Motif-based zwitterionic peptides impact their structure and immunogenicity.

The linkage of zwitterionic peptides containing alternating glutamic acid (E) and lysine (K) amino acids exhibits protective effects on protein drugs due to their high hydration capacity. Previously, short EK peptides covering the surface of a protein drug showed significant protective effects and low immunogenicity. However, for high-molecular-weight single-chain (HMWSC) zwitterionic peptides, the incorporation of structure-disrupting amino acids such as proline (P), serine (S), and glycine (G) is necessary to improve their protective ability. Herein, we first probe the immunogenicity of eight EK-containing motif-based peptides, six of which incorporate structure-disrupting amino acids P, S, and G, linked to keyhole limpet hemocyanin (KLH). These studies uncover two sequence motifs, EKS and EKG, which show uniquely higher immunogenicity, while the other motifs, especially those containing P, exhibit lower immunogenicity. Additionally, the structure and dynamics of these sequence motifs are computationally modeled by Rosetta protein predictions and molecular dynamics (MD) simulations to predict properties of higher and lower immunogenicity peptides. These simulations revealed peptides with higher immunogenicity, namely EKS and EKG, exhibit regions of charge imbalance. Then, HMWSC zwitterionic sequences were linked to a typical protein drug, interferon-alpha 2a (IFN), which showed consistent immunogenic behaviors. Finally, epitope mapping and alanine scanning experiments using the serum collected from mice injected with HMWSC sequences also implicated a link between charge imbalance and peptide immunogenicity.