Legumain and cathepsin-L expression in human unstable carotid plaque

Objective: The cysteine protease, legumain, is thought to have a role in the processing and activation of proteases such as cathepsin-L, which have been implicated in plaque rupture. This study aimed to determine: if legumain activity is up-regulated in unstable areas of plaque; the effect of legumain overexpression on the activity of cathepsin-L and the effect of mutation of the legumain RGD sequence on its cellular location. Methods and results: Legumain was measured in human carotid plaque extracts (n = 17) using a novel ELISA and modified activity assay. Unstable regions of plaque contained more than twice the amount of legumain protein (P < 0.001) and activity (P < 0.03) compared with stable regions of the same plaque. Overexpression of legumain in THP-1 macrophages using an adenoviral construct resulted in the processing of cathepsin-L from its 30 kDa to its 25 kDa form compared with controls. Conclusion: Unstable regions of plaque contain increased levels of active legumain. Over-expression of legumain in macrophages alters intracellular processing of cathepsin-L to its mature 25 kDa form. This may be a means by which legumain could contribute to plaque instability. (C) 2009 Elsevier Ireland Ltd. All rights reserved.