The cysteine protease legumain - from cell biology to cardiovascular disease

Abstract

The cysteine protease legumain is reported to be involved in the pathologies of several inflammatory conditions. The subcellular localization of legumain is thought to be mainly lysosomal. However, translocation to other cell compartments as well as secretion to the extracellular environment and appearance in serum has been reported. Being widespread in the body, legumain is suggested to have multiple functions, both intra- and extracellularly. Dysregulation of legumain is suggested to be of consequence for proteolytic degradation and activation of several proteins. Furthermore, high legumain expression is correlated with cancer progression and malignancy as well as atherosclerotic plaque instability, and pharmacological intervention targeting legumain could be beneficial. The aim of this thesis was to investigate posttranscriptional glycosylation, localization and regulation of legumain in inflammatory conditions (paper I-III), with a focus on atherosclerosis and cardiovascular disease (CVD). Atherosclerosis is a persistent inflammatory process, whereas alteration in N-linked glycosylation, a major posttranslational modification, is associated with inflammation and malignant conditions. The glycosylation of legumain and cystatin E/M were investigated for the first time. The sugar moieties on legumain were shown to be of high or hybrid type, whereas cystatin E/M is complex mannose-linked. The results suggest that glycosylation of prolegumain is necessary for correct processing to active forms and internalization, whereas the inhibitory property of cystatin E/M is independent of the glycosylation status (paper I). Also, for the first time, legumain was quantified in plasma from patients with carotid atherosclerosis (paper II). Patients had increased levels of legumain in plasma and plaques, with the highest level in patients with symptomatic disease. Within the atherosclerotic lesion, legumain was co-localized with macrophages, and in vitro, pro-inflammatory M1 macrophages secreted higher levels of legumain after stimulation with cholesterol crystals. Together with the proposed role of legumain in matrix degradation, these findings suggest a role of legumain as a potential biomarker for atherosclerotic disease. At present, data on regulation of legumain during acute cardiovascular events and in patients with CVD are scarce. The results presented in this thesis show elevated circulating levels of legumain in two large patient populations with stable or acute CVD (paper III). High levels of legumain in acute event patients correlated with improved outcome. Macrophages, platelets and alterations in glycosylation promote extracellular localization of legumain (paper I-III). Furthermore, depending of glycosylation status, legumain is internalized and processed by cells (paper I) to mediate anti-inflammatory effects through platelet and macrophage interactions to modulate plaque stability (paper III). Also, legumain is present in thrombus material obtained at the site of vascular occlusion in STEMI patients and patients with acute ischemic stroke (paper III). In conclusion, the results presented in this thesis contribute to new knowledge on regulation of the cysteine protease legumain, especially during atherosclerosis. The studies indicate a shift of paradigm from the classical malign and pro-inflammatory association of this protease to a novel player in modulation of plaque stability and potentially mediating anti-inflammatory effects, and/or potentially as a new biomarker in CVD.