i. Peptide Slicing¶

Peptides of length 9–12 amino acids were generated from both the selected human protein and the microbial proteome. These peptides were used as input for downstream binding prediction and molecular mimicry screening. To balance biological relevance and broad coverage, two complementary peptide slicing strategies were applied:

Rule-free peptide slicing
Anchor-rule-based peptide slicing

The rule-free approach ensures broad exploration of the peptide space by generating all possible 9–12-mer peptides without applying any binding constraints. This allows potential mimicry candidates to be identified without bias. On the other hand, the anchor-rule-based approach applies known HLA-B27 anchor residue preferences to enrich for peptides with a higher likelihood of binding to the MHC groove.

Both strategies were applied consistently to human and microbial proteins, ensuring that all peptides were generated and evaluated under identical rules. This combined approach increases the robustness and credibility of downstream binding prediction and similarity analysis.

Hint

Why 9–12 mers?

HLA class-I molecules, including HLA-B27, preferentially bind short peptides presented in the antigen-binding groove. Among these, 9-mer peptides are the most commonly observed binders, while 10–12-mer peptides are also accommodated with minor conformational flexibility. Therefore, peptides of length 9–12 amino acids were selected to capture canonical binders while allowing limited variability Tipu (2013), .

What are anchor residues?

Anchor residues are specific amino acids at defined positions within a peptide that enhance binding to the HLA groove by fitting into conserved binding pockets. For HLA-B27, a positively charged residue (Arg or Lys) is typically preferred at position 2, and a hydrophobic or aromatic residue is favored at the C-terminal position (PΩ). These anchor preferences increase the likelihood of stable peptide–MHC interactions Barnea et al. (2017).

Peptide Slicing Rules¶

a. Rule-Free Peptide Slicing

In this approach, peptides were generated using a simple sliding window method without applying any positional constraints. This strategy allows unbiased sampling of all possible 9–12 mer peptides from the protein sequences. The goal of this method was to ensure that no potential mimicry candidate was missed during the initial screening.

b. Anchor-Rule-Based Peptide Slicing

In this approach, peptides were generated or sliced according to known HLA-B27 anchor residue preferences rule. This method enriches for peptides that are more likely to bind to HLA-B27 and anchor rules also helps focus the analysis on biologically plausible binders while reducing the total search space.

References¶

Tipu, H. (2013). Mini Review: HLA B27 and its Immunogenetics in Ankylosing Spondylitis. Journal of Genetic Disorders and Genetic Reports, 02. 10.4172/2327-5790.1000104
Barnea, E., Melamed Kadosh, D., Haimovich, Y., Satumtira, N., Dorris, M. L., Nguyen, M. T., Hammer, R. E., Tran, T. M., Colbert, R. A., Taurog, J. D., & Admon, A. (2017). The Human Leukocyte Antigen (HLA)-B27 Peptidome in Vivo, in Spondyloarthritis-susceptible HLA-B27 Transgenic Rats and the Effect of Erap1 Deletion*. Molecular & Cellular Proteomics, 16(4), 642–662. https://doi.org/10.1074/mcp.M116.066241