Biochemical Properties Of Periaxin-PDZ Domain

Myelin is formed by the spiral wrapping of the cell membrane of Schwann cells in the peripheral nervous system. It regulates the distribution of neuronal axon and ion channels, adjusts neurofilament phosphorylation and secretes a variety of neurotrophins to promote neuronal survival. Periaxin which is one of proteins from noncompact myelin in the PNS is related with myelin ductility.The loss-of-function mutation of the gene encoding Periaxin can cause demyelinating Charcot-Marie-Tooth （CMT4F）disease.Because this protein is primarily localized in the periaxonal membranes of myelinating Schwann cells, it is named periaxin. Together with dystroglycan-dystrophin-related protein2（DRP2） and DG, periaxin form complexes in the periaxonal membranes, which play an important role in myelin integrity and stability. Periaxin contains PDZ domain,NLS domain,repeat domain and acidic domain.PDZ domain is regarded as the main domain to mediate the interaction with various proteins involving in physiological function of the cells. The gene of periaxin-PDZ was amplified by PCR with pBluescrip+SK（II）-periaxin as the template and inserted into the vector pETM-3C or pGEX-4T-2to form recombinant plasmids pETM-3C-pdz or pGEX-4T-2-pdz.The recombinant protein was overexpressed in Escherichia coli BL21（DE3） by IPTG induction.The fusion protein was purified by Ni²⁺-NTA column,GST Sepharose4B column and Sephacryl S-200column.The Periaxin-PDZ was analyzed by Protein Lipid Overlay assay, non-reducing and reducing SDS-PAGE, fluorescence spectroscopy,reduction induction by DTT and oxidation induction by H²O²and so on.The results were shown that Periaxin-PDZ has the lipid membrane binding capacity and the strong binding capacity of calcium ions.The further research indicated the oxidative stress can induce the dimer of PDZ and the lipid binding capacity of Periaxin-PDZ was decreased by oxidation.In addition,the lipid binding of Periaxin-PDZ was increased by addition of calcium ions.