(Received for publication, July 6, 1994; and in revised form, October 14, 1994)
From the
The extracellular domains of the two human tumor necrosis factor
(TNF) receptors critical for binding TNF- were examined by
deletion mapping. The ligand binding capability of full-length and
truncated recombinant soluble TNF receptors (TNFRs) was assessed by
ligand blot analysis and their binding affinity determined by Scatchard
analysis. The results showed that deletion of the fourth cysteine-rich
domain of the p55 receptor (TNFR-1) did not alter ligand binding
affinity significantly. Deletion of domains 3 and 4 of TNFR-1 resulted
in no ligand binding, suggesting that domain 3, but not 4, of TNFR-1
binds directly to ligand. Deletion of domain 4 of TNFR-2 resulted in
drastically reduced protein yield and 3-fold reduction in ligand
binding affinity, while deletion of both domains 4 and 3 yielded no
protein. Thus, the domain 4 of TNFR-2, but not that of TNFR-1, appears
to be involved directly in binding TNF, although it is also possible
that the domain 4 of TNFR-2 is involved in the correct folding of other
domains. These results suggest that the modes of interaction between
TNF-
and its dual receptors are different, providing opportunity
to modulate each receptor specifically for research and therapeutic
purposes.
Tumor necrosis factor (TNF-) (
)and lymphotoxin
(TNF-
or LT-
), secreted mainly by activated macrophage and
lymphocytes, respectively, are similar in structure and exert a wide
range of similar biological activities, some beneficial (e.g. antitumor, anti-infectious), others detrimental (e.g. septic shock and fever) (see (1, 2, 3, 4, 5) for
reviews). Understanding how TNF exerts its multiple effects is crucial
for developing TNF-based therapeutic agents against diseases mediated
or modulated by TNF.
The active form of human TNF- is a
homotrimer composed of 17-kDa
subunits(6, 7, 8, 9) . The crystal
structures of human TNF-
and TNF-
(LT-
) have been
determined(10, 11, 12) , and three elongated
antiparallel
-pleated monomers were shown to associate tightly
about a three-fold axis of symmetry to form a compact bell-shaped
trimer with three intersubunit grooves on the surface. Under different
conditions, a non-symmetrical TNF trimer was also observed in a crystal
structure (11) . The biological activities of TNF are mediated
through multiple high affinity specific cell surface
receptors(12, 13, 14) . Two TNF receptor
(TNFR) genes have now been cloned from human and mouse cells (15, 16, 17, 18) . They belong to a
receptor gene superfamily that includes nerve growth factor (NGF)
receptor, CD27, CD30, CD40, Fas antigen, and a TNFR-related receptor
protein for membrane-bound LT-
(19, 20) . Except
for NGF, ligands of the NGF/TNF receptor gene superfamily share
sequence homology with TNF. The intracellular domains of the two TNF
receptors share no similarity. They may interact with different
intracellular entities to trigger different signal transduction
pathways(21, 22) . Members of TNF/NGF receptor
superfamily have three to six conserved cysteine-rich repeat motifs in
their extracellular domain. Of the four cysteine repeats in both TNF
receptors, the fourth motif and its downstream sequence up to the
transmembrane domain (14) are the least similar.
Mutational
analyses of TNF indicated that the intersubunit grooves of TNF trimer
form at least part of the receptor binding
site(6, 23) . Recently, Banner et al.(24) determined the crystal structure of the soluble
hTNFR-1TNF-
complex, which showed three receptor fragments
bound to a TNF-
trimer at its intersubunit grooves. The receptor
fragments in the complex showed that the four sequence domains defined
by the cysteine repeat motifs were arranged, end to end, in a linear
fashion with little overlap. A direct involvement of the second and the
third cysteine-rich domains in ligand binding was also shown. The
fourth cysteine-rich domain and its downstream sequence (domain 4) of
hTNFR-1 in the complex is rather disordered and appears not to be
involved in ligand binding. However, results from two independent
peptide mapping studies suggested otherwise; a 20-amino acid synthetic
peptide derived from the domain 4 sequence of TNFR-1 and a 21-amino
acid synthetic peptide derived from the last 9 amino acids of domain 3
plus 12 amino acids in the beginning of domain 4 were found to inhibit
both TNF-
receptor binding and in vitro cytotoxic
activity(25, 26) . These two peptides cover a region
apparently not involved in hTNFR-1 binding in the crystal structure.
These data suggest that TNF-
and TNF-
may have different
modes of binding TNFR-1. The binding domain of TNFR-2 has not been
defined for either TNF-
or TNF-
. To compare the mode of
ligand-receptor interaction between TNF-
and its dual receptors,
we expressed full-length and serial C-terminal domain-truncated
recombinant soluble receptors of the two human TNFRs in baculovirus
expression system and compared their ligand binding capability. The
data suggested that the domain 4 of TNFR-2, but not that of TNFR-1, are
involved directly in binding TNF-
. Domain 4 of TNFR-2 may be also
involved in the correct folding of other domains. These results also
suggested the existence of a unique TNFR-2 binding site on the TNF
molecule susceptible to manipulation for specific TNF receptor
modulation for the development of better TNF-based therapeutic agents.
After removal of primers using size selecting filters (Centricon 30 from Amicon, Beverly, MA, or Ultrafree-MC from Millipore, Bedford, MA), PCR products were cut with BamHI/XbaI and ligated with BamHI/XbaI-cut transfer vectors pBacPAK9. The 5` primers have a BamHI site introduced upstream to ATG start codon followed by the signal peptide sequence of the hTNFRs. The 3` primers have an XbaI site introduced downstream to a stop codon placed at the end of the extracellular domain sequence or at the end of domain 3 or 2 sequence for expressing truncated soluble receptors.
Expression with Sf9 cells was performed according to the manual provided by Invitrogen (San Diego, CA): Sf-9 cells maintained in suspension culture at 27 °C in TNM-FH medium with 10% fetal calf serum (Sigma) were used to produce recombinant viruses. Transfer vector DNAs and linear BacPAK6 were co-transfected into log phase Sf-9 cells, and recombinant viruses were purified by at least two rounds of plaque assay followed by PCR screening using flanking primers.
For
recombinant protein production, High-Five cells were seeded at 5
10
cells/265-cm
flask in 35 ml of
serum-free Ex-cell 400 medium (JRH Biosciences, Lenexa, KS) and grown
to about 80% confluence (about 3
10
cells/flask in
3 days) before infecting with recombinant virus at a mutiplicity of
infection of 5. Seventy-two hours after viral infection, culture
supernatants were collected and clarified by centrifugation at 500
g for 10 min, followed by passing through a 0.2-µm
filter. Recombinant receptors were analyzed by SDS-PAGE, followed by
silver staining or Coomassie Blue staining, and purified by ligand
affinity chromatography as described below.
Clarified infected High-Five cell culture supernatant from one T265 flask was passed through a 0.5-ml TNF affinity column by gravity. After washing with 10 ml of PBS, the column was eluted with seven 500-µl aliquots of 100 mM glycine, 100 mM NaCl, pH 2.6, into microcentrifuge tubes containing 50 µl of 1 M Tris-HCl, pH 7.5. Fractions were analyzed on a 12% Laemmli gel followed by staining with Coomassie Blue.
Figure 1: SDS-PAGE analysis of full-length and truncated recombinant soluble TNF receptors. Culture supernatants (40 µl each) of High-Five cells infected with recombinant virus were fractionated on a 12% reducing SDS-PAGE followed by silver staining as described under ``Experimental Procedures.'' Lane 1, 0.2 µg/band of molecular weight markers; lanes2 and 3, full-length sTNFR-1; lanes4 and 5, domain 4-deleted sTNFR-1; lanes6 and 7, full-length sTNFR-2; lanes8 and 9, domain 4-deleted sTNFR-2. Each pair of samples were from supernatants of culture cells infected with two independent recombinant viruses derived from each expression transfer vector construct.
Fig. 2shows the SDS-PAGE/silver
staining (panel A) and ligand blot (panelB)
analyses of full-length and truncated sTNFR-1. sTNFR-1 with domain 4
deleted or with both domain 4 and 3 deleted were produced at about one
third of the full-length sTNFR-1 (panelA, lanes
1-6). While domain 4-deleted and full-length sTNFR-1 bind
equally well to ligand (panel B, lanes1-4), sTNFR-1 with domain 3 and 4 deleted shows no I-TNF binding (panel B, lanes 5 and 6). This result suggests that domain 3 but not domain 4 of
sTNFR-1 is involved in ligand binding. On the other hand, domain
4-deleted sTNFR-2 was produced in greatly reduced yield as compared to
the full-length counterpart (Fig. 1, lanes8 and 9). No recombinant protein was detectable when both
domain 4 and 3 of TNFR-2 were deleted (data not shown).
Figure 2: SDS-PAGE and ligand blot analysis of full-length and truncated sTNFR-1. Silver-stained gel (panelA) and ligand blot (panelB) were derived from two identical non-reducing 12% SDS-PAGE gels as described under ``Experimental Procedures.'' Except for lanes1 and 2, each pair of lanes contained 10 µl of culture supernatant from High-Five cells infected with two independent recombinant viruses. Lanes1 and 2, 3.3 µl of culture supernatants containing full-length sTNFR-1; lanes3 and 4, sTNFR-1 with domain 4 deleted; lanes5 and 6: sTNFR-1 with both domain 3 and 4 deleted.
Ligand blot
analysis using crude culture supernatants suggested that domain
4-deleted sTNFR-2 bound TNF- poorly, and that sTNFR-2 with both
domains 3 and 4 deleted did not bind TNF-
(data not shown). To
better assess the ligand binding capability of full-length and domain
4-deleted sTNFR-2, they were affinity-purified before ligand blot
analysis. Contrary to the results for domain 4-deleted and full-length
sTNFR-1, Fig. 3(panelB) showed that domain
4-deleted sTNFR-2 (lanes 3 and 4) binds poorly to
ligand as compared to the full-length sTNFR-2 (panelB, lane1 and 2). An identical
SDS-PAGE gel stained with silver on the left (Fig. 3, panelA) assured that comparable amount of
full-length (lanes 1 and 2) and domain 4-deleted
sTNFR-2 (lanes 3 and 4) have been loaded in SDS-PAGE
gel used for ligand blot analysis.
Figure 3: SDS-PAGE and ligand blot analysis of affinity-purified full-length and domain 4-deleted sTNFR-2. Non-reducing SDS-PAGE gels were prepared and run as described in the legend to Fig. 2. Panels A and B represent two identical gels analyzed by silver staining and ligand blot analysis, respectively. Ligand blot was performed as described in the legend of Fig. 2. Each pair of lanes contained culture supernatant from High-Five cells infected with two independent recombinant viruses derived from an expression transfer vector construct. Lanes 1 and 2, 250 ng of purified full-length sTNFR-2; lanes 3 and 4, 250 ng of purified domain 4-deleted sTNFR-2.
Figure 4: Ligand binding assay of full-length and domain 4deleted sTNFRs. In vitro solid phase ligand binding assays were performed as described under ``Experimental Procedures.'' Panels A, B, E, and F are binding curves. PanelsC, D, G, and H are Scatchard plots derived from them, respectively. PanelsA and C, full-length sTNF-R1; panelsB and D, domain 4-deleted sTNFR-1; panelsE and G, full-length sTNF-R2; panelsF and H, domain 4-deleted sTNFR-2.
The crystal structure determined by Banner et al.(24) shows that the hTNF-hTNFR-1 complex has three
receptor molecules bound symmetrically to one TNF trimer. The receptor
fragment, a very elongated end to end assembly of four similar domains,
each representing one of the four conserved extracellular cysteine-rich
domains, binds in the groove between two adjacent TNF-
subunits.
The complex also shows that domain 2 and a small region of domain 3
bind ligand, while domain 4 is further from TNF. Domain 4 is described
as disordered, which is unfortunate since synthetic peptides derived
from the domain 4 sequence of hTNFR-1, and polyclonal antibodies
against the synthetic peptides, were able to inhibit
I-TNF binding to cell surface receptors and TNF in
vitro cytotoxocity(25, 26) , suggesting that
domain 4 of TNFR-1 also bind TNF.
Our data reported here suggested
that domain 4 of TNFR-2, but not that of TNFR-1, is involved directly
in ligand binding. Our data also suggest that domain 3 of TNFR-1 is
involved in ligand binding, since its deletion resulted in truncated
receptor unable to bind ligand (Fig. 2). However, if domain 4 of
TNFR-1 is involved in ligand binding as peptide mapping results
suggested, it may be that the interaction between TNF- and domain
4 is much weaker than the sum of its interactions with other domains,
thus escaping detection by our methods, even though it may be
detectable with a very high concentration of proper peptides derived
from domain 4 sequence of TNFR-1. The structure of
TNF-
TNFR-1 complex implied that domain 4 is away from TNF if
the four domains of the receptor were arranged in a linear fashion as
assumed; therefore, unless the structure of TNF-
TNFR-1
differs substantially from that of the reported structure of
TNF-
TNFR-1(24) , in order for domain 4 to bind
TNF-
or -
, it would have to wrap around the same TNF trimer
or bind to another TNF trimer in the crystal. Because the structure of
domain 4, either in the ligand-receptor complex or in the uncomplexed
receptor, has not been determined, this possibility cannot be ruled
out.
The data also suggested that not all domains of sTNFR-2 are
folded independently, since the deletion of domain 4 of sTNFR-2
resulted in drastically reduced yield of sTNFRs secreted from
recombinant virus infected insect cells. Improper folding during
biosynthesis may result in proteins more prone to protease digestion.
In addition, the results reported here suggested that the domain 4 of
TNFR-2, but not that of TNFR-1, is involved in binding rhTNF-,
either directly and/or through indirect effect on the conformation of
the rest of the molecule. If the domain 4 of TNFR-2 is involved
directly in ligand binding, TNFR-2 may interact with TNF-
differently from TNFR-1. Since cell surface and soluble TNFR-2 bind
TNF-
with higher affinity than TNFR-1(31) , there may be a
unique domain on TNF-
, but not on TNF-
, that interacts
specifically with TNFR-2, thereby contributing additional free energy
of binding to increase the binding affinity of TNF-
for TNFR-2
severalfold over its binding affinity for TNFR-1 (see K
ratio in Table 1). In fact, monoclonal antibody against
TNF-
has been used to show recognition of distinct regions of
TNF-
by different tumor cell receptors(32) . This is
possible since the domain 4 of TNFR-2, including the fourth
cysteine-rich motif and the downstream spacer region up to the
transmembrane domain, is much longer than that of TNFR-1. This region,
therefore, may not only confer more flexibility on cell surface for
TNFR-2, but also enhance its binding affinity for TNF-
. Moreover,
although TNF-
and TNF-
share a similar folding pattern, the
two are not identical in structural details. Particularly noticeable
are longer loops present at the top of the bell-shaped molecule of
TNF-
, which extends upward from the area in which TNF-
was
shown to bind domain 3 of TNFR-1 in the crystal structure of
TNF-
sTNFR-1 complex(24) . Six amino acids in these
loops in TNF-
have been shown to confer an unique lectin-like
binding activity(33, 34) , which may be the basis for
some of the differential biological activities of TNF-
as compared
to TNF-
(35, 36, 37, 38) .
Conceivably, the extra loop region may also provide additional binding
sites for the domain 4 of TNFR-2, if not TNFR-1. It should also be
mentioned that TNF-
and TNF-
share only limited sequence
identity (about 50% similarity and 30% identity). Such is also the case
with the extracellular domains of hTNFR-1 and hTNFR-2. It is therefore
expected that the residues interacting in the four types of complexes
formed between the two pairs of ligands and receptors may not all be
identical, resulting in differential binding affinities. Recent
mutational analyses of TNF-
found examples of single surface
residue substitutions that resulted in the loss of binding capability
to both receptors without obvious changes in the conformation of
TNF-
(39, 40, 41) , suggesting that the
receptor binding site(s) on TNF-
for the two TNF receptors
overlap. The finding of several TNF mutants binding preferentially to
either one of the two human TNFRs (39, 40, 41) also supports the notion that
TNF-
interacts differently with the two receptors. Of particular
interest are the findings that mutations of contiguous residues on
human TNF-
molecule resulted in opposite receptor binding
preference(39, 41) . It has been suggested that TNFR-1
mediates in vivo tumor killing, while TNFR-2 mediates
TNF-induced systemic toxicity, which currently limits the therapeutic
usefulness of TNF as an anticancer agent ((40) , and references
cited therein). Determination of the crystal structures of
TNF-
sTNFR-1, TNF-
sTNFR-2, and
TNF-
sTNFR-2 complexes and comparison with the known
structure of TNF-
sTNFR-1 may reveal critical differences in
the molecular interaction between each ligand and the dual receptors to
guide future design of useful TNF receptor-specific therapeutic
agent(s) with diminished side effects.