Study on the binding of lactotransferrin (lactoferrin) to human PHA- activated lymphocytes and non-activated platelets: Localisation and description of the receptor-binding site

Abstract

Fluorescein isothiocyanate derivatization of human lactotransferrin on Lys-264 as well as covalent addition of sulfosuccinimidyl 2-(p- azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD)* on Lys-74 inhibits the binding of the glycoprotein to both human PHA-activated lymphocytes and non- activated platelets. This suggests that the cell binding site of lactotransferrin is located in the vicinity of the lysine residues 74 and 264 and does not occur either through electrostatic or lectin interactions. In contrast, the derivatization of lactotransferrin using sulfosuccinimidyl 6- (4'-azido-2'-nitrophenyl-amino) hexanoate (sulfo-SANPAH), on Lys-281 does not modify the binding parameters of lactotransferrin to the cells. Molecular modeling showed the position of SASD, sulfo-SANPAH and fluorescein molecules at the surface of the protein and suggested that SASD and fluorescein could mask the two loop-containing regions of human lactotransferrin (residues 28- 34 and 38-45). Elsewhere, a 6 kDa peptide covering the peptide chain from residues 4 to 52 was isolated and its inhibitory effect on the binding of lactotransferrin to both human PHA-activated lymphocytes and non-activated platelets was demonstrated. Inhibition of ADP-induced platelet aggregation by lactotransferrin (50% inhibition = 10 nM) was also found with the N-t fragment of lactotransferrin (residues 3-281; 50% inhibition = 2 μM) and with two synthetic peptides: KRDS tetrapeptide (50% inhibition = 350 μM) and CFQWQRNMRKVRGPPVSC octodecapeptide (50% inhibition = 20 μM) corresponding to the lactotransferrin amino acid sequence 39-42 and 20-37, respectively.