Background This study examines the surface activity and resistance to phospholipase degradation of a fully-synthetic lung surfactant containing a novel diether phosphonolipid (DEPN-8) plus a 34 amino acid peptide (Mini-B) related to native surfactant protein (SP)-B. Activity studies used adsorption, pulsating bubble, and captive bubble methods to assess a range of surface behaviors, supplemented by molecular studies using Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD), and plasmon resonance. Calf lung surfactant extract (CLSE) was used as a positive control. Results DEPN-8+1.5% (by wt.) Mini-B was fully resistant to degradation by phospholipase A2 (PLA2) in vitro, while CLSE was severely degraded by this enzyme. Mini-B interacted with DEPN-8 at the molecular level based on FTIR spectroscopy, and had significant plasmon resonance binding affinity for DEPN-8. DEPN-8+1.5% Mini-B had greatly increased adsorption compared to DEPN-8 alone, but did not fully equal the very high adsorption of CLSE. In pulsating bubble studies at a low phospholipid concentration of 0.5 mg/ml, DEPN-8+1.5% Mini-B and CLSE both reached minimum surface tensions <1 mN/m after 10 min of cycling. DEPN-8 (2.5 mg/ml)+1.5% Mini-B and CLSE (2.5 mg/ml) also reached minimum surface tensions <1 mN/m at 10 min of pulsation in the presence of serum albumin (3 mg/ml) on the pulsating bubble. In captive bubble studies, DEPN-8+1.5% Mini-B and CLSE both generated minimum surface tensions <1 mN/m on 10 successive cycles of compression/expansion at quasi-static and dynamic rates. Conclusions These results show that DEPN-8 and 1.5% Mini-B form an interactive binary molecular mixture with very high surface activity and the ability to resist degradation by phospholipases in inflammatory lung injury. These characteristics are promising for the development of related fully-synthetic lipid/peptide exogenous surfactants for treating diseases of surfactant deficiency or dysfunction.
References
[1]
Notter RH (2000) Lung surfactants: Basic science and clinical applications. New York: Marcel Dekker, Inc.
[2]
Kim DK, Fukuda T, Thompson BT, Cockrill B, Hales C, et al. (1995) Bronchoalveolar lavage fluid phospholipase A2 activities are increased in human adult respiratory distress syndrome. Am J Physiol 269: L109–L118.
[3]
Holm BA, Kelcher L, Liu M, Sokolowski J, Enhorning G (1991) Inhibition of pulmonary surfactant by phospholipases. J Appl Physiol 71: 317–321.
[4]
Touqui L, Arbibe L (1999) A role for phospholipase A2 in ARDS pathogenesis. Molec Med Today 5: 244–249.
[5]
Vadas P (1984) Elevated plasma phospholipase A2 levels: correlation with the hemodynamic and pulmonary changes in Gram-negative septic shock. J Lab Clin Med 104: 873–881.
[6]
Kostopanagiotou G, Routs C, Smyrniotis V, Lekka ME, Kitsiouli E, et al. (2003) Alterations in bronchoalveolar lavage fluid during ischemia-induced acute hepatic failure in the pig. Hepatology 37: 1130–1138.
[7]
Attalah HL, Wu Y, Alaoui-El-Azher M, Thouron F, Koumanov K, et al. (2003) Induction of type-IIA secretory phospholipase A2 in animal models of acute lung injury Eur Respir J 21: 1040–1045.
[8]
Nakos G, Kitsiouli E, Hatzidaki E, Koulouras V, Touqui L, et al. (2003) Phospholipases A2 and platelet-activating–factor acetylhydrolase in patients with acute respiratory distress syndrome. Crit Care Med 33: 772–779.
[9]
Ackerman SJ, Kwatia MA, Doyle CB, Enhorning G (2003) Hydrolysis of surfactant phospholipids catalyzed by phospholipase A2 and eosinophil lysophospholipases causes surfactant dysfunction: A mechanism for small airway closure in asthma. Chest 123: 255S.
[10]
Hall SB, Lu ZR, Venkitaraman AR, Hyde RW, Notter RH (1992) Inhibition of pulmonary surfactant by oleic acid: Mechanisms and characteristics. J Appl Physiol 72: 1708–1716.
[11]
Wang Z, Notter RH (1998) Additivity of protein and non-protein inhibitors of lung surfactant activity. Am J Respir Crit Care Med 158: 28–35.
[12]
Holm BA, Wang Z, Notter RH (1999) Multiple mechanisms of lung surfactant inhibition. Pediatr Res 46: 85–93.
[13]
Lewis JF, Brackenbury A (2003) Role of exogenous surfactant in acute lung injury. Crit Care Med 31: supplS324–S328.
[14]
Notter RH, Finkelstein JN, Holm BA, editors (2005) Lung injury: Mechanisms, pathophysiology and therapy. Boca Raton: Taylor Francis Group, Inc.
[15]
Griese M (1999) Pulmonary surfactant in health and human lung diseases: State of the art. Eur Respir J 13: 1455–1476.
[16]
Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, et al. (1994) The American-European Consensus Conference on ARDS: Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am J Respir Crit Care Med 149: 818–824.
[17]
Artigas A, Bernard GR, Carlet J, Dreyfuss D, Gattinoni L, et al. (1998) The American-European consensus conference on ARDS, Part 2: Ventilatory, pharmacologic, supportive therapy, study design strategies and issues related to recovery and remodeling. Intensive Care Med 24: 378–398.
[18]
Goss CH, Brower RG, Hudson LD, Rubenfeld GD, ARDS Network (2003) Incidence of acute lung injury in the United States. Crit Care Med 31: 1607–1611.
[19]
Rubenfeld GD (2003) Epidemiology of acute lung injury. Crit Care Med 31: supplS276–S284.
[20]
Chess P, Finkelstein JN, Holm BA, Notter RH (2005) Surfactant replacement therapy in lung injury. In: Notter RH, Finkelstein JN, Holm BA, editors. Lung injury: Mechanisms, pathophysiology, and therapy. Boca Raton: Taylor Francis Group, Inc. pp. 617–663.
[21]
Wang Z, Holm BA, Matalon S, Notter RH (2005) Surfactant activity and dysfunction in lung injury. In: Notter RH, Finkelstein JN, Holm BA, editors. Lung injury: Mechanisms, pathophysiology, and therapy. Boca Raton: Taylor Francis Group, Inc. pp. 297–352.
[22]
Turcotte JG, Lin WH, Pivarnik PE, Sacco AM, Bermel MS, et al. (1991) Chemical synthesis and surface activity of lung surfactant phospholipid analogs. II. Racemic N-substituted diether phosphonolipids. Biochim Biophys Acta 1084: 1–12.
[23]
Turcotte JG, Sacco AM, Steim JM, Tabak SA, Notter RH (1977) Chemical synthesis and surface properties of an analog of the pulmonary surfactant dipalmitoyl phosphatidylcholine analog. Biochim Biophys Acta 488: 235–248.
[24]
Lin WHC (1989) Synthesis and purification of novel diether and ether-amide analogs of dipalmitoyl phosphatidylcholine [PhD]. Kingston, RI: University of Rhode Island.
[25]
Waring AJ, Walther FJ, Gordon LM, Hernandez-Juviel JM, Hong T, et al. (2005) The role of charged amphipathic helices in the structure and function of surfactant protein B (SP-B). J Peptide Res 66: 364–374.
[26]
Notter RH, Wang Z (1997) Pulmonary surfactant: Physical chemistry, physiology and replacement. Rev Chem Eng 13: 1–118.
[27]
Gordon LM, Mobley PW, Pilpa R, Sherman MA, Waring AJ (2002) Conformational mapping of the N-terminal peptide of HIV-1 gp41 in membrane environments using 13C-enhanced Fourier transform infrared spectroscopy. Biochem Biophys Acta 1559: 96–120.
[28]
Sreerama N, Venyaminov SY, Woody RW (1999) Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy. Prot Sci 8: 370–380.
[29]
Caillon E, Lubochinsky B, Rigomier D (1983) Occurrence of dialkyl ether phospholipids in Stigmatella aurantiaca DW4. J Bacteriology 153: 1348–1351.
[30]
Wang Z, Schwan AL, Lairson LL, O'Donnell JS, Byrne GF, et al. (2003) Surface activity of a synthetic lung surfactant containing a phospholipase-resistant phosphonolipid analog of dipalmitoyl phosphatidylcholine. Am J Physiol 285: L550–L559.
[31]
Holm BA, Notter RH, Finkelstein JH (1985) Surface property changes from interactions of albumin with natural lung surfactant and extracted lung lipids. Chem Phys Lipids 38: 287–298.
[32]
Seeger W, Grube C, Günther A, Schmidt R (1993) Surfactant inhibition by plasma proteins: Differential sensitivity of various surfactant preparations. Eur Respir J 6: 971–977.
[33]
Hall SB, Venkitaraman AR, Whitsett JA, Holm BA, Notter RH (1992) Importance of hydrophobic apoproteins as constituents of clinical exogenous surfactants. Am Rev Respir Dis 145: 24–30.
[34]
Notter RH, Wang Z, Egan EA, Holm BA (2002) Component-specific surface and physiological activity in bovine-derived lung surfactants. Chem Phys Lipids 114: 21–34.
[35]
Chang Y, Wang Z, Notter RH, Wang Z, Long Q, et al. (2004) Synthesis and interfacial behavior of sulfur-containing analogs of lung surfactant dipalmitoyl phosphatidylcholine. Bioorg Med Chem Lett 14: 5983–5986.
[36]
Chang Y, Wang Z, Schwan AL, Wang Z, Holm BA, et al. (2005) Surface properties of sulfur- and ether-linked phosphonolipids with and without purified hydrophobic lung surfactant proteins Chem Phys Lipids 137: 77–93.
[37]
Wang Z, Chang Y, Schwan AL, Notter RH (2007) Activity and inhibition resistance of a phospholipase-resistant synthetic surfactant in rat lungs. Am J Respir Cell Mol Biol: In Press.
[38]
Willson DF, Thomas NJ, Markovitz BP, DiCarlo JV, Pon S, et al. (2005) Effect of exogenous surfactant (calfactant) in pediatric acute lung injury: a randomized controlled trial. JAMA 293: 470–476.
[39]
Johansson J, Gustafsson M, Zaltash S, Robertson B, Curstedt T (1998) Synthetic surfactant protein analogs. Biol Neonate 74: Suppl9–14.
[40]
Oosterlaken-Dijksterhuis MA, Haagsman HP, van Golde LM, Demel RA (1991) Characterization of lipid insertion into monomolecular layers mediated by lung surfactant proteins SP-B and SP-C. Biochemistry 30: 10965–10971.
[41]
Oosterlaken-Dijksterhuis MA, Haagsman HP, van Golde LM, Demel RA (1991) Interaction of lipid vesicles with monomolecular layers containing lung surfactant proteins SP-B or SP-C. Biochemistry 30: 8276–8281.
[42]
Curstedt T, Jornvall H, Robertson B, Bergman T, Berggren P (1987) Two hydrophobic low-molecular-mass protein fractions of pulmonary surfactant: Characterization and biophysical activity. Eur J Biochem 168: 255–262.
[43]
Wang Z, Gurel O, Baatz JE, Notter RH (1996) Differential activity and lack of synergy of lung surfactant proteins SP-B and SP-C in surface-active interactions with phospholipids. J Lipid Res 37: 1749–1760.
[44]
Yu SH, Possmayer F (1988) Comparative studies on the biophysical activities of the low-molecular-weight hydrophobic proteins purified from bovine pulmonary surfactant. Biochim Biophys Acta 961: 337–350.
[45]
Seeger W, Günther A, Thede C (1992) Differential sensitivity to fibrinogen inhibition of SP-C- vs. SP-B-based surfactants. Am J Physiol 261: L286–L291.
[46]
Sarin VK, Gupta S, Leung TK, Taylor VE, Ohning BL, et al. (1990) Biophysical and biological activity of a synthetic 8.7 kDa hydrophobic pulmonary surfactant protein SP-B. Proc Natl Acad Sci USA 87: 2633–2637.
[47]
Revak SD, Merritt TA, Degryse E, Stefani L, Courtney M, et al. (1988) The use of human low molecular weight (LMW) apoproteins in the reconstitution of surfactant biological activity. J Clin Invest 81: 826–833.
[48]
Waring A, Faull L, Leung C, Chang-Chien A, Mercado P, et al. (1996) Synthesis, secondary structure and folding of the bend region of lung surfactant protein B. Peptide Res 9: 28–31.
[49]
Waring A, Taeusch W, Bruni R, Amirkhanian J, Fan B, et al. (1989) Synthetic amphipathic sequences of SP-B mimic several physicochemical and in vitro properties of native pulmonary surfactant proteins. Peptide Res 2: 308–313.
[50]
Gordon LM, Horvath S, Longo ML, Zasadzinski JAN, Taeusch HW, et al. (1996) Conformation and molecular topography of the N-terminal segment of surfactant protein B in structure-promoting environments. Protein Sci 5: 1662–1675.
[51]
Gordon LM, Lee KYC, Lipp MM, Zasadzinski JM, Walther FJ, et al. (2000) Conformational mapping of the N-terminal segment of surfactant protein B in lipid using 13C-enhanced Fourier transform infrared spectroscopy. J Peptide Res 55: 330–347.
[52]
Notter RH, Wang Z, Wang Z, Davy J, Schwan AL (2007) Synthesis and surface activity of diether-linked phosphoglycerols: Potential applications for exogenous lung surfactants. Bioorg Med Chem Lett 17: 113–117.
[53]
Touchstone JC, Chen JC, Beaver KM (1980) Improved separation of phospholipids in thin-layer chromatography. Lipids 15: 61–62.
[54]
Fields CG, Lloyd DH, McDonald RL, Ottenson KM, Nobel RL (1991) HBTU activation for automated Fmoc solid-phase peptide synthesis. Peptide Res 4: 95–101.
[55]
Carpino LA, Chao H-G, Chassemi S, Mansour EME, Riemer C, et al. (1995) Novel carboxylic acid and carboxamide protective groups based on the exceptional stabilization of the cyclopropylmethyl cation. J Org Chem 60: 7718–7719.
[56]
Applied Biosystems Manual (1990) Introduction to cleavage techniques. Foster City: California Applied Biosystems. pp. 10–13.
[57]
Pace NC, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Science 4: 2411–2423.
[58]
Wang Z, Hall SB, Notter RH (1995) Dynamic surface activity of films of lung surfactant phospholipids, hydrophobic proteins, and neutral lipids. J Lipid Res 36: 1283–1293.
[59]
Wang Z, Hall SB, Notter RH (1996) Roles of different hydrophobic constituents in the adsorption of pulmonary surfactant. J Lipid Res 37: 790–798.
[60]
Hall SB, Wang Z, Notter RH (1994) Separation of subfractions of the hydrophobic components of calf lung surfactant. J Lipid Res 35: 1386–1394.
[61]
Enhorning G, Shumel B, Keicher L, Sokolowski J, Holm BA (1992) Phospholipases introduced into the hypophase affect the surfactant film outlining a bubble. J Appl Physiol 73: 941–945.
[62]
Ames BN (1966) Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol 8: 115–118.
[63]
Byler DM, Susi H (1986) Examination of the secondary structure of protein by deconvolved FTIR spectra. Biopolymers 25: 469–487.
[64]
Johnson WCJ (1990) Protein secondary structure and circular dichroism: a practical guide. Proteins 7: 205–214.
[65]
Notter RH, Smith S, Taubold RD, Finkelstein JN (1982) Path dependence of adsorption behavior of mixtures containing dipalmitoyl phosphatidylcholine. Pediatr Res 16: 515–519.
Enhorning G (1977) Pulsating bubble technique for evaluation of pulmonary surfactant. J Appl Physiol 43: 198–203.
[68]
Hall SB, Bermel MS, Ko YT, Palmer HJ, Enhorning GA, et al. (1993) Approximations in the measurement of surface tension with the oscillating bubble surfactometer. J Appl Physiol 75: 468–477.
[69]
Schoel WM, Schurch S, Goerke J (1994) The captive bubble method for the evaluation of pulmonary surfactant: surface tension, area, and volume calculations. Biochim Biophys Acta 1200: 281–290.
[70]
Malcolm JD, Elliott CD (1980) Interfacial tension from height and diameter of a single profile drop of captive bubble. Can J Chem Eng 58: 151–153.
