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

Smith, Robert; Johansen, Harald T.; Nilsen, Hilde; Haugen, Mads H.; Pettersen, Solveig J.; Maelandsmo, Gunhild M.; Abrahamson, Magnus LU and Solberg, Rigmor (2012) In Biochimie 94(12). p.2590-2599
Abstract
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 microenvironrnent 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... (More)
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 microenvironrnent 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. (C) 2012 Elsevier Masson SAS. All rights reserved. (Less)
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author
organization
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type
Contribution to journal
publication status
published
subject
keywords
Asparaginyl endopeptidase, AEP, Legumain, Cystatin E/M, Cathepsin B, Cathepsin L
in
Biochimie
volume
94
issue
12
pages
2590 - 2599
publisher
Elsevier
external identifiers
  • wos:000312517800016
  • scopus:84868196191
ISSN
1638-6183
DOI
10.1016/j.biochi.2012.07.026
language
English
LU publication?
yes
id
271ed8d4-890a-484e-ad07-d2031fec8895 (old id 3512186)
date added to LUP
2013-03-01 07:44:02
date last changed
2017-10-08 03:10:49
@article{271ed8d4-890a-484e-ad07-d2031fec8895,
  abstract     = {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 microenvironrnent 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. (C) 2012 Elsevier Masson SAS. All rights reserved.},
  author       = {Smith, Robert and Johansen, Harald T. and Nilsen, Hilde and Haugen, Mads H. and Pettersen, Solveig J. and Maelandsmo, Gunhild M. and Abrahamson, Magnus and Solberg, Rigmor},
  issn         = {1638-6183},
  keyword      = {Asparaginyl endopeptidase,AEP,Legumain,Cystatin E/M,Cathepsin B,Cathepsin L},
  language     = {eng},
  number       = {12},
  pages        = {2590--2599},
  publisher    = {Elsevier},
  series       = {Biochimie},
  title        = {Intra- and extracellular regulation of activity and processing of legumain by cystatin E/M},
  url          = {http://dx.doi.org/10.1016/j.biochi.2012.07.026},
  volume       = {94},
  year         = {2012},
}