Surface activity in vitro: role of surfactant proteins

Pattle, who provided some of the initial direct evidence for the presence of pulmonary surfactant in the lung, was also the first to show surfactant was susceptible to proteases such as trypsin. Pattle concluded surfactant was a lipoprotein. Our group has investigated the roles of the surfactant proteins (SP-) SP-A, SP-B, and SP-C using a captive bubble tensiometer. These studies show that SP-C>SP-B>SP-A in enhancing surfactant lipid adsorption (film formation) to the equilibrium surface tension of approximately 22-25 mN/m from the 70 mN/m of saline at 37 degrees C. In addition to enhancing adsorption, surfactant proteins can stabilize surfactant films so that lateral compression induced through surface area reduction results in the lowering of surface tension (gamma) from approximately 25 mN/m (equilibrium) to values near 0 mN/m. These low tensions, which are required to stabilize alveoli during expiration, are thought to arise through exclusion of fluid phospholipids from the surface monolayer, resulting in an enrichment in the gel phase component dipalmitoylphosphatidylcholine (DPPC). The results are consistent with DPPC enrichment occurring through two mechanisms, selective DPPC adsorption and preferential squeeze-out of fluid components such as unsaturated phosphatidylcholine (PC) and phosphatidylglycerol (PG) from the monolayer. Evidence for selective DPPC adsorption arises from experiments showing that the surface area reductions required to achieve gamma near 0 mN/m with DPPC/PG samples containing SP-B or SP-A plus SP-B films were less than those predicted for a pure squeeze-out mechanism. Surface activity improves during quasi-static or dynamic compression-expansion cycles, indicating the squeeze-out mechanism also occurs. Although SP-C was not as effective as SP-B in promoting selective DPPC adsorption, this protein is more effective in promoting the reinsertion of lipids forced out of the surface monolayer following overcompression at low gamma values. Addition of SP-A to samples containing SP-B but not SP-C limits the increase in gamma(max) during expansion. It is concluded that the surfactant apoproteins possess distinct overlapping functions. SP-B is effective in selective DPPC insertion during monolayer formation and in PG squeeze-out during monolayer compression. SP-A can promote adsorption during film formation, particularly in the presence of SP-B. SP-C appears to have a superior role to SP-B in formation of the surfactant reservoir and in reinsertion of collapse phase lipids.     

Content-dependent activity of lung surfactant protein B in mixtures with lipids

The content-dependent activity of surfactant protein (SP)-B was studied in mixtures with dipalmitoyl phosphatidylcholine (DPPC), synthetic lipids (SL), and purified phospholipids (PPL) from calf lung surfactant extract (CLSE). At fixed SP-B content, adsorption and dynamic surface tension lowering were ordered as PPL/SP-B approximately SL/SP-B > DPPC/SP-B. All mixtures were similar in having increased surface activity as SP-B content was incrementally raised from 0.05 to 0.75% by weight. SP-B had small but measurable effects on interfacial properties even at very low levels < or =0.1% by weight. PPL/SP-B (0.75%) had the highest adsorption and dynamic surface activity, approaching the behavior of CLSE. All mixtures containing 0.75% SP-B reached minimum surface tensions <1 mN/m in pulsating bubble studies at low phospholipid concentration (1 mg/ml). Mixtures of PPL or SL with SP-B (0.5%) also had minimum surface tensions <1 mN/m at 1 mg/ml, whereas DPPC/SP-B (0.5%) reached <1 mN/m at 2.5 mg/ml. Physiological activity also was strongly dependent on SP-B content. The ability of instilled SL/SP-B mixtures to improve surfactant-deficient pressure-volume mechanics in excised lavaged rat lungs increased as SP-B content was raised from 0.1 to 0.75% by weight. This study emphasizes the crucial functional activity of SP-B in lung surfactants. Significant differences in SP-B content between exogenous surfactants used to treat respiratory disease could be associated with substantial activity variations.     

Comparative studies on the biophysical activities of the low-molecular-weight hydrophobic proteins purified from bovine pulmonary surfactant

Two low-molecular-weight hydrophobic proteins with nominal molecular weights Mr = 15,000 and Mr = 3,500 have been isolated from the lipid extracts of bovine pulmonary surfactant by several methods, including (a) dialysis plus silicic acid chromatography, (b) elution from Waters SEP-PAK silica cartridges with a variety of solvent mixtures, and (c) ultrafiltration. As detailed in the text, these proteins have been designated surfactant-associated protein-BC (SP-BC) (15 kDa: nonreduced), and SP-C (3.5 kDa). The biophysical activities of reconstituted surfactant containing these proteins and the phospholipids present in lung surfactant have been compared with the biophysical activities of bovine lipid extract surfactant on a pulsating bubble surfactometer using a phospholipid concentration of 10 mg/ml. At this concentration, unmodified lipid extract surfactant reduces the surface tension of the pulsating bubble to near 0 within 10 pulsations at 20 cycles per min. Similar biophysical properties were observed with modified lipid extract surfactant in which the relative concentration of hydrophobic protein had been reduced from 1 to 0.4% (W/W) of the phospholipids by addition of dipalmitoylphosphatidylcholine (DPPC) or DPPC plus phosphatidylglycerol. Reconstituted surfactants, which contained partially delipidated SP-BC (15 kDa: nonreduced) obtained by method (a) at a relative concentration of 0.1%, were also capable of reducing the surface tension to near 0 mN/m. Preparations of SP-BC (15 kDa: nonreduced) obtained by method (b), which had been subjected to very low pH levels during isolation and were extensively delipidated, exhibited full biophysical activity only at higher protein concentrations and with prolonged pulsation. Extensively delipidated samples of SP-BC obtained by method (c) exhibited impaired biophysical activities, even when prepared with neutral organic solvents. Reconstituted surfactant samples containing SP-C (3.5 kDa) obtained by any of the methods listed above were only able to reduce the surface tension at minimum bubble radius to approx. 20 mN/m. The biophysical activity of SP-C (3.5 kDa) was not significantly affected by low pH or extensive delipidation. Reconstituted samples containing mixtures of SP-BC (15 kDa: nonreduced) and SP-C (3.5 kDa) were more effective than samples containing either protein alone. Furthermore, with samples containing both hydrophobic proteins the final surface tensions at maximum bubble radius were attained within a few bubble pulsations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biophysical inhibition of synthetic lung surfactants

The biophysical activity and inhibition of a series of synthetic surfactant mixtures was studied and correlated with physiological effectiveness in restoring pressure-volume (P-V) mechanics of excised lungs. Results showed that several simple mixtures of dipalmitoyl phosphatidylcholine (DPPC) with fatty acids or diacylglycerols could be formulated to give good adsorption facility and dynamic surface tension lowering to less than 1 mN/m in pulsating bubble measurements at 37 degrees C. However, although biophysical activity approached that of natural lung surfactant (LS) and a related surfactant extract (CLSE) under normal conditions, surface properties were sharply inhibited by relatively small amounts of the plasma protein albumin (2 mg/ml) with minimum surface tensions greater than 30 nM/m even at high surfactant concentrations (5-20 mg lipids/ml). This sensitivity to biophysical inhibition was markedly increased compared to LS and CLSE, and had direct consequences for physiological efficacy: in spite of initially high activity, synthetic surfactants did not exert beneficial effects on P-V mechanics when instilled into surfactant-deficient excised rat lungs. Endogenous protein material was shown to be present upon surfactant recovery by lavage, and bubble measurements confirmed surface activity well below pre-instillation levels. Moreover, full biophysical activity was restored when lavage fluid was extracted to separate the synthetic surfactants from endogenous inhibitors. These results show that it is important to define relative sensitivity to biophysical inhibition in the development of effective lung surfactant substitutes. In addition, the existence of inhibition effects can generate an apparent lack of correspondence between initial biophysical activity and ultimate physiological actions of exogenous surfactant mixtures.     

Surface activity and film formation from the surface associated material of artificial surfactant preparations

Surfactant proteins B and C (SP-B and SP-C) are present in natural derived surfactant preparations used for treatment of respiratory distress syndrome. Herein the surface activity of an SP-C analogue (SP-C(LKS)), a hybrid peptide between SP-C and bacteriorhodopsin (SP-C/BR) and a model peptide (KL(4)) was studied with a captive bubble surfactometer (CBS). The peptides were mixed with either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidylglycerol (PG) (7:3, by weight) or DPPC/PG/palmitic acid (68:22:9, by weight) at a concentration of 1 mg/ml in HEPES buffer, pH 6.9 and a polypeptide/lipid weight ratio of 0.02--0.03. In some lipid/peptide preparations also 2% of SP-B was included. Adsorption, monitored as surface tension vs. time for 10 min after bubble formation did not show discernible differences for the whole set of preparations. Equilibrium surface tensions of approximately 25 mN/m were reached after 5--10 min for all preparations, although those with SP-C/BR appeared not to reach end point of adsorption within 10 min. Area compression needed to reach minimum surface tension of 0.5--2.0 mN/m was least for the KL(4) preparation, about 13% in the first cycle. 3% SP-C(LKS) in DPPC:PG (7:3, by weight) reached minimum surface tension upon 27% compression in the first cycle. If DPPC:PG:PA (68:22:9, by weight) was used instead only 16% area compression was needed and 14% if also 2% SP-B was included. 3% SP-C(LKS) in DPPC:PG (7:3, by weight)+2% SP-B needed 34% compression to reach minimum surface tension. The replenishment of material from a surface associated surfactant reservoir was estimated with subphase depletion experiments. With the 2% KL(4) preparation incorporation of excess material took place at a surface tension of 25--35 mN/m during stepwise bubble expansion and excess material equivalent to 4.3 monolayers was found. When 2% SP-B was added to 3% SP-C(LKS) in DPPC:PG (7:3, by weight) the number of excess monolayers increased from 1.5 to 3.6 and the incorporation took place at 30--40 mN/m. When SP-B was added to 3% SP-C(LKS) in DPPC:PG:PA (68:22:9, by weight) the number of excess monolayers increased from 0.5 to 3.4 and incorporation took place at 40--50 mN/m. With 2% SP-C/BR incorporation took place at 40--45 mN/m, frequent instability clicks were observed and excess material of approximately 1.1 monolayer was estimated.     

Thermodynamic effects of the hydrophobic surfactant proteins on the early adsorption of pulmonary surfactant

We determined the influence of the two hydrophobic proteins, SP-B and SP-C, on the thermodynamic barriers that limit adsorption of pulmonary surfactant to the air-water interface. We compared the temperature and concentration dependence of adsorption, measured by monitoring surface tension, between calf lung surfactant extract (CLSE) and the complete set of neutral and phospholipids (N&PL) without the proteins. Three stages generally characterized the various adsorption isotherms: an initial delay during which surface tension remained constant, a fall in surface tension at decreasing rates, and, for experiments that reached approximately 40 mN/m, a late acceleration of the fall in surface tension to approximately 25 mN/m. For the initial change in surface tension, the surfactant proteins accelerated adsorption for CLSE relative to N&PL by more than ten-fold, reducing the Gibbs free energy of transition (DeltaG(O)) from 119 to 112 kJ/mole. For the lipids alone in N&PL, the enthalpy of transition (DeltaH(O), 54 kJ/mole) and entropy (-T. DeltaS, 65 kJ/mole at 37 degrees C) made roughly equal contributions to DeltaG(O). The proteins in CLSE had little effect on -T. DeltaS(O) (68 kJ/mole), but lowered DeltaG(O) for CLSE by reducing DeltaH(O) (44 kJ/mole). Models of the detailed mechanisms by which the proteins facilitate adsorption must meet these thermodynamic constraints.     

Surfactant protein B inhibits secretory phospholipase A2 hydrolysis of surfactant phospholipids

Hydrolysis of surfactant phospholipids (PL) by secretory phospholipases A(2) (sPLA(2)) contributes to surfactant damage in inflammatory airway diseases such as acute lung injury/acute respiratory distress syndrome. We and others have reported that each sPLA(2) exhibits specificity in hydrolyzing different PLs in pulmonary surfactant and that the presence of hydrophilic surfactant protein A (SP-A) alters sPLA(2)-mediated hydrolysis. This report tests the hypothesis that hydrophobic SP-B also inhibits sPLA(2)-mediated surfactant hydrolysis. Three surfactant preparations were used containing varied amounts of SP-B and radiolabeled tracers of phosphatidylcholine (PC) or phosphatidylglycerol (PG): 1) washed ovine surfactant (OS) (pre- and postorganic extraction) compared with Survanta (protein poor), 2) Survanta supplemented with purified bovine SP-B (1-5%, wt/wt), and 3) a mixture of dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) (DPPC:POPC:POPG, 40:40:20) prepared as vesicles and monomolecular films in the presence or absence of SP-B. Hydrolysis of PG and PC by Group IB sPLA(2) (PLA2G1A) was significantly lower in the extracted OS, which contains SP-B, compared with Survanta (P = 0.005), which is SP-B poor. Hydrolysis of PG and PC in nonextracted OS, which contains all SPs, was lower than both Survanta and extracted OS. When Survanta was supplemented with 1% SP-B, PG and PC hydrolysis by PLA2G1B was significantly lower (P < 0.001) than in Survanta alone. When supplemented into pure lipid vesicles and monomolecular films composed of PG and PC mixtures, SP-B also inhibited hydrolysis by both PLA2G1B and Group IIA sPLA2 (PLA2G2A). In films, PLA2G1B hydrolyzed surfactant PL monolayers at surface pressures ≤30 mN/m (P < 0.01), and SP-B lowered the surface pressure range at which hydrolysis can occur. These results suggest the hydrophobic SP, SP-B, protects alveolar surfactant PL from hydrolysis mediated by multiple sPLA(2) in both vesicles (alveolar subphase) and monomolecular films (air-liquid interface).     

Enhancement of biophysical activity of lung surfactant extracts and phospholipid-apoprotein mixtures by surfactant protein A

The effects of surfactant protein (SP)-A on the dynamic surface tension lowering and resistance to inhibition of dispersions of calf lung surfactant extract (CLSE) and mixtures of synthetic phospholipids combined with SP-B,C hydrophobic apoproteins were studied at 37 degrees C and rapid cycling rate (20 cycles/min). Addition of SP-A to CLSE, which already contains SP-B and -C, gave a slight improvement in the time course of surface tension lowering on an oscillating bubble apparatus in the absence of inhibitory protein molecules such as albumin or hemoglobin. However, when these proteins were present at concentrations of 10-50 mg/ml, SP-A substantially improved the resistance of CLSE to their inhibitory effects. The beneficial effect of SP-A required the presence of Ca2+ ions, and disappeared when EDTA was substituted for this divalent cation in the subphase. The effect was also retained when SP-A was heated to 50 degrees C prior to addition to CLSE, but was abolished by heating SP-A to 99 degrees C. Additional studies showed that similar improvements in resistance to inhibition were found when SP-A was added to synthetic mixtures of dipalmitoyl phosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (80:20 by weight) reconstituted with 1% SP-B or SP-B and -C, but not to phospholipid mixtures containing only SP-C. The requirements for SP-B and calcium for the beneficial effects of SP-A on surface activity suggest that the formation of ordered, larger phospholipid-apoprotein aggregates may be involved in the process. The finding that SP-A enhances the ability of CLSE and other surfactant mixtures containing SP-B to resist inhibition is an advantage that will need to be weighed against other factors such as increased antigenicity and heat sensitivity in therapeutic applications in surfactant replacement therapy.     

Interfacial adsorption of simple lipid mixtures combined with hydrophobic surfactant protein from pig lung.

Hydrophobic pulmonary surfactant protein enriched in SP-C has been mixed in amounts up to 10% by weight with various phospholipids. The lipids used were dipalmitoyl phosphatidylcholine (DPPC), or DPPC plus unsaturated phosphatidylglycerol (PG), or phosphatidylinositol (PI) in molar ratios of 9:1 and 7:3. The protein enhanced the rate and extent of adsorption of each lipid preparation into the air-water interface, and its respreading after compression on a surface balance. Maximum surface pressures attained on compression of monolayers of mixtures of lipids were slightly higher in the presence of protein. The effects on rate and extent of adsorption were proportional to the amount of protein present. Mixtures containing 30 mol% PG or PI adsorbed more readily into the interface than those containing 10% acidic lipid or DPPC alone. Mixtures containing 30% PI were slightly more rapidly adsorbed than those containing 30% PG. The results suggest that mixtures of DPPC with either acidic lipid in the presence of surfactant protein could be effective in artificial surfactants.     

Studies on lung surfactant replacement in respiratory distress syndrome. Rapid film formation from binary mixed liposomes

Binary mixed liposomes were prepared from dipalmitoylphosphatidylcholine (DPPC) and a minor compound, e.g., egg phosphatidylglycerol (PG) at a ratio of 9:1. Using different preparative techniques, large unilamellar vesicles (LUV), small unilamellar vesicles (SUV) or multilamellar vesicles (MLV) were obtained and were studied with an electron microscope for morphology, with a Wilhelmy balance for spreading and surface tension lowering potential, and in the surfactant-depleted isolated rat lung for their ability to restore expiratory lung capacity. Only the simultaneous investigation of phospholipids by negative staining and thin sectioning allows unequivocal classification of liposomes. The surface-active structures prepared with the technique of Bangham et al. (Bangham, A.D., Hill, M.W. and Miller, N.G.A. (1974) in Methods in Membrane Biology (Korn, E., ed.), Vol. 1, pp. 1-68, Plenum Press, New York) at room temperature are LUV. LUV containing DPPC:PG at a ratio of 9:1 rapidly spread to a film with high surface tension lowering potential. Within 5 min after injection into the subphase they rise to the surface and form a film at the air/liquid interface able to lower the surface tension to less than 1 mN/m at compression. SUV of the same chemical composition, however, are immediately surface-active only when spread directly onto the surface. MLV exhibit poor surface activity. LUV or pure DPPC, applied onto the surface, are weakly surface active within 5 min. DPPC vesicles injected into the subphase at 37 degrees C do not adsorb to any film with surface tension lowering potential in this time. The minor compounds PE, PI, PS, PA, lysoPC enable DPPC to form surface-active films after application on saline at 37 degrees C. Removal of surfactant decreases the expiratory lung capacity of the isolated rat lung from 49.7 to 12.4% at 4 cmH2O. After substitution with natural surfactant, the expiratory lung capacity is twice that of the washed lung (25.9%), but the original distensibility of the native lung is not restituted. The effect of LUV containing DPPC:PG at a ratio of 9:1 is also remarkable (21.2%).     

Dextran restores albumin-inhibited surface activity of pulmonary surfactant extract.

We examined the effect of dextran (molecular weight 71,000) in counteracting the surfactant inhibitory action of plasma albumin. The surface adsorption time of 0.5 mg/ml modified natural surfactant (MNS; porcine lung extract consisting of phospholipids and hydrophobic surfactant proteins) with 7.5 mg/ml albumin decreased from 681 to 143 s by addition of dextran at a concentration of 10 mg/ml (P < 0.01). The minimum surface tension of 2.0 mg/ml MNS with 30 mg/ml albumin decreased from over 21 mN/m to below 3 mN/m when dextran was added at a concentration of 10 mg/ml (P < 0.01). Surfactant-deficient newborn rabbits given 10 ml/kg of a liquid containing 2.0 mg/ml MNS with 30 mg/ml albumin had a mean tidal volume 13 ml/kg (P < 0.05). Although the underlying mechanism remains to be elucidated, we conclude that dextran restores the albumin-inhibited surface activity of MNS.     

Adsorption of pulmonary surfactant protein SP-A to monolayers of phospholipids containing hydrophobic surfactant protein SP-B or SP-C: potential differential role for tertiary interaction of lipids, hydrophobic proteins, and SP-A

Surface balance techniques were used to study the interactions of surfactant protein SP-A with monolayers of surfactant components preformed at the air-water interface. SP-A adsorption into the monolayers was followed by monitoring the increase in the surface pressure Deltapi after injection of SP-A beneath the films. Monolayers of dipalmitoylphosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (8:2, mol/mol) spread at initial surface pressure pi(i) = 5 mN/m did not promote the adsorption of SP-A at a subphase concentration of 0.68 microg/mL as compared to its adsorption to the monolayer-free surface. Surfactant proteins, SP-B or SP-C, when present in the films of DPPC:PG spread at pi(i) = 5 mN/m, enhanced the incorporation of SP-A in the monolayers to a similar extent; the Deltapi values being dependent on the levels of SP-B or SP-C, 3-17 wt %, in the lipid films. Calcium in the subphase did not affect the intrinsic surface activity of SP-A but reduced the Deltapi values produced by the adsorption of the protein to all the preformed films independently of their compositions and charges. The divalent ions likely modified the interaction of SP-A with the monolayers through their effects on the conformation, self-association, and charge state of SP-A. Values of Deltapi produced by adsorption of SP-A to the films of DPPC:PG with or without SP-B or SP-C were a function of the initial surface pressure of the films, pi(i). In the range of pressures 5 相似文献   

Lipid oxidation and signalling in programmed cell death

The pulmonary surfactant lines as a complex monolayer of lipids and proteins the alveolar epithelial surface. The monolayer dynamically adapts the surface tension of this interface to the varying surface areas during inhalation and exhalation. Its presence in the alveoli is thus a prerequisite for a proper lung function. The lipid moiety represents about 90% of the surfactant and contains mainly dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG). The surfactant proteins involved in the surface tension adaption are called SP-A, SP-B and SP-C. The aim of the present investigation is to analyse the properties of monolayer films made from pure SP-C and from mixtures of DPPC, DPPG and SP-C in order to mimic the surfactant monolayer with minimal compositional requirement. Pressure-area diagrams were taken. Ellipsometric measurements at the air-water interface of a Langmuir film balance allowed measurement of the changes in monolayer thickness upon compression. Isotherms of pure SP-C monolayers exhibit a plateau between 22 and 25 mN/m. A further plateau is reached at higher compression. Structures of the monolayer formed during compression are reversible during expansion. Together with ellipsometric data which show a stepwise increase in film thickness (coverage) during compression, we conclude that pure SP-C films rearrange reversibly into multilayers of homogenous thickness.

Lipid monolayers collapse locally and irreversibly if films are compressed to approximately 0–4 nm²/molecule. In contrast, mixed DPPG/SP-C monolayers with less than 5 mol% protein collapse in a controlled and reversible way. The pressure-area diagrams exhibit a plateau at 20 mN/m, indicating partial demixing of SP-C and DPPG. The thickness isotherm obtained by ellipsometry indicates a transformation into multilayer structures. In DPPC/DPPG/SP-C mixtures again a reversible collapse was observed but without a drastic increase in surface layer thickness which may be due to the formation of protrusion under the surface. Thus lipid monolayers containing small amounts of SP-C may mimic the lung surfactant.     

Chemical synthesis and surface activity of lung surfactant phospholipid analogs. III. Chiral N-substituted ether-amide phosphonolipids

A homologous series of chiral (R) ether-amide phosphonolipid analogs of naturally occurring (R) glycerophospholipids were synthesized and characterized for their interfacial behaviors. The phosphonolipids possess isoteric ether, amide, and phosphonate functions at positions corresponding to the sn-1, sn-2, and sn-3 ester functions, respectively, of naturally occurring glycerophospholipids. All compounds were synthesized with disaturated C16:0 alkyl/acyl moieties to give structural analogy with dipalmitoyl phosphatidylcholine (DPPC), the major glycerophospholipid component of lung surfactant. Further substitutions at the headgroup nitrogen were also used to generate differences in headgroup size and polarity in the synthetic compounds. The surface activity of the ether-amide phospholipids was investigated in terms of adsorption to the air-water interface, together with studies of dynamic respreading after monolayer collapse and surface tension lowering in dynamically compressed spread films and dispersions. Results showed that several ether-amide phosphonolipids had more rapid adsorption and improved dynamic respreading behavior compared to DPPC, plus the ability to lower surface tension into the range of less than 1 to 4 mN/m in spread films and in dispersions under dynamic conditions. In combination with a series of diether phosphonolipids synthetized in a companion study [1], these ether-amide compounds are useful in the development of molecular structure-surface activity correlates for lung surfactant-related materials, and should assist in investigating the specificity of interactions between phospholipids and other pulmonary biological molecules.     

Interactions of pulmonary surfactant protein SP-A with monolayers of dipalmitoylphosphatidylcholine and cholesterol: roles of SP-A domains

Pulmonary surfactant protein A (SP-A) is an oligomeric glycoprotein that binds dipalmitoylphosphatidylcholine (DPPC). Interactions of rat SP-A and recombinant SP-As with pure and binary monolayers of DPPC and cholesterol were studied using a rhomboid surface balance at 37 degrees C. A marked inflection at equilibrium surface tension (23 mN/m) in surface tension-area isotherm of a pure DPPC film was abolished by rat SP-A. The inflection was decreased and shifted to 18 mN/m with wild-type recombinant SP-A (SP-Ahyp). Both rat SP-A and SP-Ahyp decreased surface area reduction required for pure DPPC films to reach near zero surface tension from 30 to 25%. SP-Ahyp, E195Q,R197D, mutated in carbohydrate recognition domain (CRD) known to be essential for SP-A-vesicle interactions, conveyed a detrimental effect on DPPC surface activity. SP-ADeltaG8-P80, with deletion of collagen-like domain, had little effect. Both SP-Ahyp, C6S (Ser substitution for Cys6) and SP-Ahyp,DeltaN1-A7 (N-terminal segment deletion) which appear mainly as monomers on non-reducing SDS-PAGE analysis, increased required surface area reduction for minimal surface tension. All SP-As reduced collapse surface tension of a pure cholesterol film from 27 to 23 mN/m in the presence of Ca2+. When mixed films were formed by successive spreading of DPPC/SP-A/cholesterol, rat SP-A, SP-Ahyp, or SP-ADeltaG8-P80 blocked the interaction of cholesterol with DPPC; SP-Ahyp,E195Q,R197D could not impede the interaction; SP-Ahyp,C6S or SP-Ahyp,DeltaN1-A7 only partially blocked the interaction, and cholesterol appeared to stabilize SP-Ahyp,C6S-DPPC association. These results demonstrate the importance of CRD and N-terminal dependent oligomerization in SP-A-phospholipid associations. The findings further indicate that SP-A-cholesterol interactions differ from SP-A-DPPC interactions and may be nonspecific.     

Phospholipid binding and biophysical activity of pulmonary surfactant-associated protein (SAP)-35 and its non-collagenous COOH-terminal domains

Surfactant-associated protein of Mr = 35,000, SAP-35, is the major glycoprotein present in mammalian pulmonary surfactants. In this study, canine SAP-35 and several of its COOH-terminal peptides were purified and characterized by amino acid composition and NH2-terminal sequencing analysis. These proteins were then studied in terms of their specific lipid-binding characteristics and surface activity when combined with a synthetic phospholipid mixture, SM, chosen as an approximation of lung surfactant phospholipids. Purified, delipidated SAP-35 bound SM strongly. In contrast, SAP-21 (a non-collagenous fragment generated by collagenase digestion) bound phospholipid weakly; SAP-18 (an acidic COOH-terminal fragment comprising residues Gly-118 to Phe-231) did not bind phospholipid, demonstrating the importance of hydrophobic amino acid residues Gly-81 to Val-117 and the NH2-terminal collagenous domain in interaction of the SAP-35 with phospholipids. In surface activity experiments, purified SAP-35 enhanced the adsorption of SM phospholipids in terms of both rate and overall surface tension lowering. However, the adsorption facility of the SM-SAP-35 mixture did not approach that of either whole surfactant or the surfactant extract preparations, calf lung surfactant extract or surfactant-TA, used in exogenous surfactant replacement therapy for the neonatal respiratory distress syndrome. In addition, the dynamic surface activity of the SM-SAP-35 mixture was well below that of natural surfactant or surfactant extracts. This was also true of mixtures of SM phospholipids combined with the SAP-18 and SAP-21 fragments of SAP-35.     

Distinct steps in the adsorption of pulmonary surfactant to an air-liquid interface

To investigate the mechanisms by which vesicles of pulmonary surfactant adsorb to an air-liquid interface, we measured the effect of different phospholipids and of their concentration both in the subphase and at the interface on this process. Adsorbing vesicles contained the hydrophobic surfactant proteins mixed with the following four sets of surfactant phospholipids that varied the content of anionic headgroups and mixed acyl chains independently: the complete set of purified phospholipids (PPL) from calf surfactant; modified PPL (mPPL) from which the anionic phospholipids were removed; a mixture of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) (9:1, mol:mol); and DPPC alone. The initial reduction in surface tension depended strongly on the anionic phospholipids and the subphase concentration. The acyl groups had no effect. Adsorption beyond the initial stage depended more on the mixed acyl groups, became increasingly independent of subphase concentration, and was determined instead by the interfacial concentration of the surface film. The different constituents produced the same effects in vesicles adsorbing to a clean interface or in a preexisting film to which vesicles of SP:DPPC adsorbed. Adsorption for vesicles of SP:PPL adsorbing to DPPC or of SP:DPPC to PPL above a certain threshold surface concentration followed exactly the same isotherm. Our results fit best with a two-step model for adsorption. The anionic phospholipids first promote the initial juxtaposition of vesicles to the interface. Compounds with mixed acyl constituents at the point of contact between vesicle and interface then facilitate fusion with the surface.     

Surface respreading after collapse of monolayers containing major lipids of pulmonary surfactant

Isotherms have been obtained near 37 degrees C for a series of repetitive compressions and expansions of monolayers that contain major components of lung surfactant. The minimum surface tension or maximum surface pressure which could be achieved under conditions of dynamic compression, and the rate of return of lipid from excluded phase to the monolayers were measured. Monolayers of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or of DPPC plus 10 or 30 mol% of the calcium salt of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG) (POPG-Ca) achieved very high surface pressures or low surface tensions (near 0 mN m-1), but they showed no return of material from the collapse phases under the test conditions. Monolayers of POPG-Ca alone collapsed at relatively low surface pressures (high surface tensions), but showed good return of material from the collapse phase into the monolayer. Monolayers containing more complex mixtures of lipids (DPPC, phosphatidylglycerol (PG), unsaturated phosphatidylcholine (PC), cholesterol (chol] in ratios similar to those found in surfactant achieved minimum surface tensions intermediate between those of monolayers with less complex compositions. These more complex mixtures showed a better rate of return of lipids from the collapse phases to the monolayer than did simple DPPC-POPG mixtures. 31P-NMR and differential scanning calorimetric investigations of the mixture DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POP G/DPPG/chol (10:4:2:1:3) showed that in the bulk phase at 37 degrees C, it was in bilayers in the liquid-crystalline state.     

Differential effects of lysophosphatidylcholine on the adsorption of phospholipids to an air/water interface

To determine how the hydrophobic surfactant proteins promote insertion of the surfactant lipids into an air/water interface, we measured the effect of lysophosphatidylcholine (LPC) on adsorption. Existing models contend that the proteins function either by disordering the lipids or by stabilizing a negatively curved structure located between the adsorbing vesicle and the interface. Because LPC produces greater disorder but positive curvature, the models predict opposite effects. With vesicles containing either dioleoyl phosphatidylcholine (DOPC) or the neutral and phospholipids isolated from calf surfactant, LPC increased the initial rate at which surface tension fell. The final surface tension, however, remained well above the value of approximately 25 mN/m expected for a saturated surface. With two preparations, dioleoyl phosphatidylethanolamine and gramicidin A-DOPC, which form the negatively curved hexagonal-II (H(II)) phase and adsorb rapidly, LPC instead had little effect on initial adsorption but delayed the fall of surface tension below approximately 30 mN/m. LPC produced a similar inhibition of the late adsorption for extracted calf surfactant. Unlike dioleoyl phosphatidylethanolamine and gramicidin A-DOPC, small-angle x-ray scattering and (31)P-nuclear magnetic resonance for extracted calf surfactant detected no evidence for the H(II) phase. Our results indicate that although LPC can promote the initial adsorption of vesicles containing only lamellar lipids, it inhibits the facilitation by the hydrophobic proteins of late adsorption. Our findings support a model in which the surfactant proteins accelerate adsorption by producing a focal tendency to stabilize a negatively curved kinetic intermediate without a general shift to the H(II) phase.     

Synthesis and activity of a novel diether phosphonoglycerol in phospholipase-resistant synthetic lipid:peptide lung surfactants()

This paper reports the chemical synthesis and purification of a novel phospholipase-resistant C16:0, C16:1 diether phosphonoglycerol with structural analogy to ester-linked anionic phosphatidylglycerol (PG) in endogenous pulmonary surfactant. This diether phosphonoglycerol (PG 1) is studied for phospholipase A(2) (PLA(2)) resistance and for surface activity in synthetic exogenous surfactants combined with Super Mini-B (S-MB) peptide and DEPN-8, a previously-reported diether phosphonolipid analog of dipalmitoyl phosphatidylcholine (DPPC, the major zwitterionic phospholipid in native lung surfactant). Activity experiments measured both adsorption and dynamic surface tension lowering due to the known importance of these surface behaviors in lung surfactant function in vivo. Synthetic surfactants containing 9 : 1 DEPN-8:PG 1 + 3% S-MB were resistant to degradation by PLA(2) in chromatographic studies, while calf lung surfactant extract (CLSE, the substance of the bovine clinical surfactant Infasurf?) was significantly degraded by PLA(2). The 9 : 1 DEPN-8:PG 1 + 3% S-MB mixture also had small but consistent increases in both adsorption and dynamic surface tension lowering ability compared to DEPN-8 + 3% S-MB. Consistent with these surface activity increases, molecular dynamics simulations using Protein Modeller, GROMACS force-field, and PyMOL showed that bilayers containing DPPC and palmitoyl-oleoyl-PC (POPC) as surrogates of DEPN-8 and PG 1 were penetrated to a greater extent by S-MB peptide than bilayers of DPPC alone. These results suggest that PG 1 or related anionic phosphono-PG analogs may have functional utility in phospholipase-resistant synthetic surfactants targeting forms of acute pulmonary injury where endogenous surfactant becomes dysfunctional due to phospholipase activity in the innate inflammatory response.