Bovine Lactoferrin Structures Promoting Corneal Epithelial Wound Healing In Vitro

PURPOSE. To use an in vitro alkali-induced wound model to identify structures of bovine lactoferrin (BLF) that contribute to the promotion of human corneal epithelial healing. METHODS. BLF N-lobe and C-lobe were separated using limited proteolysis and purified by preparative chromatography. Isoforms of BLF were separated according to serine protease activity. Catalytic activities of isoforms and lobes were quantified by hydrolysis of a synthetic serine protease substrate. The promotion of healing by cognate moieties, lactoferricin-B, BLF isoforms, and BLF in various forms-iron-free, iron-saturated, deglycosylated, zwitterionic detergent exposed, chaotrope denatured, disulfide reduced-was assessed on alkali wounding of confluent monolayers of human corneal epithelial cells. RESULTS. The C-lobe of BLF (6.4-128 mu M) promoted greater wound healing than native-BLF or N-lobe. BLF (12.8 mu M) promoted wound closure in an iron-free, iron-bound, or deglycosylated state or after exposure to zwitterionic detergent. Healing was not stimulated by chaotropically denatured or disulfide reduced BLF (12.8 mu M) or by lactoferricin-B (12.8 mu M). Proteolytically active BLF (0.6 mu M) promoted wound closure at a lower concentration than proteolytically inactive BLF (12 mu M). This proteolytic activity was localized to the N-lobe. CONCLUSIONS. The C-lobe is the primary promoter of BLF-stimulated corneal epithelial wound closure in vitro and is effective at concentrations >= 6.4 mu M. Increased healing from BLF occurs with the native conformation and is unaffected by glycosylation or iron saturation. To a lesser extent, proteolytic activity of the N-lobe also improves healing rates. The BLF C-lobe may be a novel treatment for corneal lesions with delayed healing. (Invest Ophthalmol Vis Sci. 2011; 52: 2719-2726) DOI:10.1167/iovs.10-6352