Yeast display of scFv-D1.3-GFP11.

<p>The scFv-D1.3-GFP11 containing both the GFP11 peptide as well as the peptide epitope recognized by SV5 was displayed on the surface of yeast cells. Biotinylated lysozyme was detected using Alexa 633 labeled streptavidin, and display levels were assessed using either GFP1-10 or phycoerythrin labeled SV5. Labeling by GFP complementation was essentially as effective in normalizing display levels as the use of PE labeled SV5.</p

Split-GFP labeling of scFvs.

<p><b>A. GFP complementation vectors.</b> The scFv-D1.3-GFP11 construct was cloned into a pEP based vector (left) or a yeast display vector (right). The GFP11 peptide is cloned at the C-terminus of the scFv in order not to interfere with the scFv/antigen-binding activity. <b>B. Linear representation of the scFv-GFP11 molecule.</b> The sequence of the SV5 tag, followed by the GFP11 sequence and the six histidine tag (6xHis tag) found at the C terminus of the scFv is shown. <b>C. Split GFP fragment complementation.</b> The scFv is fused to the small GFP fragment (strand 11, residues 215-230). The complementary GFP fragment (1-10, residues 1-214) is expressed separately. Neither fragment alone is fluorescent. When mixed, the small and large GFP fragments spontaneously associate, resulting in reconstitution of the fluorophore and fluorescence. <b>D. Split GFP fragment complementation in scFv yeast display.</b> The scFv in fusion with the GFP11 strand is displayed on yeast cells. It is still able to bind the specific antigen and fluorescence is restored when yeast cells are incubated with the GFP1-10 fragment.</p

Affinity determination.

<p><b>A. Affinity determination by multiplexed assay using purified protein.</b> After 2 hours of complementation with the GFP1-10 fragment, the scFv-D1.3-GFP11 shows specific binding to its antigen, chicken lysozyme, and no binding activity to the negative control, myoglobin. Fluorescence values are plotted versus the different concentrations of purified scFv-D1.3-GFP11 giving a Kd value of 29±4 nM. Data are fit with a nonlinear least squares regression and are visualized with the concentration values on a log scale. <b>B. </b><b>Affinity determination by multiplexed assay using serial dilution of bacterial lysate (POP culture extract) obtained from induced cells.</b> Unpurified scFv-D1.3-GFP11 obtained from induced bacteria shows specific binding to lysozyme, but not to myoglobin or IgE receptor. Fluorescence values are plotted versus the different concentrations of scFv-D1.3-GFP11 present in crude bacteria extract calculated by reference to the GFP complementation standard (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025727#pone-0025727-g004" target="_blank">figure 4</a>), resulting in a Kd value of 38±7 nM. Data are fit with a nonlinear least squares regression and are plotted with the concentration values on a log scale.</p

GFP1-10 standard curves to evaluate protein concentration.

<p>A GFP11 standard plot was constructed using increasing amounts of sulfite reductase GFP11 (SR-GFP11) and scFv-D1.3-GFP11 (ranging from 1.5 to 160 pmol) in a molar excess of GFP1-10. The quantity of GFP11 tagged scFv in the cell extract was calculated according to the equations presented in Canbantou et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025727#pone.0025727-Cabantous3" target="_blank">[31]</a>. In all cases fluorescence (a.u. arbitrary units) was measured after overnight incubation at 4°C.</p

Progress curves analysis of the complemented fluorescence.

<p><b>A. Progress curves of complemented fluorescence using purified protein.</b> The increasing fluorescence over time was determined (a.u arbitrary units) for the complementation of scFv-D1.3-GFP11 samples at different concentrations (300, 150, 75 pmol) incubated with an excess of GFP1-10 (800 pmol). Fluorescence (λ_exc = 488 nm/λ_em = 530 nm) was monitored at 3 minutes intervals for 20 h at RT. <b>B. Progress curves of complemented fluorescence using serial dilutions of crude bacteria extract.</b> A similar trend of increasing fluorescence was measured (a.u arbitrary units) directly using serial dilution of crude bacterial culture extract, albeit at lower fluorescent values. Fluorescence (λ_exc = 488 nm/λ_em = 530 nm) was monitored at 3 minutes intervals for 10 hours at RT.</p

Comparison of antigen binding activity assessed by ELISA.

<p>The antigen binding activity was analyzed by ELISA using different concentrations of scFv-D1.3 and scFv-D1.3-GFP11 scFvs (ranging from 0.6 to 300 nM). The scFv-D1.3-GFP11 construct showed good binding activity for lysozyme (lys) even after incubation with the GFP1-10 complementing protein, indicating the absence of steric hindrance between the restored GFP protein and the anti-SV5 antibody used for detection. Myoglobin (myo) was used as negative control.</p

Disulfide Bonds within the C2 Domain of RAGE Play Key Roles in Its Dimerization and Biogenesis

<div><h3>Background</h3><p>The receptor for advanced glycation end products (RAGE) on the cell surface transmits inflammatory signals. A member of the immunoglobulin superfamily, RAGE possesses the V, C1, and C2 ectodomains that collectively constitute the receptor's extracellular structure. However, the molecular mechanism of RAGE biogenesis remains unclear, impeding efforts to control RAGE signaling through cellular regulation.</p> <h3>Methodology and Result</h3><p>We used co-immunoprecipitation and crossing-linking to study RAGE oligomerization and found that RAGE forms dimer-based oligomers. Via non-reducing SDS-polyacrylamide gel electrophoresis and mutagenesis, we found that cysteines 259 and 301 within the C2 domain form intermolecular disulfide bonds. Using a modified tripartite split GFP complementation strategy and confocal microscopy, we also found that RAGE dimerization occurs in the endoplasmic reticulum (ER), and that RAGE mutant molecules without the double disulfide bridges are unstable, and are subjected to the ER-associated degradation.</p> <h3>Conclusion</h3><p>Disulfide bond-mediated RAGE dimerization in the ER is the critical step of RAGE biogenesis. Without formation of intermolecular disulfide bonds in the C2 region, RAGE fails to reach cell surface.</p> <h3>Significance</h3><p>This is the first report of RAGE intermolecular disulfide bond.</p> </div

Design of tripartite split GFP complementation to study RAGE dimerization in the ER.

<p>(<b>A</b>) Illustration of general tripartite split GFP complementation strategy. GFP s10 and s11 are used to tag test proteins whereas GFPs1-9 functions as a detector. When tagged test proteins interact with each other to bring s10 and s11 sufficiently close that they interact with s1-9 to generate green fluorescence. (<b>B</b>) Illustration of tripartite split GFP complementation to detect RAGE dimerization in the ER. GFPs1-9 is targeted to the ER with RAGE signal peptide (black bar). Upon entering the ER, the signal peptide is cleaved and GFPs1-9 is glycosylated (magenta chain), and complementation occurs only when s10 and s11-tagged RAGE molecules dimerise. Double disulfide bridge-linked RAGE dimers then leave the ER-Golgi for the cell surface. (<b>C</b>) Targeting GFPs1-9 to the ER. Glycosylation of GFPs1-9 confirms that GFPs1-9 is localized in the ER.</p

Cell surface expression of RAGE and RAGE cysteine-to-serine mutants.

<p>FLAG-tagged RAGE and RAGE mutants were transfected to CHO-CD14 cells. After overnight incubation, the transfected cells (10⁶) were stained with anti-FLAG antibodies and subjected to flow cytometry analyses. Non-transfected cells with same staining were used as negative controls. All values were expressed as mean ± SEM, and the data were from independent transfections (<i>n</i> = 3). The <i>p</i> value for presented data is <0.01 (ANOVA).</p

RAGE(C259S/C301S) is unstable and deglycosylated.

<p>(<b>A</b>) Cyclohaximide chase and IB to compare the protein decay of RAGE(WT) and RAGE(C259S/C301S) in the cells. For FLAG-RAGE transfected cells, 5 µg of lysates were used; whereas for FLAG-RAGE(C259S/C301S) transfceted cells, 10 µg of lysates were used due to the lower expression. After IB with anti-FLAG antibodies, the blot was striped, and reprobed with anti-β-actin antibodies as a loading control. CHX: cyclohaximide. (<b>B</b>) Intracellular decay rate of RAGE and RAGE(C259S/C301S) calculated from two CHX chase experiments. The blot intensity was measured with a Kodak Gel Logic 2200 Imaging System and processed with molecular imaging software. The starting point was used as 100% and blot intensity from each time point was calculated relative to the 0 time point. The intensity value of each point was expressed as mean ± SEM, and d_1/2 was calculated when 50% of the protein I decayed. <i>C</i>, RAGE cysteine-to-serine mutants are deglycosylated. Cell lysates from FLAG-tagged RAGE and RAGE cysteine-to-serine mutants were treated with PNGase F, as described, and resolved on a SDS 4–12% NuPAGE gel.</p