RESUMO
Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.
Assuntos
Proteínas de Anfíbios/metabolismo , Proteínas de Transporte/metabolismo , Proliferação de Células/fisiologia , Células-Tronco Mesenquimais/metabolismo , Salamandridae/metabolismo , Sequência de Aminoácidos , Proteínas de Anfíbios/classificação , Proteínas de Anfíbios/genética , Animais , Western Blotting , Células COS , Proteínas de Transporte/genética , Proliferação de Células/genética , Células Cultivadas , Chlorocebus aethiops , Cisteína/genética , Cisteína/metabolismo , Células HEK293 , Humanos , Mutação , Filogenia , Ligação Proteica , Fase S/genética , Fase S/fisiologia , Salamandridae/genética , Homologia de Sequência de AminoácidosRESUMO
We show that after tail amputation in Ambystoma mexicanum (Axolotl) the correct number and spacing of dorsal root ganglia are regenerated. By transplantation of spinal cord tissue and nonclonal neurospheres, we show that the central spinal cord represents a source of peripheral nervous system cells. Interestingly, melanophores migrate from preexisting precursors in the skin. Finally, we demonstrate that implantation of a clonally derived spinal cord neurosphere can result in reconstitution of all examined cell types in the regenerating central spinal cord, suggesting derivation of a cell with spinal cord stem cell properties.