The N-terminal Arg(2), Arg(3) and Arg(4) of human lactoferrin interact with sulphated molecules but not with the receptor present on Jurkat human lymphoblastic T-cells

We previously characterized a 105 kDa receptor for human lactoferrin (hLf) on Jurkat human lymphoblastic T-cells. To delineate the role of the basic cluster Arg(2)-Arg(3)-Arg(4)-Arg(5) of hLf in the interaction with Jurkat cells, we isolated N-terminally deleted hLf species of molecular mass 80 kDa lacking two, three or four N-terminal residues (hLf(-2N), hLf(-3N) and hLf(-4N)) from native hLf that had been treated with trypsin. Native hLf bound to 102 000 sites on Jurkat cells with a dissociation constant (K-d) of 70 nM. Consecutive removal of N-terminal arginine residues from hLf progressively increased the binding affinity but decreased the number of binding sites on the cells. A recombinant hLF mutant lacking the first five N-terminal residues (rhLf(-5N)) bound to 17000 sites with a K-d of 12 nM, The binding parameters of bovine lactoferrin (Lf) and native hLf did not significantly differ, whereas the binding parameters of murine Lf (8000 sites; K-d 30 nM) resembled those of rhLf(-5N). Culture of Jurkat cells in the presence of chlorate, which inhibits sulphation, decreased the number of binding sites for both native hLf and hLf(-3N) but not for rhLf(-5N), indicating that the hlf-binding sites include sulphated molecules, We propose that the interaction of hLf with a large number of binding sites (approx. 80000 per cell) on Jurkat cells is dependent on Arg(2)-Arg(3)-Arg(4), but not on Arg(5). Interaction with approx. 20 000 binding sites per cell, presumably the hLf receptor, does not require the first N-terminal basic cluster of hLf Moreover, the affinity of hLf for the latter binding site is enhanced approx, 6-fold after removal of the first basic cluster. Thus N-terminal proteolysis of hLf in vivo might serve to modulate the nature of its binding to cells and thereby its effects on cellular physiology.