Multistep Autoactivation of Asparaginyl Endopeptidase in Vitro and in Vivo*
- Division of Cell Biology & Immunology, Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom
- § To whom correspondence should be addressed. Tel.: 44-1382-344233; Fax: 44-1382-345783; E-mail: c.watts{at}dundee.ac.uk.
Abstract
Mammalian asparaginyl endopeptidase (AEP) or legumain is a recently discovered lysosomal cysteine protease that specifically cleaves after asparagine residues. How this unusually specific lysosomal protease is itself activated is not fully understood. Using purified recombinant pro-enzyme, we show that activation is autocatalytic, requires sequential removal of C- and N-terminal pro-peptides at different pH thresholds, and is bimolecular. Removal of the N-terminal propeptide requires cleavage after aspartic acid rather than asparagine. Cellular processing, either of exogenously added AEP precursor or of pulse-labeled endogenous precursor, introduces at least one further cleavage to yield the final mature lysosomal enzyme. We also provide evidence that in living cells, there is clear compartmental heterogeneity in terms of AEP activation status. Moreover, we show that human monocyte-derived dendritic cells harbor inactive proforms of AEP that become activated upon maturation of dendritic cells with lipopolysaccharide.
Footnotes
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↵1 The abbreviations used are: AEP, asparagine endopeptidase; VPE, vacuolar processing enzyme; MHC, major histocompatibility complex; CHO, Chinese hamster ovary; Z, benzyloxycarbonyl; NHMec, 7-(4-methyl)coumarylamide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DC, dendritic cell(s); LPS, lipopolysaccharide; DHFR, dihydrofolate reductase.
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↵2 C. Moss and C. Watts, unpublished observation.
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↵3 B. Manoury and S. P. Matthews, unpublished observation.
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↵* This work was supported by Medivir UK Ltd. and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ These authors contributed equally to this work.
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- Received June 5, 2003.
- Revision received July 9, 2003.
- The American Society for Biochemistry and Molecular Biology, Inc.
