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INTRODUCTION |
The attachment of mycobacteria to fibronectin is well documented
(1, 2). All tested species including Mycobacterium bovis
strain BCG, Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium avium, Mycobacterium leprae, and Mycobacterium
smegmatis were observed to attach to fibronectin (3-5). In
studies on M. bovis BCG-mediated immunotherapy, which is the
treatment of choice for superficial bladder cancer, fibronectin
attachment was shown to be necessary for the expression of antitumor
activity (2). In addition, the attachment and internalization of
M. bovis BCG, M. avium, and M. leprae
by epithelial cells and Schwann cells also were shown to be dependent
on bacterial attachment to fibronectin (4, 6, 7). Thus, an
understanding of the interaction between mycobacterial proteins
mediating attachment to fibronectin is needed.
Two distinct mycobacterial proteins or protein complexes, fibronectin
attachment protein (FAP)1 and
proteins of the antigen 85 complex, have been linked to mycobacterial attachment to fibronectin (8, 9). The interaction of proteins from the
antigen 85 complex with fibronectin has been characterized using
recombinant 85A, 85B, and 85C proteins (10-12). The best characterized
of the complex is the 85B protein. Using in vitro assays,
the binding of 85B to fibronectin was observed to depend on a FEWYYQ
binding motif (12). This motif is highly conserved among all antigen 85 proteins. Characterization of the interaction of antigen 85 proteins
with fibronectin has been limited to studies defining the binding of
recombinant proteins to fibronectin. The role of antigen 85 proteins
and the FEWYYQ sequence in binding of viable bacteria to fibronectin is
not known.
FAP proteins constitute a family of highly homologous proteins of the
mycobacteria. Polyclonal antibodies to purified Mycobacterium vaccae FAP (FAP-V) also recognize FAP proteins of M. leprae (FAP-L), M. avium (FAP-A), M. bovis
BCG (FAP-B), M. kansasii, M. tuberculosis, and
M. smegmatis (FAP-S) (4, 5, 7). To date, FAP proteins from
four mycobacterial species including M. leprae (FAP-L),
M. avium (FAP-A), M. bovis BCG (FAP-B), and
M. smegmatis (FAP-S) have been cloned and characterized (4,
5).2 Using recombinant FAP-A
and FAP-A peptides, two non-continuous fibronectin binding regions
(amino acids 177-201 (FAP-A-(177-201)) and amino acids 269-292
(FAP-A-(269-292))) were observed to possess fibronectin binding
activity (5). Both sequences are highly conserved among all identified
FAP proteins except FAP-S, which contains only the region homologous to
FAP-A-(269-292) (5). In vitro studies on FAP-A-(269-292)
or its homologue in M. leprae, FAP-L-(240-263) showed this
fibronectin binding region to be sufficient to block the attachment of
all tested mycobacteria (M. avium, M. bovis BCG,
and M. smegmatis) to fibronectin-coated surfaces and also to
abrogate fibronectin-opsonized mycobacterial attachment to epithelial
cells and Schwann cells (5). These data suggest an important role for
FAP-A-(269-292) in FAP-mediated fibronectin binding.
In the present study, the amino acids required for fibronectin binding
of the FAP-A-(269-292) peptide were determined using Ala substitutions
in synthetic peptides and site-directed mutagenesis of the recombinant
protein. The data show that four amino acids, RWFV, are necessary for
fibronectin binding function.
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EXPERIMENTAL PROCEDURES |
Synthesis of FAP-A Peptides--
The FAP-A peptides were
synthesized and purified by high performance liquid chromatography as
described (5).
Peptide-FN Binding Assay--
The FN binding to FAP-A peptides
was as described (5). Briefly, immulon 2 microtiter 96-well plates were
coated with FAP peptides, 0.36 mM for Fig. 1 and the given
concentration for Fig. 2. Nonspecific sites were blocked with bovine
serum albumin, and 1 µg of FN or laminin (LN) in wash buffer
(phosphate-buffered saline, 0.1% bovine serum albumin, 0.05% Tween
20) was added per well. After a 3-h incubation at 25° C, wells were
washed with wash buffer and bound FN and LN were detected using rabbit
polyclonal anti-FN or anti-LN, respectively. Bound antibody was
detected using anti-rabbit Ig coupled to horseradish peroxidase.
Site-directed Mutagenesis of FAP-A Gene--
Site-directed
mutagenesis by overlap extension was used to generate mutant FAP-A DNA
(13, 14). For each mutant FAP-A DNA, one pair of complementary
oligonucleotide primers containing GCC (code for alanine) substitution
or nucleotide deletion at the desired site were used to generate two
DNA fragments having overlapping ends with primary polymerase chain
reaction. Then these fragments were combined for secondary polymerase
chain reaction. The resultant mutant FAP-A DNAs were cloned into
pBluescript SK vector (Stratagene, La Jolla, CA) and sequenced using
the Taq DyeDeoxy Termination Cycle Kit and the Applied
Biosystems 373A DNA sequencer (Applied Biosystems, Foster City, CA).
Expression and Purification of FAP-A Mutants--
All mutant
FAP-A DNAs were digested with BamHI and EcoRI and
then ligated into the expression vector pTrcHisB. The mutant FAP-A
fusion proteins containing poly-His tag were expressed and purified
using a Ni2+ affinity column under denaturing conditions
according to the manufacturer's protocol (Invitrogen, Carlsbad, CA).
The cell lysates and purified fusion proteins were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by
Coomassie Blue staining.
In Vitro Attachment of M. bovis BCG to FN-coated Surface--
M.
bovis BCG attachment was performed as described previously (5). Immulon
4 microtiter plates (96-well) were coated overnight at 4° C with 100 µl of 60 µg/ml fibronectin or 120 µg/ml human serum albumin.
After blocking nonspecific sites with human serum albumin, a total of
2 × 106 colony forming units of fluorescein
isothiocyanate-labeled M. bovis BCG were added in a volume
of 50 µl of 0.05 M Tris buffer (pH 6.2). In blocking
experiments, 1 µM recombinant FAP-A protein was added in
a volume of 50 µl of 0.05 M Tris buffer before the addition of M. bovis BCG. After a 90-min incubation at
37° C, the wells were washed with 0.05 M Tris buffer and
bound fluorescein isothiocyanate-labeled M. bovis BCG were
detected using a FL500 Fluorescence Plate Reader (Bio-Tek Instruments,
Frederick, MD).
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RESULTS |
Localization of the Binding Site on FAP-A--
Previous studies
identified two regions in FAP-A that possessed the capacity to
bind fibronectin (5). These regions consisted of amino acids 177-201
(FAP-A-(177-201)) and amino acids 269-292 (FAP-A-(269-292)).
Characterization of the peptides showed that FAP-A-(269-292) was
sufficient to block the interaction of mycobacteria with
fibronectin-coated surfaces, mycobacterial attachment to epithelial cells, and M. bovis BCG-mediated antitumor
activity (4, 5).2 Therefore we initiated studies on
FAP-A-(269-292) to determine the amino acids required for fibronectin
binding. Initially we tested synthetic peptides of varying lengths to
determine the minimal peptide length necessary for binding to
fibronectin. Truncation of FAP-A-(269-292) from the amino-terminal end
abrogated binding activity quickly suggesting that this region played
an important role in fibronectin binding (Fig.
1A). Truncation from the
carboxyl-terminal end showed that the minimal peptide length supporting
fibronectin binding was amino acids 269-280 (FAP-A-(269-280), Fig.
1B). No binding function was observed for a peptide
containing amino acids 269-277.

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Fig. 1.
Determination of the minimal peptide length
required for fibronectin binding activity. Synthetic peptides of
varying length derived from the previously determined fibronectin
binding peptide (FAP-A-(269-292)) were tested for fibronectin binding
as described under "Experimental Procedures" (5).
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Further studies were performed to identify the essential amino acids
within FAP-A-(269-280). To determine the essential amino acids,
peptides were synthesized that contained single Ala substitutions as
shown in Fig. 2A. Each peptide
was tested for binding capacity to fibronectin as described under
"Experimental Procedures" (Fig. 2). The data show that amino acids
273-276 are essential for fibronectin binding (Fig. 2, B
and C). Ala substitution of any one of these amino acids
completely abrogated fibronectin binding activity. Substitution of
amino acids 269-272, 277, and 279 had no effect on the fibronectin
binding capacity of FAP-A-(269-280) (Fig. 2C). Ala
substitution of amino acid 278 produced partial inhibition of binding
and amino acid 280 reproducibly enhanced the fibronectin binding
capacity of the FAP-A-(269-280) peptide (Fig. 2C).
Comparison of binding regions for cloned FAP proteins shows 100%
homology among the essential amino acids (RWFV; Fig.
2D).

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Fig. 2.
Effect of Ala substitution on fibronectin
binding of FAP-(269-280) peptides. A,
sequence of peptides with Ala substitutions that were used to identify
amino acids required for binding. B, Ala substitutions that
completely abrogate peptide fibronectin binding ability. C,
substitutions with no effect, partial effects, or complete abrogation
of fibronectin binding. D, comparison of regions homologous
to FAP-A-(269-292) for all cloned FAP peptides. Bold
letters show the amino acids identified as necessary for
fibronectin binding activity.
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Effect of Synthetic Peptides with Ala Substitution on M. bovis BCG
Attachment to Fibronectin--
We previously reported the ability of
the synthetic FAP-A-(269-292) peptide to inhibit the attachment of
M. bovis BCG to fibronectin-coated surfaces (5). Here we
tested the wild type minimal binding peptide (FAP-A-(269-280)) and
FAP-A-(269-280) with individual Ala substitution (at amino acid 269, 273, 274, 275, 276, or 280) for their ability to inhibit M. bovis BCG attachment to fibronectin. When compared with wild type
peptide (FAP-A-(269-292)), peptides with Ala substitution within the
active region identified above either partially
(Ala274-276) or completely (Ala273) abrogated
the ability of the respective peptides to block M. bovis BCG
attachment (Fig. 3, A and
B). The relative activity of peptides with Ala substitutions
at 274-276 varied from experiment to experiment although the data
consistently showed partial abrogation of activity for each. Therefore,
we show the mean percent inhibition of all experiments (three in
number) in Fig. 3B. These data show the reproducibility of
the effects of Ala substitution. In all experiments control peptides
containing Ala substitution at either amino acid 269 or 280 had no
significant effect on peptide function (Fig. 3, A and
B).

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Fig. 3.
Inhibition of M. bovis BCG
attachment to fibronectin by synthetic FAP-A-(269-292)
peptides containing Ala substitutions. Control peptide is a random
sequence of amino acids in FAP-A-(269-292). A, one
representative experiment showing actual M. bovis BCG
attachment. Each point is the mean ± S.D. of triplicate wells.
B, percent inhibition was calculated for three individual
experiments and reported as mean percent inhibition. Student's
t test showed the p values for
Ala273, Ala274, Ala275, and
Ala276 to equal 0.015, 0.015, 0.013, and 0.022, respectively.
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Effect of Site-directed Mutagenesis of FAP-A on M. bovis BCG
Attachment to Fibronectin--
To determine the effect of Ala
substitution on fibronectin binding activity at the FAP-A fusion
protein level, a truncated form of FAP-A consisting of amino acids
210-381 (FAP-A-(210-381)), which contains the FAP-A-(269-292)
binding region, was used. Residues 269, 273, 274, 275, 276, and 280 were selected for individual Ala substitution (Fig.
4A). In addition the four
identified active amino acids, RWFV (amino acids 273-276), were either
deleted en bloc or substituted en bloc with Ala.
As a control for the RWFV deletion and the en bloc Ala
substitution, four residues outside the identified fibronectin binding
region, NGQI (amino acids 255-257), were either deleted or substituted
with Ala. The sequences of all mutated FAP-A DNAs were confirmed by DNA
sequencing. FAP-A mutants were subcloned into the pTrcHis expression
vector and expressed, and the resultant fusion proteins were purified
as described (4, 5). Coomassie Blue staining showed the induced fusion
proteins to correspond to the correct molecular weight (data not
shown).

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Fig. 4.
Inhibition of M. bovis BCG
attachment to fibronectin by mutant FAP-A proteins with Ala
substitution or amino acid deletion. Control J (Con J)
is recombinant thioredoxin produced and purified as described for
FAP-A. A, amino acid sequence of FAP-A-(210-381). The
previously identified fibronectin binding region, FAP-A-(269-292), is
underlined. The selected residues used for alanine
substitution are shown in bold. B, one
representative experiment showing actual M. bovis BCG
attachment. Each point is the mean ± S.D. of triplicate wells. C,
percent inhibition was calculated from three individual experiments and
reported as mean percent inhibition. p values for
Ala273, Ala274, Ala275, and
Ala276 = 0.024, 0.024, 0.048, and 0.043, respectively.
p values for 4ala-(273-274) and 4D-(273-274) = 0.0023 and
0.0047, respectively, whereas for controls 4ala-(255-258) and
4D-(255-258) are 0.069 and 0.091, respectively.
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The effect of mutant FAP-A proteins on M. bovis BCG
attachment to fibronectin was tested, and a representative experiment showing M. bovis BCG attachment is provided in Fig.
4B. Fig. 4C shows the mean percent inhibition
from three independent experiments of M. bovis BCG binding
by the FAP-A mutants. Mutant FAP-A with a single Ala substitution at
either amino acid 269 (FAP-A269) or 280 (FAP-A280) showed no alteration in peptide function (Fig.
4, B and C). In contrast FAP-A mutants with Ala
substitutions at amino acids 273 (FAP-A273A), 274 (FAP-A274A), 275 (FAP-A275A), and 276 (FAP-A276A) showed partial abrogation of the ability of
each peptide to inhibit M. bovis BCG attachment to
fibronectin. When the four essential amino acids (RWFV) were either all
changed to Ala (FAP-A4ala-(273-276)) or deleted (FAP-A4d-(273-276)),
no functional activity of the respective peptides was observed (Fig. 4,
B and C). The control mutants in which the four
amino acids outside the fibronectin binding region were substituted
with Ala (NGQI, amino acids 255-258, FAP-A4ala-(255-258)) or deleted
(FAP-A4d-(255-258)) retained functional activity. These data indicate
that one Ala substitution within the RWFV motif is not sufficient to
completely abrogate fibronectin binding function in the recombinant
FAP-A protein; however, the en bloc deletion of
RWFV or Ala substitution of the RWFV motif resulted in complete loss of
fibronectin binding activity. These data demonstrate RWFV to be
critical to the fibronectin binding function of mycobacterial FAP.
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DISCUSSION |
We performed studies to identify the amino acids necessary for
fibronectin binding of FAP. Because previous studies demonstrated the
FAP-A-(269-292) fibronectin binding peptide to be sufficient to block
mycobacterial interaction with fibronectin, this site in FAP-A was
characterized. The minimal binding sequence of FAP-A for fibronectin,
consisting of 12 amino acids (amino acids 269-280), was identified.
Using this binding sequence as a base, a panel of synthetic peptides
containing single Ala substitutions was used to determine which amino
acids were necessary for fibronectin binding (Fig. 2, B and
C). Characterization of the functional effects of single Ala
substitutions in the synthetic peptides showed that substitution at
amino acids 273-276 abrogated peptide binding to fibronectin (Fig. 2,
B and C). In a more stringent test of the
biological effects of Ala substitutions at amino acids 273-276,
Ala-modified synthetic peptides were tested for their ability to block
attachment of M. bovis BCG to fibronectin. Single Ala
substitutions at Trp, Phe, or Val partially inhibited M. bovis BCG attachment whereas Ala substitution at the Arg site
completely abrogated M. bovis BCG binding (Fig. 3,
A and B). The amino acids RWFV are 100%
conserved in all FAP molecules identified to date (Fig. 2D).
These data show that the RWFV motif is essential for peptide binding to
fibronectin and also is important in the M. bovis
BCG/fibronectin interaction.
To validate the synthetic peptide data, additional studies were
performed in which mutant recombinant proteins containing Ala
substitutions or amino acid deletions were tested for fibronectin binding function. In these studies the ability of mutated recombinant FAP-A-(210-381) to inhibit M. bovis BCG binding to FN was
compared with wild type FAP-A-(210-381). FAP-A mutants with single Ala substitutions at amino acids 273-276 showed partial inhibition of
M. bovis BCG attachment to fibronectin. The Ala mutation of Arg at position 273 in recombinant FAP-(210-381) did not result in
complete abrogation of function as was seen in the synthetic peptide.
This suggests that the function of Arg273 was exaggerated
in the shorter synthetic peptide.
FAP-A mutants in which all four relevant amino acids (273-276) were
simultaneously mutated to Ala or were simultaneously deleted showed
complete loss of function. Control proteins in which Ala deletion or
substitution was performed at a site outside the fibronectin binding
region (amino acids 255-258, NGQI) retained function. Taken together,
these data demonstrate that the RWFV sequence is a critical functional
motif in FAP binding to fibronectin. Moreover, because the RWFV motif
was shown to be important in modulating M. bovis BCG
attachment to fibronectin, the data suggest an important role for the
motif in the M. bovis BCG/fibronectin interaction.
A second protein family, the antigen 85 complex, also has been
implicated in the fibronectin binding of mycobacteria. Abou-Zeid et al. (8) first reported the interaction between
antigen 85 complex proteins and fibronectin using culture supernatants
of mycobacteria. The participation of the antigen 85 complex was established by probing Western blots of mycobacterial culture supernatants with fibronectin (8). Subsequently, in vitro
studies were performed demonstrating the binding of recombinant 85A,
-B, and -C proteins to fibronectin (10-12, 15). A highly conserved motif among the antigen 85 complex proteins, FEWYYQ, was identified as
an important sequence in the binding of recombinant 85 complex proteins
to fibronectin (12). While the interaction between antigen 85 complex
proteins and fibronectin has been demonstrated in in vitro
binding assays, the role of these proteins in the attachment of viable
bacteria to fibronectin has not been established.
The relationship between the antigen 85 complex and FAP in the
mycobacterial interaction with fibronectin is not known. The minimal
amino acid sequence that will bind fibronectin was identified for the
85B protein and consists of 11 amino acids (FEWYYQSGLSV) (12). The
amino acid composition of this binding peptide consists of 8 polar and
3 non-polar amino acids with a neutral net charge. Essential amino
acids in the minimal binding sequence were identified as FEWYYQ, which
has a net negative charge. FAP contains no region of homology with the
identified 85B binding region. Furthermore, the composition of the
minimal amino acid sequence of the FAP fibronectin binding region,
which consists of 12 amino acids, is quite distinct from that of the
85B sequence. The FAP minimal binding peptide consists of 7 polar and 5 non-polar amino acids with a net positive charge. Moreover, the
essential binding motif of this binding region, RWFV, bears little
resemblance to the 85B sequence, FEWYYQ. The FAP sequence is highly
non-polar and is positively charged while the 85B sequence is
negatively charged and is comprised of 2 non-polar and 3 polar amino
acids. The composition of the respective binding sites for antigen 85B
and FAP suggest distinct function. In this regard, a previous report
localized the binding site on fibronectin for 85B to be in the collagen binding region at the amino-terminal end of fibronectin (11). Consistent with this observation, the interaction between 85B and
fibronectin was inhibited by gelatin. In contrast, our unpublished studies show that both FAP and intact M. bovis BCG attach to
the carboxyl-terminal region of fibronectin containing the heparin binding site. In collaborative studies with Dr. James McCarthy (University of Minnesota), we have shown M. bovis BCG and
FAP to bind to the 33/66 fragment of a trypsin/cathepsin D digest of
fibronectin. In addition, previous studies showed M. bovis BCG attachment to fibronectin to be inhibited by heparin but not gelatin (2).
Comparison of FAP and protein 85B with other bacterial fibronectin
attachment proteins suggests that FAP exhibits unique properties, whereas protein 85B shares many characteristics with these proteins. The best characterized of the fibronectin-binding proteins is that of
Staphylococcus. The primary fibronectin binding sequence of
Staphylococcus aureus is located in the 37-48 amino acid
sequence that is repeated four times in the binding protein (16). The essential binding motif is a negatively charged hydrophilic motif, DFEEDT, and is shared by other Gram-positive cocci for which
fibronectin attachment proteins have been identified (17). The negative charge and the hydrophilic nature of the Staphylococcus
fibronectin binding protein is similar to the characteristics of the
identified fibronectin binding sequence for the mycobacterial 85B
protein. In addition, the region in the fibronectin molecule to which
the Staphylococcus protein binds is located in the same
collagen binding region as 85B (11, 16).
The studies we report here identify and characterize a fibronectin
binding region of FAP-A. The data show that FAP-A binding depends on
the RWFV sequence, which possesses characteristics distinct from
antigen 85B and fibronectin-binding proteins of Gram-positive cocci.
Further studies are needed to characterize the respective roles of FAP
and the antigen 85 complex in mycobacterial attachment to fibronectin.