Efficient delipidation of a recombinant lung surfactant lipopeptide analogue by liquid-gel chromatography

Autoři: Oihana Basabe-Burgos aff001;  Jakub Zebialowicz Ahlström aff001;  Pavol Mikolka aff001;  Michael Landreh aff003;  Jan Johansson aff001;  Tore Curstedt aff004;  Anna Rising aff001
Působiště autorů: Department of Neurobiology, Care Sciences and Society, Division for Neurogeriatrics, Karolinska Institutet, Huddinge, Sweden aff001;  Biomedical Center Martin and Department of Physiology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia aff002;  Science for Life Laboratory, Department of Microbiology, Tumour and Cell Biology, Karolinska Institutet, Tomtebodavägen, Stockholm, Sweden aff003;  Department of Molecular Medicine and Surgery, Karolinska Institutet at Karolinska University Hospital, Stockholm, Sweden aff004;  Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden aff005
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.pone.0226072


Pulmonary surfactant preparations extracted from natural sources have been used to treat millions of newborn babies with respiratory distress syndrome (RDS) and can possibly also be used to treat other lung diseases. Due to costly production and limited supply of animal-derived surfactants, synthetic alternatives are attractive. The water insolubility and aggregation-prone nature of the proteins present in animal-derived surfactant preparations have complicated development of artificial surfactant. A non-aggregating analog of lung surfactant protein C, SP-C33Leu is used in synthetic surfactant and we recently described an efficient method to produce rSP-C33Leu in bacteria. Here rSP-C33Leu obtained by salt precipitation of bacterial extracts was purified by two-step liquid gel chromatography and analyzed using mass spectrometry and RP-HPLC, showing that it is void of modifications and adducts. Premature New Zealand White rabbit fetuses instilled with 200mg/kg of 2% of rSP-C33Leu in phospholipids and ventilated with a positive end expiratory pressure showed increased tidal volumes and lung gas volumes compared to animals treated with phospholipids only. This shows that rSP-C33Leu can be purified from bacterial lipids and that rSP-C33Leu surfactant is active against experimental RDS.

Klíčová slova:

Lipids – Phospholipids – Rabbits – Salting out – Surfactants – Tidal volume – Reversed-phase high performance liquid chromatography – Size-exclusion chromatography


1. Goerke J. Pulmonary surfactant: functions and molecular composition. Biochim Biophys Acta. 1998;1408(2–3):79–89. doi: 10.1016/s0925-4439(98)00060-x 9813251

2. Parra E, Perez-Gil J. Composition, structure and mechanical properties define performance of pulmonary surfactant membranes and films. Chem Phys Lipids. 2015;185:153–75. doi: 10.1016/j.chemphyslip.2014.09.002 25260665

3. Whitsett JA, Weaver TE. Hydrophobic surfactant proteins in lung function and disease. N Engl J Med. 2002;347(26):2141–8. doi: 10.1056/NEJMra022387 12501227

4. Curstedt T, Jornvall H, Robertson B, Bergman T, Berggren P. Two hydrophobic low-molecular-mass protein fractions of pulmonary surfactant. Characterization and biophysical activity. Eur J Biochem. 1987;168(2):255–62. doi: 10.1111/j.1432-1033.1987.tb13414.x 3665923

5. Almlen A, Walther FJ, Waring AJ, Robertson B, Johansson J, Curstedt T. Synthetic surfactant based on analogues of SP-B and SP-C is superior to single-peptide surfactants in ventilated premature rabbits. Neonatology. 2010;98(1):91–9. doi: 10.1159/000276980 20110733

6. Sardesai S, Biniwale M, Wertheimer F, Garingo A, Ramanathan R. Evolution of surfactant therapy for respiratory distress syndrome: past, present, and future. Pediatr Res. 2017;81(1–2):240–8. doi: 10.1038/pr.2016.203 27706130

7. Curstedt T, Johansson J. Different effects of surfactant proteins B and C—implications for development of synthetic surfactants. Neonatology. 2010;97(4):367–72. doi: 10.1159/000297767 20551705

8. Haitsma JJ, Lachmann U, Lachmann B. Exogenous surfactant as a drug delivery agent. Adv Drug Deliv Rev. 2001;47(2–3):197–207. doi: 10.1016/s0169-409x(01)00106-5 11311992

9. Hidalgo A, Cruz A, Perez-Gil J. Barrier or carrier? Pulmonary surfactant and drug delivery. Eur J Pharm Biopharm. 2015;95(Pt A):117–27. doi: 10.1016/j.ejpb.2015.02.014 25709061

10. Dushianthan A, Cusack R, Goss V, Postle AD, Grocott MP. Clinical review: Exogenous surfactant therapy for acute lung injury/acute respiratory distress syndrome—where do we go from here? Crit Care. 2012;16(6):238. doi: 10.1186/cc11512 23171712

11. Baer B, Souza LMP, Pimentel AS, Veldhuizen RAW. New insights into exogenous surfactant as a carrier of pulmonary therapeutics. Biochemical Pharmacology. 2019;164:64–73. doi: 10.1016/j.bcp.2019.03.036 30928674

12. Johansson J, Curstedt T. Synthetic surfactants with SP-B and SP-C analogues to enable worldwide treatment of neonatal respiratory distress syndrome and other lung diseases. J Intern Med. 2019;285(2):165–86. doi: 10.1111/joim.12845 30357986

13. Johansson J, Curstedt T, Jornvall H. Surfactant protein B: disulfide bridges, structural properties, and kringle similarities. Biochemistry. 1991;30(28):6917–21. doi: 10.1021/bi00242a015 1648964

14. Curstedt T, Johansson J, Persson P, Eklund A, Robertson B, Lowenadler B, et al. Hydrophobic surfactant-associated polypeptides: SP-C is a lipopeptide with two palmitoylated cysteine residues, whereas SP-B lacks covalently linked fatty acyl groups. Proc Natl Acad Sci U S A. 1990;87(8):2985–9. doi: 10.1073/pnas.87.8.2985 2326260

15. Johansson J, Szyperski T, Curstedt T, Wuthrich K. The NMR structure of the pulmonary surfactant-associated polypeptide SP-C in an apolar solvent contains a valyl-rich alpha-helix. Biochemistry. 1994;33(19):6015–23. doi: 10.1021/bi00185a042 8180229

16. Johansson J, Szyperski T, Wuthrich K. Pulmonary surfactant-associated polypeptide SP-C in lipid micelles: CD studies of intact SP-C and NMR secondary structure determination of depalmitoyl-SP-C(1–17). FEBS Lett. 1995;362(3):261–5. doi: 10.1016/0014-5793(95)00216-v 7729509

17. Gustafsson M, Thyberg J, Naslund J, Eliasson E, Johansson J. Amyloid fibril formation by pulmonary surfactant protein C. FEBS Lett. 1999;464(3):138–42. doi: 10.1016/s0014-5793(99)01692-0 10618493

18. Johansson J, Some M, Linderholm BM, Almlen A, Curstedt T, Robertson B. A synthetic surfactant based on a poly-Leu SP-C analog and phospholipids: effects on tidal volumes and lung gas volumes in ventilated immature newborn rabbits. J Appl Physiol (1985). 2003;95(5):2055–63.

19. Hosia W, Johansson J, Griffiths WJ. Hydrogen/deuterium exchange and aggregation of a polyvaline and a polyleucine alpha-helix investigated by matrix-assisted laser desorption ionization mass spectrometry. Mol Cell Proteomics. 2002;1(8):592–7. doi: 10.1074/mcp.m200042-mcp200 12376574

20. Kallberg Y, Gustafsson M, Persson B, Thyberg J, Johansson J. Prediction of amyloid fibril-forming proteins. J Biol Chem. 2001;276(16):12945–50. doi: 10.1074/jbc.M010402200 11134035

21. Nilsson G, Gustafsson M, Vandenbussche G, Veldhuizen E, Griffiths WJ, Sjovall J, et al. Synthetic peptide-containing surfactants—evaluation of transmembrane versus amphipathic helices and surfactant protein C poly-valyl to poly-leucyl substitution. Eur J Biochem. 1998;255(1):116–24. doi: 10.1046/j.1432-1327.1998.2550116.x 9692909

22. Yousefi-Salakdeh E, Johansson J, Stromberg R. A method for S- and O-palmitoylation of peptides: synthesis of pulmonary surfactant protein-C models. Biochem J. 1999;343 Pt 3:557–62.

23. Ricci F, Murgia X, Razzetti R, Pelizzi N, Salomone F. In vitro and in vivo comparison between poractant alfa and the new generation synthetic surfactant CHF5633. Pediatr Res. 2017;81(2):369–75. doi: 10.1038/pr.2016.231 27973472

24. Sweet DG, Turner MA, Stranak Z, Plavka R, Clarke P, Stenson BJ, et al. A first-in-human clinical study of a new SP-B and SP-C enriched synthetic surfactant (CHF5633) in preterm babies with respiratory distress syndrome. Arch Dis Child Fetal Neonatal Ed. 2017;102(6):F497–F503. doi: 10.1136/archdischild-2017-312722 28465315

25. Kronqvist N, Sarr M, Lindqvist A, Nordling K, Otikovs M, Venturi L, et al. Efficient protein production inspired by how spiders make silk. Nat Commun. 2017;8:15504. doi: 10.1038/ncomms15504 28534479

26. Sarr M, Kronqvist N, Chen G, Aleksis R, Purhonen P, Hebert H, et al. A spidroin-derived solubility tag enables controlled aggregation of a designed amyloid protein. FEBS J. 2018;285(10):1873–85. doi: 10.1111/febs.14451 29604175

27. Gustafsson M, Curstedt T, Jornvall H, Johansson J. Reverse-phase HPLC of the hydrophobic pulmonary surfactant proteins: detection of a surfactant protein C isoform containing Nepsilon-palmitoyl-lysine. Biochem J. 1997;326 (Pt 3):799–806.

28. Stark M, Wang Y, Danielsson O, Jornvall H, Johansson J. Determination of proteins, phosphatidylethanolamine, and phosphatidylserine in organic solvent extracts of tissue material by analysis of phenylthiocarbamyl derivatives. Anal Biochem. 1998;265(1):97–102. doi: 10.1006/abio.1998.2856 9866713

29. Almlen A, Stichtenoth G, Linderholm B, Haegerstrand-Bjorkman M, Robertson B, Johansson J, et al. Surfactant proteins B and C are both necessary for alveolar stability at end expiration in premature rabbits with respiratory distress syndrome. J Appl Physiol (1985). 2008;104(4):1101–8.

30. Calkovska A, Linderholm B, Haegerstrand-Bjorkman M, Pioselli B, Pelizzi N, Johansson J, et al. Phospholipid Composition in Synthetic Surfactants Is Important for Tidal Volumes and Alveolar Stability in Surfactant-Treated Preterm Newborn Rabbits. Neonatology. 2016;109(3):177–85. doi: 10.1159/000442874 26757268

31. Scherle W. A simple method for volumetry of organs in quantitative stereology. Mikroskopie. 1970;26(1):57–60. 5530651

32. Davis AJ, Jobe AH, Hafner D, Ikegami M. Lung function in premature lambs and rabbits treated with a recombinant SP-C surfactant. Am J Respir Crit Care Med. 1998;157(2):553–9. doi: 10.1164/ajrccm.157.2.97-08019 9476872

33. Walther FJ, Waring AJ, Hernandez-Juviel JM, Ruchala P, Wang Z, Notter RH, et al. Surfactant protein C peptides with salt-bridges ("ion-locks") promote high surfactant activities by mimicking the alpha-helix and membrane topography of the native protein. Peerj. 2014;2:e485. doi: 10.7717/peerj.485 25083348

34. Lukovic D, Cruz A, Gonzalez-Horta A, Almlen A, Curstedt T, Mingarro I, et al. Interfacial behavior of recombinant forms of human pulmonary surfactant protein SP-C. Langmuir. 2012;28(20):7811–25. doi: 10.1021/la301134v 22530695

35. Lukovic D, Plasencia I, Taberner FJ, Salgado J, Calvete JJ, Perez-Gil J, et al. Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity. Biochim Biophys Acta. 2006;1758(4):509–18. doi: 10.1016/j.bbamem.2006.03.005 16631109

Článok vyšiel v časopise


2019 Číslo 12