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Iron traffics in circulation bound to a siderocalin (Ngal)–catechol complex

Nature Chemical Biology volume 6, pages 602–609 (2010)Cite this article

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Abstract

The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal (also known as neutrophil gelatinase associated lipocalin, siderocalin, lipocalin 2) sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH-sensitive mechanism. As catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal–catechol–Fe(III) complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal–siderophore interactions but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.

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Figure 1: Determination of the affinity of catechol–Fe(III) in complex with Scn-Ngal.
Figure 2: UV-visible spectra of complexes of Scn-Ngal, siderophores and iron.
Figure 3: Scn-Ngal binds to catechol–Fe(III) as well as to 4-Methylcatechol–Fe(III).
Figure 4: The formation and distribution of the Scn-Ngal–catechol–Fe(III) complex in vivo.
Figure 5: Release of ligands from Scn-Ngal as a result of acidification.

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References

  1. Theil, E.C. & Goss, D.J. Living with iron (and oxygen): questions and answers about iron homeostasis. Chem. Rev. 109, 4568–4579 (2009).

    Article  CAS  Google Scholar 

  2. Iwahashi, H., Morishita, H., Ishii, T., Sugata, R. & Kido, R. Enhancement by catechols of hydroxyl-radical formation in the presence of ferric ions and hydrogen peroxide. J. Biochem. 105, 429–434 (1989).

    Article  CAS  Google Scholar 

  3. Rouault, T.A. & Tong, W.H. Iron-sulfur cluster biogenesis and human disease. Trends Genet. 24, 398–407 (2008).

    Article  CAS  Google Scholar 

  4. Li, J.Y. et al. Scara5 is a ferritin receptor mediating non-transferrin iron delivery. Dev. Cell 16, 35–46 (2009).

    Article  CAS  Google Scholar 

  5. Levy, J.E., Jin, O., Fujiwara, Y., Kuo, F. & Andrews, N.C. Transferrin receptor is necessary for development of erythrocytes and the nervous system. Nat. Genet. 21, 396–399 (1999).

    Article  CAS  Google Scholar 

  6. Gunshin, H. et al. Slc11a2 is required for intestinal iron absorption and erythropoiesis but dispensable in placenta and liver. J. Clin. Invest. 115, 1258–1266 (2005).

    Article  CAS  Google Scholar 

  7. Akerstrom, B., Flower, D.R. & Salier, J.P. Lipocalins: unity in diversity. Biochim. Biophys. Acta 1482, 1–8 (2000).

    Article  CAS  Google Scholar 

  8. Goetz, D.H. et al. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol. Cell 10, 1033–1043 (2002).

    Article  CAS  Google Scholar 

  9. Holmes, M.A., Paulsene, W., Jide, X., Ratledge, C. & Strong, R.K. Siderocalin (Lcn 2) also binds carboxymycobactins, potentially defending against mycobacterial infections through iron sequestration. Structure 13, 29–41 (2005).

    Article  CAS  Google Scholar 

  10. Flo, T.H. et al. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432, 917–921 (2004).

    Article  CAS  Google Scholar 

  11. Berger, T. et al. Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury. Proc. Natl. Acad. Sci. USA 103, 1834–1839 (2006).

    Article  CAS  Google Scholar 

  12. Mori, K. et al. Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J. Clin. Invest. 115, 610–621 (2005).

    Article  CAS  Google Scholar 

  13. Mishra, J. et al. Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 365, 1231–1238 (2005).

    Article  CAS  Google Scholar 

  14. Devireddy, L.R., Gazin, C., Zhu, X. & Green, M.R. A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake. Cell 123, 1293–1305 (2005).

    Article  CAS  Google Scholar 

  15. Wishart, D.S. et al. HMDB: the Human Metabolome Database. Nucleic Acids Res. 35, D521–D526 (2007).

    Article  CAS  Google Scholar 

  16. Hoette, T.M. et al. The role of electrostatics in siderophore recognition by the immunoprotein Siderocalin. J. Am. Chem. Soc. 130, 17584–17592 (2008).

    Article  CAS  Google Scholar 

  17. Sánchez, P. et al. Catechol releases iron (III) from ferritin by direct chelation without iron (II) production. Dalton Trans. 4, 811–813 (2005).

    Article  Google Scholar 

  18. Karpishin, T.B., Gebhard, M.S., Solomon, E.I. & Raymond, K.N. Spectroscopic studies of the electronic structure of Fe(III) tris(catecholates). J. Am. Chem. Soc. 113, 2977–2984 (1991).

    Article  CAS  Google Scholar 

  19. Abergel, R.J. et al. The Siderocalin/Enterobactin interaction: A link between mammalian immunity and bacterial iron transport. J. Am. Chem. Soc. 130, 11524–11534 (2008).

    Article  CAS  Google Scholar 

  20. Goetz, D.H. et al. Ligand preference inferred from the structure of neutrophil gelatinase associated lipocalin. Biochemistry 39, 1935–1941 (2000).

    Article  CAS  Google Scholar 

  21. Hvidberg, V. et al. The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake. FEBS Lett. 579, 773–777 (2005).

    Article  CAS  Google Scholar 

  22. Leheste, J.R. et al. Megalin knockout mice as an animal model of low molecular weight proteinuria. Am. J. Pathol. 155, 1361–1370 (1999).

    Article  CAS  Google Scholar 

  23. Rodríguez, J., Parra, C., Contreras, F.J. & Baeza, J. Dihydroxybenzenes: driven Fenton reactions. Water Sci. Technol. 44, 251–256 (2001).

    Article  Google Scholar 

  24. Setsukinai, K. et al. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J. Biol. Chem. 278, 3170–3175 (2003).

    Article  CAS  Google Scholar 

  25. Levinson, R.S. et al. Foxd1-dependent signals control cellularity in the renal capsule, a structure required for normal renal development. Development 132, 529–539 (2005).

    Article  CAS  Google Scholar 

  26. Li, J.Y. et al. Detection of intracellular iron by its regulatory effect. Am. J. Physiol. Cell Physiol. 287, C1547–C1559 (2004).

    Article  CAS  Google Scholar 

  27. Booth, A.N., Robbins, D.J., Masri, M.S. & DeEds, F. Excretion of catechol after ingestion of quinic and shikimic acids. Nature 187, 691 (1960).

    Article  CAS  Google Scholar 

  28. Martin, A.K. The origin of urinary aromatic compounds excreted by ruminants. 3. The metabolism of phenolic compounds to simple phenols. Br. J. Nutr. 48, 497–507 (1982).

    Article  CAS  Google Scholar 

  29. Lang, R., Mueller, C. & Hofmann, T. Development of a stable isotope dilution analysis with liquid chromatography-tandem mass spectrometry detection for the quantitative analysis of di- and trihydroxybenzenes in foods and model systems. J. Agric. Food Chem. 54, 5755–5762 (2006).

    Article  CAS  Google Scholar 

  30. Carmella, S.G., La, V.E.J. & Hecht, S.S. Quantitative analysis of catechol and 4-methylcatechol in human urine. Food Chem. Toxicol. 20, 587–590 (1982).

    Article  CAS  Google Scholar 

  31. Bakke, O.M. Urinary simple phenols in rats fed purified and nonpurified diets. J. Nutr. 98, 209–216 (1969).

    Article  CAS  Google Scholar 

  32. Rennick, B. & Quebbemann, A. Site of excretion of catechol and catecholamines: renal metabolism of catechol. Am. J. Physiol. 218, 1307–1312 (1970).

    Article  CAS  Google Scholar 

  33. Jones, R.L., Peterson, C.M., Grady, R.W. & Cerami, A. Low molecular weight iron-binding factor from mammalian tissue that potentiates bacterial growth. J. Exp. Med. 151, 418–428 (1980).

    Article  CAS  Google Scholar 

  34. Sawahata, T. & Neal, R.A. Biotransformation of phenol to hydroquinone and catechol by rat liver microsomes. Mol. Pharmacol. 23, 453–460 (1983).

    CAS  PubMed  Google Scholar 

  35. Parke, D.V. & Williams, R.T. Studies in detoxication. 54. The metabolism of benzene. (a) The formation of phenylglucuronide and phenylsulphuric acid from [14C]benzene. (b) The metabolism of [14C]phenol. Biochem. J. 55, 337–340 (1953).

    Article  CAS  Google Scholar 

  36. Smith, A.A. Origin of urinary pyrocatechol. Nature 190, 167 (1961).

    Article  CAS  Google Scholar 

  37. Bäckhed, F., Ley, R.E., Sonnenburg, J.L., Peterson, D.A. & Gordon, J.I. Host-bacterial mutualism in the human intestine. Science 307, 1915–1920 (2005).

    Article  Google Scholar 

  38. Marrubini, G. et al. Direct analysis of phenol, catechol and hydroquinone in human urine by coupled-column HPLC with fluorimetric detection. Chromatographia 62, 25–31 (2005).

    Article  CAS  Google Scholar 

  39. Qu, Q. et al. Validation of biomarkers in humans exposed to benzene: urine metabolites. Am. J. Ind. Med. 37, 522–531 (2000).

    Article  CAS  Google Scholar 

  40. Zager, R.A. Combined mannitol and deferoxamine therapy for myohemoglobinuric renal injury and oxidant tubular stress. Mechanistic and therapeutic implications. J. Clin. Invest. 90, 711–719 (1992).

    Article  CAS  Google Scholar 

  41. Paller, M.S. & Hedlund, B.E. Role of iron in postischemic renal injury in the rat. Kidney Int. 34, 474–480 (1988).

    Article  CAS  Google Scholar 

  42. Baliga, R., Ueda, N. & Shah, S.V. Increase in bleomycin-detectable iron in ischaemia/reperfusion injury to rat kidneys. Biochem. J. 291, 901–905 (1993).

    Article  CAS  Google Scholar 

  43. Jones, R.L. et al. Effects of iron chelators and iron overload on Salmonella infection. Nature 267, 63–65 (1977).

    Article  CAS  Google Scholar 

  44. Freestone, P.P., Walton, N.J., Haigh, R.D. & Lyte, M. Influence of dietary catechols on the growth of enteropathogenic bacteria. Int. J. Food Microbiol. 119, 159–169 (2007).

    Article  CAS  Google Scholar 

  45. Anderson, M.T. & Armstrong, S.K. Norepinephrine mediates acquisition of transferrin-iron in Bordetella bronchiseptica. J. Bacteriol. 190, 3940–3947 (2008).

    Article  CAS  Google Scholar 

  46. Richardson, D.R. 24p3 and its receptor: dawn of a new iron age? Cell 123, 1175–1177 (2005).

    Article  CAS  Google Scholar 

  47. Frisch, M.J.T. et al. Gaussian 03, Revision C.02 (Gaussian, Inc., Wallingford Connecticut, USA, 2004).

  48. Kuzmic, P. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal. Biochem. 237, 260–273 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Abergel, A. Zawadzka and Q. Al-Awqati for helpful discussion. We are grateful to the Gordon lab (Washington University) for gnotobiotic urines and E.I. Christensen (Aarhus University) and T.E. Willnow (Max Delbruck Center for Molecular Medicine) for megalin knockout urines. We salute the classes of 2010 and 2013 of the College of Physicians and Surgeons of Columbia University for donating urine for this study. This work was supported by US National Institutes of Health grants AI117448 (K.N.R.), AI59432 (R.K.S.) and the Emerald Foundation, the March of Dimes and US National Institutes of Health grants DK-55388 and DK-58872 (J.B.).

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  1. Guanhu Bao and Matthew Clifton: These authors contributed equally to this work.

Authors and Affiliations

  1. College of Physicians and Surgeons of Columbia University, New York, New York, USA

    Guanhu Bao, Shi-Xian Deng, Andong Qiu, Melanie Viltard, David Williams, Neal Paragas, Thomas Leete, Ritwij Kulkarni, Xiangpo Li, Belinda Lee, Avtandil Kalandadze, Adam J Ratner, Donald W Landry & Jonathan Barasch

  2. Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Matthew Clifton, Juan Carlos Pizarro & Roland K Strong

  3. Department of Chemistry, University of California, Berkeley, California, USA

    Trisha M Hoette & Kenneth N Raymond

  4. Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Trisha M Hoette & Kenneth N Raymond

  5. Kyoto University Graduate School of Medicine, Kyoto, Japan

    Kiyoshi Mori

  6. Max Delbruck Center for Molecular Medicine, Berlin, Germany

    Kai M Schmidt-Ott

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Contributions

G.B. identified siderophores, studied the complex in different models and performed catechol chemistry. K.M., A.K. and B.L. initiated these studies. M.C. and R.K.S. identified the structure of Scn-Ngal and the critical sites of molecular recognition defining Scn-Ngal function; T.M.H., X.L., S.-X.D., D.W.L., A.J.R., R.K., J.C.P. and K.N.R. studied catechol and catechol–Fe chemistry, binding affinity and pH sensitivity; N.P., A.Q., T.L., K.M.S.-O. and M.V. designed and performed cell biology experiments; D.W. performed radioautography, J.B. designed and analyzed experiments and, with contributions from all authors, wrote the paper.

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Correspondence to Roland K Strong or Jonathan Barasch.

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Supplementary Methods, Supplementary Figures 1–15, and Supplementary Tables 1 & 2 (PDF 4826 kb)

Supplementary Dataset 1 (PDF 278 kb)

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Bao, G., Clifton, M., Hoette, T. et al. Iron traffics in circulation bound to a siderocalin (Ngal)–catechol complex. Nat Chem Biol 6, 602–609 (2010). https://doi.org/10.1038/nchembio.402

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