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Neurite fasciculation mediated by complexes of axonin-1 and Ng cell adhesion molecule.

Kunz S, Spirig M, Ginsburg C, Buchstaller A, Berger P, Lanz R, Rader C, Vogt L, Kunz B, Sonderegger P - J. Cell Biol. (1998)

Bottom Line: In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding.These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM.The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.

ABSTRACT
Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

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Binding of axonin-1 Ig1234-Cκ and axonin-1 ΔIg1234-Cκ  to wild-type NgCAM and the two series of domain deletion mutants. (A) Schematic representation of the soluble axonin-1-fragments fused to mouse Ig Cκ domain (filled bars). As in Fig. 2, Ig  domains are represented by half-circles and FnIII-like domains  by empty bars. The molecules were expressed in stably transfected myeloma cells and purified from the supernatants by  immunoaffinity-chromatography using an anti-mouse Ig Cκ monoclonal antibody. (B) Purified proteins were subjected to SDS-PAGE and visualized by silver staining. Lane 1, axonin-1 Ig1234-Cκ;  lane 2, axonin-1 ΔIg1234-Cκ; lane 3, native axonin-1 purified  from the vitreous fluid of E14 chicken embryos; lane Mr represents the molecular mass standard. (C) The soluble fusion proteins axonin-1 Ig1234-Cκ and axonin-1 ΔIg1234-Cκ were coupled  to TRITC-conjugated Covaspheres and tested for binding to the  NgCAM mutants expressed on COS cells. The experiment was carried out as described in Fig. 2. Again, green fluorescing cells that  bound four or more red fluorescing Covaspheres were scored as  positive cells. Binding of Ig1234-Cκ is represented by the empty  bars, ΔIg1234-Cκ by the filled bars. Cells that did not express NgCAM were used as internal negative controls and were found to  bind on the average 0.1 Covaspheres per cell for Ig1234-Cκ and  0.5 Covaspheres per cell in case of ΔIg1234-Cκ. Each column corresponds to four independent experiments (± SD).
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Figure 5: Binding of axonin-1 Ig1234-Cκ and axonin-1 ΔIg1234-Cκ to wild-type NgCAM and the two series of domain deletion mutants. (A) Schematic representation of the soluble axonin-1-fragments fused to mouse Ig Cκ domain (filled bars). As in Fig. 2, Ig domains are represented by half-circles and FnIII-like domains by empty bars. The molecules were expressed in stably transfected myeloma cells and purified from the supernatants by immunoaffinity-chromatography using an anti-mouse Ig Cκ monoclonal antibody. (B) Purified proteins were subjected to SDS-PAGE and visualized by silver staining. Lane 1, axonin-1 Ig1234-Cκ; lane 2, axonin-1 ΔIg1234-Cκ; lane 3, native axonin-1 purified from the vitreous fluid of E14 chicken embryos; lane Mr represents the molecular mass standard. (C) The soluble fusion proteins axonin-1 Ig1234-Cκ and axonin-1 ΔIg1234-Cκ were coupled to TRITC-conjugated Covaspheres and tested for binding to the NgCAM mutants expressed on COS cells. The experiment was carried out as described in Fig. 2. Again, green fluorescing cells that bound four or more red fluorescing Covaspheres were scored as positive cells. Binding of Ig1234-Cκ is represented by the empty bars, ΔIg1234-Cκ by the filled bars. Cells that did not express NgCAM were used as internal negative controls and were found to bind on the average 0.1 Covaspheres per cell for Ig1234-Cκ and 0.5 Covaspheres per cell in case of ΔIg1234-Cκ. Each column corresponds to four independent experiments (± SD).

Mentions: Because the four NH2-terminal Ig domains of axonin-1 were previously found to form a domain conglomerate that is necessary and sufficient for NgCAM binding (Rader et al., 1996), we also tested the binding of the domain deletion mutants of NgCAM with two truncated forms of axonin-1, axonin-1 Ig1234-Cκ, and axonin-1 ΔIg1234-Cκ. The soluble axonin-1 fragments were produced as fusion proteins with the mouse Cκ domain, as described by Rader et al. (1996), coupled to fluorescent microspheres, and analyzed for their NgCAM binding as described for wild-type axonin-1 (Fig. 5). The binding pattern of axonin-1 Ig1234-Cκ to the NgCAM mutants (Fig. 5 C) was very similar to that of wild type axonin-1 (Fig. 2). In contrast, the complementary axonin-1 fragment axonin-1 ΔIg1234-Cκ did not show any binding to the NgCAM mutants (Fig. 5 C). This result provides experimental evidence that the binding of axonin-1 to both, Ig2-4 and Fn3 of NgCAM is mediated by the four NH2-terminal Ig domains of axonin-1. The loss of binding of axonin-1 Ig1234-Cκ to NgCAM by deletion of one of the domains Ig2-4 and Fn3 indicates that the domain conglomerate formed by the four NH2-terminal Ig domains of axonin-1 must interact simultaneously with Ig2-4 and Fn3 of NgCAM.


Neurite fasciculation mediated by complexes of axonin-1 and Ng cell adhesion molecule.

Kunz S, Spirig M, Ginsburg C, Buchstaller A, Berger P, Lanz R, Rader C, Vogt L, Kunz B, Sonderegger P - J. Cell Biol. (1998)

Binding of axonin-1 Ig1234-Cκ and axonin-1 ΔIg1234-Cκ  to wild-type NgCAM and the two series of domain deletion mutants. (A) Schematic representation of the soluble axonin-1-fragments fused to mouse Ig Cκ domain (filled bars). As in Fig. 2, Ig  domains are represented by half-circles and FnIII-like domains  by empty bars. The molecules were expressed in stably transfected myeloma cells and purified from the supernatants by  immunoaffinity-chromatography using an anti-mouse Ig Cκ monoclonal antibody. (B) Purified proteins were subjected to SDS-PAGE and visualized by silver staining. Lane 1, axonin-1 Ig1234-Cκ;  lane 2, axonin-1 ΔIg1234-Cκ; lane 3, native axonin-1 purified  from the vitreous fluid of E14 chicken embryos; lane Mr represents the molecular mass standard. (C) The soluble fusion proteins axonin-1 Ig1234-Cκ and axonin-1 ΔIg1234-Cκ were coupled  to TRITC-conjugated Covaspheres and tested for binding to the  NgCAM mutants expressed on COS cells. The experiment was carried out as described in Fig. 2. Again, green fluorescing cells that  bound four or more red fluorescing Covaspheres were scored as  positive cells. Binding of Ig1234-Cκ is represented by the empty  bars, ΔIg1234-Cκ by the filled bars. Cells that did not express NgCAM were used as internal negative controls and were found to  bind on the average 0.1 Covaspheres per cell for Ig1234-Cκ and  0.5 Covaspheres per cell in case of ΔIg1234-Cκ. Each column corresponds to four independent experiments (± SD).
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Figure 5: Binding of axonin-1 Ig1234-Cκ and axonin-1 ΔIg1234-Cκ to wild-type NgCAM and the two series of domain deletion mutants. (A) Schematic representation of the soluble axonin-1-fragments fused to mouse Ig Cκ domain (filled bars). As in Fig. 2, Ig domains are represented by half-circles and FnIII-like domains by empty bars. The molecules were expressed in stably transfected myeloma cells and purified from the supernatants by immunoaffinity-chromatography using an anti-mouse Ig Cκ monoclonal antibody. (B) Purified proteins were subjected to SDS-PAGE and visualized by silver staining. Lane 1, axonin-1 Ig1234-Cκ; lane 2, axonin-1 ΔIg1234-Cκ; lane 3, native axonin-1 purified from the vitreous fluid of E14 chicken embryos; lane Mr represents the molecular mass standard. (C) The soluble fusion proteins axonin-1 Ig1234-Cκ and axonin-1 ΔIg1234-Cκ were coupled to TRITC-conjugated Covaspheres and tested for binding to the NgCAM mutants expressed on COS cells. The experiment was carried out as described in Fig. 2. Again, green fluorescing cells that bound four or more red fluorescing Covaspheres were scored as positive cells. Binding of Ig1234-Cκ is represented by the empty bars, ΔIg1234-Cκ by the filled bars. Cells that did not express NgCAM were used as internal negative controls and were found to bind on the average 0.1 Covaspheres per cell for Ig1234-Cκ and 0.5 Covaspheres per cell in case of ΔIg1234-Cκ. Each column corresponds to four independent experiments (± SD).
Mentions: Because the four NH2-terminal Ig domains of axonin-1 were previously found to form a domain conglomerate that is necessary and sufficient for NgCAM binding (Rader et al., 1996), we also tested the binding of the domain deletion mutants of NgCAM with two truncated forms of axonin-1, axonin-1 Ig1234-Cκ, and axonin-1 ΔIg1234-Cκ. The soluble axonin-1 fragments were produced as fusion proteins with the mouse Cκ domain, as described by Rader et al. (1996), coupled to fluorescent microspheres, and analyzed for their NgCAM binding as described for wild-type axonin-1 (Fig. 5). The binding pattern of axonin-1 Ig1234-Cκ to the NgCAM mutants (Fig. 5 C) was very similar to that of wild type axonin-1 (Fig. 2). In contrast, the complementary axonin-1 fragment axonin-1 ΔIg1234-Cκ did not show any binding to the NgCAM mutants (Fig. 5 C). This result provides experimental evidence that the binding of axonin-1 to both, Ig2-4 and Fn3 of NgCAM is mediated by the four NH2-terminal Ig domains of axonin-1. The loss of binding of axonin-1 Ig1234-Cκ to NgCAM by deletion of one of the domains Ig2-4 and Fn3 indicates that the domain conglomerate formed by the four NH2-terminal Ig domains of axonin-1 must interact simultaneously with Ig2-4 and Fn3 of NgCAM.

Bottom Line: In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding.These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM.The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.

ABSTRACT
Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

Show MeSH
Related in: MedlinePlus