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Huntingtin (Htt) mutation causes Huntington’s disease.Sequence analysis of Htt revealed apossible thrombin cleavage site in the N-terminal region of Htt.In order to investigate if thrombin can eleaveHtt,we expressed the N-terminal fragment (1-969) of wild-type (wt) Htt (Htt 1-969) in MCF-7 cells andstudied its cleavage pattern by thrombin in vitro.An expression plasmid pcDNA3-Htt-18Q-969 was used totransfect MCF-cells and Htt 1-969 expression was confirmed with immunofluorescence.Cell lysates wereincubated with thrombin (1 U/ml, 10 U/ml,and 30 U/ml) for 1 h in the presence or absence of hirudin,athrombin inhibitor.Htt fragments were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and detected with anti-Htt antibodies. An Htt fragment with molecular mass of approximately80 kDa was produced after incubation with thrombin.The size of this Htt fragment was anticipated bymolecular mass generated from thrombin-mediated cleavage at the amino acid 183 in the Htt.Production ofan 80 kDa fragment was inhibited by hirudin. This study provides the first evidence that Htt is cleaved bythrombin in vitro at amino acid 183.If endogenous thrombin cleaves Htt in vivo,the physiological significanceof thrombin-mediated cleavage of Htt should be further investigated.
Huntingtin (Htt) mutation causes Huntington’s disease. Sequencing analysis of Htt exposed apostte thrombin cleavage site in the N-terminal region of Htt. Order to investigate if thrombin can electHtt, we expressed the N-terminal fragment (1-969) of wild -type (wt) Htt (Htt 1-969) in MCF-7 cells and studied its cleavage pattern by thrombin in vitro. Ana expression plasmid pcDNA3-Htt-18Q-969 was used totransfect MCF-cells and Htt 1-969 expression was confirmed with immunofluorescence. Cell lysates wereincubated with thrombin (1 U / ml, 10 U / ml, and 30 U / ml) for 1 h in the presence or absence of hirudin, athrombin inhibitor. Htt fragments were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected with anti-Htt antibodies. An Htt fragment with molecular mass of approximately 80 kDa was produced after incubation with thrombin. The size of this Htt fragment was anticipated by molecular mass generated from thrombin-mediated cleavage at the amino acid 183 in the Htt . Production ofan 80 kDa fragment was inhibited by hirudin. This study provides the first evidence that Htt is cleaved by thrombin in vitro at amino acid 183.If endogenous thrombin cleaves Htt in vivo, the physiological significance of thrombin-mediated cleavage of Htt should be further investigated .