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. 2000 Nov;79(5):2657–2666. doi: 10.1016/S0006-3495(00)76504-0

Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract.

L A Worthman 1, K Nag 1, N Rich 1, M L Ruano 1, C Casals 1, J Pérez-Gil 1, K M Keough 1
PMCID: PMC1301146  PMID: 11053138

Abstract

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 microg/ml SP-A and 0, 1.64, or 5 mM CaCl(2). In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca(2+). Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures (pi approximately 5 mN/m) and the domains grew in size with increasing pi. Above 25 mN/m, the domain size decreased with increasing pi. The amount of observable dark phase was maximal at 18% of the total film area at pi approximately 25 mN/m, then decreased to approximately 3% at pi approximately 40 mN/m. The addition of 0.16 microg/ml SP-A with 0 or 1.64 mM Ca(2+) in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to approximately 25% between pi of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca(2+) and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca(2+). PSLE films were spread on a subphase containing 0.16 microg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca(2+), TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca(2+) resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.

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Selected References

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  1. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  2. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  3. Benson B. J., Williams M. C., Sueishi K., Goerke J., Sargeant T. Role of calcium ions the structure and function of pulmonary surfactant. Biochim Biophys Acta. 1984 Mar 27;793(1):18–27. doi: 10.1016/0005-2760(84)90048-1. [DOI] [PubMed] [Google Scholar]
  4. Casals C., Miguel E., Perez-Gil J. Tryptophan fluorescence study on the interaction of pulmonary surfactant protein A with phospholipid vesicles. Biochem J. 1993 Dec 15;296(Pt 3):585–593. doi: 10.1042/bj2960585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chung J., Yu S. H., Whitsett J. A., Harding P. G., Possmayer F. Effect of surfactant-associated protein-A (SP-A) on the activity of lipid extract surfactant. Biochim Biophys Acta. 1989 Apr 26;1002(3):348–358. doi: 10.1016/0005-2760(89)90349-4. [DOI] [PubMed] [Google Scholar]
  6. Discher B. M., Maloney K. M., Grainger D. W., Sousa C. A., Hall S. B. Neutral lipids induce critical behavior in interfacial monolayers of pulmonary surfactant. Biochemistry. 1999 Jan 5;38(1):374–383. doi: 10.1021/bi981386h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Discher B. M., Maloney K. M., Schief W. R., Jr, Grainger D. W., Vogel V., Hall S. B. Lateral phase separation in interfacial films of pulmonary surfactant. Biophys J. 1996 Nov;71(5):2583–2590. doi: 10.1016/S0006-3495(96)79450-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fleming B. D., Keough K. M. Surface respreading after collapse of monolayers containing major lipids of pulmonary surfactant. Chem Phys Lipids. 1988 Nov;49(1-2):81–86. doi: 10.1016/0009-3084(88)90067-9. [DOI] [PubMed] [Google Scholar]
  9. Goerke J., Gonzales J. Temperature dependence of dipalmitoyl phosphatidylcholine monolayer stability. J Appl Physiol Respir Environ Exerc Physiol. 1981 Nov;51(5):1108–1114. doi: 10.1152/jappl.1981.51.5.1108. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Haagsman H. P., White R. T., Schilling J., Lau K., Benson B. J., Golden J., Hawgood S., Clements J. A. Studies of the structure of lung surfactant protein SP-A. Am J Physiol. 1989 Dec;257(6 Pt 1):L421–L429. doi: 10.1152/ajplung.1989.257.6.L421. [DOI] [PubMed] [Google Scholar]
  14. Hawco M. W., Coolbear K. P., Davis P. J., Keough K. M. Exclusion of fluid lipid during compression of monolayers of mixtures of dipalmitoylphosphatidylcholine with some other phosphatidylcholines. Biochim Biophys Acta. 1981 Aug 6;646(1):185–187. doi: 10.1016/0005-2736(81)90286-8. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Hawgood S. Pulmonary surfactant apoproteins: a review of protein and genomic structure. Am J Physiol. 1989 Aug;257(2 Pt 1):L13–L22. doi: 10.1152/ajplung.1989.257.2.L13. [DOI] [PubMed] [Google Scholar]
  17. Hildebran J. N., Goerke J., Clements J. A. Pulmonary surface film stability and composition. J Appl Physiol Respir Environ Exerc Physiol. 1979 Sep;47(3):604–611. doi: 10.1152/jappl.1979.47.3.604. [DOI] [PubMed] [Google Scholar]
  18. Johansson J., Curstedt T. Molecular structures and interactions of pulmonary surfactant components. Eur J Biochem. 1997 Mar 15;244(3):675–693. doi: 10.1111/j.1432-1033.1997.00675.x. [DOI] [PubMed] [Google Scholar]
  19. Keough K. M., Kariel N. Differential scanning calorimetric studies of aqueous dispersions of phosphatidylcholines containing two polyenoic chains. Biochim Biophys Acta. 1987 Aug 7;902(1):11–18. doi: 10.1016/0005-2736(87)90130-1. [DOI] [PubMed] [Google Scholar]
  20. Keough K. M., Parsons C. S., Phang P. T., Tweeddale M. G. Interactions between plasma proteins and pulmonary surfactant: surface balance studies. Can J Physiol Pharmacol. 1988 Sep;66(9):1166–1173. doi: 10.1139/y88-192. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. King R. J., Clements J. A. Surface active materials from dog lung. II. Composition and physiological correlations. Am J Physiol. 1972 Sep;223(3):715–726. doi: 10.1152/ajplegacy.1972.223.3.715. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Korfhagen T. R., Bruno M. D., Ross G. F., Huelsman K. M., Ikegami M., Jobe A. H., Wert S. E., Stripp B. R., Morris R. E., Glasser S. W. Altered surfactant function and structure in SP-A gene targeted mice. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9594–9599. doi: 10.1073/pnas.93.18.9594. [DOI] [PMC free article] [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. Kuroki Y., Voelker D. R. Pulmonary surfactant proteins. J Biol Chem. 1994 Oct 21;269(42):25943–25946. [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. McCormack F. X., Kuroki Y., Stewart J. J., Mason R. J., Voelker D. R. Surfactant protein A amino acids Glu195 and Arg197 are essential for receptor binding, phospholipid aggregation, regulation of secretion, and the facilitated uptake of phospholipid by type II cells. J Biol Chem. 1994 Nov 25;269(47):29801–29807. [PubMed] [Google Scholar]
  29. Meyboom A., Maretzki D., Stevens P. A., Hofmann K. P. Reversible calcium-dependent interaction of liposomes with pulmonary surfactant protein A. Analysis by resonant mirror technique and near-infrared light scattering. J Biol Chem. 1997 Jun 6;272(23):14600–14605. doi: 10.1074/jbc.272.23.14600. [DOI] [PubMed] [Google Scholar]
  30. Nag K., Boland C., Rich N., Keough K. M. Epifluorescence microscopic observation of monolayers of dipalmitoylphosphatidylcholine: dependence of domain size on compression rates. Biochim Biophys Acta. 1991 Sep 30;1068(2):157–160. doi: 10.1016/0005-2736(91)90204-l. [DOI] [PubMed] [Google Scholar]
  31. Nag K., Keough K. M. Epifluorescence microscopic studies of monolayers containing mixtures of dioleoyl- and dipalmitoylphosphatidylcholines. Biophys J. 1993 Sep;65(3):1019–1026. doi: 10.1016/S0006-3495(93)81155-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nag K., Perez-Gil J., Ruano M. L., Worthman L. A., Stewart J., Casals C., Keough K. M. Phase transitions in films of lung surfactant at the air-water interface. Biophys J. 1998 Jun;74(6):2983–2995. doi: 10.1016/S0006-3495(98)78005-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nielson D. W., Goerke J., Clements J. A. Alveolar subphase pH in the lungs of anesthetized rabbits. Proc Natl Acad Sci U S A. 1981 Nov;78(11):7119–7123. doi: 10.1073/pnas.78.11.7119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pérez-Gil J., Nag K., Taneva S., Keough K. M. Pulmonary surfactant protein SP-C causes packing rearrangements of dipalmitoylphosphatidylcholine in spread monolayers. Biophys J. 1992 Jul;63(1):197–204. doi: 10.1016/S0006-3495(92)81582-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pérez-Gil J., Tucker J., Simatos G., Keough K. M. Interfacial adsorption of simple lipid mixtures combined with hydrophobic surfactant protein from pig lung. Biochem Cell Biol. 1992 May;70(5):332–338. doi: 10.1139/o92-051. [DOI] [PubMed] [Google Scholar]
  36. Ruano M. L., Miguel E., Perez-Gil J., Casals C. Comparison of lipid aggregation and self-aggregation activities of pulmonary surfactant-associated protein A. Biochem J. 1996 Jan 15;313(Pt 2):683–689. doi: 10.1042/bj3130683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ruano M. L., Nag K., Worthman L. A., Casals C., Pérez-Gil J., Keough K. M. Differential partitioning of pulmonary surfactant protein SP-A into regions of monolayers of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol. Biophys J. 1998 Mar;74(3):1101–1109. doi: 10.1016/s0006-3495(98)77828-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schürch S., Possmayer F., Cheng S., Cockshutt A. M. Pulmonary SP-A enhances adsorption and appears to induce surface sorting of lipid extract surfactant. Am J Physiol. 1992 Aug;263(2 Pt 1):L210–L218. doi: 10.1152/ajplung.1992.263.2.L210. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Takahashi A., Fujiwara T. Proteolipid in bovine lung surfactant: its role in surfactant function. Biochem Biophys Res Commun. 1986 Mar 13;135(2):527–532. doi: 10.1016/0006-291x(86)90026-4. [DOI] [PubMed] [Google Scholar]
  41. Taneva S., McEachren T., Stewart J., Keough K. M. Pulmonary surfactant protein SP-A with phospholipids in spread monolayers at the air-water interface. Biochemistry. 1995 Aug 15;34(32):10279–10289. doi: 10.1021/bi00032a023. [DOI] [PubMed] [Google Scholar]
  42. Udenfriend S., Stein S., Böhlen P., Dairman W., Leimgruber W., Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972 Nov 24;178(4063):871–872. doi: 10.1126/science.178.4063.871. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Wang Z., Hall S. B., Notter R. H. Dynamic surface activity of films of lung surfactant phospholipids, hydrophobic proteins, and neutral lipids. J Lipid Res. 1995 Jun;36(6):1283–1293. [PubMed] [Google Scholar]
  45. 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]
  46. Yu S. H., Possmayer F. Adsorption, compression and stability of surface films from natural, lipid extract and reconstituted pulmonary surfactants. Biochim Biophys Acta. 1993 Apr 23;1167(3):264–271. doi: 10.1016/0005-2760(93)90228-2. [DOI] [PubMed] [Google Scholar]
  47. Yu S. H., Possmayer F. Effect of pulmonary surfactant protein A and neutral lipid on accretion and organization of dipalmitoylphosphatidylcholine in surface films. J Lipid Res. 1996 Jun;37(6):1278–1288. [PubMed] [Google Scholar]

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