Eph Receptor Clustering by Chondroitin Sulfate Inhibits Axon Regeneration

Abstract

Chondroitin sulfate proteoglycans (CSPGs) play important roles in the developing and mature nervous system, where they guide axons, maintain stable connections, restrict synaptic plasticity, and prevent axon regeneration following CNS injury. The chondroitin sulfate glycosaminoglycan (CS GAG) chains that decorate CSPGs are essential for their functions. Through these sugar chains, CSPGs are able to bind and regulate the activity of a diverse range of proteins and through these interactions can regulate neuronal growth. These CS-protein interactions depend on specific sulfation patterns within the CS GAG chains, and accordingly, particular CS sulfation motifs are upregulated during development, in the mature nervous system, and in response to CNS injury. Thus, spatiotemporal regulation of CS GAG biosynthesis may provide an important mechanism to control the functions of CSPGs and modulate intracellular signaling pathways. Here, we will discuss these sulfation-dependent processes and highlight how the CS sugars on CSPGs contribute to neuronal growth, axon guidance, and plasticity in the nervous system. Chondroitin sulfate proteoglycans (CSPGs) are a major barrier to regenerating axons in the central nervous system (CNS), exerting their inhibitory effect through their polysaccharide side chains. Chondroitin sulfate (CS) potently inhibits axon regeneration through modulation of inhibitory signaling pathways induced by carbohydrate binding to protein ligands and receptors. Here, we identify a novel carbohydrate-protein interaction between CS and EphA4 that inhibits axon regrowth. We characterize the mechanism of activation and demonstrate how carbohydrate binding induces phosphorylation of the intracellular kinase domain through clustering of cell surface EphA4. Collectively, our studies present a novel mechanism of EphA4 activation by CS independent of the canonical ephrin ligands and uncover the role of this interaction in inhibition of neurite regrowth after injury. Our results underscore a mechanism of action by which carbohydrates can function as direct, activating ligands for protein receptors and provide mechanistic insights into the inhibition of axon growth by CS following injury to the CNS. Chondroitin sulfate proteoglycans (CSPGs) regulate neuronal plasticity, as well as axon regeneration and guidance through their ability to bind protein ligands and cell surface receptors. In this way, extracellular CSPGs can modulate the activity of intracellular signaling pathways. Here, a computational analysis of EphA4-CS interactions is performed to characterize the importance of key arginine and lysine residues towards CS binding, and to identify structural differences in CS-A, CS-C, CS-D, and CS-E docking to EphA4. Carbohydrate-induced Eph receptor clustering could be a general mechanism of Eph receptor activation. To identify additional Eph receptors that interact with CS, CS-E was docked to all EphA and EphB family members to predict relative binding affinities. The relative strengths of the predicted binding energies are: EphB4 > EphA8 > EphA1 > EphA3 > EphB1 > EphB3 > EphA7 > EphA5 > EphA4 > EphA6 > EphB2 > EphB6 > EphA2. In addition, the arginine and lysine residues that mediate CS binding are identified for each Eph receptor. These computational predictions provide mechanistic insights into Eph receptor activation by chondroitin sulfate and have implications for inhibition of axon regeneration following injury to the nervous system and axon guidance during development.