Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Nov 15;328(Pt 1):145–151. doi: 10.1042/bj3280145

N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA.

P H van Berkel 1, M E Geerts 1, H A van Veen 1, M Mericskay 1, H A de Boer 1, J H Nuijens 1
PMCID: PMC1218898  PMID: 9359845

Abstract

Human lactoferrin (hLF), a protein involved in host defence against infection and excessive inflammation, interacts with heparin, the lipid A moiety of bacterial lipopolysaccharide, human lysozyme (hLZ) and DNA. To determine which region of the molecule is important in these interactions, solid-phase ligand binding assays were performed with hLF from human milk (natural hLF) and N-terminally deleted hLF variants. Iron-saturated and natural hLF bound equally well to heparin, lipid A, hLZ and DNA. Natural hLF lacking the first two N-terminal amino acids (Gly1-Arg2) showed reactivities of one-half, two-thirds, one-third and one-third towards heparin, lipid A, hLZ and DNA respectively compared with N-terminally intact hLF. A lack of the first three residues (Gly1-Arg2-Arg3) decreased binding to the same ligands to one-eighth, one-quarter, one-twentieth and one-seventeenth respectively. No binding occurred with a mutant lacking the first five residues (Gly1-Arg2-Arg3-Arg4-Arg5). An anti-hLF monoclonal antibody (E11) that reacts to an N-lobe epitope including Arg5 completely blocked hLF-ligand interaction. These results show that the N-terminal stretch of four consecutive arginine residues, Arg2-Arg3-Arg4-Arg5, has a decisive role in the interaction of hLF with heparin, lipid A, hLZ and DNA. The role of limited N-terminal proteolysis of hLF in its anti-infective and anti-inflammatory properties is discussed.

Full Text

The Full Text of this article is available as a PDF (322.5 KB).

Selected References

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

  1. Anderson B. F., Baker H. M., Norris G. E., Rice D. W., Baker E. N. Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution. J Mol Biol. 1989 Oct 20;209(4):711–734. doi: 10.1016/0022-2836(89)90602-5. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bellamy W., Takase M., Yamauchi K., Wakabayashi H., Kawase K., Tomita M. Identification of the bactericidal domain of lactoferrin. Biochim Biophys Acta. 1992 May 22;1121(1-2):130–136. doi: 10.1016/0167-4838(92)90346-f. [DOI] [PubMed] [Google Scholar]
  4. Bennett R. M., Davis J. Lactoferrin interacts with deoxyribonucleic acid: a preferential reactivity with double-stranded DNA and dissociation of DNA-anti-DNA complexes. J Lab Clin Med. 1982 Jan;99(1):127–138. [PubMed] [Google Scholar]
  5. Broxmeyer H. E., Williams D. E., Hangoc G., Cooper S., Gentile P., Shen R. N., Ralph P., Gillis S., Bicknell D. C. The opposing actions in vivo on murine myelopoiesis of purified preparations of lactoferrin and the colony stimulating factors. Blood Cells. 1987;13(1-2):31–48. [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. Ellison R. T., 3rd, Giehl T. J. Killing of gram-negative bacteria by lactoferrin and lysozyme. J Clin Invest. 1991 Oct;88(4):1080–1091. doi: 10.1172/JCI115407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Erdei J., Forsgren A., Naidu A. S. Lactoferrin binds to porins OmpF and OmpC in Escherichia coli. Infect Immun. 1994 Apr;62(4):1236–1240. doi: 10.1128/iai.62.4.1236-1240.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gerstein M., Anderson B. F., Norris G. E., Baker E. N., Lesk A. M., Chothia C. Domain closure in lactoferrin. Two hinges produce a see-saw motion between alternative close-packed interfaces. J Mol Biol. 1993 Nov 20;234(2):357–372. doi: 10.1006/jmbi.1993.1592. [DOI] [PubMed] [Google Scholar]
  10. Goldman A. S., Garza C., Schanler R. J., Goldblum R. M. Molecular forms of lactoferrin in stool and urine from infants fed human milk. Pediatr Res. 1990 Mar;27(3):252–255. doi: 10.1203/00006450-199003000-00009. [DOI] [PubMed] [Google Scholar]
  11. Harmsen M. C., Swart P. J., de Béthune M. P., Pauwels R., De Clercq E., The T. H., Meijer D. K. Antiviral effects of plasma and milk proteins: lactoferrin shows potent activity against both human immunodeficiency virus and human cytomegalovirus replication in vitro. J Infect Dis. 1995 Aug;172(2):380–388. doi: 10.1093/infdis/172.2.380. [DOI] [PubMed] [Google Scholar]
  12. He J., Furmanski P. Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA. Nature. 1995 Feb 23;373(6516):721–724. doi: 10.1038/373721a0. [DOI] [PubMed] [Google Scholar]
  13. Hutchens T. W., Henry J. F., Yip T. T. Structurally intact (78-kDa) forms of maternal lactoferrin purified from urine of preterm infants fed human milk: identification of a trypsin-like proteolytic cleavage event in vivo that does not result in fragment dissociation. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):2994–2998. doi: 10.1073/pnas.88.8.2994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Koczan D., Hobom G., Seyfert H. M. Genomic organization of the bovine alpha-S1 casein gene. Nucleic Acids Res. 1991 Oct 25;19(20):5591–5596. doi: 10.1093/nar/19.20.5591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Legrand D., van Berkel P. H., Salmon V., van Veen H. A., Slomianny M. C., Nuijens J. H., Spik G. 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. Biochem J. 1997 Nov 1;327(Pt 3):841–846. doi: 10.1042/bj3270841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Machnicki M., Zimecki M., Zagulski T. Lactoferrin regulates the release of tumour necrosis factor alpha and interleukin 6 in vivo. Int J Exp Pathol. 1993 Oct;74(5):433–439. [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Meilinger M., Haumer M., Szakmary K. A., Steinböck F., Scheiber B., Goldenberg H., Huettinger M. Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor and transport to endosomes. FEBS Lett. 1995 Feb 20;360(1):70–74. doi: 10.1016/0014-5793(95)00082-k. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Miyazawa K., Mantel C., Lu L., Morrison D. C., Broxmeyer H. E. Lactoferrin-lipopolysaccharide interactions. Effect on lactoferrin binding to monocyte/macrophage-differentiated HL-60 cells. J Immunol. 1991 Jan 15;146(2):723–729. [PubMed] [Google Scholar]
  23. Nuijens J. H., Huijbregts C. C., van Mierlo G. M., Hack C. E. Inactivation of C-1 inhibitor by proteases: demonstration by a monoclonal antibody of a neodeterminant on inactivated, non-complexed C-1 inhibitor. Immunology. 1987 Jul;61(3):387–389. [PMC free article] [PubMed] [Google Scholar]
  24. Nuijens J. H., van Berkel P. H., Schanbacher F. L. Structure and biological actions of lactoferrin. J Mammary Gland Biol Neoplasia. 1996 Jul;1(3):285–295. doi: 10.1007/BF02018081. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Qian Z. Y., Jollès P., Migliore-Samour D., Fiat A. M. Isolation and characterization of sheep lactoferrin, an inhibitor of platelet aggregation and comparison with human lactoferrin. Biochim Biophys Acta. 1995 Jan 18;1243(1):25–32. doi: 10.1016/0304-4165(94)00126-i. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Shau H., Kim A., Golub S. H. Modulation of natural killer and lymphokine-activated killer cell cytotoxicity by lactoferrin. J Leukoc Biol. 1992 Apr;51(4):343–349. [PubMed] [Google Scholar]
  29. Wang D., Pabst K. M., Aida Y., Pabst M. J. Lipopolysaccharide-inactivating activity of neutrophils is due to lactoferrin. J Leukoc Biol. 1995 Jun;57(6):865–874. doi: 10.1002/jlb.57.6.865. [DOI] [PubMed] [Google Scholar]
  30. Wu H. F., Lundblad R. L., Church F. C. Neutralization of heparin activity by neutrophil lactoferrin. Blood. 1995 Jan 15;85(2):421–428. [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Zou S., Magura C. E., Hurley W. L. Heparin-binding properties of lactoferrin and lysozyme. Comp Biochem Physiol B. 1992 Dec;103(4):889–895. doi: 10.1016/0305-0491(92)90210-i. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. van Berkel P. H., van Veen H. A., Geerts M. E., de Boer H. A., Nuijens J. H. Heterogeneity in utilization of N-glycosylation sites Asn624 and Asn138 in human lactoferrin: a study with glycosylation-site mutants. Biochem J. 1996 Oct 1;319(Pt 1):117–122. doi: 10.1042/bj3190117. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES