Control of Antigen Presentation by a Single Protease Cleavage Site

Abstract

Protein antigens require limited proteolytic processing to generate peptides for binding to class II MHC molecules, but the proteases and processing sites involved are largely unknown. Here we analyze the effect of eliminating the three major asparagine endopeptidase (AEP)-processing sites in the microbial antigen tetanus toxin C fragment. The mutant antigen is highly resistant to proteolysis by AEP and crude lysosomal extracts and is dramatically impaired in its ability to be processed and presented to T cells. Remarkably, processing at a single asparagine residue (1219) is obligatory for optimal presentation of many T cell epitopes in this antigen. These studies demonstrate that cleavage at a single processing site can be crucial for effective antigen presentation.

Disulfide bonds in merozoite surface protein 1 of the malaria parasite impede efficient antigen processing and affect the in vivo antibody response

“…Here we demonstrate that the two tightly folded EGF domains of MSP1 19 are largely resistant to in vitro cleavage by AEP, a crucial enzyme for the class II antigen processing pathway [27], despite the fact that the sequence contains several asparagine and aspartic acid residues, the peptide bonds of which are readily cleaved when the protein is reduced and alkylated. Several of these residues are predicted to be exposed on the surface of the folded protein (by homology with P. falciparum MSP1 19 ; personal communication, Dr. W. Morgan), confirming that AEP specificity is less dependent on the amino acid sequence surrounding any given asparagine than on its structural context [33,34]. The native, but not the R/A, MSP1 19 was also resistant to the proteolytic activity of whole lysosomal extracts, suggesting that other proteases present cannot substitute for the activity of AEP under these conditions.…”

An in vitro immune response model to determine tetanus toxoid antigen (vaccine) specific immunogenicity: Selection of sensitive assay criteria

“…Good Manufacturing Practice -resulting in a more consistent product -and the availability of new immunochemical, physicochemical and biochemical techniques bring the consistency concept within reach of conventionally produced bacterial vaccines [3,4]. Minute changes in antigen structure can be missed by chemical analysis, but if these are important to the processing and presentation of the antigen, they will be detectable in terms of the immune response against the antigen [5]. Therefore, functional immunological tests are essential support for chemical analyses, to confirm the biological activity of the vaccine (antigen).…”

The expression of legumain, an asparaginyl endopeptidase that controls antigen processing, is reduced in endotoxin-tolerant monocytes

“…The elimination of legumainprocessing sites within this protein causes it to become highly resistant to proteolysis by other lysosomal enzymes and to not be presented to CD4 þ cells. 17,18 Legumain is a cysteine endopeptidase with strict specificity for protein cleavage on the C-terminal side of asparagines. 19,20 The activation of legumain is triggered by acidic pH and is autocatalytic.…”

“…As deduced from real-time PCR on reverse transcribed mRNA (RT-PCR), monocytes clearly expressed legumain, although this expression was much lower than that of the other tested proteases that play a role in antigen processing, cathepsin B, cathepsin H, cathepsin L, cathepsin F and cathepsin S (Figure 1a). This low expression and the fact that legumain activates lysosomal cathepsins and unlocks the antigens for splitting 18,25 suggests that the level and activity of legumain regulates the antigen cleavage. Figure 1 The expression of legumain is reduced in endotoxintolerant monocytes.…”

“…17,18 This decreased expression apparently leads to a decrease in the splitting of antigens and a dearth of available peptides for the MHC class II loading in endotoxintolerant monocytes. This dearth of available peptides, together with the decreased HLA-DM expression, 13 was reflected by increased amounts of CLIP-loaded MHC class II on the monocytic cell surface.…”