Legumain in Acute Coronary Syndromes: A Substudy of the PLATO (Platelet Inhibition and Patient Outcomes) Trial.

Background The cysteine protease legumain is increased in patients with atherosclerosis, but its causal role in atherogenesis and cardiovascular disease is still unclear. The aim of the study was to investigate the association of legumain with clinical outcome in a large cohort of patients with acute coronary syndrome. Methods and Results Serum levels of legumain were analyzed in 4883 patients with acute coronary syndrome from a substudy of the PLATO (Platelet Inhibition and Patient Outcomes) trial. Levels were analyzed at admission and after 1 month follow-up. Associations between legumain and a composite of cardiovascular death, spontaneous myocardial infarction or stroke, and its individual components were assessed by multivariable Cox regression analyses. At baseline, a 50% increase in legumain level was associated with a hazard ratio (HR) of 1.13 (95% CI, 1.04-1.21), P=0.0018, for the primary composite end point, adjusted for randomized treatment. The association remained significant after adjustment for important clinical and demographic variables (HR, 1.10; 95% CI, 1.02-1.19; P=0.013) but not in the fully adjusted model. Legumain levels at 1 month were not associated with the composite end point but were negatively associated with stroke (HR, 0.62; 95% CI, 0.44-0.88; P=0.0069), including in the fully adjusted model (HR, 0.57; 95% CI, 0.37-0.88; P=0.0114). Conclusions Baseline legumain was associated with the primary outcome in patients with acute coronary syndrome, but not in the fully adjusted model. The association between high levels of legumain at 1 month and decreased occurrence of stroke could be of interest from a mechanistic point of view, illustrating the potential dual role of legumain during atherogenesis and acute coronary syndrome. Registration URL: https://www.clini​caltr​ials.gov; Unique identifier: NCT00391872.

Bradykinin analysis revived--a validated method for determination of its stable metabolite in whole blood by LC-MS/MS.

Investigation of bradykinin involvement in diseases like hereditary angioedema has been greatly hindered by its rapid metabolism and generation, induced by sampling. Because of this, reliable measurements of bradykinin have yet to be introduced in clinical practice. Prevention of bradykinin generation during sampling, and determination of the in vivo generated stable metabolite BK1-5, should allow a reliable indirect measure of bradykinin involvement. An LC-MS/MS method has been developed to determine BK1-5 in human whole blood. The method inactivates metabolizing enzymes with ethanol, followed by solid phase extraction (C18), separation (C8) and detection (linear ion trap) through the transitions 287.25→320.20 (y3, quantifier), 408.20 (b4, qualifier) for BK1-5, and 292.17→330.20 (y3, quantifier), 408.20 (b4, qualifier) for the heavy labelled internal standard. The method showed acceptable linearity (n=3, r(2)=0.994), intra-run precision (CV<15%), inter-run precision (CV<15%) and accuracy (CV<14%), without matrix effects. LLOQ was 265.5 pmol L(-1) and LOD was 35.4 pmol L(-1). The method was used on blood samples from patients with hereditary angioedema, where BK1-5 was measured during attacks and in remision. The samples showed elevated concentrations (up to 1.7 nmol L(-1) during attacks and 265.5 pmol L(-1) in remission) compared to healthy volunteers (<35.4 pmol L(-1)). This is the first time BK1-5 in hereditary angioedema patients has been measured.

Nuclear legumain activity in colorectal cancer.

The cysteine protease legumain is involved in several biological and pathological processes, and the protease has been found over-expressed and associated with an invasive and metastatic phenotype in a number of solid tumors. Consequently, legumain has been proposed as a prognostic marker for certain cancers, and a potential therapeutic target. Nevertheless, details on how legumain advances malignant progression along with regulation of its proteolytic activity are unclear. In the present work, legumain expression was examined in colorectal cancer cell lines. Substantial differences in amounts of pro- and active legumain forms, along with distinct intracellular distribution patterns, were observed in HCT116 and SW620 cells and corresponding subcutaneous xenografts. Legumain is thought to be located and processed towards its active form primarily in the endo-lysosomes; however, the subcellular distribution remains largely unexplored. By analyzing subcellular fractions, a proteolytically active form of legumain was found in the nucleus of both cell lines, in addition to the canonical endo-lysosomal residency. In situ analyses of legumain expression and activity confirmed the endo-lysosomal and nuclear localizations in cultured cells and, importantly, also in sections from xenografts and biopsies from colorectal cancer patients. In the HCT116 and SW620 cell lines nuclear legumain was found to make up approximately 13% and 17% of the total legumain, respectively. In similarity with previous studies on nuclear variants of related cysteine proteases, legumain was shown to process histone H3.1. The discovery of nuclear localized legumain launches an entirely novel arena of legumain biology and functions in cancer.

Intra- and extracellular regulation of activity and processing of legumain by cystatin E/M.

Legumain, an asparaginyl endopeptidase, is up-regulated in tumour and tumour-associated cells, and is linked to the processing of cathepsin B, L, and proMMP-2. Although legumain is mainly localized to the endosomal/lysosomal compartments, legumain has been reported to be localized extracellularly in the tumour microenvironment and associated with extracellular matrix and cell surfaces. The most potent endogenous inhibitor of legumain is cystatin E/M, which is a secreted protein synthesised with an export signal. Therefore, we investigated the cellular interplay between legumain and cystatin E/M. As a cell model, HEK293 cells were transfected with legumain cDNA, cystatin E/M cDNA, or both, and over-expressing monoclonal cell lines were selected (termed M38L, M4C, and M3CL, respectively). Secretion of prolegumain from M38L cells was inhibited by treatment with brefeldin A, whereas bafilomycin A1 enhanced the secretion. Cellular processing of prolegumain to the 46 and 36 kDa enzymatically active forms was reduced by treatment with either substance alone. M38L cells showed increased, but M4C cells decreased, cathepsin L processing suggesting a crucial involvement of legumain activity. Furthermore, we observed internalization of cystatin E/M and subsequently decreased intracellular legumain activity. Also, prolegumain was shown to internalize followed by increased intracellular legumain processing and activation. In addition, in M4C cells incomplete processing of the internalized prolegumain was observed, as well as nuclear localized cystatin E/M. Furthermore, auto-activation of secreted prolegumain was inhibited by cystatin E/M, which for the first time shows a regulatory role of cystatin E/M in controlling both intra- and extracellular legumain activity.

Marasmius oreades agglutinin (MOA) is a chimerolectin with proteolytic activity.

The Marasmius oreades mushroom lectin (MOA) is well known for its exquisite binding specificity for blood group B antigens. In addition to its N-terminal carbohydrate-binding domain, MOA possesses a C-terminal domain with unknown function, which structurally resembles hydrolytic enzymes. Here we show that MOA indeed has catalytic activity. It is a calcium-dependent cysteine protease resembling papain-like cysteine proteases, with Cys215 being the catalytic nucleophile. The possible importance of MOA's proteolytic activity for mushroom defense against pathogens is discussed.

Cystatin E/M suppresses legumain activity and invasion of human melanoma.

BACKGROUND: High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied. METHODS: A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay. RESULTS: Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay. CONCLUSIONS: These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells.

Collagen I regulates matrix metalloproteinase-2 activation in osteosarcoma cells independent of S100A4.

This work investigates the effect of cell-collagen I interactions on the synthesis and activation of MMP-2, as well as synthesis of MT1-MMP and TIMP-1, by using an in vitro model with 3D fibrillar and 2D monomeric collagen. In order to reveal whether the metastasis-associated protein S100A4 can influence the cell's response to the two forms of collagen, osteosarcoma cell lines with high and low endogenous levels of S100A4 were used. Attachment of osteosarcoma cells to 3D fibrillar and 2D monomeric collagen resulted in opposite effects on MMP-2 activation. Attachment to 3D fibrillar collagen decreased activation of proMMP-2, with a corresponding reduction in MT1-MMP. By contrast, attachment to monomeric collagen increased the amount of fully active MMP-2. This was caused by a reduction in TIMP-1 levels when cells were attached to monomeric 2D collagen. The effect of collagen on proMMP-2 activation was independent of endogenous S100A4 levels, whereas synthesis of TIMP-1 was dependent on S100A4. When cells were attached to monomeric collagen, cells with a high level of S100A4 showed a greater reduction in the synthesis of TIMP-1 than did those with a low level of S100A4. Taken together, this study shows that synthesis and activation of MMP-2 is affected by interactions between osteosarcoma cells and collagen I in both fibrillar and monomeric form.

Characterization of cysteine proteases in Malian medicinal plants.

Extracts form 10 different Malian medicinal plants with a traditional use against schistosomiasis were investigated for their possible content of proteolytic activity. The proteolytic activity was studied by measuring the hydrolysis of two synthetic peptide substrates Z-Ala-Ala-Asn-NHMec and Z-Phe-Arg-NHMec. Legumain- and papain-like activities were found in all tested crude extracts except those from Entada africana, with the papain-like activity being the strongest. Cissus quadrangularis, Securidaca longepedunculata and Stylosanthes erecta extracts showed high proteolytic activities towards both substrates. After gel filtration the proteolytic activity towards the substrate Z-Ala-Ala-Asn-NHMec in root extract of Securidaca longepedunculata appeared to have Mr of 30 and 97kDa, while the activity in extracts from Cissus quadrangularis was at 39kDa. Enzymatic activity cleaving the substrate Z-Phe-Arg-NHMec showed apparent Mr of 97 and 26kDa in extracts from roots and leaves of Securidaca longepedunculata, while in Cissus quadrangularis extracts the activity eluted at 39 and 20kDa, with the highest activity in the latter. All Z-Phe-Arg-NHMec activities were inhibited by E-64 but unaffected by PMSF. The legumain activity was unaffected by E-64 and PMSF. The SDS-PAGE analysis exhibited five distinct gelatinolytic bands for Cissus quadrangularis extracts (115, 59, 31, 22 and 20kDa), while two bands (59 and 30kDa) were detected in Securidaca longepedunculata extracts. The inhibition profile of the gelatinolytic bands and that of the hydrolysis of the synthetic substrates indicate the cysteine protease class of the proteolytic activities. Several cysteine protease activities with different molecular weights along with a strong variability of these activities between species as well as between plant parts from the same species were observed.

Evidence that unrestricted legumain activity is involved in disturbed epidermal cornification in cystatin M/E deficient mice.

Homozygosity for Cst6 null alleles causes the phenotype of the ichq mouse, which is a model for human harlequin ichthyosis (OMIM 242500), a genetically heterogeneous group of keratinization disorders. Here we report evidence for the mechanism by which deficiency of the cysteine protease inhibitor cystatin M/E (the Cst6 gene product) leads to disturbed cornification, impaired barrier function and dehydration. Absence of cystatin M/E causes unrestricted activity of its target protease legumain in hair follicles and epidermis, which is the exact location where cystatin M/E is normally expressed. Analysis of stratum corneum proteins revealed a strong decrease of soluble loricrin monomers in skin extracts of ichq mice, although normal levels of loricrin were present in the stratum granulosum and stratum corneum of ichq mice, as shown by immunohistochemistry. This suggested a premature or enhanced crosslinking of loricrin monomers in ichq mice by transglutaminase 3 (TGase 3). In these mice, we indeed found strongly increased levels of TGase 3 that was processed into its activated 30 and 47 kDa subunits, compared to wild-type mice. This study shows that cystatin M/E and legumain form a functional dyad in epidermis in vivo. Disturbance of this protease-antiprotease balance causes increased enzyme activity of TGase 3 that could explain the observed abnormal cornification.