Effects of oligomerization and secondary structure on the surface behavior of pulmonary surfactant proteins SP-B and SP-C

The relationship among protein oligomerization, secondary structure at the interface, and the interfacial behavior was investigated for spread layers of native pulmonary surfactant associated proteins B and C. SP-B and SP-C were isolated either from butanol or chloroform/methanol lipid extracts that were obtained from sheep lung washings. The proteins were separated from other components by gel exclusion chromatography or by high performance liquid chromatography. SDS gel electrophoresis data indicate that the SP-B samples obtained using different solvents showed different oligomerization states of the protein. The CD and FTIR spectra of SP-B isolated from all extracts were consistent with a secondary structure dominated by alpha-helix. The CD and FTIR spectra of the first SP-C corresponded to an alpha-helical secondary structure and the spectra of the second SP-C corresponded to a mixture of alpha-helical and beta-sheet conformation. In contrast, the spectra of the third SP-C corresponded to antiparallel beta-sheets. The interfacial behavior was characterized by surface pressure/area (pi-A) isotherms. Differences in the oligomerization state of SP-B as well as in the secondary structure of SP-C all produce significant differences in the surface pressure/area isotherms. The molecular cross sections determined from the pi-A isotherms and from dynamic cycling experiments were 6 nm(2)/dimer molecule for SP-B and 1.15 nm(2)/molecule for SP-C in alpha-helical conformation and 1.05 nm(2)/molecule for SP-C in beta-sheet conformation. Both the oligomer ratio of SP-B and the secondary structure of SP-C strongly influence organization and behavior of these proteins in monolayer assemblies. In addition, alpha-helix --> beta-sheet conversion of SP-C occurs simply by an increase of the summary protein/lipid concentration in solution.     

Effects of surfactant-associated protein SP-B synthetic analogs on the structure and surface activity of model membrane bilayers.

The effect of several synthetic peptides based on the sequence of human pulmonary surfactant-associated protein B (SPB) on the molecular packing of model membrane lipids (7:1 dipalmitoyl phosphatidylcholine (DPPC)/dipalmitoyl phosphatidylglycerol (DPPG)) was studied using fluorescence anisotropy. This information was then correlated with complementary biophysical data obtained on both a modified Wilhelmy-Langmuir balance and a pulsating bubble surfactometer. The SP-B peptides examined in these studies are synthetic human SP-B Phe1-Ser78 (SP-B 1-78, full-length sequence), synthetic human SP-B Phe1-Thr60 (SP-B 1-60), synthetic human SP-B Phe1-Ala20 (SP-B 1-20), synthetic human SP-B Ala20-Thr60 (SP-B 20-60), synthetic human SP-B Leu27-Ser78 (SP-B 27-78), synthetic human SP-B Leu40-Thr60 (SP-B 40-60) and synthetic human SP-B Tyr53-Ser78 (SP-B 53-78). trans-parinaric acid was utilized to detect changes in ordering of lipids within the interior upon incorporation of synthetic SP-B peptide, whereas 1-hexadecanoyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)-a min ohexanoyl] phosphatidylcholine (6-NBD-PC) and 1-acyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)aminohexanoyl ] phosphatidylglycerol (6-NBD-PG) were utilized to determine alterations in lipid order at the surface of the model membrane bilayer. With the exception of SP-B 40-60, which corresponds to the most hydrophobic segment of the full-length SP-B, none of the other peptide significantly perturbed the interior bilayer as determined by fluorescence anisotropy of trans-parinaric acid. Incorporation of any of the peptides with the exception of SP-B 40-60, resulted in an increase in anisotropy of NBD-PC. The most significant enhancements resulted from the addition of SP-B 1-78, SP-B 1-20, SP-B 27-78 or SP-B 53-78. The magnitude of anisotropy increase with these peptides is similar to that observed with an equivalent molar ratio of native SP-B isolated from a bovine source. These observations suggest that these four synthetic peptides have the structural and compositional characteristics required for surface ordering of the membrane bilayer in a manner similar to that observed with native SP-B, thereby facilitating the surfactant-like properties of phospholipid mixtures.     

Microstructure and dynamic surface properties of surfactant protein SP-B/dipalmitoylphosphatidylcholine interfacial films spread from lipid-protein bilayers

 Suspensions of dipalmitoylphosphatidylcholine (DPPC) bilayers containing 5, 10 or 20% (w/w) surfactant protein SP-B have been reconstituted and spread at air-liquid interfaces. Compression isotherms of DPPC/SP-B monolayers spread from these preparations were qualitatively comparable to the isotherms of the corresponding DPPC/SP-B monolayers spread from solvents. SP-B was squeezed-out at higher pressures from vesicle-spread films than from solvent-spread monolayers. SP-B caused a marked decrease on the rate of relaxation of DPPC collapse phases to equilibrium pressures in all the lipid/protein films assayed. This stabilizing effect was higher in vesicle-spread than in solvent-spread monolayers. Inclusion in the films of traces of the fluorescent probe NBD-PC (1 mol%) and use of a fluorescent derivative of SP-B labeled with a rhodamine derivative, Texas Red, allowed for direct observation of protein and lipid domains at the interface by epifluorescence microscopy. Upon compression, SP-B altered the packing of phospholipids in the bilayer-spread films, observed as a SP-B-induced reduction of the area of liquid-condensed domains, in a way similar to its effect in solvent-spread monolayers. SP-B was not associated with condensed regions of the films. Fluorescence images from vesicle-spread films showed discrete fluorescent aggregates that could be consistent with the existence of lipid-protein vesicles in close association with the monolayer. Both the retention of SP-B at higher surface pressures and the greater stability of collapse phases of DPPC/SP-B films prepared by spreading from liposomes in comparison to those spread from solvents can be interpreted as a consequence of formation of complex bilayer-monolayer interacting systems. Received: 1 December 1999 / Revised version: 2 March 2000 / Accepted: 2 March 2000     

Lung surfactant protein SP-B promotes formation of bilayer reservoirs from monolayer and lipid transfer between the interface and subphase

We investigated the possible role of SP-B proteins in the function of lung surfactant. To this end, lipid monolayers at the air/water interface, bilayers in water, and transformations between them in the presence of SP-B were simulated. The proteins attached bilayers to monolayers, providing close proximity of the reservoirs with the interface. In the attached aggregates, SP-B mediated establishment of the lipid-lined connection similar to the hemifusion stalk. Via this connection, a lipid flow was initiated between the monolayer at the interface and the bilayer in water in a surface-tension-dependent manner. On interface expansion, the flow of lipids to the monolayer restored the surface tension to the equilibrium spreading value. SP-B induced formation of bilayer folds from the monolayer at positive surface tensions below the equilibrium. In the absence of proteins, lipid monolayers were stable at these conditions. Fold nucleation was initiated by SP-B from the liquid-expanded monolayer phase by local bending, and the proteins lined the curved perimeter of the growing fold. No effect on the liquid-condensed phase was observed. Covalently linked dimers resulted in faster kinetics for monolayer folding. The simulation results are in line with existing hypotheses on SP-B activity in lung surfactant and explain its molecular mechanism.     

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

The effects of pulmonary surfactant protein SP-B on the properties of monolayers of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), and a mixture of DPPC:DPPG (7:3, mol:mol) were studied using spread films at the air-water interface. The addition of SP-B to the phospholipid monolayers gave positive deviations from additivity of the mean areas in the films. At low protein concentrations (less than 45% amino acid residues which corresponds to 0.5 mol% or 10 weight% SP-B) monolayers of SP-B/DPPC, SP-B/DPPG and SP-B/(DPPC:DPPG) collapsed at surface pressures of about 70 mN.m-1, comparable to those of the lipids alone. At higher concentrations of SP-B in the protein-lipid monolayers, kink points appeared in the isotherms at about 40-45 mN.m-1, implying possible exclusion of material from the films, hence, changes in the original monolayer compositions. Calculated analyses of the monolayer compositions as a function of surface pressure indicated that nearly pure SP-B, associated with small amounts of phospholipid (2-3 lipid molecules per SP-B dimer), was lost from SP-B/DPPC, SP-B/DPPG, and SP-B/(DPPC:DPPG) films at surface pressures higher than 40-45 mN.m-1. The results are consistent with a low effectiveness of SP-B in removing saturated phospholipids, DPPC or DPPG, from the spread SP-B/phospholipid films.     

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   

Inhibition of mixtures of surfactant lipids and synthetic sequences of surfactant proteins SP-B and SP-C

The respiratory distress syndrome of premature infants is caused by both surfactant deficiency and surfactant inhibition by capillary-alveolar leakage of serum factors. Dispersions of a standard surfactant lipid mixture, with and without various synthetic peptides, modeled on human surfactant proteins SP-B (residues 1-25, 49-66, 1-78) and SP-C (residues 1-10), were evaluated for inhibition by serum and by plasma constituents using a pulsating bubble surfactometer. Inhibition was derived from the changes in surface properties of these mixtures after addition of human serum or plasma constituents. Modified bovine surfactant (TA) containing native SP-B and SP-C was used as a control. In the absence of serum inhibitors, mixtures with synthetic peptides gave results similar to surfactant TA. However, inhibition was more evident in the dispersions with synthetic peptides when compared with surfactant TA. The peptide/phospholipid mixture with the entire sequence of SP-B and the first 10 residues of SP-C were more resistant to inhibition than mixtures with synthetic peptides containing fewer domains. Addition of calcium reduced the inhibitory effects of serum both in mixtures containing synthetic peptides and in surfactant TA. Therefore, synthetic SP-B and SP-C peptides in surfactant lipids, in cooperation with calcium, permit resistance to inhibition by several plasma constituents that probably inactivate surfactant by a variety of different mechanisms.     

Reactive oxygen species inactivation of surfactant involves structural and functional alterations to surfactant proteins SP-B and SP-C

Exposing bovine lipid extract surfactant (BLES), a clinical surfactant, to reactive oxygen species arising from hypochlorous acid or the Fenton reaction resulted in an increase in lipid (conjugated dienes, lipid aldehydes) and protein (carbonyls) oxidation products and a reduction in surface activity. Experiments where oxidized phospholipids (PL) were mixed with BLES demonstrated that this addition hampered BLES biophysical activity. However the effects were only moderately greater than with control PL. These results imply a critical role for protein oxidation. BLES oxidation by either method resulted in alterations in surfactant proteins SP-B and SP-C, as evidenced by altered Coomassie blue and silver staining. Western blot analyses showed depressed reactivity with specific antibodies. Oxidized SP-C showed decreased palmitoylation. Reconstitution experiments employing PL, SP-B, and SP-C isolated from control or oxidized BLES demonstrated that protein oxidation was more deleterious than lipid oxidation. Furthermore, addition of control SP-B can improve samples containing oxidized SP-C, but not vice versa. We conclude that surfactant oxidation arising from reactive oxygen species generated by air pollution or leukocytes interferes with surfactant function through oxidation of surfactant PL and proteins, but that protein oxidation, in particular SP-B modification, produces the major deleterious effects.     

Secondary structure in lung surfactant SP-B peptides: IR and CD studies of bulk and monolayer phases

Pulmonary surfactant protein SP-B is known to facilitate adsorption and spreading of surfactant components across the air/water interface. This property appears essential for in vivo function in the alveolar subphase and at the air/alveolar surface. Three peptides with amino acid sequences based on SP-B containing predicted alpha-helical regions (SP-B(1--20), SP-B(9--36A), SP-B(40--60A)) have been synthesized to probe structure-function relationships and protein-lipid interaction in bulk phase and monolayer environments. IR and CD studies are reported along with traditional surface pressure-molecular area (pi-A) isotherms and IR reflection-absorption spectroscopy (IRRAS) investigations conducted at the air/water interface. In bulk phase, helix-promoting environments (methanol and aqueous dispersions of lipid vesicles), SP-B(1--20) and SP-B(9--36A) contained significant amounts of alpha-helical structure, whereas varying degrees of alpha-helix, random coil, and beta-sheet were observed in aqueous solutions and monolayers. The most striking behavior was observed for SP-B(9--36A), which displayed reversible surface pressure-induced beta-sheet formation. Bulk phase lipid melting curves and monolayer experiments with peptide-lipid mixtures showed subtle differences in the degree of bulk phase interaction and substantial differences in peptide surface activity. The uniqueness of IRRAS is emphasized as the importance of evaluating secondary structure in both bulk phase and monolayer environments for lung surfactant peptide mimics is demonstrated.     

Pulmonary surfactant proteins SP-B and SP-C in spread monolayers at the air-water interface: III. Proteins SP-B plus SP-C with phospholipids in spread monolayers.

Spread binary monolayers of surfactant-associated proteins SP-B and SP-C were formed at the air-water interface. Surface pressure measurements showed no interactions between the hydrophobic proteins. The effects of a mixture of SP-B plus SP-C (2:1, w/w) on the properties of monolayers of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and DPPC:DPPG (7:3, mol:mol) were studied. During compression of ternary and quaternary films, containing less than 0.4 mol% or 5 weight% total protein, the proteins were not squeezed out and appeared to remain associated with the film until collapse at surface pressures of about 65-70 mN.m-1. At initial concentrations of total protein of about 0.9 mol% or 10 weight%, exclusion of protein-lipid complexes was observed at 40-50 mN.m-1. Larger amounts of phospholipid were removed by proteins from (SP-B:SP-C)/DPPG films than from (SP-B:SP-C)/DPPC ones. Separate squeeze-out of SP-B (or SP-B plus DPPC) at about 40 mN.m-1, followed by exclusion of SP-C (or SP-C plus DPPC) at about 50 mN.m-1, was observed in (SP-B:SP-C)/DPPC films. This led to a conclusion that there was independent behavior of SP-B and SP-C in (SP-B:SP-C)/DPPC monolayers. The quaternary (SP-B:SP-C)/(DPPC:DPPG) films showed qualitatively similar process of squeeze-out of the proteins. In the ternary mixtures of SP-B plus SP-C with DPPG separate exclusion of SP-B was not detected; rather, the data was consistent with exclusion of a (SP-B:SP-C)/DPPG complex at about 50 mN.m-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pulmonary surfactant proteins SP-B and SP-C and calcium ions on the surface properties of hydrophobic fractions of lung surfactant

This study focused on two hydrophobic fractions (HF-A and HF-B) isolated from porcine lung surfactant (LS) that had similar phospholipid composition, but HF-A consisted of the hydrophobic LS specific proteins (SP-B and SP-C), in contrast to HF-B. Monolayers spread in a Langmuir trough were formed at the air/water interface of both fractions and the rate of adsorption-desorption and the respreading potential of the LS constituents was studied during six consecutive compression/decompression cycles of the monolayers. By drawing a comparison between the behavior of HF-A and HF-B monolayers on the subphase of 150 mm NaCl, either with or without additional Ca²⁺, we estimated the role of hydrophobic LS proteins and Ca²⁺ ions for LS surface activity. The results demonstrated much higher ability of the HF-A sample, compared to HF-B, to maintain lower surface tension (γ) during monolayer compression and its better respreading capacity during decompression. For instance, at a surface concentration corresponding to 80 Ų per phospholipid molecule, the HF-A monolayers showed a much lower γ _max value (surface tension at 100% of the trough area), being ca. 31.0 mN/m, compared to the HF-B monolayers (γ _max? 62.0 mN/m). The surface tension after compression to 20% of the initial area (γ _min) reached ca. 7.0 and 19.0 mN/m in the HF-A and HF-B monolayers, respectively. Better respreading of the HF-A monolayers compared to the HF-B monolayers was due to the faster adsorption and spreading of LS phospholipids during decompression, facilitated by the hydrophobic proteins. As the phospholipid composition of both fractions was similar, we showed that the hydrophobic surfactant proteins were responsible also for the prevention of the irreversible loss of material from the surface during monolayer compression/decompression. The effects observed demonstrated also that the hydrophobic surfactant proteins were the stronger determinant, compared with Ca²⁺ ions, for the surface tension decrease and respreading of the monolayers during film compression/decompression. For instance, when the HF-A monolayers were spread on a subphase with an additional 5 mm Ca²⁺ ion content, no significant changes were detected in the γ _min and γ _max values between the first and sixth cycle, compared to the monolayers spread on a subphase of 150 mm NaCl only. However, in the absence of positively charged SP-B and SP-C (HF-B sample) in highly compressed monolayers, Ca²⁺ ions were able to cause the effects shown by SP-B and SP-C, although to a less extent. The role of the electrostatic and hydrophobic interactions is discussed for the better respreading of LS components in the presence of LS proteins and Ca²⁺ ions.     

External reflection absorption infrared spectroscopy study of lung surfactant proteins SP-B and SP-C in phospholipid monolayers at the air/water interface.

The interactions of the hydrophobic pulmonary surfactant proteins SP-B and SP-C with 1,2-dipalmitoylphosphatidylcholine in mixed, spread monolayer films have been studied in situ at the air/water interface with the technique of external reflection absorption infrared spectroscopy (IRRAS). SP-C has a mostly alpha-helical secondary structure both in the pure state and in the presence of lipids, whereas SP-B secondary structure is a mixture of alpha-helical and disordered forms. When films of SP-B/1,2-dipalmitoylphosphatidylcholine are compressed to surface pressures (pi) greater than approximately 40-43 mN/m, the protein is partially (15-35%) excluded from the surface, as measured by intensity ratios of the peptide bond amide l/lipid C==O stretching vibrations. The extent of exclusion increases as the protein/lipid ratio in the film increases. In contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids. The amide l peak of SP-C becomes asymmetric as a result of the formation of intermolecular sheet structures (1615-1630 cm-1) suggestive of peptide aggregation. The power of the IRRAS experiment for determination of film composition and molecular structure, i.e., as a direct test of the squeeze-out hypothesis of pulmonary surfactant function, is evident from this work.     

Intrinsic structural and functional determinants within the amino acid sequence of mature pulmonary surfactant protein SP-B

Pulmonary surfactant protein SP-B is absolutely required for proper function of surfactant in the alveoli, and is an important component of therapeutical surfactant preparations used to treat respiratory pathologies. To explore inherent structural and functional determinants within the amino acid sequence of mature SP-B, porcine SP-B has been subjected to extensive disulfide reduction under highly denaturing conditions and to cysteine carboxyamidomethylation, and the structure, lipid-protein interactions, and surface activity of this modified form have been characterized. Refolding of the reduced protein yielded a form (SP-Br) with secondary structure practically identical to that of the native disulfide-linked SP-B dimer. Reduced SP-Br exhibited higher structural flexibility than native SP-B, as indicated by a higher susceptibility of fluorescence emission to quenching by acrylamide and biphasic behavior during interaction of the protein with lipid bilayers and monolayers. SP-Br had, however, effects similar to those of native SP-B on the thermotropic properties of dipalmitoylphosphatidylcholine (DPPC) bilayers. SP-Br was more effective than native SP-B in promoting interfacial adsorption of phospholipid bilayers into interfacial films, presumably because of its higher structural flexibility, and retained the ability of native SP-B to stabilize DPPC interfacial films compressed to pressures near collapse against spontaneous relaxation. SP-Br also mimicked the behavior of native SP-B in lipid-protein films subjected to dynamic compression-expansion cycling in a captive bubble surfactometer, but only in the presence of phosphatidylglycerol (PG), the main anionic phospholipid in surfactant. The presence of PG appears to be required for SP-Br to acquire the appropriate tertiary folding to produce progressively more efficient lipid-protein films capable of reaching very high pressures upon limited compression with almost no hysteresis.     

Interaction of lipid vesicles with monomolecular layers containing lung surfactant proteins SP-B or SP-C

Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air-fluid interface of the lung. The Wilhelmy plate method was used to study the involvement of SP-B and SP-C in the formation of phospholipid monolayers. The proteins were either present in the phospholipid vesicles which were injected into the subphase or included in a preformed phospholipid monolayer. In agreement with earlier investigators, we found that SP-B and SP-C, present in phospholipid vesicles, were able to induce the formation of a monolayer, as became apparent by an increase in surface pressure. However, when the proteins were present in a preformed phospholipid monolayer (20 mN/m) at similar lipid to protein ratios, the rate of surface pressure increase after injection of pure phospholipid vesicles into the subphase at similar vesicle concentrations was 10 times higher. The process of phospholipid insertion from phospholipid vesicles into the protein-containing monolayers was dependent on (1) the presence of (divalent) cations, (2) the phospholipid concentration in the subphase, (3) the size of the phospholipid vesicles, (4) the protein concentration in the preformed monolayer, and (5) the initial surface pressure at which the monolayers were formed. Both in vesicles and in preformed monolayers, SP-C was less active than SP-B in promoting the formation of a phospholipid monolayer. The use of preformed monolayers containing controlled protein concentrations may allow more detailed studies on the mechanism by which the proteins enhance phospholipid monolayer formation from vesicles.     

Effect of Pulmonary Surfactant Protein SP-B on the Micro- and Nanostructure of Phospholipid Films

Monolayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3, w/w) in the absence or in the presence of 2, 5, 10, or 20 weight percent of porcine surfactant protein SP-B were spread at the air-liquid interface of a surface balance, compressed up to surface pressures in the liquid-expanded/liquid-condensed (LE-LC) plateau of the isotherm, transferred onto mica supports, and analyzed by scanning force microscopy. In the absence of protein, the films showed micrometer-sized condensed domains with morphology and size that were analogous to those observed in situ at the air-liquid interface by epifluorescence microscopy. Scanning force microscopy permits examination of the coexisting phases at a higher resolution than previously achieved with fluorescent microscopy. Both LE and LC regions of DPPC films were heterogeneous in nature. LC microdomains contained numerous expanded-like islands whereas regions apparently liquid-expanded were covered by a condensed-like framework of interconnected nanodomains. Presence of increasing amounts of pulmonary surfactant protein SP-B affected the distribution of the LE and LC regions of DPPC and DPPC/DPPG films both at the microscopic and the nanoscopic level. The condensed microdomains became more numerous but their size decreased, resulting in an overall reduction of the amount of total LC phase in both DPPC and DPPC/DPPG films. At the nanoscopic level, SP-B also caused a marked reduction of the size of the condensed-like nanodomains in the LE phase and an increase in the length of the LE/LC interface. SP-B promotes a fine nanoscopic framework of lipid and lipid-protein nanodomains that is associated with a substantial mechanical resistance to film deformation and rupture as observed during film transference and manipulation. The effect of SP-B on the nanoscopic structure of the lipid films was greater in DPPC/DPPG than in pure DPPC films, indicating additional contributions of electrostatic lipid-protein interactions. The alterations of the nanoscopic structures of phospholipid films by SP-B provide the structural framework for the protein simultaneously sustaining structural stability as well as dynamical flexibility in surfactant films at the extreme conditions imposed by the respiratory mechanics. SP-B also formed segregated two-dimensional clusters that were associated with the boundaries between LC microdomains and the LE regions of DPPC and DPPC/DPPG films. The presence of these clusters at protein-to-lipid proportions above 2% by weight suggests that the concentration of SP-B in the surfactant lipid-protein complexes may be close to the solubility limit of the protein in the lipid films.     

Structure-function relationships of bovine pulmonary surfactant proteins: SP-B and SP-C

Pulmonary surfactant contains at least three unique proteins: SP-A, SP-B and SP-C. SP-B and SP-C from bovine surfactant are markedly hydrophobic and have molecular masses between 3 and 26 kDa. We identify surfactant proteins under nonreducing conditions on polyacrylamide gels with approximate molecular mass of 5, 14, 26 kDa (SP-5, 14, 26) when organic solvent-soluble material is eluted from a Sephadex LH-20 size exclusion column followed by separation on a high-performance reverse-phase chromatography system. These bands correspond to monomeric SP-C, oligomeric SP-C and oligomeric SP-B, respectively. Computer analysis (Eisenberg-hydrophobic moment) of sequences for these proteins suggests that SP-B contains surface-seeking amphiphilic segments. In contrast, SP-C resembles a more hydrophobic transmembrane anchoring peptide. Dispersions containing dipalmitoylphosphatidylcholine, phosphatidylglycerol, palmitic acid and multimeric SP-B and SP-C duplicate the surface activity of natural surfactant when assayed in a pulsating bubble surfactometer. We speculate that oligomers of SP-B and monomers and oligomers of SP-C may act cooperatively in affecting surfactant function. An important function of SP-B and SP-C may be to affect the ordering of surfactant lipids so that rates of transport of surfactant lipids to the hypophase surface in the alveoli are enhanced.     

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.     

Combinations of fluorescently labeled pulmonary surfactant proteins SP-B and SP-C in phospholipid films

Hydrophobic pulmonary surfactant (PS) proteins B (SP-B) and C (SP-C) modulate the surface properties of PS lipids. Epifluorescence microscopy was performed on solvent-spread monolayers of fluorescently labeled porcine SP-B (R-SP-B, labeled with Texas Red) and SP-C (F-SP-C, labeled with fluorescein) in dipalmitoylphosphatidylcholine (DPPC) (at protein concentrations of 10 and 20 wt%, and 10 wt% of both) under conditions of cyclic compression and expansion. Matrix-assisted laser desorption/ionization (MALDI) spectroscopy of R-SP-B and F-SP-C indicated that the proteins were intact and labeled with the appropriate fluorescent probe. The monolayers were compressed and expanded for four cycles at an initial rate of 0.64 A2 x mol(-1) x s(-1) (333 mm2 x s x [-1]) up to a surface pressure pi approximately 65 mN/m, and pi-area per residue (pi-A) isotherms at 22 +/- 1 degrees C were obtained. The monolayers were microscopically observed for the fluorescence emission of the individual proteins present in the film lipid matrix, and their visual features were video recorded for image analysis. The pi-A isotherms of the DPPC/protein monolayers showed characteristic "squeeze out" effects at pi approximately 43 mN/m for R-SP-B and 55 mN/m for F-SP-C, as had previously been observed for monolayers of the native proteins in DPPC. Both proteins associated with the expanded (fluid) phase of DPPC monolayers remained in or associated with the monolayers at high pi (approximately 65 mN/m) and redispersed in the monolayer upon its reexpansion. At comparable pi and area/molecule of the lipid, the proteins reduced the amounts of condensed (gel-like) phase of DPPC monolayers, with F-SP-C having a greater effect on a weight basis than did R-SP-B. In any one of the lipid/protein monolayers the amounts of the DPPC in condensed phase were the same at equivalent pi during compression and expansion and from cycle to cycle. This indicated that only minor loss of components from these systems occurred between compression-expansion cycles. This study indicates that hydrophobic PS proteins associate with the fluid phase of DPPC in films, some proteins remain at high surface pressures in the films, and such lipid-protein films can still attain high pi during compression.     

Deficiency of SP-B reveals protective role of SP-C during oxygen lung injury.

Although the surface properties of surfactant protein (SP)-B and SP-C are similar, the contributions that either protein may make to lung function have not been identified in vivo. Mutations in SP-B cause lethal respiratory failure at birth; however, SP-B null mice are deficient in both SP-B and SP-C. To identify potential contributions of SP-C to lung function in vivo, the following transgenic mice were generated and exposed to 95% O(2) for 3 days: (SP-B(+/+),SP-C(+/+)), (SP-B(+/+), SP-C(-/-)), (SP-B(+/-),SP-C(+/+)), (SP-B(+/-),SP-C(+/-)), and (SP-B(+/-),SP-C(-/-)). Hyperoxia altered pressure-volume curves in mice that were heterozygous for SP-B, and these values were further decreased in (SP-B(+/-),SP-C(-/-)) mice. Likewise, alveolar interleukin (IL)-6 and IL-1 beta were maximally increased by O(2) exposure of (SP-B(+/-),SP-C(-/-)) mice compared with the other genotypes. Lung hysteresivity was lower in the (SP-B(+/-),SP-C(-/-)) mice. Surfactant isolated from (SP-B(+/+),SP-C(-/-)) and (SP-B(+/-),SP-C(-/-)) mice failed to stabilize the surface tension of microbubbles, showing that SP-C plays a role in stabilization or recruitment of phospholipid films at low bubble radius. Genetically decreased levels of SP-B combined with superimposed O(2)-induced injury reveals the distinct contribution of SP-C to pulmonary function in vivo.