Analysis Of The Interaction Mode Between Ezrin And L-periaxin

Periaxin, a protein of noncompact myelin, is specifically expressed in the PNS. It is comprised 16% of PNS myelin protein by weight, and playing an important role in initially myelin formation. During Schwann cell myelination, the intracellular location of Periaxin in Schwann cells is different all the time. L-PRX is first localized in the adaxonal plasma membrane, and then in the abaxonal plasma membrane. In addition, the expression quantity of Periaxin also gradually reduces. L-periaxin protein is one of the isoforms of periaxin protein, and includes four domains, a PDZ domain, a NLS domain, a repeat domain and a C-terminal acid amino domain. PDZ domains can recognise and bind the carboxy termini of their target proteins. Ezrin is a cytoskeleton protein and expressed in several specialized tissues. Ezrin, which is one of the ERM protein family, was highly expressed in the myelinated Schwann cells. Ezrin contains three domains:FERM domain, α-helical domain and C-terminal domain. Ezrin was regulated by an autoinhibitory mechanism, with the inactive protein being held in a closed and inactive conformation by head-to-tail like intramolecular interactions. However, phosphorylation at Thr567 contributes to ezrin activation. Except co-expression in Schwann cell, L-periaxin and Ezrin exist in a macromolecular complex with proteins involved in membrane organization, and play an important role in maturation and assembling.In this study, we mapped the interaction region between L-periaxin and ezrin. To verify the co-localization of endogenous Periaxin and ezrin in RSC96 by the immunofluorescent staining, the results show that they can co-locate in the cell membrane and cytoplasm. In order to analysis the two specific regions of interaction between L-periaxin and ezrin, immunofluorescence, bimolecular fluorescence complementation （BiFC） and co-immunoprecipitation performed. The results indicated that interaction mode between L-periaixn and ezrin is called "head to head and tail to tail" through the interaction between NLS of L-periaxin（104-200aa） with FERM of ezrin（1-296aa）, and between carboxyl terminal of L-periaxin （1368-146 1aa） with carboxyl terminal of ezrin （475-585aa）. Because NLS contains NLS1/NLS2/NLS3, co-localization and GST-pull down revealed that NLS3 of L-periaxin interacted with FERM of Ezrin.Secondly, we make mutant forms of ezrin, ezrin T567D and T567A, maintains the protein phosphorylation and dephosphorylation. Then, the immunofluorescent staining results showed that ezrin（T567D） and L-periaxin co-localized on the cell membrane, but ezrin （T567A）. It indicates that phosphorylation at Thr⁵⁶⁷ may guide L-periaxin on the cell membrane. But the phosphorylation of Thr has no influence on the interaction between Periaxin and Ezrin. So we thought that phosphorylating the Thr⁵⁶⁷ of ezrin may affect the localization of ezrin and Periaxin, but will not affect the interaction of them.To obtain the periaxin knockout cell lines, periaxin gene in Schwanns was knocked out by transcription activator-like effector nucleases （TALENs）.Furthermore, we constructed one pair of PRX-TALEN vector and transfected into RSC96. Sequencing results demonstrated that TALEN was available for PRX knockout. By picking monoclonal, the periaxin gene knockout cell line was screened out. One of the nucleotides deletes 23bp, and the other one deletes 10bp.They all were frameshift mutation, which could provide the possibility in production of PRX knockout. This cell line will provide the experimental base for the future research.