Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 1. Flow cytometry sorting of the combinatorial Scl library. (A) Library expression was monitored by staining with a phycoerythrin- conjugated antibody binding to a primary anti-c-Myc antibody. The purple rectangle gate indicates cells with the highest expression. (B) The library was incubated with 5 nM LRP4, and the low-affinity library fraction was collected (purple triangle). (C) The low-affinity library was incubated with 650 nM LRP6, and the purple rectangle represents clones that bind to LRP6.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Flow Cytometry, Expressing, Staining, Binding Assay, Incubation, Clone Assay

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 2. Identification of affinity-reducing mutations. Heat maps demonstrating significantly enriched Scl variants in (A) LRP4LOW library compared to SclNAIVE library fractions; (B) LRP4LOWLRP6 library compared to LRP4LOW library fractions; (C) LRP4LOW library compared to SclNAIVE library fractions that overlap with the LRP4LOWLRP6 library. The heat maps present the log2 transformation of the ER (red scale bar on the right-hand side) and highlight single mutations that significantly (A) reduce the binding affinity to LRP4, (B) reduce the binding affinity to LRP4 and retain binding to LRP6, and (C) overlap in (A) and (B). The substituting amino acids are shown on the X-axis, and the substituted positions are shown on the Y-axis. Statistical significance was determined by a two-sided Poisson exact test and multi-test corrected by the Benjamini–Hochberg FDR.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Transformation Assay, Binding Assay

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 4. YSD binding of SclWT and the selected single-mutation variants to LRP4. Geometric mean fluorescence intensity (Geo MFI) is presented as a fold change. Recombinant yeast cells expressing SclWT or its variants were incubated with (A) 1 nM, (B) 10 nM, or (C) 50 nM soluble LRP4. The binding signal of each Scl variant was normalized first to the expression signal of the corresponding variant and then to the binding signal of SclWT at the respective LRP4 concentration. Each experiment was repeated at least three times, and the results are presented as means SD. Statistical significance was assessed using an unpaired, two-tailed Student’s t-test. *P ≤0.05; **P ≤0.01; ***P ≤0.001.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Binding Assay, Mutagenesis, Recombinant, Expressing, Incubation, Variant Assay, Concentration Assay, Two Tailed Test

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 7. SPR analysis of binding of purified SclWT and Scl single-mutation variants to LRP4. SPR data showing binding of (A) SclWT, (B) SclK75Q, (C) SclK75E, and (D) SclV136D to 3 μg of immobilized LRP4 receptor. Different protein concentrations are represented by different colors.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Binding Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function

doi: 10.1074/jbc.m110.190330

Figure Lengend Snippet: FIGURE 3. LRP4 is expressed in human bone, and Lrp4 silencing blunts sclerostin-mediated inhibition of in vitro bone mineralization. A, LRP4 protein is expressed in human osteoblasts (arrowheads) and osteocytes (arrows). Immunohistochemistry of LRP4 in human femoral neck from a male subject aged 75 is shown. Scale bar, 50 m. B, LRP4 RNA is expressed in samples from human femoral neck. RNA was extracted from a female subject aged 65 and a male subject aged 80. PTH1R, LRP4, SOST, LRP5, and LRP6 RNA levels were assessed by real-time quantitative PCR and normalized with GAPDH, and relative expression compared with PTH1R is shown. C, knockdown of LRP4 in Kusa-A1 blocks the inhibitory effect of sclerostin on in vitro mineralization activity. Kusa-A1 cells were transducedwithlentiviralparticlesharboringshRNAagainstLrp4.ReductionofLrp4RNAlevelswasassessedbyreal-timequantitativePCR,priortotheaddition of an osteoblastic differentiation medium. Calcium content was assessed 4 days later. Error bars, S.E.

Article Snippet: Recombinant human DKK1 and LRP6 were purchased from R&D Systems.

Techniques: Inhibition, In Vitro, Immunohistochemistry, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Activity Assay

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Relationship between LAIR1 expression and intraleukocytic haemozoin. (a) Pairwise comparisons of LAIR1 transcript levels between children with and without pigment-containing monocytes (PCM) and pigment-containing neutrophils (PCN) in the non-SMA and SMA groups were determined using Mann-Whitney U test. Data are presented as box-plots, where the box represents the interquartile range, the line through the box is the median, and whiskers show the 10th and 90th percentiles. LAIR1 transcript levels were lower in children with PCM compared to those without, while LAIR1 transcript levels were comparable between absence/presence of PCN. (b) Temporal kinetics of LAIR1 transcript levels in response to Pf Hz treatment of PBMC from malaria-naïve donors ( n = 6, measured in triplicate). Pairwise comparisons were determined using Student t -test. Significant ( P < .05) differences in transcript levels between no treatment and Pf Hz treatment (10 μg/mL) groups represented by *at 0.5, 2, and 4 h time points. Data represent average of individuals ( n = 3) with each condition performed in triplicate (error bars represent SEM). (c) Immunoblot analysis of non-treated (baseline) and Pf Hz-treated (10 μg/mL) PBMC lysates for 12, 24, and 48 h. (d) Densitometric analysis of normalised cellular LAIR1 protein production presented as mean ± SEM. Across group comparisons analysed using ANOVA. Pairwise comparisons analysed using Student t-test. Data represent average of individuals ( n = 6) with each condition performed in triplicate (error bars represent SEM). LAIR1 protein levels were lower in Pf Hz-treated PBMC lysates compared to no treatment at 12, 24, and 24 h.

Article Snippet: Soluble LAIR1 was measured in serum of study participants using the human LAIR1 ELISA matched antibody pair, and recombinant human LAIR1 (Creative Diagnostics, 45–16 Ramsey Rd, Shirley, NY 11967; Cat No. ABPR-0519).

Techniques: Expressing, MANN-WHITNEY, Western Blot

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Effect of intraleukocytic Pf Hz on LAIR1 and SHP-1 phosphorylation. Temporal kinetics of the signalling pathway in response to the different treatment conditions was determined in PBMCs from malaria-naïve donors (n = 6, measured in triplicate). (a) Human Phospho-immunoreceptor array results showing LAIR1 and SHP-1 phosphorylation upon no treatment (baseline), C1q (25 μg/mL), Pf Hz (10 μg/mL), and Pf Hz + C1q. Phosphorylation (spots) in the different conditions with control represented by blue boxes, LAIR1 by red boxes, and SHP-1 by green boxes. (b) Densitometric analysis of human phosphor-immunoreceptor array data. Data presented as (mean ± SEM). Across group comparisons analysed using ANOVA. *indicates significant differences ( P < .05) determined by Student t-test in pLAIR1 and pSHP-1 compared to C1q treatment. Phagocytosis of Pf Hz resulted in a reduction of pLAIR1 relative to C1q (alone) treatment. Similarly, ingestion of Pf Hz also caused a marked decrease in pSHP-1 levels relative to C1q treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Soluble LAIR1 was measured in serum of study participants using the human LAIR1 ELISA matched antibody pair, and recombinant human LAIR1 (Creative Diagnostics, 45–16 Ramsey Rd, Shirley, NY 11967; Cat No. ABPR-0519).

Techniques:

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: LAIR1 signalling pathway in malaria. The LAIR1 pathway is activated by attachment of collagen and collagenous ligands (C1q) to LAIR1 extracellular surface. This results in phosphorylation of intracellular LAIR1 ITIM tyrosine residues by Src family kinases. Phosphorylated ITIMs serve as docking sites for recruitment of SHP-1 and SHP-2 phosphatases. SHP-1 and SHP-2 become localized to phosphorylated ITIMs through their regulatory SH2 domains, subsequently inducing their phosphatase activity. Activated SHP-1 has been shown to block activation and nuclear translocation of nuclear factor nuclear factor-kappa beta (NF-κB) through de-phosphorylation of inhibitor of kappa-beta kinase complex (IKK). SHP-1 phosphatase activity also inhibits activation and translocation of IRFs from the cytoplasm to the nucleus by preventing TANK-binding kinase 1 (TANK-1) phosphorylation of interferon regulatory factors (IRFs). These events subsequently block transcription of inflammatory mediator encoding genes with response elements for IRFs and NF-κB. SHP-2 inhibits activation of IRF 8 and blocks expression of phagocyte NADPH oxidase (gp91 PHOX ). LAIR1 inhibitory signalling is regulated by soluble LAIR1 and LAIR2 (soluble homolog of LAIR1) through competition for collagenous ligands. Children with SMA had increased circulating levels of sLAIR1 and sLAIR2 indicative of enhanced receptor shedding. C1q was reduced in children with SMA, thereby, limiting ligand availability. Phagocytosis of Pf Hz antagonizes LAIR1 signalling through down-regulation of LAIR1 ITIM and SHP-1 phosphorylation, but does not alter SHP-2 phosphorylation. Leukocytic ingestion of Pf Hz also decreases LAIR1 transcripts and protein. These events result in NF-κB activation and the consequent production of pro-inflammatory mediators that enhance the pathogenesis of SMA. Red arrows represent ligand-receptor interaction, black solid arrows represent activation, and dashed black arrows represent blockade. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Soluble LAIR1 was measured in serum of study participants using the human LAIR1 ELISA matched antibody pair, and recombinant human LAIR1 (Creative Diagnostics, 45–16 Ramsey Rd, Shirley, NY 11967; Cat No. ABPR-0519).

Techniques: Activity Assay, Blocking Assay, Activation Assay, Translocation Assay, De-Phosphorylation Assay, Binding Assay, Expressing

Journal: Journal for immunotherapy of cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity.

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: Figure 2 In vitro characterization of anti-GARP antibody PIIO-1. (A) GARP expression on human regulatory T cells and platelets was evaluated by flow cytometry after staining with PIIO-1 at 10 µg/mL. (B) 293 FT cell line was transfected with empty vector (EV), human GARP (hGARP)-expressing vector only or co-transfected with hGARP and latent TGFβ1 expression vectors. GARP expression on indicated cell line was detected by flow cytometry after staining with PIIO-1 at 10 µg/mL. (C) Human GARP sequence was replaced by murine GARP according to the schematic diagram to generate the chimeric constructs of human and murine GARP that were tagged with HA (hemagglutinin) epitope. Transfection efficiency was determined using anti- HA antibody. All constructs were transfected into 293 FT cells. (D) Crystal structure of the GARP (green)-LTGFβ (gray) complex (PDB DOI: 10.2210/pdb6GFF/pdb). The region of PIIO-1 recognition is orange and the residues interacting with LTGFβ are cyan. LTGFβ occludes approximately 30% of the potential antibody binding site and may sterically or allosterically restrict access of the antibody to GARP in the LTGFβ-complexed state. Modeling was carried out using Pymol. (E) Jurkat cell line, made to overexpress hGARP, was incubated with LTGFβ1 along with mIgG1 or PIIO-1 at indicated concentration for 30 min at 37℃. Human LAP expression was detected by flow cytometry. All data are representative of 2–6 independent experiments. GARP, Glycoprotein-A repetitions predominant; LAP, latency-associated peptide.

Article Snippet: Generation of anti-human GARP (hGARP) antibodies The generation of anti- hGARP antibody has been described previously.19 BALB/c mice was immunized with recombinant human GARP (R&D Systems) in Freund’s complete adjuvant and followed by boosting with SP2/0hGARP cells for 2–3 times.

Techniques: In Vitro, Expressing, Flow Cytometry, Staining, Transfection, Plasmid Preparation, Sequencing, Construct, Binding Assay, Incubation, Concentration Assay

Journal: Journal for immunotherapy of cancer

Article Title: Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8 + T cell antitumor immunity.

doi: 10.1136/jitc-2022-005433

Figure Lengend Snippet: Figure 3 PIIO-1 enhanced antitumor efficacy of anti-PD-1 ICB in GARP+ TNBC. (A) Experimental scheme. BALB/c mice were injected with 1×105 4T1-hGARP cells in the mammary fat pad, followed by i.p. injection of 200 µg/mouse of PIIO-1 and/or 150 µg/mouse anti-PD-1 every 3 days. (B) Primary tumor growth curve. (C) Overall survival of mice. (D) Summary of the incidence of tumor-free mice among groups. (E) Lungs were collected and sectioned at experimental end point, then stained with H&E. Representative images from each group of mice are shown. Scale bar, 20 µm. The numbers of visible lung metastatic nodules are quantified. (F) Summary of the incidence of metastasis among groups. (G) Tumors were collected at end point and stained by IHC for pSMAD3, α-SMA. Representative images of tumor tissues from four groups of mice are shown (left). Scale bar, 50 µm. Quantification of the IHC staining is shown (right). (H) Sera from each mouse was collected at end point. Total and active TGFβ level in the sera were assessed by ELISA. (I) Mice with tumor regression following combination therapy were monitored for 300 days, then rechallenged with 5×105 wild-type 4T1 mammary tumor in contralateral mammary fat pad. Naive BALB/c mice without pre-exposure to tumor were used as control. Shown is the overall survival. Tumor curve analysis was performed using repeated measures 2-way analysis of variance. Overall survival is analyzed by log-rank (Mantel-Cox) test. (E, G) were analyzed by paired t-test according to the tumor collection time points. Other data were analyzed by two-tailed Student’s t-test. B, C were corrected for multiple testing using the Tukey procedure. All data are presented as mean±SEM. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001.

Article Snippet: Generation of anti-human GARP (hGARP) antibodies The generation of anti- hGARP antibody has been described previously.19 BALB/c mice was immunized with recombinant human GARP (R&D Systems) in Freund’s complete adjuvant and followed by boosting with SP2/0hGARP cells for 2–3 times.

Techniques: Injection, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test

Journal: Journal of applied physiology (Bethesda, Md. : 1985)

Article Title: Leptin receptors in human skeletal muscle.

doi: 10.1152/japplphysiol.01313.2006

Figure Lengend Snippet: Fig. 2. Determination of the expression of human leptin receptor (OB-R) in human skeletal muscle. Protein extracts were prepared from muscle, SAT, and hypothalamus (HIP), and OB-R, perilipin A, and -tubulin protein expression was analyzed by Western blot. A: representative immunoblot assay after incubation with a polyclonal rabbit anti-OB-R antibody specifically raised against the long isoform. B: representative Western blot after incubation with a polyclonal rabbit anti-perilipin A antibody in the same samples used in A. C: representative immunoblot analysis after incubation with the monoclonal mouse anti--tubulin antibody in the same samples used in A. D: densitometric immunosignal values (arbitrary units of band densities) of OB-R bands relative to those obtained for -tubulin.

Article Snippet: The recombinant human (RH) leptin R/Fc chimera, generated from DNA containing the extracellular domain of OB-R (amino acid residues 1-839) fused to the Fc region of human IgG1, was obtained from R&D Systems (McKinley Place).

Techniques: Expressing, Western Blot, Incubation

Journal: Journal of applied physiology (Bethesda, Md. : 1985)

Article Title: Leptin receptors in human skeletal muscle.

doi: 10.1152/japplphysiol.01313.2006

Figure Lengend Snippet: Fig. 3. The anti-OB-R antibody recognized specifically the three OB-R bands detected in the muscle protein extracts. Increasing amounts of recombinant human (RH) leptin R/Fc (RH OB-R) chimera (0, 10, 100, 500 ng) were preincubated with anti-OB-R antibody (1:2,000). OB-R protein expression from muscle extracts was analyzed by immunoblotting using the preincubation solution. A: representative Western blot analysis with different preincubation solutions in the same muscle protein extract (50 g). B: representative immunoblot with the -tubulin antibody as a loading control. C: densitometric percentage of OB-R immnunostaining values (band quenching) in presence of increasing amounts (10, 100, 500 ng) of RH OB-R relative to those observed for a control (0 ng of RH OB-R). *P 0.01 vs. 0 ng of RH OB-R.

Article Snippet: The recombinant human (RH) leptin R/Fc chimera, generated from DNA containing the extracellular domain of OB-R (amino acid residues 1-839) fused to the Fc region of human IgG1, was obtained from R&D Systems (McKinley Place).

Techniques: Recombinant, Expressing, Western Blot, Control

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 1. Preparation of recombinant murine leptin and its PASylated fusion proteins. (A) Expression plasmids are based on the generic pASK75 vector comprising the chemically inducible tetp/o promoter, an OmpA signal sequence for periplasmic secretion, a His6-tag for affinity purification, a short linker allowing insertion of the PAS gene cassette, and the coding region for mature murine leptin. The preparation of PASylated leptin versions from E. coli in a functional monomeric form was confirmed by SDS−PAGE (B) and ESI-TOF mass spectrometry (C). (D) The correct fold of the leptin moiety and the secondary structure of the attached PAS polypeptide were investigated by CD spectroscopy.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Recombinant, Expressing, Plasmid Preparation, Sequencing, Functional Assay, SDS Page, Mass Spectrometry, Circular Dichroism

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 2. Size measurements for PASylated leptin versions. The effect of the genetic fusion of leptin with the conformationally disordered PAS polypeptide was investigated by analytical size exclusion chromatography (SEC) and dynamic light scattering (DLS). (A) SEC in the presence of phosphate-buffered saline (PBS) resulted in a single peak with decreasing elution volume for PAS fusions with increasing number of amino acid residues. Comparison with a half- logarithmic calibration curve (inset) led to the apparent molecular masses listed in Table 1. (B) Absolute and relative molecular sizes determined by both SEC and DLS measurements in the presence of PBS vs the length of the PAS tag.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Size-exclusion Chromatography, Saline, Comparison

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 3. Investigation of receptor binding activity for PASylated leptin by real-time SPR analysis and in a cell culture reporter assay. (A) Dilution series of leptin versions from 64 to 1 nM were applied in PBS/T to a sensor chip charged with a murine LepRb-Fc fusion protein. Biacore sensorgrams were fitted to a 1:1 Langmuir binding model, and resulting dissociation constants and kinetic parameters are listed in Table 1. (B) Visualization of the parameters from plot A in a kon/koff plot. (C) Leptin versions were applied to HEK cells transiently co-transfected with an LepRb-expression vector and a STAT3- responsive Photinus luciferase gene. The leptin versions were applied in a dilution series in duplicate, and after incubation for 18 h luciferase activity was assayed (N = 4). Luminescence values (normalized against constitutively coexpressed Renilla luciferase) for each test protein were fitted to a sigmoidal dose−response curve; resulting EC50 values are listed in Table 1.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Binding Assay, Activity Assay, Cell Culture, Reporter Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Incubation

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 4. PK of recombinant murine leptin and its PASylated versions in mouse plasma. C57BL6/J mice (N = 9) were injected ip with a protein dose of 287 nmol·kg−1 bw. The leptin concentration in plasma was quantified in a sandwich ELISA with antibodies directed against the adipokine, using appropriate calibration curves, and data were plotted in a semilogarithmic fashion against the time of sampling post injection. The PK profiles show distinct resorption and elimination phases; deduced terminal half-lives, clearance, and AUC parameters are listed in Table 1. Plot of the same data in a linear fashion (inset) illustrates the strongly increased AUC, indicating high bioavailability of PASylated leptins.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Recombinant, Clinical Proteomics, Injection, Concentration Assay, Sandwich ELISA, Sampling

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 5. Hypothalamic receptor signaling of PASylated murine leptin versions. The PD of unmodified recombinant leptin and its PASylated versions was investigated in lean C57BL/6J mice by quantifying STAT3 phosphorylation in the hypothalamus after ip injection of 287 nmol·kg−1 bw for each test protein. (A) Downstream signaling in neurons of the hypothalamus was examined by immunostaining of phosphorylated (pSTAT3) and total STAT3 (tSTAT3) on a Western blot. For determination of basal STAT3 phosphorylation, a group of mice that had only received PBS injections was included (samples from these mice are labeled on the Western blot as “0”). The bands were quantified by densitometry (N = 3), and the change in the pSTAT3/tSTAT3 ratio relative to the mock samples is shown in the lower panel. (B) The area under the curve from the test proteins shown in panel A as well as some other PASylated leptin versions (see Table 1) was plotted against the corresponding terminal plasma half- life (N = 3), revealing a linear correlation.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Recombinant, Phospho-proteomics, Injection, Immunostaining, Western Blot, Labeling, Clinical Proteomics

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 6. Physiological effects of PASylated murine leptin versions. The PD of unmodified recombinant leptin, as well as the superactive mouse leptin antagonist (SMLA), and its PASylated versions was investigated in mice with regard to daily food consumption (A) and body weight change (B) using a phenotyping device (N = 8). Lean C57BL/6J mice were injected with a single ip dose of 287 nmol·kg−1 bw test protein on day 0.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Recombinant, Injection

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG improved glucose tolerance in the absence of leptin in Lep ob mice and exhibited no effect in LepR-deficient Lepr db mice. ( A ) Body weight of Lep ob male mice in groups before and after treatment with Veh (PBS, white bar) or EREG (50 ng/g body weight (BW), black bar) for 26 days. Mice were on regular chow diet. Unpaired t -test, n = 7/group. ns, not significant. ( B , C ) Fat ( B ) and lean body ( C ) composition in same groups of mice at the end of the study was measured by Echo-MRI. Fat and lean mass are shown as % of the total weight (100%). ( D , E ) Glucose tolerance test (GTT) was performed in fasted Lep ob mice treated with PBS (Veh, open circles) or EREG (closed circles) ( n = 7 per group). GTT kinetics ( D ) and area under the curve (AUC) ( E ) are shown. Statistical significance was examined by ANOVA ( D ) and Student’s t -test ( E ). ( F ) Insulin levels in plasma in both mouse groups were measured by ELISA. Unpaired student’s t -test. ( G ) Weight before and after treatment of Lepr db male mice with Veh (PBS, white bar) or EREG (50 ng/g body weight (BW), black bar) for 4 weeks ( n = 6 per treatment). Mice were on regular chow. Unpaired Student’s t -test, n = 6/group. ( H , I ) Fat ( H ) and lean body ( I ) composition (% of total weight) in the same groups of mice at the end of the study were measured by Echo-MRI. ( J , K ) GTT kinetics ( J ) and AUC ( K ) were obtained from Lepr db mice treated with PBS (Veh, open circles) or EREG (closed circles). ANOVA ( J ) and Student’s t -test ( K ). ( L ) Insulin levels in plasma in both mouse groups were measured by ELISA. Unpaired student’s t -test.

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG regulated glucose uptake via binding with LepR in Lep ob mice. ( A ) EREG and insulin tolerance test in Lep ob mice ( n = 5 per group) treated with a single intraperitoneal injection of insulin (0.012 IU/g BW, triangle dashed line) or EREG (80 ng/g BW, closed circles. Asterisks, significant (* p < 0.05) compared to glucose levels before EREG treatment. # Hashtag, significant difference in glucose levels 30 min after treatment with EREG or insulin. Unpaired Student’s t -test. ( B ) Area under the curve (AUC) quantification of insulin (hatched bar) and EREG (black bar) tolerance tests. Unpaired Student’s t -test, ns . ( C ) GTT kinetics were measured in Lep ob mice ( n = 5 per treatment) treated with a single injection of PBS (Veh, open circles) or EREG (closed circles). Student’s t -test. * p < 0.05 from comparison between control and EREG treated mice at each time point. ( D ) Area under the curve (AUC) quantification of insulin (hatched bar) and EREG (black bar) tolerance tests. Unpaired Student’s t -test. ( E , F ). Immunoprecipitation of LepR was performed with anti-EREG antibody using homogenates from subcutaneous fat ( C ) and visceral fat ( D ). Fat tissue was isolated from non-treated Lep ob (Veh) as well as Lep ob mice 15 min after injection of EREG (50 ng/mL).

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Binding Assay, Injection, Comparison, Control, Immunoprecipitation, Isolation

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG-stimulated glucose uptake was dependent on LepR but independent of EGFR. ( A , B ) Fluorescently-labelled (FD) glucose uptake was measured in stromal vascular fraction (SVF) cells isolated from visceral tissues of Lepr db ( A ) or Lep ob mice ( B ). Cells were treated with either Veh (PBS), mouse EREG (50 ng/mL), human insulin (Ins, 10 µg/mL), or mouse leptin (Lep, 200 ng/mL) for 80 min. For inhibition experiment, Lep ob SVF cells were pre-treated with EGFR inhibitor (EGFR-I, AST-1306, 10 µM) or vehicle (Veh, DMSO) for 40 min. Data are shown as a percentage of Veh-treated control (100%, n = 8 per treatment). Unpaired Student’s t -test. ( C – E ) FD-glucose uptake was measured in mouse 3T3-L3 preadipocytes. ( C ) Preadipocytes were treated with vehicle, human insulin (Ins, 10 µg/mL), and mouse EREG (50 ng/mL) for 30 min (mean ± SEM, n = 6, t -test). ( D ) Time-dependent uptake of FD-glucose in 3T3-L1 preadipocytes stimulated with human insulin (Ins, 10 µg/mL), mouse leptin (Lep, 200 ng/mL), and mouse EREG (50 ng/mL). Data are shown (mean ± SEM, n = 8, t -test) as % of glucose uptake compared to control cells at the same time point (Veh, 100%). ( E ) Concentration-dependent increase in FD-glucose uptake by 3T3-L1 preadipocytes stimulated with different concentrations of mouse EREG. Data are shown as a percentage of Veh-treated control (100%, n = 6 per concentration). * p < 0.05, significant differences compared to the vehicle group, one-way ANOVA). ( F ) NIH-3T3 preadipocytes were transiently transfected with pB- Glut4 -7myc-GFP and stimulated with vehicle, Ins (10 µg/mL), EREG (50 ng/mL) for 60 min. Data show representative fluorescent images of GFP-labeled GLUT4 selected from three independent experiments. 10× magnification. Yellow arrow shows GFP-labeled GLUT4 that was translocated to the cellular membrane. ( G ) Quantification of GFP was performed in adipocytes of similar size ( n = 10) in each group.

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Isolation, Inhibition, Control, Concentration Assay, Transfection, Labeling, Membrane

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG mediates glucose uptake via PI3K with transient activation of ERK. ( A ) FD-glucose uptake in 3T3-L3 preadipocytes treated with or without EREG (50 ng/mL) and in the presence of inhibitors for EGFR-I (AG1478, 10 µM), EGFR and ErbB2 (AST-1306 or CI-1033 10 µM), dual IR/IGF-1R inhibitor (BMS 536924, 1 µM), and SRC-I, AZM475271, 1 µM) for 30 min. Cells were starved for 90 min before stimulation. Dashed line shows glucose uptake in the presence of insulin (Ins, 10 µg/mL). ( B ) FD-glucose uptake was measured in mouse 3T3-L1 preadipocytes with or without EREG (50 ng/mL) and inhibitors of MEK1/2 and PI3K (MEK1/2-I, U0126 10 μM, and PI3K-I, wortmannin 200 nM). Data (mean ± SD, n = 6) are shown as a percentage of control (Veh 100%). Unpaired Student’s t -test. ( C ) 3T3-L1 preadipocytes were stimulated with EREG at different concentrations (0–100 ng/mL) for 5 or 15 min. The total and phosphorylated levels of AKT, STAT3, STAT5, and ERK were measured by Western blot in duplicates. Data are shown in a representative Western blot. ( D ) The kinetics of pERK expression was quantified based on the Western blots. pAKT, p-STAT3, and p-STAT5 analysis are described in . Pearson correlation analysis. ( E ) 3T3-L1 preadipocytes were stimulated with or without EREG or EGF (50 ng/mL, each) for 30 min in the presence and absence of EGFR inhibitor AST1306 (100 nM), and antibody against mouse LepR (Invitrogen, PA1-053, 10 μg/mL). For inhibition, cells were pre-treated 30 min before EREG and EGF stimulation. ( F ) FD-glucose uptake was measured in mouse 3T3-L3 preadipocytes pre-treated with either Veh (DMSO) or ERK inhibitors (U0126, SCH772984, or DEL 22379, each 10 µM in DMSO) for 40 min. Then, cells were treated with either Veh (PBS), mouse EREG (50 ng/mL), or mouse leptin (Lep, 200 ng/mL) for 80 min. Data are shown as a percentage of Veh-treated control (100%, n = 7 per group). Unpaired Student’s t -test. ns , not significant ( p > 0.05).

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Activation Assay, Control, Western Blot, Expressing, Inhibition

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: Kinetics of the changes in LepR film thickness in the presence of leptin ( A ) or EREG ( B ). Film thickness was measured using QCM and quantified based on the binding kinetics to a gold sensor.

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Binding Assay

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: Evolutionary analysis of EREG binding to LepR. ( A – E ) EREG docking to LepR. Evolutionary analysis of 175 open The dependence of EREG-mediated glucose uptake on the ERK phosphorylation cascade was examined using (1) a specific inhibitor of ERK1/2 SCH772984 , (2) an inhibitor of ERK dimerization DEL-22379 , and (3) a selective inhibitor of MEK1 and MEK2 U0126 . All inhibitors increased basal glucose uptake, which was further increased by leptin ( F). The inhibition of ERK1/2 and MEK1/2 as well as ERK dimerization prevented stimulatory effect of EREG on FD-glucose uptake but did not decrease it beyond the levels seen in the control cells. Although transient ERK phosphorylation occurred in response to EREG stimulation, this pathway was dispensable for glucose uptake and dependent on PI3K and may be other pathways ( B and ). ( F ) Hypothetic mechanism suggesting EREG as an alternative ligand for both EGFR and LepR. The canonic leptin/LepR response can induce JAK/STAT3 signaling and required the long form of LepR. The alternative binding of EREG to LepR can induce ERK and PI3K activation increasing GLUT4 translocation and glucose uptake, but not the other canonic effects of leptin, including the regulation of appetite and energy expenditure.

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Binding Assay, Phospho-proteomics, Inhibition, Control, Activation Assay, Translocation Assay

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Production of leptin in normal and OA osteoblasts . The expression of leptin was first determined by qPCR. Confluent osteoblasts (Ob) were lized in TRIzol and RNA extracted as described in Material and methods. RNA (1 μg) was reversed transcribed followed by qPCR amplification of 100 ng cDNA using specific primers for leptin and GAPDH. The data were processed with the GeneAmp 5700 SDS software and given as threshold cycle (Ct), corresponding to the PCR cycle at which an increase in reporter fluorescence above baseline signal can first be detected. The Ct was converted to the number of molecules and the values for each sample calculated as the ratio of the number of molecules of the target gene/number of molecules of GAPDH. A ) Quantification of leptin mRNA using Lep1 and Lep2 primers. Results are given as the mean value of markers relative to GAPDH ± SEM of n = 4 OA preparations. B ) Quantification of leptin mRNA levels in normal and OA Ob using Lep1 primers. Results are the mean ± SEM of n = 5 normal and n = 15 OA individual Ob preparations. C ) OA Ob were exposed to increasing doses of leptin and lepin mRNA levels were determined using Lep1 primers. Results are the mean ± SEM of n = 4 preparations. The protein production of leptin was next detected using a very selective ELISA. Conditioned-media of confluent normal and OA Ob incubated in HAM's F12/DMEM media containing 0.5% FBS for their last 48 hours of culture were recuperated and stored at -80°C. D ) Aliquots were taken to measure leptin using a very sensitive ELISA. Results are the mean ± SEM of n = 5 normal and n = 6 OA individual Ob preparations.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Expressing, Amplification, Software, Fluorescence, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Production of leptin receptors (OB-Rb) in normal and OA osteoblasts . The expression of leptin receptors was first determined by qPCR. A ) Confluent Ob were lized in TRIzol and RNA extracted as described in Material and Methods. RNA was reversed transcribed followed by PCR amplification of 100 ng cDNA as described in Figure 1 using OB-Rb and GAPDH primers. Results are the mean ± SEM of n = 7 normal and n = 19 OA Ob preparations, P < 0.004 vs normal and OA. B ) OA Ob were incubated for 24 hours with increasing concentrations of exogenous leptin. Cells were then lyzed and used for PCR amplification of OB-Rb as in A. Results are the mean ± SEM of n = 6 OA Ob preparations. Second, the production of leptin receptors was determined by Western blot analysis. C ) Confluent Ob were treated for 48 hours with or without 1,25(OH) 2 D 3 (50 nM), leptin (100 ng/ml), TGF-β1 (10 ng/ml) or HGF (10 ng/ml). The cells were then lized in RIPA buffer prior to separation using SDS-PAGE and Western blotting using specific antibodies to OB-Rb.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Expressing, Amplification, Incubation, Western Blot, SDS Page

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Cellular proliferation and intracellular signaling of OA osteoblasts in response to leptin . OA osteoblasts were plated at 10,000 cells/cm 2 and allowed to attach overnight in HAM's F12/DMEM media containing 10% FBS. Cells were then treated with the same media with 0.5% FBS for 24 hours prior to receiving increasing doses of leptin (10 ng/ml, 100 ng/ml, 1 mg/ml or 10 mg/ml) or the vehicle in the same media for another incubation of 24 hours. Cell proliferation was assessed by the incorporation of BrdU or MTT assay. A ) Incorporation of BrdU by OA Ob in response to leptin; B ) Proliferation of OA Ob by MTT assay; C) Representative phospho p42/44 Western blot analysis in response to increasing doses of leptin in OA Ob. D ) Determination of phospho p42/44 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program . E) Representative phospho p38 Western blot analysis in response to increasing doses of leptin in OA Ob. F) Determination of phospho p38 levels using the NIH Image program developed at the U.S. National Institutes of Health with the Scion Image 1.63 program . Values are the mean ± SEM of at least four separate experiments; * P < 0.05, ** P < 0.01.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Incubation, MTT Assay, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Modulation of alkaline phosphatase, osteocalcin and collagen type 1 in OA Ob by inactivating leptin signaling . Confluent normal and OA Ob were treated for their last two days of culture with either media alone containing 0.5% FBS with or without 1,25(OH) 2 D 3 (50 nM) as per indicated for the individual markers. Cells were treated with either exogenous leptin, antibodies against leptin, tyrphostin (AG490, 100 μM) or Piceatannol (Pce, 75 μM) for 30 minutes prior to the addition of 1,25(OH) 2 D 3 except for CICP that was performed in the absence of 1,25(OH) 2 D 3 . At the end of the 48 h incubation, the supernatant was kept for osteocalcin and for collagen production, and cells were lyzed in ALPase buffer prior to measuring alkaline phosphatase activity by substrate hydrolysis. A ) Results of alkaline phosphatase activity for normal OB; B ) Results of alkaline phosphatase activity for OA OB; C ) Results of osteocalcin release by OA Ob; D ) Results of CICP production; E ) Confluent OA Ob were incubated in Ham's F12/DMEM media without serum and containing 1% ITS. Cells were treated with or without exogenous leptin, tyrphostin (AG490, 100 μM) or Piceatannol (Pce, 75 μM) for their last 48 hours of culture. Results of TGF-β1 levels in supernatants are shown. The results are the mean ± SEM of n = 4 normal and n = 9 OA Ob preparations.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Incubation, Activity Assay

Journal: Arthritis Research & Therapy

Article Title: Local leptin production in osteoarthritis subchondral osteoblasts may be responsible for their abnormal phenotypic expression

doi: 10.1186/ar2925

Figure Lengend Snippet: Modulation of alkaline phosphatase and osteocalcin release in OA Ob by inactivating leptin or leptin signaling . OA Ob were treated with siRNA for either leptin or OB-Rb or a scrambled RNA as described in Material and methods. Cells were then used to determine alkaline phosphatase activity and osteocalcin release. A ) Results of alkaline phosphatase activity in response to leptin or OB-Rb siRNA treatments. B ) Results of osteocalcin release in response to leptin or OB-Rb siRNA treatments. C ) Leptin expression in response to siRNA. D ) OB-Rb expression in response to siRNA. Results are the mean ± SEM of n = 6 OA Ob preparations.

Article Snippet: For the determiniation of phenotypic markers, cells were either treated with 1 μg/ml recombinant human leptin (rhleptin, Calbiochem, San Diego, California, USA), 10 μg/ml recombinant human leptin R/Fc chimera (R&D Systems, Minneapolis, MN, USA) that neutralizes the activity of rhleptin, 100 μM Tyrphostin (AG490, Sigma-Aldrich), 75 μM piceatannol (Pce, Sigma-Aldrich), or the vehicle.

Techniques: Activity Assay, Expressing