Isolated rat hepatocytes differentially bind and internalize bovine lactoferrin N- and C-lobes.
Isolated rat hepatocytes bind and internalize bovine lactoferrin (Lf) and its bound iron in a Ca2+-dependent manner. In this study, we determined if one or both halves of Lf (N- and C-lobes) were responsible for the interaction of Lf with hepatocytes. We isolated three tryptic fragments of bovine Lf. Cleavage at Arg284-Ser285 generated two fragments: N-terminal pp36 that contained 80% of Lf N-lobe and C-terminal pp51. A second cleavage at Arg338-Ala339 generated a smaller fragment (pp44) that contained all of the C-lobe with no N-lobe elements. Hepatocytes bound Lf and pp51 in a Ca2+-dependent manner with the same affinity (Kd approx. 75 nM) and to nearly identical extents (approx. 10(6) sites per cell). Lf and pp51 competed with each other for binding to cells over a similar titration range. Hepatocytes internalized Lf at a faster rate than pp51 (kin=0.28 and 0.19 min-1 respectively), but cells degraded pp51 at approx. twice the rate of native Lf. pp44 competed with 125I-labelled Lf for binding to Ca2+-dependent binding sites on hepatocytes as well as native Lf or pp51. In contrast, hepatocytes bound pp36 (Kd=90 nM, <=5x10(6) sites per cell) but did not internalize or degrade it appreciably. Moreover, pp36 binding to cells was not Ca2+-dependent, and pp36 competed poorly with native Lf and pp51 for binding to cells. We conclude from these findings that the Lf determinants responsible for binding to the Ca2+-dependent receptor on hepatocytes is present within the C-lobe of Lf.
Sitaram
M P
MP
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556-5645, USA.
McAbee
D D
DD
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D016264:Dextran Sulfate / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004705:Endocytosis; D007501:Iron / Q000378:metabolism; D007781:Lactoferrin / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000378:metabolism*
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C038340:lactoferrin receptors; D004533:Egtazic Acid; D016264:Dextran Sulfate; D007501:Iron; D007781:Lactoferrin; D002118:Calcium