Structural Basis for Single-Site Cleavage of Lactoferrin by Diverse Proteases for Prolonged Antibacterial Action: Structure of the Chymotrypsin-Cleaved Lactoferrin C-Lobe.

The stable lactoferrin C-lobe offers strong potential for therapeutic applications as an antibacterial agent. Lactoferrin is a 78 kDa (Ala1Arg689) iron-binding glycoprotein which is composed of two homologous N- and C-lobes, connected by an 11-residue α-helical linker (Thr334Arg344). The limited proteolysis of lactoferrin, carried out using chymotrypsin, generated a 40 kDa, fully functional C-lobe. The structure determination revealed that the protein chain consisted of residues from Thr343 to Leu680 together with a disulfide-linked tripeptide, Ala683Cys684Ala685. It showed that the cleavage occurred specifically at the Tyr342Thr343 peptide bond within the inter-lobe 11-residue-long peptide. Remarkably, previous studies using proteinase K, trypsin, and pepsin also produced an identical C-lobe. Thus, the inter-lobe region seems to be stereochemically designed by nature for the single-site cleavage by multiple digestive enzymes. The proteolytically generated C-lobe, with three observed glycosylation sites, remains stable for 3 days in the presence of digestive enzymes. The stable C-lobe continues to sequester iron, thus showing a prolonged antibacterial property. This is a unique example of evolutionary convergence whereby multiple digestive enzymes cleave a native protein into a stable half molecule with full antibacterial action.