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. 2008;3(10):e3452.
doi: 10.1371/journal.pone.0003452. Epub 2008 Oct 20.

A ligand peptide motif selected from a cancer patient is a receptor-interacting site within human interleukin-11

Marina Cardó-Vila  1 , Amado J Zurita, Ricardo J Giordano, Jessica Sun, Roberto Rangel, Liliana Guzman-Rojas, Cristiane D Anobom, Ana P Valente, Fábio C L Almeida, Johanna Lahdenranta, Mikhail G Kolonin, Wadih Arap, Renata Pasqualini
Affiliations

A ligand peptide motif selected from a cancer patient is a receptor-interacting site within human interleukin-11

Marina Cardó-Vila et al. PLoS One. 2008.

Abstract

Interleukin-11 (IL-11) is a pleiotropic cytokine approved by the FDA against chemotherapy-induced thrombocytopenia. From a combinatorial selection in a cancer patient, we isolated an IL-11-like peptide mapping to domain I of the IL-11 (sequence CGRRAGGSC). Although this motif has ligand attributes, it is not within the previously characterized interacting sites. Here we design and validate in-tandem binding assays, site-directed mutagenesis and NMR spectroscopy to show (i) the peptide mimics a receptor-binding site within IL-11, (ii) the binding of CGRRAGGSC to the IL-11R alpha is functionally relevant, (iii) Arg4 and Ser8 are the key residues mediating the interaction, and (iv) the IL-11-like motif induces cell proliferation through STAT3 activation. These structural and functional results uncover an as yet unrecognized receptor-binding site in human IL-11. Given that IL-11R alpha has been proposed as a target in human cancer, our results provide clues for the rational design of targeted drugs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Binding of IL-11-like peptides to IL-11Rα.
(A) CGRRAGGSC-phage binding to the individual receptor components of the IL-11 receptor complex: IL-11Rα and gp130. Leptin receptor and BSA served as negative controls for binding. (B) Concentration-dependent binding inhibition of CGRRAGGSC-phage to IL-11Rα by the cognate synthetic peptide. Bars represent mean±standard error of the mean (SEM).
Figure 2
Figure 2. Structural basis of the interaction between CGRRAGGSC and IL-11Rα.
(A) Effect of the CGRRAGGSC peptide concentration upon biding to IL-11Rα. Amide region of 1D-1H-NMR spectra of increasing molar concentrations of the peptide CGRRAGGSC binding to IL-11Rα (6 µM) is shown (blue). CGRRAGGSC peptide alone (400 µM) and the spectrum of IL-11Rα alone are also shown (in red and black, respectively). The appearance of arginine side-chain resonances (not seen in the spectrum of the peptide alone) is indicated (*). (B) Chemical shift changes induced on the CGRRAGGSC resonances by binding to IL-11Rα. The 2D-1H-NMR TOCSY spectra of CGRRAGGSC (400 µM) either alone (black) or in the presence of 6 µM IL-11Rα (red) are shown. Single-headed and double-headed arrows indicate chemical shift changes and the appearance of new spin-systems, respectively. (C) Composition of amide region of 1D-1H-NMR and corresponding 2D-1H-NMR TOCSY spectra of the CGRRAGGSC peptide at 5°C (left), at 25°C (middle) and at 25°C in the presence of IL-11Rα (right). The circles with dotted lines and the arrow indicate arginine resonances.
Figure 3
Figure 3. Binding of wild-type or site-directed mutants of IL-11 to IL-11Rα.
(A) Purified recombinant proteins were analyzed by Coomassie staining. (B) Western blot analyses with polyclonal anti-IL-11 and anti-GST antibodies. (C) Recombinant GST fusion proteins (alanine scan mutants of residues 112–117 of IL-11, wild-type IL-11, GST alone, or rhIL-11) were coated in triplicate overnight and incubated with IL-11Rα. Binding was detected with anti-Fc antibody. Bars represent mean±standard error of the mean (SEM).
Figure 4
Figure 4. Biological effects of the IL-11-like peptide CGRRAGGSC on IL-11-responsive cells.
(A) Concentration-dependent proliferative response to CGRRAGGSC is shown on IL-11Rα-expressing human TF-1 leukemia cells in the absence or presence of IL-11 (left panel). No response is observed on non-IL-11Rα-expressing control cells (right panel). (B) Soluble IL-11Rα-mediated inhibition of the proliferative effect induced by 150 µM CGRRAGGSC peptide (and by IL-11). * t-test, P<0.005. Bars represent mean±standard error of the mean (SEM).
Figure 5
Figure 5. The IL-11-Like peptide CGRRAGGSC induces STAT3 activation upon binding to IL-11Rα in a concentration-dependent manner.
(A) Proliferation of human TF-1 leukemia cells induced by CGRRAGGSC is associated with STAT3 phosphorylation, as assessed with an anti-STAT3-P-Tyr705 antibody. (B) CGRRAGGSC peptide-induced activation of STAT3 is concentration dependent. No effect is observed with a control peptide. To avoid inter-experimental variation, the lysate was divided in two: one-half of the lysate was immunoblotted with an anti-STAT3-P-Tyr705 antibody whereas the other half served to determine the total amount of STAT3 (as a surrogate for protein loading) with a specific anti-STAT3 antibody. Note that the commercial HeLa cell extracts serving as controls display only the ∝-STAT3 band.
Figure 6
Figure 6. Residues identified within known human IL-11 binding sites.
Arrowheads indicate human IL-11 residues predicted (through site-directed mutagenesis) to be ligand-receptor binding sites; solid colors specify residues that are critical for binding –. Residues 112–117 corresponding to the IL-11-like motif , described in this work are highlighted in yellow. The basic scheme of human IL-11, as shown here, has been modified from reference .
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