Environmental Science and Pollution Research

, Volume 24, Issue 15, pp 13474–13483 | Cite as

Interaction of TiO2 nanoparticles with proteins from aquatic organisms: the case of gill mucus from blue mussel

  • Adeline BourgeaultEmail author
  • Véronique Legros
  • Florence Gonnet
  • Regis Daniel
  • Aurélie Paquirissamy
  • Clémence Bénatar
  • Olivier Spalla
  • Corinne Chanéac
  • Jean-Philippe Renault
  • Serge Pin
Research Article


To better understand the mechanisms of TiO2 nanoparticle (NP) uptake and toxicity in aquatic organisms, we investigated the interaction of NPs with the proteins found in gill mucus from blue mussels. Mucus is secreted by many aquatic organisms and is often their first line of defense against pathogens, xenobiotics, and other sources of environmental stress. Here, five TiO2 NPs and one SiO2 NP were incubated with gill mucus and run out on a one-dimensional polyacrylamide gel for a comparative qualitative analysis of the free proteins in the mucosal solution and the proteins bound to NPs. We then used nanoscale liquid chromatography coupled with tandem mass spectrometry to identify proteins of interest. Our data demonstrated dissimilar protein profiles between the crude mucosal solution and proteins adsorbed on NPs. In particular, extrapallial protein (EP), one of the most abundant mucus proteins, was absent from the adsorbed proteins. After thermal denaturation experiments, this absence was attributed to the EP content in aromatic amino acids that prevents protein unfolding and thus adsorption on the NP. Moreover, although the majority of the protein corona was qualitatively similar across the NPs tested here (SiO2 and TiO2), a few proteins in the corona showed a specific recruitment pattern according to the NP oxide (TiO2 vs SiO2) or crystal structure (anatase TiO2 vs rutile TiO2). Therefore, protein adsorption may vary with the type of NP.

Graphical abstract

Proteins with adsorption selectivity as identified from isolated bands


Mussel Gill mucus Protein Adsorption TiO2 nanoparticles Interaction nanoLC-MS/MS 1D PAGE 



The present study was supported by the Programme Transversal de Toxicologie at the CEA. We would like to thank Drs. Jean Labarre and Géraldine Klein for their assistance with the 1D gels.

Supplementary material

11356_2017_8801_MOESM1_ESM.docx (4.2 mb)
ESM 1 Details on thermal denaturation experiment. Spectral power of the light used during some exposures. List of identified proteins from selected bands on 1D gels. 1D gels and associated protein names obtained after testing the effect of UV and thermal denaturation. Amino acid composition of proteins of interest. (DOCX 4308 kb)
11356_2017_8801_MOESM2_ESM.xlsx (13 kb)
ESM 2 (XLSX 12.5 kb)


  1. Al-Sid-Cheikh M, Rouleau C, Pelletier E (2013) Tissue distribution and kinetics of dissolved and nanoparticulate silver in Iceland scallop (Chlamys islandica). Mar Environ Res 86:21–28CrossRefGoogle Scholar
  2. Allouni ZE, Gjerdet NR, Cimpan MR, Høl PJ (2015) The effect of blood protein adsorption on cellular uptake of anatase TiO2 nanoparticles. Int J Nanomedicine 10:687–695Google Scholar
  3. Battin TJ, Kammer FVD, Weilhartner A, Ottofuelling S, Hofmann T (2009) Nanostructured TiO2: transport behavior and effects on aquatic microbial communities under environmental conditions. Environ Sci Technol 43:8098–8104CrossRefGoogle Scholar
  4. Bourgeault A, Cousin C, Geertsen V, Cassier-Chauvat C, Chauvat F, Chanéac C, Spalla O (2015) The challenge of studying TiO2 nanoparticles bioaccumulation at environmental concentrations: crucial use of a stable isotope tracer. Environ Sci Technol 49:2451–2459CrossRefGoogle Scholar
  5. Bradford MM (1976) Rapid and sansitive method for quantitation of microgram quantities of protein utilizing pinciple of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  6. Campos B, Rivetti C, Rosenkranz P, Navas JM, Barata C (2013) Effects of nanoparticles of TiO2 on food depletion and life-history responses of Daphnia magna. Aquat Toxicol 130-131:174–183CrossRefGoogle Scholar
  7. Canesi L, Ciacci C, Fabbri R, Marcomini A, Pojana G, Gallo G (2012) Bivalve molluscs as a unique target group for nanoparticle toxicity. Mar Environ Res 76:16–21CrossRefGoogle Scholar
  8. Canesi L, Ciacci C, Fabbri R, Balbi T, Salis A, Damonte G, Cortese K, Caratto V, Monopoli MP, Dawson K, Bergami E, Corsi I (2016) Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: role of soluble hemolymph proteins. Environ Res 150:73–81CrossRefGoogle Scholar
  9. Cassar L, Pepe C (2002) Hydraulic binder and cement compositions containing photocatalyst particles. Google PatentsGoogle Scholar
  10. Chen HB, Su XD, Neoh SB, Choe W-S (2006) QCM-D analysis of binding mechanism of phage particles displaying a constrained heptapeptide with specific affinity to SiO2 and TiO2. Anal Chem 78:4872–4879CrossRefGoogle Scholar
  11. de la Fuente M, Csaba N, Garcia-Fuentes M, Alonso MJ (2008) Nanoparticles as protein and gene carriers to mucosal surfaces. Nanomedicine (Lond) 3:845–857CrossRefGoogle Scholar
  12. Dessombz A, Chiche D, Davidson P, Panine P, Chaneac C, Jolivet J-P (2007) Design of liquid-crystalline aqueous suspensions of rutile nanorods: evidence of anisotropic photocatalytic properties. J Am Chem Soc 129:5904–5909CrossRefGoogle Scholar
  13. Devineau S 2013: Protein adsorption on nanomaterials. Biochemistry and physical-chemistry of a new stress. Université Paris Sud - Paris XIGoogle Scholar
  14. Devineau S, Zanotti JM, Loupiac C, Zargarian L, Neiers F, Pin S, Renault JP (2013) Myoglobin on silica: a case study of the impact of adsorption on protein structure and dynamics. Langmuir 29:13465–13472CrossRefGoogle Scholar
  15. Dickerson MB, Jones SE, Cai Y, Ahmad G, Naik RR, Kroger N, Sandhage KH (2008) Identification and design of peptides for the rapid, high-yield formation of nanoparticulate TiO2 from aqueous solutions at room temperature. Chem Mater 20:1578–1584CrossRefGoogle Scholar
  16. Dufour F, Pigeot-Remy S, Durupthy O, Cassaignon S, Ruaux V, Torelli S, Mariey L, Maugé F, Chanéac C (2015) Morphological control of TiO2 anatase nanoparticles: what is the good surface property to obtain efficient photocatalysts? Appl Catal B Environ 174–175:350–360CrossRefGoogle Scholar
  17. Easy RH, Ross NW (2009) Changes in Atlantic salmon (Salmo salar) epidermal mucus protein composition profiles following infection with sea lice (Lepeophtheirus salmonis). Comp Biochem Physiol Part D Genomics Proteomics 4:159–167CrossRefGoogle Scholar
  18. Federici G, Shaw BJ, Handy RD (2007) Toxicity of titanium dioxide nanoparticles to rainbow trout (Oncorhynchus mykiss): gill injury, oxidative stress, and other physiological effects. Aquat Toxicol 84:415–430CrossRefGoogle Scholar
  19. Geertsen V, Tabarant M, Spalla O (2014) Behavior and determination of titanium dioxide nanoparticles in nitric acid and river water by ICP spectrometry. Anal Chem 86:3453–3460CrossRefGoogle Scholar
  20. Goldberg ED, Bowen VT, Farrington JW, Harvey G, Martin JH, Parker PL, Risebrough RW, Robertson W, Schneider E, Gamble E (1978) The mussel watch. Environ Conserv 5:1–25CrossRefGoogle Scholar
  21. Gomes T, Pereira CG, Cardoso C, Bebianno MJ (2013) Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag. Aquat Toxicol 136:79–90CrossRefGoogle Scholar
  22. Gondikas AP, Kammer FVD, Reed RB, Wagner S, Ranville JF, Hofmann T (2014) Release of TiO2 nanoparticles from sunscreens into surface waters: a one-year survey at the old Danube recreational Lake. Environ Sci Technol 48:5415–5422CrossRefGoogle Scholar
  23. Guo C, Holland GP (2014) Investigating lysine adsorption on fumed silica nanoparticles. J Phys Chem C 118:25792–25801CrossRefGoogle Scholar
  24. Hu M, Lin D, Shang Y, Hu Y, Lu W, Huang X, Ning K, Chen Y, Wang Y (2017). CO2-induced pH reduction increases physiological toxicity of nano-TiO2 in the mussel Mytilus coruscus. Scientific Reports 7Google Scholar
  25. Joubert Y, Pan JF, Buffet PE, Pilet P, Gilliland D, Valsami-Jones E, Mouneyrac C, Amiard-Triquet C (2013) Subcellular localization of gold nanoparticles in the estuarine bivalve Scrobicularia plana after exposure through the water. Gold Bull 46:47–56CrossRefGoogle Scholar
  26. Kitadai N, Yokoyama T, Nakashima S (2009) ATR-IR spectroscopic study of L-lysine adsorption on amorphous silica. J Colloid Interface Sci 329:31–37CrossRefGoogle Scholar
  27. Klein G, Mathé C, Biola-Clier M, Devineau S, Drouineau E, Hatem E, Marichal L, Alonso B, Gaillard J-C, Lagniel G, Armengaud J, Carrière M, Chédin S, Boulard Y, Pin S, Renault J-P, Aude J-C, Labarre J (2016) RNA-binding proteins are a major target of silica nanoparticles in cell extracts. Nanotoxicology 10:1555–1564CrossRefGoogle Scholar
  28. Koenig T, Menze BH, Kirchner M, Monigatti F, Parker KC, Patterson T, Steen JJ, Hamprecht FA, Steen H (2008) Robust prediction of the MASCOT score for an improved quality assessment in mass spectrometric proteomics. J Proteome Res 7:3708–3717CrossRefGoogle Scholar
  29. Lee N, Sverjensky DA, Hazen RM (2014) Cooperative and competitive adsorption of amino acids with Ca2+ on rutile (α-TiO2). Environ Sci Technol 48:9358–9365CrossRefGoogle Scholar
  30. Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA (2008) Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc Natl Acad Sci U S A 105:14265–14270CrossRefGoogle Scholar
  31. Lynch I, Cedervall T, Lundqvist M, Cabaleiro-Lago C, Linse S, Dawson KA (2007) The nanoparticle-protein complex as a biological entity; a complex fluids and surface science challenge for the 21st century. Adv Colloid Interface Sci 134–135:167–174CrossRefGoogle Scholar
  32. Lynch I, Dawson KA (2008) Protein-nanoparticle interactions. Nano Today 3:40–47CrossRefGoogle Scholar
  33. Lynch I, Salvati A, Dawson KA (2009) Protein-nanoparticle interactions: what does the cell see? Nat Nanotechnol 4:546–547CrossRefGoogle Scholar
  34. Marucco A, Fenoglio I, Turci F, Fubini B (2013) Interaction of fibrinogen and albumin with titanium dioxide nanoparticles of different crystalline phases. J Phys Conf Ser 429Google Scholar
  35. Mathé C, Devineau S, Aude J-C, Lagniel G, Chédin S, Legros V, Mathon M, Renault J-P, Pin S, Boulard Y, Labarre J (2013) Structural determinants for protein adsorption/non adsorption to silica surface. PLoS One 8:e81346CrossRefGoogle Scholar
  36. Monopoli MP, Walczyk D, Campbell A, Elia G, Lynch I, Baldelli Bombelli F, Dawson KA (2011) Physical-chemical aspects of protein corona: relevance to in vitro and in vivo biological impacts of nanoparticles. J Am Chem Soc 133:2525–2534CrossRefGoogle Scholar
  37. Monopoli MP, Aberg C, Salvati A, Dawson KA (2012) Biomolecular coronas provide the biological identity of nanosized materials. Nat Nanotechnol 7:779–786CrossRefGoogle Scholar
  38. Nel AE, Mädler L, Velegol D, Xia T, Hoek EMV, Somasundaran P, Klaessig F, Castranova V, Thompson M (2009) Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater 8:543–557CrossRefGoogle Scholar
  39. Norde W (2008) My voyage of discovery to proteins in flatland …and beyond. Colloids Surf B Biointerfaces 61:1–9CrossRefGoogle Scholar
  40. Oberholzer MR, Lenhoff AM (1999) Protein adsorption isotherms through colloidal energetics. Langmuir 15:3905–3914CrossRefGoogle Scholar
  41. OECD 2016: Nanomaterials in waste streams: current knowledge on risks and impacts., OECD Publishing, Paris
  42. Oliva FY, Avalle LB, Cámara OR, De Pauli CP (2003) Adsorption of human serum albumin (HSA) onto colloidal TiO2 particles. J Colloid Interface Sci 261:299–311CrossRefGoogle Scholar
  43. Pales Espinosa E, Perrigault M, Allam B (2010) Identification and molecular characterization of a mucosal lectin (MeML) from the blue mussel Mytilus edulis and its potential role in particle capture. Comp Biochem Physiol A Mol Integr Physiol 156:495–501CrossRefGoogle Scholar
  44. Planchon M, Ferrari R, Guyot F, Gelabert A, Menguy N, Chaneac C, Thill A, Benedetti MF, Spalla O (2013) Interaction between Escherichia coli and TiO2 nanoparticles in natural and artificial waters. Colloids Surf B Biointerfaces 102:158–164CrossRefGoogle Scholar
  45. Roach P, Farrar D, Perry CC (2006) Surface tailoring for controlled protein adsorption: effect of topography at the nanometer scale and chemistry. J Am Chem Soc 128:3939–3945CrossRefGoogle Scholar
  46. Rocha TL, Gomes T, Sousa VS, Mestre NC, Bebianno MJ (2015) Ecotoxicological impact of engineered nanomaterials in bivalve molluscs: an overview. Mar Environ Res 111:74–88CrossRefGoogle Scholar
  47. Rocher B, Bultelle F, Chan P, Le Foll F, Letendre J, Monsinjon T, Olivier S, Péden R, Poret A, Vaudry D, Knigge T (2015) 2-DE mapping of the blue mussel gill proteome: the usual suspects revisited. Proteomes 3:3–41CrossRefGoogle Scholar
  48. Spalla O, Lyonnard S, Testard F (2003) Analysis of the small-angle intensity scattered by a porous and granular medium. J Appl Crystallogr 36:338–347CrossRefGoogle Scholar
  49. Sun TY, Gottschalk F, Hungerbuehler K, Nowack B (2014) Comprehensive probabilistic modelling of environmental emissions of engineered nanomaterials. Environ Pollut 185:69–76CrossRefGoogle Scholar
  50. Taggart LE, McMahon SJ, Butterworth KT, Currell FJ, Schettino G, Prise KM (2016) Protein disulphide isomerase as a target for nanoparticle-mediated sensitisation of cancer cells to radiation. Nanotechnology 27:215101CrossRefGoogle Scholar
  51. Tentorio A, Canova L (1989) Adsorption of α-amino acids on spherical TiO2 particles. Colloids and Surfaces 39:311–319CrossRefGoogle Scholar
  52. Tenzer S, Docter D, Rosfa S, Wlodarski A, Kuharev J, Rekik A, Knauer SK, Bantz C, Nawroth T, Bier C, Sirirattanapan J, Mann W, Treuel L, Zellner R, Maskos M, Schild H, Stauber RH (2011) Nanoparticle size is a critical physicochemical determinant of the human blood plasma corona: a comprehensive quantitative proteomic analysis. ACS Nano 5:7155–7167CrossRefGoogle Scholar
  53. Tenzer S, Docter D, Kuharev J, Musyanovych A, Fetz V, Hecht R, Schlenk F, Fischer D, Kiouptsi K, Reinhardt C, Landfester K, Schild H, Maskos M, Knauer SK, Stauber RH (2013) Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat Nanotechnol 8:772–781CrossRefGoogle Scholar
  54. Tomanek L, Zuzow MJ (2010) The proteomic response of the mussel congeners Mytilus galloprovincialis and M. trossulus to acute heat stress: implications for thermal tolerance limits and metabolic costs of thermal stress. J Exp Biol 213:3559–3574CrossRefGoogle Scholar
  55. Weir A, Westerhoff P, Fabricius L, Hristovski K, von Goetz N (2012) Titanium dioxide nanoparticles in food and personal care products. Environ Sci Technol 46:2242–2250CrossRefGoogle Scholar
  56. Yang SP, Bar-Ilan O, Peterson RE, Heideman W, Hamers RJ, Pedersen JA (2013) Influence of humic acid on titanium dioxide nanoparticle toxicity to developing zebrafish. Environ Sci Technol 47:4718–4725CrossRefGoogle Scholar
  57. Zhu XS, Chang Y, Chen YS (2010) Toxicity and bioaccumulation of TiO2 nanoparticle aggregates in Daphnia magna. Chemosphere 78:209–215CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Adeline Bourgeault
    • 1
    Email author
  • Véronique Legros
    • 2
    • 3
  • Florence Gonnet
    • 2
    • 3
  • Regis Daniel
    • 2
    • 3
  • Aurélie Paquirissamy
    • 1
  • Clémence Bénatar
    • 1
  • Olivier Spalla
    • 1
  • Corinne Chanéac
    • 4
  • Jean-Philippe Renault
    • 1
  • Serge Pin
    • 1
  1. 1.LIONS, NIMBE, CEA, CNRSUniversité Paris-SaclayGif-sur-YvetteFrance
  2. 2.CNRS, UMR 8587Laboratoire Analyse et Modélisation pour la Biologie et l’EnvironnementEvryFrance
  3. 3.Laboratoire Analyse et Modélisation pour la Biologie et l’EnvironnementUniversité Evry-Val-d’EssonneEvryFrance
  4. 4.Collège de France, Laboratoire de Chimie de la Matière Condensée de ParisSorbonne Universités, UPMC Univ Paris 06, CNRSParisFrance

Personalised recommendations