Journal: The Journal of Biological Chemistry

Article Title: Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

doi: 10.1074/jbc.RA120.012723

Figure Lengend Snippet: Generation and characterization of scR1antTNF-Fc protein. A, schematic structure modeling of scR1antTNF-Fc protein. Two TNFR1 antagonistic proteins were fused with human IgG-Fc. N, N-terminal. Amino acid sequences and domain information of scR1antTNF-Fc are described in Fig. S1A. B, X-ray structure modeling of the scR1antTNF-TNFR1 complex. scR1antTNF bound to the homotrimer of TNFR1. Red, TNFR1; green, TNFR1 interaction domain of scR1antTNF; orange, peptide linker for forming the single-chain structure. C, schematic pCAG-based mammalian expression vector for scR1antTNF-Fc protein. The cDNA was composed to express scR1antTNF-Fc whereby triple R1antTNF domains fused by peptide linkers (GGGSGGG) were further fused to a human–IgG Fc domain (Ch2 and Ch3). Signal sequence peptide genes derived from a mouse IgG Vh (Vhss) or human IL-2 (IL-2ss) were linked at the 5′-terminal of scR1antTNF-Fc cDNA. D, supernatants of cultured medium (left side) and purified proteins (right side) 7 days after transfection were assessed by SDS-PAGE following Coomassie Brilliant Blue staining. Arrowhead shows an ∼75-kDa band of the scR1antTNF-Fc monomer. E, each recombinant protein expressed with Vhss and IL-2ss was purified by size-exclusion chromatography. F, the molecular weight of monomeric scR1antTNF-Fc protein was confirmed by Western blotting with an anti-human IgG-Fc antibody.

Article Snippet: The resultant gene was inserted to a pCAG-Hyg human IgG-Fc fusion vector (Fujifilm Wako) to generate the scR1antTNF-Fc expression vector.

Techniques: Expressing, Plasmid Preparation, Sequencing, Derivative Assay, Cell Culture, Purification, Transfection, SDS Page, Staining, Recombinant, Size-exclusion Chromatography, Molecular Weight, Western Blot

Journal: bioRxiv

Article Title: Dimeric prion protein ligand activates Adgrg6 but does not rescue myelinopathy of PrP-deficient mice

doi: 10.1101/2020.07.07.191452

Figure Lengend Snippet: a) Design of FT 2 Fc. FT was fused to mIgG1-Fc at the hinge, thereby replacing the antigen-binding fragment (Fab) and forming a homodimeric Fc-fusion protein. b) FT 2 Fc was secreted by 293-F cells after transient transfection and was present in the cell culture supernatant as a homodimer with a size of 56 kD. Under reducing conditions (+DTT), FT 2 Fc was present as a monomer. FT 2 Fc was detected in western blotting by antibodies targeting the Fc-fragment and a Fab specific for FT (Fab83). Supernatant from non-transfected cells (NT) was used as negative control. c) Sandwich ELISA of serially diluted cell culture supernatant from cells transfected with FT 2 Fc showed a sigmoidal curve. The optical-density (OD) - dilution curves were fitted using the four-parameter logistic nonlinear regression model (dashed lines, R 2 = 0.94 for FT 2 Fc). Only background signal was detected in supernatant from cells transfected with the empty vector control (EVC) or non-transfected cells (NT), or when the plate was coated with BSA instead of Fab83. d) Design of the immunoprecipitation assay (left). Western blots of eluates and beads boiled in sample loading buffer (right). FT 2 Fc was captured in cell culture supernatant by beads coated with Fab83 and was eluted with a peptide specifically competing for the Fab83 binding site, but not with a non-competing peptide. The western blot from the beads confirmed that FT 2 Fc (size of specific bands marked with *) was bound to the beads in all conditions. e) In the thermal shift assay, the unfolding of FT 2 Fc with increasing temperature was monitored using a fluorescent dye. The curve was fitted to the Boltzmann equation (dashed line, R 2 = 0.99). The inflection point at 74.5 °C corresponds to the melting temperature of FT 2 Fc in 20 mM sodium phosphate, pH 7.

Article Snippet: The mammalian expression vector for FT 2 Fc was obtained from ATUM (vector pD2610-v1).

Techniques: Binding Assay, Transfection, Cell Culture, Western Blot, Negative Control, Sandwich ELISA, Plasmid Preparation, Immunoprecipitation, Thermal Shift Assay

Journal: bioRxiv

Article Title: Dimeric prion protein ligand activates Adgrg6 but does not rescue myelinopathy of PrP-deficient mice

doi: 10.1101/2020.07.07.191452

Figure Lengend Snippet: a) FT and FT 2 Fc elicited an increase in cAMP levels in SW10 WT cells, but not in SW10 ΔAdgrg6 cells. cAMP levels were measured 20 min after treatment with 5 μM FT or 2.5 μM FT 2 Fc. To account for variability in cAMP levels across cell lines, cAMP was expressed as x-fold change to the average of the controls (PBS and 20 mM sodium phosphate buffer) for each of the 4 independent experiments. One-way ANOVA for selected comparisons, Sidak’s multiple comparisons test: in SW10 WT FT vs. PBS p = 0.0392, FT 2 Fc vs. buffer p = 0.0035. In SW10 ΔAdgrg6 FT and FT 2 Fc vs. controls p > 0.05. b) SW10 WT cells were incubated with increasing concentrations of FT 2 Fc. cAMP levels increased up to a maximum of a 9-fold change when compared to untreated cells. Nonlinear regression analysis revealed an EC 50 of 3.49 μM. For the curve fitting with the four-parameter logistic regression model (dashed line, R2 = 0.78) only values from 0-8 μM (2-3 replicates per concentration) were used, because the strongly reduced activity at 9 and 10 μM would have confounded the analysis. c) SW10 WT , but not SW10 ΔAdgrg6 cells showed a time-dependent increase in AKT phosphorylation upon treatment with 1 μM FT 2 Fc. Quantification of 5 independent experiments (left), representative western blot of cell lysates (right). Comparison of SW10 WT to SW10 ΔAdgrg6 cells with two-way ANOVA followed by Sidak’s multiple comparisons test: at 10 min, 15 min and 30 min p < 0.0001, at 5 min and 10 min p > 0.05. ns = not significant.

Article Snippet: The mammalian expression vector for FT 2 Fc was obtained from ATUM (vector pD2610-v1).

Techniques: Incubation, Concentration Assay, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Dimeric prion protein ligand activates Adgrg6 but does not rescue myelinopathy of PrP-deficient mice

doi: 10.1101/2020.07.07.191452

Figure Lengend Snippet: a) AKT phosphorylation increased in sciatic nerves of ZH3 mice 30 min after injection of 600 μg FT. As control (ctrl), mice were injected with an inactive peptide, in which lysine residues have been replaced with alanine residues . In mice injected with FT 2 Fc (10 mg/Kg), no significant change in AKT phosphorylation was detected after 30 min when compared to mIgG injected mice. Square brackets in western blots indicate left and right sciatic nerves taken from one mouse. The average value was used for quantification. b) AKT phosphorylation did not increase after six consecutive FT 2 Fc injections. c,d) ZH3 mice exhibited an age-dependent decrease in sciatic nerve Egr2 levels and a concomitant increase in GFAP and c-Jun levels. Western blots of sciatic nerve lysates are shown in (c) , result of densitometry in (d) . e) ZH3 mice were i.v. ( n = 2) or i.p. ( n = 2) injected with FT 2 Fc (5 mg/Kg) and FT 2 Fc serum levels were monitored by western blotting with Fab83 at 1, 6 and 24 h after injection. A serum sample from a mouse injected with mIgG (5 mg/Kg) was used as negative control. The early serum level – time course was similar in i.v. and i.p. injected mice. f) ZH3 mice were i.p. injected with 10 mg/Kg FT 2 Fc and blood samples were collected from 1 to 8 days post injection. The elimination of FT 2 Fc from serum followed first order elimination kinetics. g) Serum level values (normalized to the level at 23 h post injection) were plotted on a semi-logarithmic graph and fitted with nonlinear least-squares analysis (dashed line, R 2 = 0.92) to calculate the terminal serum half-life of FT 2 Fc. The serum half-life was estimated to be 45 h with a 95% confidence interval of 37-58 h. The number of mice investigated per time point ( n ) is indicated above the points.

Article Snippet: The mammalian expression vector for FT 2 Fc was obtained from ATUM (vector pD2610-v1).

Techniques: Injection, Western Blot, Negative Control

Journal: bioRxiv

Article Title: Dimeric prion protein ligand activates Adgrg6 but does not rescue myelinopathy of PrP-deficient mice

doi: 10.1101/2020.07.07.191452

Figure Lengend Snippet: a) Design of the prophylactic treatment experiment in ZH3 and WT mice. Starting at 1 month of age, ZH3 and WT mice were injected with FT 2 Fc (or control treatment) 3 times per week until the age of 5 months. Bars with grey shades show expected changes in protein levels with dark and light grey meaning high and low levels, respectively. b) FT 2 Fc serum levels were measured 2 days after the last injection. Representative western blot showing serum levels for 4 ZH3 mice after 1 and 2 months of treatment. FT 2 Fc serum levels did not decrease after 2 months of treatment when compared to the level at 1 month ( n = 8, paired t-test, p = 0.3529), suggesting that FT 2 Fc half-life was unaltered. c) Bodyweight was not significantly different between treatment groups (as analysed by two-way repeated measures ANOVA, p > 0.05). Body weight was recorded at every injection time point for all the mice in the chronic treatment study and is here shown for week 1 to 6 of treatment as percentage of the body weight at treatment start (reference). d) Representative western blots and quantification for levels of GFAP, c-Jun and Egr2 in sciatic nerves. GFAP levels were significantly higher in ZH3 mice compared to WT mice, but no significant change was induced by FT 2 Fc compared to buffer treatment (one-way ANOVA for selected comparisons, Bonferroni’s multiple comparisons test: ZH3 vs. WT for buffer treated mice p = 0.0345, for FT 2 Fc treated mice p = 0.0343. FT 2 Fc vs. buffer treated mice, both ZH3 and WT p > 0.05). C-Jun levels were not decreased in ZH3 compared to WT mice, and no significant change in c-Jun levels was induced by FT 2 Fc treatment (one-way ANOVA for selected comparisons, Bonferroni’s multiple comparisons test, p > 0.05). Egr2 levels were significantly lower in ZH3 mice compared to WT mice, but FT 2 Fc treatment did not induce a significant change in any genotype (one-way ANOVA for selected comparisons, Bonferroni’s multiple comparisons test: ZH3 vs. WT for buffer treated mice p < 0.0001, for FT 2 Fc treated mice p = 0.0044. FT 2 Fc vs. buffer treated mice, both ZH3 and WT p > 0.05). Protein levels were expressed as fold change the average level in buffer treated ZH3 mice. e) Toluidine-blue stained semithin sections of sciatic nerves. No difference in fibre morphology was detected between treatment groups in our ZH3 mouse specimens. Scale bar 50 μM. f) Number of myelinated axons per mm 2 in sciatic nerves of ZH3 mice revealed no difference between treatment groups (comparison of FT 2 Fc and IgG treated to buffer treated mice by one-way ANOVA followed by Dunnett’s multiple comparisons test, p > 0.05). ns = not significant.

Article Snippet: The mammalian expression vector for FT 2 Fc was obtained from ATUM (vector pD2610-v1).

Techniques: Injection, Western Blot, Staining

Journal: bioRxiv

Article Title: Dimeric prion protein ligand activates Adgrg6 but does not rescue myelinopathy of PrP-deficient mice

doi: 10.1101/2020.07.07.191452

Figure Lengend Snippet: All tests and calculations were done with the examiners masked as to treatment and strain allocation. a) Motor nerve conduction velocities (NCV) for WT and ZH3 mice. There was no significant difference between WT and ZH3 mice, or between FT 2 Fc treated and buffer treated mice (one-way ANOVA for indicated comparisons, Bonferroni’s multiple comparisons test, p > 0.05). b) Compound sensory NCV (csNCV) for WT and ZH3 mice. Again, there was no significant difference between WT and ZH3 mice, or FT 2 Fc treated and buffer treated mice (one-way ANOVA for indicated comparisons, Bonferroni’s multiple comparisons test, p > 0.05). c) The ratio of proximal to distal compound muscle action potential (CMAP) amplitude was not significantly different when comparing ZH3 to WT mice or FT 2 Fc to buffer treated mice (one-way ANOVA for indicated comparisons, Bonferroni’s multiple comparisons test, p > 0.05). ns = not significant.

Article Snippet: The mammalian expression vector for FT 2 Fc was obtained from ATUM (vector pD2610-v1).

Techniques:

Journal: bioRxiv

Article Title: Dimeric prion protein ligand activates Adgrg6 but does not rescue myelinopathy of PrP-deficient mice

doi: 10.1101/2020.07.07.191452

Figure Lengend Snippet: a) Hierarchical clustering analysis based on the 100 genes with the highest variance across all samples showed a separation between FT 2 Fc treated mice and control mice ( n = 4 per treatment). b) Heatmap of the selected repair Schwann cell genes. The red-blue colour key is based on the row-wise Z-scores. 4 months old ZH3 mice ( n = 3) showed a mild, 13-15 months old ZH3 mice ( n = 4) a pronounced upregulation of these genes when compared to age-matched WT mice. No difference was detected in FT 2 Fc treated ZH3 mice ( n = 4) compared to buffer treated mice ( n = 4). c) Volcano plot showing differentially expressed genes in sciatic nerves of FT 2 Fc treated compared to buffer treated mice. Genes with FDR < 0.05 were considered significantly up- or downregulated (43 downregulated genes, 1 upregulated gene). Genes involved in actin binding are labelled in the plot. Log2FC = log2 fold change.

Article Snippet: The mammalian expression vector for FT 2 Fc was obtained from ATUM (vector pD2610-v1).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Dimeric prion protein ligand activates Adgrg6 but does not rescue myelinopathy of PrP-deficient mice

doi: 10.1101/2020.07.07.191452

Figure Lengend Snippet: a) Log2 fold change (log2FC) for significantly downregulated genes ( n = 43, based FDR < 0.05) in sciatic nerves of FT 2 Fc treated compared to buffer treated mice and corresponding log2FC in tibialis anterior muscle of PrP overexpressing mice. * marks genes which were also significantly downregulated in muscle based on FDR < 0.05 ( n = 15). Grouping of genes according to common functions was based on a manual search in the UniProt database. b) Scatterplot comparing gene expression changes in muscle of PrP overexpressing mice and sciatic nerves of FT 2 Fc treated mice. 1229 and 43 genes were significantly (FDR < 0.05) downregulated in muscle and nerve, respectively, with 15 of these genes overlapping. Grey line represents linear regression. Pearson’s correlation coefficient r = 0.09. c) 10 μM Gomori trichrome stained frozen sections of gastrocnemius muscle from FT 2 Fc and buffer treated ZH3 mice. Muscle fibre morphology was similar in buffer and FT 2 Fc treated mice. No myopathic changes were detected in FT 2 Fc treated mice. Scale bar 20 μM.

Article Snippet: The mammalian expression vector for FT 2 Fc was obtained from ATUM (vector pD2610-v1).

Techniques: Expressing, Staining