Bovine lactoferrin binds to insulin-like growth factor-binding protein-3.
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 125I-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 [125I]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 [125I]bLf to rhIGFBP-3 was shown, but [125I]bTf did not show binding to rhIGFBP-3. While [125I]rhIGF-II does not bind to bLf, unlabeled rhIGF-II was shown to compete with [125I]bLf for rhIGFBP-3 binding. More detailed analysis by dot blot showed that Lf competes (ED(50)=3 microg/ml) or displaces (ED(50)=1mg/ml) bound [125I]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 [125I]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.
Baumrucker
C R
CR
Department of Dairy and Animal Science, The Pennsylvania State University, 302 Henning Building, University Park, PA 16802, USA. crb@psu.edu
Gibson
C A
CA
Schanbacher
F L
FL
D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002417:Cattle / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004847:Epithelial Cells / Q000648:ultrastructure; D005260:Female; D006801:Humans; D007163:Immunosorbent Techniques; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D007457:Iodine Radioisotopes; D007781:Lactoferrin / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000648:ultrastructure; D051379:Mice; D008861:Microsomes / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology
D017077:Culture Media, Conditioned; D018972:Insulin-Like Growth Factor Binding Protein 3; D018975:Insulin-Like Growth Factor Binding Protein 5; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D014212:Tretinoin; D007335:Insulin-Like Growth Factor II; D007781:Lactoferrin