Journal: The Journal of Biological Chemistry

Article Title: Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response

doi: 10.1074/jbc.RA118.004420

Figure Lengend Snippet: Characterization of anti-HER3 antibodies. A, sequences of anti-HER3 Fabs. Sequences of CDR positions randomized in the library are shown and are numbered according to the IMGT standards (72). B, IgG specificity for ErbB family members assessed by ELISA. Error bars represent the standard deviation of three independent experiments, and each point the mean of one experiment. C, Western blots of lysates from SKBR3 cells treated with the indicated IgG (5 μg/ml) for 1 h prior to treatment with NRG1 (2 nm) for 10 min. Blots were developed with the antibodies to the proteins indicated on the right. The data are representative of three independent experiments. D, cell proliferation assays with SKBR3 cells treated with the indicated antibodies. Relative confluence was measured after control cells doubled 1.5 times (∼55 h). Error bars represent the standard deviation of three independent experiments. *, p < 0.05; **, p < 0.005, one-way ANOVA (see “Experimental procedures”). E, in vivo xenograft assays. Subcutaneous BxPC3 xenografts were established in CB-17 SCID mice and treated with the indicated antibodies. Tumor size was measured at the indicated time points (n = 10 for the PBS control, and n = 9 for all other treatment groups). **, p < 0.0001; *, p = 0.0001, two-way ANOVA.

Article Snippet: Sensor-captured IgG 95 was exposed to 200 n m HER3/HER2-Fc, 200 n m HER3-His (Sino Biological), or 3-fold serial dilutions in PBS, 1% bovine serum albumin, 0.05% Tween 20.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Western Blot, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response

doi: 10.1074/jbc.RA118.004420

Figure Lengend Snippet: Kinetic constants for IgG 95 binding monomeric or heterodimeric HER3 Values are representative of 3 independent experiments.

Article Snippet: Sensor-captured IgG 95 was exposed to 200 n m HER3/HER2-Fc, 200 n m HER3-His (Sino Biological), or 3-fold serial dilutions in PBS, 1% bovine serum albumin, 0.05% Tween 20.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response

doi: 10.1074/jbc.RA118.004420

Figure Lengend Snippet: Comparison of IgGs 95 and MOR09825. A, Western blots of BT474 cell lysates immunoprecipitated with the antibodies indicated above the lanes. Cells were untreated (−DTSSP) or treated with a protein cross-linking agent (+DTSPP) prior to lysis and immunoprecipitation, and blots were developed with antibodies to the proteins indicated to the right. HER2 band intensities are indicated below the lanes. Representatives of three independent experiments are shown. B, ELISA for detection of NRG1 binding to immobilized HER3-Fc preblocked with the indicated IgG. Error bars represent the standard deviation of three independent experiments, and each point the mean of one experiment. *, p < 0.005, one-way ANOVA. C, proliferation assays for BT474 cells treated with 15 μg/ml of the indicated IgG or mixture (7.5 μg/ml of each antibody) for 7 days. Error bars represent the standard deviation of three independent experiments, and each point the mean of one experiment. *, p < 0.005, one-way ANOVA.

Article Snippet: Sensor-captured IgG 95 was exposed to 200 n m HER3/HER2-Fc, 200 n m HER3-His (Sino Biological), or 3-fold serial dilutions in PBS, 1% bovine serum albumin, 0.05% Tween 20.

Techniques: Western Blot, Immunoprecipitation, Lysis, Enzyme-linked Immunosorbent Assay, Binding Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response

doi: 10.1074/jbc.RA118.004420

Figure Lengend Snippet: Effects of IgG 95 on HER3 internalization and ubiquitination. A, Western blots of lysates from SKBR3 cells treated with the indicated IgGs for 90 min. The following concentrations were used for IgG 95: 0.04, 0.2, 1, or 5 μg/ml, and 5 μg/ml was used for IgG control and trastuzumab. The indicated cells were stimulated with 2 nm NRG1 for 10 min before harvest. Blots were developed with the antibodies indicated to the right. The data are representative of 3 independent experiments. B, Western blots of lysates from SKBR3 cells treated with 100 μm chloroquine (chlor) or 50 nm bortezomib (bort) for 1 h prior to treatment with 1 μg/ml of IgG 95 for 2 h, as indicated. Blots were developed with antibodies to the proteins indicated to the right. Representative of three independent experiments. C, internalization of IgG 95. SKBR3 cells were treated with 100 μm chloroquine or 50 nm bortezomib for 1 h prior to treatment with 1 μg/ml of IgG 95 for 2 h, as indicated. The cells were then processed for flow cytometry by staining with IgG 95. Error bars represent the S.D. of 4–5 independent experiments, and each point the relative median fluorescent intensity of a single experiment. *, p < 0.005, one-way ANOVA. D, Western blots for assessment of ubiquitination of HER3. The indicated cells were treated with 100 μm chloroquine for 30 min followed by treatment with 1 μg/ml of IgG 95 for 30 min, as indicated. Lysates were subjected to immunoprecipitation with IgG 95, followed by development with antibodies to the proteins indicated to the right. The data are representative of three independent experiments.

Article Snippet: Sensor-captured IgG 95 was exposed to 200 n m HER3/HER2-Fc, 200 n m HER3-His (Sino Biological), or 3-fold serial dilutions in PBS, 1% bovine serum albumin, 0.05% Tween 20.

Techniques: Western Blot, Flow Cytometry, Staining, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response

doi: 10.1074/jbc.RA118.004420

Figure Lengend Snippet: RNF41 depletion protects HER3 protein from degradation induced by IgG 95. A, quantification by Western blotting of HER3 in lysates from SKBR3 cells stably expressing the indicated shRNA. Error bars represent the S.D. of three independent experiments, and each point the value of one experiment. *, p < 0.05; **, p < 0.005, t test. B, quantification by Western blotting of HER3 in lysates from SKBR3 cells stably expressing the indicated shRNA and treated with 1 μg/ml of IgG 95 or control IgG for 2.5 h. Error bars represent the S.D. of at least five independent experiments, and each point the value of one experiment. *, p < 0.05, one-way ANOVA. C, Western blots of IgG 95 immunoprecipitates from SKBR3 cells stably expressing the indicated shRNA and pretreated with 100 μm chloroquine for 30 min, and then with 1 μg/ml of control or IgG 95 for 30 min. Representative data from one of three independent experiments are shown. D, Western blots of lysates from SKBR3 cells stably expressing the indicated shRNA and treated with 5 μg/ml of the indicated IgG for 2.5 h. The data are representative of three independent experiments.

Article Snippet: Sensor-captured IgG 95 was exposed to 200 n m HER3/HER2-Fc, 200 n m HER3-His (Sino Biological), or 3-fold serial dilutions in PBS, 1% bovine serum albumin, 0.05% Tween 20.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: Amino acid sequence of the dimeric HER2-binding Affibody (Z HER2 ) 2:L -Cys. The protein construct is designed to be made up of two domains with three-helix folds connected by a 15-residue linker containing a unique cysteine, underlined in the sequence, for simple labeling of the construct with thiol-reactive compounds. The dimer has three N-terminal glycines and carries a C-terminal sortase A recognition motif (LPETG) to make the construct a substrate for sortase A-mediated cyclization. The hexahistidine tag in the linear construct is used for IMAC purification of the Escherichia coli -expressed construct, but is cleaved off during the cyclization reaction. Helical regions in a crystal structure of the parental monomeric Z HER2:342 Affibody are indicated by helices 1–3 and 4–6, respectively .

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: Sequencing, Binding Assay, Construct, Labeling, Purification

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: ( A ) Homology model of (Z HER2 ) 2:L -Cys generated with the SWISS-MODEL protein modelling server based on a crystal structure of two tandem B-domains connected by a conserved linker (PDB code: 4NPF ). Residues 1–3, i.e., the three N-terminal glycines, and C-terminal residues 135–154, containing the sortase A recognition site and the His6-tag, were not included in this homology model. The unique cysteine, C 70 , is predicted to be situated in the linker region between domains 1 and 2. ( B ) Schematic structure of the cyclic Z HER2 -dimer, (Z HER2 ) 2:C . Sortase A-mediated cyclization of the dimeric protein results in the formation of a native peptide bond between Gly 1 and Thr 146 . ( C ) Schematic illustrations of three different approaches to intramolecular crosslinking or backbone cyclization of Affibody molecules previously published by our group and others. The HER2-binding Z HER2:CL was synthesized using solid phase peptide synthesis (SPPS) and has an intramolecular thioether bond going from a cysteine residue in the loop between helices 1 and 2 and the chloroacetyl-modified side chain of the C-terminal lysine residue . Z min is a truncated version of the Z-domain, in which the two IgG-binding helices 1 and 2 are joined by a peptide bond. Z min was prepared using SPPS and backbone-cyclized using natural chemical ligation . Lasso is a recombinantly expressed Z-domain dimer. The two IgG-binding Z-domains are joined by flexible linkers and the construct is backbone-cyclized using split-intein technology . The schematic drawings of Z HER2:CL , Z min and lasso are based on information found in references [ , , ].

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: Generated, Binding Assay, Synthesized, Modification, Ligation, Construct

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: SDS-PAGE analysis of sortase A-mediated cyclization of the Z HER2 -dimer. M: Molecular weight marker. Lane 1: Sortase A 3 *, lane 2: (Z HER2 ) 2:L , and lane 3: unpurified sample taken after a 20 h sortase A-mediated cyclization reaction.

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: SDS Page, Molecular Weight, Marker

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: Theoretical and observed molecular weights of proteins in this study.

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques:

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: ( A ) Circular dichroism (CD) expressed as molar ellipticity of linear (Z HER2 ) 2:L (blue) and (Z HER2 ) 2:C (red) before (solid line) and after (dashed line) refolding after thermal melt at 90 °C. Inserted is a close-up of the CD spectra in the 205–230 nm region. ( B ) Fraction folded protein as a function of temperature for (Z HER2 ) 2:L (blue) and (Z HER2 ) 2:C (red). The melting temperature, T m , of the linear dimer was 65 °C and it was elevated to 68 °C in the cyclic dimer.

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques:

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: Evaluation of kinetic titration series of ( A ) the linear dimer (Z HER2 ) 2:L and ( B ) the cyclic dimer (Z HER2 ) 2:C binding to immobilized HER2-Fc.

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: Titration, Binding Assay

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: In vitro digestion of (Z HER2 ) 2:L and (Z HER2 ) 2:C . ( A ) Digestion with trypsin (0.9 µg/mL) plus chymotrypsin (0.4 µg/mL). ( B ) Digestion with carboxypeptidase A (0.5 µM).

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: In Vitro

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: Fluorescent microscopy images of either SKOV-3 cells or MCF-7 cells with DAPI-stained nuclei (blue) treated with 2.4 nM of (Z HER2 ) 2:C -DL594 (red). ( A ) SKOV-3 cells treated with (Z HER2 ) 2:C -DL594 as a positive control in the first row. In the second row, SKOV-3 cells pre-incubated with unlabeled (Z HER2 ) 2:C (1.2 µM) before addition of (Z HER2 ) 2:C -DL594. ( B ) MCF-7 cells treated with (Z HER2 ) 2:C -DL594. ( C ) SKOV-3 cells treated with (Z HER2 ) 2:C -DL594 as a positive control in the first row. The second row presents SKOV-3 cells pre-incubated with trastuzumab (Herceptin; 1 mg/mL) before the addition of (Z HER2 ) 2:C -DL594.

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: Microscopy, Staining, Positive Control, Incubation