(Received for publication, February 3, 1995; and in revised form, May 29, 1995 )
From the
The location of biologically relevant epitopes on human tumor
necrosis factor (hTNF-
) was evaluated by testing the
immunoreactivity of anti-TNF-
antibodies against 149 sequential,
overlapping octamer peptides. A goat polyclonal antibody raised against
recombinant hTNF-
, which neutralizes hTNF-
biological
activities, reacted with oligopeptides corresponding to hTNF-
residues 7-11, 17-23, 30-39, 42-49,
106-112, and 135-142. A possible assembled epitopic region
(residues 25, 27, and 144) for neutralizing murine monoclonal
antibodies designated 11D7G4 and 9C4G5 was deduced from the fact that
they bound to tripeptides, mimicking a discontinuous epitope. These
antigenic regions were found to include or adjoin poorly conserved
amino acids and they were located in the turns between
-sheets on
the surface of the molecule. Three of the sequential epitopic regions
and an assembled region were closely related to the receptor binding
sites proposed in several other studies. These antibodies appear to
neutralize TNF-
activities by directly masking receptor binding
sites.
Tumor necrosis factor (TNF-
) (
)with a
molecular weight of 17,000 was originally characterized by its ability
to cause hemorrhagic necrosis in certain transplanted tumors (1) . This factor, produced mainly by activated macrophages,
has been shown to have a wide variety of biological activities and is
considered to be an acute phase inflammatory cytokine(2) . The
biological functions of the molecule are mediated by high affinity
binding to receptors expressed on many cell
types(3, 4, 5) . The three-dimensional
structure of human TNF-
(hTNF-
) has been
determined(6, 7) , and such studies suggest that the
trimeric quaternary structure corresponds to the most stable state of
the molecule and that the TNF monomer folds to form a sandwich of two
-pleated sheets.
Several attempts have been made to assign
functions to particular regions of the TNF- molecule. By
mutational analysis(8, 9) , several sites crucial for
hTNF-
biological activity have been localized in loops at the base
of the molecule. Furthermore, residues 32, 36, 84, and 86(9) ,
31-35, 84-87, and 143-148(10) , 31, 35, 87,
95, 133, and 147(11) , 11-13, 37-42, 49-57,
and 155-157 (7) are putative receptor binding sites.
Single amino acid substitutions at positions 29 and 32 reduce binding
activities with the p75 receptor, although they still interact with the
p55 receptor(12) . Banner et al.(13) reported the complex structure of the extra-cellular
domain of human 55-kDa TNF receptor and TNF-
. Their x-ray
crystallographic study revealed that two
-turns in the TNF-
monomer form three grooves between each monomer on the trimer. These
grooves were demonstrated to be the interaction sites of the p55
receptor(13) .
Monoclonal antibodies (mAbs) against
hTNF- have been reported
previously(14, 15, 16, 17) . mAbs
bind discrete epitopes on the molecule and affect its binding to the
receptors. Epitope analysis for mAbs against whole TNF molecules could
be useful for elucidating the receptor binding sites. The screening of
short, sequential overlapping peptides for antibody reactivity as
described by Geisen et al.(18, 19) permits
the simultaneous screening of entire molecules for linear
immunoreactive epitopes. This approach has been effective in mapping
epitopes of the outer protein VP1 of foot-and-mouth disease virus (18, 20) and human
-interferon(21) .
Using Geisen's methods, possible epitopic regions for two
types of mAbs (neutralizing and non-neutralizing) and a goat polyclonal
antibody were investigated to determine their relationship with the
receptor binding sites on the three-dimensional structure of
hTNF-.
Natural
hTNF- was secreted from a human myelomonocytic cell THP-1 (24) , kindly provided by Dr. T. Watanabe (Kyushu University,
Fukuoka, Japan). The cells were incubated at a density of 1
10
cells/ml for 24 h using serum-free RPMI 1640 that
contained 100 ng/ml phorbol 12-myristate 13-acetate (Sigma). The
culture supernatant was concentrated using a YM-10 ultrafiltration
membrane (Amicon, Danvers, MA), then stored at -80 °C until
use.
Murine tumor necrosis serum (mTNS) was prepared as described by
Green et al.(25) . BALB/c mice (Japan SLC, Hamamatsu,
Japan) were primed with 1 mg/animal of Corynebacterium porvum (Ribi Immunochem Research, Hamilton, MT). Then, 2 weeks later, 10
µg/animal of lipopolysaccharide from E. coli O55B5 (Difco)
was injected into the primed mice. Two hours later, sera were collected
and stored at -80 °C. RhTNF- was purchased from Bender
Medsystems (Vienna, Austria).
The binding of
antibodies to rhTNF- was performed using the immunoblot
technique(28) . After separation by SDS-PAGE, the proteins were
electrophoretically (50 V for 1.5 h) transferred to nitrocellulose
membranes (Bio-Rad). After transferring, the sheets were incubated in
Tris buffer (20 mM Tris, 0.5 M NaCl, pH 7.5)
containing 3% gelatin at 25 °C for 2 h, washed twice with Tris
buffer containing 0.05% Tween 20 (TBS-T), and incubated in TBS-T
containing 1% gelatin and 1/3000 diluted test antiserum or 2.0
µg/ml of test mAbs at 25 °C for 16 h. After washing, the sheets
were incubated with horseradish peroxidase-conjugated anti-goat IgG
rabbit antibody (Bio-Rad) for a test serum or anti-mouse IgG rabbit
antibody (Bio-Rad) for test mAbs dissolved in TBS-T containing 1%
gelatin at 25 °C for 2 h, washed twice with TBS-T, and subjected to
color development using 4-chloro-1-naphthol (Bio-Rad).
In the neutralization experiments, antibodies were incubated with antigen for 1 h at 37 °C prior to the assay. The incubated mixture was then added to the L929 cell culture.
Peptides corresponding to selected sequences of hTNF- were
synthesized using a peptide synthesizer (model 430A, Applied
Biosystems, Foster City, CA) and purified to homogeneity by
reverse-phase high-performance liquid chromatography. The amino acid
sequences of the peptides were confirmed using an amino acid sequencer
(model 471A, Applied Biosystems).
Figure 1:
Effects of
anti-rhTNF- antiserum on L929 fibroblast cytolytic activities of
TNFs. L929 cells were exposed to the indicated dilution series of
rhTNF-
(3 µg/ml) (a), 40 times concentrated THP-1
cell culture supernatant (b), rhTNF-
(10 µg/ml) (c), or mTNS (d) in RPMI 1640 containing 5% fetal
bovine serum and 1 µg/ml of actinomycin D at 37 °C for 17 h.
Each antigen was incubated with (closed circles) or without (open circles) 1/100 (a, c) or 1/1000 (b, d)
diluted goat anti-rhTNF-
antiserum for 1 h at 37 °C prior to
the assay. Viabilities were determined by crystal violet staining of
viable cells compared with control
cultures.
The antiserum
also inhibited the cytolytic activity of natural hTNF- secreted
from a human myelomonocytic cell THP-1 stimulated by phorbol
12-myristate 13-acetate (Fig. 1b). The neutralization
of natural hTNF-
was also seen by adding neutralizing mAbs. (
)This indicates that the topography of the site related to
the cytolytic activity on rhTNF-
in relation to the epitopes
recognized by these antibodies is almost the same for natural
hTNF-
. Therefore, it is likely that the tertiary structure of
rhTNF-
is similar to that of natural hTNF-
, at least for the
region concerning the function of cytolytic activity. However, the
antiserum cross-reacted with neither mTNS nor hTNF-
(Fig. 1, c and d). Similar specificity was
observed using neutralizing mAbs. (
)Therefore, the
antibodies showed species-specific reactivity to TNF-
, and they
also distinguished hTNF-
from hTNF-
.
Figure 2:
RhTNF- immunoblot probed with the
antiserum and monoclonal antibodies. a, RhTNF-
(0.15 mg)
was mixed with 50 µl of 1/50 diluted human serum (lane A)
or 100 µl of E. coli lysate (6.4 mg/ml) (lane B).
Samples were subjected to SDS-PAGE on a 4-20% gradient gel, and
the proteins were transferred to nitrocellulose. Blots were probed with
goat anti-rhTNF-
antiserum. b, RhTNF-
in E. coli lysate immunoblot probed with 11D7G4 (lane C) and with
1F12A7 (lane D) in the same procedure as a. The
position of the TNF-
is shown by the arrow, which
indicates the estimated molecular mass in
kilodaltons.
Fig. 2b shows the mAbs 1F12A7 and 11D7G4 bound to
SDS-denatured rhTNF- in the presence of E. coli lysates.
The same specificities of these two mAbs were shown by the other mAbs. (
)
Figure 3:
Antibody reactivity to hTNF- octamer
peptides. Bindings to octapeptides of (a) the goat polyclonal
antibody in the antiserum (1/1000 dilution), (b) 1F12A7 (2.3
µg/ml) were detected in an ELISA. The absorbance is represented as
the height of the vertical bar for each octapeptide whose
amino-terminal residue is designated by the peptide number. The
numerical assignment of the peptide is such that peptide number 1
represents residues 1-8, number 2 represents residues 2-9, etc., to the last peptide number 150 that represents residues
150-157. Results are the mean values from duplicate
samples.
A murine non-neutralizing mAb, 1F12A7, recognized one
octapeptide with initial residue 104, one of the six major areas of
binding by the polyclonal antibody (Fig. 3b). In cases
of overlapping sequential hexamer peptides, this mAb demonstrated
exactly the same reactivity. The only hexapeptide this mAb recognized
had initial residue 106. ()
On the other hand, neutralizing mAbs, 11D7G4, 9C4G5, and 1G7D3, did not bind to any sequential octamer peptide, even when the concentration of mAb was 10-fold higher.
Figure 4:
Inhibition of binding of mAb 1F12A7 to
rhTNF- by a synthetic peptide composed of hTNF-
residues
98-127. mAb 1F12A7 was preincubated for 1 h at 37 °C with
various amounts of a peptide composed of residues 98-127 and
tested by the same ELISA procedure for selection of antibodies. Ratios
of the mAb 1F12A7 bound to hTNF-
were calculated from changes in
absorbance at 405 nm per minute.
One set of 380 dipeptides made
from the L- and D-optical isomers of common amino
acids was synthesized. An amino acid derivatives set for synthesis
provided by the manufacturer included L- and D-isomer
of the common amino acids except for D--cysteine. All
combinations of dipeptides and following extended tripeptides, except
for peptides containing D-
-cysteine, were synthesized and
treated with neutralizing mAbs. Table 2summarizes the major
reactions of 11D7G4, 9C4G5 and the polyclonal antibody. The dipeptide L-Phe-D-Gln (F-q) was found to give the greatest
binding activity to both neutralizing mAbs. The defined pair F-q was
chosen for further extension.
Figure 5:
Antibody reactivity to F-q-based extended
trimer peptides. Based on the reacting pair F-q, two sets of
tripeptides were synthesized with the general formulae
acetyl-@-F-q-solid support and acetyl-F-q-@-solid
support. The symbol ``@'' represents all L-
and D--amino acids. Each set was composed of 38
tripeptides. The antibody binding activities were determined by
reacting each of the 76 tripeptides in an ELISA with the mAbs 11D7G4 (a), 9C4G5 (b), and the goat polyclonal antibody (c). The vertical bars are proportional to the ELISA
absorbance. Every group of 76 bars corresponds to a 38-trimer set for
@-F-q and another 38-trimer set for F-q-@. Within each
group of 38 bars, the left-hand bar corresponds to L-
-alanine (A)-containing tripeptide, and the successive
19 bars are then in alphabetic order according to the single letter
code for the amino acids. The following 18 bars correspond to D-
-amino acids in the same order as L-
-amino acids. Several highly reactive peptides are
indicated in the figure.
The present study was conducted to characterize the antibody
binding to hTNF-. By scanning the entire sequence of hTNF-
for immunoreactivity of sequential, overlapping octapeptides, we have
identified six major sequential epitopic areas that were recognized by
the goat polyclonal antibody against rhTNF-
. One of them was also
recognized by non-neutralizing mAb 1F12A7.
As sequential epitopes,
non-conservative regions in amino acid sequences of TNF- between
human and other species were to be recognized. The high degree of amino
acid conservation among the various TNF-
sequences has been
studied(30) . Thus, the non-conservative regions are highly
limited. All of the epitopic regions were found to either contain or
adjoin to poorly conserved sequences by comparing amino acid sequences
of murine(31) , goat(30) , and human (32) TNF-
. Therefore, it is likely that species-specific
regions are recognized as sequential epitopes.
Those sequential
epitopes were thought to be positioned on the surface area of the
TNF- molecule through examination of the three-dimensional
structure of rhTNF-
(Fig. 6). Eck and Sprang (7) determined the three-dimensional structure of hTNF-
at
2.6 Å resolution by x-ray crystallography, and the atomic
coordinates have been deposited in the Brookhaven Protein Data Bank by
the same authors (identification code 1TNF). The locations of all the
sequential epitopes on the tertiary structure of the TNF monomer were
examined using the BIOGRAF software (Molecular Simulation Inc.,
Pasadena, CA). They were all positioned on the surface area of the
TNF-
monomer (Fig. 6). These results support the work of
Sprang and Eck(30) , describing that all but 12 of the 45
poorly conserved residues correspond to solvent-accessible amino acids
in the three-dimensional structure of the hTNF trimer. The resulting
six epitopic regions were revealed to contain or adjoin those
solvent-accessible poorly conserved amino acids. In addition, five of
six epitopic regions are positioned on short polypeptide turn
structures between antiparallel
-strands. The rest (residue
numbers 30-39) include a turn and a short
-strand. Similar
results were obtained from studies of antibody recognition sites of
gp120, the external envelope protein of human immunodeficiency virus
type 1. The sequence Gly-Pro-Gly, a candidate for a polypeptide
-turn in the RP135 disulfide loop within gp120, has been proposed
as an epitope of neutralizing antibodies(33) . Therefore, it is
likely that those sequential epitopes which are located in
-turns
correspond to antibody recognition sites.
Figure 6:
HTNF- trimer showing the deduced
epitopic regions recognized by goat polyclonal antibody. A, a
view of the hTNF-
trimer. The positions of C-
carbon atoms of
three monomers of the TNF trimer are traced in yellow, aqua, and purple lines. All atoms of the epitopic
regions on the yellow subunit are shown in orange (residues 7-11), magenta (residues 17-23), light blue (residues 30-39), red (residues
42-49), cyan (residues 106-112), and green (residues 135-142). All the epitopic regions are positioned
on turn structures between
-sheets. B, a
Corey-Pauling-Koltun model of the hTNF-
monomer. All atoms of the
epitopic regions are shown in the same color as Fig. 7A. All the epitopic regions include
solvent-accessible amino acids on the surface area of an hTNF-
monomer molecule.
Figure 7:
A possible assembled epitopic region for
neutralizing mAbs 11D7G4 and 9C4G5. a, a view of hTNF-
showing Phe
(cyan) and Gln
,
Gln
, and Gln
(magenta) on a
Corey-Pauling-Koltun model of a monomer. The C-
carbon atoms of
the other two monomers are traced in aqua and purple
lines. b, a stick model showing Phe
(cyan) positioned onto a
-turn of the g-h loop
apart from Gln
, Gln
, and Gln
(magenta) onto the other
-turn of the a-a` loop. c, superposition of F-q-q tripeptide (orange) on
Phe
, Gln
, and Gln
shows that it
may mimic an assembled epitope. The tripeptide was built, superposed
mainly on the side chains of Phe
, Gln
, and
Gln
, and its energy was minimized to find a conformation
that corresponded to the nearest local minimum in the potential energy
surface using BIOGRAF.
A non-neutralizing mAb
1F12A7 recognized a linear epitope spanning residues 106-111 (Fig. 3b). This was supported by the fact that a
peptide composed of residues 98-127 inhibited the binding of
1F12A7 to rhTNF- in a dose-dependent fashion (Fig. 4). mAb
1F12A7 did not inhibit hTNF-
binding to its receptor. These
results suggest that residues 106-111 are not involved in the
TNF-receptor interaction. The peptide composed of residues 98-127
had neither rhTNF-
agonistic nor antagonistic activity at
concentrations up to 0.15 mM. (
)
The goat
polyclonal antibody neutralizes the biological activity of hTNF-.
Thus, the activity was inhibited by bound antibodies. Therefore, some
of the epitopic regions might be closely related to the functional
domains of this molecule. In these epitopes, except for the one common
to mAb 1F12A7, there might be sequential epitopes for neutralizing
antibodies. As for the receptor binding site of hTNF-
, several
common areas have been proposed. Residues 32 and 36(9) ,
31-35 (10) , 31 and 35(11) , and 37-42 (7) are candidates for the receptor binding site, which are
consistent with the two epitopic regions for the polyclonal antibody,
residues 31-39 and 42-49. Owing to the direct binding to
these possible receptor binding sites, this antibody might neutralize
TNF-
activities by interfering with the interaction between
TNF-
and its receptor.
Neutralizing mAbs 11D7G4, 9C4G5, and
1G7D3 failed to react with any peptide of 149 sequential, overlapping
octamer peptides. This indicates that these mAbs might bind assembled
or discontinuous epitopes that are dependent on the tertiary folding.
Testing combinations of dipeptides and the extended tripeptides based
on the dipeptide F-q, mAbs 11D7G4 and 9C4G5 have been revealed to react
with tripeptides, including phenylalanine and glutamine residues. The
possibility that these tripeptides could mimic an assembled epitope has
been shown by the close positions of Phe and
Gln
, Gln
, and Gln
on the
tertiary structure of the hTNF-
monomer (Fig. 7, a and b). The monomer contains four Phe residues and ten
Gln residues. Among these, Phe
has been revealed to be
the closest to Gln
, Gln
, and
Gln
. Moreover, all these are positioned onto two
-turns of two independent loops and on the solvent accessible
surface of the trimer molecule. The simulation of the F-q-q tripeptide
superposing on Phe
, Gln
, and Gln
(Fig. 7c) shows that it may mimic a certain
portion of the TNF molecule. Gln
of hTNF-
, which
aligns with Glu
of murine TNF-
, is not conserved
between the two species. Several species specificities are also
observed on residues 19-27. These show that the region including
these residues possibly forms an assembled epitope for neutralizing
mAbs.
Replacements at positions 29 and 146 clearly reduced
cytotoxicity only when poorly conserved alterations were
induced(9) . From the results of a number of experiments using
site-directed mutagenesis, Jones et al.(10) reported
the two hot spot regions situated on separate sides of the TNF monomer,
consisting of residues 31-35, 84-87, and 143-148.
Banner et al.(13) reported that the receptor fragment
binds in the groove between two adjacent TNF- subunits of a trimer
molecule. This groove formed between the d-e loop (
)(residues 105-110 which align with residues
84-89 in hTNF-
) of one subunit and the a-a"
region (residues 36-52 which align with residues 19-35 in
hTNF-
) of the next. Additionally, there is the upper contact
region flanked by the end of strand g (His
and
Asp
which align with Gln
and Asn
in hTNF-
) of one subunit and the a-a` loop (residues
37-40 which align with residues 20-23 in hTNF-
) of the
next. Three-dimensional structures of TNF-
and -
(34) are strikingly similar and they bind to the same
receptors. It is, therefore, reasonable to deduce the regions in
TNF-
from the alignment of amino acids with TNF-
. The
receptor binding site and contact regions of TNF-
are in agreement
with the two hot spot regions reported by Jones et
al.(10) , the assembled epitopic region for neutralizing
mAbs, and three sequential epitopic regions for the polyclonal antibody
(residues 17-23, 30-39, and 135-142). Therefore,
recognizing species-specific regions that are in close proximity to the
receptor binding sites, neutralizing antibodies tested appear to
neutralize TNF activities by directly masking the receptor binding
sites.
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