Journal of Bacteriology, August 1999, p. 5094-5098, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Demonstration by Mass Spectrometry that Purified
Native Treponema pallidum Rare Outer Membrane Protein 1 (Tromp1) Has a Cleaved Signal Peptide
David R.
Blanco,1,2,*
Julian P.
Whitelegge,3,4
James N.
Miller,1 and
Michael A.
Lovett1,2
Department of Microbiology, Immunology, and
Molecular Genetics1 and Department of
Medicine,2 School of Medicine,
The Pasarow Mass Spectrometry Laboratory,3 and
Department of Chemistry and
Biochemistry,4 University of California at Los
Angeles, Los Angeles, California 90095
Received 11 March 1999/Accepted 10 June 1999
 |
ABSTRACT |
Purified native Tromp1 was subjected to mass spectrometric analysis
in order to determine conclusively whether this protein possesses a
cleaved or uncleaved signal peptide. The molecular masses of Tromp1,
three Treponema pallidum lipoproteins, and a bovine serum
albumin (BSA) control were determined by matrix-assisted laser
desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The
molecular masses of all of the T. pallidum lipoproteins and BSA were within 0.7% of their respective calculated masses. The molecular mass of Tromp1 was 31,510 Da, which is consistent with a
signal-less form of Tromp1, given a calculated mass of unprocessed Tromp1 of 33,571 Da, a difference of 2,061 Da (a 6.5% difference). Purified native Tromp1 was also subjected to MALDI-TOF analysis in
comparison to recombinant Tromp1 following cyanogen bromide cleavage,
which further confirmed the identity of Tromp1 and showed that native
Tromp1 was not degraded at the carboxy terminus. These studies confirm
that Tromp1 is processed and does not contain an uncleaved signal
peptide as previously reported.
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TEXT |
Treponema pallidum subsp.
pallidum, the etiologic agent of venereal syphilis, has for
over a decade been known to possess a unique outer membrane containing
an extremely low density of membrane-spanning surface-exposed protein
(17, 20). It is believed that these T. pallidum
rare outer membrane proteins, termed TROMPs (6), are the
only surface-exposed antigens on this organism and therefore represent
the key surface targets for protective host immune mechanisms which
develop during syphilitic infection.
In our previous attempts to identify potential TROMP candidates, two
proteins of 31 and 28 kDa were found to be markedly enriched in outer
membranes isolated from T. pallidum (5). The
31-kDa protein, termed Tromp1, was found to have properties consistent with those of an outer membrane porin protein, including amphiphilicity following phase separation in the detergent Triton X-114 and electrical conductivity when analyzed in planar lipid bilayers (3). It was also determined that recombinant Tromp1, when expressed, exported, and targeted to Escherichia coli outer membranes, also
exhibited porin activity similar to that measured for native Tromp1
(4). Tromp1 has also been found to have 26 to 28% sequence
identity to adhesin proteins found in the streptococcal family
(12), suggesting a potential role as a virulence determinant.
While our studies have shown that Tromp1 is a porin protein, recent
studies by Hardham et al. (10) have found that Tromp1 is
also part of an operon which possesses similarities to ABC transporter
systems and that Tromp1, also called TroA in these studies, has 28%
sequence identity to periplasmic binding proteins of these ABC
transporter operons. This apparent disparity between the demonstrated
outer membrane location and porin activity of Tromp1 and the suggestion
that TroA is a periplasmic binding protein from homology
comparisons is an area of research which is currently being
investigated. It has also been recently reported by Akins et al.
(2) that Tromp1 possesses an uncleaved signal peptide, which
these investigators conclude anchors Tromp1 to the inner membrane and
accounts for its demonstrated hydrophobicity. Because Tromp1 possesses
an N terminus blocked to Edman sequencing, the conclusion that Tromp1
is uncleaved was based upon a sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE)-based size comparison
of native Tromp1 and that generated from an in vitro translation
product. Thus, the findings of these studies have resulted in one
current view that Tromp1 is not an outer membrane protein but rather a
periplasmic binding protein anchored to the inner membrane by an
uncleaved signal peptide.
In order to begin to address the controversy surrounding Tromp1, we
have isolated and purified the hydrophobic form of native Tromp1 for
mass spectrometric analysis to determine conclusively whether this
protein possesses a cleaved or uncleaved signal peptide. The findings
reported here demonstrate conclusively that Tromp1 has a cleaved signal
peptide and is therefore not anchored to the inner membrane by an
uncleaved signal peptide as previously reported (2). Also
implicit from these findings is that the hydrophobicity of Tromp1 is
not due to an uncleaved signal peptide, as reported previously, but is
rather an inherent property of this protein, which we believe is
consistent with its outer membrane location.
Isolation and purity of native Tromp1 and three other
T. pallidum hydrophobic proteins.
Native hydrophobic
forms of Tromp1, the 47-kDa lipoprotein, the MglB homolog lipoprotein
(41 kDa), and the TmpC lipoprotein (35 kDa) were isolated from
approximately 2 × 1011 T. pallidum cells
as follows. T. pallidum subsp. pallidum (Nichols strain) was extracted in phosphate-buffered saline (PBS; pH 7.2) from
20 intratesticularly infected rabbits as previously described (13). Approximately 800 ml of treponemal extract was
centrifuged twice at 400 × g for 10 min each time in
order to pellet gross tissue debris and then at 20,000 × g for 30 min in order to pellet the treponemes. The treponemal
pellet was washed in 200 ml of PBS and then recentrifuged at
20,000 × g for 30 min. The final treponemal pellet was
resuspended in 26 ml of ice-cold PBS to which was added 4 ml of
ice-cold 10% hydrogenated Triton X-114 (Calbiochem, San Diego, Calif.)
to yield a final detergent concentration of 2%. The suspension was
next incubated on a rocker at 4°C for 2 h in order to solubilize
the outer membrane. After this incubation, the suspension was
centrifuged at 20,000 × g in order to remove T. pallidum protoplasmic cylinders and the supernatant was removed and warmed to 37°C for 5 min, which resulted in cloud formation of
the detergent. The suspension was then centrifuged at 3,000 × g in order to yield separated hydrophobic (bottom) and aqueous (top) phases. The hydrophobic phase was recovered (approximately 2 ml),
extracted twice with 40 ml of warmed PBS, and then centrifuged as
described above. The final extracted hydrophobic phase (approximately 2 ml) was then combined with 30 ml of ice-cold acetone, incubated for
2 h at 4°C, and then centrifuged at 10,000 × g
for 30 min to recover precipitated protein. The protein pellet was then
subjected to two-dimensional SDS-PAGE as previously described
(5). After electrophoresis, proteins in the gel were
transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore,
Bedford, Mass.) as previously described (18), stained with
0.2% amido black in water for 20 min, and destained by using distilled
water. Protein spots corresponding to Tromp1, the 47-kDa lipoprotein,
the MglB lipoprotein, and the TmpC lipoprotein were identified based
upon their respective molecular weights and isoelectric points as
previously described (15).
In order to elute the proteins from the membrane, the section of the
PVDF membrane corresponding to each of these proteins was cut out and
placed in 250 µl of 2% SDS-1% hydrogenated Triton X-100-50 mM
Tris, pH 9.0 (1). The suspensions were incubated on a shaker
for 16 h at room temperature and then centrifuged at
13,000 × g for 10 min. Samples from each of the
supernatants were analyzed by SDS-PAGE (12% polyacrylamide) and
identified by silver staining (Bio-Rad). The total amount of protein
recovered from each of the eluted samples was estimated to be 3 to 5 µg.
As shown in Fig. 1, 1/25 (approximately
200 ng) of each sample analyzed showed a single band corresponding to a
molecular mass consistent with that of Tromp1 (31 kDa), the 47-kDa
lipoprotein, the 41-kDa MglB homolog lipoprotein, and the 35-kDa TmpC
lipoprotein. In addition, Tromp1 was further analyzed by
two-dimensional immunoblotting using specific anti-Tromp1 serum and
enhanced chemiluminescence as previously described (4). As
shown in Fig. 2, the amido black-stained
immunoblot showed a doublet at 31 kDa (Fig. 2A) corresponding to the
molecular mass and pI (6.6) reported previously for Tromp1
(3). The stained 31-kDa doublet reacted specifically with
anti-Tromp1 serum (Fig. 2B), confirming that the isolated protein was
Tromp1.
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FIG. 1.
SDS-PAGE, followed by silver staining, of isolated
native Tromp1, the 47-kDa lipoprotein (47-kDa LP), the 41-kDa Mglb
homolog lipoprotein (Mglb), and the 35-kDa TmpC lipoprotein (TmpC).
Approximately 200 ng of each isolated protein was combined with sample
buffer containing 2% SDS and 5% 2-mercaptoethanol and boiled prior to
electrophoresis. The values on the left indicate the positions of
molecular weight standards (103).
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FIG. 2.
Two-dimensional immunoblot analysis of isolated native
Tromp1. Approximately 500 ng of isolated native Tromp1 was separated in
the first dimension by denaturing isoelectric focusing (IEF) and then
in the second dimension by SDS-PAGE. Proteins were then transferred to
a PVDF membrane. (A) Amido black-stained PVDF membrane showing the
detection of only Tromp1. (B) Membrane in panel A probed with specific
anti-Tromp1 serum, confirming the detection of Tromp1. The values on
the left indicate the positions of molecular weight standards
(103).
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MALDI mass spectrometry of purified native Tromp1.
To obtain
an accurate measurement of the molecular mass of Tromp1, its mass
spectrum was recorded by using matrix-assisted laser desorption
ionization (MALDI) time-of-flight (TOF) mass spectrometry. MALDI-TOF
mass spectrometry was performed by using a Voyager RP machine
(Perceptive Biosystems, Framingham, Mass.) operating in linear mode.
The three other T. pallidum membrane lipoproteins and bovine
serum albumin (BSA) were also analyzed as controls. For sample
preparation, 0.3 µl of chloroform-methanol-precipitated protein
(21) (1 to 10 pmol) dissolved in 60% HCOOH (2 to 4 µl) was mixed with 0.5 µl of a 20-mg/ml matrix solution
(2,5-dihydroxybenzoic acid; Aldrich, Milwaukee, Wis.) in 50%
HCOOH-50% isopropanol and dried immediately on the MALDI plate.
The measured masses of the lipoproteins and BSA, as well as their
calculated masses based upon their gene sequences and posttranslational modifications, are presented in Table 1.
Reasonable agreement between the measured and calculated masses was
obtained for the three control lipoproteins and BSA, with discrepancies
of 0.7% or less. Mass accuracy of 0.1% is expected for the MALDI-TOF
technique using internal calibration, as we used for these experiments, and thus it was concluded that the proteins had been modestly modified
during the isolation process. Methionine oxidation and acrylamide
adducts of cysteine are expected on proteins exposed to SDS-PAGE.
Nevertheless, the measured masses support the structural assignment of
N-palmitoyl,
S-[2,3-bis(palmitoyloxy)-propyl] to the N-terminal
cysteine of these lipoproteins and support translational termination at
the first stop codon in the case of the 47-kDa lipoprotein.
As shown in Fig. 3, the MALDI-TOF mass
spectrum of Tromp1 showed strong ions for the singly charged protein
(M+H+) and the doubly charged ion (M+2H+); the
triply charged ion was also visible (M+3H+). The measured
mass of Tromp1 was found to be, whether in the presence or absence of
an internal BSA standard, 31,510 Da (Fig. 3), which is in disagreement
with the calculated mass of 33,571 Da for an unprocessed translation
product (6.5% error). This mass determination for Tromp1 was
consistent and reproducible following several separate experiments. A
much closer agreement between the measured and calculated masses was
obtained for a cleaved Tromp1 translation product (Table 1). A better
match between masses is achieved if the THA leader peptidase I cleavage
site in the Tromp1 signal peptide is invoked, although the calculated mass (31,182 Da) is lower than the measured mass (1% error). Arbitrary cleavage N terminal to Thr20 of the THA motif improves the match considerably (0.1% error). These results demonstrate that the native
Tromp1 protein is cleaved to size from its original translation product. However, the level of accuracy in this analysis precludes assignment of the true N terminus and, formally, cleavage of the C
terminus must also be considered.
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FIG. 3.
Molecular mass spectrum of native Tromp1. The mass
spectrum was recorded with a Perceptive Biosystems Voyager RP with
laser intensity sufficient to achieve efficient ionization. The singly
and doubly charged ions of BSA were used to calibrate the mass
spectrometer, and similar results were obtained whether an internal
calibration was used or not. dhb, 2,5-dihydroxybenzoic acid matrix
solution.
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MALDI mass spectrometric comparison of native and recombinant
Tromp1.
Native Tromp1 and a signal-less form of recombinant Tromp1
were treated with CNBr, and the fragments were probed by MALDI-TOF mass
spectrometry in order to compare the observed peptide maps with
theoretical ones based upon the known gene sequence and potential cleavage sites.
A signal-less form of recombinant Tromp1, corresponding to the signal
peptide cleavage site of alanine-phenylalanine-glycine (AFG), was
generated as follows. An N-terminal primer
(5'-CGCCATATGAGCAAGGATGCCGCAGCAGAC-3'; the
underlined region is the tromp1 gene sequence)
corresponding to signal peptide cleavage after AFG was generated
containing an NdeI restriction endonuclease site at the 5'
end (Gibco BRL, Gaithersburg, Md.). This construct results in a single
methionine residue placed ahead of the site of signal peptide cleavage
for the purpose of translation. A C-terminal primer consisting of 5'-CGCGGATCCCTAGCGAGCCAACGCAGCAA-3' and
corresponding to the end of the tromp1 gene was generated
containing a BamHI restriction endonuclease site at the 5'
end. A PCR was performed with these primers as described previously
(4). The tromp1 PCR product was ligated into
pET17b (Novagen, Inc.), which had previously been digested with
NdeI and BamHI. The resulting construct was transformed into E. coli BL21 DE3/pLysE (Novagen, Inc.)
using cells made competent by CaCl2. Expression and
fast-performance liquid chromatography purification of recombinant
Tromp1 were performed as described previously (4).
For CNBr treatment of native and recombinant Tromp1,
chloroform-methanol-precipitated protein pellets were dissolved in 35 µl of a 1-g/ml saturated solution of CNBr (Sigma, St. Louis, Mo.) in
90% formic acid (Fisher, Fair Lawn, N.J.) to which was added 15 µl
of water. A final CNBr/Met molar ratio of 500 was exceeded in all
digestions. CNBr digestion was carried out for 4 h in the dark at
room temperature. The reaction mixture was then dried (SpeedVac),
redissolved in 250 µl of 0.1% trifluoroacetic acid (Pierce,
Rockford, Ill.), and dried again. For MALDI-TOF mass spectrometry, 0.3 µl of the CNBr-digested samples (1 to 2 pmol) dissolved in 60% HCOOH
(2 to 4 µl) was mixed with 0.5 µl of a 10-mg/ml matrix solution
(
-hydroxycinnamic acid; Aldrich) in 0.1% trifluoroacetic acid-70%
CH3CN and dried immediately on the MALDI plate. Bovine
insulin was used as a standard.
Table 2 summarizes the results of
MALDI-TOF mass spectrometric analysis of CNBr-treated recombinant and
native Tromp1. Central to the interpretation of the mapping experiments
is the appearance of peptides derived from the N and C termini of the
protein. The C-terminal peptide (peptide 8) was observed in both cases,
providing conclusive evidence that any processing was not at the C
terminus. Internal peptides (peptides 3, 4, and 6) were well
represented in both proteins, proving that the protein analyzed was
Tromp1. In addition, the set of the native Tromp1 peptides used to
search the National Center for Biotechnology Information protein
databases was found to match only Tromp1 and TroA, confirming that the
peptides analyzed were from Tromp1. In native Tromp1, N-terminal data
set peptides with masses agreeing well with those of peptide 2 and hybrid peptide 2/3 were observed, whereas only peptide 2 was observed in the recombinant protein. In the case of native Tromp1, the masses of
the peptides that were observed support a model with cleavage between
residues 19 and 20 generating a novel set of CNBr fragments that were
not observed in the map of the uncleaved protein.
The results presented here, in contrast to the previous report by Akins
et al. (2), conclusively demonstrate that Tromp1 has a
processed and cleaved signal peptide. Mass spectrometry analysis of
native Tromp1 resulted in a molecular mass of 31,510 Da, consistent
with a processed form of this protein given a calculated mass of
unprocessed Tromp1 of 33,571 Da, a difference of 2,061 Da, which is the
average size of a 19-residue signal peptide (19). This
result was also reproducible whether an internal BSA protein control
was included in or excluded from the sample containing Tromp1. Further,
mass spectrometry peptide analysis of CNBr-treated Tromp1 confirmed
that the sample tested was Tromp1 and that it was not degraded from the
carboxy terminus. In addition, three T. pallidum
lipoproteins, isolated the same way as Tromp1 from the hydrophobic
phase of a Triton X-114 detergent extract, were also analyzed in order
to confirm the validity of the molecular mass results obtained for
T. pallidum proteins isolated by this method. The molecular
masses obtained for the three lipoproteins were all within 0.7% of
their calculated values given the addition for each of these
lipoproteins of a covalent association of glycerol and three
molecules of the fatty acid palmitate. Therefore, mass spectrometry
analysis, which provides the most reliable assessment of molecular
mass, has proven that native Tromp1 possesses a cleaved signal peptide.
Of the three potential leader peptidase I cleavage sites present at the
carboxy terminus of the Tromp1 signal peptide, which include (from the
N to the C terminus) threonine-histidine-alanine (THA),
alanine-phenylalanine-glycine (AFG), and alanine-alanine-alanine (AAA),
theoretical cleavage following THA results in a calculated molecular
mass of the mature protein having the closest agreement with the mass
spectrometry result of native Tromp1 (1% error). THA is also the
cleavage site predicted for the Tromp1 signal peptide by the SignalP
analysis program (14). However, given that a mass accuracy
of 0.1% is expected for the MALDI-TOF technique, prediction of the
cleavage site based upon this level of accuracy would place the
cleavage site N terminal to the threonine of the THA motif. It is
unlikely that this is the actual N terminus of the mature protein
because this would require the upstream sequence of
threonine-glycine-phenylalanine to be recognized as the cleavage motif,
which is not a classic leader peptidase I cleavage recognition site
(19).
Purified native Tromp1 and a signal-less form of recombinant Tromp1
were also analyzed in this study by mass spectrometry following CNBr
cleavage. The intact-protein mass data combined with the CNBr mapping
data provide overwhelming evidence that the observed protein is indeed
Tromp1 and that again it is cleaved to a significantly shorter length
than that predicted from the uncleaved protein. CNBr mapping data can
be accommodated with cleavage between amino acids 19 and 20 or
thereabout, bearing in mind the N-terminal modification implied by
blockage to Edman sequencing. Definitive structural assignment of the N
terminus of Tromp1 requires further biochemical analyses. Central to
these analyses will be electrospray-ionization mass spectrometry, which has recently been applied to membrane proteins (23) and has, indeed, been used to measure the molecular weight of recombinant Tromp1
(22). Resolution by electrospray-ionization mass
spectrometry should solve the heterogeneity of Tromp1 apparent in gels.
In the previous study by Akins et al. (2), their conclusion
that Tromp1 possesses an uncleaved signal peptide was used to explain
the hydrophobicity of Tromp1 and suggest its anchoring to the inner
membrane. In contrast, our latest studies have indicated that the
hydrophobicity of Tromp1 is due to reasons other than an uncleaved
signal peptide. Further, our previous studies showed that treatment of
T. pallidum with low concentrations of the nonionic detergent Triton X-114, which completely solubilizes the T. pallidum outer membrane without solubilizing the inner membrane
(7, 16), resulted in the complete release of Tromp1 with no
residual detection of Tromp1 in the inner membrane protoplasmic
cylinder complex (3). Such findings are consistent with the
idea that this hydrophobic protein has an outer membrane origin rather
than being anchored to the inner membrane.
It was also reported by Akins et al. (2), who used a
purified recombinant, that Tromp1 did not show any porin activity by
the liposome swelling assay (8). Similarly, we have also found that with the exception of recombinant Tromp1 targeted to E. coli outer membranes (4), no soluble
recombinant form of Tromp1 tested has shown porin activity when planar
lipid bilayers were used. It should be emphasized, however, that this
is in direct contrast to purified native Tromp1, which we have found to
have consistent and reproducible porin activity following isolation by
isoelectric focusing (3) and more recently by
fast-performance liquid chromatography (9). We believe that
the difference in this demonstrable porin activity between the native
and recombinant proteins may be due to conformation.
In summary, the findings presented in this study conclusively show that
native Tromp1 does, indeed, possess a processed signal peptide and is
therefore an exported protein. These findings, therefore, indicate that
Tromp1 is not anchored to the inner membrane and support the
possibility that Tromp1 is a bona fide outer membrane protein of
T. pallidum.
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ACKNOWLEDGMENTS |
We thank Xiao-Yang Wu for his excellent technical assistance. We
also thank Kym Faull for his support and encouragement.
This work was supported by U.S. Public Health Service grants AI-21352
and AI-12601 to M. A. Lovett and AI-37312 to J. N. Miller. Funds from the UCLA Jonsson Comprehensive Cancer Center (P30-A16042) were used toward the purchase of the MALDI-TOF mass spectrometer used.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Immunology, and Molecular Genetics, School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095. Phone:
(310) 206-6510. Fax: (310) 206-3865. E-mail:
DBLANCO{at}microimmun.medsch.ucla.edu.
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Journal of Bacteriology, August 1999, p. 5094-5098, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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