducing end of all bacteria polysialic acids, but has a definite preference for
(2-8) acceptors. Gangliosides containing neuNAc
(2-8)neuNAc are elongated, whereas monsialylated gangliosides are not. Disialylgangliosides are better acceptors than short oligosaccharides, suggesting a lipid-linked oligosaccharide may be preferred in the elongation reaction. These studies show that the K92 polysialyltransferase catalyzes an elongation reaction that involves transfer of sialic acid from CMPsialic acid to the nonreducing end of two different acceptor substrates.
Key words: E. coli/polysialic acid/polysialyltransferase
 |
Introduction
|
---|
Capsular polysaccharides are important virulence factors for many pathogenic bacteria. The type 2 capsular polysaccharides of Escherichia coli (Jann and Jann, 1992
) are negatively charged and form structures on the bacterial surface distinct from the O-antigen. In several cases capsular polysaccharides consist of polysialic acid. The polysialic acid capsule of E. coli K1 is an
2-8 homopolymer of sialic acid (McGuire and Binkley, 1964
) and is identical to the capsule of Neisseria meningitidis serogroup B. In vertebrates this polysialic structure is found on neural tissue and tumors (Roth et al., 1993
). The N. meningitidis serogroup C capsular polysaccharide is a homopolymer of
(2-9) sialic acid. The biosynthesis of capsular polysaccharides of E. coli is encoded by a cluster of genes consisting of three distinct groups of genes. The central group of genes in E. coli K1 and K92, neuDBACES, have been suggested to be sufficient to produce a polysialic acid in the bacterial cytosol (Annunziato et al., 1995
). Although gene clusters encoding production of several capsular polysaccharides have been described (Roberts, 1996
; Whitfield and Roberts, 1999
), many of the details of the biochemical mechanism of capsular polysaccharide synthesis in these Gram-negative bacteria have not been elucidated. We have chosen E. coli K92, which contains both
(2-8)- and
(2-9)-linked sialic acid (Egan et al., 1997
) as a model system to investigate the biosynthesis of Gram-negative capsular polysaccharides.
The E. coli polysialyltransferase (PST), unlike the vertebrate enzyme, is a peripheral membrane protein and has no sequence homology to known vertebrate sialyltransferases (Angata et al., 2000
). The E. coli K1 and K92 polysialyltransferases are encoded by the neuS gene (Steenbergen et al., 1992
). These polysialyltransferases have 92% amino acid sequences homology, which suggests that these two proteins may operate by the same or very similar mechanisms of action (Steenbergen et al., 1992
). The E. coli K1 PST transfers sialic acid from cytidine 5'-monophosphate (CMP)sialic acid to the nonreducing end of the growing chain in processive fashion (Troy, 1992
).
The elongation reaction of the K92 polysialyltransferase has been investigated by studying its acceptor specificity in two recent reports (Chao et al., 1999
; Shen et al., 1999
). The results in these reports are in conflict and therefore do not provide a clear understanding of the requirements of the K92 PST elongation reaction. Chao et al. (1999)
suggest that K92 and K1 polysaccharides are preferred acceptors, whereas Shen et al. (1999)
suggest that the K1
(2-8) polysialic acid is not an acceptor. Furthermore, Shen et al. (1999)
reported monomeric sialic acid as an acceptor, which is contrary to earlier observations (Troy and McCloskey, 1979
). Chao et al. (1999)
and Vann (1995)
reported that the N. meningitidis serogroup C
(2-9) polysialic acid is not an acceptor but rather inhibits K92 polysaccharide formation. These experiments with group C polysaccharide were performed with membrane preparation containing endogenous polysaccharide acceptor and could be explained by competition of a poor substrate with endogenous synthesis. In this article we describe the properties of the elongation reaction catalyzed by recombinant K92 PST in membranes free of endogenous polymer synthesis. Troy (1992)
suggested the involvement of undecaprenol phosphate in the polymerization process. Indeed, these authors identified a putative lipid binding motif in the neuE gene product, implicating neuE in the polymerization process. In this study we investigate the role of the neuE gene in the reaction catalyzed by the K92 PST.
 |
Results and discussion
|
---|
The K92 neuS gene product synthesizes both
(2-8) and
(2-9) neuNAc linkages
In this article we want to define the reactions catalyzed by the K92 neuS gene product. First we would like to determine whether this enzyme synthesizes both linkages independent of the presence of a
(2-8) neuNAc PST. It has been reported that cells harboring the K92 neuS gene product can form de novo an
(2-8)/
(2-9) alternating polymer. De novo synthesis of the alternating polysialic acid has not been directly demonstrated but has been inferred from either antibody binding to radiolabeled product (Steenbergen et al., 1992
) or a recent report the nuclear magnetic resonance (NMR) spectrum of polysaccharide (Shen et al., 1999
) isolated from a strain containing both the K1 and K92 neuS genes. In the latter report it is unclear what role the PSTs encoded by the K1 and K92 neuS genes are playing in the synthesis of the product isolated. Are both the K1 and K92 enzymes involved in the synthesis of the product that these authors isolated? It was also not clear from the data presented in the report of Shen et al. (1999)
whether the observed spectra were due to a polysaccharide mixture or alternating linkages because the strain used could potentially synthesize more than one polysaccharide. Indeed the 1H-NMR spectra suggest an unequal mixture of polysaccharides. To clarify this point, we transformed the K92 neuS containing plasmid into the neuS negative K1 strain EV240. Because EV240 cannot synthesize
(2-8)-linked polysialic acid, capsular polysaccharide isolated from this construct would be a product of the K92 neuS gene expression alone. Polysaccharide isolated from this strain gave a 13C-NMR spectrum identical to that expected for an
(2-8)/
(2-9) alternating polymer (Figure 1). This is evidenced by the characteristic pair of signal for C-2, C-3, and C-5 carbons. The signals in each pair are of approximate equal intensity as is expected for the alternating repeating unit of
(2-8)/
(2-9)neuNAc.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1. 13C-NMR spectra of polysaccharides isolated from (A) the construct EV240:pWV213, which is neuSK1 and neuSK92+, and (B) the wild-type K92 strain Bos-12. Spectra were recorded at 25°C on polysaccharide dissolved in D2O at 7 mg/ml.
|
|
Acceptor dependence of E. coli K92 PST
Polysialic acid chains are elongated by a reaction that involves the transfer of sialic acid from CMPsialic acid to a polysialic acid acceptor. To define the reactions catalyzed by the neuS gene product we investigated the details of the acceptor in the PST reaction. Chao et al. (1999)
and our laboratory (Vann, 1995
) have shown that membranes of fully encapsulated E. coli K92 can elongate both
(2-8) and
(2-9) polysialic acids, but synthesis is inhibited by
(2-9) polysialic acid. In these experiments it is not clear what role the PST is playing in the formation of polymer nor the influence that endogenous polysaccharide has on the overall reaction. To define the catalytic requirements of the K92 neuS gene product, PST, we constructed strains that lacked other genes in the K92 gene cluster (Boulnois and Roberts, 1990
). The membranes of these strains were therefore free of polysialic acid or sialylated oligosaccharides. Thus, reactions observed with these membrane preparations are either due to de novo synthesis or elongation of exogenously added polysaccharide acceptors. As can be seen from the data in Table I the K92 polysialyltransferase alone does not catalyze de novo synthesis of polysaccharide in the absence of other capsule gene products. Membranes were prepared from acapsular hosts, such as DH5
cells harboring plasmids containing the K92 neuS gene. These membrane preparations required the addition of polymeric sialic acid to detect incorporation of radiolabeled sialic acid into chromatographically immobile product (Table I). Other charged nonsialylated polysaccharides, such as E. coli K5 and K13, did not act as acceptor for the recombinant K92 PST.
View this table:
[in this window]
[in a new window]
|
Table I. Activity of K92 PST in membrane fractions isolated from E. coli DH5 harboring the plasmids with neuE (pK92 and pWV210) and without (pWV213) the neuE gene. These membrane fractions were assayed in the presence and absence of K92 polysaccharide. Reaction products were separated by paper chromatography as described in Materials and methods
|
|
In a recent report (Shen et al., 1999
) it was suggested that the K92 PST is active in a soluble fraction when prepared using a high-level expression system. Although such a system would provide a convenient means of studying the PST, it is contrary to the experience with the K1 PST (Steenbergen et al., 1992
). We expressed PST under the control of a strong promoter in the plasmid pWV213. In our experiments sialyltransferase activity was found only in membrane fractions and was not detected in the soluble portion of cell lysates. This was the case when the K92 PST was assayed for activity at both high and low CMPsialic acid concentrations
Linkage requirement for K92 PST
The K92 polysaccharide is elongated from the nonreducing end, thus the PST might transfer to either an
2-8 or
2-9 disaccharide acceptor of exogenously added K92 polysialic acid. To determine whether there is a preference for acceptor linkage by K92 PST, a series of polysialic acid acceptors with differing linkages were tested for the ability to act as acceptors for polysialic sialic acid free membranes. The data in Table II show that all of the three polysialic acids,
2-8/29 neuNAc,
2-8 neuNAc, and
2-9 neuNAc, can serve as acceptor for the K92 PST. This is in contrast to the results reported previously for membranes containing competing endogenous polysaccharide (Chao et al., 1999
) or the report that recombinant polysialyltransferase (Shen et al., 1999
) does not transfer to
(2-8) polysialic acid. As suggested by Chao et al. (1999)
the highest amount of activity was observed with the homologous K92 polysaccharide. N. meningitidis group C
-2-9 linked polysialic acid is the least preferred. Transfer to the
(2-9) polysialic acid acceptor substrate was confirmed by high percentage sodium dodceyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) of the reaction products. The results of these experiments suggest that the binding site for the acceptor has a preference for the
-2,8 linkage at its nonreducing end. The K92 transferase, unlike its counterpart from the K1 system, can use capsular polysaccharide from other sources (Troy and McCloskey, 1979
).
View this table:
[in this window]
[in a new window]
|
Table II. Polysialyltransferase activity E. coli DH5a:pWV213 membranes with bacterial polysialic acids as acceptors. The E. coli K1 and K92 and the meningococcal group C polysialic acids were included in assay mixtures as acceptors
|
|
Elongation of oligosaccharide acceptors
Because the
-(2-9) neuNAc polymer is a poor acceptor we tested oligosaccharides ending with the disaccharide
-(2-9)neuNAc
-(2-8)neuNAc derived from the K92 polysaccharide. Oligosaccharides were prepared by digestion with the
-(2-8) neuNAcspecific endoneuraminidase. Because this endoneuraminidase cleaves specifically at the
-(2-8) neuNAc, the resulting product will have an
-(2-9)neuNAc nonreducing end. These oligosaccharides were purified after bacteriophage digestion as single species by ion exchange chromatography. Radiolabeled polymeric material could be detected in the paper chromatography assay when K1F phagedigested oligosaccharides were added to a reaction mixture containing membranous K92 PST. As is shown in Figure 2 14C-neuNAc is incorporated into oligosaccharides with
(2-9) neuNAc at the nonreducing ends, which are larger than a hexamer. The hexadecamer, unlike the hexamer, appears to be elongated in this experiment because the radiolabeled products of the hexdecamer reaction migrate as a diffuse streak. A tetradecamer of the
2-9-linked meningococcal group C polysaccharide is an acceptor in the paper chromatography assay and produced high molecular weight product as detected by gel electrophoresis. In a similar experiment oligosaccharides prepared from
(2-8) linked polysialic acid were incubated with membranous K92 PST and the reaction products analyzed by high percentage gel electrophoresis. Radiolabled neuNAc was transferred to all of the oligosaccharides assayed greater than a hexamer. This is contrary to an earlier report on recombinant K92 PST, which suggested that this enzyme does not transfer to colominic acid oligomers.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 2. Gel electrophoresis of reaction mixtures with K92 polysaccharide acceptors. Membrane preparations of E. coli DH5 :pWV213 were incubated with substrate (CMP-[14C]-sialic acid) and varying concentrations of polysaccharide acceptor. The reaction mixtures were extracted with phenol and the aqueous phase subjected to gel electrophoresis on 25% polyacrylamide gels. Lanes: None, no acceptor was added to membranes; K92 1, 10, 100, E. coli K92 polysaccharide (1 µg, 10 µg, 100 µg); K92 (16) 1, 10, 100, E. coli K92 hexadecamer (1 µg, 10 µg, 100 µg); K92(6) 1, 10, 100, E. coli K92 hexamer (1 µg, 10 µg, 100 µg).
|
|
Because biosynthesis of a polysialic acid chain must begin by the addition of an initial sialic acid residue we tested the ability of mono- and disialyl compounds to act as acceptor substrates. Shen et al. (1999)
suggest that the K92 PST preferentially transfers to monomeric sialic acid. Contrary to this observation, we were unable to detect formation of a polymeric product with sialic acid as acceptor. Oligosaccharides possessing a terminal monomeric sialic acid, such as sialyllactose and the ganglioside GM1 or neuNAc
(2-8)neuNAc, were not acceptors (Figure 3, Table III). These acceptor requirements are in contrast to those reported for vertebrate polysialyltransferase by Angata et al. (2000)
.
Elongation of gangliosides by K92 PST
Although the free disaccharide neuNAc
(2-8)neuNAc is not an acceptor substrate for the K92 PST, it had been previously demonstrated that disialyl gangliosides (Table IV) could serve as acceptors for the K1 PST (Cho and Troy, 1994
). Table V presents the activity observed with the K92 PST and the
-2,8-disialyl containing gangliosides, Gt1b, Gd3, Gd1b, as acceptor substrates. All three are excellent acceptors for the recombinant K92 polysialyltransferase. Note that in Figure 3 GM1 was not an acceptor substrate. The product of disialylganglioside reactions did not migrate in paper chromatography or thin-layer chromatography nor enter PAGE gels suggesting the formation of high molecular weight product (Figure 4). The reactions appear to result in high molecular weight product by addition to the
-2,8 disialic acid of a ganglioside. In contrast, the K92 PST only adds a single residue to a hexasaccharide acceptor (Figure 3). This result suggests that the enzyme prefers a lipid-linked acceptor.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 4. Thin-layer chromatography of K92 neuS reaction with gangliosides. Membrane preparations of E. coli DH5 :pWV213 were incubated with substrate (CMP-[14C]-sialic acid) and varying concentrations of acceptor. The reaction mixtures were extracted with phenol and the aqueous phase subjected to thin-layer chromatography (6:1:2 n-propanol:ammonium hydroxide:water) Spots: None, no acceptor was added to membranes; K92, E. coli K92 polysaccharide; GT1b, ganglioside GT1b; GD3, ganglioside GD3; GD1b, ganglioside GD1b; Mix, equal mixture of gsanglioside GT1b,GD1b, and GD3.
|
|
Because the disialylated gangliosides served as acceptors, we tested a ganglioside analogue possessing a fluorescent hydrophobic aglycon instead of the ceramide. The disialylated analogue supported the incorporation of radiolabeled sialic acid in the paper chromatography assay. Chromatography of reaction product of a nonradioactive assay revealed the presence of several species of slow-migrating flourescent materials. The major species were isolated and analyzed by matrix-assisted laser desorption ionization (MALDI) to determine the number of sialic acid residues added. As can be seen in Figure 5, sialic acid residues were added to increase the molecular weight of the starting material by increments of m/z 291. These results further implicate the role of a hydrophobic aglycon in the acceptor.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5. MALDI mass spectra of products K92 PST elongation of the ganglioside analogue FCHASE GD3. The ganglioside analogue FCHASE GD3 (333 nmoles) and CMP-neuNAc (1560 nmoles) were incubated with E. coli DH5 :pWV213 membranes in 4 ml 50 mM Tris, 25 mM MgCl2, pH 8.0, for 24 h at 37°C. The reaction products were purified and treated as described Materials and methods prior to analysis by MALDI in the negative ion mode. The expected m/z for the FCHASE GD3 = 1486. An increment of 291 m/z is interpreted as the increase in mass due to the addition of a sialic acid residue.
|
|
Comparison between K92 PST expressed with neuE and in the absence of neuE
Steenbergen et al. (1992)
identified an open reading frame, designated neuE, upstream from the neuS gene. The putative product of this gene has been postulated to bind to isoprenyl groups and may play a role in the polymerization reaction. There has been no direct biochemical data to support either of these claims. To address the role of the neuE gene product in the K92 PST reaction we constructed plasmids that would contain both the putative neuE gene and neuS gene, pWV210, or the neuS gene alone, pWV213. Membranous K92 PST prepared from cells harboring these two plasmids were compared. The results in Table I demonstrate that neuE does not alleviate the requirement for addition of an exogenous polysaccharide acceptor to observe polymerization catalyzed by the neuS gene product. Membranes prepared from cells harboring the plasmids and pWV210, which contains both the neuE and neuS genes, are inactive without addition of polysaccharide. The lower level of PST activity in the strain harboring pWV210 may be due to a difference in gene expression because this plasmid construct contains the neuE gene and additional DNA upstream of the neuS gene. In the construct pWV213, the neuS gene is adjacent to the lac promoter.
The experiments above suggest that the K92 PST can elongate and synthesize a polymer with alternating linkages. This requires at least three reactions, the first two being the transfer of sialic acid to two different acceptor hydroxyls, then the translocation of the newly elongated chain. The biosynthesis of the alternating linkages at the nonreducing end of a homopolymer is reminiscent of cellulose biosynthesis (Koyama et al., 1997
). In cellulose formation, the ß-1,4 glucan grows by the addition of a glucose unit in alternating orientations because of the three-dimensional structure of the chains. Several authors have proposed dual substrate sites to accommodate this mechanistic requirement (Saxena et al., 1995
; Koyama et al., 1997
; Albersheim et al., 1997
). The acceptor requirement of the K92 PST can be explained by assuming a mechanism similar to that postulated for bacterial cellulose (Koyama et al., 1997
). In this model (Scheme 01), because there is a strong preference for acceptors containing
(2-8) linkage there would be an
(2-8) acceptor site. The
(2-9) acceptor binds poorly to this site although
(2-9)
(2-8) works well as acceptor. This latter observation can be explained if there are two sites for linkage formation with the
(2-9) site adjacent to the acceptor site. The
(2-9) polymer could serve as an acceptor by binding to the
(2-9) catalytic site and accepting transfer of neuNAc from the
(2-8) catalytic site. The
(2-9)
(2-8) works well as an acceptor because it would bind to both the acceptor site and the adjacent
(2-9) catalytic site. This model would require that two molecules of CMP-neuNAc bind and transfer neuNAc prior to translocation and would suggest three sites on the single polypeptide. This arrangement would ensure the fidelity of an alternating linkage.
An alternative to the three-site model is to assume a single sugar nucleotide site and an acceptor site. In this model the
(2-8) acceptor and
(2-9) acceptor would bind to the same site but in different orientations such that the appropriate hydroxyl is in the catalytic site (Scheme 02). Thus, the linkage that is formed is determined by the orientation of the acceptor at the acceptor site. To accommodate the observation of a linkage preference one would postulate that free enzyme binds
(2-8) acceptor better but is then converted to a form that binds the
(2-9) acceptor. The model in Scheme 01 predicts two separate catalytic sites and is testable in future experiments by affinity labeling and site directed mutagenesis.
 |
Materials and methods
|
---|
Bacterial strains and plasmids
The plasmid pK92 containing a fragment of the neuE gene and the complete neuS gene of K92 harbored in E. coli DH5
were obtained from Dr. Eric Vimr, University of Illinois (Urbana). The plasmid pWV210 was constructed using the primers neuCfor-GAG CAG ATA GAT GTT GATT and pK92r with chromosomal K92 DNA as a template. The resulting fragment containing the complete neuE and neuS genes were ligated into pCR-XL-TOPO. Similarly, the neuS gene was amplified from chromosomal K92 DNA using the primers pK92forII-AGG AGC AAA GCT AAT GAT ATT TGA TGC TAG TTT AAA GAA GTT GAG G and pK92rev stopCTA CTC CCC CAA GAA AAT CCT TTT and ligated into the pCR-XL-TOPO. This latter plasmid was designated pWV213 and contained only the neuS gene.
Polysaccharides and oligosaccharides
The K92 polysialic acid was isolated and purified from E. coli Bos 12 (Vann and Freese, 1994
; Egan et al., 1997
). Meningococcal C oligosaccharide was a gift from Biocine Chiron. CMPsialic acid [sialic-4,5,6,7,8,9-14C] (14C CMP-NeuAc) was purchased from New England Nuclear with a specific activity of 114 mCi/mmole. Oligosaccharides of
(2-8)-sialic acid (dimer, trimer, tetramer, pentamer, and hexamer) were purchased from CalBiochem. The flourescent oligosaccharides labeled with FCHASE (6-(5-fluorescein-carboxamido)-hexanoic acid succimidyl ester), GD3-FCHASE, and GM1-FCHASE were a gift of Warren W. Wakarchuk and Micheal Glibert, National Research Council of Canada (Ottawa, Ontario). NMR spectra were recorded as described previously (Vann and Freese, 1994
).
Preparation of membranes possessing PST
Bacterial cultures of DH5
:pK92 were grown on 1.5 L Luria-Bertani media supplemented with 100 µg/ml ampicillin at 37°C until late log phase of growth was obtained (14 h). Strains harboring the plasmids pWV210 and pWV213 were grown to A600 = 0.6 and induced with 1 mM isopropylthiogalactopyronoside (IPTG) for 2 h. The cells were then harvested by centrifugation; resuspended in 50 mM TrisHCl, 25 mM MgCl2, pH 8.0 (buffer A); and lysed in a French pressure cell (American Instrument Co.) at 16,000 lbs./in2. The lysate is cleared of debris by centrifugation at 10,700 x g and the membranes isolated from the resulting supernatant by ultracentrifugation at 100,000 x g. The membrane pellet was resuspended with a ground glass tissue grinder in 3 ml of buffer A.
K92 PST assay
Each reaction mixture contained 50 µl of membrane preparation (612 mg protein/ml), 0.5 nmol of CMP-14C- NeuAc, and a known amount of acceptor in 100 µl of buffer A (Vann, 1995
). The reaction mixture was then incubated at 37°C for 1 h. As a negative control, in parallel, a reaction mixture with all components except acceptor and CMP-14C- NeuAc was boiled for 1 min. After boiling, acceptor and CMP-14C- NeuAc were added and the mixture incubated at 37°C for 1 h. A reaction mixture was also incubated without acceptor to demonstrate the necessity of exogenous acceptor for this construct. The reaction was quenched by spotting 30 µl onto Whatman 3M chromatography paper. The chromatogram was developed overnight with a 7:3 ethanol:1 M ammonium acetate, pH 7.0, solvent system and dried. The radiolabel incorporated at the origin was quantitated by liquid scintillation counting (Anoroc Scientific Envirosafe).
Phenol extraction of reaction products
Polysaccharide produced in enzyme reaction mixtures was extracted as follows. Thirty microliters of buffered phenol (50 g of recrystallized phenol in 14 ml of 10% saturated sodium acetate) was added to 70 µl of PST reaction and mixed vigorously. The resulting emulsion was separated into aqueous and organic layers by centrifugation in a microfuge for 20 min at 14,000 r.p.m.. The upper aqueous layer was immediately removed and reserved for further analysis. Any aqueous material not used immediately was stored at 20°C.
PAGE of PST reaction products
High percentage polyacrylamide gels (25%) were prepared as described by Pelkonen et al. (1988)
. The samples were mixed with 0.1 volume of 2 M sucrose in 0.089 M Tris-borate-EDTA buffer. Dyes of defined molecular size, which have been determined to correspond to degree of polymerization of polysialic acid were used as markers. These dyes are 0.05% trypan blue (degree of polymerization [DP] 100), 0.02% xylene cyanol (DP52), bromphenol blue (DP19), bromcresol purple (DP 11.5), and phenol red (DP 4). Samples (15 µl) were electrophoresed at 4°C at 40 V for 1418 h. The resulting gels were analyzed by a Molecular Dynamics phosphorimager screen and the gel bands visualized by autoradiography.
Preparation of K92 oligosaccharides
K92 polysialic acid (50 mg) dissolved in 5 ml of 10 mM TrisHCl, pH 7.4, was digested at 37°C for 24 h with 0.5 ml of crude endoneuraminidase (6.13 mg/ml protein) prepared as a K1f phage lysate. A second aliquot of crude endoneuraminidase was added and the reaction allowed to proceed for an additional 24 h at 37°C. After the limit digest was reached, the reaction mixture was filtered through a 0.8-µm syringe unit to remove precipitated protein. The filtrate was then applied to a Pharmacia Q Sepharose column (2.5 x 11 cm) that was washed with 20 mM TrisHCl, pH 7.5, until absorbance at 280 nm returned to baseline. The oligosaccharides were eluted with a linear gradient of 01.0 M NaCl in 20 mM TrisHCl, pH 7.5. The dimer was eluted at 10%, tetramer at 18% and decamer at 30% of the gradient. Fractions were analyzed by a modified version of the thiobarbituic acid assay developed by Warren as described below. Oligomers were desalted by passing the individual peaks from the previous column over gel filtration resin, Sephadex G-10 (Pharmacia), with water as eluent. The size of the oligosaccharide was determined by ion exchange chromatography on a Pharmacia mono-Q resin. The column was calibrated against oligosaccharide of defined length (monomer, dimer, trimer, tetramer, and hexamer) as well as colominic acid. Samples were loaded and washed with 20 mM TrisHCl, pH 7.5, followed by elution with a compound gradient of 00.6 M NaCl subsequently up to 1.0 M NaCl.
Determination of protein concentration
The protein concentration in membraneous fractions was determined by a modified Enhanced BCA Assay (Pierce). The membranes were diluted in 2% SDS and boiled for 5 min prior to mixing with BCA to ensure solubilization and to minimize lipid interference. Bovine serum albumin was used as a protein standard.
Preparation and analysis of FCHASE reaction products
The ganglioside analogue FCHASE GD3 (333 nmoles) and CMP-neuNAc (1560 nmoles) were incubated with E. coli DH5
:pWV213 membranes in 4 ml 50 mM Tris, 10 mM MgCl2, pH 8.0, for 24 h at 37°C. The soluble portion of reaction mixture was recovered by centrifugation at 100,000 x g for 2 h and the supernatant lyophilized. Reaction products in the lyophilized residue were detected as yellow-green fluorescent spots on paper chromatograms. Sialylated FCHASE GD3 was purified by paper chromatography developed as described above. Several products migrating slower than the starting material FCHASE were detected. The major fraction was eluted with water and treated with a second round of membranes and CMP-sialic acid.
The reaction products were analyzed by MALDI mass spectrometry using a Voyager DE-RP time-of-flight system (PerSeptive Biosystems, Framingham, MA) equipped with GRAMS/386 software. The samples were combined with
-cyano-4-hydroxycinnamic acid (10 mg/ml) in 50% acetonitrile, 0.1% trifluoroacetic acid, ionized with a nitrogen laser at 337 nm and analyzed in the negative ion mode.
 |
Acknowledgments
|
---|
The authors thank the following members of the Center for Biologics Research and Evaluation, Food and Drug Administration (Bethesda, MD): Robert Boykins for performing mass spectral analysis, Andrzej Wilk for NMR analysis, and Lee Pitts, a guest worker, for assistance in isolation of polysaccharides. The authors also thank Eric Vimr, University of Illinois (Urbana) for kindly donating strains and Warren W. Wakarchuk and Micheal Gilbert, National Research Council of Canada (Ottawa, Ontario) for ganglioside analogues.
 |
Abbreviations
|
---|
CMP, cytidine 5'-monophosphate; DP, degree of polymerization; FCHASE, 6-(5-fluorescein-carboxamido)-hexanoic acid succimidyl ester labeled oligosaccharides; MALDI, matrix-assisted laser desorption ionization; NMR, nuclear magnetic resonance; PST, polysialyltransferase; SDSPAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis.
 |
Footnotes
|
---|
1 To whom correspondence should be addressed 
 |
References
|
---|
Albersheim, P., Darvill, A., Roberts, K., Staelin, L.A., and Varner, J.E. (1997) Do the structures of cell wall polysaccharides define the mode of synthesis? Plant Physiol., 113, 13.[Free Full Text]
Angata, K., Suzuki, M., McAuliffe, J., Ding, Y., Hindsgaul, O., and Fukuda, M. (2000) Differential biosynthesis of polysialic acid on neural cell adhesion molecule (NCAM) and oligosaccharide acceptors by three distinct a2, 8 sialyltransferases, ST8Sia IV (PST), ST8Sia II (STX), and ST8Sia III. J. Biol. Chem., 275, 18594186601.[Abstract/Free Full Text]
Annunziato, P.W., Wright, L.F., Vann, W.F., and Silver, R.P. (1995) Nucleotide sequence and genetic analysis of the neuD and neuB genes in region 2 of the polysialic acid gene cluster of Escherichia coli K1. J. Bacteriol., 177, 312319.[Abstract]
Boulnois, G.J., and Roberts, I.S. (1990) Genetics of capsular polysaccharide production in bacteria. Curr. Top. Microbiol. Immunol., 150, 118.[ISI][Medline]
Chao, C.-F., Chaung, H.-C., Chiou, S.-T., and Liu, T.-Y. (1999) On the biosynthesis of alternating
-2, 9/
-2, 8 heteropolymer of sialic acid catalyzed by the sialyltransferase of Escherichia coli Bos-12. J. Biol. Chem., 274, 1820618212.[Abstract/Free Full Text]
Cho, J-W., and Troy, F.A. (1994) Polysialic acid engineering: synthesis of polysialylated neoglycosphingolipids by using the polysialyltransferase from neuroinvasive Escherichia coli K1. Proc. Natl. Acad. Sci. USA, 91, 1142711431.[Abstract/Free Full Text]
Egan, W., Liu, T.-Y., Dorrow, D., Cohen, J.S., Robbins, J.D., Gotschlich, E.C., and Robbins, J.B. (1997) Structure studies on the sialic acid polysaccharide antigen of Escherichia coli strain Bos-12. Biochemistry, 19, 36873692.
Jann, B., and Jann, K. (1992) Capsular polysaccharides of Escherichia coli, expression and biological significance. Can. J. Microbiol., 38, 701710.
Koyama, M., Helbert, W., Imai, T., Sugiyama, J., and Henrissat, B. (1997) Parallel-up structure evidences the molecular directionality during biosynthesis of bacterial cellulose. Proc. Natl Acad. Sci. USA, 94, 90919095.[Abstract/Free Full Text]
McGuire, E.J., and Binkley, S.B. (1964) The structure and chemistry of colominic acid. Biochemistry, 3, 247251.[ISI]
Pelkonen, S., Hayrinen, J., and Finne, J. (1988) Polyacrylamide gel electrophoresis of the capsular polysaccharides of Escherichia coli K1 and other bacteria. J. Bacteriol., 170, 26462653.[ISI][Medline]
Roberts, I.S. (1996) The biochemistry and genetics of capsular polysaccharide production in bacteria. Annu. Rev. Microbiol., 50, 285315.[ISI][Medline]
Roth, J., Rutishauser, U., and Troy, F.A. (1993) Polysialic Acid: From Microbes to Man. Birkhauser Verlag, Basel.
Saxena, I.M., Brown, R.M. Jr., Fevre, M., Geremia, R.A., and Henrissat, B. (1995) Multidomain architecture of ß-glycosyltransferases: Implications for mechanism of action. J. Bacteriol., 177, 14191424.[Free Full Text]
Shen, G.-W., Datta, A.K., Izumi, M., Koeller, K.M., and Wong, C.-H. (1999) Expression of
-2, 9/
-2, 8 polysialyltransferase from Escherichia coli K92: characterization of the enzyme and its reaction products. J. Biol. Chem., 274, 3513935146.[Abstract/Free Full Text]
Steenbergen, S.M., Wrona, T.J., and Vimr, E.R. (1992) Functional analysis of the sialyltransferase complexes in Escherichia coli K1 and K92. J. Bacteriol., 174, 10991108.[Abstract]
Troy, F.A. II (1992) Polysialylation: from bacteria to brains. Glycobiology, 2, 523.[ISI][Medline]
Troy, F.A., and McCloskey, M.A. (1979) Role of a membraneous sialyltransferase complex in the synthesis of surface polymers containing sialic acid in Escherichia coli: temperature induced alterations in the assembly process. J. Biol. Chem., 254, 73777387.[Abstract]
Vann, W.F. (1995) A filter assay for polysialyltransferase. FEMS Microbiol. Lett., 128, 163165.[ISI]
Vann, W.F., and Freese, S.J. (1994) Purification of Escherichia coli K antigens. Methods Enzymol., 235, 304311.[ISI][Medline]
Whitfield, C., and Roberts, I.S. (1999) Structure, assembly, and regulation of expression of capsules of Escherichia coli. Mol. Microbiol., 31, 13071319.[ISI][Medline]