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Research
Peptides identify multiple hotspots within the ligand binding domain of the TNF receptor 2
Ku-chuan Hsiao , Renee E Brissette , Pinger Wang , Paul W Fletcher , Vanessa Rodriguez , Michael Lennick , Arthur J Blume and Neil I Goldstein
DGI BioTechnologies, Inc., 40 Talmadge Road, Edison NJ 08818, USA
Proteome Science 2003 1:1
The electronic version of this article is the complete one and can be found online at: proteomesci.com
Received 12 November 2002
Accepted 24 January 2003
Published 24 January 2003
© 2003 Hsiao et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
Abstract
Background
Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response.
Results
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, TNFá and TNFâ and induced an unexpected biological response in a TNFR2-specific manner.
Conclusions
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. TNFá and TNFâ 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 TNFá and TNFâ 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 [13], metastasis [14] and chronic autoimmune disorders such as rheumatoid arthritis [15,16], multiple sclerosis [17] and Crohn's disease [18]. It is worth noting that TNFá 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 [13]. These observations indicated that finding modulators specific for each of the TNF receptors is indeed possible.
While TNFá 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 TNFá [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 TNFá. In a biological model for TNFá 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.
Figures
Figure 1
Competitive Displacement Of Phage Clones Expressing hTNFR2-Hotspot Peptide Ligands By Human TNF (Alpha And Beta) Or Soluble TNF Receptor (R1 Or R2) Fusions
Figure 2
Inhibition Of Hotspot Peptide Ligand(s) Binding To Recombinant Human Soluble TNFR2 By Anti-TNFR2 Antibody, mAb226
Figure 3
Hotspot Peptide Ligand Kcf12 Competes TNF Binding To Endogenous TNFR2 Receptors
Figure 4
Cytotoxic Effect induced By The HotSpot Peptide Ligands In SK-N-BE Cells
Tables
Table 1
Receptor-Specific Hotspot Ligands Are Identified By Biopanning TNFR2.
Table 2
Phage Competition ELISAs
Table 3
Summary Of HotSpot Ligand Interactions With TNFR2
Human TNFR2 was panned with random 20 mer and 40 mer phage display peptide libraries of high diversity [2,3,28]. After four rounds of panning, four phage clones were isolated with specificity for TNFR2. The peptides were initially divided into two groups: a 40 mer designated KcF12 and three 20 mers designated KcC7, KcD11 and KcF6 (Table 1). From the amino acid sequences, KcC7, KcD11 and KcF12 were found to have a pair of cysteine residues suggesting the possibility of intra-chain disulfide bonding. In addition, a putative motif, WxEYxxRGG, was found for peptides KcF6 and KcD11 (Table 1). It was interesting that this motif was related to a sequence WxExxxxxGG found at the C-terminal (amino acids 190–198) of TNF-á. No appreciable sequence homology was seen between either of the natural ligands, TNFá or TNFâ, and KcF12 although the spacing of cysteine loop was close to that seen in the cysteine rich domains of TNFR2 [29]. At present, it is not known whether these structural elements play a role in binding of the peptides to TNFR2 and it may be that the peptides mimic rather than recapitulate the contact domains of the natural ligands. ELISA analysis showed that all four phage clones bound specifically to human TNFR2 and did not react with either human or murine TNFR1 or murine TNFR2 (data not shown).
Competition ELISAs were performed using TNFá and TNFâ vs. the four phage clones at their ED50 values (Figure 1a,1b,1c,1d). More TNFá and TNFâ were required to compete KcF12 than the other phage clones suggesting that certain intrinsic properties of KcF12 (such as its length and/or amino acid content) may contribute to a higher relative affinity (Table 2). In addition, the synthetic forms of KcC7, KcD11, KcF6 and the recombinant KcF12 (rKcF12) were able to compete the binding of TNFá and TNFâ, as well as KcF6 and KcF12 phage clones in the micromolar range (unpublished data). Interestingly, the three 20 mer HSPLs but not KcF12 were competed by an anti-TNFR2 monoclonal antibody, mAb226, (Figure 2) suggesting that KCF12 binds to a hotspot on TNFR2 distinct from the other 3 peptides. A summary of the phage/peptide competition data is shown in Table 3.
Binding of KcF12 to native TNFR2 was measured in Jurkat and Jiyoye cells using a radioreceptor assay. Jurkat cells have been reported to have approximately equal numbers of TNFR1 and TNFR2 per cell [27] whereas Jiyoye cells express mainly TNFR2 (unpublished data). KcF12 inhibits TNFá binding to Jiyoye cells with a Ki of about 2 uM whereas the Ki for the peptide in Jurkat cells was found to be >30 uM (Figure 3). Since TNFá binding to Jurkat probably involves both TNFR1 and TNFR2, the data offers additional confirmation of KcF12's selectivity for TNFR2.
It has been previously reported that TNFá induced a TNFR2-specific proliferation of the neuroblastoma cell line SK-N-BE [30]. KcF12, KcC7 and KcF6 inhibited the proliferative response to TNF-á in this cell line indicating that they could act as antagonists (data not shown). In addition, the same three peptides were found to be cytotoxic to SK-N-BE cells in the absence of TNFá (Figure 4a). KcF12 possessed the strongest killing activity with an IC50 of ~700 nM whereas the IC50s for KcC7 and KcF6 were 2 uM and 7 uM, respectively. None of the peptides induced a cytotoxic response in cell lines not expressing TNFR2 (data not shown). Interestingly, KcD11, which shares a motif with KcF6 (Table 1), had no effect on SK-N-BE cells. The absence of activity by KcD11 did not appear to be due to breakdown of the peptide during culturing since mass spectral analyses on aliquots retrieved from culture media showed relatively no degradation of peptides between the initial and the final time points (data not shown). Therefore, the proliferation data suggests evidence of a hotspot on TNFR2 recognized by KcF12, KcC7 and KcF6 that is linked to this unique biological effect. The inhibition of the KcC7 and KcF6 cytotoxic effect with mAb226 (Figure 4b) confirms the relationship between the peptide-induced cytotoxic event and TNFR2 because of the antibody's specificity for TNFR2. Overall, these data indicate that the surface topology of the TNFR2 binding site is very complex and may be composed of multiple hotspots capable of different regulatory functions.
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