From the Department of Oral Bacteriology, Nagasaki University School of Dentistry, 1-7-1 Sakamoto, Nagasaki 852, Japan
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ABSTRACT |
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The binding motifs of the immunodominant antigen
(Ag) -Ag (Ag 85 complex B) of Mycobacterium kansasii for
human fibronectin were examined using digested fragments. We defined
two fibronectin-binding epitopes on 27 amino acids from 84 to 110 and
on 20 amino acids from 211 to 230. The epitopes were almost conserved
in the closely related Ag 85 complex of other mycobacteria species.
Inhibition of fibronectin binding to intact
-Ag molecules was
observed with peptide-(84-110), but not with peptide-(211-230).
Peptide-(84-110) could also inhibit fibronectin binding to all
components of the Ag 85 complex of Bacillus Calmette-Guérin (Ag
85A, Ag 85B, and Ag 85C). Further study with synthetic peptides defined
11 residues from 98 to 108 as the minimum motif. Six residues
(98FEWYYQ103) were critical for interacting
with fibronectin. The motif revealed no homology to other known
prokaryotic and eukaryotic fibronectin-binding proteins. The defined
motif of
-Ag is novel and unique for mycobacteria.
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INTRODUCTION |
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In recent years, there has been a dramatic increase in mycobacterial disease even in some developed countries (1). The incidence has been associated with an increase in the number of individuals infected with human immunodeficiency virus-1 or patients whose immune systems have been compromised by immunosuppressive agents used to treat other diseases (2). Mycobacterium tuberculosis, Mycobacterium avium-intracellulare complex and Mycobacterium kansasii are the most frequently isolated mycobacteria from AIDS patients (3), and disseminated infection due to M. kansasii is a well established feature of immunocompromised patients (4, 5). Furthermore, M. kansasii has been implicated in pulmonary disease and has been reported as a cause of cutaneous infections and osteomyelitis (6, 7).
The elucidation of the mechanism of these infections and interactions
with host immune systems is needed. We have been particularly interested in proteins secreted by mycobacteria. These proteins may
play important roles not only in the establishment, progress, and
continuation of the infection, but also in the host defense system
since live mycobacteria can provoke protective immunity against
tuberculosis, but killed organisms cannot (8). -Antigen (Ag),1 also known as Ag 85B
(9), Ag 6 (10), and MPT59 (11), is one of the most dominant secretary
proteins (12) and is a major stimulant of cellular and humoral immunity
(11, 13-15). It is widely distributed among M. tuberculosis, Bacillus Calmette-Guérin (BCG) isolated from
Mycobacterium bovis, and atypical mycobacteria (16). This Ag
belongs to the Ag 85 complex, which consists of three structurally
related components, Ag 85A, Ag 85B (
-Ag), and Ag 85C. The complex is
characterized by the ability to bind to human fibronectin (FN) (9) and
has recently been defined as a mycolyltransferase, which is an
important enzyme for unique mycobacterial cell wall synthesis (17).
-Ag induces interferon-
synthesis (18, 19) and protective
immunity against M. tuberculosis infection (20-22) and
mediates attachment of whole bacteria to FN-coated surfaces (23-26).
It has been suggested that binding to FN may represent the first step
in the attachment and entry of mycobacteria into host cells.
-Ag has
been regarded as an important molecule for BCG-mediated antitumor
activity in the treatment of superficial bladder carcinoma (23).
Interestingly, this Ag is a stimulus for human monocytes to induce
tumor necrosis factor-
and this stimulatory effect may be mediated
through plasma FN (27). We have cloned and sequenced the genes encoding
-Ag of BCG (28), M. kansasii (29), M. avium
(30), M. intracellulare (31), and Mycobacterium
scrofulaceum (32). As a continuation of this work, we attempted to
delineate the specificity of the interaction of
-Ag and FN
molecules. The novel motif required for FN binding and the contribution
of the individual residues were investigated.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strain-- BCG substrain Tokyo was used in this study. It was grown at 37 °C in Sauton medium (33). Transformed BCG was maintained with kanamycin (20 µg/ml).
Media and Reagents-- Peroxidase-conjugated swine anti-rabbit immunoglobulins was purchased from Dako A/S Co. (Glostrup, Denmark). FN was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Trypsin, which was modified by reductive alkylation and treated with tosylphenylalanyl chloromethyl ketone, was purchased from Promega (Madison, WI). Reagents used for synthesis and analysis were reagent-grade. Amino acid derivatives were purchased from Watanabe Chemical (Hiroshima, Japan).
Antigens--
M. kansasii -Ag was purified from
culture filtrate (CF) of transformed BCG harboring plasmid pIJK-1,
which contains the gene encoding M. kansasii
-Ag (34).
BCG
-Ag (Ag 85B), Ag 85A, Ag 85C, and MPB51 (35) were purified from
CF derived from BCG. The procedure to purify antigens was developed on
the basis of the purification technique for MPT59 as described
previously (36).
Antibodies-- Rabbit antibodies raised against FN were prepared as described previously (24).
Digestion of M. kansasii -Ag--
Purified M. kansasii
-Ag (1 mg/ml) was digested by overnight incubation at
room temperature with CNBr (10 mg/ml) in 70% formic acid. The reaction
mixture was dried under a stream of N2. M. kansasii
-Ag (125 µg/ml) was also digested with trypsin (5 µg/ml) at 37 °C for 36 h. The digested mixture was
precipitated with 10% (w/v) trichloroacetic acid. These digests were
stored at
20 °C.
Binding of FN to CF Proteins and Digested Fragments of M. kansasii -Ag--
The CF proteins (40 µg) were separated by
two-dimensional gel electrophoresis as described previously (37). The
digested fragments were separated by Tricine
(N-tris(hydroxymethyl)methyl glycine/SDS-polyacrylamide gel
electrophoresis (38) and two-dimensional gel electrophoresis. Gels were
stained with Coomassie Brilliant Blue or transblotted onto Immobilon
membranes (Millipore Corp., Bedford, MA). The membranes were blocked
with 3% bovine serum albumin in phosphate-buffered saline (BSA/PBS)
for 1 h at 37 °C and probed with FN at 10 µg/ml in BSA/PBS
for 1 h at 37 °C. Membranes were washed with 0.05% Tween 20 in
PBS at 37 °C, and bound FN was detected with anti-FN antibodies
followed by peroxidase-conjugated swine anti-rabbit immunoglobulins.
Enzyme activity was visualized with 3,3
-diaminobenzidine and hydrogen
peroxide in 0.05 M Tris-HCl, pH 7.5.
Protein Sequencing-- The blotted membranes were stained with Coomassie Brilliant Blue. The stained bands and spots were cut out and applied to an Applied Biosystems 477A gas-phase protein sequencer (Applied Biosystems, Foster, CA). Then, the sequence of the five N-terminal amino acid residues of each sample was determined.
Synthesis of Peptides-- The peptides were synthesized using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry (39). The synthesized peptides were purified by high pressure liquid chromatography with a RESOURCE RPC reversed-phase column (6.4 × 30 mm, 1 ml; Pharmacia Biotech, Inc., Uppsala, Sweden). Elution was carried out with a linear gradient of 16-32% acetonitrile in 0.1% NH4HCO3, pH 8.3, for 20 min and was monitored at 220 nm. For purification of only peptide-(211-230), 0.1% trifluoroacetic acid was used as buffer. The final products were identified by amino acid analysis. The peptides were lyophilized, and the concentrations were defined by weight.
Enzyme-linked Immunosorbent Assay (ELISA) for FN Binding-- The FN binding to proteins was examined by solid-phase ELISA. SUMILON 96-well ELISA plates (Sumitomo Bakelite Co., Ltd. Tokyo, Japan) were coated for 2 h at 37 °C with 0.5 µg of proteins/well in carbonate buffer, pH 9.6. Nonspecific sites were blocked by incubation with BSA/PBS. After washing, the plates were incubated with 2 µg of FN/well in BSA/PBS for 1 h at 37 °C. Bound FN was determined by rabbit anti-FN antibodies and peroxidase-conjugated swine anti-rabbit immunoglobulins and was subsequently developed with o-phenylendiamine dihydrochloride. The abilities of synthetic peptides to bind to FN were determined as follows. SUMILON 96-well ELISA plates were coated with 100 µl of 6.28 µM peptides in carbonate buffer, pH 9.6, for 24 h at 37 °C. After blocking nonspecific sites with BSA/PBS, the quantity of FN (2 µg/well in BSA/PBS) bound after 1 h of incubation at 37 °C was assayed as described above.
Peptide Inhibition Assay: FN and -Ag--
The capacity of
synthetic peptides to interfere with protein binding to FN was
examined. SUMILON 96-well ELISA plates were coated for 2 h at
37 °C with 0.5 µg of proteins/well in PBS, pH 7.2. Then,
nonspecific sites were blocked by incubation with BSA/PBS as described
above. FN in BSA/PBS (20 µg/ml) was preincubated with the peptide at
15 µM for 1 h at 37 °C. Then, 100 µl of
FN/peptide mixture was added to the wells. FN bound to solid-phase
proteins was then assayed as described above.
Sequence Data Analysis-- The programs FASTA, TFASTA, and BLAST in DDBJ (Shizuoka, Japan) were used to search amino acid sequence homologies in the DDBJ, EMBL, GenBankTM, PIR, and SWISS-PROT data bases. The deduced amino acid sequence alignment was performed using the program ODEN in DDBJ.
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RESULTS |
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FN Binding to CF Proteins-- Fig. 1A shows the two-dimensional gel electrophoresis of CF proteins derived from BCG. The labeled spots were identified as described previously (35). FN bound not only to the Ag 85 complex, but also to MPB51. There were some additional spots around the Ag 85 complex. These spots might be due to streaking of the Ag 85 complex occurring in the first dimension.
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Mapping of the FN-binding Epitope on M. kansasii
-Ag--
Purified M. kansasii
-Ag was digested by
CNBr or trypsin to investigate the FN-binding sites. The digested
fragments were analyzed by Tricine/SDS-polyacrylamide gel
electrophoresis. CNBr digestion generated three bands from M. kansasii
-Ag that had molecular masses of 10.5, 6.2, and 5.8 kDa (Table I, CNBr-1, CNBr-2, and
CNBr-3). Trypsin digestion generated three bands that had molecular
masses of 8.0, 6.1, and 5.7 kDa (Table I, trypsin-1, trypsin-2, and
trypsin-3). The other fragments were too small to see on
Tricine/SDS-polyacrylamide gel electrophoresis. FN bound to the CNBr-3
and trypsin-1 bands (Fig. 1C). In control experiments, M. kansasii
-Ag and the digested bands did not react with
anti-FN antibodies. The sequence of the first five N-terminal residues of each digested band was determined. From the sequence data of the
trypsin-1 band, it became clear that it contained two fragments. The
band was separated into two spots by two-dimensional gel
electrophoresis (Fig. 1D, spots A and
B). FN bound to both spots (Fig. 1E). The amino acid
sequence of M. kansasii
-Ag had already been determined, so N-terminal sequence analysis allowed the identification of the bands
and spots based on their predicted size and cleavage sites with CNBr
and trypsin (Table I). Thus, the suggested key residues for FN binding
were 27 residues (amino acids 84-110) and 4 residues (amino acids
216-219) (Fig. 2).
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Inhibition of FN Binding by Peptides--
We tested whether the
peptides could inhibit the binding of FN to the M. kansasii
-Ag molecule. Peptide-(84-110) inhibited the binding of FN to
M. kansasii
-Ag (Fig. 4).
On the other hand, peptide-(211-230) had no effect on the interaction
of FN and M. kansasii
-Ag even with the peptide
concentration raised to 150 µM (data not shown).
Peptide-(84-110) could also inhibit the binding of FN to BCG
-Ag,
Ag 85A, Ag 85C, and MPB51 (Fig. 4).
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Limiting FN-binding Motif--
Using a series of systematically
shortened lengths, a fine FN-binding motif was identified (Fig.
5). A significantly decreased inhibition
of FN binding to M. kansasii -Ag was associated with the
removal of Phe98. Deletion of Val108 from the C
terminus resulted in complete loss of inhibition. The critical residues
required for FN binding were 11 residues (FEWYYQSGLSV) that
corresponded to peptide-(98-108).
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Analysis of the Motif by Single Residue-substituted Analogues of
Peptide-(98-108)--
A series of peptides containing a single
substitution with alanine was prepared to identify the residues within
amino acids 98-108 that were critical for binding to FN. Fig.
6 shows the inhibition of FN binding to
M. kansasii -Ag by these peptides. Substitutions at
positions 98-103 dramatically reduced inhibition. The
Glu99
Ala and Tyr102
Ala peptides
reduced the inhibitory activity by 15-17%. The Phe98
Ala, Trp100
Ala, Tyr101
Ala, and
Gln103
Ala peptides gave no inhibition. The
Ser104
Ala, Leu106
Ala,
Ser107
Ala, and Val108
Ala peptides
reduced inhibition slightly. No marked difference was observed in the
Gly105
Ala peptide.
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DISCUSSION |
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Pathogenic bacteria attach to their preferred host target by
molecules on the bacterial surface, termed adhesins, that recognize cognate host-cell receptors (40). -Ag and its families have been
suggested to be one of the bacterial ligands that can achieve attachment to a bridging ligand of host origin extracellular matrix FN.
This Ag is specific for mycobacteria, and its interaction with FN might
also enable the pathogen to reach a natural pathway determined by the
bridging ligand FN-host receptor interaction. We examined the
interaction of
-Ag with FN to elucidate its role in mycobacterial
infection.
At least two different FN-binding epitopes were defined in M. kansasii -Ag, amino acids 84-110 and 211-230. There is no
sequence similarity between defined epitopes. These epitopes have no
homology to other known prokaryotic and eukaryotic FN-binding proteins except the Ag 85 complex. Therefore, the Ag 85 complex might have unique abilities to bind to FN.
In the peptide inhibition assay, only peptide-(84-110) could inhibit
the binding of FN to intact M. kansasii -Ag. Each peptide had no effect on the binding of FN to the other (data not shown). The
relative positions of these two epitopes within the three-dimensional structure of M. kansasii
-Ag are still unknown, but the
results indicate that the multiple epitopes of M. kansasii
-Ag may work separately for binding to FN. Amino acids 211-230
might be hidden in the intact natural molecule and not hidden in the
denatured state. In contrast, amino acids 84-110 might be exposed on
the surface of the molecule and work as a major domain to bind to FN.
The multiple FN-binding regions had been previously reported in -Ag
of Mycobacterium leprae (25) and M. bovis (41),
but their role in the natural
-Ag molecule in FN binding had not yet
been examined. In this study, we defined a new region (amino acids
84-110) that might play a very important role in FN binding to the
-Ag molecule. This region and its surrounding sequence are almost
identical among mycobacterial species including the other structurally
related components, Ag 85A and Ag 85C. Peptide-(84-110) could inhibit
the binding of FN to all components of the Ag 85 complex of BCG. The
defined region may contain the common motif of the Ag 85 complex for
binding to FN.
We attempted successively to determine the common motif of -Ag using
a series of peptides that truncated at the N and C termini. The peptide
inhibition assay was performed to exclude the artifacts that might
result from a different efficiency in peptide fixation to the ELISA
plates. Concerning deletion at Ala97, ELISA could not
detect the binding of FN to the wells that were coated with the peptide
(data not shown), but the same peptide showed the full binding
inhibition of FN as an original peptide. Ala97 might work
to interact with the ELISA plates, but not with FN. The results
indicated that the binding motif contained 11 residues, 98FEWYYQSGLSV108.
Further study defined critical amino acid residues in this motif using analogous peptides that were substituted with alanines. Substitution with alanine allowed essential residues of the motif for binding to FN to be determined. The negatively charged residue Glu99, the polar residue Gln103, and the aromatic residues Phe98, Trp100, Tyr101, and Tyr102 seem to be essential for binding to FN. Peptide-(98-108) also could inhibit FN binding to the Ag 85 complex of BCG (data not shown). Comparing the amino acid sequences among these antigens revealed that the defined FN-binding motif allowed some amino acid substitutions. The aromatic residues Trp100 and Tyr102 can change place with the negatively charged residues Glu and Asp, respectively (Fig. 7). These positions might be substituted with other negatively charged residues.
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The substitution Val108 Ala did not affect the
inhibition ability. However, the deletion of this residue removed the
binding ability completely. Therefore, the residue at this position is absolutely required, although it can be replaced with other hydrophobic residues.
This is the first report on the ability of MPB51 to bind to FN. The secreted protein MPB51 is one of the major proteins in the CF derived from BCG and immunologically cross-reacts with the Ag 85 complex. We have defined the complete sequence of this Ag. MPB51 showed 37-43% homology to the components of the Ag 85 complex (35). MPB51 could bind to FN. Peptide-(84-110) could inhibit the binding of FN to MPB51. Interestingly, there is no sequence similarity between the peptide and MPB51. The Ag 85 complex and MPB51 might share the same binding position on FN. It is very meaningful to analyze their roles in pathogenesis and host immunity.
The motif does not overlap with the region that corresponds to the
monoclonal antibody HYT27 binding determinant, amino acids 111-119
(42). There is good agreement with previous studies that HYT27 failed
to block -Ag binding to FN (23). Immunoglobulins that recognize the
FN-binding motif may enhance mycobacterial binding to FN. The knowledge
presented in this study might be useful in the control of mycobacterial
infection. We propose the motif, particularly the key residues
98FEWYYQ103, as a possible candidate component
of a subunit synthetic vaccine against mycobacterial infection.
Studies to examine the binding sites on FN molecules that interact with
-Ag are currently underway in our laboratory. Our findings
may contribute to clarification of the roles of
-Ag in the
mycobacteria-host cell relationship.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Niidome for helpful technical support, suggestions, and discussions. We also thank Dr. H. Kitaura, Dr. M. Takano-Shirai, and H. Yukitake for suggestions and encouragement.
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FOOTNOTES |
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* This work was supported in part by grants from the Human Science Foundation and the Sasakawa Memorial Health Foundation.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.
To whom correspondence and reprint requests should be addressed.
Tel.: 81-95-849-7649; Fax: 81-95-849-7650; E-mail: mnaito{at}net.nagasaki-u.ac.jp.
1 The abbreviations used are: Ag, antigen; BCG, Bacillus Calmette-Guérin; FN, fibronectin; CF, culture filtrate; Tricine, N-tris(hydroxymethyl)methylglycine; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
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