An asparaginyl endopeptidase processes a microbial antigen for class II MHC presentation

Foreign protein antigens must be broken down within endosomes or lysosomes to generate suitable peptides that will form complexes with class II major histocompatibility complex molecules for presentation to T cells. However, it is not known which proteases are required for antigen processing. To inv estigate this, we exposed a domain of the microbial tetanus toxin antigen ( TTCF) to disrupted lysosomes that had been purified from a human B-cell lin e. Here we show that the dominant processing activity is not one of the kno wn lysosomal cathepsins, which are generally believed to be the principal e nzymes involved in antigen processing, but is instead an asparagine-specifi c cysteine endopeptidase. This enzyme seems similar or identical to a mamma lian homologue(1) of the legumain/haemoglobinase asparaginyl endopeptidases found originally in plants' and parasites(3). We designed competitive pept ide inhibitors of B-cell asparaginyl endopeptidase (AEP) that specifically block its proteolytic activity and inhibit processing of TTCF in vitro. In vivo, these inhibitors slow TTCF presentation to T cells, whereas preproces sing of TTCF with AEP accelerates its presentation, indicating that this en zyme performs a key step in TTCF processing. We also show that N-glycosylat ion of asparagine residues blocks AEP action in vitro. This indicates that N-glycosylation could eliminate sites of processing by AEP in mammalian pro teins, allowing preferential processing of microbial antigens.