Abstract
SPG4 gene encodes Spastin, and its mutations are the main cause of hereditary spastic paraplegia (HSP). There are more than 50 gene mutations in HSP, and mutations of Spastin account for more than 40%, so SPG4 is the key gene that causes the disease. In order to clarify the effect on microtubule stability after inactivation of AAA functional domain caused by mutations of Spastin, we carried out PCR amplification and C413Y site-directed mutation of the target gene according to the sequence of SPG4 in PUBMED gene bank and constructed GFP-Spastin and GFP-Spastin C413Y recombinant plasmids. The recombinant plasmids were introduced into COS7 cells, and the expression of recombinant plasmids in COS7 cells and changes of microtubule stability were observed. The results of colony PCR and gene identification showed that recombinant plasmids were successfully constructed. Western blotting showed that GFP-Spastin and GFP-Spastin C413Y could be expressed normally in COS7 cells. Immunofluorescence assay showed that the distribution of GFP-Spastin was punctate in the cells and GFP-Spastin had strong microtubule cleavage ability, while GFP-Spastin C413Y was almost distributing in the nucleus, and the ability of microtubule cleavage was weakened and the microtubule was in a stable state. Therefore, we concluded that Spastin C413Y changed the distribution of Spastin in cells and weakened the ability of microtubule cleavage. Spastin C413Y accumulated in the nucleus which could not cut microtubules effectively.
Highlights
SPG4 gene encodes spastin, and its gene mutations are the main cause of hereditary spastic paraplegia (HSP) [1]
There are more than 50 gene mutations in HSP, and Spastin mutations account for more than 40%
According to the designed primers, the PCR products of GFP-Spastin and GFP-Spastin C413Y were obtained and ligated to the pEGFP-C1 vector digested by BGIII and EcoR I
Summary
SPG4 gene encodes spastin, and its gene mutations are the main cause of hereditary spastic paraplegia (HSP) [1]. It is the key gene that causes the disease, and most of the mutations are located in the AAA region of Spastin [4]. Spastin contains several main functional regions, including the N-terminal MIT region (microtubuleinteracting and trafficking domain), MBD (microtubule-binding domain), and C-terminal AAA enzyme catalysis. Spastin is highly conserved in the process of evolution, especially the AAA region, but the AAA region is a high incidence region of mutations in HSP. Mutations in this region lead to changes in the function of Spastin protein [7], resulting in corresponding clinical symptoms in patients with HSP
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