Molecular interactions between human lactotransferrin and the phytohemagglutinin-activated human lymphocyte lactotransferrin receptor lie in two loop-containing regions of the N-terminal domain I of human lactotransferrin.
Fluorescein isothiocyanate derivatization of the human lactotransferrin on Lys-264 inhibits the binding of the protein of human PHA-activated lymphocytes [Legrand, D., Mazurier, J., Maes, P., Rochard, E., Montreuil, J., & Spik, G. (1991) Biochem. J. 276, 733-738], indicating that part of the receptor-binding site is located in the N-terminal domain I of lactotransferrin. In the present study, a 6-kDa peptide (residues 4-52) was isolated from the N-terminal lobe of human lactotransferrin which inhibited the binding of the protein to its cell receptor. In addition, lactotransferrin was derivatized using sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) and sulfosuccinimidyl 6-((4'-azido-2'-nitrophenyl)amino)hexanoate (sulfo-SANPAH), two heterobifunctional reagents generally used for receptor-ligand cross-linking. The azide group of these two reagents was inactivated by photolysis, and only the succinimidyl ester group was allowed to react with lysine residues of the protein. The binding of the derivatized lactotransferrins to the human lymphocyte receptor was assayed. SASD, which binds to Lys-74, was able to inhibit the binding of lactotransferrin to the cell receptor, in contrast to Lys-281-binding sulfo-SANPAH. 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 residues 4-6 and two loop-containing regions of human lactotransferrin (residues 28-34 and 38-45). The comparison of the primary and tertiary structures of human lactotransferrin and serotransferrin, which bind to specific cell receptors, shows that the above-mentioned regions, which are likely involved in protein-receptor interactions, possess specific structural features.
Legrand
D
D
Laboratoire de Chimie Biologique, Université des Sciences et Techniques de Lille Flandres-Artois, Villeneuve d'Ascq, France.
Mazurier
J
J
Elass
A
A
Rochard
E
E
Vergoten
G
G
Maes
P
P
Montreuil
J
J
Spik
G
G
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007700:Kinetics; D007781:Lactoferrin / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000502:physiology*; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010835:Phytohemagglutinins; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*
D010446:Peptide Fragments; D010835:Phytohemagglutinins; D011956:Receptors, Cell Surface; C038340:lactoferrin receptors; D007781:Lactoferrin; D016650:Fluorescein-5-isothiocyanate