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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board Member: Buchner, Johannes / Ludwig, Stephan / Sies, Helmut / Turk, Boris / Wittinghofer, Alfred

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Activation of Progelatinase A by Mammalian Legumain, a Recently Discovered Cysteine Proteinase

J.-M. Chen / M. Fortunato / R.A.E. Stevens / A.J. Barrett

Citation Information: Biological Chemistry. Volume 382, Issue 5, Pages 777–783, ISSN (Print) 1431-6730, DOI: 10.1515/BC.2001.093, June 2005

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The activation of progelatinase A to gelatinase A requires cleavage of an asparaginyl bond to form the Nterminus of the mature enzyme. We have asked whether the activation can be mediated by legumain, the recently discovered lysosomal cysteine proteinase that is specific for hydrolysis of asparaginyl bonds. Addition of purified legumain to the concentrated conditioned medium from HT1080 cell culture that contained both progelatinases A and B caused the conversion of the 72 kDa progelatinase A to the 62 kDa form. The progelatinase B in the medium was unaffected. Incubation of recombinant progelatinase A with legumain resulted in an almost instantaneous activation as judged by the fluorometric assay with a specific gelatinase A substrate, McaProLeuGly LeuDpaAlaArgNH[2]. Legumain also activated progelatinase A when it was in complex with TIMP-2. Zymographic analysis and Nterminal sequencing revealed that legumain cleaved the 72 kDa progelatinase A at the bonds between Asn[109]Tyr[110] or Asn[111] Phe[112] to produce the 62 kDa mature enzyme, and that further cleavage at Asn[430] also occurred to generate a 36 kDa active form. More 62 kDa gelatinase A was detected in cultures of C13 cells that overexpressed legumain than in those of the control HEK293 cells. We conclude that legumain is clearly capable of processing progelatinase A to the active enzyme in vitro and in cultured cells.

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