Isolated rat hepatocytes differentially bind and internalize bovine lactoferrin N- and C-lobes Available to Purchase

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 (K_d approx. 75 nM) and to nearly identical extents (approx. 10⁶ 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 (k_in=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 (K_d = 90 nM, ⩽ 5×10⁶ 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.