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.
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.
Ziere
G J
GJ
Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, University of Leiden, The Netherlands.
Bijsterbosch
M K
MK
van Berkel
T J
TJ
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
D001057:Apolipoproteins E; D008079:Lipoproteins, VLDL; D010446:Peptide Fragments; D000626:Aminopeptidases; D018826:CD13 Antigens; D007781:Lactoferrin