Bovine lactoferrin binds to insulin-like growth factor-binding protein-3

Abstract

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) has been shown to have IGF independent actions that appear to be mediated by specific IGFBP-3 binding proteins located on cell membranes. We show here using Western ligand blotting, a number of mammary membrane proteins that bind 125 I -labeled rhIGFBP-3. Immunoprecipitation studies demonstrated that the >70 kDa protein was identified from bovine mammary microsomes as bovine lactoferrin (bLf). In addition to being a secretory protein, Lf is tightly associated with cellular membranes. Labeled rhIGFBP-3 was shown to bind to commercially purchased and processed apo- or holo-human or bLf, but not bovine transferrin (bTf). Binding of [ 125 I ]rhIGFBP-3 to other positively charged proteins was not detected nor was binding to rhIGFBP-5 or other mammary-secreted IGFBPs observed. Reciprocal specific binding of [ 125 I ]bLf to rhIGFBP-3 was shown, but [ 125 I ]bTf did not show binding to rhIGFBP-3. While [ 125 I ]rhIGF-II does not bind to bLf, unlabeled rhIGF-II was shown to compete with [ 125 I ]bLf for rhIGFBP-3 binding. More detailed analysis by dot blot showed that Lf competes (ED 50=3 μg/ml) or displaces (ED 50=1 mg/ml) bound [ 125 I ]rhIGF-II from dot blotted rhIGFBP-3. In vitro studies with a bovine primary mammary epithelial cell culture showed that all- trans-retinoic acid stimulates the appearance of bovine IGFBP-3 and bLf in the conditioned media and that [ 125 I ]rhIGFBP-3 could be utilized to detect conditioned media bLf. These findings reveal a novel role for bLf, binding to IGFBP-3 and perhaps disassociating IGFBP-3:IGF when in high concentration.