©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping the Domain(s) Critical for the Binding of Human Tumor Necrosis Factor- to Its Two Receptors (*)

(Received for publication, July 6, 1994; and in revised form, October 14, 1994)

Paul Chih-Hsueh Chen (1)(§) Garrett C. DuBois (2) Mann-Jy Chen (1)(¶)

From the  (1)Departments of Microbiology and Immunology and (2)Pharmacology, Thomas Jefferson University, Jefferson Medical College and Jefferson Cancer Institute, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The extracellular domains of the two human tumor necrosis factor (TNF) receptors critical for binding TNF-alpha 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-alpha and its dual receptors are different, providing opportunity to modulate each receptor specifically for research and therapeutic purposes.


INTRODUCTION

Tumor necrosis factor (TNF-alpha) (^1)and lymphotoxin (TNF-beta or LT-alpha), 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-alpha is a homotrimer composed of 17-kDa subunits(6, 7, 8, 9) . The crystal structures of human TNF-alpha and TNF-beta (LT-alpha) have been determined(10, 11, 12) , and three elongated antiparallel beta-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-beta(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-1bulletTNF-beta complex, which showed three receptor fragments bound to a TNF-beta 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-alpha 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-alpha and TNF-beta may have different modes of binding TNFR-1. The binding domain of TNFR-2 has not been defined for either TNF-alpha or TNF-beta. To compare the mode of ligand-receptor interaction between TNF-alpha 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-alpha. 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.


EXPERIMENTAL PROCEDURES

Cell Lines and Reagents

The Spodoptera frugiperda (Sf9) and High-Five insect cell lines were obtained from Invitrogen (San Diego, CA). BacPAK6, genomic DNA from a derivative of Autographa california (AcMNPV) baculovirus, and transfer vector pBacPAK9 were from Clontech (Palo Alto, CA). Recombinant hTNF-alpha was iodinated with NaI (Amersham Corp., ICN, or DuPont NEN) and IODOGEN (Pierce) as described(6) . All plasmid DNA samples for transfection or DNA sequencing were prepared with Qiagen plasmid preparation kits (Chatsworth, CA). Polymerase chain reaction (PCR) DNA amplifications were performed using a Perkin-Elmer kit and a model 9600 DNA Thermal Cycler (Perkin-Elmer). Human TNFR cDNAs were kind gifts of Immunex (Seattle, Washington).

Construction of Expression Transfer Vectors and the Expression of rsTNFRs

The four extracellular domains of hTNFRs are defined here as the four cysteine-rich motifs defined by Banner et al.(24) except that the domain 4 includes downstream sequence up to the transmembrane domain. Expression vectors of the full-length and serial C-terminal domain truncated forms of the two hTNFRs were generated by PCR amplification using the following oligonucleotide primer pairs: full-length sTNFR-1 (P1/P2), domain 4-deleted sTNFR-1 (P1/P3), domain 3- and 4-deleted sTNFR-1 (P1/P4); full-length sTNFR-2 (P5/P6), domain 4-deleted sTNFR-2 (P5/P7), domain 3- and 4-deleted sTNFR-2 (P5/P8). Starting from the 5` end, the sequences of the primers are the following: P1, CATAGGGATCCACCATGGGCCTCTCCACCGTGC; P2, TGACCAGGGTCTAGAGTTATGTGGTGCCTGAGTCCTC; P3, AAGAGCTTCTAGATTTAGGTGCACACGGTGTTCTG; P4, TTGTCCTTCTAGATTTAGCCACACACGGTGTCCCG; P5, CCCGCGGATCCACCATG-GCGCCCGTCGCCGTC; P6, CAACTGGTCTAGATTAGTCGCCAG-TGCTCCCTTCAG; P7, CCTCGGTCTAGATTACTTGCACACCAC-GTCTGAT; P8, ACCAGATTCTAGATTAGGTGCAGATGCGGTTCTGTT.

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 times 10^6 cells/265-cm^2 flask in 35 ml of serum-free Ex-cell 400 medium (JRH Biosciences, Lenexa, KS) and grown to about 80% confluence (about 3 times 10^7 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 times 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.

Expression and Purification of rhTNF-alpha

PUC118tnf human TNF-alpha mutagenesis/expression vector has been described(6) . PUC118tnf-transformed E. coli MV1190 was pelleted by centifugation and resuspended in 25 mM ammonium acetate, pH 6.0, with 1.0 mM each of DTT, EDTA, and phenylmethylsulfonyl fluoride at 0.3 g/ml. The suspension was sonicated extensively at 4 °C and clarified by centrifugation at 200,000 times g for 1 h at 4 °C. After passing through a Nalgene 0.8 µM cellulose acetate membrane, the supernatant was applied to an S-Sepharose column (Pharmacia Biotech Inc.) equilibrated with 10 mM ammonium acetate, pH 6.0, 1 mM DTT. The column was then washed with 5-10 column volumes of 0.2 M ammonium acetate, pH 6.0, and eluted with a linear gradient (0.2-1.0 M ammonium acetate, 1 mM DTT) at a flow rate of 2.3 ml/min. Fractions containing TNF-alpha were identified by Western blot analysis as described(6) . The pooled fractions were dialyzed against 10 mM Tris-HCl, pH 7.5, with 1 mM DTT and applied to a Q-Sepharose column (Pharmacia) previously equilibrated with dialysis buffer. The column was then washed with 5 column volumes of the same buffer and eluted with a linear gradient of NaCl (10-200 mM). Fractions containing TNF were pooled, dialyzed against PBS, aliquoted, and stored at -80 °C, or else dialyzed against 10 mM ammonium bicarbonate, aliquoted, lyophilized, and stored at -20 °C.

Protein Quantification by SDS-PAGE Analyses

Recombinant soluble receptors, affinity-purified or in crude culture supernatant, were fractionated on a reducing Laemmli SDS-polyacrylamide gel with 2% beta-mercaptoethanol in the loading samples followed by Coomassie Blue or silver staining as described(6) . Silver stain kits were from Boehringer Mannheim or LabLogix (Belmont, CA) and were used according to the supplier's manual.

Purification of rshTNFRs by TNF Affinity Chromatography

Purified rhTNF-alpha was coupled to CNBr-activated Sepharose 4B according to the protocol provided by Pharmacia (capacity 16-32 mg/g) with the following modifications. The gel was swollen with 1 mM of HCl and washed three times for 5 min each, using a total of 200 ml of 1 mM HCl/g of gel. rTNF was dissolved at 7.5 mg/3 ml of phosphate-buffered saline and dialyzed three times against 500 ml of 0.1 M NaHCO(3), pH 8.3, 0.5 M NaCl. The volume of the ligand was made up to 5 ml with the same buffer and mixed with the washed gel (2.5 mg of rTNF/ml of swollen gel slurry) by rotating gently overnight at 4 °C. Excess ligand was washed away with the same buffer, and the remaining active groups were blocked with 0.1 M Tris-HCl, pH 8.0 for 2 h at room temperature. The gel was then washed with 3 cycles of alternative pH, using 0.1 M sodium acetate buffer, pH 4.0, and 0.1 M Tris-HCl, pH 8.0, each containing 0.5 M NaCl. The conjugate was stored in PBS at 4 °C.

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.

Ligand Blot Analysis

Supernatants from recombinant virus-infected Sf9 cell cultures were passed through Sephadex G-15 column (gel swollen in H(2)O), lyophilized, and resuspended in 0.1 volume of H(2)O. Aliquots (10 µl) of High-Five cell culture supernatant or 10-fold concentrated Sf9 cell culture supernatant were fractionated on a 12% non-reducing SDS-PAGE and electroblotted onto a nitrocellulose filter. After blocking with 5% non-fat milk in 50 mM Tris-HCl, pH 7.5, 140 mM NaCl, 5 mM EDTA, 0.02% NaN(3), the filter was incubated with 1 µCi of I-hTNF-alpha (400-800 Ci/mmol) at room temperature for 4 h, washed three times with PBS, and exposed to x-ray films for 1-3 days.

Protein Amino Acid Sequencing

The N-terminal sequence of the purified sTNFRs were analyzed as follows: purified receptors were subjected to SDS-PAGE, electroblotted onto Immobilon membrane filters (Millipore, Belford, MA), and the protein visualized by Coomassie Blue staining as described above. The protein bands were excised from the membrane, placed in a reaction cartridge, and 20-30 cycles of Edman degradation were performed using standard protocols on an Applied Biosystems 477A-120A protein sequencer. Purified rhTNF-alpha (>50 pmol) was subjected to end sequence analysis directly and the data used to calculate the concentration of the TNF sample. Homogeneous rhTNF-alpha in PBS has an optical density of 2 A/mg protein.

Solid Phase Ligand Binding Assay and Scatchard Analysis

Direct solid phase ligand binding assays were performed as follows; 96-well microtiter plates (Immunolon-4, Removawell, Dynatech) were coated overnight at 4 °C with 25 ng and 100 ng/100 µl/well of affinity-purified full-length or truncated sTNFR-1 and sTNFR-2, respectively. Each sample was diluted at least 100-fold in PBS beforehand. After blocking for 2 h with 1% bovine serum albumin, 0.02% NaN(3) in PBS, the wells were washed with PBS and incubated with increasing amount of I-TNF in the same buffer. Control assays contained 300-fold excess of cold TNF. The wells were then washed four times with 0.1% bovine serum albumin in PBS and counted in a -counter after binding for 4 h at room temperature. Dissociation constants (K(d)) were determined by Scatchard analysis(30) .


RESULTS

Expression and Ligand Binding Capability of Full-length and Truncated sTNFRs

SDS-PAGE and ligand blot analyses were used to determine the level of rsTNFRs expression and their ligand binding capability. Fig. 1shows the result of SDS-PAGE analyses of High-Five cell secreted full-length (24-28-kDa bands in lanes2 and 3) and domain 4-deleted sTNFR-1 (20-24-kDa bands in lanes4 and 5), as well as full-length (32-37-kDa bands in lanes6 and 7) and domain 4-deleted sTNFR-2 (16-17-kDa bands in lanes8 and 9). Each pair of lanes represents results obtained with two independent recombinant virus isolates. The molecular mass values of the core proteins predicted from the DNA sequence of the expression constructs are 20 kDa (182 amino acids) and 26 kDa (235 amino acids) for sTNFR-1 and sTNFR-2, respectively. Glycosylation thus contributes at least 4-8 and 6-11 kDa to the molecular weight of sTNFR-1 and sTNFR-2, respectively. With the context of the DNA sequence surrounding the ribosomal binding site modified according to Kozak's rules (28) in the expression vector, the recombinant baculovirus infected High-Five cells, 3 times 10^7 cells in a 265-cm^2 ridged T-flask in 35 ml of culture medium, secreted 60-90 and 30-60 µg/ml full-length sTNFR-1 and sTNFR-2, respectively, 20-30 µg/ml domain 4-deleted sTNFR-1 (sTNFR-1DeltaD4), and about 3-5 µg/ml domain 4-deleted sTNFR-2 (sTNFR-2DeltaD4), into the medium after 3-5 days in culture, as estimated from silver-stained SDS-PAGE gels (Fig. 1). Recombinant sTNFR-1 and sTNFR-2 constitute about 10 and 5% of the total secreted proteins, respectively. The levels of expression of the full-length soluble receptors are at least 10-fold higher than those obtained from Sf9 cells previously used by us and by others(29) .


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-alpha poorly, and that sTNFR-2 with both domains 3 and 4 deleted did not bind TNF-alpha (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.



Comparison of Ligand Binding Affinity of Full-length and Domain 4-deleted sTNFR-1 and sTNFR-2 by Scatchard Analysis

A direct solid phase ligand binding assay was used to determine the binding affinity of purified sTNFRs by Scatchard analysis(30) .Results for one of the four ligand binding experiments are shown in Fig. 4. Panels A-D on the left are binding curves and Scatchard plots of full-length and domain 4-deleted sTNFR-1, and panelsE-H are those of sTNFR-2. Ligand binding dissociation constants (K(d)) were estimated from the slopes of the Scatchard plots and average values from four separate experiments, using four different preparations of I-TNF and soluble receptor preparations, are listed in Table 1. Full-length sTNFR-2 (average K(d) of 0.35 nM) binds TNF 3 times better than sTNFR-1 (average K(d) of 1.23 nM). The K(d) ratio for the domain 4-deleted sTNFR-1 to the full-length sTNFR-1 was 0.93, indicating that deletion of domain 4 of sTNFR-1 did not affect binding capacity substantially. These data suggested that domain 4 of sTNFR-1 is not involved directly in ligand binding. On the contrary, deletion of domain 4 of sTNFR-2 reduced binding affinity by 3-fold (see K(d) ratio in Table 1), suggesting that the domain 4 of TNFR-2 may be also involved directly in ligand binding and/or the folding of the ligand binding domain.


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.






DISCUSSION

The crystal structure determined by Banner et al.(24) shows that the hTNF-betabullethTNFR-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-beta 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-alpha 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-betabulletTNFR-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-alphabulletTNFR-1 differs substantially from that of the reported structure of TNF-betabulletTNFR-1(24) , in order for domain 4 to bind TNF-alpha or -beta, 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-alpha, 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-alpha differently from TNFR-1. Since cell surface and soluble TNFR-2 bind TNF-alpha with higher affinity than TNFR-1(31) , there may be a unique domain on TNF-alpha, but not on TNF-beta, that interacts specifically with TNFR-2, thereby contributing additional free energy of binding to increase the binding affinity of TNF-alpha for TNFR-2 severalfold over its binding affinity for TNFR-1 (see K(d) ratio in Table 1). In fact, monoclonal antibody against TNF-alpha has been used to show recognition of distinct regions of TNF-alpha 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-alpha. Moreover, although TNF-alpha and TNF-beta 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-alpha, which extends upward from the area in which TNF-beta was shown to bind domain 3 of TNFR-1 in the crystal structure of TNF-betabulletsTNFR-1 complex(24) . Six amino acids in these loops in TNF-alpha 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-alpha as compared to TNF-beta(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-alpha and TNF-beta 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-alpha 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-alpha(39, 40, 41) , suggesting that the receptor binding site(s) on TNF-alpha 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-alpha interacts differently with the two receptors. Of particular interest are the findings that mutations of contiguous residues on human TNF-alpha 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-alphabulletsTNFR-1, TNF-alphabulletsTNFR-2, and TNF-betabulletsTNFR-2 complexes and comparison with the known structure of TNF-betabulletsTNFR-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.


FOOTNOTES

*
This study was supported in part by Grant CA-59832 (to M.-J. C.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
This work was performed in partial fulfillment of the requirements for the Ph.D. degree.

To whom correspondence should be addressed.

(^1)
The abbreviations used are: TNF, tumor necrosis factor; LT, lymphotoxin; TNFR, TNF receptor; NGF, nerve growth factor; PCR, polymerase chain reaction; DTT, dithiothreitol; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Irene Weber for critical reading of the manuscript and Sherry Song for technical assistance.


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