Human Cysteine Cathepsins Degrade Immunoglobulin G In Vitro in a Predictable Manner

Abstract

Cysteine cathepsins are critical components of the adaptive immune system involved in the generation of epitopes for presentation on human leukocyte antigen (HLA) molecules and have been implicated in degradation of autoantigens. Immunoglobulin variable regions with somatic mutations and random complementarity region 3 amino acid composition are inherently immunogenic. T cell reactivity towards immunoglobulin variable regions has been investigated in relation to specific diseases, as well as reactivity to therapeutic monoclonal antibodies. Yet, how the immunoglobulins, or the B cell receptors, are processed in endolysosomal compartments of professional antigen presenting cells has not been described in detail. Here we present in silico and in vitro experimental evidence suggesting that cysteine cathepsins S, L and B may have important roles in generating peptides fitting HLA class II molecules, capable of being presented to T cells, from monoclonal antibodies as well as from central nervous system proteins including a well described autoantigen. By combining neural net models with in vitro proteomics experiments, we further suggest how such degradation can be predicted, how it fits with available cellular models, and that it is immunoglobulin heavy chain variable family dependent. These findings are relevant for biotherapeutic drug design as well as to understand disease development. We also suggest how these tools can be improved, including improved machine learning methodology.

Keywords: B cell; antigen presenting cell; bioinformatics; cathepsin; endolysosome; endosome; in silico model; protease; protease cleavage prediction.

Figure 1

Peptide lengths resulting from in vitro cathepsin digestion of central nervous system proteins. Distribution of peptide lengths after digestion of alpha-synuclein (aSyn), recombinant myelin basic protein (rMBP) isoforms 2 and 6, and tau with either cathepsin B, L, or S at 6, 24, or 30 h at pH 6. Each data point represents one identified peptide at the given time point. Black lines with annotations indicate the mean size of peptides. Purple and green areas indicate peptide sizes fitting HLA class I and II, respectively. * aSyn 6-h sample for cathepsin L was lost due to technical error.

Figure 2

Comparison of predicted and observed cleavage of CNS proteins. All potential cleavage site octamers (CSOs) within alpha-synuclein, recombinant myelin basic protein isoforms 2 and 6, and tau were binned into ranges of 0.2 based on the predicted cleavage probability (X-axis). Intra-protein z-standardized number of observed cuts after 24 h at corresponding CSOs are depicted on the Y-axis. The p-values indicate Welch ANOVA significance for cathepsin B/L/S (F(4, 1.53/13.03/12.24)) and differing letters indicate binned groups that have significant difference in mean number of observed cleavages (Tukey–Kramer, HSD). Whiskers are outlier box-plots.

Figure 3

Digestion of monoclonal antibodies at pH 6 by cathepsins S, L and B. Detected peptides after digestion of 1200 nM alemtuzumab, rituximab, natalizumab, or 2400 nM adalimumab, infliximab, or ocrelizumab with either cathepsin B, L, or S at 6, 24, or 30 h at pH 6. (A) Cathepsin S yields significantly more detectable peptides than cathepsins L and B at pH 6, after 24 h of incubation. The bars indicate average number of peptides detected. Significance as determined by ANOVA testing and Tukey–Kramer HSD (different red letters indicate significant difference between groups). (B) Distribution of peptide lengths (x-axis). Each data point represents one identified peptide at the given time point. Black lines with annotations indicate the mean size of peptides. Purple and green areas indicate peptide sizes fitting HLA class I and II, respectively. The length range is cropped to display 99% of the peptides.

Figure 4

Observed pattern of cathepsin S cuts in monoclonal antibodies. The monoclonal antibodies adalimumab, alemtuzumab, infliximab, natalizumab, ocrelizumab, and infliximab were incubated with cathepsin s for 24 h at pH 6. Non-standardized number of observed cuts in light (A/B) and heavy chains (C/D) identified by nano-liquid chromatography mass spectrometry. Cuts are presented by their location in sequence (A/C) or summarized by region (B/D). For alignment purposes, the variable region position is assigned by the relative position of P1′ in the cleavage site octamer to the cysteine (0) of CDR3. The constant regions are aligned to start at position 30.

Figure 5

Detected peptides overlap with predicted cleavage sites. Predicted cleavage probability (x-axis) by cathepsin S in variable (upper panel) and constant heavy 2 (CH2) (lower panel) region of alemtuzumab. The vertical bars indicate the predicted position of P1′ of a P1-P1′ cleavage bond, and thus the first amino acid after a cut. Horizontal bars each indicate unique peptides detected starting at a P1′ and ending at a P1, as identified by nLCMS after 6 (blue), 24 (green), and 30 (purple) hours.

Figure 6

Evaluation of cleavage accuracy for monoclonal antibody variable regions. Cleavage probability by cathepsin S for all possible cleavage site octamers (CSOs) within (A) heavy and (B) light chain variable regions of rituximab, infliximab, ocrelizumab, natalizumab, alemtuzumab, and adalimumab were binned into ranges of 0.2 (X-axis). Intra-chain z-standardized number of observed cuts after 24 h at pH 6 are depicted on the Y-axis. p-values indicate Welch ANOVA significance (F(4, 5.16/9.05) for heavy and light respectively), and differing red letters indicate significant differences between groups (Tukey–Kramer, HSD). Whiskers are outlier box-plots.

Figure 7

Adalimumab digestion by cathepsin S, L and B at pH 4, 5, and 6. Distribution of peptide lengths after digestion of 2400 nM adalimumab with either cathepsin B, L, S at 6, 24, or 30 h at pH 4, 5, or 6. Each data point represents one identified peptide at the given time point. Black lines with annotations indicate the mean size of peptides. Purple and green areas indicate peptide sizes fitting HLA class I and II, respectively. (Note: For pH 6, the data for cathepsins S and B are the same as in Figure 3B).

Figure 8

Predicted cleavage patterns of IGHV families using GenBank IGHV set. Approximately 16,000 curated IGHV sequences were divided by their V-family and analyzed with the cathepsin cleavage models. All possible cleavage site octamers (CSO) for each IGHV were aligned according the relative position of P1′ to the CDR3 (yellow) region cysteine (x-axis). Mean predicted probability for CSO cleavage at each position by cathepsin S (A) or cathepsin B (B) is shown on the y-axis. Superimposed are aligned IGHV peptides described by Khodadoust et al. (31), eluted from HLA class II of mantle cell lymphoma: MCL065 (purple) and MCL052 (red).