Abstract
Our work centers on understanding how the extracellular matrix molecule tenascin-C regulates neuronal growth. We have found that the region of tenascin-C containing only alternately spliced fibronectin type-III repeat D, called fnD, when used by itself, dramatically increases neurite outgrowth in culture. We used overlapping synthetic peptides to localize the neurite outgrowth-promoting site within fnD to a 15 amino acid sequence, called D5. An antibody against D5 blocked promotion of neurite outgrowth by fnD as well as tenascin-C, indicating that this peptide sequence is functional in the context of the native molecule. Further testing of shorter synthetic peptides restricted the neurite outgrowth-promoting site to eight amino acids, VFDNFVLK. Of these, "FD" and "FV" are conserved in tenascin-C sequences derived from all the species available in the GenBank. To investigate the hypothesis that FD and FV are critical for the interaction with neurons, we tested a recombinant fnD protein and synthetic peptides with alterations in FD and/or FV. These molecules did not facilitate process extension, suggesting that the conserved amino acids are required for formation of the active site in fnD. We next investigated whether VFDNFVLK could be used as a reagent to overcome the neurite outgrowth inhibitory properties of chondroitin sulfate proteoglycans, the major inhibitory molecules in the glial scar. The peptide significantly enhanced outgrowth on proteoglycans and was more effective than laminin-1, L1-Fc, or intact tenascin-C, thus demonstrating the potential applicability of tenascin-C regions as therapeutic reagents.
Highlights
Our work centers on understanding how the extracellular matrix molecule tenascin-C regulates neuronal growth
We have demonstrated previously that fnA-D can promote both neurite outgrowth and axonal guidance through different parts of the molecule: the fibronectin type III (FN-III) repeat C appears to be essential for promotion of neurite guidance (Meiners et al, 1999a), whereas fnD is responsible for promotion of neurite
A short linear amino acid sequence in fnD promotes neurite outgrowth We investigated the hypothesis that a short linear amino acid sequence is responsible for neurite outgrowth promotion by fnD, much as the sequence SIKVAV is responsible for neurite outgrowth promotion by the ␣1 helical domain of laminin-1 (Sephel et al, 1989)
Summary
Our work centers on understanding how the extracellular matrix molecule tenascin-C regulates neuronal growth. To investigate the hypothesis that FD and FV are critical for the interaction with neurons, we tested a recombinant fnD protein and synthetic peptides with alterations in FD and/or FV These molecules did not facilitate process extension, suggesting that the conserved amino acids are required for formation of the active site in fnD. Further experiments supported the hypothesis that this sequence is functional in native tenascin-C and that the amino acids “FD” and “FV,” which are conserved in tenascin-C derived from all vertebrate species, are essential for the formation of the neurite outgrowth-promoting motif in fnD. This is the most precise localization of a neurite outgrowth regulatory region within tenascin-C. CSPGs are upregulated in the glial scar and are strongly repellant to growing axons (McKeon et al, 1991; Pindzola et al, 1993; Zuo et al, 1998; Fernaud-Espinosa et al, 1998)
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