Peptides identify multiple hotspots within the ligand binding domain of the TNF receptor 2
© Hsiao et al; licensee BioMed Central Ltd. 2003
Received: 12 November 2002
Accepted: 24 January 2003
Published: 24 January 2003
Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response.
Here we describe the use of complex and high diversity phage display libraries to isolate peptides (called Hotspot Ligands or HSPLs) which sub-divide the ligand binding domain of the tumor necrosis factor receptor 2 (TNFR2; p75) into multiple hotspots. We have shown that these libraries could generate HSPLs which not only subdivide hotspots on protein and non-protein targets but act as agonists or antagonists. Using this approach, we generated peptides which were specific for human TNFR2, could be competed by the natural ligands, TNFα and TNFβ and induced an unexpected biological response in a TNFR2-specific manner.
To our knowledge, this is the first report describing the dissection of the TNFR2 into biologically active hotspots with the concomitant identification of a novel and unexpected biological activity.
Hotspots have been defined as the minimal functional protein:protein interaction domains through which biological activity can be modulated [1–3]. A novel strategy called Phenogenix® has been developed as a means for studying these interactions. To enable Phenogenix®, we use large and diverse phage display libraries consisting of randomized 20 mer and 40 mer amino acid peptides with > 1011 independent clones. Because of their ability to modulate protein: protein interactions, the resulting peptides are called Hotspot Ligands (HSPLs). Using this approach, we have successfully identified peptide agonists and antagonists for a number of biologically important molecules including growth hormone receptor, insulin receptor and the insulin-like growth factor receptor [2, 3].
In this report, we describe the use of Phenogenix® to identify the critical protein:protein interactions underlying the TNF/TNFR axis. TNFα and TNFβ have been extensively studied and are involved in immune and pro-inflammatory responses, playing an important role in host defenses against infection and other disease states [4–7]. The biological effects of TNFα and TNFβ are mediated through the two membrane associated receptors, TNFR1 (p55) and TNFR2 (p75), that are expressed on the target cells [8, 9]. The postulated pathogenic roles for TNF include sepsis and other bacterial and viral pathologies [10–12], certain cancers , metastasis  and chronic autoimmune disorders such as rheumatoid arthritis [15, 16], multiple sclerosis  and Crohn's disease . It is worth noting that TNFα mutants have been identified that selectively bind TNFR1. One such mutant induced less systemic toxicity yet showed no compromise in its anti-tumor activity in nude mice . These observations indicated that finding modulators specific for each of the TNF receptors is indeed possible.
While TNFα binds equally well to both receptors, the majority of biologic responses attributed to TNF are mediated via TNFR1 although there is evidence that activation of TNFR2 is responsible for some adverse effects of TNFα [19–24]. Thus, the differential regulation of these two receptors may require drugs that specifically target either TNFR1 or TNFR2 allowing therapeutic efficacy without the expected toxicity profile. One obvious possibility then is to search for Surrogates that selectively bind and regulate the individual receptors. To date several antagonistic protein reagents have been isolated for treating diseases with an underlying TNF etiology. These include neutralizing monoclonal antibodies and soluble forms of TNFR1 and TNFR2 [25–27]. However, both antibodies and extracellular receptors are large proteins and not amenable to oral administration. They also carry the unwanted risk of stimulating an auto-immune response during chronic use.
By panning our peptide libraries against the TNFR2, we isolated four HSPL peptides binding specifically to human TNFR2 that allow the subdivision of the receptor into a minimum of at least three independent hotspots involved in its interaction with TNFα. In a biological model for TNFα activation of TNFR2, three of the four peptides induced cytotoxic response in the target cell line suggesting that at least one of these domains induced a unique TNFR2-specific biological response. Overall, these data show the utility of using the HSPL peptides for subdividing protein:protein interacting domains and for defining unique novel biological activities not observed through the natural ligand/receptor interaction.
Receptor-Specific Hotspot Ligands Are Identified By Biopanning TNFR2.
GLQRWC RWAESAC GDSGPLR
ATW QEY FRRGG SC SLAESC G
CWRW IEY WSRGG QVGRRAGA
W xEY xxRGG
SVVAWTGALRPSWVELRC LREVSDSGKMHPAWLDYC GELS
Phage Competition ELISAs
Competitor IC50 (M)
Summary Of HotSpot Ligand Interactions With TNFR2
Competed by TNF-alpha & TNF-Beta
Competed by Peptides
Competed by Anti-TNFR2 (mAb226)
Cytotoxic Response On SK-N-E cells
Cytotoxic Response Inhibited by
Peptide libraries have been successfully used for identifying novel peptides mimicing natural molecules in terms of the critical protein:protein interactions involved in biological activity. Yanofsky et al.  described a peptide antagonist to IL-1 that was active in the nM range and Wrighton et al. and Livnah et al. [32, 33] identified peptides binding to the erythropoietin (EPO) receptor with full agonist activity in vivo. Neither the EPO agonist nor the IL-1 antagonist peptides show any significant sequence homology to the natural ligand suggesting that they act as mimics of the natural ligand. In the case of the EPO agonists, X-ray crystallographic analysis revealed that one of these peptides spontaneously formed non-covalent homodimers that allowed the dimerization of two EPO receptors. Two families of small peptides binding to the human thrombopoietin receptor (TPOR) were isolated that competed with the natural ligand TPO . The peptide with the highest affinity was subsequently dimerized and shown to function as a potent TPO agonist in vivo. Recently, a small, non-peptide organic molecule binding to one chain of the EPO receptor was multimerized and shown to mimic the biological activities of the EPO .
In this report we show that HSPLs can be isolated that bind to human TNFR2 at its ligand-binding site and subdivide it into multiple hotspots. All of the peptides were competed by both TNFα and TNFβ implying they recognized a hotspot that mapped either within or at least overlapping the ligand-binding domain of TNFR2. The HSPLs KcC7, KcD11, KcF6 but not KcF12 were also inhibited by the neutralizing antibody mAb226 suggesting the 20 mers but not KcF12 interact with a hotspot overlapping with the one recognized by antibody. The fact that recombinant KcF12 peptide competed all of the phage expressed peptides (Table 3) indicated that the four peptides also recognize a common hotspot which may be the one responsible for TNF ligand binding to the receptor. In addition, the biological data suggests that KcD11 binds to a hotspot that can be further separated from KcF12, KcF6 and KcC7. While we cannot rule out possible allosteric interactions between the peptides, the data suggests that the HSPLs were able to dissect the ligand-binding region of TNFR2 into several structural and/or functional hotspots. In addition, the HSPLs were specific for TNFR2 unlike the native ligands TNF-α and TNF-β which bind to both receptors. These results demonstrate that the approach has the ability to dissect critical protein:protein interaction domains in terms of both structural and functional elements. Using a similar approach, we recently reported on the subdivision of the ligand binding domain on the insulin receptor into two separate and non-overlapping hotspots with different biological activities .
It has been shown that TNFα induced proliferation of the undifferentiated neuroblastoma cell line, SK-N-BE, resulted from activation of TNFR2 . Proliferation of SK-N-BE cells could be suppressed by the pre-incubation of cells with anti-TNFR2 blocking antibodies without concomitant cytotoxicity (data not shown). While all of the peptides blocked the agonist effects of TNFα indicating that they can act as antagonists, KcC7, KcF6 and KcF12 by themselves induced a cytotoxic response by killing SK-N-BE cells in the absence of TNFα. This is surprising because these peptides share a hotspot with the natural ligand TNFα (which is proliferative rather than-cytotoxic) and KcC7 and KcF6 bind to a hotspot common to the neutralizing but non-cytotoxic, antibody mAb226. Overall, these data suggest that activation (agonism or cytotoxicity) of TNFR2 is mediated through unique hotspots on the receptor that can be defined by the peptides. Under the present experimental conditions, it is unclear whether the natural ligand, TNFα, touches the hotspot responsible for cytotoxicity although one can speculate that, if it does, the cytotoxic effect might be nullified by the sum of the natural ligand:receptor interactions. Competition experiments showing that mAb 226 reversed the effects of KcC7 and KcF6 confirm that these two peptides induce their effects though their interaction with TNFR2. These findings suggest that TNFR2 has multiple biological functions. In addition to transferring TNFα to TNFR1 as proposed by the ligand passing model , TNFR2 may have a role in modulating proliferative responses in both positive and negative manner. Ongoing studies will attempt to elucidate the mechanism of action behind the phenomenon such as the effects on downstream signaling pathways including NFkB, and apoptosis. However, it is possible that peptide-induced cytotoxicity works through a mechanism similar to TNF mediated apoptosis in T-cells .
In summary, HSPLs were isolated with which we were able to dissect the ligand-binding domain of a natural receptor and generate information about novel and unexpected bioactivities. This technology has also been successfully used for known protein targets, such as the insulin receptor  and TNF-β  as well as non-protein targets such as disease-specific RNAs . We have also shown that the HSPL libraries identify hotspots on novel genes of unknown function and identify functional interaction pathways (manuscript in preparation). Overall, the use of HSPLs have the potential to become a general approach for scanning and understanding complex protein:protein interactions.
E. coli, strain TG1 (genotype = K12Δ(lac-pro), supE, thi, hsdΔ5/F' [traD36, proAB, lacI q , lacZΔM15]) was obtained from Pharmacia (Piscataway, NJ). Recombinant human Tumor Necrosis Factor-α (rhTNFα) was purchased from R&D Systems, Inc. (Minneapolis, MN) or PeproTech Inc. (Rocky Hill, NJ). Monoclonal antibodies against recombinant human soluble TNF receptor RI (mAb225) and RII (mAb226) were purchased from R&D Systems, Inc. (Minneapolis, MN). WST-1 was purchased from Roche (Nutley, NJ). 125I-TNFα (NEX 257) with a specific activity of 97 μCi/μg (1698 Ci/mmol) was purchased from NEN Life Science Products, Inc (Boston, MA). Beckman Ready Safe™ scintillation fluid was purchased from Beckman Coulter, Inc. (Fullerton, CA). MultiSreen™ assay filter plate system and 96-well MultiScreen® MAFC filter plates were from Millipore (Bedford, MA). RPMI 1640 media, fetal bovine serum, and GlutaMAX™-I were obtained from Life Technologies (Rockville, MD). Polyvinylpyrrolidone (PVP), Tween-20R™, phosphate-buffered saline (PBS), and Dulbecco's phosphate-buffered saline (containing no calcium or magnesium) were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals and reagents used were analytical grade.
SK-N-BE neuroblastoma cells, Jurkat (TIB-152) and Jiyoye (CCL-87) cells were obtained from the ATCC (Manassas, VA). SK-N-BE and Jurkat cells were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and 2 mM GlutaMAX™-I, while Jiyoye cells were maintained in RPMI 1640 media supplemented with 15% FBS and 2 mM GlutaMAX™.
Design of the Primary Peptide Libraries
Subtractive Panning and ELISA
Panning was done as described [3, 28], except that only 6 wells of a 96-well microtiter plate were coated with target at concentrations ranging from 10–100 ng/well. The cross reactive phages from the eluted phage were then subtracted by panning sequentially against human TNFR1, TNFα and TNFβ as a way to obtain peptides specific to the original target. ELISA was done as previous described [3, 28].
Phage Competition ELISA Studies
Target was coated on MaxiSorp plates and blocked with NFM-PBS as described [3, 28]. HSPLs (phage clones or synthetic peptides) are added in the presence of varying concentrations of competitor (e.g., TNF ligand or soluble receptor). IC50s are determined by probing for phage or peptide binding to target in the presence of competitor.
Receptor Binding and Competition Assay
The binding of TNFα to TNFR2 on intact cells was measured in a radioareceptor assay using Jurkat and Jiyoye cells and the MultiSreen™ assay filter plate system as described . Data analysis was performed with an add-in for Microsoft Excel, Xlfit™ (version 2.0) from ID Business Solutions (Guildford, UK), which allows curve fitting using nonlinear regression. To determine binding kinetic parameters, the Four-Parameter logistic equation y = Bottom + (Top – Bottom)/(1 + (IC50/x)Hill slope) was used to calculate IC50 values and Hill slopes. Ki values were calculated according to the equation, Ki = IC50/(1 + [ligand]/Kd), as described by Cheng and Prusoff .
Proliferation cell assay
SK-N-BE cells were seeded loosely in 96 well plates in SK-N-BE medium and allowed to grow to 75–80% confluency. Cells were starved overnight in media containing 1% serum before each cell experiment. Briefly, cells were first treated with a human TNFR1 blocking mAb, mAb225 (10 ug/ml), for at least 30 min at 37 C prior to the addition of agents. All agents used in the cell experiment were serial diluted at 1:2 and mixed in a separate plate before transferring into the actual cell plate. The starting highest final concentration of each peptide was at 30 uM or at its highest possible concentration. Cells were then returned into the incubator for 40–45 hrs before the addition of 1/10 vol of WST-1. The plates were further incubated and developed according to the manufacturer's instruction.
The authors would like to thank Huong Voung for biopanning; Dariusz Galkowski and Leigh-Ann Ricks for sequencing; Nicole McGuire for purification of rKcF12; Krystina Zagorska for cell assays; Joanna Llanas and Urvashi Patel for the radioreceptor assays.
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