title: Removal of 14 N-terminal amino acids of lactoferrin enhances its affinity for parenchymal liver cells and potentiates the inhibition of beta- very low density lipoprotein binding.
abstract: Lactoferrin inhibits the hepatic uptake of lipoprotein remnants, and we showed earlier that arginine residues of lactoferrin are involved. In this study, lactoferrin was treated with aminopeptidase M (APM), which resulted in removal of 14 N-terminal amino acids, including 4 clustered arginine residues at positions 2-5 (APM-lactoferrin). After intravenous injection into rats, 125I-labeled APM-lactoferrin was cleared within 10 min by the liver parenchymal cells (74.7% of the dose). In contrast to native lactoferrin, APM-lactoferrin was rapidly internalized after liver association (> 80% of the liver-associated radioactivity was internalized within 10 min). Binding of APM-lactoferrin to isolated parenchymal liver cells was saturable with a Kd of 186 nM (750,000 sites/cell). This is in striking contrast to the binding of native lactoferrin (Kd 10 microM; 20 x 10(6) sites/cell). Preinjection of rats with 20 mg of APM-lactoferrin/kg of body weight reduced the liver association of beta-very low density lipoprotein (beta-VLDL) by 50%, whereas lactoferrin had no effect at this dose. With isolated parenchymal liver cells, APM-lactoferrin was a more effective competitor for beta-VLDL binding than native lactoferrin (50% inhibition at 0.5 mg/ml versus 8.0 mg/ml). Selective modification of the arginines of APM-lactoferrin with 1,2-cyclohexanedione reduced the liver association by approximately 60% and abolished the capacity of APM-lactoferrin to inhibit the binding of 125I-labeled beta-VLDL in vitro. In conclusion, our data indicate that the four-arginine cluster of lactoferrin at positions 2-5 is involved in its massive, low affinity association of lactoferrin with the liver, possibly to proteoglycans, but is not essential for the inhibition of lipoprotein remnant uptake. The Arg-Lys sequence at positions 25-31, which resembles the binding site of apolipoprotein E, may mediate the high affinity binding of lactoferrin and block the binding of beta-VLDL to the remnant receptor with high efficiency.
authors.affiliation: Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, University of Leiden, The Netherlands.
authors.forename: G J
authors.initials: GJ
authors.lastname: Ziere
authors.forename: M K
authors.initials: MK
authors.lastname: Bijsterbosch
authors.forename: T J
authors.initials: TJ
authors.lastname: van Berkel
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D001057:Apolipoproteins E / Q000378:metabolism; D018826:CD13 Antigens; D002478:Cells, Cultured; D006801:Humans; D007781:Lactoferrin / Q000097:blood / Q000378:metabolism*; D008079:Lipoproteins, VLDL / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D017384:Sequence Deletion
chemical_list: D001057:Apolipoproteins E; D008079:Lipoproteins, VLDL; D010446:Peptide Fragments; D000626:Aminopeptidases; D018826:CD13 Antigens; D007781:Lactoferrin