Study On Interaction Between Microtubules Associated Protein 2 And Prion Protein

Prion diseases include a series of diseases such as CJD,GSS,FFI,Kuru of human and Scrapie,BSE,CWD of animals. Prion diseases are characterized by neuronal celldeath, glial proliferation and deposition of prion peptide aggregates. An abnormal misfolded isoform of the prion protein (PrPsc) is considered to be responsible for this neurodegeneration.Neuron protein PrP and tau are considered to play roles in some transmissible spo- ngiform encephalopathies (TSEs) such as certain GSS andâ…¡type CJD. MAP2 and tau are same in strcture and function, and there are about 60%~80% amino acids of the repeated sequence in the microtubule binding domain are same. It suggested that MAP2 may have some relationships with Prion diseases. To address the possible molecular linkage of PrP an MAP2, total RNA of human neuron cell line SF126 was extracted and the cDNA encoding the microtubule binding region of MAP2 was synthesized and amplified by RT-PCR. After verified by sequence analysis and enzyme digested identification, the segment of MAP2 was cloned into an expressing vector pET32a, generating recombinant plasmid pET32a-MAP2. After induced by IPTG, a 43 ku soluble MAP2-fusion protein was expressed in E.Coli BL21 and purified by affinity chromatography.In order to identify the biological functions of the recombinant protein, the native tubulin was purified from rabbit's brains. Immunoprecipitation and ELISA tests revealed a remarkable molecular interaction between the recombinant MAP2 and native tubulin purified from rabbit's brains in vitro. Our work proved that the recombinant MAP2 hold some biological functions of native MAP2 such as binding tubulin.To address the possible molecular linkage of MAP2 and Prion disease, different fragments of PrP was expressed in E. coli M15 and purified by affinity chromatography. Using immunoprecipitation, microtubules binding region of MAP2 was confirmed to be able to interact with full length PrP23-231and PrP91-231, similar results were observed in ELISA tests. On this basis, PrPc was etracted from healthy hamster brain, immunoprecipitation proved they can form protein complexes.In summary, the recombinant microtubules binding region of MAP2 could interact with PrP in our experimental system. Our work has provided a good basis for the further research on the biological meanings of MAP2 in Prion diseases and other neurodegeneration diseases.