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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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Axonin-1 binding  to wild-type NgCAM and  the domain deletion mutants  ΔIg56, ΔIg6Fn1, ΔFn12, ΔIg56  Fn1, ΔIg6Fn12, and ΔIg56Fn12.  (A) Analysis of the molecular masses of the NgCAM  domain deletion mutants  ΔIg56Fn1, ΔIg6Fn12, and  ΔIg56Fn12 transiently expressed in COS7 cells. Western blot analysis was performed as described in Fig. 1,  using a polyclonal anti-NgCAM antibody followed  by enhanced chemiluminescence for detection. Molecular masses are indicated. (B)  Binding of axonin-conjugated TRITC Covaspheres to  COS cells expressing wild-type NgCAM and the domain deletion mutants ΔIg56,  ΔIg6Fn1, ΔFn12, ΔIg56Fn1,  ΔIg6Fn12, and ΔIg56Fn12.  The detection of NgCAM  expressing cells and the  quantification was carried  out as described in Fig. 2.  Each column corresponds to  three independent experiments (± SD).
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Figure 3: Axonin-1 binding to wild-type NgCAM and the domain deletion mutants ΔIg56, ΔIg6Fn1, ΔFn12, ΔIg56 Fn1, ΔIg6Fn12, and ΔIg56Fn12. (A) Analysis of the molecular masses of the NgCAM domain deletion mutants ΔIg56Fn1, ΔIg6Fn12, and ΔIg56Fn12 transiently expressed in COS7 cells. Western blot analysis was performed as described in Fig. 1, using a polyclonal anti-NgCAM antibody followed by enhanced chemiluminescence for detection. Molecular masses are indicated. (B) Binding of axonin-conjugated TRITC Covaspheres to COS cells expressing wild-type NgCAM and the domain deletion mutants ΔIg56, ΔIg6Fn1, ΔFn12, ΔIg56Fn1, ΔIg6Fn12, and ΔIg56Fn12. The detection of NgCAM expressing cells and the quantification was carried out as described in Fig. 2. Each column corresponds to three independent experiments (± SD).

Mentions: The axonin-1 binding properties of ΔIg5, ΔIg6, and ΔFn12 show clearly that the domains Ig5, Ig6, Fn1, and Fn2, which link the NgCAM domains Ig2-4 and Fn3, are not essential for axonin-1 binding. However, a marked reduction of axonin-1 binding was observed with the mutant ΔIg56 (Fig. 3 B). Because the deletions of Ig5 and Ig6 individually do not reduce axonin-1 binding, the possibility of an involvement of Ig5 or Ig6 in the axonin-1–NgCAM interaction must be excluded (Fig. 2 B). The effect of the deletion of the domain tandem Ig5+Ig6 cannot be ascribed to a reduced spacing between Ig2-4 and Fn3, because no comparable effect was observed with the deletion mutants ΔIg6Fn1 and ΔFn12. The mutants ΔIg56Fn1, ΔIg6Fn12, which lack three domains, and ΔIg56Fn12, which lacks all four domains between Ig2-4 and Fn3 exhibit axonin-1 binding comparable to ΔIg56 (Fig. 3 B). This indicates that the domains Ig5-Fn2 do not participate directly in axonin-1 binding but rather provide the conformational flexibility required for an optimal arrangement of Ig2-4 and Fn3 with respect to the corresponding binding areas on axonin-1.


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)

Axonin-1 binding  to wild-type NgCAM and  the domain deletion mutants  ΔIg56, ΔIg6Fn1, ΔFn12, ΔIg56  Fn1, ΔIg6Fn12, and ΔIg56Fn12.  (A) Analysis of the molecular masses of the NgCAM  domain deletion mutants  ΔIg56Fn1, ΔIg6Fn12, and  ΔIg56Fn12 transiently expressed in COS7 cells. Western blot analysis was performed as described in Fig. 1,  using a polyclonal anti-NgCAM antibody followed  by enhanced chemiluminescence for detection. Molecular masses are indicated. (B)  Binding of axonin-conjugated TRITC Covaspheres to  COS cells expressing wild-type NgCAM and the domain deletion mutants ΔIg56,  ΔIg6Fn1, ΔFn12, ΔIg56Fn1,  ΔIg6Fn12, and ΔIg56Fn12.  The detection of NgCAM  expressing cells and the  quantification was carried  out as described in Fig. 2.  Each column corresponds to  three independent experiments (± SD).
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Figure 3: Axonin-1 binding to wild-type NgCAM and the domain deletion mutants ΔIg56, ΔIg6Fn1, ΔFn12, ΔIg56 Fn1, ΔIg6Fn12, and ΔIg56Fn12. (A) Analysis of the molecular masses of the NgCAM domain deletion mutants ΔIg56Fn1, ΔIg6Fn12, and ΔIg56Fn12 transiently expressed in COS7 cells. Western blot analysis was performed as described in Fig. 1, using a polyclonal anti-NgCAM antibody followed by enhanced chemiluminescence for detection. Molecular masses are indicated. (B) Binding of axonin-conjugated TRITC Covaspheres to COS cells expressing wild-type NgCAM and the domain deletion mutants ΔIg56, ΔIg6Fn1, ΔFn12, ΔIg56Fn1, ΔIg6Fn12, and ΔIg56Fn12. The detection of NgCAM expressing cells and the quantification was carried out as described in Fig. 2. Each column corresponds to three independent experiments (± SD).
Mentions: The axonin-1 binding properties of ΔIg5, ΔIg6, and ΔFn12 show clearly that the domains Ig5, Ig6, Fn1, and Fn2, which link the NgCAM domains Ig2-4 and Fn3, are not essential for axonin-1 binding. However, a marked reduction of axonin-1 binding was observed with the mutant ΔIg56 (Fig. 3 B). Because the deletions of Ig5 and Ig6 individually do not reduce axonin-1 binding, the possibility of an involvement of Ig5 or Ig6 in the axonin-1–NgCAM interaction must be excluded (Fig. 2 B). The effect of the deletion of the domain tandem Ig5+Ig6 cannot be ascribed to a reduced spacing between Ig2-4 and Fn3, because no comparable effect was observed with the deletion mutants ΔIg6Fn1 and ΔFn12. The mutants ΔIg56Fn1, ΔIg6Fn12, which lack three domains, and ΔIg56Fn12, which lacks all four domains between Ig2-4 and Fn3 exhibit axonin-1 binding comparable to ΔIg56 (Fig. 3 B). This indicates that the domains Ig5-Fn2 do not participate directly in axonin-1 binding but rather provide the conformational flexibility required for an optimal arrangement of Ig2-4 and Fn3 with respect to the corresponding binding areas on axonin-1.

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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Related in: MedlinePlus