Diverse Proteases Cleave Lactoferrin at Single Site, Yielding Stable C-Lobe with Prolonged Antibacterial Action

Lactoferrin is a 78 kDa iron-binding glycoprotein with established antibacterial properties, but its full-length form can be susceptible to rapid degradation by digestive enzymes, limiting its therapeutic potential for sustained action. The challenge lies in developing stable, active fragments that retain efficacy in harsh biological environments. Its two-lobe structure, particularly the C-lobe, has shown promise, but the precise mechanism of its proteolytic generation and stability for prolonged antibacterial activity needed elucidation. This study investigates how specific, single-site cleavage yields a stable, potent C-lobe.

Researchers performed limited proteolysis of full-length lactoferrin (78 kDa, Ala1Arg689) using chymotrypsin. The resulting fragments were analyzed, and the structure of the generated C-lobe was determined. This was compared to previous findings where proteinase K, trypsin, and pepsin also produced an identical C-lobe. The stability of the proteolytically generated C-lobe, with its observed glycosylation sites, was assessed over 3 days in the presence of digestive enzymes, and its iron-sequestering and antibacterial properties were monitored.

Limited proteolysis of lactoferrin by chymotrypsin specifically generated a 40 kDa, fully functional C-lobe. Structural determination revealed this C-lobe consisted of residues from Thr343 to Leu680, together with a disulfide-linked tripeptide (Ala683Cys684Ala685). The cleavage occurred precisely at the Tyr342Thr343 peptide bond within the 11-residue inter-lobe linker (Thr334Arg344).