Study on the binding of lactotransferrin (lactoferrin) to human PHA-activated lymphocytes and non-activated platelets. Localisation and description of the receptor-binding site.
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 microM) and with two synthetic peptides: KRDS tetrapeptide (50% inhibition = 350 microM) and CFQWQRNMRKVRGPPVSC octodecapeptide (50% inhibition = 20 microM) corresponding to the lactotransferrin amino acid sequence 39-42 and 20-37, respectively.
Mazurier
J
J
Université des Sciences et Technologies de Lille, Laboratoire de Chimie Biologique (Unité Mixte de Recherche no 111 du CNRS, Villeneuve d'Ascq, France.
Legrand
D
D
Leveugle
B
B
Rochard
E
E
Montreuil
J
J
Spik
G
G
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D007781:Lactoferrin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D010835:Phytohemagglutinins / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism*
D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D010835:Phytohemagglutinins; D010975:Platelet Aggregation Inhibitors; D011956:Receptors, Cell Surface; C038340:lactoferrin receptors; D000244:Adenosine Diphosphate; D007781:Lactoferrin; D008239:Lysine