Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions

Abstract

| Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost-and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.

Antigen Processing and Presentation in Multiple Sclerosis

“…By binding directly to MHC in the absence of costimulation, they are expected to induce tolerance to the epitope. Some groups have attempted to increase stability of the tolerizing peptide by circularising it or changing key residues to eliminate proteolytic cleavage sites (Burster et al 2007b;Tselios et al 2002).…”

Processing and presentation of (pro)‐insulin in the MHC class II pathway: the generation of antigen‐based immunomodulators in the context of type 1 diabetes mellitus

“…Interestingly, exchanging one amino acid at the TCR contact site (F to P, red letter) of the MBP-derived APL (MBP85-99 Mu1) is sufficient to reduce T cell proliferation by 50% using the MBP86-98-reactive T cell clone. The binding capacity of these APL is decreased up to 75% after the incorporation of Q for N (blue letter) [57]. Overall, the functional outcome of the peptide/MHC-complex can be dramatically altered by the introduction of a single amino acid substitution.…”

Cathepsin A is expressed in primary human antigen-presenting cells

“…These factors may account for the variations in peptide digestion that were observed in the different studies. In our studies, the lack of CatA-mediated digestion of the proline rich peptide, MBPMu4, which is not digested by endoproteases [30], supports the intolerance for a proline residue in the P1 and P1 position [19]. Analysis of the peptides DCins8 or DCins10, however, identified the release of single amino acids by recombinant CatA or BLC-derived lysosomal cathepsins.…”

“…The peptide substrates MBPMu4 and DCins10 were then added to each of the reactions. The MBPMu4 peptide was chosen because its endoprotease cleavage sites were eliminated, as previously described [30]. The resulting fragments were then identified by HPLC and mass spectrometry.…”

“…VVHPFPPIVTPPPP (MBPMu4, [30]), FVNQHLCGSHLV (DCins8), and FVNQHLCGSHLVEAL (DCins10) peptides were synthesized by the solid phase Fmoc strategy on the multiple peptide synthesizer Syro II (MultiSynTech, Witten, Germany). Activation and coupling were performed with TBTU, HOBT, and NMM (Merck, Darmstadt, Germany).…”