Huntingtin cleavage induced by thrombin in vitro

Huntingtin (Htt) mutation causes Huntington's disease.Sequence analysis of Htt revealed a possible thrombin cleavage site in the N-terminal region of Htt.In order to investigate if thrombin can eleave Htt,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.An expression plasmid pcDNA3-Htt-18Q-969 was used to transfect MCF-cells and Htt 1-969 expression was confirmed with immunofluorescence.Cell lysates were incubated with thrombin (1 U/ml, 10 U/ml,and 30 U/ml) for 1 h in the presence or absence of hirudin,a thrombin 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 of an 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.