Study Of The State Of Polymerization Of S-periaxin

Periaxin is expressed by myelinating Schwann cells which play an essential role in stabilizing the Schwann cell-axon.Periaxin mutations cause demyelinating peripheral neuropathies,Charcot-Marie-Tooth 4F.Owing to alternative intron rentention,periaxin gene encodes L-periaxin and a truncated isoform, S-periaxin,which are 1461-and 147-amino acid residues in size,respectively.Both proteins have N-terminal PDZ protein-binding domains.In addition,L-periaxin contains Nuclear localization signal,Repeat domain,Acidic domain. Altuough nearly 20 years have been passed since the first report on periaxin,the structure and function of S-periaxin is still unclear.Using the cDNA of RSC96 cells as template,the DNA sequence encoding S-periaxin was amplified by PCR and inserted into the vector pET-M-3C to form a recombinant plasmid pET-M-3C-S-periaxin.The mutations of S-periaxin were generated by replacing Cys at 88,97 and 139 of S-periaxin with Gly,respectively or in defferent combination. Both S-periaxin and its mutants were expressed in E.coli BL-21 with 0.3 mM IPTG.The fusion proteins were purified by Ni-NTA and Sephacry S-200. S-periaxin is easy to form the different degree of polymerization by glutaraldehyde crosslinking in vitro.Co-immunoprecipitation experiment indicated that S-periaxin could homodimerize.In addition,His-S-periaxin and its mutants were incubated with H2O2 or DTT and analyzed by non-reducing SDS-PAGE, the results were showed that S-periaxin proteins could form reversible, intermolecular disulfide bonds under mild oxidation conditions and the dimer could be fully converted to the monomer via addition of reductant DTT in vitro. Both Cysss and Cys139 in S-periaxin was involved in intermolecular disulfide bond formation,but Cys97 was relatively more inert to oxidation.The mCherry proteins were split at the positions between amino acids sites 159 and 160. The N-and C-terminal coding regions of mCherry were amplified by PCR and then inserted into the pQE-30, pET-28a to construct prokaryotic BiFC system. To assess the reliability of prokaryotic BiFC system for studying protein interactions in vivo, GFP which has a propensity for dimerization was used as a test model. The DNA sequence of S-periaxin (WT and its mutants) was inserted into pCherryl-159 or pCherry160-237,the results showed that S-periaxin can form dimer via Cys.In Addition, mCherry1-159 and mCherry 160-237 were also inserted into the pEGFP-N1、pEGFP-Cl to yield eukaryptic BiFC system, which also demonstrated an interaction between S-periaxin protein in RSC96 cells.