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Zusammenfassung:
Axon guidance cues are crucial signals for neurons to build complex networks during early developmental stages. Netrin is one of the main guidance cues that in most cases attracts neurons to their destinations. Distinct Netrins and Netrin receptors have been identified to carry out the guidance function. However, no direct Netrin modulator has been described yet. In this study, we used AVEXIS (Avidity based Extracellular Interaction Screen), a large-scale protein-protein interaction screen assay, to identify novel binding partners for Netrin proteins, and zebrafish as model organism to study the function of the newly identified interactions. We found Draxin, a secreted axon guidance protein, to directly interact with Netrin. The novel interaction is conserved for the orthologous human proteins. Furthermore, Draxin is able to outcompete Netrin receptors for Netrin-1 binding in a biochemical competition assay. The binding of Netrin receptors to Netrin-1 is reduced or even abolished in the presence of Draxin, indicating an inhibitory function of Draxin by breaking the communication between Netrin and its receptors. By generating Netrin truncations, I narrowed down the interaction interface to the third EGF domain of Netrin-1, the domain described to be necessary for Netrin-Netrin receptor interactions. I further narrowed down the interaction interface to a highly conserved 22 amino acid fragment within Draxin. This Draxin 22 amino acid fragment is sufficient for binding to Netrin-1 and the protein sequence is conserved across vertebrate species including chick, mouse and human. Furthermore, the 22 amino acid peptide fused to the Fc protein (the constant region of an immunoglobulin molecule) is able to outcompete Netrin receptors for Netrin binding. Expression analysis in zebrafish embryos shows that both draxin and netrin mRNAs are expressed in the neural tube. They are co-expressed in regions surrounding the forebrain commissures. In the spinal cord, draxin is mainly expressed dorsally whereas netrin is located ventrally. Since both Draxin and Netrin are secreted proteins and have been reported to act as long-range guidance cues, the expression patterns suggests that reciprocal gradients of active Draxin and Netrin proteins can form along the dorsal-ventral axis of the developing spinal cord to regulate the correct formation of spinal cord commissures. Two independent embryonic binding assays revealed that Draxin and Netrin are able to interact in zebrafish embryos. Thus, we propose a model in which Draxin functions as a secreted Netrin signaling modulator influencing vertebrate axon pathfinding. Draxin might shape the functional extracellular Netrin gradient by sequestering Netrin proteins. Since human Netrin-1 serves as a survival factor for specific tumors, Draxin—or the Netrin binding fragment of Draxin—could be potentially used to activate cell death in human cancer cells.
