From the Institute of Microbiology of the Academy of
Sciences of the Czech Republic, 14220 Prague 4, Czech Republic and
§ Department of Medicinal Chemistry, University of
Washington, Seattle, Washington 98195
Received for publication, July 20, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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The capacity of adenylate cyclase toxin (ACT) to
penetrate into target cells depends on post-translational
fatty-acylation by the acyltransferase CyaC, which can palmitoylate the
conserved lysines 983 and 860 of ACT. Here, the in vivo
acylating capacity of a set of mutated CyaC acyltransferases was
characterized by two-dimensional gel electrophoresis and mass
spectrometric analyses of the ACT product. Substitutions of the
potentially catalytic serine 20 and histidine 33 residues ablated
acylating activity of CyaC. Conservative replacements of alanine 140 by
glycine (A140G) and valine (A140V) residues, however, affected
selectivity of CyaC for the two acylation sites on ACT. Activation by
the A140G variant of CyaC generated a mixture of bi- and monoacylated
ACT molecules, modified either at both Lys-860 and Lys-983, or
only at Lys-860, respectively. In contrast, the A140V CyaC produced a
nearly 1:1 mixture of nonacylated pro-ACT with ACT monoacylated almost
exclusively at Lys-983. The respective proportion of toxin molecules
acylated at Lys-983 correlated well with the cell-invasive activity of
both ACT mixtures, which was about half of that of ACT fully acylated
on Lys-983 by intact CyaC. These results show that acylation of Lys-860
alone does not confer cell-invasive activity on ACT, whereas acylation
of Lys-983 is necessary and sufficient.
The whooping cough agent, Bordetella pertussis,
secretes a 1706-residue-long RTX1 adenylate cyclase
toxin-hemolysin (ACT, AC-Hly, or CyaA),
which can invade a variety of eukaryotic cells (1, 2). ACT delivers into cells a catalytic adenylate cyclase domain (AC) that is activated by intracellular calmodulin and catalyzes unregulated conversion of ATP
to cAMP (3-6). This impairs microbicidal functions of immune effector
cells and induces apoptosis of lung macrophages (7, 8). In
addition, ACT has the capacity to form small cation-selective membrane
channels that account for its weak hemolytic activity (7-13).
The capacity of ACT to form hemolytic channels and to penetrate target
cell membranes and deliver the AC domain (cell-invasive activity)
depends on a covalent post-translational fatty-acyl modification
(14-16). This is catalyzed by a dedicated protein acyltransferase,
CyaC, which can acylate the Heterogeneity in extent and nature of acyl residues linked to lysines
983 and 860 was observed previously for ACT from different sources.
Native ACT produced by the Bordetella pertussis strain 338 (Bp-ACT) was initially found to be acylated exclusively by palmitoyl residues and only on the lysine 983 (16). In contrast, acylation by a mixture of palmitoleil (cis In a previous study, we have shown that Lys-860 is by itself important
for ACT function and that a conservative substitution of Lys-860 by an
arginine residue drastically reduced the capacity of ACT to insert into
and translocate across target cell membrane (28). This substitution,
however, also prevented the acylation of Lys-860 and it could not be
rigorously excluded that this acylation itself, independent of the
K860R substitution, was important for cell-invasive activity of ACT. In
this study we have generated a set of mutant CyaC acyltransferases,
aiming at a definition of residues that are critical to the process of
ACT acylation. It is shown, by the use of monoacylating variants of
CyaC, that in contrast to acylation of Lys-983, the acylation of
Lys-860 alone does not confer cell-invasive activity on ACT.
Bacterial Strains, Growth Conditions, and Plasmids--
The
E. coli K12 strain XL1-Blue (Stratagene) was used throughout
this work for DNA manipulation and for expression of ACT. Bacteria were
grown at 37 °C in LB medium supplemented with 150 µg/ml
ampicillin. pT7CACT1 is a construct derived from pCACT3 (29), which was
designed for enhanced production of recombinant CyaC-activated ACT in
E. coli (r-Ec-ACT) under control of the IPTG-inducible lacZp promoter (28). To construct
pT7NFLAG-CACT1, a synthetic oligonucleotide,
5'-TATGGATTATAAAGATGACGATGACAAATCACG, encoding the FLAG epitope
(DYKDDDDK), was inserted in-frame into the unique NdeI site
encompassing the ATG start codon of cyaC. The fusion was
verified by DNA sequencing.
Site-directed Mutagenesis of cyaC--
The individual
substitutions were introduced into the cyaC gene by a
two-step PCR mutagenesis procedure using Taq DNA polymerase and appropriate mutagenic and amplification primer pairs listed in
Table I. In a first step, two
complementary mutagenic primers were used in two separate amplification
reactions on the cyaC template DNA to introduce the mutation
into two partially overlapping PCR products. These were purified on
agarose gel and used as a template in a second PCR amplification with
an assembly pair of primers (5'-GGAGATATACATATGGATTATAAAG and
5'-TCTAGAGGATCCTTAGGCGGT) to assemble the whole mutagenized
cyaC gene with the desired substitution. The final product
was recloned as an NdeI-BamHI fragment into the
appropriately digested pT7NFLAG-CACT1, to replace the original cyaC allele. The presence of desired substitutions and the
absence of any additional secondary site mutations were systematically verified by resequencing the entire mutagenized cyaC alleles
within the pT7NFLAG-CACT construct.
Plasmids for coexpression of the truncated ACT Production and Purification of the CyaA-derived
Proteins--
The ACT and ACT High Resolution Two-dimensional Gel Electrophoresis--
The
two-dimensional gel electrophoresis assay for acylation of the ACT Mass Spectrometric Analysis of Protein Acylation--
The
location and identity of the acyl modifications on ACT produced in the
presence of certain NFLAG-CyaC variants were analyzed by LC/MS/MS and
MALDI-TOF MS techniques as described previously (16, 17, 28). Tryptic
and Asp-N fragments were generated according to standard protocols and
separated on 50-µm inner diameter × 12-cm capillary column
packed with Magic 5-µm, 200-Å C18 material (Michrom BioResources,
Auburn, CA), at a flow rate of 150 nl·min
Positive-ion MALDI-TOF mass spectra were measured on a Bruker
BIFLEX-III reflectron time-of-flight mass spectrometer
(Bruker-Franzen, Bremen, Germany) equipped with a SCOUT-384 inlet and
gridless delayed extraction ion source. Ion acceleration voltage was 19 kV, and the reflectron (ion mirror) voltage was set to 20 kV. For
delayed extraction, a 4-kV potential difference between the probe and
the extraction lens was applied with a time delay in the range of
200-400 ns after each laser pulse. Samples were irradiated at a
frequency of 5 Hz by 337-nm photons from a pulsed Laser Science (Cambridge, MA) nitrogen laser. Typically, 20-50 shots were summed into a single mass spectrum. 4-Hydroxy-
Identities of the C16:0 and C16:1 cis Assay of Adenylate Cyclase, Cell Binding, Cell-invasive, and
Hemolytic Activities--
Adenylate cyclase (AC) activities were
measured as described previously in the presence of 1 µM
calmodulin (36). One unit of AC activity corresponds to 1 µmol of
cAMP formed per min at 30 °C, pH 8.0. Cell-invasive AC was
determined as the amount of AC that became protected against externally
added trypsin upon internalization into erythrocytes within 30 min of
incubation (9). The hemolytic activity was measured as the hemoglobin released upon incubation of washed sheep erythrocytes (5 × 108/ml) with the toxins for 270 min, respectively (9).
Erythrocyte binding of the toxins was determined as described in detail
previously (37).
Substitutions of Ser-20 and His-33 Residues Ablate Activity of
CyaC--
The acyl-ACP-dependent acyltransferases
activating RTX toxins exhibit a very high degree of sequence
conservation. This makes identification of functionally essential
residues of CyaC difficult. Substitution of several potentially
catalytic serine, histidine, and cysteine residues of CyaC was,
therefore, performed to probe their functional importance (Fig.
1).
The substitutions were introduced into a CyaC construct tagged by a
FLAG epitope at its N-terminal end (N-FLAG-CyaC). This allowed tracking
of mutant CyaC in vivo and discrimination between loss of
function and enhanced degradation of CyaC. The N-FLAG-CyaC, carrying
various substitutions, were coexpressed in E. coli with the
full-length pro-ACT to assess the capacity of CyaC variants to confer
cell-invasive activity on ACT. In parallel, the capacity of the CyaC
variants to acylate in vivo a C-terminally truncated pro-ACT
As summarized in Table II and documented in Fig.
2A (fields 1-4),
the N-terminal addition of the FLAG epitope did not alter the toxin
activation and acylation capacity of CyaC. Replacement of the highly
conserved cysteine 67 by a serine residue (Cys-67 Alanine 140 Is a Key Structural Residue of CyaC Involved in
Selection of Acylation Sites on ACT--
In 11 of the 13 known toxin
acyltransferases, a Gly-Lys dipeptide is conserved at the position
corresponding to residues 140 and 141 of CyaC, where an Ala-Arg
dipeptide is found (Fig. 1). Moreover, Guzmán-Verri and
colleagues (38) have recently reported that the conservative Gly-128
As shown in Fig. 2B (fields 5-12), both
conservative substitutions of the alanine 140 residue of CyaC by
glycine (A140G) and valine residues (A140V) resulted in nearly complete
loss of production of the biacylated form of ACT
Therefore, the full-length ACT produced in cells expressing intact,
A140G and the A140V variants of CyaC, respectively, were purified close
to homogeneity (Fig. 3) and analyzed by
mass spectrometry (28) to clarify the acylation status of Lys-860 and
Lys-983. For each ACT preparation, the relative abundance of acylated
versus nonacylated peptides covering the two acylation sites
were estimated semiquantitatively, from the relative intensities of
selected ions in reconstructed ion current chromatograms. The identity of the peptides and the character of the linked acyl chains was further
determined by partial sequencing of the peptides upon collisionally
induced dissociation (CID) and analysis of daughter ion spectra, as
illustrated by a typical example of spectra shown in Fig.
4.
In both preparations of ACT activated by either the A140G or the A140V
variant of CyaC, respectively, a similar degree of acylation at the
lysine 983 residue was detected, with about 50% of the peptides
containing lysine 983 acylated by palmitic (C16:0) and/or palmitoleic
(C16:1) fatty acyl groups, and the rest being nonacylated, as
summarized in Table III. However, in the
same ACT preparations a striking difference in the extent of acylation of the lysine 860 residue was observed. Although about 90% of Lys-860
residues were found acylated in ACT activated by A140G-CyaC, only about
10% of Lys-860 residues were acylated in ACT activated by A140V-CyaC.
Almost no nonacylated pro-ACT was present in the preparation acylated
by A140G-CyaC. In contrast, about half of the ACT preparation activated
by the A140V variant of CyaC consisted of nonacylated pro-ACT
molecules, whereas the ACT molecules making the second half of the
preparation were monoacylated almost exclusively on the Lys-983
residue. Hence, both the A140G and A140V substitutions caused a
moderate reduction in the capacity of ACT to acylate the Lys-983
residue, whereas the A140V substitution selectively caused a strong
reduction in acylation of Lys-860 of ACT. Because even such
conservative substitutions differentially affected the capacity of CyaC
to acylate ACT, this suggests that alanine 140 is an important
structural residue involved in interaction of CyaC with the two
acylation sites of the protoxin.
Acylation of Only the Lysine 983 of ACT Is Necessary and Sufficient
for Cell-invasive Activity of the Toxin--
When half of the ACT
preparation consisted of ACT monoacylated on Lys-860 (activation by
A140G-CyaC), or when half of it was nonacylated pro-ACT (activation by
A140V-CyaC), the membrane insertion and cell-invasive AC activities of
both the ACT preparations were very similar, as shown in Table II.
These activities were close to 50% of the activity of ACT acylated by
intact CyaC, and there was a good correlation between the proportion of
ACT molecules acylated on the Lys-983 residue and the cell-invasive
activity of the ACT preparation, as defined by the capacity of ACT
molecules to insert into the membrane of erythrocytes and to deliver
the catalytic AC domain into a compartment where it was protected against externally added trypsin (cf. Tables II and III).
These results clearly show that ACT molecules acylated uniquely at the Lys-860 residue were as noninvasive as the nonacylated pro-ACT molecules. Therefore, only ACT molecules acylated on Lys-983
contributed to cell-invasive activity.
The results reported here suggest that the Ala-140 residue plays
an important structural role in activity of CyaC. The acylation pattern
of Lys-983 and Lys-860 residues of coexpressed ACT was strongly
affected by subtle changes to Ala-140, which consisted of removal of
one methyl group in the A140G variant or addition of two methyl groups
by the A140V substitution. The most straightforward explanation is that
Ala-140 of CyaC directly interacts with the acylation sites on ACT.
Substitutions of Ala-140 could then differently affect binding and/or
the catalytic efficiency of CyaC at the two sites, and this might well
account for the observed acylation differences at Lys-983 and Lys-860
of ACT. It is conceivable that both glycine and valine side chains
introduced at position 140 may cause a similar loss of binding and/or
acylation efficiency of CyaC at the Lys-983 acylation site, whereas
only the shorter glycine side chain may still allow productive CyaC
interaction at the Lys-860 site. The bulkier valine side chain could
then selectively interfere with CyaC binding and/or acylation at the sterically different Lys-860 site.
By analogy to the reaction mechanism of HlyC, CyaC is expected to form
an intermediary acyl-ACP·CyaC complex prior to interaction with the
pro-ACT substrate (23, 25). It cannot be excluded that the Ala-140
substitutions in CyaC may also affect the capacity of CyaC to form
these complexes. However, a simultaneous decrease in acylation at both
the Lys-983 and Lys-860 sites, proportional to the respective
affinities of the complex for these two sites, would be expected if the
substitutions in CyaC would affect formation and availability of the
acyl-ACP·CyaC complexes. This was clearly not the case, because the
A140V and A140G substitutions in CyaC caused simultaneously a similar
decrease of acylation at Lys-983 and a very different effect on
acylation at Lys-860. It is, therefore, more likely that the Ala-140
substitutions affect interaction and/or acyl transfer efficiency of
CyaC at the Lys-983 and Lys-860 sites and not the formation of
acyl-ACP·CyaC complex.
The results further show that the cell-invasive activity of ACT did not
depend on acylation of Lys-860 but did correlate with the extent
of acylation at the Lys-983 residue. Therefore, it can be concluded
that acylation of the lysine 983 is necessary and sufficient at least
for the capacity of ACT to insert into and translocate across the model
target membrane of sheep erythrocytes. These results provide a
rationale for the previous observations, that various preparations of
native and/or recombinant ACT exhibited identical capacity to insert
into the target membranes and to deliver the AC domain into cells,
despite a variable extent of Lys-860 acylation (17). A stable and
essentially complete acylation of Lys-983 by intact CyaC was, indeed,
observed in those preparations. Moreover, recent mass spectrometric
analyses show a batch to batch variation in the degree of Lys-860
acylation even for the native ACT produced by the same
Bordetella strain, suggesting the influence of growth
conditions on the extent of Lys-860 acylation of ACT by
CyaC.2 The sum of the
available data suggests that CyaC and/or its acyl·ACP·CyaC complex
may have a higher affinity for the Lys-983 acylation site of ACT, as
compared with the Lys-860 site, and this may account for the
preferential acylation of Lys-983 under certain physiological conditions. Altogether, these results question the role of Lys-860 acylation in the biological activity of ACT and are in good agreement with our previous observation, that, regardless of its acylation status, Lys-860 is by itself an important structural residue, involved
in toxin interaction with target membranes (28). In this respect, it is
interesting that the sequence of the Lys-860 acylation site is better
conserved among the known RTX toxins, relative to that of the Lys-983
acylation site. It will be important to determine in other target cell
models, such as primary lung macrophages and neutrophils, whether the
variable acylation of the conserved lysine 860 contributes to the
biological activity of the AC toxin, or whether it is just an
evolutionary relict.
We have previously suggested that the acylation of Lys-860 might
account for the observed difference in the propensity of membrane-inserted recombinant (r-Ec-ACT) and native
(Bp-ACT) to oligomerize and to form channels (17). In
contrast to other RTX toxins, such as HlyA, however, the primary
biological activity of ACT appears to consist of delivery of an enzyme
into cells and their intoxication by production of cAMP rather than in
the formation of membrane channels (39). This may suggest why double acylation of HlyA at Lys-564 and Lys-690 is optimal for channel forming
(cytotoxic) activity of HlyA, whereas a single acylation at the
conserved Lys-983 residue appears to be sufficient for membrane
insertion and cell-invasive activity of B. pertussis ACT.
ACT has recently gained a lot of attention as a candidate protective
antigen for acellular pertussis vaccines and as a novel vector for
delivery of viral and tumoral antigens into major histocompatibility complex class I antigen-presenting cells and induction of specific cellular immune responses (29, 40-45). It will be important to identify the factors determining the variability of ACT acylation and
to define the relations between differences in acylation and the
capacity of ACT to interact with various target cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino groups of two internal lysine
residues of ACT, Lys-983 and Lys-860, located within conserved RTX
acylation sites (14-17). The mechanism of this novel type of protein
acylation was recently analyzed in substantial detail for the prototype
RTX toxin-activating and acyl-ACP-dependent protein
acyltransferase HlyC, which acylates the homologous lysines 564 and 690 of the Escherichia coli
-hemolysin HlyA (18-21). Several
residues, including Ser-20 and His-23, were identified as being
potentially involved in acyl transfer catalysis by HlyC (22-24). A
model of the reaction mechanism was proposed, and formation of an
intermediary acyl-ACP·HlyC complex was demonstrated (22-25).
Various acyl-ACP-carrying fatty acids, including the most common
in E. coli, the palmitoyl (C16:0) and pamitoleil (C16:1) residues, were found to be efficiently used in vitro as acyl
donors for modification of HlyA (18, 26). In vivo, however,
HlyC exhibits a high selectivity for C14:0 myristic acid, which
represents only about 2% of total E. coli fatty acids but
was found to constitute about 68% of the acyl chains covalently linked
to lysines 564 and 690 of native HlyA (27). Moreover, the extremely
rare odd carbon-saturated C15:0 and C17:0 fatty-acyl residues
constituted the rest of the in vivo acylation of HlyA from
two different E. coli strains (27). The functional
consequences of this particular in vivo acylation of HlyA
remain unknown.
9 C16:1), palmitoyl (C16:0), and myristoyl (C14:0) fatty-acyl residues was found for the
recombinant r-Ec-ACT activated by CyaC in E. coli
(17, 28). Moreover, in addition to acylation of Lys-983, an incomplete
(60%) acylation was found at Lys-860 of r-Ec-ACT (17, 28).
r-Ec-ACT exhibited also a lower capacity to induce
protective immune response against Bordetella infection in
mice and a significantly lower channel-forming and hemolytic activity,
as compared with Bp-ACT (13, 15, 17, 29, 30). Interestingly,
however, the capacity of both forms of ACT to insert into target
membranes and to deliver the AC domain into cells (cell invasiveness)
was identical (15, 17). We have suggested earlier that acylation of
Lys-860 might be an artifact of recombinant expression of ACT in
E. coli and that it may selectively affect the propensity of
r-Ec-ACT to form the oligomeric channels and to lyse
erythrocytes (17). Recently, however, the r-Bp-ACT produced
by a recombinant B. pertussis 18323 strain was found to be
fully palmitoylated also on Lys-860, whereas its hemolytic activity was
higher than that of r-Ec-ACT (47). This indicates that the
extent of Lys-860 acylation may vary as a function of the strain and
physiological state of the producing bacteria and the impact of Lys-860
acylation on biological activity of ACT needs to be established.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
List of PCR primers used for mutagenesis and assembly of cyaC
alleles
protein together with
the mutant NFLAG-CyaC proteins were obtained as described previously,
by introducing a TAA stop codon at position 1009 of the cyaA
gene. This resulted in production of a truncated ACT protein (ACT
)
with a C-terminal Asp-1008 residue (28). The nonacylated variants of
ACT and ACT
were expressed from plasmids from which the
cyaC gene was deleted as an NdeI-BamHI fragment.
proteins were produced in the
presence or absence of the various N-FLAG-CyaC acyltransferase
derivatives, using the E. coli strain XL1-Blue (Stratagene)
transformed with the appropriate plasmid derivative of pT7NFLAG-CACT.
For ACT production, exponential 500-ml cultures were induced with IPTG
(1 mM), and the extracts of insoluble cell debris after
sonication were prepared in 8 M urea, 50 mM
Tris-HCl, pH 8.0, 0.2 mM CaCl2, as described previously (15). The differently acylated full-length ACT proteins were
further purified by ion-exchange chromatography on DEAE-Sepharose and
phenyl-Sepharose (Amersham Pharmacia Biotech) as described previously
(31). In the final step, the proteins were eluted with 8 M
urea, 50 mM Tris-HCl, pH 8.0, and stored frozen.
has previously been established and was performed as described in
detail elsewhere (28). It allows separation of the non-, mono-, and
biacylated forms of ACT
in whole cell extracts, due to the loss of
one and/or two positive charges upon acylation of the Lys-860 and/or
the Lys-983 residues, respectively, which causes alteration of the
isoelectric point of the protein. Briefly, whole cell extracts of the
respective clones expressing the truncated ACT
protein, together
with the different CyaC acyltransferase variants, were prepared from
exponentially growing cultures induced by IPTG (1 mM) for
3 h. Total protein samples (20 µg) were analyzed by large format
two-dimensional (IEF/SDS-PAGE) gel electrophoresis (32) using the
Investigator system (Oxford Glycosystems). The gels were stained with
Coomassie Blue. We have previously demonstrated that the
two-dimensional gel electrophoresis of whole cell samples provides a
convenient and unambiguous semiquantitative assay for assessment of
ACT
acylation in vivo. It has further been shown by
MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) and tandem mass spectrometry of purified preparations that acylation of
ACT
reflects well the acylation of full-length ACT produced under
similar experimental conditions (28).
1, with a
2-95% acetonitrile gradient (1% acetic acid) over 40 min. Peptide
analysis by microcapillary high pressure liquid chromatography coupled
to a Finnigan TSQ 7000 electrospray tandem quadrupole mass spectrometer
was performed as described in detail elsewhere (33, 34).
-cyano-cinnamic acid was used
as the MALDI matrix. Spectra were calibrated externally using the
monoisotopic [M+H]+ ion of a peptide standard (bombesin,
Aldrich) and reprocessed by Bruker XMASS 5.0 software.
9 fatty acyl groups were
confirmed by capillary GC/MS with electron impact ionization after a
one-step extraction and derivatization procedure (35).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (40K):
[in a new window]
Fig. 1.
Alignment of three conserved portions of the
RTX toxin-activating acyltransferases. The amino acid
sequences of 13 homologous RTX-activating protein
acyltransferases were aligned using the Lasergene software (DNASTAR)
and employing the Clustal method. Three highly conserved sequence
blocks in which amino acid substitutions were introduced are shown, and
the substituted residues are indicated by arrows. The
numbering corresponds to residue positions in the CyaC sequence. The
highly conserved consensus residues are shaded. The
following sequences were aligned. BpCyaC, CyaC of B. pertussis 338, GenBankTM accession number 95513; EcHlyC1, HlyC of
E. coli J96, accession number 123192; EcHlyC2, HlyC of
E. coli 2001, accession number 123195; EcHlyC3, HlyC of
E. coli EDL 933, accession number 2118623; EcHlyC4, HlyC of
E. coli/pHly152, accession number 123208; PhLktC1, LktC of
Pasteurella cf. hemolytica "5943B," accession number
1708224; PhLktC2, LktC of P. hemolytica A1, accession number
126360; PhLktC4, LktC of P. hemolytica A11, accession number
1708217; AaLktC, LktC of Actinobacillus
actinomycetemcomitans JP2, accession number 126359; ApClyC1,
CLY-IC of A. pleuropneumoniae 4074, accession number
1710798; ApClyC2, CLY-IIIC of A. pleuropneumoniae 405, accession number 1173323; AsClyC3, CLY-IIC of A. suis 3714, accession number 1710799.
derivative of ACT was determined by a two-dimensional gel
electrophoresis IEF/SDS-PAGE assay (28). We have previously demonstrated that this allows a straightforward and reliable assessment of the relative abundance of the differently charged non-mono- and
biacylated forms of ACT
in whole cell extracts and that pro-ACT
undergoes identical acylation as the
full-length pro-ACT (28).
Biological activities of the ACT activated in vivo by the CyaC variants
Ser) and of the
serine 68 by a threonine residue (Ser-68
Thr), respectively, had no
observable effect on the toxin-activating capacity of N-FLAG-CyaC
in vivo, as documented in Table II and illustrated for the
S68T-CyaC in Fig. 2A (fields 5 and 6).
In contrast, the ACT
proteins produced in the presence of the Ser-30
Arg (S30R), Ser-30
Trp (S30W), His-33
Asp (H33D), and
His-33
Ser (H33S) variants of CyaC, respectively, migrated in
two-dimensional gels at the position of nonacylated pro-ACT
and
resolved quantitatively from the more acidic monoacylated ACT
K860R
and biacylated ACT
standards (28), when these were added to the
samples (Fig. 2A, fields 7-15). In agreement
with a complete loss of fatty-acyl activation, the toxin activity of
full-length ACT produced in vivo in the presence of the same
CyaC variants was nil, as shown in Table II. However, normal levels of
CyaC proteins carrying the substitutions of Ser-30 and His-33 were
detected in cellular extracts by Western blotting (data not shown).
Collectively, these results suggest that the Ser-30 and His-33 residues
are specifically required for the acylating activity of CyaC. Indeed,
while this work was in progress, the same conclusion on the role of the
corresponding Ser-20 and His-23 residues of HlyC was drawn from an
extensive in vitro characterization of intact and mutant
HlyC variants (22-24).
View larger version (11K):
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Fig. 2.
Analysis of ACT
acylation in vivo by different CyaC variants
using two-dimensional electrophoresis. 50 µg of total protein
extracts of IPTG-induced cells producing the ACT
proteins in the
presence or absence of the various CyaC variants were prepared and
analyzed by two-dimensional (IEF/SDS-PAGE) electrophoresis, as
previously described (28) and outlined under "Experimental
Procedures." Binary or tertiary mixtures of extracts were prepared by
directly mixing equal volumes of extracts containing similar amounts of
total cell proteins. The gels were stained with Coomassie Blue, and gel
sections comprising only the spots of the various forms of
ACT
-derived proteins are shown. The sections were aligned in columns
so that the position of the spots relative to the invariant cell proteins
(positioning standards) in the original individual gels was respected.
Therefore, the relative position of the spots in the columns reflects
their relative position in respect to the anode (+) and cathode (
) in
the original pH gradients after equilibrium isoelectric focusing in the
first dimension. As demonstrated previously (28), the shift of the
ACT
spot toward the anode (+) in respect to position of the
nonacylated pro-ACT
reflects the loss of one or two positive charges
in ACT
due to CyaC-mediated acylation of one or both of the Lys-860
and Lys-983 residues of ACT
, respectively. The arrowheads
labeled b, m, and n indicate the
migration positions of the bi-, mono-, and nonacylated ACT
,
K860R-ACT
, and pro-ACT
standard proteins, respectively.
A, two-dimensional gel electrophoretic analysis of acylation
of ACT
by the CyaC variants carrying substitutions of Ser-30,
His-33, and Thr-68. 1, wt, biacylated ACT
;
2, wt + pro-ACT
, biacylated ACT
plus nonacylated
pro-ACT
; 3, wt +
K860R, biacylated ACT
plus
monoacylated K860R-ACT
; 4, wt +
K860R + pro-ACT
;
5, S68T, ACT
acylated by the S68T-CyaC; 6,
S68T +
K860R; 7, S30R, ACT
activated by S30R-CyaC;
8, S30R + pro-ACT
; 9, wt +
K860R + S30R;
10, S30W, ACT
activated by S30W-CyaC; 11, S30W + pro-ACT
; 12, H33S, ACT
activated by H33S-CyaC;
13, H33S + pro-ACT
; 14, H33D, H33D-CyaC
activated ACT
; 15, wt +
K860R + S30R. B,
two-dimensional gel electrophoretic analysis of acylation of ACT
by
the A140V and A140G variants of CyaC. 1, wt,
ACT
; 2, wt + pro-ACT
; 3, wt +
K860R;
4, wt +
K860R + pro-ACT
; 5, A140G, ACT
acylated by the A140G-CyaC; 6, A140G + wt; 7,
A140G +
K860R; 8, A140G + pro-ACT
; 9,
A140V, ACT
acylated by A140V-CyaC; 10, A140V + wt;
11, A140V +
K860R; 12, A140V + pro-ACT
.
Val and Lys-129
Arg substitutions in HlyC affected the capacity
of HlyC to perform the double acylation of HlyA in vivo. It
was important to examine the impact of substitutions of the
corresponding Ala-140 and Arg-141 residues of CyaC on its in
vivo acylating capacity.
. The ACT
preparations acylated in vivo by the A140G-CyaC consisted
predominantly of monoacylated ACT
and of a small amount (10-15% of
total) of biacylated ACT
, whereas no nonacylated pro-ACT
was
detected by two-dimensional gel electrophoresis (Fig. 2B,
field 5). In contrast, activation by the A140V-CyaC yielded
a mixture of monoacylated ACT
with the nonacylated pro-ACT
(Fig.
2B, field 9).
View larger version (53K):
[in a new window]
Fig. 3.
SDS-PAGE analysis of purified full-length ACT
proteins acylated by different CyaC variants. The proteins were
expressed in the presence of the variants of the activating protein
CyaC in recombinant E. coli K12 strains and purified from
urea extracts of cell debris by DEAE- and phenyl-Sepharose
chromatography as described previously (31). Proteins (2 µg) were
separated on 7.5% acrylamide gel and visualized by Coomassie Blue
staining. Lane 1, ACT acylated by intact N-FLAG-CyaC;
lane 2, ACT acylated by the A140V variant of CyaC;
lane 3, ACT acylated by the A140G variant of CyaC.
View larger version (31K):
[in a new window]
Fig. 4.
Analysis of ACT acylation by mass
spectrometric analysis of proteolytic fragments. CID mass spectra
of the [M+2H]2+ ion (m/z 811) of the
palmitoylated peptide (A) EGVATQTTAYGKC16:0R
() and of its nonacylated analogue EGVATQTTAYGK (B).
The yi and bi series ions (see
Ref. 46 for nomenclature) confirm the presence or absence of epsilon
amino-linked palmitic residues in the tryptic peptide. A difference of
238 mass units (C16:0 palmitate residue) was observed between
the acylated and nonacylated peptides, leading to differing values for
the y ion (C-terminal) series, which is most prominent in
both peptides. The m/z values of fragment ions in the
parentheses refer to the nonacylated peptide. The prominent ion at
m/z 72 is indicative of the small, hydrophobic amino acid
residues Ala, Ile, Leu, or Val.
Semiquantitative mass spectrometric assessment of Lys-983 and Lys-860
acylation in ACT activated by the CyaC variants in vivo
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank William Howald for the GC/MS analysis and Weibin Chen for his assistance with the TSQ 7000 mass spectrometer and the MALDI-TOF.
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FOOTNOTES |
---|
* This work was supported by Grant QLK2-CT-1999-00556 from the 5th Framework Program of the European Union, Grants 310/95/0432 and 310/96/K102 from the Grant Agency of the Czech Republic, Grant A5020907 of the Grant Agency of the Czech Academy of Sciences, and Grants ME167 and VS96149 of the Ministry of Education Youth and Sports of the Czech Republic (to P. S.). Additional funding was provided by the Departments of Biochemistry and Medicinal Chemistry and the School of Pharmacy, University of Washington (to M.H.). The MALDI-TOF was funded under shared instrumentation Grant S10 RR12939-01A1from the National Institutes of Health.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.
¶ Present address: Institute of Microbiology of the Academy of Sciences of the Czech Republic, Prague, Czech Republic.
To whom correspondence should be addressed: Institute of
Microbiology CAS, Videnska 1083, CZ-142 20 Prague 4, Czech Republic. Tel.: 42-02-475-2762; Fax: 42-02-475-2152; E-mail:
sebo@biomed.cas.cz.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M006463200
2 M. Hackett et al., unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
RTX, Repeat in ToXin family protein;
AC, adenylate cyclase;
ACT, adenylate cyclase toxin;
CID, collisionally
induced dissociation;
Hly, hemolysin;
IPTG, isopropyl--D-thiogalactopyranoside;
MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight;
MS, mass
spectrometry;
IEF, isoelectric focusing;
PAGE, polyacrylamide
gel electrophoresis;
LC/MS/MS, liquid chromatography-tandem mass
spectrometry;
GC, gas chromatography.
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REFERENCES |
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