Glycosylation is important for legumain localization and processing to active forms but not for cystatin E/M inhibitory functions

Lunde, Ngoc Nguyen; Haugen, Mads H.; Larsen, K.; Damgaard, Ingrid; Pettersen, Solveig; Kasem, Roya; Rut, Wioletta; Drąg, Marcin; Poręba, Marcin; Johansen, Harald Thidemann; Solberg, Rigmor

doi:10.1016/j.biochi.2017.05.009

Cited by 23 publications

(22 citation statements)

References 285 publications

(400 reference statements)

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“…Native cystatin E harbors an N -glycosylation site on its L2 loop (Asn 112 ). Because both glycosylated and nonglycosylated hCE were previously reported in vivo , we were interested in the relevance of glycosylation for hCE dimerization ( 20 , 42 ). The crystal structure of the hCE dimer suggested no negative effect of glycosylation on dimer formation ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural and functional analysis of cystatin E reveals enzymologically relevant dimer and amyloid fibril states

Dall

Hollerweger

Dahms

et al. 2018

Journal of Biological Chemistry

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Protein activity is often regulated by altering the oligomerization state. One mechanism of multimerization involves domain swapping, wherein proteins exchange parts of their structures and thereby form long-lived dimers or multimers. Domain swapping has been specifically observed in amyloidogenic proteins, for example the cystatin superfamily of cysteine protease inhibitors. Cystatins are twin-headed inhibitors, simultaneously targeting the lysosomal cathepsins and legumain, with important roles in cancer progression and Alzheimer's disease. Although cystatin E is the most potent legumain inhibitor identified so far, nothing is known about its propensity to oligomerize. In this study, we show that conformational destabilization of cystatin E leads to the formation of a domain-swapped dimer with increased conformational stability. This dimer was active as a legumain inhibitor by forming a trimeric complex. By contrast, the binding sites toward papain-like proteases were buried within the cystatin E dimer. We also showed that the dimers could further convert to amyloid fibrils. Unexpectedly, cystatin E amyloid fibrils contained functional protein, which inhibited both legumain and papain-like enzymes. Fibril formation was further regulated by glycosylation. We speculate that cystatin amyloid fibrils might serve as a binding platform to stabilize the pH-sensitive legumain and cathepsins in the extracellular environment, contributing to their physiological and pathological functions.

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Section: Resultsmentioning

confidence: 99%

Structural and functional analysis of cystatin E reveals enzymologically relevant dimer and amyloid fibril states

Dall

Hollerweger

Dahms

et al. 2018

Journal of Biological Chemistry

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“…AEP is glycosylated and this modification mediates its sorting from the ER into lysosomes (34,35). To examine whether transfected GST-AEP is indeed glycosylated, we transfected GST-AEP into HEK293 cells, followed by treatment with tunicamycin, an inhibitor of N-linked glycosylation, and PNGase F and Endo H were used to characterize the glycosylation types.…”

Section: Resultsmentioning

confidence: 99%

BDNF inhibits neurodegenerative disease–associated asparaginyl endopeptidase activity via phosphorylation by AKT

Wang

Kang

et al. 2018

JCI Insight

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AEP is an age-dependent lysosomal asparaginyl endopeptidase that cleaves numerous substrates including tau and α-synuclein and mediates their pathological roles in neurodegenerative diseases. However, the molecular mechanism regulating this critical protease remains incompletely understood. Here, we show that Akt phosphorylates AEP on residue T322 upon brain-derived neurotrophic factor (BDNF) treatment and triggers its lysosomal translocation and inactivation. When BDNF levels are reduced in neurodegenerative diseases, AEP T322 phosphorylation is attenuated. Consequently, AEP is activated and translocates into the cytoplasm, where it cleaves both tau and α-synuclein. Remarkably, the unphosphorylated T322A mutant increases tau or α-synuclein cleavage by AEP and augments cell death, whereas phosphorylation mimetic T322E mutant represses these effects. Interestingly, viral injection of T322E into Tau P301S mice antagonizes tau N368 cleavage and tau pathologies, rescuing synaptic dysfunction and cognitive deficits. By contrast, viral administration of T322A into young α-SNCA mice elicits α-synuclein N103 cleavage and promotes dopaminergic neuronal loss, facilitating motor defects. Therefore, our findings support the notion that BDNF contributes to the pathogenesis of neurodegenerative diseases by suppressing AEP via Akt phosphorylation.

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“…In platelets, both the pro-and mature form were detected by immunoblotting, with highest level of the mature form. Notably, the mature form is active as it binds the legumain-selective ABP MP-L01 [15]. Active legumain in platelets might indicate lysosomal storage, as legumain is a lysosomal protease and low pH is required for activation and proteolytic activity of legumain and other proteases, and shown for cathepsin D in platelets [18].…”

Section: Discussionmentioning

confidence: 99%

“…Active legumain in platelets might indicate lysosomal storage, as legumain is a lysosomal protease and low pH is required for activation and proteolytic activity of legumain and other proteases, and shown for cathepsin D in platelets [18]. However, after secretion, prolegumain can be activated in less acidic environments when stabilized by the cell surface αvβ 3 integrin, glycosaminoglycans or cystatins present extracellularly [15,19]. Also, the inflamed arterial wall in atherosclerosis creates a local acidic milieu that could activate prolegumain extracellularly.…”

Section: Discussionmentioning

confidence: 99%

“…2B). The mature form was shown to be active, by binding to the legumain-selective activity-based probe (ABP) MP-L01 [15]. Furthermore, when platelet-rich plasma (PRP) was exposed to the PAR-1 agonist SFLLRN, a rapid and significant release (40% increase within 10 min) of legumain was induced (Fig.…”

Section: Legumain Is Co-localized With Platelets In Carotid Plaques Amentioning

confidence: 99%

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Legumain is upregulated in acute cardiovascular events and associated with improved outcome - potentially related to anti-inflammatory effects on macrophages

et al. 2020

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Glycosylation is important for legumain localization and processing to active forms but not for cystatin E/M inhibitory functions

Cited by 23 publications

References 285 publications

Structural and functional analysis of cystatin E reveals enzymologically relevant dimer and amyloid fibril states

Structural and functional analysis of cystatin E reveals enzymologically relevant dimer and amyloid fibril states

BDNF inhibits neurodegenerative disease–associated asparaginyl endopeptidase activity via phosphorylation by AKT

Legumain is upregulated in acute cardiovascular events and associated with improved outcome - potentially related to anti-inflammatory effects on macrophages

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