Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

<p>Abstract</p> <p>Background</p> <p>The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings.</p> <p>Results</p> <p>After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2), it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence.</p> <p>Conclusion</p> <p>We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development.</p

Generation of a single chain antibody variable fragment (scFv) to sense selectively RhoB activation.

Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display Kd values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation

scFvE3 is a selective sensor of RhoB activation in HeLa cells.

<p>A, CBD-pulldown experiments on nucleotides loaded HeLa cell extracts showing the specificities of the selected scFvs. HeLa cell extracts were loaded with either GDP (1 mM) or GTPγS (100 µM) and incubated with scFvs F7, D10 and E3 fixed on chitin beads. CBD-pulldowns were analyzed by Western blotting using anti-RhoA, anti-RhoB and anti-RhoC antibodies. Total extract used for CBD-pulldown is indicated as <i>input</i> and examined by western blotting with the same antibodies. Western Blot is representative of 4 independent experiments. B, RhoB and RhoA activation were assessed by GST-pulldown (RBD) and CBD-pulldown (E3) experiments with cell lysate from HeLa cells transiently transfected with plasmids expressing Myc-tagged XPLN or GFP under the control of CMV promotor. XPLN was detected by using an anti-c-myc antibody. C, HeLa cells were serum-starved for 24 h and treated with EGF (2.5 ng/mL) for 10 min before lysis then RhoB and RhoA activation were assessed by GST-pulldown (RBD) and CBD-pulldown (E3) experiments. Beads-bound proteins were analyzed by Western blotting using anti-RhoA and anti-RhoB antibodies. Total cell extracts are indicated as <i>input</i> and examined by western blotting with the same antibodies. Western Blots are representatives of 2 independent experiments.</p

ScFvE3 preferentially binds to RhoB active conformation.

<p>A, Binding affinities of two selected scFvs (A5 and E3) for 6xHis-RhoAL63 (AL63), 6xHis-RhoBL63 (BL63) were measured by competitive ELISA on dilution of 6his-RhoL63 displayed in molar logarithmic scale (Log M), as described in experimental procedures. K_d values were determined by nonlinear regression and listed in the insert table (mean ± SD, n = 3 each). B, The specificity of the purified scFvE3 for the active form of the recombinant wild type GST-RhoB loaded with either GDP or GTPγS was assessed by ELISA. Results are expressed as absorbance at 450 nm (mean ± SD, Mann-Whitney test, n = 4).</p

Structure comparison between RhoA and RhoB.

<p>A, The filling structure of active RhoAV14-GTPγS (Pdb code 1A2B) was created by UCSF-Chimera software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111034#pone.0111034-Pettersen1" target="_blank">[53]</a>. Switch I & II are depicted in red and blue, respectively. The RhoA/RhoB differences in amino acids sequence are shown in green with the residue 29 indicated by an arrow. B, Structural divergence between RhoA and RhoB in the β2-β3 région. The <i>Matchmaker tool</i> in <i>UCSF-Chimera</i> (<a href="http://www.cgl.ucsf.edu/chimera" target="_blank">http://www.cgl.ucsf.edu/chimera</a>) was used to generate the structural superposition. RhoA (Green, Pdb code 1DPF) & RhoB (Yellow, Pdb code 2FV8) structures are shown in ribbon model and the substrate GDP in stick. Both proteins are in the inactive form with only GDP in the active site.</p

Affinity maturation revealed the possibility to obtain binders distinguishing RhoA and RhoC from RhoB active conformations.

<p>A, Improvement in apparent affinity throughout the rounds of selection was evaluated by a polyclonal phage ELISA on dilution of GST-RhoAL63 displayed in molar logarithmic scale (Log M). B, Three purified scFvs (F7, H9 and D10) were analyzed for their binding specificity towards L63 active mutants of recombinant GST-RhoA, RhoB and RhoC by ELISA. Purified scFvC1 was used as a control. C, Affinities of two scFvs (F7 and D10) for 6xHis-RhoAL63 (AL63), 6xHis-RhoBL63 (BL63) were measured by competitive ELISA as described in experimental procedures. K_d values were determined by nonlinear regression and listed in the insert table (mean ± SD, n = 3 each). D, The specificity of purified scFvF7 and scFvD10 for the active form of the recombinant wild type GST-RhoA and GST-RhoB loaded with either GDP or GTPγS were assessed by ELISA. Results are expressed as normalized absorbance of the scFvs to the total amount of coated GST-Rho quantified by the use of commercial antibodies (mean ± SD, Mann-Whitney test, n = 4 each).</p

Real-time binding of scFvs F7, D10 and E3 by Surface Plasmon Resonance on immobilized GST fusion Rho active mutant proteins.

<p>Single Cycle Kinetics analysis was performed on immobilized GST fusion proteins RhoAL63, RhoBL63 and RhoCL63 (1000 RU) with five injections of analyte at 6.25nM, 12.5nM, 25nM, 50nM, and 100nM. Analyte injections lasted for 120 s each and were separated by 180 s dissociation phases. An extended dissociation period of 10 min followed the last injection. The two sensograms recorded for a given analyte were fitted globally to a 1∶1 interaction (data not show). Each sensogram represents a differential response where reference channel of immobilized GST protein has been substracted and is expressed in RU as a function of time in second. Bottom: table summarizing kinetic constant parameters. ND means not determined. § means that kinetics parameters were obtained by fitting curves with too low resonance units to give accurate values.</p

Selection of a RhoB active conformation specific scFv.

<p>A, Strategy of phage display selection. B, The enrichment of clones specific of the RhoB active form throughout the selection procedure was assessed by polyclonal phage ELISA on captured GST-Rho proteins from crude extract. Total amount of coated GST-Rho and active form of GST-RhoL63 were quantified with commercial anti-RhoA (Ab-RhoA) and anti-RhoB (Ab-RhoB) antibodies, and phageF7 (F7) and phageD10 (D10), respectively. GDP-bound GST-Rho (wt) was included as controls. C, (top panel) 88 individual clones were analyzed for their binding to GST-RhoBL63 and GST-RhoAL63 by phage ELISA. Results are expressed as the ratio of absorbance against GST-RhoBL63 <i>vs.</i> GST-RhoAL63. PhageF7 (black circle) and phageD10 (black diamond) were included as controls. Arrows indicate the clones E3 and A5 further selected. (Bottom panel) 26 clones were further analyzed for their binding to GST-RhoBL63 and GST-RhoBwt-GDP. Results are expressed as the ratio of absorbance against GST-RhoBL63 <i>vs.</i> GST-RhoBwt. Arrows indicate the clones E3 and A5. E3 was the best conformational sensor selective of active RhoB-GTP.</p