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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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Analysis of the molecular masses of wild-type NgCAM and NgCAM domain deletion mutants transiently expressed in COS7  cells. Total protein of nontransfected COS cells (COS) and COS cells transfected with wild-type NgCAM (wild-type) and the and NgCAM single (A) and double (B) domain deletion mutants was separated by SDS-PAGE, transferred to nitrocellulose and immunostained using a polyclonal anti-NgCAM antibody followed by enhanced chemiluminescence detection. Molecular masses are indicated.
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Figure 1: Analysis of the molecular masses of wild-type NgCAM and NgCAM domain deletion mutants transiently expressed in COS7 cells. Total protein of nontransfected COS cells (COS) and COS cells transfected with wild-type NgCAM (wild-type) and the and NgCAM single (A) and double (B) domain deletion mutants was separated by SDS-PAGE, transferred to nitrocellulose and immunostained using a polyclonal anti-NgCAM antibody followed by enhanced chemiluminescence detection. Molecular masses are indicated.

Mentions: Entire domains of NgCAM, as defined by their homology to the Ig domains or the type-III domains of fibronectin (Grumet et al., 1991; Buchstaller et al., 1996), were deleted. Two different sets of deletion mutants were generated, a complete set of single domain deletions and a complete set of double deletions of adjacent domains. This strategy was based on the observation that in many IgFnIII class molecules some of the folding units comprise two adjacent domains (Huber et al., 1994; Vaughn and Bjorkman, 1995). The NgCAM mutants were cloned into the NgCAM expression vector pSCTNgCAM. Transfection of COS7 cells by electroporation resulted in 30–40% of the cells expressing heterologous protein. The cells were solubilized 48 h after transfection and analyzed by immunoblotting using a polyclonal anti-NgCAM antibody. The molecular masses of the NgCAM domain deletion mutants were in agreement with the expected values (Fig. 1). In accordance with the localization of the natural proteolytic cleavage site of NgCAM in the third FnIII domain (Burgoon et al., 1991), proteolytic cleavage was absent in all mutants lacking the third FnIII domain. All mutants, except ΔFn45, were expressed in similar quantities at the cell surface, as revealed by immunofluorescence analysis. 2 d after electroporation the cells were incubated with axonin-1–conjugated polystyrene microspheres (Covaspheres). After washing and fixation the NgCAM-expressing cells were identified by immunostaining. The deletion of each of the Ig domains Ig2-4 resulted in an almost complete loss of axonin-1 binding. Deletion of Fn2 and Fn3 resulted in strong reduction of axonin-1 binding, deletion of Fn4 in a much weaker, but still significant, reduction (Fig. 2 B). The axonin-1 binding capacities of the mutants with deleted Ig2, Ig3, Ig4, and Fn3, were in accordance with the data from the double deletion mutants lacking Ig1+2, Ig2+3, Ig3+4, Ig4+5, and the membrane proximal domain tandems Fn2+3 and Fn3+4 (data not shown). In contrast, the lack of axonin-1 binding by ΔFn2 contradicts the observation that axonin-1 binding is unaffected by the deletion of Fn1 and Fn2 in ΔFn12 (Fig. 2 B). In consideration of the latter finding, a direct involvement of Fn2 in the binding to axonin-1 must be excluded. Interestingly, x-ray crystallographic analysis of the domain-tandem Fn1+2 of neuroglian, the homologue of NgCAM in Drosophila melanogaster, revealed an extended hydrophobic contact area between the two domains, which is conserved in the vertebrate isologues (Huber et al., 1994). The deletion of Fn2 places Fn1 adjacent to the putative binding domain Fn3. It is conceivable that, in the absence of the cognate area on Fn2, the hydrophobic contact area of Fn1 prevents a correct folding of Fn3. Thus, we conclude that the Ig domains 2-4 and the third FnIII domain of NgCAM are essential for axonin-1 binding. The deletion of each of these domains resulted in a loss of binding. This indicates that Ig2-4 and Fn3 do not represent two alternative binding sites, but must engage cooperatively in a direct or indirect manner in order to establish a binding of 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)

Analysis of the molecular masses of wild-type NgCAM and NgCAM domain deletion mutants transiently expressed in COS7  cells. Total protein of nontransfected COS cells (COS) and COS cells transfected with wild-type NgCAM (wild-type) and the and NgCAM single (A) and double (B) domain deletion mutants was separated by SDS-PAGE, transferred to nitrocellulose and immunostained using a polyclonal anti-NgCAM antibody followed by enhanced chemiluminescence detection. Molecular masses are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132982&req=5

Figure 1: Analysis of the molecular masses of wild-type NgCAM and NgCAM domain deletion mutants transiently expressed in COS7 cells. Total protein of nontransfected COS cells (COS) and COS cells transfected with wild-type NgCAM (wild-type) and the and NgCAM single (A) and double (B) domain deletion mutants was separated by SDS-PAGE, transferred to nitrocellulose and immunostained using a polyclonal anti-NgCAM antibody followed by enhanced chemiluminescence detection. Molecular masses are indicated.
Mentions: Entire domains of NgCAM, as defined by their homology to the Ig domains or the type-III domains of fibronectin (Grumet et al., 1991; Buchstaller et al., 1996), were deleted. Two different sets of deletion mutants were generated, a complete set of single domain deletions and a complete set of double deletions of adjacent domains. This strategy was based on the observation that in many IgFnIII class molecules some of the folding units comprise two adjacent domains (Huber et al., 1994; Vaughn and Bjorkman, 1995). The NgCAM mutants were cloned into the NgCAM expression vector pSCTNgCAM. Transfection of COS7 cells by electroporation resulted in 30–40% of the cells expressing heterologous protein. The cells were solubilized 48 h after transfection and analyzed by immunoblotting using a polyclonal anti-NgCAM antibody. The molecular masses of the NgCAM domain deletion mutants were in agreement with the expected values (Fig. 1). In accordance with the localization of the natural proteolytic cleavage site of NgCAM in the third FnIII domain (Burgoon et al., 1991), proteolytic cleavage was absent in all mutants lacking the third FnIII domain. All mutants, except ΔFn45, were expressed in similar quantities at the cell surface, as revealed by immunofluorescence analysis. 2 d after electroporation the cells were incubated with axonin-1–conjugated polystyrene microspheres (Covaspheres). After washing and fixation the NgCAM-expressing cells were identified by immunostaining. The deletion of each of the Ig domains Ig2-4 resulted in an almost complete loss of axonin-1 binding. Deletion of Fn2 and Fn3 resulted in strong reduction of axonin-1 binding, deletion of Fn4 in a much weaker, but still significant, reduction (Fig. 2 B). The axonin-1 binding capacities of the mutants with deleted Ig2, Ig3, Ig4, and Fn3, were in accordance with the data from the double deletion mutants lacking Ig1+2, Ig2+3, Ig3+4, Ig4+5, and the membrane proximal domain tandems Fn2+3 and Fn3+4 (data not shown). In contrast, the lack of axonin-1 binding by ΔFn2 contradicts the observation that axonin-1 binding is unaffected by the deletion of Fn1 and Fn2 in ΔFn12 (Fig. 2 B). In consideration of the latter finding, a direct involvement of Fn2 in the binding to axonin-1 must be excluded. Interestingly, x-ray crystallographic analysis of the domain-tandem Fn1+2 of neuroglian, the homologue of NgCAM in Drosophila melanogaster, revealed an extended hydrophobic contact area between the two domains, which is conserved in the vertebrate isologues (Huber et al., 1994). The deletion of Fn2 places Fn1 adjacent to the putative binding domain Fn3. It is conceivable that, in the absence of the cognate area on Fn2, the hydrophobic contact area of Fn1 prevents a correct folding of Fn3. Thus, we conclude that the Ig domains 2-4 and the third FnIII domain of NgCAM are essential for axonin-1 binding. The deletion of each of these domains resulted in a loss of binding. This indicates that Ig2-4 and Fn3 do not represent two alternative binding sites, but must engage cooperatively in a direct or indirect manner in order to establish a binding of 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.

Show MeSH
Related in: MedlinePlus