The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Harald Thidemann Johansen - One of the best experts on this subject based on the ideXlab platform.
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mammalian Legumain a lysosomal cysteine protease with extracellular functions
Biochimie, 2019Co-Authors: Ngoc Nguyen Lunde, Rigmor Solberg, Tatjana Bosnjak, Harald Thidemann JohansenAbstract:The cysteine protease Legumain (asparaginyl endopeptidase, AEP) plays important roles in normal physiology but is also associated with several disorders, such as atherosclerosis, osteoporosis, cancer and neurodegenerative diseases. The functional roles of Legumain have mainly been associated with the presence in lysosomes where Legumain is active and mediates processing of multiple proteins, such as the conversion of single to double chain forms of cysteine cathepsins. However, in recent years, a number of studies point to extracellular roles of Legumain in addition to the pivotal roles in the lysosomes. In this review, recent knowledge on novel extracellular functions of this protease will be addressed and new discoveries in relation to the diseases mentioned above will be presented.
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lansoprazole inhibits the cysteine protease Legumain by binding to the active site
Basic & Clinical Pharmacology & Toxicology, 2019Co-Authors: Tatjana Bosnjak, Rigmor Solberg, Abbas Jafari, Paya Diana Hemati, Moustapha Kassem, Harald Thidemann JohansenAbstract:Proton pump inhibitors (PPIs) are prodrugs used in the treatment of peptic ulcer diseases. Once activated by acidic pH, the PPIs subsequently inhibit the secretion of gastric acid by covalently forming disulphide bonds with the SH groups of the parietal proton pump, that is the H+ /K+ -ATPase. Long-term use of PPIs has been associated with numerous adverse effects, including bone fractures. Considering the mechanism of activation, PPIs could also be active in acidic micro-environments such as in lysosomes, tumours and bone resorption sites. We suggested that the SH group in the active site of cysteine proteases could be susceptible for inhibition by PPIs. In this study, the inhibition by lansoprazole was shown on the cysteine proteases Legumain and cathepsin B by incubating purified proteases or cell lysates with lansoprazole at different concentrations and pH conditions. The mechanism of Legumain inhibition was shown to be a direct interaction of lansoprazole with the SH group in the active site, and thus blocking binding of the Legumain-selective activity-based probe MP-L01. Lansoprazole was also shown to inhibit both Legumain and cathepsin B in various cell models like HEK293, monoclonal Legumain over-expressing HEK293 cells (M38L) and RAW264.7 macrophages, but not in human bone marrow-derived skeletal (mesenchymal) stem cells (hBMSC-TERT). During hBMSC-TERT differentiation to osteoblasts, lansoprazole inhibited Legumain secretion, alkaline phosphatase activity, but had no effects on in vitro mineralization capacity. In conclusion, lansoprazole acts as a direct covalent inhibitor of cysteine proteases via disulphide bonds with the SH group in the protease active site. Such inhibition of cysteine proteases could explain some of the off-target effects of PPIs.
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glycosylation is important for Legumain localization and processing to active forms but not for cystatin e m inhibitory functions
Biochimie, 2017Co-Authors: Ngoc Nguyen Lunde, Mads H Haugen, Solveig Pettersen, Kristina Broe Bodin Larsen, Ingrid Damgaard, Roya Kasem, Wioletta Rut, Marcin Drag, Marcin Poreba, Harald Thidemann JohansenAbstract:The asparaginyl endopeptidase Legumain and its inhibitor cystatin E/M are endogenously glycosylated. However, little is known about the nature of the carbohydrate groups and whether they affect the functions of these proteins. In this study both glycosylated and unglycosylated forms of Legumain and cystatin E/M were studied. HEK293 cell lines stably over-expressing Legumain or cystatin E/M, and HCT116 cells were used as cell models, and mature Legumain was purified from bovine kidneys. To obtain unglycosylated proteins, cells were treated with tunicamycin, an inhibitor of N-linked glycosylation, whereas PNGase F and Endo H were used to characterize the glycosylation types. Cells were incubated with glycosylated, unglycosylated proteins and/or Legumain selective activity-based probe, and Legumain and/or cystatin E/M was studied by activity measurement, ELISA or immunoblotting in cell lysates or conditioned media. Legumain and probe in whole cells were studied by immunofluorescence. The carbohydrates on Legumain were shown to be of the hybrid or high mannose type, whereas cystatin E/M was characterized as complex mannose-linked. While glycosylated proLegumain was able to autoactivate, the unglycosylated form was not, and addition of glycosaminoglycans did not facilitate autoactivation of unglycosylated proLegumain. Glycosylated proLegumain was internalized and processed to the mature active form, but no internalization of unglycosylated proLegumain was observed. A Cy5-labelled Legumain specific activity-based probe (MP-L09) was synthesized and shown to be a novel tool to study intracellular Legumain. Also, internalization of mature Legumain (36 kDa) was visualized both alone and complexed with probe. Contrary to the importance of Legumain glycosylation, both glycosylated and unglycosylated cystatin E/M showed similar capacity to inhibit Legumain. In conclusion, 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.
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Simvastatin inhibits glucose metabolism and Legumain activity in human myotubes. PLoS One 2014
2016Co-Authors: Robert Smith, Rigmor Solberg, Linn Løkken Jacobsen, Anette Larsen Vorel, Christian Rustan, Hege G. Thoresen, Harald Thidemann JohansenAbstract:Simvastatin, a HMG-CoA reductase inhibitor, is prescribed worldwide to patients with hypercholesterolemia. Although simvastatin is well tolerated, side effects like myotoxicity are reported. The mechanism for statin-induced myotoxicity is still poorly understood. Reports have suggested impaired mitochondrial dysfunction as a contributor to the observed myotoxicity. In this regard, we wanted to study the effects of simvastatin on glucose metabolism and the activity of Legumain, a cysteine protease. Legumain, being the only known asparaginyl endopeptidase, has caspase-like properties and is described to be involved in apoptosis. Recent evidences indicate a regulatory role of both glucose and statins on cysteine proteases in monocytes. Satellite cells were isolated from the Musculus obliquus internus abdominis of healthy human donors, proliferated and differentiated into polynuclear myotubes. Simvastatin with or without mevalonolactone, farnesyl pyrophosphate or geranylgeranyl pyrophosphate were introduced on day 5 of differentiation. After 48 h, cells were either harvested for immunoblotting, ELISA, cell viability assay, confocal imaging or enzyme activity analysis, or placed in a fuel handling system with [14C]glucose or [3H]deoxyglucose for uptake and oxidation studies. A dose-dependent decrease in both glucose uptake and oxidation were observed in mature myotubes after exposure to simvastatin in concentrations not influencing cell viability. In addition, simvastatin caused a decrease in maturation and activity of Legumain. Dysregulation of glucose metabolism and decreased Legumain activity by simvastatin points out new knowledge about the effects of statin
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particulate yeast β glucan is internalized by raw 264 7 macrophages and reduces the activity of the tumor associated protease Legumain
Bioactive Carbohydrates and Dietary Fibre, 2015Co-Authors: Lise Berven, Harald Thidemann Johansen, Frode Miltzow Skjeldal, Kristian Prydz, Layla Mohammad Khader Zubaidi, Simon Ballance, Anne Berit SamuelsenAbstract:Abstract Yeast-derived β-glucans (Y-BG) are potential immune-modulating compounds able to activate macrophages. We investigated whether Y-BG affects the activity of a proteolytic enzyme, Legumain, which is expressed in macrophages and involved in inflammatory conditions as well as in cancer development. RAW 264.7 macrophages were stimulated with either particulate (BG-P), water soluble (BG-S) or partly water soluble (BG-PS) Y-BG for 48 h, and cellular Legumain activity was determined using a fluorogenic peptide substrate. BG-P inhibited Legumain activity in a dose-dependent manner, while BG-S and BG-PS did not. Immunoblotting indicated that the observed inhibition of Legumain activity was caused by reduced proLegumain synthesis within the cells, rather than inhibition of Legumain proform activation. Blocking of the C-type lectin dectin-1 receptor with antibodies abolished the effect, revealing that this receptor was involved, and it was further demonstrated by confocal microscopy that fluorescently labeled BG-P was internalized into macrophages. RAW 264.7 macrophages stimulated with Y-BG for 6 h were shown to secrete TNF-α, and BG-P was more potent than BG-PS and BG-S in this respect. Y-BG is shown to inhibit Legumain activity after internalization into macrophages, providing new insight into the mode of action of β-glucans.
Rigmor Solberg - One of the best experts on this subject based on the ideXlab platform.
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mammalian Legumain a lysosomal cysteine protease with extracellular functions
Biochimie, 2019Co-Authors: Ngoc Nguyen Lunde, Rigmor Solberg, Tatjana Bosnjak, Harald Thidemann JohansenAbstract:The cysteine protease Legumain (asparaginyl endopeptidase, AEP) plays important roles in normal physiology but is also associated with several disorders, such as atherosclerosis, osteoporosis, cancer and neurodegenerative diseases. The functional roles of Legumain have mainly been associated with the presence in lysosomes where Legumain is active and mediates processing of multiple proteins, such as the conversion of single to double chain forms of cysteine cathepsins. However, in recent years, a number of studies point to extracellular roles of Legumain in addition to the pivotal roles in the lysosomes. In this review, recent knowledge on novel extracellular functions of this protease will be addressed and new discoveries in relation to the diseases mentioned above will be presented.
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lansoprazole inhibits the cysteine protease Legumain by binding to the active site
Basic & Clinical Pharmacology & Toxicology, 2019Co-Authors: Tatjana Bosnjak, Rigmor Solberg, Abbas Jafari, Paya Diana Hemati, Moustapha Kassem, Harald Thidemann JohansenAbstract:Proton pump inhibitors (PPIs) are prodrugs used in the treatment of peptic ulcer diseases. Once activated by acidic pH, the PPIs subsequently inhibit the secretion of gastric acid by covalently forming disulphide bonds with the SH groups of the parietal proton pump, that is the H+ /K+ -ATPase. Long-term use of PPIs has been associated with numerous adverse effects, including bone fractures. Considering the mechanism of activation, PPIs could also be active in acidic micro-environments such as in lysosomes, tumours and bone resorption sites. We suggested that the SH group in the active site of cysteine proteases could be susceptible for inhibition by PPIs. In this study, the inhibition by lansoprazole was shown on the cysteine proteases Legumain and cathepsin B by incubating purified proteases or cell lysates with lansoprazole at different concentrations and pH conditions. The mechanism of Legumain inhibition was shown to be a direct interaction of lansoprazole with the SH group in the active site, and thus blocking binding of the Legumain-selective activity-based probe MP-L01. Lansoprazole was also shown to inhibit both Legumain and cathepsin B in various cell models like HEK293, monoclonal Legumain over-expressing HEK293 cells (M38L) and RAW264.7 macrophages, but not in human bone marrow-derived skeletal (mesenchymal) stem cells (hBMSC-TERT). During hBMSC-TERT differentiation to osteoblasts, lansoprazole inhibited Legumain secretion, alkaline phosphatase activity, but had no effects on in vitro mineralization capacity. In conclusion, lansoprazole acts as a direct covalent inhibitor of cysteine proteases via disulphide bonds with the SH group in the protease active site. Such inhibition of cysteine proteases could explain some of the off-target effects of PPIs.
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Simvastatin inhibits glucose metabolism and Legumain activity in human myotubes. PLoS One 2014
2016Co-Authors: Robert Smith, Rigmor Solberg, Linn Løkken Jacobsen, Anette Larsen Vorel, Christian Rustan, Hege G. Thoresen, Harald Thidemann JohansenAbstract:Simvastatin, a HMG-CoA reductase inhibitor, is prescribed worldwide to patients with hypercholesterolemia. Although simvastatin is well tolerated, side effects like myotoxicity are reported. The mechanism for statin-induced myotoxicity is still poorly understood. Reports have suggested impaired mitochondrial dysfunction as a contributor to the observed myotoxicity. In this regard, we wanted to study the effects of simvastatin on glucose metabolism and the activity of Legumain, a cysteine protease. Legumain, being the only known asparaginyl endopeptidase, has caspase-like properties and is described to be involved in apoptosis. Recent evidences indicate a regulatory role of both glucose and statins on cysteine proteases in monocytes. Satellite cells were isolated from the Musculus obliquus internus abdominis of healthy human donors, proliferated and differentiated into polynuclear myotubes. Simvastatin with or without mevalonolactone, farnesyl pyrophosphate or geranylgeranyl pyrophosphate were introduced on day 5 of differentiation. After 48 h, cells were either harvested for immunoblotting, ELISA, cell viability assay, confocal imaging or enzyme activity analysis, or placed in a fuel handling system with [14C]glucose or [3H]deoxyglucose for uptake and oxidation studies. A dose-dependent decrease in both glucose uptake and oxidation were observed in mature myotubes after exposure to simvastatin in concentrations not influencing cell viability. In addition, simvastatin caused a decrease in maturation and activity of Legumain. Dysregulation of glucose metabolism and decreased Legumain activity by simvastatin points out new knowledge about the effects of statin
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counter selection substrate library strategy for developing specific protease substrates and probes
Chemistry & Biology, 2016Co-Authors: Ngoc Nguyen Lunde, Rigmor Solberg, Wioletta Rut, Marcin Poreba, Paulina Kasperkiewicz, Scott J Snipas, Marko Mihelic, Dusan TurkAbstract:Legumain (AEP) is a lysosomal cysteine protease that was first characterized in leguminous seeds and later discovered in higher eukaryotes. AEP upregulation is linked to a number of diseases including inflammation, arteriosclerosis, and tumorigenesis. Thus this protease is an excellent molecular target for the development of new chemical markers. We deployed a hybrid combinatorial substrate library (HyCoSuL) approach to obtain P1-Asp fluorogenic substrates and biotin-labeled inhibitors that targeted Legumain. Since this approach led to probes that were also recognized by caspases, we introduced a Counter Selection Substrate Library (CoSeSuL) approach that biases the peptidic scaffold against caspases, thus delivering highly selective Legumain probes. The selectivity of these tools was validated using M38L and HEK293 cells. We also propose that the CoSeSuL methodology can be considered as a general principle in the design of selective probes for other protease families where selectivity is difficult to achieve by conventional sequence-based profiling.
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Legumain expression activity and secretion are increased during monocyte to macrophage differentiation and inhibited by atorvastatin
Biological Chemistry, 2015Co-Authors: Rigmor Solberg, Robert L Smith, Hilde Nilsen, Maria Almlof, Eyassu Tewolde, Harald Thidemann JohansenAbstract:Macrophages express several lysosomal cysteine proteases such as cathepsins and Legumain. In this study, we assessed the expression, activity and secretion of legu- main in cellular models of monocytes/macrophages. Macrophages were derived from M-CSF- or GM-CSF/IFN γ - stimulated human primary monocytes (M2 and M1, respec- tively), PMA-treated human THP-1 cells, or murine RAW264.7 macrophages. In both primary monocytes and THP-1 cells, monocyte-to-macrophage differentiation caused highly increased cellular expression and activity of Legumain. Also, secretion of Legumain from macrophages, but not from monocytes, was observed. Notably, M2 macrophages expressed significantly higher levels of active Legumain than M1 macrophages, which are not previously reported. Legumain mRNA has been shown to be down-regulated in monocytes isolated from patients treated with the HMG-CoA reductase inhibitor atorvastatin. Interestingly, in our study, the active Legumain produced by M2 macrophages was found to be inhibited by atorvastatin, which was reflected in aberrant cellular expression and processing.
Hans Brandstetter - One of the best experts on this subject based on the ideXlab platform.
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structural and functional studies of arabidopsis thaliana Legumain beta reveal isoform specific mechanisms of activation and substrate recognition
Journal of Biological Chemistry, 2020Co-Authors: Elfriede Dall, Florian B Zauner, Chiara Cabrele, Wai Tuck Soh, Fatih Demir, Sven O Dahms, Pitter F Huesgen, Hans BrandstetterAbstract:The vacuolar cysteine protease Legumain plays important functions in seed maturation and plant programmed cell death. Because of their dual protease and ligase activity, plant Legumains have become of particular biotechnological interest, e.g. for the synthesis of cyclic peptides for drug design or for protein engineering. However, the molecular mechanisms behind their dual protease and ligase activities are still poorly understood, limiting their applications. Here, we present the crystal structure of Arabidopsis thaliana Legumain isoform β (AtLEGβ) in its zymogen state. Combining structural and biochemical experiments, we show for the first time that plant Legumains encode distinct, isoform-specific activation mechanisms. Whereas the autocatalytic activation of isoform γ (AtLEGγ) is controlled by the latency-conferring dimer state, the activation of the monomeric AtLEGβ is concentration independent. Additionally, in AtLEGβ the plant-characteristic two-chain intermediate state is stabilized by hydrophobic rather than ionic interactions, as in AtLEGγ, resulting in significantly different pH stability profiles. The crystal structure of AtLEGβ revealed unrestricted nonprime substrate binding pockets, consistent with the broad substrate specificity, as determined by degradomic assays. Further to its protease activity, we show that AtLEGβ exhibits a true peptide ligase activity. Whereas cleavage-dependent transpeptidase activity has been reported for other plant Legumains, AtLEGβ is the first example of a plant Legumain capable of linking free termini. The discovery of these isoform-specific differences will allow us to identify and rationally design efficient ligases with application in biotechnology and drug development.
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structural analyses of arabidopsis thaliana Legumain γ reveal differential recognition and processing of proteolysis and ligation substrates
Journal of Biological Chemistry, 2018Co-Authors: Florian B Zauner, Elfriede Dall, Chiara Cabrele, Brigitta Elsasser, Hans BrandstetterAbstract:Legumain is a dual-function protease–peptide ligase whose activities are of great interest to researchers studying plant physiology and to biotechnological applications. However, the molecular mechanisms determining the specificities for proteolysis and ligation are unclear because structural information on the substrate recognition by a fully activated plant Legumain is unavailable. Here, we present the X-ray structure of Arabidopsis thaliana Legumain isoform γ (AtLEGγ) in complex with the covalent peptidic Ac-YVAD chloromethyl ketone (CMK) inhibitor targeting the catalytic cysteine. Mapping of the specificity pockets preceding the substrate-cleavage site explained the known substrate preference. The comparison of inhibited and free AtLEGγ structures disclosed a substrate-induced disorder–order transition with synergistic rearrangements in the substrate-recognition sites. Docking and in vitro studies with an AtLEGγ ligase substrate, sunflower trypsin inhibitor (SFTI), revealed a canonical, protease substrate–like binding to the active site–binding pockets preceding and following the cleavage site. We found the interaction of the second residue after the scissile bond, P2′–S2′, to be critical for deciding on proteolysis versus cyclization. cis-trans-Isomerization of the cyclic peptide product triggered its release from the AtLEGγ active site and prevented inadvertent cleavage. The presented integrative mechanisms of proteolysis and ligation (transpeptidation) explain the interdependence of Legumain and its preferred substrates and provide a rational framework for engineering optimized proteases, ligases, and substrates.
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crystal structure of plant Legumain reveals a unique two chain state with ph dependent activity regulation
The Plant Cell, 2018Co-Authors: Florian B Zauner, Elfriede Dall, Christof Regl, Luigi Grassi, Chrisitian G Huber, Chiara Cabrele, Hans BrandstetterAbstract:The vacuolar cysteine protease Legumain can cleave and selectively rebuild peptide bonds, thereby vastly expanding the sequential repertoire of biomolecules. In this context, plant Legumains have recently attracted particular interest. Furthermore, Legumains have important roles in many physiological processes, including programmed cell death. Their efficient peptide bond ligase activity has gained tremendous interest in the design of cyclic peptides for drug design. However, the mechanistic understanding of these dual activities is incomplete and partly conflicting. Here, we present the crystal structure of a plant Legumain, Arabidopsis thaliana isoform-γ (AtLEGγ). Employing a conserved Legumain fold, the plant Legumain AtLEGγ revealed unique mechanisms of autoactivation, including a plant-specific two-chain activation state, which remains conformationally stable at neutral pH, which is a prerequisite for full ligase activity and survival in different cell compartments. The charge distribution around the α6-helix mediates the pH-dependent dimerization and serves as a gatekeeper for the active site, thus regulating its protease and ligase activity.
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distinct roles of catalytic cysteine and histidine in the protease and ligase mechanisms of human Legumain as revealed by dft based qm mm simulations
ACS Catalysis, 2017Co-Authors: Brigitta Elsasser, Florian B Zauner, Elfriede Dall, Wai Tuck Soh, Johann Messner, Hans BrandstetterAbstract:The cysteine protease enzyme Legumain hydrolyzes peptide bonds with high specificity after asparagine and under more acidic conditions after aspartic acid [Baker, E. N. J. Mol. Biol. 1980, 141, 441−484; Baker, E. N.; J. Mol. Biol. 1977, 111, 207–210; Drenth, J.; Biochemistry 1976, 15, 3731–3738; Menard, R.; J. Cell. Biochem. 1994, 137; Polgar, L. Eur. J. Biochem. 1978, 88, 513–521; Storer, A. C.; Methods Enzymol. 1994, 244, 486–500. Remarkably, Legumain additionally exhibits ligase activity that prevails at pH > 5.5. The atomic reaction mechanisms including their pH dependence are only partly understood. Here we present a density functional theory (DFT)-based quantum mechanics/molecular mechanics (QM/MM) study of the detailed reaction mechanism of both activities for human Legumain in solution. Contrasting the situation in other papain-like proteases, our calculations reveal that the active site Cys189 must be present in the protonated state for a productive nucleophilic attack and simultaneous rupture of...
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Distinct Roles of Catalytic Cysteine and Histidine in the Protease and Ligase Mechanisms of Human Legumain As Revealed by DFT-Based QM/MM Simulations
2017Co-Authors: Brigitta Elsässer, Florian B Zauner, Elfriede Dall, Wai Tuck Soh, Johann Messner, Hans BrandstetterAbstract:The cysteine protease enzyme Legumain hydrolyzes peptide bonds with high specificity after asparagine and under more acidic conditions after aspartic acid [Baker, E. N. J. Mol. Biol. 1980, 141, 441−484; Baker, E. N.; J. Mol. Biol. 1977, 111, 207–210; Drenth, J.; Biochemistry 1976, 15, 3731–3738; Menard, R.; J. Cell. Biochem. 1994, 137; Polgar, L. Eur. J. Biochem. 1978, 88, 513–521; Storer, A. C.; Methods Enzymol. 1994, 244, 486–500. Remarkably, Legumain additionally exhibits ligase activity that prevails at pH > 5.5. The atomic reaction mechanisms including their pH dependence are only partly understood. Here we present a density functional theory (DFT)-based quantum mechanics/molecular mechanics (QM/MM) study of the detailed reaction mechanism of both activities for human Legumain in solution. Contrasting the situation in other papain-like proteases, our calculations reveal that the active site Cys189 must be present in the protonated state for a productive nucleophilic attack and simultaneous rupture of the scissile peptide bond, consistent with the experimental pH profile of Legumain-catalyzed cleavages. The resulting thioester intermediate (INT1) is converted by water attack on the thioester into a second intermediate, a diol (INT2), which is released by proton abstraction by Cys189. Surprisingly, we found that ligation is not the exact reverse of the proteolysis but can proceed via two distinct routes. Whereas the transpeptidation route involves aminolysis of the thioester (INT1), at pH 6 a cysteine-independent, histidine-assisted ligation route was found. Given Legumain’s important roles in immunity, cancer, and neurodegenerative diseases, our findings open up possibilities for targeted drug design in these fields
Rong Xiang - One of the best experts on this subject based on the ideXlab platform.
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Legumain an asparaginyl endopeptidase mediates the effect of m2 macrophages on attenuating renal interstitial fibrosis in obstructive nephropathy
Kidney International, 2018Co-Authors: Dekun Wang, Rong Xiang, Ze Liu, Junbo Gong, Min Xiong, Chuanai Chen, Yuzhi Shi, Mianzhi Zhang, Xiangrong Song, Ergang LiuAbstract:Two distinct macrophage phenotypes contribute to kidney injury and repair during the progression of renal interstitial fibrosis; proinflammatory (M1) and antiinflammatory (M2) macrophages. Legumain, an asparaginyl endopeptidase of the cysteine protease family, is overexpressed in macrophages in some pathological conditions. However, the macrophage subtype and function of macrophage-derived Legumain remains unclear. To resolve this we tested whether M2 macrophages contribute to the accumulation of Legumain in the unilateral ureteral obstruction model. Legumain-null mice exhibited more severe fibrotic lesions after obstruction compared with wild-type control. In vitro, IL4-stimulated M2 polarization led to the overexpression and secretion of Legumain. The levels of fibronectin and collagen I/III, major components of the extracellular matrix, were reduced in the conditioned medium of TGF-β1-stimulated tubular epithelial cells or fibroblasts after treatment with Legumain or conditioned medium from IL4-stimulated macrophages. Administration of the Legumain inhibitor RR-11a exacerbated fibrotic lesions following obstruction. Therapeutically, adoptive transfer of Legumain-overexpressing macrophages or IL4-stimulated macrophages ameliorated the deposition of collagen and fibronectin induced by ureteral obstruction, either in the wild-type mice or in lgmn–/– mice. Thus, M2 macrophages overexpress and secret Legumain and Legumain mediates the anti-fibrotic effect of M2 macrophages in obstructive nephropathy.
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m2 tumour associated macrophages contribute to tumour progression via Legumain remodelling the extracellular matrix in diffuse large b cell lymphoma
Scientific Reports, 2016Co-Authors: Long Shen, Rong Xiang, Junbo Gong, Dekun Wang, Yuzhi Shi, Jing Xun, Sifan Zhou, Xiaoyue TanAbstract:Effects of M2 tumour-associated macrophages on the pathogenesis of diffuse large B cell lymphoma (DLBCL) are still controversial. Our data showed that the number of CD163-positive M2 macrophages correlated negatively with DLBCL prognosis. Macrophage depletion by clodronate liposomes significantly suppressed tumour growth in a xenograft mouse model of DLBCL using OCI-Ly3 cells. Moreover, M2 polarization of macrophages induced Legumain expression in U937 cells. Exogenous Legumain promoted degradation of fibronectin and collagen I, which was abolished by administration of a Legumain inhibitor RR-11a. Overexpression of Legumain in Raw 264.7 cells also induced tube formation of endothelial cells in matrigel. In the xenograft mouse model of DLBCL, decreased fibronectin and collagen I, as well as increased Legumain expression and angiogenesis were found at the late stage tumours compared with early stage tumours. Co-localization of Legumain and fibronectin was observed in the extracellular matrix of tumour tissues. Administration of the Legumain inhibitor to the xenograft DLBCL model suppressed tumour growth, angiogenesis and collagen deposition compared with the control. Taken together, our results suggest that M2 tumour-associated macrophages affect degradation of the extracellular matrix and angiogenesis via overexpression of Legumain, and therefore play an active role in the progression of DLBCL.
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Prognostic value of Legumain in uveal melanoma
Molecular medicine reports, 2016Co-Authors: Lei Sun, Rong Xiang, Weining Rong, Fengyuan Sun, Ningli WangAbstract:The present study aimed to assess the expression of Legumain in uveal melanoma (UM) cell lines and primary UM specimens, and to determine the possible association between Legumain expression and clinical as well as pathological characteristics to reveal its impact on the prognosis of patients with UM. Records of primary UM cases treated at Beijing Tongren Hospital and Tianjin Eye Hospital between 1996 and 2005 were retrieved for analysis and a total of 82 patients with uveal melanoma were included in the study. The expression of Legumain in the formalin‑fixed and paraffin‑embedded surgical specimens of these 82 patients was determined using immunohistochemical analysis. In addition, the expression of Legumain was examined in two uveal melanoma cell lines using polymerase chain reaction and western blot analyses. The association of Legumain expression with clinical/pathological characteristics was analyzed using the χ2 and Fisher's exact test. In addition, the impact of Legumain on the prognosis of patients with uveal melanoma was examined. Upregulation of Legumain was more predominant in the highly invasive uveal melanoma cell line MUM‑2B compared with that in the MUM‑2C with low invasiveness. Of 82 primary uveal melanoma tissues, 35 exhibited high expression of Legumain, while the other 47 specimens exhibited low or negative expression of Legumain. High Legumain expression was primarily associated with local invasion of UM. Overall survival analysis revealed that the patients with high Legumain expression exhibited poorer survival than patients with low/negative Legumain expression. These findings suggested that upregulation of Legumain is associated with malignant behavior of UM and that Legumain may be used as an negative prognostic factor as well as a therapeutic target.
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alginic acid coated chitosan nanoparticles loaded with Legumain dna vaccine effect against breast cancer in mice
PLOS ONE, 2013Co-Authors: Ze Liu, Rong Xiang, Shu Liu, Junbo Gong, Da Wang, Min Xiong, Xiaoniao Chen, Xiaoyue TanAbstract:Legumain-based DNA vaccines have potential to protect against breast cancer. However, the lack of a safe and efficient oral delivery system restricts its clinical application. Here, we constructed alginic acid-coated chitosan nanoparticles (A.C.NPs) as an oral delivery carrier for a Legumain DNA vaccine. First, we tested its characteristic in acidic environments in vitro. DNA agarose electrophoresis data show that A.C.NPs protected DNA better from degradation in acidic solution (pH 1.5) than did chitosan nanoparticles (C.NPs). Furthermore, size distribution analysis showed that A.C.NPs tended to aggregate and form micrometer scale complexes in pH<2.7, while dispersing into nanoparticles with an increase in pH. Mice were intragastrically administrated A.C.NPs carrying EGFP plasmids and EGFP expression was detected in the intestinal Peyer’s patches. Full-length Legumain plasmids were loaded into different delivery carriers, including C.NPs, attenuated Salmonella typhimurium and A.C.NPs. A.C.NPs loaded with empty plasmids served as a control. Oral vaccination was performed in the murine orthotopic 4T1 breast cancer model. Our data indicate that tumor volume was significantly smaller in groups using A.C.NPs or attenuated Salmonella typhimurium as carriers. Furthermore, splenocytes co-cultured them with 4T1 cells pre-stimulated with CoCl2, which influenced the translocation of Legumain from cytoplasm to plasma membrane, showed a 4.7 and 2.3 folds increase in active cytotoxic T lymphocytes (CD3+/CD8+/CD25+) when treated with A.C.NPs carriers compared with PBS C.NPs. Our study suggests that C.NPs coated with alginic acid may be a safe and efficient tool for oral delivery of a DNA vaccine. Moreover, a Legumain DNA vaccine delivered orally with A.C.NPs can effectively improve autoimmune response and protect against breast cancer in mice.
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Legumain a biomarker for diagnosis and prognosis of human ovarian cancer
Journal of Cellular Biochemistry, 2012Co-Authors: Lina Wang, Si Chen, Mingna Zhang, Yanan Chen, Yanhua Liu, Yixuan Yang, Dwayne G Stupack, Rong XiangAbstract:Legumain is a member of the asparaginyl endopeptidase family that is over-expressed in response to hypoxic stress on mammary adenocarcinoma, colorectal cancer, proliferating endothelial cells, and tumor-associated macrophages (TAMs). Here, we demonstrate that elevated expression of Legumain in ovarian cancer by a proteomic approach using isobaric tags for relative and absolute quantification (iTRAQ) followed by liquid chromatography-mass spectrometry (LC-MS/MS). To investigate the relationship between Legumain expression and ovarian cancer development, we tested Legumain expression in malignant human ovarian tumors (n = 60), borderline ovarian tumors (n = 20), benign ovarian tumors (n = 20), and normal ovary samples (n = 20) using immunohistochemical assay (IHC). A correlation between Legumain expression, and clinocopathologic and biological variables was also established. Importantly, increased Legumain expression was validated by real-time PCR and Western blots, correlated positively with an increased malignancy of ovarian tumors (P < 0.01). In fact, patients with strong Legumain expression had a worse prognosis (P = 0.03). In addition, results of in vitro experiments revealed that over-expression of Legumain correlates with increased cell migration and invasion of ovarian cancer cells. Although Legumain's functional role and clinical utility remain to be established, our results indicated that a sensitive assay for early expression of Legumain may serve as both a potential biomarker and a molecular target for treatment of ovarian cancer.
Mads H Haugen - One of the best experts on this subject based on the ideXlab platform.
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glycosylation is important for Legumain localization and processing to active forms but not for cystatin e m inhibitory functions
Biochimie, 2017Co-Authors: Ngoc Nguyen Lunde, Mads H Haugen, Solveig Pettersen, Kristina Broe Bodin Larsen, Ingrid Damgaard, Roya Kasem, Wioletta Rut, Marcin Drag, Marcin Poreba, Harald Thidemann JohansenAbstract:The asparaginyl endopeptidase Legumain and its inhibitor cystatin E/M are endogenously glycosylated. However, little is known about the nature of the carbohydrate groups and whether they affect the functions of these proteins. In this study both glycosylated and unglycosylated forms of Legumain and cystatin E/M were studied. HEK293 cell lines stably over-expressing Legumain or cystatin E/M, and HCT116 cells were used as cell models, and mature Legumain was purified from bovine kidneys. To obtain unglycosylated proteins, cells were treated with tunicamycin, an inhibitor of N-linked glycosylation, whereas PNGase F and Endo H were used to characterize the glycosylation types. Cells were incubated with glycosylated, unglycosylated proteins and/or Legumain selective activity-based probe, and Legumain and/or cystatin E/M was studied by activity measurement, ELISA or immunoblotting in cell lysates or conditioned media. Legumain and probe in whole cells were studied by immunofluorescence. The carbohydrates on Legumain were shown to be of the hybrid or high mannose type, whereas cystatin E/M was characterized as complex mannose-linked. While glycosylated proLegumain was able to autoactivate, the unglycosylated form was not, and addition of glycosaminoglycans did not facilitate autoactivation of unglycosylated proLegumain. Glycosylated proLegumain was internalized and processed to the mature active form, but no internalization of unglycosylated proLegumain was observed. A Cy5-labelled Legumain specific activity-based probe (MP-L09) was synthesized and shown to be a novel tool to study intracellular Legumain. Also, internalization of mature Legumain (36 kDa) was visualized both alone and complexed with probe. Contrary to the importance of Legumain glycosylation, both glycosylated and unglycosylated cystatin E/M showed similar capacity to inhibit Legumain. In conclusion, 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.
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proteolysis mediated by cysteine cathepsins and Legumain recent advances and cell biological challenges
Protoplasma, 2015Co-Authors: Klaudia Brix, Mads H Haugen, Tripti Tamhane, Joseph Mcinnes, Alaa Alhashimi, Maren RehdersAbstract:Proteases play essential roles in protein degradation, protein processing, and extracellular matrix remodeling in all cell types and tissues. They are also involved in protein turnover for maintenance of homeostasis and protein activation or inactivation for cell signaling. Proteases range in function and specificity, with some performing distinct substrate cleavages, while others accomplish proteolysis of a wide range of substrates. As such, different cell types use specialized molecular mechanisms to regulate the localization of proteases and their function within the compartments to which they are destined. Here, we focus on the cysteine family of cathepsin proteases and Legumain, which act predominately within the endo-lysosomal pathway. In particular, recent knowledge on cysteine cathepsins and their primary regulator Legumain is scrutinized in terms of their trafficking to endo-lysosomal compartments and other less recognized cellular locations. We further explore the mechanisms that regulate these processes and point to pathological cases which arise from detours taken by these proteases. Moreover, the emerging biological roles of specific forms and variants of cysteine cathepsins and Legumain are discussed. These may be decisive, pathogenic, or even deadly when localizing to unusual cellular compartments in their enzymatically active form, because they may exert unexpected effects by alternative substrate cleavage. Hence, we propose future perspectives for addressing the actions of cysteine cathepsins and Legumain as well as their specific forms and variants. The increasing knowledge in non-canonical aspects of cysteine cathepsin- and Legumain-mediated proteolysis may prove valuable for developing new strategies to utilize these versatile proteases in therapeutic approaches.
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high expression of the cysteine proteinase Legumain in colorectal cancer implications for therapeutic targeting
European Journal of Cancer, 2015Co-Authors: Mads H Haugen, Kjetil Boye, Jahn M Nesland, Solveig Pettersen, Eivind Valen Egeland, Tripti Tamhane, Klaudia Brix, Gunhild Mari MaelandsmoAbstract:Abstract Background The cysteine proteinase Legumain is highly expressed in cancer. Legumain is a potential biomarker and has been suggested to be utilised for prodrug activation in cancer therapy. However, to define the suitability of Legumain for such purposes, detailed knowledge of cell type-specific and subcellular expression together with proteolytic activity patterns in tumour tissue is necessary. Methods Expression of Legumain was examined in a panel of 277 primary tumours from colorectal cancer (CRC) patients using immunohistochemistry. Tumour (cytoplasmic diffuse, cytoplasmic granulated, and nuclear) and stromal cell expression of Legumain was quantified, and associations with clinicopathological parameters and outcome were analysed. Additionally, normal colon tissue and spontaneous mouse tumours were stained for Legumain. Results Legumain was highly expressed in tumour and stromal cells. Nuclear Legumain was detected in 30% of the tumours. In colon cancer patients, high Legumain expression was associated with overall and metastasis-free survival (OS; MFS) in uni- and multivariate analysis. Nuclear Legumain was associated with poor OS, but not MFS in the colon cancer subgroup. Cytoplasmic granulated or diffuse expression was not associated with OS or MFS. Normal epithelial cells exhibited granulated Legumain mainly at the apical pole, and Legumain was highly expressed in CD68 positive macrophages. Conclusions Legumain is a highly expressed proteinase in CRC and associated with poor outcome in colon cancer. Diversified localisation of Legumain expression in tumour and stromal cells suggests multiple functions in CRC, representing both a challenge and an opportunity for use in therapeutic targeting.
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abstract 1093 Legumain in colorectal cancer unorthodox localization and trafficking
Cancer Research, 2014Co-Authors: Mads H Haugen, Eivind Valen Egeland, Tripti Tamhane, Harald Thidemann Johansen, Rigmor Solberg, Kjersti Flatmark, Ingrid Damgaard, Birgit Engesaeter, Siri Tveito, Solveig PettersenAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The cysteine proteinase Legumain is unique in cleaving substrates N-terminally of asparagine, and recent observations also revealed carboxypeptidase activity. This specificity combined with its up-regulation in a number of solid tumors prompts to take advantage of Legumain for cancer therapy and as a prognostic biomarker. Traditionally Legumain was thought to reside within the endo-lysosomes, but the proteinase has also been reported expressed elsewhere in the cell, and we recently described nuclear localization of proteolytically active Legumain in colorectal cancer cells. Presently, trafficking of Legumain in the cell remains enigmatic, particularly when not routed to the endo-lysosomes. Our recent analysis of Legumain expression in tissue from primary colorectal cancer (n=277) revealed that the subcellular localization pattern in tumor cells was diversified between the cases. Some had mostly dispersed and others more granulated cytoplasmic expression, while nuclear localization could be observed in about one third of the patients. We have thus established the association of Legumain expression with clinicopathological parameters and outcome, and found that high levels of total or nuclear Legumain in colon cancer cells were significantly negative for overall survival. Although an overall lower expression, nuclear localized Legumain was also observed in normal tissue while in cytoplasm it was found polarized primarily to the apical side of the epithelial layer. We have previously observed Legumain in the extracellular media of cancer cells and are in the process of analyzing wether it can also be found in exosomes. The unexpected observations in cellular localization are presumably due to altered trafficking. We have therefore analyzed Legumain transport in colorectal cancer cells treated with tunicamycin to block N-glycosylation of de novo synthesized Legumain, which had a striking effect on the trafficking and activation of the proteinase. Regarding functionality, we hypothesize that nuclear Legumain may be acting as a transcription factor and have made fusion constructs of Legumain and HaloTag® to identify potential genomic targets. The fusion protein has so far been verified to be highly expressed in the nuclei and will be used for chromatin capture. While the unique features of Legumain make it an attractive target to exploit for therapy and diagnosis of cancer, our results reveal that location and function of Legumain in normal and cancerous tissue is not completely elucidated. Such factors may be of outmost importance in attempts to utilize the protease activity for diagnostic and/or therapeutic purposes and may also represent novel cancer treatment opportunities. Citation Format: Mads H. Haugen, Kjersti Flatmark, Ingrid Damgaard, Tripti Tamhane, Birgit Engesaeter, Siri Tveito, Eivind V. Egeland, Rigmor Solberg, Harald T. Johansen, Solveig J. Pettersen, Kjetil Boye, Klaudia Brix, Gunhild M. Maelandsmo. Legumain in colorectal cancer: Unorthodox localization and trafficking. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1093. doi:10.1158/1538-7445.AM2014-1093
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nuclear Legumain activity in colorectal cancer
PLOS ONE, 2013Co-Authors: Mads H Haugen, Solveig Pettersen, Klaudia Brix, Gunhild Mari Maelandsmo, Harald Thidemann Johansen, Rigmor Solberg, Kjersti FlatmarkAbstract: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.