Limits...
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 to the NgCAM mutants NgCAM  PM1-3, NgCAM ΔP871-V888, and wild-type NgCAM. (A) Schematic representation of the point mutations introduced into positions R865, R864, and R862 of NgCAM PM1, NgCAM PM2, and  NgCAM PM3. Amino acid exchanges are underlined and the site  of cleavage marked by an arrow. The relative positions of the residues with respect to the cleavage site are indicated. (B) Western  blot analysis. The NgCAM mutants were transiently expressed in  COS7 cells, total protein separated by SDS-PAGE, transferred  to nitrocellulose and immunostained using a monoclonal anti-NgCAM antibody against the 135-kD fragment followed by enhanced chemiluminescence for detection. Molecular masses are  indicated. (C) Binding of axonin-conjugated TRITC Covaspheres to COS cells expressing wild-type NgCAM and the NgCAM mutants NgCAM PM1-3, NgCAM ΔP871-V888. The identification of NgCAM expressing cells and the quantification of  the binding of axonin-1 Covaspheres was carried out as described  in Fig. 2. Each column corresponds to three independent experiments (± SD).
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Figure 4: Binding of axonin-1 to the NgCAM mutants NgCAM PM1-3, NgCAM ΔP871-V888, and wild-type NgCAM. (A) Schematic representation of the point mutations introduced into positions R865, R864, and R862 of NgCAM PM1, NgCAM PM2, and NgCAM PM3. Amino acid exchanges are underlined and the site of cleavage marked by an arrow. The relative positions of the residues with respect to the cleavage site are indicated. (B) Western blot analysis. The NgCAM mutants were transiently expressed in COS7 cells, total protein separated by SDS-PAGE, transferred to nitrocellulose and immunostained using a monoclonal anti-NgCAM antibody against the 135-kD fragment followed by enhanced chemiluminescence for detection. Molecular masses are indicated. (C) Binding of axonin-conjugated TRITC Covaspheres to COS cells expressing wild-type NgCAM and the NgCAM mutants NgCAM PM1-3, NgCAM ΔP871-V888. The identification of NgCAM expressing cells and the quantification of the binding of axonin-1 Covaspheres was carried out as described in Fig. 2. Each column corresponds to three independent experiments (± SD).

Mentions: The cleavage site that is localized within the third FnIII domain is preceded by conserved Arg residues at positions −1 (R865), −2 (R864), and −4 (R862). This pattern of conserved residues resembles the recognition sequences for furin-type proteases (Burgoon et al., 1991; Hosaka et al., 1991; Grumet and Sakurai, 1996). To generate NgCAM variants deficient in proteolytic cleavage we introduced the following point mutations: NgCAM PM1, R865; NgCAM PM2, R865A and R864S; and NgCAM PM3, R865A, R864S, and R862A (Fig. 4 A). In another construct, the segment P871-V888 that is localized COOH terminally to the cleavage site was deleted. Sequence alignments revealed that this Pro-rich sequence occurs only in NgCAM but not in its putative mammalian homologue L1, which is found primarily in the uncleaved 200-kD form (Grumet et al., 1991). Based on comparisons of the sequences of NgCAM and L1 (Grumet et al., 1991) and considering the recently published hypothetical outline structure of Fn3 of mammalian L1 (Bateman et al., 1996), we expected the deletion of the sequence P871-V888 to have an effect on proteolytic cleavage without extensive changes in the tertiary structure of Fn3.


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 to the NgCAM mutants NgCAM  PM1-3, NgCAM ΔP871-V888, and wild-type NgCAM. (A) Schematic representation of the point mutations introduced into positions R865, R864, and R862 of NgCAM PM1, NgCAM PM2, and  NgCAM PM3. Amino acid exchanges are underlined and the site  of cleavage marked by an arrow. The relative positions of the residues with respect to the cleavage site are indicated. (B) Western  blot analysis. The NgCAM mutants were transiently expressed in  COS7 cells, total protein separated by SDS-PAGE, transferred  to nitrocellulose and immunostained using a monoclonal anti-NgCAM antibody against the 135-kD fragment followed by enhanced chemiluminescence for detection. Molecular masses are  indicated. (C) Binding of axonin-conjugated TRITC Covaspheres to COS cells expressing wild-type NgCAM and the NgCAM mutants NgCAM PM1-3, NgCAM ΔP871-V888. The identification of NgCAM expressing cells and the quantification of  the binding of axonin-1 Covaspheres was carried out as described  in Fig. 2. Each column corresponds to three independent experiments (± SD).
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Figure 4: Binding of axonin-1 to the NgCAM mutants NgCAM PM1-3, NgCAM ΔP871-V888, and wild-type NgCAM. (A) Schematic representation of the point mutations introduced into positions R865, R864, and R862 of NgCAM PM1, NgCAM PM2, and NgCAM PM3. Amino acid exchanges are underlined and the site of cleavage marked by an arrow. The relative positions of the residues with respect to the cleavage site are indicated. (B) Western blot analysis. The NgCAM mutants were transiently expressed in COS7 cells, total protein separated by SDS-PAGE, transferred to nitrocellulose and immunostained using a monoclonal anti-NgCAM antibody against the 135-kD fragment followed by enhanced chemiluminescence for detection. Molecular masses are indicated. (C) Binding of axonin-conjugated TRITC Covaspheres to COS cells expressing wild-type NgCAM and the NgCAM mutants NgCAM PM1-3, NgCAM ΔP871-V888. The identification of NgCAM expressing cells and the quantification of the binding of axonin-1 Covaspheres was carried out as described in Fig. 2. Each column corresponds to three independent experiments (± SD).
Mentions: The cleavage site that is localized within the third FnIII domain is preceded by conserved Arg residues at positions −1 (R865), −2 (R864), and −4 (R862). This pattern of conserved residues resembles the recognition sequences for furin-type proteases (Burgoon et al., 1991; Hosaka et al., 1991; Grumet and Sakurai, 1996). To generate NgCAM variants deficient in proteolytic cleavage we introduced the following point mutations: NgCAM PM1, R865; NgCAM PM2, R865A and R864S; and NgCAM PM3, R865A, R864S, and R862A (Fig. 4 A). In another construct, the segment P871-V888 that is localized COOH terminally to the cleavage site was deleted. Sequence alignments revealed that this Pro-rich sequence occurs only in NgCAM but not in its putative mammalian homologue L1, which is found primarily in the uncleaved 200-kD form (Grumet et al., 1991). Based on comparisons of the sequences of NgCAM and L1 (Grumet et al., 1991) and considering the recently published hypothetical outline structure of Fn3 of mammalian L1 (Bateman et al., 1996), we expected the deletion of the sequence P871-V888 to have an effect on proteolytic cleavage without extensive changes in the tertiary structure of Fn3.

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