Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Nov 1;327(Pt 3):841–846. doi: 10.1042/bj3270841

The N-terminal Arg2, Arg3 and Arg4 of human lactoferrin interact with sulphated molecules but not with the receptor present on Jurkat human lymphoblastic T-cells.

D Legrand 1, P H van Berkel 1, V Salmon 1, H A van Veen 1, M C Slomianny 1, J H Nuijens 1, G Spik 1
PMCID: PMC1218865  PMID: 9581564

Abstract

We previously characterized a 105 kDa receptor for human lactoferrin (hLf) on Jurkat human lymphoblastic T-cells. To delineate the role of the basic cluster Arg2-Arg3-Arg4-Arg5 of hLf in the interaction with Jurkat cells, we isolated N-terminally deleted hLf species of molecular mass 80 kDa lacking two, three or four N-terminal residues (hLf-2N, hLf-3N and hLf-4N) from native hLf that had been treated with trypsin. Native hLf bound to 102000 sites on Jurkat cells with a dissociation constant (Kd) of 70 nM. Consecutive removal of N-terminal arginine residues from hLf progressively increased the binding affinity but decreased the number of binding sites on the cells. A recombinant hLF mutant lacking the first five N-terminal residues (rhLf-5N) bound to 17000 sites with a Kd of 12 nM. The binding parameters of bovine lactoferrin (Lf) and native hLf did not significantly differ, whereas the binding parameters of murine Lf (8000 sites; Kd 30 nM) resembled those of rhLf-5N. Culture of Jurkat cells in the presence of chlorate, which inhibits sulphation, decreased the number of binding sites for both native hLf and hLf-3N but not for rhLf-5N, indicating that the hLf-binding sites include sulphated molecules. We propose that the interaction of hLf with a large number of binding sites (approx. 80000 per cell) on Jurkat cells is dependent on Arg2-Arg3-Arg4, but not on Arg5. Interaction with approx. 20000 binding sites per cell, presumably the hLf receptor, does not require the first N-terminal basic cluster of hLf. Moreover, the affinity of hLf for the latter binding site is enhanced approx. 6-fold after removal of the first basic cluster. Thus N-terminal proteolysis of hLf in vivo might serve to modulate the nature of its binding to cells and thereby its effects on cellular physiology.

Full Text

The Full Text of this article is available as a PDF (342.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Appelmelk B. J., An Y. Q., Geerts M., Thijs B. G., de Boer H. A., MacLaren D. M., de Graaff J., Nuijens J. H. Lactoferrin is a lipid A-binding protein. Infect Immun. 1994 Jun;62(6):2628–2632. doi: 10.1128/iai.62.6.2628-2632.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bi B. Y., Liu J. L., Legrand D., Roche A. C., Capron M., Spik G., Mazurier J. Internalization of human lactoferrin by the Jurkat human lymphoblastic T-cell line. Eur J Cell Biol. 1996 Mar;69(3):288–296. [PubMed] [Google Scholar]
  3. Birgens H. S., Karle H., Hansen N. E., Ostergaard Kristensen L. Lactoferrin receptors in normal and leukaemic human blood cells. Scand J Haematol. 1984 Sep;33(3):275–280. doi: 10.1111/j.1600-0609.1984.tb02228.x. [DOI] [PubMed] [Google Scholar]
  4. Bläckberg L., Hernell O. Isolation of lactoferrin from human whey by a single chromatographic step. FEBS Lett. 1980 Jan 14;109(2):180–183. doi: 10.1016/0014-5793(80)81081-7. [DOI] [PubMed] [Google Scholar]
  5. Djeha A., Brock J. H. Effect of transferrin, lactoferrin and chelated iron on human T-lymphocytes. Br J Haematol. 1992 Feb;80(2):235–241. doi: 10.1111/j.1365-2141.1992.tb08906.x. [DOI] [PubMed] [Google Scholar]
  6. Elass-Rochard E., Roseanu A., Legrand D., Trif M., Salmon V., Motas C., Montreuil J., Spik G. Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide. Biochem J. 1995 Dec 15;312(Pt 3):839–845. doi: 10.1042/bj3120839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Huettinger M., Retzek H., Hermann M., Goldenberg H. Lactoferrin specifically inhibits endocytosis of chylomicron remnants but not alpha-macroglobulin. J Biol Chem. 1992 Sep 15;267(26):18551–18557. [PubMed] [Google Scholar]
  8. Hutchens T. W., Henry J. F., Yip T. T., Hachey D. L., Schanler R. J., Motil K. J., Garza C. Origin of intact lactoferrin and its DNA-binding fragments found in the urine of human milk-fed preterm infants. Evaluation by stable isotopic enrichment. Pediatr Res. 1991 Mar;29(3):243–250. doi: 10.1203/00006450-199103000-00005. [DOI] [PubMed] [Google Scholar]
  9. Keller K. M., Brauer P. R., Keller J. M. Modulation of cell surface heparan sulfate structure by growth of cells in the presence of chlorate. Biochemistry. 1989 Oct 3;28(20):8100–8107. doi: 10.1021/bi00446a021. [DOI] [PubMed] [Google Scholar]
  10. Legrand D., Mazurier J., Elass A., Rochard E., Vergoten G., Maes P., Montreuil J., Spik G. 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. Biochemistry. 1992 Sep 29;31(38):9243–9251. doi: 10.1021/bi00153a018. [DOI] [PubMed] [Google Scholar]
  11. Legrand D., Mazurier J., Metz-Boutigue M. H., Jolles J., Jolles P., Montreuil J., Spik G. Characterization and localization of an iron-binding 18-kDa glycopeptide isolated from the N-terminal half of human lactotransferrin. Biochim Biophys Acta. 1984 May 31;787(1):90–96. doi: 10.1016/0167-4838(84)90111-0. [DOI] [PubMed] [Google Scholar]
  12. Legrand D., Salmon V., Coddeville B., Benaissa M., Plancke Y., Spik G. Structural determination of two N-linked glycans isolated from recombinant human lactoferrin expressed in BHK cells. FEBS Lett. 1995 May 22;365(1):57–60. doi: 10.1016/0014-5793(95)00441-b. [DOI] [PubMed] [Google Scholar]
  13. Leveugle B., Mazurier J., Legrand D., Mazurier C., Montreuil J., Spik G. Lactotransferrin binding to its platelet receptor inhibits platelet aggregation. Eur J Biochem. 1993 May 1;213(3):1205–1211. doi: 10.1111/j.1432-1033.1993.tb17871.x. [DOI] [PubMed] [Google Scholar]
  14. MONTREUIL J., TONNELAT J., MULLET S. [Preparation and properties of lactosiderophilin (lactotransferrin) of human milk]. Biochim Biophys Acta. 1960 Dec 18;45:413–421. doi: 10.1016/0006-3002(60)91478-5. [DOI] [PubMed] [Google Scholar]
  15. Mann D. M., Romm E., Migliorini M. Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin. J Biol Chem. 1994 Sep 23;269(38):23661–23667. [PubMed] [Google Scholar]
  16. Masson P. L., Heremans J. F., Schonne E. Lactoferrin, an iron-binding protein in neutrophilic leukocytes. J Exp Med. 1969 Sep 1;130(3):643–658. doi: 10.1084/jem.130.3.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mazurier J., Legrand D., Hu W. L., Montreuil J., Spik G. Expression of human lactotransferrin receptors in phytohemagglutinin-stimulated human peripheral blood lymphocytes. Isolation of the receptors by antiligand-affinity chromatography. Eur J Biochem. 1989 Feb 1;179(2):481–487. doi: 10.1111/j.1432-1033.1989.tb14578.x. [DOI] [PubMed] [Google Scholar]
  18. McAbee D. D., Esbensen K. Binding and endocytosis of apo- and holo-lactoferrin by isolated rat hepatocytes. J Biol Chem. 1991 Dec 15;266(35):23624–23631. [PubMed] [Google Scholar]
  19. Metz-Boutigue M. H., Jollès J., Mazurier J., Schoentgen F., Legrand D., Spik G., Montreuil J., Jollès P. Human lactotransferrin: amino acid sequence and structural comparisons with other transferrins. Eur J Biochem. 1984 Dec 17;145(3):659–676. doi: 10.1111/j.1432-1033.1984.tb08607.x. [DOI] [PubMed] [Google Scholar]
  20. Miao H. Q., Ishai-Michaeli R., Atzmon R., Peretz T., Vlodavsky I. Sulfate moieties in the subendothelial extracellular matrix are involved in basic fibroblast growth factor sequestration, dimerization, and stimulation of cell proliferation. J Biol Chem. 1996 Mar 1;271(9):4879–4886. doi: 10.1074/jbc.271.9.4879. [DOI] [PubMed] [Google Scholar]
  21. Mikogami T., Heyman M., Spik G., Desjeux J. F. Apical-to-basolateral transepithelial transport of human lactoferrin in the intestinal cell line HT-29cl.19A. Am J Physiol. 1994 Aug;267(2 Pt 1):G308–G315. doi: 10.1152/ajpgi.1994.267.2.G308. [DOI] [PubMed] [Google Scholar]
  22. Pentecost B. T., Teng C. T. Lactotransferrin is the major estrogen inducible protein of mouse uterine secretions. J Biol Chem. 1987 Jul 25;262(21):10134–10139. [PubMed] [Google Scholar]
  23. Pierce A., Colavizza D., Benaissa M., Maes P., Tartar A., Montreuil J., Spik G. Molecular cloning and sequence analysis of bovine lactotransferrin. Eur J Biochem. 1991 Feb 26;196(1):177–184. doi: 10.1111/j.1432-1033.1991.tb15801.x. [DOI] [PubMed] [Google Scholar]
  24. Rey M. W., Woloshuk S. L., deBoer H. A., Pieper F. R. Complete nucleotide sequence of human mammary gland lactoferrin. Nucleic Acids Res. 1990 Sep 11;18(17):5288–5288. doi: 10.1093/nar/18.17.5288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rochard E., Legrand D., Lecocq M., Hamelin R., Crepin M., Montreuil J., Spik G. Characterization of lactotransferrin receptor in epithelial cell lines from non-malignant human breast, benign mastopathies and breast carcinomas. Anticancer Res. 1992 Nov-Dec;12(6B):2047–2051. [PubMed] [Google Scholar]
  26. Salmon V., Legrand D., Georges B., Slomianny M. C., Coddeville B., Spik G. Characterization of human lactoferrin produced in the baculovirus expression system. Protein Expr Purif. 1997 Mar;9(2):203–210. doi: 10.1006/prep.1996.0687. [DOI] [PubMed] [Google Scholar]
  27. Slater K., Fletcher J. Lactoferrin derived from neutrophils inhibits the mixed lymphocyte reaction. Blood. 1987 May;69(5):1328–1333. [PubMed] [Google Scholar]
  28. Spik G., Strecker G., Fournet B., Bouquelet S., Montreuil J., Dorland L., van Halbeek H., Vliegenthart J. F. Primary structure of the glycans from human lactotransferrin. Eur J Biochem. 1982 Jan;121(2):413–419. doi: 10.1111/j.1432-1033.1982.tb05803.x. [DOI] [PubMed] [Google Scholar]
  29. Wu H. F., Monroe D. M., Church F. C. Characterization of the glycosaminoglycan-binding region of lactoferrin. Arch Biochem Biophys. 1995 Feb 20;317(1):85–92. doi: 10.1006/abbi.1995.1139. [DOI] [PubMed] [Google Scholar]
  30. Ziere G. J., Bijsterbosch M. K., van Berkel T. J. Removal of 14 N-terminal amino acids of lactoferrin enhances its affinity for parenchymal liver cells and potentiates the inhibition of beta- very low density lipoprotein binding. J Biol Chem. 1993 Dec 25;268(36):27069–27075. [PubMed] [Google Scholar]
  31. Ziere G. J., van Dijk M. C., Bijsterbosch M. K., van Berkel T. J. Lactoferrin uptake by the rat liver. Characterization of the recognition site and effect of selective modification of arginine residues. J Biol Chem. 1992 Jun 5;267(16):11229–11235. [PubMed] [Google Scholar]
  32. van Berkel P. H., Geerts M. E., van Veen H. A., Kooiman P. M., Pieper F. R., de Boer H. A., Nuijens J. H. Glycosylated and unglycosylated human lactoferrins both bind iron and show identical affinities towards human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis. Biochem J. 1995 Nov 15;312(Pt 1):107–114. doi: 10.1042/bj3120107. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES