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

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-2

to the human immunoglobulins sequences (imgt). Sequences highlighted in red vary from the amino-acid sequence of scFv C1. Underlined sequences correspond to CDR. Asterisks indicate the missing amino-acid that would generate the usual CDR.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-7

sequence of bacterial pectate lyase that mediates secretion into the periplasmic space; : variable fragment of the heavy chain; : light chain; : 6 histidine-tag; : myc-tag; amber stop codon; : portions of the N- and C-term of phage capside protein pIII; and : primers used for sequencing the Vand Vdomain. Schematic construction of vectors encoding soluble scFv C1 and scFv C1-N1N2. The amber stop codon between the scFv and gene III in pHEN 2 was removed by mutagenesis (middle construct). The C-terminal portion of pIII was removed in the final pHEN C1-N1N2 vector (bottom construct), first by PCR amplification of pHEN C1-pIII, introducing an EcoRI site after N2, and subsequently by cloning the NcoI and EcoRI digested PCR product into the linearized pHEN C1-pIII plasmid at the NotI and EcoRI sites. : 6 histidine-tag; M: myc-tag.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

P2Y13 receptor regulates HDL metabolism and atherosclerosis in vivo.

High-density lipoprotein (HDL) is known to protect against atherosclerosis by promoting the reverse cholesterol transport. A new pathway for the regulation of HDL-cholesterol (HDL-c) removal involving F1-ATPase and P2Y13 receptor (P2Y13R) was described in vitro, and recently in mice. However, the physiological role of F1-ATPase/P2Y13R pathway in the modulation of vascular pathology i.e. in the development of atherosclerotic plaques is still unknown. We designed a specific novel agonist (CT1007900) of the P2Y13R that caused stimulation of bile acid secretion associated with an increased uptake of HDL-c in the liver after single dosing in mice. Repeated dose administration in mice, for 2 weeks, stimulated the apoA-I synthesis and formation of small HDL particles. Plasma samples from the agonist-treated mice had high efflux capacity for mobilization of cholesterol in vitro compared to placebo group. In apoE-/- mice this agonist induced a decrease of atherosclerotic plaques in aortas and carotids. The specificity of P2Y13R pathway in those mice was assessed using adenovirus encoding P2Y13R-shRNA. These results demonstrate that P2Y13R plays a pivotal role in the HDL metabolism and could also be a useful therapeutic agent to decrease atherosclerosis. In this study, the up-regulation of HDL-c metabolism via activation of the P2Y13R using agonists could promote reverse cholesterol transport and promote inhibition of atherosclerosis progression in mice

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-4

ScFvs were resolved on 12,5% SDS-PAGE, immunoblotted with c-myc antibody, and visualized by enhanced chemiluminescence as described in Methods. (2) scFvs from crude extract were analysed for binding to GST-RhoA and GST-RhoAQ63L protein immobilized on an ELISA plate. Bound scFvs were detected with horseradish peroxydase-labeled anti-c-myc using TMB as substrate. Results are expressed as absorbance at 480 nm. Graphs are representative of 6 experiments, each performed in duplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-0

sequence of bacterial pectate lyase that mediates secretion into the periplasmic space; : variable fragment of the heavy chain; : light chain; : 6 histidine-tag; : myc-tag; amber stop codon; : portions of the N- and C-term of phage capside protein pIII; and : primers used for sequencing the Vand Vdomain. Schematic construction of vectors encoding soluble scFv C1 and scFv C1-N1N2. The amber stop codon between the scFv and gene III in pHEN 2 was removed by mutagenesis (middle construct). The C-terminal portion of pIII was removed in the final pHEN C1-N1N2 vector (bottom construct), first by PCR amplification of pHEN C1-pIII, introducing an EcoRI site after N2, and subsequently by cloning the NcoI and EcoRI digested PCR product into the linearized pHEN C1-pIII plasmid at the NotI and EcoRI sites. : 6 histidine-tag; M: myc-tag.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-1

RhoB (white columns) and GST-RhoBQ63L (black columns) protein immobilized on an ELISA plate. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Helper phage was used as a control. Results are expressed as absorbance at 480 nm. : ratio of absorbance of binding to RhoBQ63L to absorbance of binding to RhoB. : Selectivity of C1 phages on WT and activated Q63L form of Rho. 10clones of C1 (black columns) and control (white columns) phage were analyzed for binding to GST-RhoA, RhoB and RhoC, both wild type (WT) and Q63L forms, immobilized on a glutathione ELISA plate. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. Concentrations of GST-Rho proteins in each well were monitored by anti-GST (not shown). The graph is representative for 3 experiments, and each binding assay was performed in duplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-3

100 μM of GDP and GTPγS at 37°C. 10clones of C1 phages were incubated with loaded (GTP or GDP) GST-RhoA-bound beads. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. The graph is representative of 2 independents experiments. GST-RhoA, B and C () and GST-RhoB, Rac1 and Cdc42 () were loaded with 200 μM of GDP or GTPγS for 30 min and purified on glutathione ELISA plates. 10clones of C1 phages were incubated in each well. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. Amount of GST protein was quantified with goat anti-GST antibody followed by horseradish peroxydase-labeled anti-goat (not shown). The graph is representative of 3 experiments, each binding assay performed in duplicate. : Specific radioactivity binding of [S] GTPγS on RhoB, Rac1 and Cdc42. GST-RhoB, Rac1 and Cdc42 were loaded with 20 nM [S] GTPγS in the presence (non specific binding) or not (total binding) of 200 μM unlabeled GTP for 30 minutes at 37°C and purified on gluthatione ELISA plates. Radioactivity was measured in each well. The difference between the total binding and the non specific binding represent the specific binding. The graph is representative of 2 experiments, each performed in triplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-6

recombinant RhoA and RhoB loaded with either GTP or GDP were incubated with C1-N1N2 fixed on Ni-beads. An irrevelant scFv was used as control. Complexes on beads were resolved by SDS-PAGE and immunobloted with anti-RhoA and anti-RhoB. Western Blot is representative of 2 independent experiments. Immunofluorescence shows that scFv C1-N1N2 specifically binds to activated HeLa cells. Suspension containing scFv C1-N1N2 was incubated with GDP-loaded RhoA(and ) or GTPγS-preloaded RhoA beads (). Twenty-four hours after seeding, HeLa cells were serum-starved for 48 h and activated with 10% SVF and EGF (100 ng/ml) for 1 hour. Cells were fixed, permabilized and incubated with supernatants from scFv C1-N1N2-Rho incubation and anti-c-myc FITC conjugate secondary antibody. ) Non-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads, ) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads ) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GTPγS-loaded RhoA beads. () Non-activated HeLa cells incubated with the commercial Rhoa antibody, () EGF-activated HeLa cells incubated with the commercial RhoA antibody, () EGF-activated HeLa cells incubated with irrelevant scFv (anti-tyroglobulin). Pictures are representative of 2 independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p