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
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ABSTRACT |
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Polysialic acid (PSA) capsules are
cell-associated homopolymers of 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 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.
Bacterial Strains, Plasmids, and Growth
Conditions--
Laboratory E. coli K-12 strain DH5 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 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'.
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 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
Exogenous Acceptor Activities of Heterologously Expressed
PolySTs--
We previously showed that when neuS is
expressed in an E. coli K-12 strain such as DH5 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.
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.
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
Fig. 4 shows the repeat structures of
PSAs from E. coli K1 and group B meningococci, E. coli K92, and group C meningococci. The 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.
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-
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).
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).
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
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
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 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 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
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 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 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
2,8-,
2,9-, or alternating
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
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,8 linkages, whereas group C
N. meningitidis and E. coli K92 express PSA
homopolymers with
2,9 or alternating
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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
GpC antiserum kindly donated by Willie Vann. H46 is specific for a
minimal epitope of approximately eight
2,8-linked Neu5Ac residues,
whereas
GpC recognizes the
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.
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).
Specific primers used for chimeric enzyme synthesis and mutagenesis by
overlap extension PCR
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 DH5
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.
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
, 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).
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.
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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.
Complementation analysis of polySTs expressed in different
polymerase-deficient mutant backgrounds
2,8/2,9 PSA synthesized by K92
neuS by its immunochemical cross-reactivity against
GpC
antiserum and susceptibility to digestion with endo-N, which is
specific for PSA oligomers with at least eight
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
2,8 and
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.
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
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
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
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- 2,8-linked sialic acid; B,
poly-
2,8/2,9-linked sialic acid; C, poly-
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.
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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.
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
2,8- or repeating
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.
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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.
Polysialic acid expression in mutants with altered K1 or K92 polySTs
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.
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
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
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
GpC antiserum (not shown).
B, results of PSA immunodetection using
GpC antiserum.
Colony 1, EV240/pSX616; colony 2, EV240/pSX617;
colony 3, EV240/pSX618; colony 4, EV240/pSX613;
and colony 5, EV240/pSX625.
-bonds from
-linked donor) that figure prominently in microbial pathogenesis,
nervous system development, and tumorigenesis (35).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
2,9-linked disaccharide and another linking these units at a site
favoring
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
2,8 and
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.
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
2,8-,
2,9-, and repeating
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.
2,8- or
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
2,9 PSA.
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
-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
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.
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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.
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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.
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.
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ABBREVIATIONS |
---|
The abbreviations used are:
Neu5Ac, N-acetylneuraminic acid;
GlcNAc, N-acetylglucosamine;
PSA, polysialic acid;
polyST, polysialyltransferase;
H46, anti-poly2,8-linked sialic acid
antiserum;
GpC, anti-poly
2,9-linked sialic acid antiserum;
endo-N, endo-N-acylneuraminidase (poly
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.
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REFERENCES |
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