From the Abteilung Zelluläre Chemie, Zentrum
Biochemie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse
1, 30625 Hannover and the ¶ Institut für Mikro-
und Molekularbiologie, Justus-Liebig-Universität Giessen,
Ludwigstrasse 23, D-35390 Giessen, Germany
Received for publication, November 26, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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Bacteriophages infecting the
neuroinvasive pathogen Escherichia coli K1 require an
endosialidase to penetrate the polysialic acid capsule of the host.
Sequence information is available for the endosialidases endoNE,
endoNF, and endoN63D of the K1-specific phages The capsule of the neuroinvasive pathogen Escherichia
coli K1 (E. coli K1), causing meningitis and sepsis in
neonates, is composed of Because of high substrate specificity, endoN is of particular
importance for studying the functional role of polySia in different organisms. In mammals, polySia is found exclusively as a
dynamically regulated post-translational modification of
the neural cell adhesion molecule (13). The presence of the
large polyanionic carbohydrate moiety attenuates the
binding properties of the neural cell adhesion molecule and promotes
plasticity of cell-cell interactions during cell migration and axonal
pathfinding (14). Specific removal of polySia by endoN demonstrated the
importance of polySia for synaptic activity involved in learning and
memory (15, 16) and its role in promoting metastasis and invasion of
polySia-positive tumor cells (17).
In contrast to bacterial and viral exosialidases, little is known about
the structure of the phage-derived endosialidases. SDS-resistant
trimers, composed of three identical subunits of 105 kDa each, have
been described for endoNF of coliphage Cloning of endoNE revealed that the gene encodes a 90-kDa protein,
whereas for the purified phage-borne and the recombinantly expressed
endoNE an apparent molecular mass between 74 and 76 kDa was observed
(8, 18, 19), indicating post-translational processing. In contrast, no
discrepancy between the apparent and calculated molecular mass was
observed for endoNF. However, in the latter case no enzymatic activity
was detected for the recombinantly expressed 102-kDa protein (11).
Here we describe the isolation of the full-length gene of endoNF
encoding a 119-kDa protein, and for the first time, active endoNF was
recombinantly expressed. The primary translation product is
proteolytically cleaved into a 101-kDa enzymatically active part and an
18-kDa C-terminal fragment playing a crucial role for trimerization and
functionality of the catalytic domain.
Materials--
Colominic acid and N-acetylneuraminic
acid were purchased from Sigma. pET expression vectors and E. coli BL21(DE3) were obtained from Novagen. The plasmid pMCL200
(21) was kindly provided by Yoshio Nakano (Kyushu University, Japan).
Propagation of Bacteriophages Cloning of EndoNF--
Based on the endoNF sequence published by
Petter and Vimr (11) a primer binding nucleotides 2652-2669
(5'-TAACGATAACGTCACTGC-3') was generated and used for direct sequencing
of purified Expression Plasmids--
Using purified Site-directed Mutagenesis and Generation of C-terminal
Truncated and Chimeric Endosialidases--
Site-directed mutagenesis
was performed by PCR using the QuikChange site-directed mutagenesis kit
(Stratagene) following the manufacturer's guidelines with the primers
given in Table I. For the generation of chimeric endosialidases an
AvrII restriction site was introduced in pT7-endoNF-His by
the silent mutation C2709A, maintaining a triplet coding for Leu-903
(primers used for PCR are given in Table I). EndoNE
contains an endogenous AvrII site at the corresponding
position, and this site was used to exchange the gene fragments
encoding the C-terminal domains. C-terminal truncated constructs were
generated by PCR using the primers given in Table I. PCR products were
ligated into the NcoI and BamHI sites of pET23d
and BamHI and XhoI sites of pET23a for endoNE and
endoNF constructs, respectively. The identity of all constructs was
confirmed by sequencing.
Expression of Recombinant Endosialidases--
Freshly
transformed E. coli BL21(DE3) were cultivated at 37 °C in
Luria-Bertani medium containing 200 µg/ml carbenicillin. At an
absorbance (A600) of 0.6 expression was
induced by adding 1 mM
isopropyl-1-thio- Affinity Purification of Recombinant EndoNF--
Bacteria
expressing T7-endoNF-His were harvested 2 h after induction,
resuspended in binding buffer (50 mM
Na2HPO4, NaH2PO4, pH
8.0, 500 mM NaCl) and lysed by sonication. The soluble
fraction was applied to a 5-ml Ni2+-loaded HiTrap chelating
column (Amersham Biosciences) according to the manufacturer's
instructions. After washing with 25 ml of binding buffer containing
10% glycerol, the protein was eluted with a linear imidazol gradient
(0-500 mM imidazol in binding buffer containing 10%
glycerol). For purification of the T7 epitope tagged N-terminal
fragment the flow through of the chelating column was adsorbed to 1 ml
of T7 tag antibody agarose (Novagen) according to the manufacturer's
protocol. Protein was eluted with 100 mM glycine, pH 2.7, and the fractions were neutralized with 1 M Tris, pH
9.0.
N-terminal Amino Acid Analysis--
Affinity-purified
His6-tagged C-terminal fragments of endoNE and endoNF were
separated by 10% SDS-PAGE and were blotted onto polyvinylidene
difluoride membrane (Millipore). Coomassie-stained protein bands were
excised, and amino acid sequencing of the immobilized protein was
performed at Giessen University (Biochemisches Institut am Klinikum).
Determination of Endosialidase Activity--
Sialyl oligomers
(colominic acid) with a degree of polymerization of 16 (22) were used
as a substrate, and endoN-catalyzed cleavage of SDS-PAGE and Immunoblotting--
SDS-PAGE was performed under
reducing conditions using 2.5% (v/v) Cloning of the Full-length Endosialidase Gene from Bacteriophage
Proteolytic Cleavage of Endosialidases--
Recombinant endoNE and
endoNF containing an N-terminal T7 epitope and a C-terminal
His6 tag (see Fig.
2B) were expressed in E. coli BL21(DE3), and enzyme expression was analyzed by Western blotting. For each enzyme three bands were detected (Fig.
2A). The top bands, visible with anti-His6
antibody, indicate the expression of full-length proteins with
molecular masses corresponding to the calculated mass of 93 and 121 kDa
for epitope-tagged endoNE and endoNF, respectively. In addition, low
molecular weight fragments of 13 (endoNE) and 19 kDa (endoNF) were
detected with anti-His6 tag antibody, demonstrating that
these fragments contain the C terminus. The corresponding N-terminal
fragments were detected exclusively with the anti-T7 antibody. These
results clearly demonstrate proteolytic cleavage of the full-length
endosialidases into a small C-terminal and a large N-terminal
fragment.
To identify the cleavage sites, the small fragments were isolated by
affinity purification on a Ni2+-chelating column and
subjected to N-terminal sequencing. Two peptide sequences
(DADXKYGISS and GERKTEPVVF for endoNE and endoNF, respectively) were identified, corresponding to
707DADHKYGISS716 in endoNE and
913GERKTEPVVF922 in endoNF. Based on this
sequence information, the cleavage sites were located between
Ser-706/Asp-707 and Asn-912/Gly-913 for endoNE and endoNF, respectively
(see Fig. 2B). As indicated in Fig. 1A and shown
in detail in the protein alignment given in Fig. 7, the identified
cleavage site is located at a similar site in both proteins with a
highly conserved serine residue in front of the cleavage site (Ser-706
and Ser-911 in endoNE and endoNF, respectively). It is very likely
that endoNF is also cleaved directly after Ser-911, releasing a
C-terminal fragment containing Asn-912 at the N terminus. Asparagine
residues, particularly in front of a glycine, are very labile and can
undergo spontaneous degradation by intramolecular succinimide-forming
reactions, leading to deamidation, isomerization, and peptide bond
cleavage (24, 25). As a consequence, Gly-913 but not Asn-912 was the
first amino acid identified by Edman degradation of the C-terminal fragment.
Enzymatic Activity Is Associated with the N-terminal
Fragment--
For identification of the enzymatically active fragment,
bacterial lysates containing N-terminal T7- and C-terminal
His6-tagged endoNF were passed through a
Ni2+-chelating column to remove the His6-tagged
full-length protein and the small C-terminal fragment. As shown in Fig.
2C (lane 1), the flow-through contained
exclusively the N-terminal fragment of 103 kDa. Although the first
fractions of the protein peak eluted from the
Ni2+-chelating Sepharose contained full-length and cleaved
protein (lane 2, Fig. 2C), later fractions
contained exclusively the C-terminal fragment (lane 3, Fig.
2C). As shown in Fig. 2D, no enzymatic activity
was detected for the purified C-terminal fragment. In contrast, high
enzymatic activity was observed in the flow-through (Fig.
2D), demonstrating that the large N-terminal fragment
contains the catalytic domain. The fact that a small part of the
N-terminal fragment lacking the C-terminal His6 tag was
adsorbed to the Ni2+-chelating Sepharose and coeluted with
the full-length protein can be explained by oligomerization of
N-terminal domains (see below). By the formation of hetero-oligomers
containing His6-tagged full-length endoNF and the cleaved
N-terminal domain, the latter fraction can be bound to the column.
The C-terminal Domain Is Essential for the Formation of an Active
Protein--
To examine the role of the small fragment, C-terminally
truncated forms of endoNE and endoNF (for schematic representation, see
Fig. 3, A and B)
were expressed in E. coli BL21(DE3), and the enzymatic
activity was monitored in soluble fractions of the bacterial lysates.
As shown in Fig. 3, C and D, all truncated forms
of endoNE and endoNF were expressed as soluble proteins with the
expected molecular mass. For truncated endoNF, several degradation
products became visible, indicating reduced stability of the truncated
proteins. Although the C-terminal domain is dispensable after cleavage
of the full-length protein, none of the truncated enzymes was active
(data not shown), suggesting that the C-terminal domain plays an
important role in the nascent protein. Interestingly, a short
C-terminal truncation of only 38 amino acids (endoNE
To address directly whether the truncated forms can be rescued by the
presence of the C-terminal fragment, truncated forms of endoNE were
coexpressed with a His6-tagged C-terminal domain encompassing amino acids 706-811 (Fig. 3A). Although both
fragments, encoded on two different plasmids, were expressed and
detected in the soluble fraction of bacterial lysates, coexpression of the C-terminal fragment could not restore the activity of the truncated
forms (data not shown). This result clearly demonstrates that in a
first step the C-terminal domain must be part of the primary
translation product to fulfill its function.
Expression of Chimeric Endosialidases--
Assuming a similar
function for the C-terminal domain of all endosialidases, we next
analyzed whether the C-terminal part of endoNE can be replaced by the
corresponding domain of endoNF and vice versa (Fig.
4A). Both chimeras, endoNE-F
and endoNF-E, were expressed in E. coli BL21(DE3) with an
N-terminal T7 and a C-terminal His6 tag. As shown in Fig.
4B, chimeric proteins were proteolytically processed, and
fragments of 78 and 19 kDa (endoNE-F) and 103 and 13 kDa (endoNF-E)
were released, indicating structural integrity of the cleavage site.
Although endoNE-F showed partial activity (70% activity of wild type
endoNE), no activity was detected for endoNF-E (see Fig.
4C). Because the C-terminal domain of endoNE is 49 amino
acids shorter than the corresponding domain in endoNF, the positioning
of functional important amino acid residues may not fulfill the
requirements of endoNF.
Proteolytic Cleavage Is Not Essential for Enzymatic
Activity--
To investigate whether proteolytic processing is
required to generate active endosialidases, several single amino acid
exchanges were introduced in endoNE and endoNF by site-directed
mutagenesis. After expression in E. coli BL21(DE3), cleavage
and enzymatic activity were analyzed in bacterial lysates. The results
are summarized in Fig. 5, A
and C, for endoNE and in Fig. 5, B and
D, for endoNF. Compared with the corresponding wild type
proteins, the amino acid exchanges P702A and P907A in endoNE and
endoNF, respectively, had no significant effect on proteolytic cleavage
or enzymatic activity. Similar results were obtained for endoNF-T910A,
but the activity of the corresponding endoNE mutant (T705A) was reduced to 24% of the wild type activity. In contrast, exchange of the serine
residue that is part of the cleavage site (S706A and S911A in endoNE
and endoNF, respectively) prevented proteolytic processing (Fig. 5,
A and B). The expressed full-length proteins were
active (Fig. 5, D and C), demonstrating that
cleavage of the C-terminal part is not a prerequisite for the formation
of active enzymes.
If, however, a conserved histidine residue located in the center of the
C-terminal domain (see Fig. 7) was replaced by alanine, proteolytic
cleavage was prevented, and enzymatic activity was completely abolished
(see mutants H747A and H954A in Fig. 5). The expressed full-length
proteins were detected predominantly in the insoluble fraction,
indicating accumulation of misfolded protein. These latter mutants
demonstrate an involvement of the C-terminal domain in the folding of
endosialidases and in combination with the previous data suggest a
potential chaperone function for the C-terminal domain.
The C-terminal Domain of EndoNF Is Essential for
Trimerization--
For the phage-borne endoNF, SDS-resistant trimers
were observed (9) which were also detectable for the recombinantly
expressed enzyme (lane 2, Fig.
6A). If samples were separated
by SDS-PAGE omitting the boiling step, the appearance of a high
molecular mass band of about 300 kDa and the disappearance of
the 103-kDa N-terminal fragment were observed, confirming the formation
of a trimer. The high molecular mass band was detected using anti-T7, but not with anti-His6 tag antibody, demonstrating that the
C-terminal domain is not part of the complex. Nevertheless, neither of
the C-terminal truncated forms (
The next question was whether the trimer provides the active form of
endoNF. Therefore, catalytically active and inactive forms of endoNF
were analyzed by SDS-PAGE as shown in Fig. 6A. Trimer
formation was observed for all endoNF mutants with enzymatic activity
(Fig. 6A). For endoNF-T910A a slightly increased expression of full-length protein was observed, and part of the unprocessed protein was detected in the complex with anti-His6 tag
antibody (see lane 6 in Fig. 6A, lower
panel). The formation of mixed trimers containing one or two
full-length proteins is additionally indicated by the appearance of
several bands above 300 kDa.
In the case of endoNF-S911A, an active mutant lacking the ability for
proteolytic cleavage, trimers composed of full-length protein were
formed. Although large amounts of monomeric 120-kDa full-length protein
were detected in the heat-treated sample (lane 7 in Fig.
6A), the corresponding band is drastically reduced in the
nonboiled sample, and a trimer detected with anti-His6
antibody appears. An additional band corresponding to a dimer of 240 kDa became visible, suggesting reduced complex stability for trimers containing full-length protein, exclusively. The enzymatically inactive
mutant endoNF-H954A, which is also expressed as a full-length protein,
lost its capacity to oligomerize (Fig. 6A, lane
10). In combination, these data demonstrate that trimerization
indicates correctly folded and thus active endoNF.
Bacteriophages specific for encapsulated bacteria have to
penetrate the capsular polysaccharide before infection, and therefore the host range is determined by the presence of specific polysaccharide depolymerases. In this study, we have characterized the endosialidases of the E. coli K1-specific phages A BLAST (26) search performed with the newly identified C-terminal part
of endoNF identified similarities to the C-terminal parts of several
tail fiber proteins of other bacteriophages (Fig. 7). The alignment includes the C-terminal
domains of the neck appendage protein gp12 of the Bacillus
subtilis phage GA-1 (EMBL accession no. X96987.2), the L-shaped
tail fiber protein of coliphage T5 (27), the K5-specific lyases of
coliphages K1E,
K1F,
and 63D, respectively. The cloned sequences share a highly conserved
catalytic domain but differ in the length of the N- and C-terminal
parts. Although the expression of active recombinant enzyme succeeded
in the case of endoNE, it failed for endoNF. Protein alignments of all
three endosialidase sequences gave rise to the assumption that
inactivity of the cloned endoNF is caused by a C-terminal truncation.
By reinvestigation of the respective gene locus in the
K1F genome,
we identified an extended open reading frame of 3195 bp, encoding a
119-kDa protein. Full-length endoNF contains the C-terminal domain
conserved in all endosialidases, which may act as an intramolecular
chaperone. Comparative studies carried out with endoNE and endoNF
demonstrate that endosialidases are proteolytically processed,
releasing the C-terminal domain. Using a mutational approach in
combination with protein analytical techniques we demonstrate that (i)
the C-terminal domain is a common feature of endosialidases and other
tail fiber proteins; (ii) the integrity of the C-terminal domain and
its presence in the nascent protein are crucial for the formation of
active enzymes; (iii) proteolytic processing is not essential for
enzymatic activity; and (iv) functional folding is a prerequisite for
trimerization of endoNF.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,8-linked sialic acid with up to 200 residues (polysialic acid;
polySia)1 (1, 2). This large
homopolymer is an important virulence factor protecting the bacterium
from the immune system, but it also serves as an attachment site for
lytic bacteriophages. Several coliphages specific for
E. coli K1 have been isolated from sewage samples (3-6).
Interestingly, administration of anti-K1 phages to E. coli
K1-infected mice and chicken was shown to prevent septicemia and
meningitis-like infections, demonstrating their potential for
antibacterial therapy (5, 7). Anti-K1 phages are lytic linear
double-stranded DNA viruses of different morphology. Although most of
the isolated phages have an isometric head and a short tail, some are
provided with a long and flexible tail (6) similar to that of
bacteriophage
. Common to all anti-K1 phages is an endosialidase
(endo-N-acetylneuraminidase; endoN) highly specific for
2,8-linked polySia (8-10) which was observed as a tail fiber protein (6, 11). In contrast to exosialidases, phage-borne endosialidases require oligomers of up to eight
2,8-linked sialic acid residues for binding, and distinct cleavage patterns have been
observed for individual enzymes isolated from different anti-K1 phages.
Although sialyl dimers were found as the main product for some
endosialidases (6), most enzymes release sialyl oligomers of three to
seven residues (12).
K1F (9). For endoNE of
K1E, hetero-oligomeric structures of 208 and 325 kDa were described,
composed of endoNE and a not as yet identified 38-kDa protein (8, 18).
So far, genes encoding endosialidases have been cloned from four
different phages:
K1F (11),
K1E (18, 19), 63D (GenBank accession
number AB015437), and the dual specificity phage
K1-5 that can
infect E. coli K1 and K5 (20). However, recombinant
expression of active enzyme has been reported only in the case of
endoNE (18, 19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
K1E and
K1F and Isolation of
Phage DNA--
The E. coli K1 strain U9/41 (O2:K1:H4) (3)
was used for propagation of bacteriophages
K1E and
K1F (3). Phage
DNA was isolated from purified phage particles as described previously (18).
K1F phage DNA. An in-frame stop codon was found by
primer walking using a primer binding nucleotides 2848-2865
(5'-GATGCTCGTATTCACTTC-3'). The newly identified 3195-bp open reading
frame (ORF) was confirmed by sequencing. Full-length endoNF
(nucleotides 1-3192 lacking the stop codon) was amplified by PCR using
purified phage DNA as a template and the primer pair MM97/MM98
(5'-CGGGATCCATGTCCACGATTACACAATTC-3'/5'-GTCCGCTCGAGCTTCTGTTCAAGAGCAGAAAG-3'), containing BamHI and XhoI restriction sites
(underlined) on the 3'- and 5'-ends, respectively, and ligated into
pET23a. The resulting expression plasmid pT7-endoNF- His encodes
full-length endoNF with an N-terminal T7 epitope tag (MASMTGGQQMG)
and a C-terminal His6 tag. The identity of this construct
was confirmed by sequencing.
K1E phage DNA as a
template, a 2.4-kb PCR fragment containing full-length endoNE with a
C-terminal factor Xa cleavage site was amplified with primers AH18
(5'-GCGGATCCATGATTCAAAGACTAGG-3') and AH26b
(5'-GAGGATCCCCTACCCTCGATACTGATTTTATTAGTGGC-3'),
including BamHI restriction sites (underlined) and the
coding sequence for a factor Xa cleavage site (italics) in the case of
AH26b. The PCR product was digested with BamHI and ligated
in the BamHI/BglII sites in front of the
His6 tag of the pQE16 vector (Qiagen). The 2.4-kb
BamHI/HindIII fragment (encompassing full-length
endoNE with a C-terminal factor Xa cleavage site followed by a
His6 tag) obtained from this plasmid was then subcloned
into pET23a. The resulting plasmid pT7-endoNE-His encodes full-length
endoNE with an N-terminal T7 epitope tag and a C-terminal
His6 tag. The plasmid pET-endoNE encoding full-length
endoNE was constructed in pET23d. Starting with purified
K1E phage
DNA, a 2.4-kb fragment was amplified by PCR with the primers MM72
(5'-CATGCCATGGTTATTCAAAGACTAGGTTCTTC-3') and AH25
(5'-GAGGATCCTTAACTGATTTTATTAGT-3'), digested with
NcoI and BamHI, and ligated into
pET23d. For coexpression experiments, the 3'-end of the endoNE gene
encoding amino acids 706-811 was amplified by PCR using the primers
given in Table I, and the PCR product was
ligated into the NdeI/XhoI sites of pET23a. The 492-bp BglII/BlpI fragment of this construct,
containing the T7 promoter, the ribosomal binding site, and the
His6-tagged C-terminal domain of endoNE, was then cloned in
pMCL200 with a PA15 origin of replication. The identity of all
constructs was confirmed by sequencing.
Oligonucleotides used for the introduction of amino acid exchanges and
C-terminal truncations
-D-galactopyranoside, and bacteria were harvested 2 h after induction. Cells were resuspended in 50 mM Tris-HCl, pH 8.0, and 2 mM EDTA and
incubated for 15 min at 30 °C in the presence of 100 µg/ml
lysozyme. Bacteria were lysed by sonication, and soluble fractions were
obtained after centrifugation (12,000 × g, 15 min).
2,8-linkages was
measured by quantification of the released reducing termini. Colominic
acid (5 mg/ml) in 0.1 M NaH2PO4 and
Na2HPO4, pH 5.1, was incubated at 37 °C in
the presence of 1/10 volume of the appropriate enzyme dilutions, and at
regular time intervals the production of free reducing ends was
determined using the thiobarbituric acid assay procedure according to
Skoza and Mohos (23). Values were corrected for the amount of
substrate-associated reducing ends determined in the absence of enzyme.
Using a standard curve with 0.5-5 µg of sialic acid, the amount of
thiobarbituric acid-reactive product was converted to nmol of free
reducing ends. One unit was defined as the amount of endoN that forms 1 nmol of reducing ends/min at 37 °C and pH 5.1.
-mercaptoethanol. Gradient
gels were prepared with ProSieve 50 gel solution (BioWhittaker) and run
in Tris/Tricine electrode buffer according to the manufacturer's
instructions for 12% resolving and 3% stacking gels. For Western blot
analysis proteins were blotted onto nitrocellulose membranes
(Schleicher & Schuell), and blots were developed as described (18). For
detection of His6-tagged proteins Penta-His antibody
(Qiagen) was used at a concentration of 1 µg/ml. T7-tagged proteins
were detected with 0.1 µg/ml anti-T7 antibody (Novagen) and
polyclonal anti-endoNE guinea pig serum (18) at a dilution of 1:1000.
For quantification of endoN in bacterial lysates, blots were developed
with the SuperSignal West Femto Luminol solution (Pierce), and signal
intensities were quantified using a Kodak image station 440 CF with
purified T7-tagged endoNF as a standard.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
K1F--
Comparison of the protein sequences of endosialidases from
bacteriophages
K1E (18, 19),
K1F (11), and 63D (accession no.
AB015437) revealed an extended region of about 650 amino acids with
high sequence similarity (indicated as gray boxes in Fig.
1A). This core domain shows
81% amino acid identity between endoNE and endoNF, 52% identity
between endoNF and endoN63D, and 50% identity between endoNE and
endoN63D. However, the N- and C-terminal parts differ in length, and
the N-terminal domains were found to possess only very low sequence
similarity. Of all cloned endosialidases, endoNF has the shortest
C-terminal part, lacking the amino acid clusters shared by endoNE and
endoN63D (indicated as black boxes in Fig. 1A).
Moreover, no enzymatic activity was observed for the recombinantly
expressed endoNF (11). We knew from earlier studies that endoNE lacking
the last 38 amino acids is completely inactive (18) and asked whether
the recombinantly expressed endoNF was inactive because of a truncated
C terminus. By direct sequencing of DNA isolated from bacteriophage
K1F, we reinvestigated the size and sequence of the endoNF gene. In contrast to the published data describing a 2763-bp ORF (11), we
identified a 3195-bp ORF. Although the first 2740 bp are identical in
both sequences, the insertion of two nucleotides (G2741 and G2744) that
caused a premature stop codon could not be confirmed. As shown in Fig.
1B, lack of these two nucleotides resulted in an elongation
of the previously described ORF by 432 bp. The newly identified ORF
encodes a potential 118.9-kDa protein, containing all conserved amino
acid clusters in the C-terminal part found in endoNE and endoN63D (see
Fig. 1A).
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Fig. 1.
A, schematic representation of
endosialidases. The endosialidases of bacteriophages K1E (endoNE),
63D (endoN63D), and
K1F (endoNF) are shown as open bars
with the amino acid numbers on top. Full-length endoNF
refers to the protein encoded by the ORF identified in this paper; the
shorter form of endoNF is the protein described by Petter and Vimr
(11). The protein part with the highest similarity among all
endosialidases is represented by a gray box, and clusters of
highly conserved amino acids in the C-terminal part are shown as
black boxes. The locations of the identified proteolytic
cleavage sites are indicated by arrows. B,
partial sequence of endoNF. The 3'-end of the newly identified 3195-bp
ORF of endoNF (accession no. AJ505988) is shown compared with the
3'-end (bp 2647-2763) of the previously identified ORF (accession no.
M63657). Base pairs 1-2646 of both coding sequences are identical and
therefore not shown in this figure. Nucleotides G2741 and G2744, marked
with asterisks, were not confirmed in the newly identified
ORF. Triplets coding for stop codons are underlined, and a
proteolytic cleavage site located in front of Asp-912 is indicated by
an arrow. Nucleotide and amino acid (bold)
numbers on the right refer to the complete ORFs,
starting with the start codon and the N-terminal methionine,
respectively. The complete coding sequence of endoNF is available in
the EMBL/GenBank/DDBJ data bases under the accession no.
AJ505988.
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Fig. 2.
Proteolytic cleavage of endosialidases.
A, endoNE and endoNF with N-terminal T7 and C-terminal
His6 tags were expressed in E. coli BL21(DE3). 2 h after induction soluble fractions of bacterial lysates were analyzed
by 14% ProSieve SDS-PAGE and immunoblotting using
anti-His6 tag antibody or a combination of
anti-His6 and anti-T7 antibodies. B, schematic
representation of fragments obtained after proteolytic processing.
Molecular masses of N-terminal and C-terminal fragments were calculated
for enzymes with T7 and His6 tags. The amino acid sequences
around the cleavage sites are shown on the right. For endoNF
we propose cleavage after the conserved Ser-911 followed by spontaneous
degradation of Asn-912 (for details, see "Results").
C, the soluble fraction of bacterial lysates containing T7-
and His6-tagged endoNF was passed through a
Ni2+-chelating column. The flow-through (lanes
1, 4, and 7) and all fractions of the eluted
protein peak were analyzed by 12% ProSieve SDS-PAGE and immunoblotting
with the indicated antibodies. The first (lanes 2,
5, and 8) and the last (lanes 3,
6, and 9) peak fraction are shown exemplarily.
Bands corresponding to full-length protein and N- and C-terminal
fragments are indicated with arrows. D,
endosialidase activity was analyzed in the flow-through of the
Ni2+-chelating column, containing exclusively the
N-terminal fragment, and in the last peak fraction, containing only the
C-terminal fragment.
38) resulted in
a noncleaved protein of 86 kDa (Fig. 3C, third
lane), indicating that the complete C-terminal domain is essential
for proper cleavage.
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Fig. 3.
C-terminal truncations of
endosialidases. Schematic representations of wild type
(wt) and C-terminal truncated forms are shown in
A and B for endoNE and endoNF, respectively. The
positions of the identified cleavage site are indicated by
arrowheads. The C-terminal amino acid and the number of
deleted ( ) amino acids are shown. In the case of the C-terminal
fragment of endoNE the numbers of the first and last amino
acid are shown. C, soluble fractions of E. coli
BL21(DE3) expressing C-terminally His6-tagged wild type or
C-terminally truncated constructs of endoNE were analyzed by 12%
ProSieve SDS-PAGE and immunoblotting. A polyclonal anti-endoNE guinea
pig serum was used for detection. A faint 75 kDa band that is also
visible in the first lane showing lysate of mock
transformed bacteria is visualized by cross-reactivity of the
polyclonal serum. Bands corresponding to full-length and cleaved wild
type endoNE are indicated with arrows. D, wild
type and C-terminally truncated endoNF containing an N-terminal T7 tag
were analyzed by 10% SDS-PAGE and immunoblotting using an anti-T7
antibody.
View larger version (26K):
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Fig. 4.
Endosialidase chimera. A,
schematic representation of T7- and His6-tagged chimeric
endosialidases composed of the N-terminal domain of endoNE and the
C-terminal domain of endoNF (endoNE-F) and vice versa
(endoNF-E). Numbers shown in this scheme indicate
the amino acid numbers of the fused fragments, with endoNF-derived
parts in gray. B, wild type and chimeric
endosialidases were expressed in E. coli BL21(DE3). Soluble
fractions of bacterial lysates were analyzed by 14% ProSieve SDS-PAGE
followed by Western blot developed with a combination of anti-T7- and
anti-His6-tag antibodies. Bands corresponding to the N- and
C-terminal fragments of endoNE and endoNF obtained after cleavage of
the wild type proteins (second and fifth
lanes) are indicated with arrows. The soluble
fraction of bacteria transformed with the expression vector was
analyzed in the first lane (mock). C, enzymatic
activities of the chimera were monitored in the soluble fractions using
the thiobarbituric acid assay. Activities were normalized to the amount
of endosialidase in the soluble fractions determined by quantification
of the corresponding bands in enhanced chemiluminescence-stained
Western blots using purified endoN as a standard. The activity of the
wild type enzymes is given as 100%.
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Fig. 5.
Expression and enzymatic activity of
endosialidase mutants. Single amino acid exchanges of the
indicated residues by alanine were introduced in T7- and
His6-tagged endoNE and endoNF by site-directed mutagenesis.
Protein expression in soluble and insoluble fractions of E. coli BL21(DE3) expressing the indicated mutants was analyzed by
14% ProSieve SDS-PAGE and Western blot using anti-T7 and
anti-His6 antibodies. Enzymatic activities were monitored
in the soluble fractions using the thiobarbituric acid assay.
Activities were normalized to the amount of endosialidase in the
soluble fractions determined by quantification of the corresponding
bands in enhanced chemiluminescence-stained Western blots using
purified endoN as a standard. The activity of the wild type enzymes is
given as 100%. Results obtained for endoNE are shown in A
and C, and those for endoNF are in B and
D.
153 and
154) was able to assemble in an oligomeric complex, and only monomeric proteins were detected (lanes 12 and 14 in Fig. 6A). Similar
results were obtained for the endoNE-F and endoNF-E chimeras (see Fig.
6B). Although SDS-resistant complexes have been described
for the phage-associated endoNE (8, 18), no oligomerization was
observed for the recombinant protein (see lane 8 in Fig.
6B). In contrast to the homotrimer formed by endoNF, endoNE
was reported to form hetero-oligomeric complexes together with a 38-kDa
protein (8, 18). The absence of this protein in the recombinant enzyme
fraction (data not shown) might explain the lack of SDS-resistant
complexes. Fusion of the C-terminal part of endoNF to the catalytic
domain of endoNE did not change the monomeric state of the protein.
Interestingly, the enzymatically inactive endoNF-E chimera lost the
ability to assemble into trimers. These results confirm the pivotal
role of the C-terminal domain for folding and/or oligomerization.
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Fig. 6.
Trimer formation of endoNF.
A, wild type and mutant endoNF were expressed in E. coli BL21(DE3), and soluble fractions of the bacterial lysates
were analyzed by 6% SDS-PAGE and Western blot. To visualize
SDS-resistant trimers, one aliquot of each sample was analyzed omitting
the boiling step before electrophoresis. The upper blot was
developed with a combination of anti-T7 and anti-His6
antibody and the lower blot with anti-His6
antibody, exclusively. EndoNF mutants with the indicated amino acid
exchanges are shown in lanes 3-10 and C-terminal truncated
forms in lanes 11-14. Bands corresponding to trimers,
N-terminal fragments, and full-length endoNF are indicated with
arrows. B, wild type endoNF, endoNE, and the
chimera endoNF-E and endoNE-F were monitored for trimer formation as
described above. Bands corresponding to an SDS-resistant complex and
the N-terminal catalytic domains of endoNF and endoNE are marked with
arrows.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
K1E and
K1F,
essential for binding and degradation of the polySia capsule of the
host. For both enzymes proteolytic processing was observed, resulting
in a large N-terminal catalytic domain and a small C-terminal fragment that may serve as an intramolecular chaperone. Although the C-terminal domain is dispensable after cleavage, its presence in the primary translation product is necessary to obtain active
endosialidases. Consequently, truncated enzymes lacking the C-terminal
domain, completely or partially, were found to be inactive. In line
with this observation, expression of the previously cloned endoNF
gene resulted in an inactive enzyme (11). Compared with the full-length gene identified in the present study, the coding sequence identified by
Petter and Vimr (11) lacks the 3'-end coding for the C-terminal domain
of endoNF. This gene truncation is most likely the result of a
sequencing error that led to the identification of a premature stop
codon (see Fig. 1B).
K1-5 (20) and
K5 (28), and the corresponding K5 lyase
of E. coli K5 (29). The protein alignment reveals that the
cleavage site identified for endoNE and endoNF (marked with an
arrowhead in Fig. 7) is highly conserved, implying that
proteolytic processing might be a common feature of all proteins listed
in Fig. 7. The serine residue that was shown in this study to be
essential for proper cleavage (Ser-706 and Ser-911 in endoNE and
endoNF, respectively) is present in all sequences, and the aspartic
acid after the cleavage site is highly conserved with the single
exception of endoNF, where an exchange by asparagine is found.
View larger version (90K):
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Fig. 7.
Multiple alignment of the C-terminal parts of
proteins that share sequence similarity with the C-terminal part of
endoNF. The alignment includes the endosialidases of the
K1-specific phages K1E (X78310),
K1-5 (AF322019),
K1F
(AJ505988), and 63D (AB015437), the neck appendage protein gp12 of the
B. subtilis phage GA-1 (NC_002649), the L-shaped tail fiber
protein of coli phage T5 (X69460), the K5 lyases of the E. coli K5-specific phages
K5 (Y10025) and
K1-5 (AF322019), and
the K5 lyase of E. coli K5 (X96495). Amino acid numbers are
shown on the left. Residues that are identical in all
sequences are shaded with black. Residues that are conserved
in five or more of the nine sequences are enclosed in boxes,
showing identical and similar amino acids in bold. The
position of the cleavage site identified for endoNE and endoNF is
indicated by an arrowhead, and the positions of amino acids
that were exchanged in this study by alanine are marked with an
asterisk. The multiple sequence alignment was performed
using the MultAlin program (37). Alignment of the N-terminal parts of
the proteins is not included in this figure because of the very low
similarity among endosialidases, gp12, L-shaped tail fiber protein
(LTF), and lyases.
Data reported on the endosialidase isolated from the K1-specific phage 63D confirm the presence of a common cleavage site. The activity of the phage-derived enzyme was reported to be associated with a 90-kDa protein (10), although the corresponding gene (GenBank accession number AB015437) encodes a protein of 108.3 kDa. Cleavage after Ser-852 at the proposed cleavage site would release fragments of 93 and 15 kDa, suggesting that in endoN63D cleavage occurs at the same position as in endoNE and endoNF.
The L-shaped tail fiber protein of the T5 phage accelerates adsorption
to the lipopolysaccharide of E. coli F (30). The ltf gene codes for a 148-kDa protein (27), and in agreement with the cleavage site proposed here, processing of a 150-kDa precursor
protein into a 125-kDa protein was reported (30). The presence of a
common cleavage site is also supported by results obtained for the
recombinantly expressed lyase of E. coli K5 which depolymerizes the capsular polysaccharide composed of repeating units
of 4-linked -N-acetylglucosamine and
-glucuronic acid (29, 31). In addition to full-length protein of 89 kDa, a 70-kDa
fragment lacking the C terminus was observed (29), corresponding to the
calculated fragment sizes of 73 and 16.7 kDa. Similar to our results
obtained for endoNE lacking the last 38 amino acids, no activity was
found for the E. coli K5 lyase truncated by only 15 amino
acids (29). Both truncations affect homologous stretches at the very C
terminus, and, obviously, the presence of the complete C-terminal
domain is essential for proper folding and subsequent cleavage of these proteins.
Whether proteolytic maturation occurs by self-cleavage or catalyzed by E. coli proteases will need further analysis. Coupled in vitro transcription-translation of the endoNE gene in an E. coli lysate system resulted in an unprocessed translation product (19). This result argues against an autoprocessing protein. However, no endosialidase activity was detected for the in vitro translation product (19), indicating that the in vitro system might be insufficient in providing a proper folding environment.
Proteolytic processing and complex formation of a phage tail protein were demonstrated recently in molecular detail for the tail lysozyme (gp5) of bacteriophage T4 (32). Cleavage was observed between Ser-351 and Ala-352 when gp5 was incorporated into the phage baseplate or stored at high concentration. Although the released N-terminal part contains the catalytic domain, the C-terminal fragment remains part of the phage particle forming the tip of the cell-puncturing device. Similar to the C-terminal part of endoNF, the C-terminal fragment of gp5 was reported to be necessary for trimerization of three copies of a hetero-oligomeric complex composed of the N- and C-terminal fragment of gp5 and gp27 (32). In contrast to gp5, the C-terminal domain of endoNF is not associated with the endosialidase complex and can be easily separated.
In the case of endoNE, hetero-oligomeric complexes with a tightly associated 38-kDa protein were observed for the phage-derived enzyme (8, 18). This protein might act as an assembly protein and in its absence, no SDS-resistant complexes were observed for the recombinantly expressed protein. Interestingly, the catalytic and the C-terminal domain of recombinant endoNE remain associated after cleavage,2 and dissociation might occur after assembling with the 38-kDa protein. During the preparation of this manuscript, Leggate et al. (33) also reported on proteolytic cleavage of endoNE. In line with our results, no SDS-resistant oligomers were detected for a recombinantly expressed glutathione S-transferase fusion protein of endoNE. However, under nondenaturing conditions, a complex above 250 kDa became visible, indicating trimer formation. Similar to our results obtained for C-terminally truncated endoNF, only monomeric protein was detectable for a 105-amino acid truncated enzyme (33), supporting our finding that the C-terminal domain of endosialidases is essential for oligomerization of the catalytic domain.
The finding that several tail fiber proteins with different functions
contain the same conserved C-terminal domain argues for horizontal
transfer of the corresponding gene fragment. Evidence for horizontal
transfer among tail fiber genes has been reported (34), and the
endosialidase gene of K1F is another example for a mosaic gene
composed of at least three different parts. Although the catalytic
domain shows no similarities to other known phage proteins, significant
similarities have been identified between the first 175 N-terminal
amino acids and the N-terminal parts of the tail fiber protein gp17 of
the coliphages T7 and T3 and the yersiniophage
YeO3 specific for
Yersinia enterocolitica serotype O:3 (35). The N-terminal
part of gp17 attaches the protein to the phage tail (36), and in endoNF
this part might have a similar function. By identification of a
C-terminal domain shared by several tail fiber proteins of distinct
phages (B. subtilis phage GA-1 and coliphages T5), we
provide further evidence for horizontal transfer of tail fiber gene
fragments across different groups of tailed bacteriophages.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Dietmar Linder for protein sequencing and Drs. Françoise Routier and Joe Tiralongo for critical remarks on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant GE 801/3-3 and by the Fonds der Chemischen Industrie.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) AJ505988.
§ To whom correspondence should be addressed. E-mail: muehlenhoff. martina{at}mh-hannover.de.
Present address: BESSY GmbH, Albert-Einstein-Strasse 15, Berlin 12469, Germany.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212048200
2 M. Mühlenhoff, M. Sauerborn, and R. Gerardy-Schahn, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
polySia, poly-2,8-sialic acid;
endoN, endo-N-acetylneuraminidase
(EC 3.2.1.129);
gp, gene product;
ORF, open reading frame;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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