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. 1993 Dec 15;296(Pt 3):585–593. doi: 10.1042/bj2960585

Tryptophan fluorescence study on the interaction of pulmonary surfactant protein A with phospholipid vesicles.

C Casals 1, E Miguel 1, J Perez-Gil 1
PMCID: PMC1137738  PMID: 8280055

Abstract

The fluorescence characteristics of surfactant protein A (SP-A) from porcine and human bronchoalveolar lavage were determined in the presence and absence of lipids. After excitation at either 275 or 295 nm, the fluorescence emission spectrum of both proteins was characterized by two maxima at about 326 and 337 nm, indicating heterogeneity in the emission of the two tryptophan residues of SP-A, and also revealing a partially buried character for these fluorophores. Interaction of both human and porcine SP-A with various phospholipid vesicles resulted in an increase in the fluorescence emission of tryptophan without any shift in the emission wavelength maxima. This change in intrinsic fluorescence was found to be more pronounced in the presence of dipalmitoyl phosphatidylcholine (DPPC) than with dipalmitoyl phosphatidylglycerol (DPPG), DPPC/DPPG (7:3, w/w) and 1-palmitoyl-sn-glycerol-3-phosphocholine (LPC). Intrinsic fluorescence of SP-A was almost completely unaffected in the presence of egg phosphatidylcholine (egg-PC). In addition, we demonstrated a shielding of the tryptophan fluorescence from quenching by acrylamide on interaction of porcine SP-A with DPPC, DPPG or LPC. This shielding was most pronounced in the presence of DPPC. In the case of human SP-A, shielding was only observed on interaction with DPPC. From the intrinsic fluorescence measurements as well as from the quenching experiments, we concluded that the interaction of some phospholipid vesicles with SP-A produces a conformational change on the protein molecule and that the interaction of SP-A with DPPC is stronger than with other phospholipids. This interaction appeared to be independent of Ca2+ ions. Physiological ionic strength was found to be required for the interaction of SP-A with negatively charged vesicles of either DPPG or DPPC/DPPG (7:3, w/w). Intrinsic fluorescence of SP-A was sensitive to the physical state of the DPPC vesicles. The increase in intrinsic fluorescence of SP-A in the presence of DPPC vesicles was much stronger when the vesicles were in the gel state than when they were in the liquid-crystalline state. The effect produced by SP-A on the lipid vesicles was also dependent on temperature. The aggregation of DPPC, DPPC/DPPG (7:3, w/w) or dimyristoyl phosphatidylglycerol (DMPG) was many times higher below the phase-transition temperature of the corresponding phospholipids. These results strongly indicate that the interaction of SP-A with phospholipid vesicles requires the lipids to be in the gel phase.

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Selected References

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  1. BEAVEN G. H., HOLIDAY E. R. Ultraviolet absorption spectra of proteins and amino acids. Adv Protein Chem. 1952;7:319–386. doi: 10.1016/s0065-3233(08)60022-4. [DOI] [PubMed] [Google Scholar]
  2. Benson B., Hawgood S., Schilling J., Clements J., Damm D., Cordell B., White R. T. Structure of canine pulmonary surfactant apoprotein: cDNA and complete amino acid sequence. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6379–6383. doi: 10.1073/pnas.82.19.6379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boggaram V., Qing K., Mendelson C. R. The major apoprotein of rabbit pulmonary surfactant. Elucidation of primary sequence and cyclic AMP and developmental regulation. J Biol Chem. 1988 Feb 25;263(6):2939–2947. [PubMed] [Google Scholar]
  4. Casals C., Herrera L., Miguel E., Garcia-Barreno P., Municio A. M. Comparison between intra- and extracellular surfactant in respiratory distress induced by oleic acid. Biochim Biophys Acta. 1989 Jun 8;1003(2):201–203. doi: 10.1016/0005-2760(89)90256-7. [DOI] [PubMed] [Google Scholar]
  5. Childs R. A., Wright J. R., Ross G. F., Yuen C. T., Lawson A. M., Chai W., Drickamer K., Feizi T. Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids. J Biol Chem. 1992 May 15;267(14):9972–9979. [PubMed] [Google Scholar]
  6. Cockle S. A., Epand R. M., Moscarello M. A. Intrinsic fluorescence of a hydrophobic myelin protein and some complexes with phospholipids. Biochemistry. 1978 Feb 21;17(4):630–637. doi: 10.1021/bi00597a011. [DOI] [PubMed] [Google Scholar]
  7. Efrati H., Hawgood S., Williams M. C., Hong K., Benson B. J. Divalent cation and hydrogen ion effects on the structure and surface activity of pulmonary surfactant. Biochemistry. 1987 Dec 1;26(24):7986–7993. doi: 10.1021/bi00398a066. [DOI] [PubMed] [Google Scholar]
  8. Eftink M. R., Ghiron C. A. Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry. 1976 Feb 10;15(3):672–680. doi: 10.1021/bi00648a035. [DOI] [PubMed] [Google Scholar]
  9. Eftink M. R., Ghiron C. A. Fluorescence quenching studies with proteins. Anal Biochem. 1981 Jul 1;114(2):199–227. doi: 10.1016/0003-2697(81)90474-7. [DOI] [PubMed] [Google Scholar]
  10. Eisinger J. Intramolecular energy transfer in adrenocorticotropin. Biochemistry. 1969 Oct;8(10):3902–3908. doi: 10.1021/bi00838a004. [DOI] [PubMed] [Google Scholar]
  11. Floros J., Phelps D. S., Taeusch H. W. Biosynthesis and in vitro translation of the major surfactant-associated protein from human lung. J Biol Chem. 1985 Jan 10;260(1):495–500. [PubMed] [Google Scholar]
  12. Gasset M., Oñaderra M., Goormaghtigh E., Gavilanes J. G. Acid phospholipid vesicles produce conformational changes on the antitumour protein alpha-sarcin. Biochim Biophys Acta. 1991 Oct 11;1080(1):51–58. doi: 10.1016/0167-4838(91)90111-c. [DOI] [PubMed] [Google Scholar]
  13. Goerke J. Lung surfactant. Biochim Biophys Acta. 1974 Dec 16;344(3-4):241–261. doi: 10.1016/0304-4157(74)90009-4. [DOI] [PubMed] [Google Scholar]
  14. Grainger D. W., Reichert A., Ringsdorf H., Salesse C. Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy. Biochim Biophys Acta. 1990 Apr 30;1023(3):365–379. doi: 10.1016/0005-2736(90)90128-b. [DOI] [PubMed] [Google Scholar]
  15. Haagsman H. P., Elfring R. H., van Buel B. L., Voorhout W. F. The lung lectin surfactant protein A aggregates phospholipid vesicles via a novel mechanism. Biochem J. 1991 Apr 1;275(Pt 1):273–276. doi: 10.1042/bj2750273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haagsman H. P., Hawgood S., Sargeant T., Buckley D., White R. T., Drickamer K., Benson B. J. The major lung surfactant protein, SP 28-36, is a calcium-dependent, carbohydrate-binding protein. J Biol Chem. 1987 Oct 15;262(29):13877–13880. [PubMed] [Google Scholar]
  17. Haagsman H. P., Sargeant T., Hauschka P. V., Benson B. J., Hawgood S. Binding of calcium to SP-A, a surfactant-associated protein. Biochemistry. 1990 Sep 25;29(38):8894–8900. doi: 10.1021/bi00490a003. [DOI] [PubMed] [Google Scholar]
  18. Haagsman H. P., van Golde L. M. Synthesis and assembly of lung surfactant. Annu Rev Physiol. 1991;53:441–464. doi: 10.1146/annurev.ph.53.030191.002301. [DOI] [PubMed] [Google Scholar]
  19. Haas C., Voss T., Engel J. Assembly and disulfide rearrangement of recombinant surfactant protein A in vitro. Eur J Biochem. 1991 May 8;197(3):799–803. doi: 10.1111/j.1432-1033.1991.tb15974.x. [DOI] [PubMed] [Google Scholar]
  20. Hawgood S., Benson B. J., Hamilton R. L., Jr Effects of a surfactant-associated protein and calcium ions on the structure and surface activity of lung surfactant lipids. Biochemistry. 1985 Jan 1;24(1):184–190. doi: 10.1021/bi00322a026. [DOI] [PubMed] [Google Scholar]
  21. Hawgood S., Benson B. J., Schilling J., Damm D., Clements J. A., White R. T. Nucleotide and amino acid sequences of pulmonary surfactant protein SP 18 and evidence for cooperation between SP 18 and SP 28-36 in surfactant lipid adsorption. Proc Natl Acad Sci U S A. 1987 Jan;84(1):66–70. doi: 10.1073/pnas.84.1.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. King R. J., Carmichael M. C., Horowitz P. M. Reassembly of lipid-protein complexes of pulmonary surfactant. Proposed mechanism of interaction. J Biol Chem. 1983 Sep 10;258(17):10672–10680. [PubMed] [Google Scholar]
  23. King R. J., Phillips M. C., Horowitz P. M., Dang S. C. Interaction between the 35 kDa apolipoprotein of pulmonary surfactant and saturated phosphatidylcholines. Effects of temperature. Biochim Biophys Acta. 1986 Oct 24;879(1):1–13. doi: 10.1016/0005-2760(86)90259-6. [DOI] [PubMed] [Google Scholar]
  24. King R. J., Simon D., Horowitz P. M. Aspects of secondary and quaternary structure of surfactant protein A from canine lung. Biochim Biophys Acta. 1989 Feb 20;1001(3):294–301. doi: 10.1016/0005-2760(89)90114-8. [DOI] [PubMed] [Google Scholar]
  25. Kuroki Y., Akino T. Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine. J Biol Chem. 1991 Feb 15;266(5):3068–3073. [PubMed] [Google Scholar]
  26. Ladbrooke B. D., Chapman D. Thermal analysis of lipids, proteins and biological membranes. A review and summary of some recent studies. Chem Phys Lipids. 1969 Dec;3(4):304–356. doi: 10.1016/0009-3084(69)90040-1. [DOI] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Lehrer S. S. Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry. 1971 Aug 17;10(17):3254–3263. doi: 10.1021/bi00793a015. [DOI] [PubMed] [Google Scholar]
  29. Lichtenberg D., Romero G., Menashe M., Biltonen R. L. Hydrolysis of dipalmitoylphosphatidylcholine large unilamellar vesicles by porcine pancreatic phospholipase A2. J Biol Chem. 1986 Apr 25;261(12):5334–5340. [PubMed] [Google Scholar]
  30. Reilly K. E., Mautone A. J., Mendelsohn R. Fourier-transform infrared spectroscopy studies of lipid/protein interaction in pulmonary surfactant. Biochemistry. 1989 Sep 5;28(18):7368–7373. doi: 10.1021/bi00444a033. [DOI] [PubMed] [Google Scholar]
  31. Ross G. F., Notter R. H., Meuth J., Whitsett J. A. Phospholipid binding and biophysical activity of pulmonary surfactant-associated protein (SAP)-35 and its non-collagenous COOH-terminal domains. J Biol Chem. 1986 Oct 25;261(30):14283–14291. [PubMed] [Google Scholar]
  32. Ross G. F., Sawyer J., O'Connor T., Whitsett J. A. Intermolecular cross-links mediate aggregation of phospholipid vesicles by pulmonary surfactant protein SP-A. Biochemistry. 1991 Jan 22;30(3):858–865. doi: 10.1021/bi00217a040. [DOI] [PubMed] [Google Scholar]
  33. Sano K., Fisher J., Mason R. J., Kuroki Y., Schilling J., Benson B., Voelker D. Isolation and sequence of a cDNA clone for the rat pulmonary surfactant-associated protein (PSP-A). Biochem Biophys Res Commun. 1987 Apr 14;144(1):367–374. doi: 10.1016/s0006-291x(87)80519-3. [DOI] [PubMed] [Google Scholar]
  34. Schlame M., Casals C., Rüstow B., Rabe H., Kunze D. Molecular species of phosphatidylcholine and phosphatidylglycerol in rat lung surfactant and different pools of pneumocytes type II. Biochem J. 1988 Jul 1;253(1):209–215. doi: 10.1042/bj2530209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schürch S., Bachofen H., Goerke J., Possmayer F. A captive bubble method reproduces the in situ behavior of lung surfactant monolayers. J Appl Physiol (1985) 1989 Dec;67(6):2389–2396. doi: 10.1152/jappl.1989.67.6.2389. [DOI] [PubMed] [Google Scholar]
  36. Suzuki Y., Fujita Y., Kogishi K. Reconstitution of tubular myelin from synthetic lipids and proteins associated with pig pulmonary surfactant. Am Rev Respir Dis. 1989 Jul;140(1):75–81. doi: 10.1164/ajrccm/140.1.75. [DOI] [PubMed] [Google Scholar]
  37. Tenner A. J., Robinson S. L., Borchelt J., Wright J. R. Human pulmonary surfactant protein (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR1-mediated phagocytosis. J Biol Chem. 1989 Aug 15;264(23):13923–13928. [PubMed] [Google Scholar]
  38. Van Golde L. M., Batenburg J. J., Robertson B. The pulmonary surfactant system: biochemical aspects and functional significance. Physiol Rev. 1988 Apr;68(2):374–455. doi: 10.1152/physrev.1988.68.2.374. [DOI] [PubMed] [Google Scholar]
  39. Voorhout W. F., Veenendaal T., Haagsman H. P., Verkleij A. J., van Golde L. M., Geuze H. J. Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice. J Histochem Cytochem. 1991 Oct;39(10):1331–1336. doi: 10.1177/39.10.1940306. [DOI] [PubMed] [Google Scholar]
  40. Voss T., Eistetter H., Schäfer K. P., Engel J. Macromolecular organization of natural and recombinant lung surfactant protein SP 28-36. Structural homology with the complement factor C1q. J Mol Biol. 1988 May 5;201(1):219–227. doi: 10.1016/0022-2836(88)90448-2. [DOI] [PubMed] [Google Scholar]
  41. White R. T., Damm D., Miller J., Spratt K., Schilling J., Hawgood S., Benson B., Cordell B. Isolation and characterization of the human pulmonary surfactant apoprotein gene. 1985 Sep 26-Oct 2Nature. 317(6035):361–363. doi: 10.1038/317361a0. [DOI] [PubMed] [Google Scholar]
  42. Williams M. C., Hawgood S., Hamilton R. L. Changes in lipid structure produced by surfactant proteins SP-A, SP-B, and SP-C. Am J Respir Cell Mol Biol. 1991 Jul;5(1):41–50. doi: 10.1165/ajrcmb/5.1.41. [DOI] [PubMed] [Google Scholar]
  43. Wright J. R., Dobbs L. G. Regulation of pulmonary surfactant secretion and clearance. Annu Rev Physiol. 1991;53:395–414. doi: 10.1146/annurev.ph.53.030191.002143. [DOI] [PubMed] [Google Scholar]
  44. van Iwaarden F., Welmers B., Verhoef J., Haagsman H. P., van Golde L. M. Pulmonary surfactant protein A enhances the host-defense mechanism of rat alveolar macrophages. Am J Respir Cell Mol Biol. 1990 Jan;2(1):91–98. doi: 10.1165/ajrcmb/2.1.91. [DOI] [PubMed] [Google Scholar]

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