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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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Western blot of brain extracts with affinity-purified antibodies against Ten-m1. 3% Triton  X-100 extracts of mouse brain  (lanes 1 and 2) were precipitated  with acetone. Dried precipitates  were dissolved in sample buffer, either for 30 min at 70°C (lane 1) or  at room temperature (lane 2). Both  samples were reduced for 5 min at  95°C before electrophoresis. Lane 3  shows recombinant Ten-m1sec  which served as a positive control  and size marker. The arrow indicates the position of a second band  occasionally occurring in the recombinant Ten-m1sec lane (also  faintly visible here), probably reflecting incompletely reduced Ten-m1sec dimers.
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Figure 6: Western blot of brain extracts with affinity-purified antibodies against Ten-m1. 3% Triton X-100 extracts of mouse brain (lanes 1 and 2) were precipitated with acetone. Dried precipitates were dissolved in sample buffer, either for 30 min at 70°C (lane 1) or at room temperature (lane 2). Both samples were reduced for 5 min at 95°C before electrophoresis. Lane 3 shows recombinant Ten-m1sec which served as a positive control and size marker. The arrow indicates the position of a second band occasionally occurring in the recombinant Ten-m1sec lane (also faintly visible here), probably reflecting incompletely reduced Ten-m1sec dimers.

Mentions: To assess protein expression of Ten-m1, a specific antiserum was produced by immunizing rabbits with the recombinant extracellular domain of Ten-m1. The antiserum was further purified by affinity chromatography with covalently immobilized immunogen. The results obtained by the specific antibodies in Western blots depended strongly on the reducing agent in the sample buffer and the treatment of the samples (see Materials and Methods). After exhaustive reduction of detergent extracts from mouse brain with dithiothreitol, two major protein bands with apparent molecular masses of 270 and 225 kD were observed (Fig. 6, lane 1). Often also higher molecular mass proteins, probably representing incompletely reduced protein dimers, were recognized by the purified antibody (Fig. 6, lane 2), occasionally even after prolonged incubations in the reducing sample buffer. Whereas the 270-kD form might correspond to the complete Ten-m1 molecule, the 225-kD form might represent an alternatively spliced or proteolytically processed molecule. It is also possible that the 225-kD form is a soluble form of the molecule, since it migrated at a similar position as the recombinant extracellular domain of Ten-m1 (Fig. 6, lane 3). A clear pattern of protein bands recognized by the purified antiserum could only be observed in brain extracts, but not in extracts of other tissue such as thymus, lung, and kidney (not shown). A lower abundance of Ten-m1 protein in tissues other than brain would be in agreement with the results of the Northern blot assay.


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)

Western blot of brain extracts with affinity-purified antibodies against Ten-m1. 3% Triton  X-100 extracts of mouse brain  (lanes 1 and 2) were precipitated  with acetone. Dried precipitates  were dissolved in sample buffer, either for 30 min at 70°C (lane 1) or  at room temperature (lane 2). Both  samples were reduced for 5 min at  95°C before electrophoresis. Lane 3  shows recombinant Ten-m1sec  which served as a positive control  and size marker. The arrow indicates the position of a second band  occasionally occurring in the recombinant Ten-m1sec lane (also  faintly visible here), probably reflecting incompletely reduced Ten-m1sec dimers.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2185078&req=5

Figure 6: Western blot of brain extracts with affinity-purified antibodies against Ten-m1. 3% Triton X-100 extracts of mouse brain (lanes 1 and 2) were precipitated with acetone. Dried precipitates were dissolved in sample buffer, either for 30 min at 70°C (lane 1) or at room temperature (lane 2). Both samples were reduced for 5 min at 95°C before electrophoresis. Lane 3 shows recombinant Ten-m1sec which served as a positive control and size marker. The arrow indicates the position of a second band occasionally occurring in the recombinant Ten-m1sec lane (also faintly visible here), probably reflecting incompletely reduced Ten-m1sec dimers.
Mentions: To assess protein expression of Ten-m1, a specific antiserum was produced by immunizing rabbits with the recombinant extracellular domain of Ten-m1. The antiserum was further purified by affinity chromatography with covalently immobilized immunogen. The results obtained by the specific antibodies in Western blots depended strongly on the reducing agent in the sample buffer and the treatment of the samples (see Materials and Methods). After exhaustive reduction of detergent extracts from mouse brain with dithiothreitol, two major protein bands with apparent molecular masses of 270 and 225 kD were observed (Fig. 6, lane 1). Often also higher molecular mass proteins, probably representing incompletely reduced protein dimers, were recognized by the purified antibody (Fig. 6, lane 2), occasionally even after prolonged incubations in the reducing sample buffer. Whereas the 270-kD form might correspond to the complete Ten-m1 molecule, the 225-kD form might represent an alternatively spliced or proteolytically processed molecule. It is also possible that the 225-kD form is a soluble form of the molecule, since it migrated at a similar position as the recombinant extracellular domain of Ten-m1 (Fig. 6, lane 3). A clear pattern of protein bands recognized by the purified antiserum could only be observed in brain extracts, but not in extracts of other tissue such as thymus, lung, and kidney (not shown). A lower abundance of Ten-m1 protein in tissues other than brain would be in agreement with the results of the Northern blot assay.

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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