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Mouse ten-m/Odz is a new family of dimeric type II transmembrane proteins expressed in many tissues.

Oohashi T, Zhou XH, Feng K, Richter B, Mörgelin M, Perez MT, Su WD, Chiquet-Ehrismann R, Rauch U, Fässler R - J. Cell Biol. (1999)

Bottom Line: Expression of fusion proteins composed of the NH2-terminal and hydrophobic domain of ten-m1 attached to the alkaline phosphatase reporter gene resulted in membrane-associated staining of the alkaline phosphatase.The extracellular domain of Ten-m1 fused to an alkaline phosphatase reporter bound to specific regions in many tissues which were partially overlapping with the Ten-m1 immunostaining.Far Western assays and electronmicroscopy demonstrated that Ten-m1 can bind to itself.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
The Drosophila gene ten-m/odz is the only pair rule gene identified to date which is not a transcription factor. In an attempt to analyze the structure and the function of ten-m/odz in mouse, we isolated four murine ten-m cDNAs which code for proteins of 2,700-2, 800 amino acids. All four proteins (Ten-m1-4) lack signal peptides at the NH2 terminus, but contain a short hydrophobic domain characteristic of transmembrane proteins, 300-400 amino acids after the NH2 terminus. About 200 amino acids COOH-terminal to this hydrophobic region are eight consecutive EGF-like domains. Cell transfection, biochemical, and electronmicroscopic studies suggest that Ten-m1 is a dimeric type II transmembrane protein. Expression of fusion proteins composed of the NH2-terminal and hydrophobic domain of ten-m1 attached to the alkaline phosphatase reporter gene resulted in membrane-associated staining of the alkaline phosphatase. Electronmicroscopic and electrophoretic analysis of a secreted form of the extracellular domain of Ten-m1 showed that Ten-m1 is a disulfide-linked dimer and that the dimerization is mediated by EGF-like modules 2 and 5 which contain an odd number of cysteines. Northern blot and immunohistochemical analyses revealed widespread expression of mouse ten-m genes, with most prominent expression in brain. All four ten-m genes can be expressed in variously spliced mRNA isoforms. The extracellular domain of Ten-m1 fused to an alkaline phosphatase reporter bound to specific regions in many tissues which were partially overlapping with the Ten-m1 immunostaining. Far Western assays and electronmicroscopy demonstrated that Ten-m1 can bind to itself.

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Binding pattern of AP-ten-m1 in tissue sections. (A) Cartoon of Ten-m1, the fusion protein composed of AP-ten-m1 and AP.  Symbols are the same as in Fig. 1 B. (B) Coomassie blue staining of the purified AP-ten-m1 and AP separated by 7% SDS-PAGE under  reducing conditions. (C–F) Frozen sections derived from cerebellum (C), hippocampus (E), and kidney (G) were incubated with AP-ten-m1. Control sections (D, F, and H) were incubated with AP. Arrowheads indicate Purkinje cells. g, granular layer; m, molecular  layer; DG, dentate gyrus; G, glomerulus. Bars, 50 μm in C, D, G, and H; 25 μm in E and F.
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Figure 8: Binding pattern of AP-ten-m1 in tissue sections. (A) Cartoon of Ten-m1, the fusion protein composed of AP-ten-m1 and AP. Symbols are the same as in Fig. 1 B. (B) Coomassie blue staining of the purified AP-ten-m1 and AP separated by 7% SDS-PAGE under reducing conditions. (C–F) Frozen sections derived from cerebellum (C), hippocampus (E), and kidney (G) were incubated with AP-ten-m1. Control sections (D, F, and H) were incubated with AP. Arrowheads indicate Purkinje cells. g, granular layer; m, molecular layer; DG, dentate gyrus; G, glomerulus. Bars, 50 μm in C, D, G, and H; 25 μm in E and F.

Mentions: Transmembrane proteins often have the capability to bind to molecules outside the cell and thereby act as receptors. To search for ligands that bind to Ten-m1, the cDNA encoding the entire extracellular domain was linked to an AP module equipped with a signal peptide resulting in the fusion protein AP-ten-m1 (Fig. 8 A). Fig. 8 B shows that AP-ten-m1 and AP alone, which was used as a control, are secreted proteins with molecular masses of ∼300 and 67 kD, respectively. As ten-m1 is expressed in many tissues (see Figs. 5 and 7), AP-ten-m1 fusion protein should be able to detect its ligand(s) directly in a variety of tissue sections. Cryosections were treated with AP-ten-m1, washed, and then tested for bound AP activity of the fusion protein using an AP substrate precipitating on the cells. Whereas each concentration of AP-ten-m1 produced a specific staining pattern in tissues (Fig. 8, C, E, and G), equimolar concentrations of unfused AP did not show any AP reaction products (Fig. 8, D, F, and H).


Mouse ten-m/Odz is a new family of dimeric type II transmembrane proteins expressed in many tissues.

Oohashi T, Zhou XH, Feng K, Richter B, Mörgelin M, Perez MT, Su WD, Chiquet-Ehrismann R, Rauch U, Fässler R - J. Cell Biol. (1999)

Binding pattern of AP-ten-m1 in tissue sections. (A) Cartoon of Ten-m1, the fusion protein composed of AP-ten-m1 and AP.  Symbols are the same as in Fig. 1 B. (B) Coomassie blue staining of the purified AP-ten-m1 and AP separated by 7% SDS-PAGE under  reducing conditions. (C–F) Frozen sections derived from cerebellum (C), hippocampus (E), and kidney (G) were incubated with AP-ten-m1. Control sections (D, F, and H) were incubated with AP. Arrowheads indicate Purkinje cells. g, granular layer; m, molecular  layer; DG, dentate gyrus; G, glomerulus. Bars, 50 μm in C, D, G, and H; 25 μm in E and F.
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Figure 8: Binding pattern of AP-ten-m1 in tissue sections. (A) Cartoon of Ten-m1, the fusion protein composed of AP-ten-m1 and AP. Symbols are the same as in Fig. 1 B. (B) Coomassie blue staining of the purified AP-ten-m1 and AP separated by 7% SDS-PAGE under reducing conditions. (C–F) Frozen sections derived from cerebellum (C), hippocampus (E), and kidney (G) were incubated with AP-ten-m1. Control sections (D, F, and H) were incubated with AP. Arrowheads indicate Purkinje cells. g, granular layer; m, molecular layer; DG, dentate gyrus; G, glomerulus. Bars, 50 μm in C, D, G, and H; 25 μm in E and F.
Mentions: Transmembrane proteins often have the capability to bind to molecules outside the cell and thereby act as receptors. To search for ligands that bind to Ten-m1, the cDNA encoding the entire extracellular domain was linked to an AP module equipped with a signal peptide resulting in the fusion protein AP-ten-m1 (Fig. 8 A). Fig. 8 B shows that AP-ten-m1 and AP alone, which was used as a control, are secreted proteins with molecular masses of ∼300 and 67 kD, respectively. As ten-m1 is expressed in many tissues (see Figs. 5 and 7), AP-ten-m1 fusion protein should be able to detect its ligand(s) directly in a variety of tissue sections. Cryosections were treated with AP-ten-m1, washed, and then tested for bound AP activity of the fusion protein using an AP substrate precipitating on the cells. Whereas each concentration of AP-ten-m1 produced a specific staining pattern in tissues (Fig. 8, C, E, and G), equimolar concentrations of unfused AP did not show any AP reaction products (Fig. 8, D, F, and H).

Bottom Line: Expression of fusion proteins composed of the NH2-terminal and hydrophobic domain of ten-m1 attached to the alkaline phosphatase reporter gene resulted in membrane-associated staining of the alkaline phosphatase.The extracellular domain of Ten-m1 fused to an alkaline phosphatase reporter bound to specific regions in many tissues which were partially overlapping with the Ten-m1 immunostaining.Far Western assays and electronmicroscopy demonstrated that Ten-m1 can bind to itself.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
The Drosophila gene ten-m/odz is the only pair rule gene identified to date which is not a transcription factor. In an attempt to analyze the structure and the function of ten-m/odz in mouse, we isolated four murine ten-m cDNAs which code for proteins of 2,700-2, 800 amino acids. All four proteins (Ten-m1-4) lack signal peptides at the NH2 terminus, but contain a short hydrophobic domain characteristic of transmembrane proteins, 300-400 amino acids after the NH2 terminus. About 200 amino acids COOH-terminal to this hydrophobic region are eight consecutive EGF-like domains. Cell transfection, biochemical, and electronmicroscopic studies suggest that Ten-m1 is a dimeric type II transmembrane protein. Expression of fusion proteins composed of the NH2-terminal and hydrophobic domain of ten-m1 attached to the alkaline phosphatase reporter gene resulted in membrane-associated staining of the alkaline phosphatase. Electronmicroscopic and electrophoretic analysis of a secreted form of the extracellular domain of Ten-m1 showed that Ten-m1 is a disulfide-linked dimer and that the dimerization is mediated by EGF-like modules 2 and 5 which contain an odd number of cysteines. Northern blot and immunohistochemical analyses revealed widespread expression of mouse ten-m genes, with most prominent expression in brain. All four ten-m genes can be expressed in variously spliced mRNA isoforms. The extracellular domain of Ten-m1 fused to an alkaline phosphatase reporter bound to specific regions in many tissues which were partially overlapping with the Ten-m1 immunostaining. Far Western assays and electronmicroscopy demonstrated that Ten-m1 can bind to itself.

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