Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: Solution binding measurements of human and rat Shh to ch5E1 Fab by isothermal titration calorimetry

Article Snippet: Chimeric 5E1 Fab fragments (ch5E1 Fab) were obtained by digestion with endoproteinase Lys C (Wako) in 0.1 m Tris, pH 8, with an enzyme:IgG ratio of 1:500 (w/w) for 1 h at 37 °C.

Techniques: Binding Assay, Titration

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: Chimeric 5E1 is functionally equivalent to murine 5E1. A, schematic of murine 5E1 (m5E1, yellow) IgG (33) and its chimeric counterpart (ch5E1), where the constant domains (CH1–3 and CL) have been replaced with the corresponding domains from the humanized antibody trastuzumab (blue) (35), leaving the variable light and heavy (VL and VH) domains of the murine 5E1 antibody intact. B, ch5E1 and m5E1 bind similarly to an endogenous Hh-expressing cell line. Flow cytometry analysis is shown of endogenous Hh in HT29 cells with the indicated concentrations of m5E1 or ch5E1 (in μg/ml). The means ± S.D. of triplicate reactions are plotted. C, ch5E1 (■) and m5E1 (●) bind similarly to stably transfected Shh-COS cells by flow cytometry analysis. The means ± S.D. of a representative duplicate experiment are shown. Isotype controls (chimeric IgG (□) or murine IgG1 (○)) show no appreciable binding. D, ch5E1 and m5E1 compete for cell surface Shh. The ability of increasing amounts of ch5E1 (■) or m5E1 (●) to compete with ∼0.69 nm (0.1 μg/ml) m5E1 for binding to Shh-expressing cells and vice versa as monitored by flow cytometry analysis is shown, normalized to 100% for no competitor after background subtraction. Isotype controls (murine IgG1 (○) or chimeric IgG (□)) are unable to compete for Shh binding. E, ch5E1 specifically detects Hh in the developing mouse embryo. E10.5 embryos were sectioned and stained with m5E1 (top) or ch5E1 (bottom), followed by Cy3-conjugated secondary antibodies (left panel and red in merged right panel) and 4′,6-diamidino-2-phenylindole (DAPI) (blue nuclear staining in right merged panels). ch5E1 is as specific as m5E1 in detecting Hh in the notochord (NC) and floor plate (FP). Scale bar is 200 μm (images taken at ×10 magnification). F, ch5E1 and m5E1 inhibit Hh signaling similarly. HT29 cells secreting Hh were co-cultured with S12 cells (C3H10T1/2 cells stably expressing a Gli-luciferase reporter (36)). Hh signaling was stimulated by serum starvation for 24 h in the presence of the indicated concentrations of ch5E1 (■), m5E1 (●), hIgG1 (▴), or mIgG1 (○) antibodies. The means ± S.D. of the luciferase signals (RLU; relative luminescence units) of triplicate measurements are plotted. This experiment is representative of multiple independent experiments.

Article Snippet: Chimeric 5E1 Fab fragments (ch5E1 Fab) were obtained by digestion with endoproteinase Lys C (Wako) in 0.1 m Tris, pH 8, with an enzyme:IgG ratio of 1:500 (w/w) for 1 h at 37 °C.

Techniques: Expressing, Flow Cytometry, Stable Transfection, Transfection, Binding Assay, Staining, Cell Culture, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: Binding kinetics of Hh ligands to ch5E1 Fab in the presence and absence of divalent ions by biolayer interferometry Binding measurements for human and rat Shh were carried out using immobilized Hh ligand and ch5E1 or m5E1 Fabs in solution. Binding measurements for human Ihh and human Dhh were carried out using immobilized ch5E1 and Hh ligand in solution as described under “Experimental Procedures.”

Article Snippet: Chimeric 5E1 Fab fragments (ch5E1 Fab) were obtained by digestion with endoproteinase Lys C (Wako) in 0.1 m Tris, pH 8, with an enzyme:IgG ratio of 1:500 (w/w) for 1 h at 37 °C.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: 5E1 binding to Shh is enhanced by divalent cations. Biolayer interferometry sensorgrams of ch5E1 Fab binding to C-terminally biotinylated Shh on streptavidin-coated biosensors are shown in the absence (A) or presence (B) of Ca2+ and Zn2+. Sensorgrams of seven 2-fold serial dilutions of ch5E1 Fab starting at 125 nm are shown, where 125 nm results in the largest response. C, isothermal titration calorimetry of human Shh with ch5E1 Fab. Experiments were carried out in the absence or presence of Ca2+ (5 μm CaCl2) and/or Zn2+ (500 μm ZnSO4) as indicated. As expected, the stoichiometry of 5E1-Shh complex formation in solution derived from the ITC data indicates 1:1 binding in all cases.

Article Snippet: Chimeric 5E1 Fab fragments (ch5E1 Fab) were obtained by digestion with endoproteinase Lys C (Wako) in 0.1 m Tris, pH 8, with an enzyme:IgG ratio of 1:500 (w/w) for 1 h at 37 °C.

Techniques: Binding Assay, Isothermal Titration Calorimetry, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: X-ray data collection and refinement statistics

Article Snippet: Chimeric 5E1 Fab fragments (ch5E1 Fab) were obtained by digestion with endoproteinase Lys C (Wako) in 0.1 m Tris, pH 8, with an enzyme:IgG ratio of 1:500 (w/w) for 1 h at 37 °C.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: Structure of 5E1 bound to Shh. A, comparison of the structures of ch5E1 Fab alone and in complex with Shh. Shh-bound ch5E1 light and heavy chains are shown as C-α ribbons and colored green and blue, respectively; both heavy and light chains of the free ch5E1 Fab are colored gray. Residues in CDR H3 differing most upon binding to Shh (not pictured) are labeled. B, comparison of the structures of Shh free (white), bound to Cdon (orange), Hhip (green), or 5E1 (yellow). The bound divalent metal cations are shown as spheres (Zn2+ in pink and Ca2+ in green). C, complex between 5E1 and Shh. The Fab is colored as in A, and Shh is in yellow, with Zn2+ and Ca2+ colored as in B.

Article Snippet: Chimeric 5E1 Fab fragments (ch5E1 Fab) were obtained by digestion with endoproteinase Lys C (Wako) in 0.1 m Tris, pH 8, with an enzyme:IgG ratio of 1:500 (w/w) for 1 h at 37 °C.

Techniques: Binding Assay, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: Detailed views of the 5E1-Shh interaction. A, “open book” view of the 5E1-Shh interface. The surface of Shh (left, oriented as in Fig. 3B) and ch5E1 Fab (right) are colored according to buried surface area upon complex formation. Residues that bury 75–100, 50–75, or 25–50% of their solvent-accessible surface area on complex formation are colored red, orange, and yellow, respectively. Residues colored red or orange are labeled. The boundary between the heavy and light chains is indicated with a dotted line. Shh residue Ser67, which is a threonine in mouse and rat Shh, is colored pink. The location of Shh residue Arg72, corresponding to Hh residue Lys132 implicated in multimerization of Hh (44), is also indicated. B, 5E1-Shh interface. Shh is shown in yellow with a transparent molecular surface. Shh residues mentioned in the text are labeled with the exception of Asp95, Asp129, and Ala179, which are obscured in this orientation. 5E1 is colored with the heavy chain in blue and the light chain in green. CDR loops are labeled, and individual 5E1 side chains mentioned in the text are labeled in italic font. Hydrogen bonds and salt bridges mentioned in the text are shown as dashed lines. The Zn2+ and Ca2+ ions are shown as pink and green spheres, respectively.

Article Snippet: Chimeric 5E1 Fab fragments (ch5E1 Fab) were obtained by digestion with endoproteinase Lys C (Wako) in 0.1 m Tris, pH 8, with an enzyme:IgG ratio of 1:500 (w/w) for 1 h at 37 °C.

Techniques: Labeling

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: Sequence alignment of human Shh, Ihh, and Dhh. The amino acid sequences of the Shh, Ihh, and Dhh N-terminal signaling domains are aligned showing the conservation of residues in the 5E1 epitope (overlined). The dashed line highlights the 5E1-protected tryptic peptide previously shown to form part of the epitope (21). Residues that contact Hhip are indicated with an asterisk. Rat and mouse sequence numbers are one higher than human Shh due to an extra amino acid in the signal sequence. Thus, Ser67 of human Shh is Thr68 in rodent Shh; His180 is Arg181 in the rat Shh immunogen. Thr68 is listed below the alignment as are the residues in the rat Shh immunogen described by Roelink and co-workers (33, 43) that differ from current data base sequences for rat Shh. Residues whose side chains or backbones coordinate the Ca2+ ions are indicated by “Ca” and those that coordinate the Zn2+ ion by “Zn”.

Article Snippet: Chimeric 5E1 Fab fragments (ch5E1 Fab) were obtained by digestion with endoproteinase Lys C (Wako) in 0.1 m Tris, pH 8, with an enzyme:IgG ratio of 1:500 (w/w) for 1 h at 37 °C.

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: Comparison of the 5E1 epitope with the Shh receptor-binding sites. A, footprint of 5E1 (left panel, blue), Hhip (middle panel, green) (21), and Cdon (right panel, orange) (11) on the molecular surface of Shh; Zn2+ and Ca2+ are shown as pink and green spheres, respectively. The location of the Ca2+ is indicated as the ions are largely hidden by the Shh molecule surfaces. The footprint is defined as Shh residues within 4.2 Å of the binding partner. Shh is oriented as in Fig. 3B. B, comparison of the binding modes of Shh with 5E1 (left panel), Hhip (center panel, green), and Cdon (right panel, orange). 5E1, Hhip and Cdon are shown as C-α ribbons. The complexes are oriented such that the Shh pseudo-active site groove runs from left to right in the plane of the figure. The 5E1 and Cdon complexes are in the same orientation. The Hhip complex has been rotated slightly to improve the visibility of Hhip.

Article Snippet: Chimeric 5E1 Fab fragments (ch5E1 Fab) were obtained by digestion with endoproteinase Lys C (Wako) in 0.1 m Tris, pH 8, with an enzyme:IgG ratio of 1:500 (w/w) for 1 h at 37 °C.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: Competition binding of ch5E1 Fab with Hhip L2 peptide binding to Shh. Biolayer interferometry sensorgrams of 250 nm Shh binding to biotinylated Hhip L2 peptide on streptavidin-coated biosensors are shown in the presence and absence of various concentrations of ch5E1 Fab. The inset shows the fraction response after binding at equilibrium versus the molar ratio of ch5E1 Fab/Shh. Because Shh and ch5E1 concentrations used are well above the KD value, ch5E1 effectively titrates Shh, having a molar ratio of 1.25 (x axis intercept), close to the predicted value of 1.0. At molar ratio 2.4, there is no observed binding of Shh to the Hhip L2 peptide.

Article Snippet: Chimeric 5E1 Fab fragments (ch5E1 Fab) were obtained by digestion with endoproteinase Lys C (Wako) in 0.1 m Tris, pH 8, with an enzyme:IgG ratio of 1:500 (w/w) for 1 h at 37 °C.

Techniques: Binding Assay