RESUMEN
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.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Animales , Asparagina/química , Ácido Aspártico/química , Células CHO , Línea Celular , Células Cultivadas , Clonación Molecular , Cricetinae , Células Dendríticas/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Lipopolisacáridos/química , Lisosomas/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Datos de Secuencia Molecular , Monocitos/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Péptidos/química , Estructura Terciaria de Proteína , Conejos , Ovinos , Factores de Tiempo , TransfecciónRESUMEN
The invariant chain (Ii) chaperone for MHC class II molecules is crucial for their effective function. Equally important is its removal. Cathepsins S or L are known to be required for the final stages of Ii removal in different APCs, but the enzymes which initiate Ii processing have not been identified. Here we show that this step can be performed in B lymphocytes by asparagine endopeptidase (AEP), which targets different asparagine residues in the lumenal domain of human and mouse invariant chain. Inhibition of AEP activity slows invariant chain processing and hinders the expression of an antigenic peptide engineered to replace the groove binding region of Ii (CLIP). However, the initiation of Ii removal can also be performed by other proteases, reflecting the importance of this step.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/metabolismo , Cisteína Endopeptidasas/fisiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/metabolismo , Linfocitos B/metabolismo , Línea Celular , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , RatonesRESUMEN
Mammalian asparaginyl endopeptidase (AEP) or legumain is a recently identified lysosomal cysteine protease belonging to clan CD. To date it has been shown to be involved in antigen presentation within class II MHC positive cells and in pro-protein processing. Further elucidation of the biological functions of the enzyme will require potent and selective inhibitors and thus we describe here new acyloxymethylketone inhibitors of AEP. The most potent of the series is 2,6-dimethyl-benzoic acid 3-benzyloxycarbonylamino-4-carbamoyl-2-oxo-butyl ester (MV026630) with a kobs/[I] value of 1.09 x 10(5) M(-1) s(-1). At low microM concentrations this compound is able to enter living cells and irreversibly inactivate AEP. We show that this results in inhibition of AEP autoactivation and in perturbation of the processing and presentation of T cell epitopes from both tetanus toxin and myelin basic protein.