Production and structural characterization of the fibronectin III domains of EGFLAM/Pikachurin, a proteoglycan involved in ribbon synapse formation

Pikachurin (also known as EGFLAM) is recently identified proteoglycan involved in the formation of ribbon synapses. This molecule is localized in synaptic clefts of retinal cells, and contributes to the stabilization of the ribbon synapse structure. Pikachurin binds to dystroglycan (DG), a cellular receptor, through its C-terminus. This interaction enables the transmission of visual signals from photoreceptor cells to dendrites. Although poorly studied, Pikachurin is essential: lack of this molecule results in misalignment of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, muscular dystrophies and eye abnormalities. Pikachurin is a multi-domain protein containing two fibronectin-type III domains (FnIII) at N-terminal, followed by repeated cluster of three laminin G-like (LG) domains alternated by two epidermal growth factor (EGF domains). Besides the function of the C-terminal LG domain in binding to dystroglycan, very little is known about the molecular roles of the other domains of Pikachurin. In particular, the N-terminal domains are particularly interesting, as they are not conserved in homologous proteoglycans and may be involved in specific interactions with yet unidentified ligands at ribbon synapses. In this work, I developed the experimental strategies for the recombinant expression and purification of the N-terminal FnIII domains of Pikachurin. The work comprised definition of cloning methodologies to generate the recombinant expression vectors, followed by over-expression of recombinant protein fragments in small and large scale. Being able to successfully express various domain fragments of Pikachurin, I performed biophysical thermofluor stability assays to identify the most suitable conditions for protein stabilization, storage and crystallization. I also set up crystallization trials, exploring numerous reagents mixtures among which I identified protein crystals that were analysed using synchrotron X-ray diffraction. This work contributed to obtaining the crystal structure of the two N-terminal domains of Pikachurin, which is currently being refined and will serve as a reference point to understand the structural organization of this complex proteoglycan.