Use of chimeric F3-NCAM molecules to explore the properties of VASE exon in modulating polysialylation and neurite outgrowth.
Differential splicing of VASE exon in the fourth immunoglobulin (Ig) domain and attachment to the fifth Ig domain of alpha 2-8 linked sialic acid (PSA) both dramatically change, in opposite manner, Neural Cell Adhesion Molecule (NCAM) functional properties. Reciprocal patterns of VASE and PSA expression suggest that they might be mutually exclusive. Here, we tested whether informations conferring polysialylation reside in NCAM-Ig domains 4 and 5 and the influence of the VASE exon encoded sequence on this process. We also examined if the VASE sequence was still able to inhibit neurite outgrowth when presented out of its normal NCAM context. Constructs have been prepared encoding NCAM-Ig domains 4 (with or without the VASE exon) and 5 fused to the F3 molecule. Stable clones expressing the chimeric molecules or wild type F3 were then obtained in the AtT-20 cell line. Although the chimeric molecules were expressed on the cell surface none of them was bearing PSA. Thus, polysialylation cannot be conferred to proteins by addition of the NCAM-Ig domains 4 and 5 modular motif and in this molecular context, the VASE sequence is not influencing the process. These chimeric molecules, either expressed at the surface of RIN or COS cells or presented as soluble forms, were examined for their effect on neurite outgrowth. In all cases, the length of neurites of sensory neurons was significantly reduced when grown in presence of the VASE containing chimera by comparison with the chimera without VASE or wild type F3. When neurons from NCAM knock-out mice were used for the assay, the VASE inhibition could not be detected. Thus VASE is able to act as a modular motif and NCAM expressed on neurons participates in transducing its effect.
Arce
V
V
Laboratoire de Génétique et Physiologie du Développement, UMR 9943 CNRS, Université Aix-Marseille II, France.
Gristina
R
R
Buttiglione
M
M
Cremer
H
H
Gennarini
G
G
Rougon
G
G
D000818:Animals; D019556:COS Cells; D015816:Cell Adhesion Molecules, Neuronal / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D058966:Contactins; D005091:Exons / Q000502:physiology*; D005727:Ganglia, Spinal / Q000166:cytology; D007136:Immunoglobulins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D019006:Neural Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D009475:Neurons, Afferent / Q000166:cytology; D009842:Oligopeptides / Q000235:genetics* / Q000494:pharmacology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis / Q000378:metabolism*
D015816:Cell Adhesion Molecules, Neuronal; D058966:Contactins; D007136:Immunoglobulins; D019006:Neural Cell Adhesion Molecules; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D012794:Sialic Acids; C086869:VASE peptide; C021319:polysialic acid; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase