ELUCIDATING THE LRRTM4-GPC5 INTERACTION IN CNS SYNAPTIC CLEFT

Among the vast plethora of molecules present in the Central Nervous System (CNS), essential for its development and functioning, synaptic adhesion molecules (SAMs) occupy an important role in forming the around 100 trillion connections, in our brain. These connections involve not only pre- and post-synaptic compartments, but they also mediate trans-synaptic recognition and signalling processes, which are essential for the synapse establishment, specification, and plasticity. While some of these proteins have been fully characterized, many of them still lack molecular structure and functional details. Our work aims at elucidating the features of a newly discovered trans-synaptic interaction occurring between the pre-synaptic glycosyl-phosphatidylinositol (GPI) anchored Glypican-5 (GPC5) and the post-synaptic Leucine Rich Repeat Transmembrane protein 4 (LRRTM4). Mutations in the gene encoding for LRRTM4 lead to neurodevelopmental and psychiatric disorders, such as schizophrenia, epilepsy, and Tourette syndrome, rendering these proteins an interesting target for diagnostics and neuropharmacological therapeutics. By exploiting a mix of chromatographic techniques, and binding assays, we could rationalize unprecedented details about the interactions occurring between the LRRTM4 globular extracellular domain and the heparan sulfate chains of GPC5. Through thus work, several purification strategies and protocols were optimized, in order to improve recombinant protein expression, purification, and analysis of recombinant LRRTM4s and GPC5. The binding profile of the two synaptic proteins was evaluated through multiple tests, which involved the purification of different soluble recombinant protein constructs. These were obtained by fusing their protein-coding sequence in a pUPE plasmid for mammalian expression. DNA propagation was conducted using E.coli TOP10 cells, while protein expression was performed by transient transfection in HEK293F cells. After recovering the conditioned medium from the cell culture, proteins of interest were purified using Immobilized Metal Affinity Chromatography (IMAC) or Heparin Sepharose columns. The first chromatographic technique exploits the 6xHis-tag fused at the N-terminus of recombinant GPC5 to retain the protein from the supernatant, while the Heparin column was used to bind LRRTM4, due to its ability to interact with long heparin chains. Once eluted, proteins were concentrated and flowed through a desalting column, to perform buffer exchange and transfer the sample in a more stable storage condition. The eluted soluble recombinant LRRTM4s and GPC5 were then concentrated again and subject Size Exclusion Chromatography (SEC), obtaining samples with high purity. During every step of our working pipeline, a fraction of each sample was collected and analyzed through Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE), to confirm the presence and/or purity of recombinant protein targets. Mass Photometry technology was then exploited to characterize the complex; individual recombinant protein samples were used as controls. Results about the molar ratio of the complex were also confirmed through further experiments, where molecules underwent cross-linking, and further measurement through Mass Photometry. This type of analysis enabled also us to look at possible oligomerization states that could interfere with the visualization of the complex. Finally, we tested the specific binding affinity of LRRTM4 towards highly sulfated sugars, using Fondaparinux as ligand. Results were obtained performing Micro Scale Thermophoresis (MST) using a recombinant version of LRRTM4 conjugated with a superfolder green fluorescent protein (sfGFP).