Structural and biophysical analysis of Netrin-1 with heparan sulfate proteoglycans

Abstract

The development of the nervous system involves the migration of axons from their origin to destination and ultimately completing the synapse formation. This process involves guidance molecules, the expression of receptors on the cell surface, and ultimately complex cell signalling. This thesis focuses on the characterization of one of the guidance molecules, Netrin-1, in solution and the impact of heparan oligosaccharides on its dynamic behaviour. Netrin-1 is a chemotropic cue involved in the attraction and repulsion of axons, cell migration, adhesion, differentiation, angiogenesis, inflammation, and cancer. Overexpression of netrin-1 is found in different forms of cancer. It acts as a survival factor for cancer cells by preventing cell apoptosis mediated via dependence receptors, including Un coordinated 5 (Unc5) and Deleted in Colorectal cancer (DCC) families. Interference with netrin-1 and its receptors interactions is associated with tumor cell death in various preclinical models. Netrin-1 interacts with glycosaminoglycan (GAG) chains of diverse heparan sulfate proteoglycans (HSPG) such as glypican. DCC also binds with heparin. It has been suggested that DCC/heparin interaction is mediated by netrin-1. However, these GAG molecule’s specific role with netrin-1 has not been demonstrated whether its interaction with netrin-1 concentrates it on the cell surface or it has some role in receptor binding. Therefore, a more detailed determination of the molecular interaction between netrin-1 and GAG molecules is of particular interest. A solution structure determined via SAXS with octasaccharide showed the appearance of a trimer that might be a seed for higher-order oligomers. The crystal structure of the complex of netrin-1 and the smallest heparan sugar unit (Sucrose Octa Sulfate, SOS) revealed a dimer of netrin-1 with one SOS per monomer unit. Distinct binding sites for two SOS suggest that GAG chains of different lengths could associate with netrin-1. Mutant studies also validated the observed netrin-1 GAG interface. Therefore, netrin-1 interaction studies with GAG molecules potentially promote new ideas for the development of small molecules targeting the GAG binding sites for the treatment of cancer. From this information, we can begin to elucidate the interaction mechanism of netrin-1 with GAG and understand its biological relevance. The GAG molecules might concentrate netrin-1 on the cell surface and help netrin-1 in bringing receptors together to form receptor clustering and further cell signalling.