The Journal of Membrane Biology

, Volume 251, Issue 4, pp 581–592 | Cite as

Using Liprotides to Deliver Cholesterol to the Plasma Membrane

  • Henriette S. Frislev
  • Janni Nielsen
  • Jesper NylandstedEmail author
  • Daniel OtzenEmail author


Cholesterol (chol) is important in all mammalian cells as a modulator of membrane fluidity. However, its low solubility is a challenge for controlled delivery to membranes. Here we introduce a new tool to deliver chol to membranes, namely, liprotides, i.e., protein–lipid complexes composed of a fatty acid core decorated with partially denatured protein. We focus on liprotides prepared by incubating Ca2+-depleted α-lactalbumin with oleic acid (OA) for 1 h at 20 °C (lip20) or 80 °C (lip80). The binding and membrane delivery properties of liprotides is compared to the widely chol transporter methyl-β-cyclodextrin (mBCD). Both lip20 and lip80 increase the solubility of chol ~ 50% more than mBCD and deliver chol to membranes with comparable efficiency. Although OA is cytotoxic at high concentrations, its effects are counterbalanced by chol. Further, cytotoxicity is strongly reduced when OA is replaced by cis-palmitoleic acid or cis-vaccenic acid. This makes liprotides good tools to deliver chol to membranes and cells.


Cholesterol Liprotides Membrane Cytotoxicity 







cis-Palmitoleic acid


cis-Vaccenic acid






Human α-lactalbumin made lethal to tumor cells


Complexes between lipids and partially denatured proteins


Liprotide prepared at 20 °C


Liprotide prepared at 80 °C, lip:chol, liprotide in complex with chol




mBCD in complex with chol


Oleic acid


Quartz crystal microbalance with dissipation


Reversed-phase high-performance liquid chromatography


Room temperature


Trifluoroacetic acid



This work is supported by a grant from the Danish Research Council | Technology and Production (DFF-4005-00479) to D.E.O. and H.S.F. We are grateful to Camilla Bertel Andersen for help with analysis of chol.

Compliance with Ethical Standards

Conflict of interest

The authors declare no competing financial interests.

Supplementary material

232_2018_34_MOESM1_ESM.docx (11.1 mb)
Supplementary Information includes figures depicting the amount of transfer of content to vesicles and the membrane destabilizing effect of liprotides (DOCX 11362 KB)


  1. Brinkmann CR, Brodkorb A, Thiel S, Kehoe JJ (2013) The cytotoxicity of fatty acid/α-lactalbumin complexes depends on the amount and type of fatty acid. Eur J Lipid Sci Technol 115(6):591–600CrossRefGoogle Scholar
  2. Brinkmann CR, Heegaard CW, Petersen TE, Jensenius JC, Thiel S (2011) The toxicity of bovine alpha-lactalbumin made lethal to tumor cells is highly dependent on oleic acid and induces killing in cancer cell lines and noncancer-derived primary cells. FEBS J 278(11):1955–1967CrossRefPubMedGoogle Scholar
  3. Brinkmann CR, Thiel S, Otzen DE (2013) Protein-fatty acid complexes: biochemistry, biophysics and function. FEBS J 280(8):1733–1749CrossRefPubMedGoogle Scholar
  4. Christian AE, Haynes MP, Phillips MC, Rothblat GH (1997) Use of cyclodextrins for manipulating cellular cholesterol content. J Lipid Res 38(11):2264–2272PubMedGoogle Scholar
  5. Davis ME, Brewster ME (2004) Cyclodextrin-based pharmaceutics: past, present and future. Nat Rev Drug Discov 3(12):1023–1035CrossRefPubMedGoogle Scholar
  6. Fang B, Zhang M, Jiang L, Jing H, Ren FZ (2012) Influence of pH on the structure and oleic acid binding ability of bovine alpha-lactalbumin. Protein J 31(7):564–572CrossRefPubMedGoogle Scholar
  7. Fontana A, Spolaore B, Polverino de Laureto P (2013) The biological activities of protein/oleic acid complexes reside in the fatty acid. Biochim Biophys Acta 1834(6):1125–1143CrossRefPubMedGoogle Scholar
  8. Frislev HS, Boye TL, Nylandsted J, Otzen D (2017) Liprotides kill cancer cells by disrupting the plasma membrane. Sci Rep 7:15129CrossRefPubMedPubMedCentralGoogle Scholar
  9. Frislev HS, Jessen CM, Oliveira CLP, Pedersen JS, Otzen DE (2016) Liprotides made of a-lactalbumin and cis fatty acids form core-shell and multi-layer structures with a common membrane-targeting mechanism. BBA Proteins Proteom 1864:847–859CrossRefGoogle Scholar
  10. Gustafsson L, Hallgren O, Mossberg AK, Pettersson J, Fischer W, Aronsson A, Svanborg C (2005) HAMLET kills tumor cells by apoptosis: structure, cellular mechanisms, and therapy. J Nutr 135(5):1299–1303CrossRefPubMedGoogle Scholar
  11. Hakansson A, Zhivotovsky B, Orrenius S, Sabharwal H, Svanborg C (1995) Apoptosis induced by a human milk protein. Proc Natl Acad Sci USA 92(17):8064–8068CrossRefPubMedGoogle Scholar
  12. Holick MF (1995) Defects in the synthesis and metabolism of vitamin D. Exp Clin Endocrinol Diabetes 103(4):219–227CrossRefPubMedGoogle Scholar
  13. Ikonen E (2006) Mechanisms for cellular cholesterol transport: defects and human disease. Physiol Rev 86(4):1237–1261CrossRefPubMedGoogle Scholar
  14. Ikonen E (2008) Cellular cholesterol trafficking and compartmentalization. Nat Rev Mol Cell Biol 9(2):125–138CrossRefPubMedGoogle Scholar
  15. Kamijima T, Ohmura A, Sato T, Akimoto K, Itabashi M, Mizuguchi M, Kamiya M, Kikukawa T, Aizawa T, Takahashi M, Kawano K, Demura M (2008) Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death. Biochem Biophys Res Commun 376(1):211–214CrossRefPubMedGoogle Scholar
  16. Kaspersen JD, Pedersen JN, Hansted JG, Nielsen SB, Sakthivel S, Wilhelm K, Nemashkalova EL, Permyakov SE, Permyakov EA, Pinto Oliveira CL, Morozova-Roche LA, Otzen DE, Pedersen JS (2014) Generic structures of cytotoxic liprotides: nano-sized complexes with oleic acid cores and shells of disordered proteins. ChemBioChem 15(18):2693–2702CrossRefPubMedGoogle Scholar
  17. Leventis R, Silvius JR (2001) Use of cyclodextrins to monitor transbilayer movement and differential lipid affinities of cholesterol. Biophys J 81(4):2257–2267CrossRefPubMedPubMedCentralGoogle Scholar
  18. Mahammad S, Parmryd I (2015) Cholesterol depletion using methyl-β-cyclodextrin. In: Owen DM (ed) Methods in membrane lipids. Humana Press, New York, pp 91–102Google Scholar
  19. Maxfield FR, Tabas I (2005) Role of cholesterol and lipid organization in disease. Nature 438(7068):612–621CrossRefPubMedGoogle Scholar
  20. Maxfield FR, van Meer G (2010) Cholesterol, the central lipid of mammalian cells. Curr Opin Cell Biol 22(4):422–429CrossRefPubMedPubMedCentralGoogle Scholar
  21. Nielsen SB, Wilhelm K, Vad B, Schleucher J, Morozova-Roche LA, Otzen D (2010) The interaction of equine lysozyme:oleic acid complexes with lipid membranes suggests a cargo off-loading mechanism. J Mol Biol 398(2):351–361CrossRefPubMedGoogle Scholar
  22. Nielsen SB, Wilhelm K, Vad BS, Schleucher J, Morozova-Roche L, Otzen DE (2010) The interaction of equine lysozyme:oleic acid complexes with lipid membranes suggests a cargo off-loading mechanism. J Mol Biol 398(4984):351–361CrossRefPubMedGoogle Scholar
  23. Olson RE (1998) Discovery of the lipoproteins, their role in fat transport and their significance as risk factors. J Nutr 128(2 Suppl):439S–443SCrossRefPubMedGoogle Scholar
  24. Pedersen JN, Frislev HS, Pedersen JS, Otzen DE (2016) Using protein-fatty acid complexes to improve vitamin D stability. J Dairy Sci 99(10):7755–7767CrossRefPubMedGoogle Scholar
  25. Pedersen JN, Pedersen JS, Otzen DE (2015) The use of liprotides to stabilize transport hydrophobic molecules. Biochemistry 54(31):4815–4823CrossRefPubMedGoogle Scholar
  26. Permyakov SE, Knyazeva EL, Khasanova LM, Fadeev RS, Zhadan AP, Roche-Hakansson H, Hakansson AP, Akatov VS, Permyakov EA (2012) Oleic acid is a key cytotoxic component of HAMLET-like complexes. Biol Chem 393(1–2):85–92PubMedGoogle Scholar
  27. Rog T, Pasenkiewicz-Gierula M, Vattulainen I, Karttunen M (2009) Ordering effects of cholesterol and its analogues. Biochim Biophys Acta 1788(1):97–121CrossRefPubMedGoogle Scholar
  28. Simons K, Vaz WL (2004) Model systems, lipid rafts, and cell membranes. Annu Rev Biophys Biomol Struct 33:269–295CrossRefPubMedGoogle Scholar
  29. Svensson M, Fast J, Mossberg AK, Duringer C, Gustafsson L, Hallgren O, Brooks CL, Berliner L, Linse S, Svanborg C (2003) Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells). Protein Sci 12(12):2794–2804CrossRefPubMedPubMedCentralGoogle Scholar
  30. Svensson M, Hakansson A, Mossberg AK, Linse S, Svanborg C (2000) Conversion of alpha-lactalbumin to a protein inducing apoptosis. Proc Natl Acad Sci USA 97(8):4221–4226CrossRefPubMedGoogle Scholar
  31. Svensson M, Mossberg AK, Pettersson J, Linse S, Svanborg C (2003) Lipids as cofactors in protein folding: stereo-specific lipid-protein interactions are required to form HAMLET (human alpha-lactalbumin made lethal to tumor cells). Protein Sci 12(12):2805–2814CrossRefPubMedPubMedCentralGoogle Scholar
  32. Ulloth JE, Almaguel FG, Padilla A, Bu L, Liu JW, De Leon M (2007) Characterization of methyl-beta-cyclodextrin toxicity in NGF-differentiated PC12 cell death. Neurotoxicology 28(3):613–621CrossRefPubMedPubMedCentralGoogle Scholar
  33. Wehbi Z, Perez MD, Sanchez L, Pocovi C, Barbana C, Calvo M (2005) Effect of heat treatment on denaturation of bovine alpha-lactalbumin: determination of kinetic and thermodynamic parameters. J Agric Food Chem 53(25):9730–9736CrossRefPubMedGoogle Scholar
  34. Wilhelm K, Darinskas A, Noppe W, Duchardt E, Mok KH, Vukojevic V, Schleucher J, Morozova-Roche LA (2009) Protein oligomerization induced by oleic acid at the solid-liquid interface–equine lysozyme cytotoxic complexes. FEBS J 276:3975–3989CrossRefPubMedGoogle Scholar
  35. Yang F Jr, Zhang M, Chen J, Liang Y (2006) Structural changes of alpha-lactalbumin induced by low pH and oleic acid. Biochim Biophys Acta 1764(8):1389–1396CrossRefPubMedGoogle Scholar
  36. Zhang M, Yang F Jr, Yang F, Chen J, Zheng CY, Liang Y (2009) Cytotoxic aggregates of alpha-lactalbumin induced by unsaturated fatty acid induce apoptosis in tumor cells. Chem Biol Interact 180:131–142CrossRefPubMedGoogle Scholar
  37. Zidovetzki R, Levitan I (2007) Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochim Biophys Acta 1768(6):1311–1324CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and GeneticsAarhus UniversityAarhus CDenmark
  2. 2.Membrane Integrity Group, Cell Death and Metabolism UnitDanish Cancer Society Research CenterCopenhagenDenmark

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