Cathepsin S and an asparagine-specific endoprotease dominate the proteolytic processing of human myelin basic proteinin vitro

Abstract

The biochemical characterization of antigen degradation is an important basis for a better understanding of both the immune response and autoimmune diseases mediated by MHC class II molecules.In this study we used high‐performance liquid chromatography and mass spectrometry to analyze the processing of myelin basic protein (MBP), a potential autoantigen implicated in the pathogenesis ofmultiple sclerosis. We resolved the kinetics of MBP processing by lysosomal extracts or purified endocytic proteases, identified the major cleavage sites during this process and assigned them to the activity of proteolytic enzymes. Proteolytic processing of MBP is mostly guided along the hydrophobic regions of the protein. It is initiated by two proteolytic steps (after N92 and S110) that are performed by an asparagine‐specific endopeptidase (AEP) and by cathepsin (Cat) S, respectively. The resulting processing intermediates are converted into more than 60 different species of 20–40‐mers due to the activity of endopeptidases including CatS, D and L. The fragments thus generated are subsequently degraded by C‐ or N‐terminal trimming. Strikingly, the initial cleavages during MBP processing affect two immunodominant regions of the potential autoantigen [MBP(85–99) and MBP(111‐129)] in an inverse manner. CatS directly generates the N terminus of the epitope MBP(111–129)in large quantities during the initial phase of processing, which might explain the immunogenicity of this region in spite of its relatively poor binding to HLA‐DR4. In contrast, the dominant cleavage by AEP mediates the destruction of MBP(85–99) unless the epitope is protected, e.g. by binding to HLA‐DR. Our results thus characterize the proteolytic events during processing of MBP on a molecular level and suggest a biochemical basis for the immunogenicity of the immunodominant epitopes, which could serve as a guideline for future therapeutic strategies.

Dual inhibition of proteasomal and lysosomal proteolysis ameliorates autoimmune central nervous system inflammation

“…It is involved in both the intracellular breakdown of li [9] and proteolytic processing of different antigens including myelin basic protein, a potential autoantigen implicated in the pathogenesis of MS [33]. CatS activity is required for induction of the MHC II-mediated collagen-induced arthritis [34].…”

Asparagine Deamidation Perturbs Antigen Presentation on Class II Major Histocompatibility Complex Molecules

“…However, it is possible that loss of processing sites (for AEP or other enzymes) as a result of post-translational protein modifications might enhance the presentation of T cell epitopes in other antigens/autoantigens. This idea is supported by recent evidence that proteolytic processing reactions, for both class I and class II major histocompatibility complex epitopes, can be destructive as well as productive (25)(26)(27)). An example of this, which also illustrates how direct and indirect effects of Asn deamidation on immune recognition can be linked, is seen in the case of another AEP substrate, myelin basic protein (MBP).…”

“…An example of this, which also illustrates how direct and indirect effects of Asn deamidation on immune recognition can be linked, is seen in the case of another AEP substrate, myelin basic protein (MBP). A T cell epitope lying between residues 85-99 in MBP is destroyed by AEP cleavage at Asn-94 (25,26). We have shown that presentation of this peptide to MBP-specific T cells is inversely proportional to AEP activity and have suggested that T cells specific for this region of MBP escape tolerization in the thymus because of this destructive processing reaction (26).…”

Lysosomal Cysteine and Aspartic Proteases Are Heterogeneously Expressed and Act Redundantly to Initiate Human Invariant Chain Degradation

“…The requirement for AEP in tetanus presentation is a robust example of enzyme-restricted Ag processing. Similarly, AEP-mediated processing of MBP has been shown to destroy an asparagine-containing immunodominant epitope, MBP peptide 85-99 (6,19). Because MBP-derived epitopes are presented intrathymically and can contribute to self-tolerance against MBP, the questions of how and whether such peptides are generated are of great importance.…”

“…Blockade of the progressive cleavage of Ii results in the accumulation of Ii intermediates and a corresponding decrease in surface expression of class II MHC products (4,5). Experiments using human B cells treated with the cathepsin S (CatS)-specific inhibitor leucine-homophenylalanine-vinyl sulfone (LHVS) (6,7) and characterization of CatS knockout mice (2,8,9) have implicated CatS in the terminal cleavage of Ii to yield CLIP (10). Further studies have demonstrated cell-specific cleavage of p10 by cathepsin L (CatL) (11) in thymic epithelial cells (8) and cathepsin F (CatF) in macrophages (9).…”