Influence of N- and O-glycosylation on structural properties and biological activity of a C-terminal LL-37 fragment.

Glycosylation represents a versatile strategy in peptide glycoengineering to modulate the structural, physicochemical, and biological properties of antimicrobial peptides (AMPs). In this study, we report the synthesis and characterization of O- and N-glycosylated analogues of the C-terminal fragment of human cathelicidin LL-37 (Ac-DFLRNLVPRTES-COOH), achieved via solid-phase peptide synthesis using Fmoc-Thr(β-d-Glc) and Fmoc-Asn(β-d-Glc) as glycosylated building blocks. The metal-binding affinity of these glycopeptides (N163 and T168) and the unmodified reference peptide (hCAP) toward Cu2+ and Mn2+ ions were evaluated by steady-state fluorescence spectroscopy. All peptides formed non-permanent complexes (Ka = 102 -104 M-1), with the native hCAP exhibiting the highest affinity. Molecular dynamics (MD) simulations revealed that glycosylation reduced conformational flexibility without significantly altering the overall shape of the peptide. Occasional sugar-metal contacts were observed, indicating transient carbohydrate participation in metal coordination. Cytotoxicity studies using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays confirmed the biocompatibility of all tested peptides, showing low toxicity against human fibroblasts, keratinocytes, and T-cells up to 100 μM. Our results demonstrate that carbohydrate conjugation subtly reshapes peptide-metal ion interactions, and structural dynamics. In this study, glycosylation serves primarily as a chemical model of post-translational modification, designed to probe how the presence of a carbohydrate moiety influences peptide conformation and coordination behaviour, rather than a strategy for enhancing antimicrobial function.