Human lactotransferrin receptors have been recently characterized on mitogen-stimulated human lymphocytes [(1989) Eur. J. Biochem. 179, 481-487]. In order to define the lactotransferrin recognition site by these receptors, the binding to lymphocytes of several tryptic fragments, isolated from human lactotransferrin by mild tryptic hydrolysis [(1984) Biochim. Biophys. Acta 787, 90-96]; has been investigated. The 30 kDa N-tryptic fragment (residues 4-281) and the re-associated N,C-tryptic complex bind to lactotansferrin lymphocyte receptor with a dissociation constant of 44 nM and 39 nM, respectively, similar to the value obtained for the native lactotransferrin (Kd = 46 riM). However, neither the N-terminal domain II (residues 91-257) nor the 50 kDa C-tryptic fragment (residues 282-703) are recognized. These results suggest that the binding site of human lactotransferrin by the lymphocyte receptor is located in the N-terminal lobe and more precisely in the N-terminal domain I (residues 4-90 and/or 258-281).

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.

Labelling of human lactotransferrin with fluorescein 5'-isothiocyanate (FITC) in an equimolar ratio inhibits the binding of the protein to phytohaemagglutinin-activated human peripheral-blood lymphocytes. Therefore it can be assumed that FITC reacts at, or near, the receptor-binding site. Three FITC-labelled peptides have been purified from a tryptic digest of the FITC-labelled lactotransferrin. The determination of their amino acid sequence and their localization on the primary structure of the protein permitted the identification of two FITC-accessible areas in the N-terminal lobe and one in the C-terminal lobe. In fact, only 10% of the total FITC was conjugated to one lysine residue (Lys579) of the C-terminal lobe, whereas most (80%) of the FITC was conjugated to three close lysine residues [Lys263 (65% of total fluorescence), Lys280 and Lys282 (15% of total fluorescence)] located in beta-turn structures, of the N-terminal domain I of human lactotransferrin. The results obtained show that the receptor-binding site should be located in the vicinity of the FITC-accessible Lys263, Lys280 and Lys282, and corroborate our preliminary results reporting the involvement of the N-terminal domain I in the binding of human lactotransferrin to mitogen-stimulated lymphocytes [Rochard, Legrand, Mazurier, Montreuil & Spik (1989) FEBS Lett. 255, 201-204]. In any case, FITC labelling is not suitable for studying the binding of lactotransferrin to activated lymphocytes and its use may lead to erroneous interpretations of cell binding experiments.

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