Study on the Binding of Lactotransferrin (Lactoferrin) to Human PHA-Activated Lymphocytes and Non-Activated Platelets

Summary

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 & 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.