Pulmonary Surfactant Protein SP-C Research Articles

Role of pulmonary surfactant protein Sp-C dimerization on membrane fragmentation: An emergent mechanism involved in lung defense and homeostasis

Surfactant protein C (SP-C) is a protein present in the pulmonary surfactant system that is involved in the biophysical properties of this lipoprotein complex, but it also has a role in lung defense and homeostasis. In this article, we propose that the link between both functions could rely on the ability of SP-C to induce fragmentation of phospholipid membranes and generate small vesicles that serve as support to present different ligands to cells in the lungs. Our results using bimolecular fluorescence complementation and tunable resistive pulse sensing setups suggest that SP-C oligomerization could be the triggering event that causes membrane budding and nanovesiculation. As shown by fluorescence microscopy and flow cytometry, these vesicles are differentially assimilated by alveolar macrophages and alveolar type II cells, indicating distinct roles of these alveoli-resident cells in the processing of the SP-C- induced vesicles and their cargo. These results depict a more accurate picture of the mechanisms of this protein, which could be relevant for the comprehension of pulmonary pathologies and the development of new therapeutic approaches.

Effect of Cholesterol and Palmitoylation on the Structure, Orientation and Lipid-Protein Interactions of Pulmonary Surfactant Protein SP-C

Pulmonary surfactant is a specialized lipid-protein complex, which essential function is to stabilize the gas exchange surface of the respiratory epithelium against physical forces tending to its collapse. Hydrophobic surfactant proteins SP-B and SP-C are critical to promote very rapid adsorption of phospholipids into the interface and to stabilize the surface active film along the compression-expansion respiratory cycles. The composition of pulmonary surfactant varies slightly between species but in general it consists of 90% lipids by weight and 8-10% proteins, including the specific surfactant proteins SP-A, SP-B, SP-C and SP-D. The main feature of protein SP-C (35 amino acids, 3.7kDa) is its extreme hidrophobicity. Structurally it consists of a transmembrane α-helical stretch and a short N-terminal segment. The positions 5 and 6 of SP-C sequence are in most species occupied by two cysteines that are palmitoylated. It has been proposed that SP-C interacts with cholesterol-rich phases of surfactant membranes and films, contributing to modulate the interfacial behavior of surfactant in the alveoli but evidence remains uncertain.Our aim in this work was to elucidate how palmitoylation influences the interaction of SP-C with lipids in different surfactant-mimetic systems, in the presence or absence of cholesterol. To this end, we have compared the secondary structure, tilting and lipid-protein interactions of native palmitoylated SP-C purified from porcine lungs with the behavior of non-palmitoylated recombinant versions of SP-C produced in bacteria, by means of ATR-FTIR. This technique is sensitive to the orientation, structure and interactions of SP-C in membranes, which can be correlated with the contribution of SP-C to stabilize compressed cholesterol-containing surfactant films.

Interfacial Behavior of Recombinant Forms of Human Pulmonary Surfactant Protein SP-C

The behavior at air-liquid interfaces of two recombinant versions of human surfactant protein SP-C has been characterized in comparison with that of native palmitoylated SP-C purified from porcine lungs. Both native and recombinant proteins promoted interfacial adsorption of dipalmitoylphosphatidylcholine bilayers to a limited extent, but catalyzed very rapid formation of films from different lipid mixtures containing both zwitterionic and anionic phospholipids. Once at the interface, the recombinant variants exhibited compression-driven structural transitions, consistent with changes in the orientation of the deacylated N-terminal segment, which were not observed in the native protein. Compression isotherms of lipid/protein films suggest that the recombinant SP-C forms promote expulsion at high pressures of a higher number of lipid molecules per mole of protein than does native SP-C. A more dynamic conformation of the N-terminal segment in recombinant SP-C forms is likely also responsible for facilitating compression-driven condensation of domains in anionic phospholipid films as observed by epifluorescence microscopy. Finally, both native palmitoylated SP-C and the phenylalanine-containing recombinant versions facilitate similarly the repetitive compression-expansion dynamics of lipid/protein films, which were able to reach maximal surface pressures with practically no hysteresis along multiple quasi-static or dynamic cycles.

Palmitoylation of Pulmonary Surfactant Protein SP-C Is Critical for Its Functional Cooperation with SP-B to Sustain Compression/Expansion Dynamics in Cholesterol-Containing Surfactant Films

Recent data suggest that a functional cooperation between surfactant proteins SP-B and SP-C may be required to sustain a proper compression-expansion dynamics in the presence of physiological proportions of cholesterol. SP-C is a dually palmitoylated polypeptide of 4.2 kDa, but the role of acylation in SP-C activity is not completely understood. In this work we have compared the behavior of native palmitoylated SP-C and recombinant nonpalmitoylated versions of SP-C produced in bacteria to get a detailed insight into the importance of the palmitic chains to optimize interfacial performance of cholesterol-containing surfactant films. We found that palmitoylation of SP-C is not essential for the protein to promote rapid interfacial adsorption of phospholipids to equilibrium surface tensions (∼22 mN/m), in the presence or absence of cholesterol. However, palmitoylation of SP-C is critical for cholesterol-containing films to reach surface tensions ≤1 mN/m at the highest compression rates assessed in a captive bubble surfactometer, in the presence of SP-B. Interestingly, the ability of SP-C to facilitate reinsertion of phospholipids during expansion was not impaired to the same extent in the absence of palmitoylation, suggesting the existence of palmitoylation-dependent and -independent functions of the protein. We conclude that palmitoylation is key for the functional cooperation of SP-C with SP-B that enables cholesterol-containing surfactant films to reach very low tensions under compression, which could be particularly important in the design of clinical surfactants destined to replacement therapies in pathologies such as acute respiratory distress syndrome.

Pulmonary Surfactant Protein SP-C Counteracts the Deleterious Effects of Cholesterol on the Activity of Surfactant Films under Physiologically Relevant Compression-Expansion Dynamics

The presence of cholesterol is critical in defining a dynamic lateral structure in pulmonary surfactant membranes. However, an excess of cholesterol has been associated with impaired surface activity of surfactant. It has also been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films. In this study, we analyzed the effect of SP-C on the thermodynamic properties of phospholipid membranes containing cholesterol, and the ability of lipid/protein complexes containing cholesterol to form and respread interfacial films capable of producing very low surface tensions upon repetitive compression-expansion cycling. SP-C modulates the effect of cholesterol to reduce the enthalpy associated with the gel-to-liquid-crystalline melting transition in dipalmitoylphosphatidylcholine (DPPC) bilayers, as analyzed by differential scanning calorimetry. The presence of SP-C affects more subtly the effects of cholesterol on the thermotropic properties of ternary membranes, mimicking more closely the lipid composition of native surfactant, where SP-C facilitates the miscibility of the sterol. Incorporation of 1% or 2% SP-C (protein/phospholipid by weight) promotes almost instantaneous adsorption of suspensions of DPPC/palmitoyloleoylphospatidylcholine (POPC)/palmitoyloleoyl-phosphatidylglycerol (POPG) (50:25:15, w/w/w) into the air-liquid interface of a captive bubble, in both the absence and presence of cholesterol. However, cholesterol impairs the ability of SP-C-containing films to achieve very low surface tensions in bubbles subjected to compression-expansion cycling. Cholesterol also substantially impairs the ability of DPPC/POPC/POPG films containing 1% surfactant protein SP-B to mimic the interfacial behavior of native surfactant films, which are characterized by very low minimum surface tensions with only limited area change during compression and practically no compression-expansion hysteresis. However, the simultaneous presence of 2% SP-C practically restores the compression-expansion dynamics of cholesterol- and SP-B-containing films to the efficient behavior shown in the absence of cholesterol. This suggests that cooperation between the two proteins is required for lipid-protein films containing cholesterol to achieve optimal performance under physiologically relevant compression-expansion dynamics.

Cholesterol modulates the exposure and orientation of pulmonary surfactant protein SP-C in model surfactant membranes

Cholesterol is the major neutral lipid in lung surfactant, accounting for up to 8–10% of surfactant mass, while surfactant protein SP-C (∼ 4.2 kDa) accounts for no more than 1–1.5% of total surfactant weight but plays critical roles in formation and stabilization of pulmonary surfactant films. It has been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films and this interaction could have a potential role on modulating surfactant function. In the present study, we have analyzed the effect of cholesterol on the structure, orientation and dynamic properties of SP-C embedded in physiologically relevant model membranes. The presence of cholesterol does not induce substantial changes in the secondary structure of SP-C, as analyzed by Attenuated Reflection Fourier Transformed Infrared spectroscopy (ATR-FTIR). However, the presence of cholesterol modifies the orientation of the transmembrane helix and the dynamic properties of the protein, as demonstrated by hydrogen/deuterium exchange kinetics. The effect of cholesterol on SP-C reconstituted in zwitterionic, entirely fluid, membranes made of POPC (palmitoyloleoylphospatidylcholine) or in anionic membranes with coexistence of ordered and disordered phases, such as those made of dipalmitoylphosphatidylcholine (DPPC):POPC:Palmitoyloleoylphosphatidylglycerol (POPG) (50:25:15) is dual. Cholesterol decreases the exposure of the protein to the aqueous environment and the tilt of its transmembrane helical segment up to a ratio Cholesterol:SP-C of 4.8 and 2.4 (mol/mol) in the two lipid systems tested, respectively, and it increases the exposure and tilt at higher cholesterol proportions. The results presented here suggest the existence of an interaction between SP-C and cholesterol-enriched phases, with consequences on the behavior of the protein, which could be of relevance for cholesterol-dependent structure–function relationships in pulmonary surfactant membranes and films.

Surface Behaviour of Peptoid Mimics of Pulmonary Surfactant Protein SP-C: Captive Bubble Surfactometry

Pulmonary surfactant lipopeptide SP-C modulates the surface properties of interfacial films required to stabilize the respiratory interface along breathing dynamics. Several attempts have been made to produce entirely synthetic analogs of SP-C suitable to develop potentially useful therapeutic preparations.In this study we have tested the potential of five different poly-N-substituted glycines, or peptoids, designed to mimic (roughly) the primary and secondary structure and hydrophobicity of SP-C, to produce acceptable surfactant-like behaviour once incorporated into lipid/peptoid suspensions and assessed in a captive bubble surfactometer (CBS). The surface activities of different peptoids were compared in two model lipid mixtures: DPPC/POPG/Palmitic acid (68/22/9), which resembles the lipid composition of several clinical surfactants currently in use, and DPPC/POPC/POPG/Chol (50/25/15/10), which mimics the balance of saturated/unsaturated and zwitterionic/anionic phospholipids and the cholesterol content of natural surfactant as purified from bronchoalveolar lavage. We have assessed the ability of the different lipid/peptoid suspensions to i) rapidly adsorb at the bubble air-liquid interface, ii) stably produce very low surface tensions upon relatively slow repetitive quasi-static compressions and iii) maintain the lowest surface tensions with minimal compression and hysteresis under rapid physiological-like compression-expansion dynamics. Significant differences were found between different peptoids differing in their backbone structure and hydrophobicity, with some of the peptoids mimicking efficiently the effect of native SP-C, usually at larger proportions of peptoids than required for the natural protein.

Effects of Palmitoylation on Dynamics and Phospholipid-Bilayer-Perturbing Properties of the N-Terminal Segment of Pulmonary Surfactant Protein SP-C as Shown by 2H-NMR

It has been proposed that palmitoylation of the N-terminal segment of surfactant protein SP-C is important for maintaining association of pulmonary surfactant complexes with interfacial films compressed to high pressures at the end of expiration. In this study, we examined surfactant membrane models containing palmitoylated and nonpalmitoylated synthetic peptides, based on the N-terminal SP-C sequence, in dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (7:3, w/w) by 2H-NMR. Perturbations of lipid properties by the peptide versions were compared in samples containing chain- and headgroup-deuterated lipid (DPPC- d 62 and DPPC- d 4 respectively). Also, deuterated peptide palmitate chains were compared with those of DPPC in otherwise identical lipid-protein mixtures. Palmitoylated peptide increased average DPPC- d 62 chain orientational order slightly, particularly for temperatures spanning gel and liquid crystalline coexistence, implying penetration of palmitoylated peptide into ordered membrane. In contrast, the nonpalmitoylated peptide had a small disordering effect in this temperature range. Both peptide versions perturbed DPPC- d 4 headgroup orientation similarly, suggesting little effect of palmitoylation on the largely electrostatic peptide-headgroup interaction. Deuterated acyl chains attached to the SP-C N-terminal segment displayed a qualitatively different distribution of chain order, and lower average order, than DPPC- d 62 in the same membranes. This likely reflects local perturbation of lipid headgroup spacing by the peptide portion interacting with the bilayer near the peptide palmitate chains. This study suggests that SP-C-attached acyl chains could be important for coupling of lipid and protein motions in surfactant bilayers and monolayers, especially in the context of ordered phospholipid structures such as those potentially formed during exhalation, when stabilization of the respiratory surface by surfactant is the most crucial.

Effect of acylation on the interaction of the N-Terminal segment of pulmonary surfactant protein SP-C with phospholipid membranes

SP-C, the smallest pulmonary surfactant protein, is required for the formation and stability of surface-active films at the air–liquid interface in the lung. The protein consists of a hydrophobic transmembrane α-helix and a cationic N-terminal segment containing palmitoylated cysteines. Recent evidence suggests that the N-terminal segment is of critical importance for SP-C function. In the present work, the role of palmitoylation in modulating the lipid–protein interactions of the N-terminal segment of SP-C has been studied by analyzing the effect of palmitoylated and non-palmitoylated synthetic peptides designed to mimic the N-terminal segment on the dynamic properties of phospholipid bilayers, recorded by spin-label electron spin resonance (ESR) spectroscopy. Both palmitoylated and non-palmitoylated peptides decrease the mobility of phosphatidylcholine (5-PCSL) and phosphatidylglycerol (5-PGSL) spin probes in dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) bilayers. In zwitterionic DPPC membranes, both peptides have a greater effect at temperatures below than above the main gel-to-liquid-crystalline phase transition, the palmitoylated peptide inducing greater immobilisation of the lipid than does the non-palmitoylated form. In anionic DPPG membranes, both palmitoylated and non-palmitoylated peptides have similar immobilizing effects, probably dominated by electrostatic interactions. Both palmitoylated and non-palmitoylated peptides have effects comparable to whole native SP-C, as regards improving the gel phase solubility of phospholipid spin probes and increasing the polarity of the bilayer surface monitored by pK shifts of fatty acid spin probes. This indicates that a significant part of the perturbing properties of SP-C in phospholipid bilayers is mediated by interactions of the N-terminal segment. The effect of SP-C N-terminal peptides on the chain flexibility gradient of DPPC and DPPG bilayers is consistent with the existence of a peptide-promoted interdigitated phase at temperatures below the main gel-to-liquid-crystalline phase transition. The palmitoylated peptide, but not the non-palmitoylated version, is able to stably segregate interdigitated and non-interdigitated populations of phospholipids in DPPC bilayers. This feature suggests that the palmitoylated N-terminal segment stabilizes ordered domains such as those containing interdigitated lipids. We propose that palmitoylation may be important to promote and facilitate association of SP-C and SP-C-containing membranes with ordered lipid structures such as those potentially existing in highly compressed states of the interfacial surfactant film.

Interaction of the N-terminal segment of pulmonary surfactant protein SP-C with interfacial phospholipid films

Pulmonary surfactant protein SP-C is a 35-residue polypeptide composed of a hydrophobic transmembrane alpha-helix and a polycationic, palmitoylated-cysteine containing N-terminal segment. This segment is likely the only structural motif the protein projects out of the bilayer in which SP-C is inserted and is therefore a candidate motif to participate in interactions with other bilayers or monolayers. In the present work, we have detected intrinsic ability of a peptide based on the sequence of the N-terminal segment of SP-C to interact and insert spontaneously into preformed zwitterionic or anionic phospholipid monolayers. The peptide expands the π–A compression isotherms of interfacial phospholipid/peptide films, and perturbs the lipid packing of phospholipid films during compression-driven liquid-expanded to liquid-condensed lateral transitions, as observed by epifluorescence microscopy. These results demonstrate that the sequence of the SP-C N-terminal region has intrinsic ability to interact with, insert into, and perturb the structure of zwitterionic and anionic phospholipid films, even in the absence of the palmitic chains attached to this segment in the native protein. This effect has been related with the ability of SP-C to facilitate reinsertion of surface active lipid molecules into the lung interface during respiratory compression–expansion cycling.

Selective Labeling of Pulmonary Surfactant Protein SP-C in Organic Solution

Pulmonary surfactant protein SP-C has been isolated from porcine lungs and treated with dansyl isothiocyanate in chloroform:methanol 2:1 (v/v) solutions,under conditions optimized to introduce a single dansyl group covalently attached to the N-terminalamine group of the protein without loss of its native thioesther-linked palmitic chains. The resulting derivative Dans-SP-C conserves the secondary structure of native SP-C as well as the ability to promote interfacial adsorption of DPPC suspensions and to affect the thermotropic behavior of DPPC bilayers. This derivative can be used to characterize lipid-protein and protein-protein interactions of a native-like SP-C in lipid/protein complexes.

Intrinsic structural differences in the N-terminal segment of pulmonary surfactant protein SP-C from different species

Predictive studies suggest that the known sequences of the N-terminal segment of surfactant protein SP-C from animal species have an intrinsic tendency to form β-turns, but there are important differences on the probable location of these motifs in different SP-C species. Our hypothesis is that intrinsic structural determinants of the sequence of the N-terminal region of SP-C could define conformation, acylation and perhaps surface properties of the mature protein. To test this hypothesis we have synthesized peptides corresponding to the 13-residue N-terminal sequence of porcine and canine SP-C, and studied their structural behaviour in solution and in phospholipid bilayers and monolayers. In these peptides, leucine at position 1 of both sequences has been replaced by tryptophan in order to allow their study by fluorescence spectroscopy. Far-u.v. circular dichroism spectra of the peptides in aqueous and organic solutions and in phospholipid micelles or vesicles are consistent with predicted conformational differences between the porcine and the canine sequences. Both families of peptides showed changes in their fluorescence emission spectra in the presence of phospholipids that were consistent with spontaneous lipid/peptide interactions. Both canine and porcine peptides were able to form monolayers at air–liquid interfaces, the canine peptides occupying lower area/molecule and being compressible to higher pressures than the porcine sequences. The peptides also shifted the isotherms and perturbed the packing of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers, the effects being always higher in anionic than in zwitterionic lipids, and also substantially higher in films containing canine peptide in comparison to porcine peptide. Acylation of cysteines at the N-terminal end of SP-C may modulate these intrinsic conformational features and the changes induced could be important for the development of its surface activity.

The surface properties of chemically synthesized peptides analogous to human pulmonary surfactant protein SP-C.

A number of peptides with different lengths corresponding to various regions of human pulmonary surfactant protein SP-C were synthesized and their activity evaluated to improve in vitro surface activities and in situ lung pressure-volume characteristics of a ternary lipid mixture composed of dipalmitoylphosphatidylcholine, phosphatidylglycerol and palmitic acid (75:25:10, w/w). SP-C (1-35), a synthetic peptide with the entire length of human SP-C, and some other peptides with various lengths of its partial sequences were remarkably active. All of these peptides shared a common core sequence of (C)CPVHLKRLLIVVVVVVLIVVVIVGAL(L). Any deletion in this core sequence resulted in reduction of activity of the peptide. SP-C (5-31) and SP-C (6-32), the minimum peptides containing the core sequence, were combined with the ternary lipid mixture at the final peptide concentration of 2% (w/w) into synthetic surfactants which showed excellent properties comparable with those of Surfacten, a commercially available modified bovine lung surfactant. In a Langmuir-Wilhelmy surface balance, the synthetic surfactant containing SP-C (6-32) spread and adsorbed quickly to reach a surface tension of 30.8 mN/m at 30-s spreading time and 41.2 mN/m at 1-min adsorption time, respectively; the presence of SP-C (6-32) significantly prevented the decrease of surface activity of the ternary lipid mixture during dynamic compression-expansion cycles. Furthermore, tracheal instillation of the synthetic surfactant containing SP-C (6-32) at the dose of 50 mg of phospholipids/kg improved lung pressure-volume characteristics of immature rabbit neonates to a level similar to that of mature neonates at term.

Structural and Functional Importance of the C-Terminal Part of the Pulmonary Surfactant Polypeptide SP-C

A synthetic non-palmitoylated form of the pulmonary surfactant protein SP-C and three peptides of different lengths corresponding to its N-terminal and middle parts were reconstituted into dipalmitoylglycerophosphocholine/phosphatidylglycerol (7:3, by mass) lipid bilayers. The adsorption properties of each reconstituted system were determined by measurement of the surface pressure after injection of the samples underneath an air/water interface. Attenuated total reflection infrared spectroscopy provided information about the structure and orientation of peptides in lipid bilayers. The hydrophobic C-terminal helix is crucial for the rapid adsorption as shortening of the C-terminal part drastically restricted this activity. The C-terminal amino acid sequence can however be replaced with that of the second transmembrane helix of bacteriorhodopsin without significantly modifying the adsorption properties. The data suggest that the hydrophobic C-terminal part allows the anchorage of SP-C in the lipid bilayer in such a manner that the N-terminal domain adopts an optimal conformation and orientation for the rapid adsorption of phospholipids at the air/water interface, and/or that a membrane-spanning helix as such is needed for this activity.

Structural Similarities between Myelin and Hydrophobic Surfactant Associated Proteins: Protein Motifs for Interacting with Bilayers

A group of predicted structural similarities exist between the hydrophobic pulmonary surfactant protein SP-B and SP-C and the hydrophobic proteins of myelin, either the myelin basic protein and the proteolipid protein of central nervous system myelin, or the P 0 protein of peripheral myelin. These similarities include, among other factors, the charge density, post-translational modification by fatty acids, some potential periodicity in primary structure and an overall hydrophobicity. The similarities suggest that these proteins might have similar means of interacting with each other and with lipid bilayers in their respective biological systems.

Deacylated pulmonary surfactant protein SP-C has different effects on the thermotroplc behaviour of bilayers of dipalmitoylphosphatidyl-glycerol (DPPG) than the native acylated protein

Conference Article| August 01 1994 Deacylated pulmonary surfactant protein SP-C has different effects on the thermotroplc behaviour of bilayers of dipalmitoylphosphatidyl-glycerol (DPPG) than the native acylated protein JESÚS PÉREZ-GIL; JESÚS PÉREZ-GIL 1Dept. Bioquímica y Biología Molecular I. Fac. Químicas, Universidad Complutense, 28040 Madrid (SPAIN) Search for other works by this author on: This Site PubMed Google Scholar JOSE L. LÓPEZ-LACOMBA; JOSE L. LÓPEZ-LACOMBA 2Instituto Pluridisciplinar. Unidad de RMN. Universidad Complutense de Madrid (SPAIN) Search for other works by this author on: This Site PubMed Google Scholar ANTONIO CRUZ; ANTONIO CRUZ 1Dept. Bioquímica y Biología Molecular I. Fac. Químicas, Universidad Complutense, 28040 Madrid (SPAIN) Search for other works by this author on: This Site PubMed Google Scholar ALEJANDRO BELDARRAÍN; ALEJANDRO BELDARRAÍN 2Instituto Pluridisciplinar. Unidad de RMN. Universidad Complutense de Madrid (SPAIN) Search for other works by this author on: This Site PubMed Google Scholar CRISTINA CASALS CRISTINA CASALS 1Dept. Bioquímica y Biología Molecular I. Fac. Químicas, Universidad Complutense, 28040 Madrid (SPAIN) Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1994) 22 (3): 372S. https://doi.org/10.1042/bst022372s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation JESÚS PÉREZ-GIL, JOSE L. LÓPEZ-LACOMBA, ANTONIO CRUZ, ALEJANDRO BELDARRAÍN, CRISTINA CASALS; Deacylated pulmonary surfactant protein SP-C has different effects on the thermotroplc behaviour of bilayers of dipalmitoylphosphatidyl-glycerol (DPPG) than the native acylated protein. Biochem Soc Trans 1 August 1994; 22 (3): 372S. doi: https://doi.org/10.1042/bst022372s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1994 Biochemical Society1994 Article PDF first page preview Close Modal You do not currently have access to this content.

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: II. Monolayers of pulmonary surfactant protein SP-C and phospholipids

The interaction of the hydrophobic pulmonary surfactant protein SP-C with dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and DPPC:DPPG (7:3, mol:mol) in spread monolayers at the air-water interface has been studied. At low concentrations of SP-C (about 0.5 mol% or 3 weight%protein) the protein-lipid films collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At initial protein concentrations higher than 0.8 mol%, or 4 weight%, the isotherms displayed kinks at surface pressures of about 50 mN.m-1 in addition to the collapse plateaux at the higher pressures. The presence of less than 6 mol%, or 27 weight%, of SP-C in the protein-lipid monolayers gave a positive deviation from ideal behavior of the mean areas in the films. Analyses of the mean areas in the protein-lipid films as functions of the monolayer composition and surface pressure showed that SP-C, associated with some phospholipid (about 8-10 lipid molecules per molecule of SP-C), was squeezed out from the monolayers at surface pressures of about 55 mN.m-1. The results suggest a potential role for SP-C to modify the composition of the monolayer at the air-water interface in the alveoli.

Lipid effects on aggregation of pulmonary surfactant protein SP-C studied by fluorescence energy transfer.

The self-association of pulmonary surfactant protein SP-C in lipid vesicles was studied using fluorescence energy transfer. Bovine SP-C was labeled with two fluorescent probes, succinimidyl 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate and eosin isothiocyanate, on the amino terminus of the protein, producing NBD-SP-C and EITC-SP-C, respectively. The N-terminus of SP-C was relatively immobile between 20 and 37 degrees C, as demonstrated by high fluorescence anisotropy of NBD-SP-C and EITC-SP-C. The mobility increased at the transition of the lipid to the fluid phase. Using fluorescence energy transfer, with NBD-SP-C as the donor and EITC-SP-C as the acceptor, a high degree of SP-C/SP-C association was found below 25 degrees C, decreasing to very little self-association above 42 degrees C in 7:1 1,2-dipalmitoylphosphatidylcholine-1,2-dipalmitoylphosphatidylglycerol (DPPC-DPPG) vesicles. The fraction of SP-C aggregated below 37 degrees C in 7:1 DPPC-DPPG was estimated from the observed energy transfer to be more than 70% of total SP-C. In various lipid mixtures, self-association of SP-C was dependent on the presence of at least some gel-phase lipids. In a lipid mixture resembling pulmonary surfactant, gradually increasing self-association was observed below 38 degrees C. The relation of the present data to the state of aggregation of SP-C in pulmonary surfactant is discussed.

Pulmonary surfactant protein SP-C causes packing rearrangements of dipalmitoylphosphatidylcholine in spread monolayers

The hydrophobic pulmonary surfactant protein SP-C has been isolated from porcine lung surfactant, and it has been incorporated into monolayers of dipalmitoylphosphatidylcholine (DPPC). The monolayers, which contained 1 mol% of a fluorescently-labeled phosphatidylcholine, were observed under various states of compression in an epifluorescence surface balance. SP-C altered the packing arrangements of DPPC in the monolayer, causing the production of many more, smaller condensed lipid domains in its presence than in its absence.

Pulmonary surfactant protein SP-C and phosphatidylcholines in bilayers and monolayers

Pulmonary surfactant lowers surface tension in the lung alveoli, reduces the work of breathing and prevents alveolar collapse at low lung volumes. The material consists of about 90% lipids, of which dipalmitoylphosphatidylcholine (DPPC) is the major component, and at least four proteins, SP-A, SP-B, SP-C, and SP-D. SP-B and SP-C, are very hydrophobic and are considered to help in the process of transferring the lipid from bilayers, the form in which it is secreted into a monolayer at the air-water interface. SP-C in saturated phosphatidylcholines in bilayers and monolayers has been studied by 2H nuclear magnetic resonance spectroscopy, high sensitivity differential scanning calorimetry, and epifluorescence of monolayers under compression. The gel to liquid-crystalline phase transition was broadened by SP-C. The T 1 relaxation times of the acyl chains were essentially unaffected by the protein. The T 2e of the chains were decreased in the liquid crystal and their temperature dependence was altered in the gel. Initial studies indicate that the first moments of the 2H NMR spectra of the head group of DPPC were only slightly affected by SP-C. SP-C affected the distribution of condensed and fluid domains in monolayers in the liquid expanded/liquid condensed region by producing more, smaller, condensed domains in the two phase region than seen for pure lipid monolayers. The results are consistent with SP-C being embedded, at least in part, in the acyl chains of the lipid, and that severe packing dislocations in PC bilayers are not produced by the protein.