Biochemistry zyxwvu 1992, 31, zyxwvu 9243-9251 9243 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? Dominique Legrand,* Joel Mazurier,* Abdelaziz Elass,# Elisabeth Rochard,* G6rard Vergoten,s Pierrette Maes,ll Jean Montreuil,* and Genevihve Spik’J Laboratoire de Chimie Biologique de I’UniversitD des Sciences et Techniques de Lille Flandres-Artois, Unite Mixte de Recherche no. I I I du CNRS, zyxwvuts 59655 Villeneuve d’Ascq Cedex, France, Laboratoire de Genie Biologique et Medical, Unite INSERM U279. Facultd de Pharmacie. rue Laguesse, 59045 Lille, France, and Service de Chimie des Biomolecules de l’lnstitut Pasteur de Lille, Unite Associee au CNRS 0-1309, I rue Calmette, 59109 Lille, France Received January IO, 1992; Revised Manuscript Received April 17, I992 ABSTRACT: Fluorescein isothiocyanate derivatization of the human lactotransferrin zyxw on Lys-264 inhibits the binding of the protein to human PHA-activated lymphocytes [Legrand, D., Mazurier, J., Maes, P., Roch- ard, E., Montreuil, J., zyxwvuts & Spik, G. (1991) Biochem. J. 276,733-7381, 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-@-azidosalicylamido)ethyl-l,3~-dithiopropionate (SASD) and sulfosuccinimidyl 6-((4/-azido-2/-nitro- pheny1)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. Human lactotransferrin (also called lactoferrin) is a bi- lobed iron-binding glycoprotein present in milk (Montreuil et al., 1960a,b; Johansson, 1960),in external secretions (Biserte et al., 1963; Masson, 1970),and in neutrophils (Masson et al., 1969). The primary structure of human lactotransferrin was first determined by Metz-Boutigue et al. (1984) and then by cDNAcloning (Powell & Ogden, 1990;Rey et al., 1990).The 3D structure of the protein is now well elucidated (Anderson et al., 1987, 1989, 1990; Baker et al., 1987). In the present paper, the molecular interactions between human lactotransferrin and the lactotransferrin receptor evidenced at the surface of the human phytohemagglutinin- stimulated peripheral blood lymphocytes (Mazurier et al., ~ f This work was supported in part by the Universitd des Sciences et Technologiesde Lille, the MinistCe de I’Education Nationale, the Cen- tre National de la Recherche Scientifique [Unite Mixte de Recherche du CNRS no. 11 1 (Director, Prof. A. Verbert) and Unit6 AssociC au CNRS D-1309 (Director, Prof. A. Tartar)], the Institut National de la Santt et de la Recherche M€dicale [Unit6 INSERM no. 279 (Director, Dr. Moschetto)] and INSERM Grant 882016, the Association pour la Recherche sur le Cancer, and the Institut Pasteur de Lille. * To whom correspondence should be addressed. Laboratoire de Chimie Biologique de l’Universit6 des Sciences et Techniques de Lille Flandres-Artois, Unit6 Mixte de Recherche no. 111 du CNRS. LaboratoiredeChimieBiologiqueetMMical, Unit6 INSERM U279, Facultd de Pharmacie. 11 Service de Chimie des Biomol€cules de I’Institut Pasteur de Lille, Unit6 AssociC au CNRS D-1309. 1989) were investigated. Preliminary results using large protein fragments obtained by tryptic hydrolysis of human lactotransferrin (Legrand et al., 1984, 1986) demonstrated that the N-terminal domain I (residues 1-91 and 251-319, numbered according to Powell and Ogden (1990) and Rey et al. (1990)) of the protein could interact with the mitogen- stimulated lymphocyte receptor (Rochard et al., 1989). Furthermore, we recently showed that preferential FITC’ de- rivatization of lactotransferrin on Lys-264 prevented binding of the protein to the lymphocyte receptor (Legrand et al., 1991). The direct involvement of Lys-264 in receptor binding remained uncertain, and because of the volume occupied in space by the fluorescein molecule, a large area of the N- terminal domain I of lactotransferrin could be masked by the fluorescent marker. In the present study, further proteolysis of the 30-kDa N- tryptic fragment of the protein (Legrand et al., 1984, 1986) was performed and a 6-kDa peptide (residues 4-52) still able to inhibit the binding of lactotransferrin to the PHA-activated lymphocyte was isolated. This result demonstrates that Lys- Abbreviations: FITC, fluorescein isothiocyanate; SASD, sulfosuc- cinimidyl 2-(p-azidosalicylamido)ethyl-1,3’-dithiopropionate; sulfo- SANPAH, sulfosuccinimidyl 6-((4’-azido-2’-nitrophenyl)amino)hex- anoate; RP-HPLC, revemphase high-performance liquid chromatography; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; Tris, tris(hydroxymethy1)ami- nomethane; PHA, phytohemagglutinin. 0006-2960/92/0431-9243$03.00/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1992 American Chemical Society