Histochemistry and Cell Biology

, 136:357

Internalization of odorant-binding proteins into the mouse olfactory epithelium

Original Paper


The detection of odorants in vertebrates is mediated by chemosensory neurons that reside in the olfactory epithelium of the nose. In land-living species, the hydrophobic odorous compounds inhaled by the airstream are dissolved in the nasal mucus by means of specialized globular proteins, the odorant-binding proteins (OBPs). To assure the responsiveness to odors of each inhalation, a rapid removal of odorants from the microenvironment of the receptor is essential. In order to follow the fate of OBP/odorant complexes, a recombinant OBP was fluorescently labeled, loaded with odorous compounds, and applied to the nose of a mouse. Very quickly, labeled OBP appeared inside the sustentacular cells of the epithelium. This uptake occurred only when the OBP was loaded with appropriate odorant compounds. A search for candidate transporters that could mediate such an uptake process led to the identification of the low density lipoprotein receptor Lrp2/Megalin. In the olfactory epithelium, megalin was found to be specifically expressed in sustentacular cells and the Megalin protein was located in their microvilli. In vitro studies using a cell line that expresses megalin revealed a rapid internalization of OBP/odorant complexes into lysosomes. The uptake was blocked by a Megalin inhibitor, as was the internalization of OBPs into the sustentacular cells of the olfactory epithelium. The results suggest that a Megalin-mediated internalization of OBP/odorant complexes into the sustentacular cells may represent an important mechanism for a rapid and local clearance of odorants.


Mouse Olfaction Mucus Clearance Megalin 


  1. Avanzini F, Bignetti E, Bordi C, Carfagna G, Cavaggioni A, Ferrari G, Sorbi RT, Tirindelli R (1987) Immunocytochemical localization of pyrazine-binding protein in bovine nasal mucosa. Cell Tissue Res 247:461–464PubMedCrossRefGoogle Scholar
  2. Bakalyar HA, Reed RR (1990) Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science 250:1403–1406PubMedCrossRefGoogle Scholar
  3. Birn H, Willnow TE, Nielsen R, Norden AG, Bonsch C, Moestrup SK, Nexo E, Christensen EI (2002) Megalin is essential for renal proximal tubule reabsorption and accumulation of transcobalamin-B(12). Am J Physiol Renal Physiol 282:F408–F416PubMedGoogle Scholar
  4. Bogdanffy MS (1990) Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach. Environ Health Perspect 85:177–186PubMedCrossRefGoogle Scholar
  5. Buck L, Axel R (1991) A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65:175–187PubMedCrossRefGoogle Scholar
  6. Burchell B (1991) Turning on and turning off the sense of smell. Nature 350:16–17PubMedCrossRefGoogle Scholar
  7. Campanacci V, Krieger J, Bette S, Sturgis JN, Lartigue A, Cambillau C, Breer H, Tegoni M (2001) Revisiting the specificity of Mamestra brassicae and Antheraea polyphemus pheromone-binding proteins with a fluorescence binding assay. J Biol Chem 276:20078–20084PubMedCrossRefGoogle Scholar
  8. Carr VM, Menco BP, Yankova MP, Morimoto RI, Farbman AI (2001) Odorants as cell-type specific activators of a heat shock response in the rat olfactory mucosa. J Comp Neurol 432:425–439PubMedCrossRefGoogle Scholar
  9. Christensen EI, Willnow TE (1999) Essential role of megalin in renal proximal tubule for vitamin homeostasis. J Am Soc Nephrol 10:2224–2236PubMedGoogle Scholar
  10. Christensen EI, Moskaug JO, Vorum H, Jacobsen C, Gundersen TE, Nykjaer A, Blomhoff R, Willnow TE, Moestrup SK (1999) Evidence for an essential role of megalin in transepithelial transport of retinol. J Am Soc Nephrol 10:685–695PubMedGoogle Scholar
  11. Dahl AR, Hadley WM, Hahn FF, Benson JM, McClellan RO (1982) Cytochrome P-450-dependent monooxygenases in olfactory epithelium of dogs: possible role in tumorigenicity. Science 216:57–59PubMedCrossRefGoogle Scholar
  12. Ding XX, Coon MJ (1990) Immunochemical characterization of multiple forms of cytochrome P-450 in rabbit nasal microsomes and evidence for tissue-specific expression of P-450 s NMa and NMb. Mol Pharmacol 37:489–496PubMedGoogle Scholar
  13. Ding X, Peng HM, Coon MJ (1992) Cytochromes P450 NMa, NMb (2G1), and LM4 (1A2) are differentially expressed during development in rabbit olfactory mucosa and liver. Mol Pharmacol 42:1027–1032PubMedGoogle Scholar
  14. Flower DR (1996) The lipocalin protein family: structure and function. Biochem J 318(Pt 1):1–14PubMedGoogle Scholar
  15. Flower DR, North AC, Sansom CE (2000) The lipocalin protein family: structural and sequence overview. Biochim Biophys Acta 1482:9–24PubMedCrossRefGoogle Scholar
  16. Garibotti M, Navarrini A, Pisanelli AM, Pelosi P (1997) Three odorant-binding proteins from rabbit nasal mucosa. Chem Sens 22:383–390CrossRefGoogle Scholar
  17. Gburek J, Birn H, Verroust PJ, Goj B, Jacobsen C, Moestrup SK, Willnow TE, Christensen EI (2003) Renal uptake of myoglobin is mediated by the endocytic receptors megalin and cubilin. Am J Physiol Renal Physiol 285:F451–F458PubMedGoogle Scholar
  18. Getchell TV, Heck GL, DeSimone JA, Price S (1980) The location of olfactory receptor sites. Inferences from latency measurements. Biophys J 29:397–411PubMedCrossRefGoogle Scholar
  19. Getchell TV, Margolis FL, Getchell ML (1984) Perireceptor and receptor events in vertebrate olfaction. Prog Neurobiol 23:317–345PubMedCrossRefGoogle Scholar
  20. Hammes A, Andreassen TK, Spoelgen R, Raila J, Hubner N, Schulz H, Metzger J, Schweigert FJ, Luppa PB, Nykjaer A, Willnow TE (2005) Role of endocytosis in cellular uptake of sex steroids. Cell 122:751–762PubMedCrossRefGoogle Scholar
  21. Hegg CC, Lucero MT (2006) Purinergic receptor antagonists inhibit odorant-induced heat shock protein 25 induction in mouse olfactory epithelium. Glia 53:182–190PubMedCrossRefGoogle Scholar
  22. Herent MF, Collin S, Pelosi P (1995) Affinities of nutty and green-smelling pyrazines and thiazoles to odorant-binding proteins, in relation with their lipophilicity. Chem Sens 20:601–608CrossRefGoogle Scholar
  23. Joshi H, Getchell ML, Zielinski B, Getchell TV (1987) Spectrophotometric determination of cation concentrations in olfactory mucus. Neurosci Lett 82:321–326PubMedCrossRefGoogle Scholar
  24. Kozyraki R, Fyfe J, Verroust PJ, Jacobsen C, Dautry-Varsat A, Gburek J, Willnow TE, Christensen EI, Moestrup SK (2001) Megalin-dependent cubilin-mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia. Proc Natl Acad Sci USA 98:12491–12496PubMedCrossRefGoogle Scholar
  25. Lakso M, Pichel JG, Gorman JR, uer B, Okamoto Y, Lee E, Alt FW, Westphal H (1996) Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc Natl Acad Sci USA 93:5860–5865PubMedCrossRefGoogle Scholar
  26. Lazard D, Tal N, Rubinstein M, Khen M, Lancet D, Zupko K (1990) Identification and biochemical analysis of novel olfactory-specific cytochrome P-450IIA and UDP-glucuronosyl transferase. Biochemistry 29:7433–7440PubMedCrossRefGoogle Scholar
  27. Lazard D, Zupko K, Poria Y, Nef P, Lazarovits J, Horn S, Khen M, Lancet D (1991) Odorant signal termination by olfactory UDP glucuronosyl transferase. Nature 349:790–793PubMedCrossRefGoogle Scholar
  28. Leheste JR, Rolinski B, Vorum H, Hilpert J, Nykjaer A, Jacobsen C, Aucouturier P, Moskaug JO, Otto A, Christensen EI, Willnow TE (1999) Megalin knockout mice as an animal model of low molecular weight proteinuria. Am J Pathol 155:1361–1370PubMedCrossRefGoogle Scholar
  29. Ling G, Gu J, Genter MB, Zhuo X, Ding X (2004) Regulation of cytochrome P450 gene expression in the olfactory mucosa. Chem Biol Interact 147:247–258PubMedCrossRefGoogle Scholar
  30. Lobel D, Marchese S, Krieger J, Pelosi P, Breer H (1998) Subtypes of odorant-binding proteins–heterologous expression and ligand binding. Eur J Biochem 254:318–324PubMedCrossRefGoogle Scholar
  31. Menco BM, Bruch RC, Dau B, Danho W (1992) Ultrastructural localization of olfactory transduction components: the G protein subunit Golf alpha and type III adenylyl cyclase. Neuron 8:441–453PubMedCrossRefGoogle Scholar
  32. Menco BP, Cunningham AM, Qasba P, Levy N, Reed RR (1997) Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study. J Neurocytol 26:691–706PubMedCrossRefGoogle Scholar
  33. Miyawaki A, Homma H, Tamura H, Matsui M, Mikoshiba K (1996) Zonal distribution of sulfotransferase for phenol in olfactory sustentacular cells. EMBO J 15:2050–2055PubMedGoogle Scholar
  34. Monte MD, Centini M, Anselmi C, Pelosi P (1993) Binding of selected odorants to bovine and porcine odorant-binding proteins. Chem Sens 18:713–721CrossRefGoogle Scholar
  35. Moulton DG, Beidler LM (1967) Structure and function in the peripheral olfactory system. Physiol Rev 47(1):1–47PubMedGoogle Scholar
  36. Nef P, Heldman J, Lazard D, Margalit T, Jaye M, Hanukoglu I, Lancet D (1989) Olfactory-specific cytochrome P-450. cDNA cloning of a novel neuroepithelial enzyme possibly involved in chemoreception. J Biol Chem 264:6780–6785PubMedGoogle Scholar
  37. Nespoulous C, Briand L, Delage MM, Tran V, Pernollet JC (2004) Odorant binding and conformational changes of a rat odorant-binding protein. Chem Sens 29:189–198CrossRefGoogle Scholar
  38. Pelosi P (1994) Odorant-binding proteins. Crit Rev Biochem Mol Biol 29:199–228PubMedCrossRefGoogle Scholar
  39. Pelosi P (1996) Perireceptor events in olfaction. J Neurobiol 30:3–19PubMedCrossRefGoogle Scholar
  40. Pelosi P (1998) Odorant-binding proteins: structural aspects. Ann N Y Acad Sci 855:281–293PubMedCrossRefGoogle Scholar
  41. Pelosi P (2001) The role of perireceptor events in vertebrate olfaction. Cell Mol Life Sci 58:503–509PubMedCrossRefGoogle Scholar
  42. Pelosi P, Baldaccini NE, Pisanelli AM (1982) Identification of a specific olfactory receptor for 2-isobutyl-3-methoxypyrazine. Biochem J 201:245–248PubMedGoogle Scholar
  43. Pes D, Pelosi P (1995) Odorant-binding proteins of the mouse. Comp Biochem. Physiol B Biochem Mol Biol 112:471–479PubMedCrossRefGoogle Scholar
  44. Pes D, Mameli M, Andreini I, Krieger J, Weber M, Breer H, Pelosi P (1998) Cloning and expression of odorant-binding proteins Ia and Ib from mouse nasal tissue. Gene 212:49–55PubMedCrossRefGoogle Scholar
  45. Pevsner J, Sklar PB, Snyder SH (1986) Odorant-binding protein: localization to nasal glands and secretions. Proc Natl Acad Sci USA 83:4942–4946PubMedCrossRefGoogle Scholar
  46. Pevsner J, Hwang PM, Sklar PB, Venable JC, Snyder SH (1988) Odorant-binding protein and its mRNA are localized to lateral nasal gland implying a carrier function. Proc Natl Acad Sci USA 85:2383–2387PubMedCrossRefGoogle Scholar
  47. Pevsner J, Hou V, Snowman AM, Snyder SH (1990) Odorant-binding protein. Characterization of ligand binding. J Biol Chem 265:6118–6125PubMedGoogle Scholar
  48. Potter SM, Zheng C, Koos DS, Feinstein P, Fraser SE, Mombaerts P (2001) Structure and emergence of specific olfactory glomeruli in the mouse. J Neurosci 21:9713–9723PubMedGoogle Scholar
  49. Reuter D, Zierold K, Schroder WH, Frings S (1998) A depolarizing chloride current contributes to chemoelectrical transduction in olfactory sensory neurons in situ. J Neurosci 18:6623–6630PubMedGoogle Scholar
  50. Schofield PR (1988) Carrier-bound odorant delivery to olfactory receptors. Trends Neurosci 11:471–472PubMedCrossRefGoogle Scholar
  51. Sousa MM, Norden AG, Jacobsen C, Willnow TE, Christensen EI, Thakker RV, Verroust PJ, Moestrup SK, Saraiva MJ (2000) Evidence for the role of megalin in renal uptake of transthyretin. J Biol Chem 275:38176–38181PubMedCrossRefGoogle Scholar
  52. Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, Costantini F (2001) Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1:4PubMedCrossRefGoogle Scholar
  53. Strotmann J, Levai O, Fleischer J, Schwarzenbacher K, Breer H (2004) Olfactory receptor proteins in axonal processes of chemosensory neurons. J Neurosci 24:7754–7761PubMedCrossRefGoogle Scholar
  54. Su T, Sheng JJ, Lipinskas TW, Ding X (1996) Expression of CYP2A genes in rodent and human nasal mucosa. Drug Metab Dispos 24:884–890PubMedGoogle Scholar
  55. Tegoni M, Pelosi P, Vincent F, Spinelli S, Campanacci V, Grolli S, Ramoni R, Cambillau C (2000) Mammalian odorant binding proteins. Biochim Biophys Acta 1482:229–240PubMedCrossRefGoogle Scholar
  56. Thornton-Manning JR, Nikula KJ, Hotchkiss JA, Avila KJ, Rohrbacher KD, Ding X, Dahl AR (1997) Nasal cytochrome P450 2A: identification, regional localization, and metabolic activity toward hexamethylphosphoramide, a known nasal carcinogen. Toxicol Appl Pharmacol 142:22–30PubMedCrossRefGoogle Scholar
  57. Vinnikov YA (1982) Chemoreceptor cells (olfactory and taste cells). In: Evolution of receptor cells. Springer, Berlin, pp 29–41Google Scholar
  58. Willnow TE, Rohlmann A, Horton J, Otani H, Braun JR, Hammer RE, Herz J (1996) RAP, a specialized chaperone, prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors. EMBO J 15:2632–2639PubMedGoogle Scholar
  59. Willnow TE, Nykjaer A, Herz J (1999) Lipoprotein receptors: new roles for ancient proteins. Nat Cell Biol 1:E157–E162PubMedCrossRefGoogle Scholar
  60. Wolf G (2007) Identification of a membrane receptor for retinol-binding protein functioning in the cellular uptake of retinol. Nutr Rev 65:385–388PubMedCrossRefGoogle Scholar
  61. Yamagata M, Ozono K, Hashimoto Y, Miyauchi Y, Kondou H, Michigami T (2005) Intraperitoneal administration of recombinant receptor-associated protein causes phosphaturia via an alteration in subcellular distribution of the renal sodium phosphate co-transporter. J Am Soc Nephrol 16:2338–2345PubMedCrossRefGoogle Scholar
  62. Zupko K, Poria Y, Lancet D (1991) Immunolocalization of cytochromes P-450olf1 and P-450olf2 in rat olfactory mucosa. Eur J Biochem 196:51–58PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Institute of PhysiologyUniversity of HohenheimStuttgartGermany

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