Interaction of pulmonary surfactant protein SP-A with DPPC/egg-PG bilayers

In the mixture of lipids and proteins which comprise pulmonary surfactant, the dominant protein by mass is surfactant protein A (SP-A), a hydrophilic glycoprotein. SP-A forms octadecamers that interact with phospholipid bilayer surfaces in the presence of calcium. Deuterium NMR was used to characterize the perturbation by SP-A, in the presence of 5 mM Ca(2+), of dipalmitoyl phosphatidylcholine (DPPC) properties in DPPC/egg-PG (7:3) bilayers. Effects of SP-A were uniformly distributed over the observed DPPC population. SP-A reduced DPPC chain orientational order significantly in the gel phase but only slightly in the liquid-crystalline phase. Quadrupole echo decay times for DPPC chain deuterons were sensitive to SP-A in the liquid-crystalline mixture but not in the gel phase. SP-A reduced quadrupole splittings of DPPC choline beta-deuterons but had little effect on choline alpha-deuteron splittings. The observed effects of SP-A on DPPC/egg-PG bilayer properties differ from those of the hydrophobic surfactant proteins SP-B and SP-C. This is consistent with the expectation that SP-A interacts primarily at bilayer surfaces.     

Method of purification affects some interfacial properties of pulmonary surfactant proteins B and C and their mixtures with dipalmitoylphosphatidylcholine

Two methods were employed for preparation of lipid extracts from porcine lung surfactant. Pulmonary surfactant proteins SP-B and SP-C were isolated from the extracts using gel-exclusion chromatography on LH-60 with chloroform:methanol acidified with hydrochloric acid. Monolayers of pure SP-B or SP-C isolated from butanol lipid extracts spread at the air-water interface showed larger molecular areas than those determined in films of SP-B or SP-C isolated from chloroform surfactant extracts. Aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) supplemented with 2.5 and 5.0 wt% of SP-B or SP-C obtained from butanol extracts adsorbed faster to the air-water interface than their counterparts reconstituted with proteins isolated from chloroform extracts. Surface pressure-area characteristics of spread monolayers of DPPC plus SP-B or SP-C did not depend on the method of isolation of the proteins. The diagrams of the mean molecular areas vs. composition for the monolayers of DPPC plus SP-B or SP-C showed positive deviations from the additivity rule, independently of the procedure used for preparation of lipid extract surfactant. Matrix-assisted laser desorption/ionization spectrometry of the proteins isolated from different extraction solvents was consistent with some differences in the chemical compositions of SP-Bs. Butylation of SP-B during extraction of surfactant pellet with butanol may account for the differences observed in the molecular masses of SP-Bs isolated by the two different extraction protocols. The study suggests that the method of purification of SP-B and SP-C may modify their ability to enhance the adsorption rates of DPPC/protein mixtures, and this may be relevant to the formulation of protein-supplemented lipids for exogenous treatment of pulmonary surfactant insufficiency.     

Surfactant protein metabolism in vivo

The surfactant components saturated phosphatidylcholine, SP-B and SP-C, are secreted together in lamellar bodies, and at least a part of the de novo synthesized SP-A is secreted independently. The surface film forms from tubular myelin and loose lipid arrays, and it generates unilamellar vesicles that lack surfactant proteins and are thought to represent catabolic forms. The half-life values for the clearance of surfactant proteins from lungs range from 6.5 to 28 h and vary with species. There is minimal information about the associations of the surfactant proteins with lipids or with each other after film formation, although all surfactant components seem to be recycled back into lamellar bodies in type II cells. The relative importance of type II cells or macrophages to the catabolism of the protein components of surfactant remains to be characterized, as do regulators of surfactant homeostasis.     

Site specificity of surfactant protein expression in airways of baboons during gestation

BACKGROUND: There are disparate reports concerning the presence of surfactant proteins in the airways of lung. The recent finding of SP-A in tracheobronchial epithelium and submucosal glands in lungs from second trimester humans has renewed interest in potential new functions of surfactant in lung biology. METHODS: In situ hybridization studies were done to determine the distribution of SP-A, SP-B, and SP-C in baboon lung specimens from 60, 90, 120, 140, 160, and 180 (term) days of gestation and adults. Lungs from gestation controls were obtained at the time of hysterotomy and adult lungs at necropsy. Riboprobes used for in situ hybridization contained the entire coding regions for human SP-A, SP-B, and SP-C. RESULTS: At 60 days, SP-C mRNA expression was evident in focal portions of primitive tubular epithelium but not bronchi. This distal pattern of SP-C mRNA expression persisted and was present in some epithelial cells of respiratory bronchioles at term. At 90 days, SP-A mRNA expression was present in the epithelium of trachea and large bronchi. SP-B mRNA expression was found in small bronchi, bronchioles, and distal tubular epithelium at 120 days of gestation. SP-A mRNA bronchiolar localization became evident at 140 days of gestation and alveolar type 2 cellular expression at 160 days of gestation. Abrupt transitions of surfactant protein expression were identified (e.g., SP-A mRNA-positive cells in the epithelium of large bronchi with adjoining SP-B mRNA expression in small bronchi and bronchioles). CONCLUSIONS: Findings in the baboon indicate that there are well-delineated sites of surfactant protein mRNA expression in bronchial and bronchiolar epithelia. mRNA expressions of SP-A and SP-B are present in both bronchial and bronchiolar epithelium but at different sites, whereas SP-C expression is seen in loci of epithelial cells in respiratory bronchioles.     

Protein-lipid interactions and enzyme requirements for light subtype generation on cycling reconstituted surfactant

Surfactant convertase is required for conversion of heavy density (H) natural surfactant to light density (L) subtype during cycling in vitro, a technique that reproduces surfactant metabolism. To study mechanisms of H to L conversion, we prepared liposomes of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG), or the phospholipids (PL) in combination with either surfactant protein A (SP-A), surfactant protein B (SP-B), or both SP-A and SP-B. Phospholipids alone showed time-dependent conversion from heavy to light subtype on cycling in the absence of convertase, which was decreased by adding SP-B, but not SP-A, to phospholipids (p < 0.01 for PL+SP-B, or PL+SP-A+SP-B vs. PL, or PL+SP-A). The ultrastructure, surface activity, buoyant density, and L subtype generation on cycling PL+SP-A+SP-B with partially purified convertase or with phospholipase D were similar to those of natural TM. In conclusion, a reconstituted surfactant mimics the behavior of natural surfactant on cycling, and reveals that interaction of SP-B with phospholipids decreases L subtype generation. In addition, esterase/ phospholipase D activity is required for conversion of heavy to light subtype on cycling.     

Calcium-triggered selective intermembrane exchange of phospholipids by the lung surfactant protein SP-A

It is shown that human lung surfactant protein (SP-A) mediates selective exchange of phospholipid probes with unlabeled phospholipid in excess vesicles in the presence of calcium and NaCl. The exchange occurs without leakage of vesicle contents, or transbilayer movement (flip-flop) of the phospholipid probes, or fusion of vesicles. Individual steps preceding the exchange are dissected by a combination of protocols, and the results are operationally interpreted in terms of a model where a calcium-dependent change in SP-A triggers aggregation of vesicles followed by probe exchange between the vesicles in contact through SP-A. The contacts remain stable in the presence of calcium; i.e., the vesicles in contact do not change their partners on the time scale of several minutes. The binding of SP-A to vesicles and the aggregation of vesicles are rapid, and the aggregation is rapidly reversed by EGTA; i.e., both the forward and reverse aggregation reactions are complete in about 1 min. The exchange rate of the various probes between aggregated vesicles below 1 mM calcium in the presence of NaCl shows selectivity, i.e., a modest dependence on the net anionic charge on vesicles and for the headgroup of the probe. Exchange with lower selectivity is seen at >2 mM Ca in the absence of NaCl. SP-A binding to vesicles does not show an absolute specificity for the phospholipid structure, but the time course of the subsequent changes does. The results suggest that SP-A contacts between phospholipid interfaces could mediate the exchange of phospholipid species (trafficking and sorting) between lung surfactant pools in the hypophase and all accessible phospholipid interfaces of the alveolar space.     

Surfactant protein A (SP-A) gene targeted mice

Mice lacking surfactant protein A (SP-A) mRNA and protein in vivo were generated using gene targeting techniques. SP-A (-/-) mice have normal levels of SP-B, SP-C and SP-D mRNA and protein and survive and breed normally in vivarium conditions. Phospholipid composition, secretion and clearance, and incorporation of phospholipid precursors are normal in the SP-A (-/-) mice. Lungs of SP-A (-/-) mice have markedly decreased tubular myelin figures and clear Group B streptococci and Pseudomonas aeruginosa less efficiently than SP-A wild type mice. These studies of SP-A (-/-) mice demonstrate that SP-A has an important role in the innate immune system of the lung in vivo.     

Specific recognition of thymic self-peptides induces the positive selection of cytotoxic T lymphocytes

Congenital alveolar proteinosis is a recently described cause of lung dysfunction and respiratory distress in term neonates. In several cases a deficiency or insufficiency of surfactant apoprotein B (SP-B) has been caused by a frameshift mutation in the gene encoding SP-B. Five full-term children in three unrelated families from The Netherlands are reported. Immunohistochemistry demonstrated large amounts of surfactant proteins A and C (SP-A and SP-C) and precursors in alveolar cells and in intra-alveolar material. Results were positive for antibovine SP-B antibody but negative for antipig SP-B1 antibody, most probably reflecting differences in the antibody specificity. The findings suggest abnormal SP-B function. In two sibs, no pre-SP-C was demonstrated in the alveoli, although it was found in considerable amounts in alveolar cells. One such case has previously been reported. In two families, the parents were heterozygous for the 121 ins 2 mutation in the SP-B gene. Our findings suggest that congenital alveolar proteinosis may result from abnormalities in one or more of the surfactant proteins.     

Tumor necrosis factor-alpha-induced apoptosis in a human keratinocyte cell line (HaCaT) is counteracted by transforming growth factor-alpha

Pulmonary surfactant is a complex mixture of lipids and proteins that functions to keep alveoli from collapsing at the end of expiration. Dipalmitoylphosphatidylcholine has been identified as the most important component for lowering surface tension at the air-liquid interface. Hydrophobic surfactant apoproteins, SP-B and SP-C, play essential roles in the biophysical functions of the surfactant phospholipids. Hydrophilic surfactant apoproteins (SP-A and SP-D) that are members of C-type lectin superfamily, interact with phospholipids and glycolipids and modulate host defense functions in the lung. SP-A also plays an important role in regulating phospholipid homeostasis in the alveolar spaces. Recent advances in genetics and molecular biology have clarified the structure-function relationship of surfactant apoproteins.     

Quantity and structure of surfactant proteins vary among patients with alveolar proteinosis

Alveolar proteinosis (AP) is an idiopathic condition characterized by excess alveolar surfactant. Although the surfactant proteins (SP) are known to be aberrant, little is known of their variation between patients or their abundance relative to the lipids. We have examined surfactant composition in lavage fluid from 16 normal subjects and 13 patients with AP, one of whom was lavaged on 11 occasions over approximately 13 mo. In this patient we have examined composition on each occasion and in each sequential lavage aliquot. Composition was constant between right and left lung, but it differed markedly between patients. The cholesterol/disaturated phospholipid ratios (CHOL/DSP) were invariably elevated, on average by approximately 7-fold, whereas the SP-A/DSP and SP-B/DSP ratios were generally elevated, in some cases by as much as approximately 40- and approximately 100-fold, respectively. Although AP lavage generally contained more non-thiol-dependent SP-A aggregates and low Mr isoforms, the two-dimensional immunochemical staining patterns varied between patients and right and left lung. In the patient lavaged on multiple occasions, the SP-A/DSP and SP-B/DSP ratios progressively decreased as the patient's condition resolved. Because the SP-B/SP-A ratio was normal in all cases, we suggest that structural changes to the proteins occurred secondarily and that caution must be used in comparing functional data derived using SP-A obtained from patients with AP.     

Differential effects in vivo of thyroid hormone on the expression of surfactant phospholipid, surfactant protein mRNA and antioxidant enzyme mRNA in fetal rat lung

Antenatal administration of triiodo-L-thyronine (T3) to late gestation rats resulted in decreased lung antioxidant enzyme (AOE) activity but increased surfactant phospholipids. In fetal rat lung explant cultures, T3 decreased the expression of surfactant proteins (SP) A and B. There have been no reported studies of the simultaneous in vivo developmental influence of T3 on both pulmonary AOE and SP gene expression. We hypothesized that antenatal T3 treatment would cause differential regulation of surfactant phospholipid, SP, and AOE genes in the late gestation fetal rat. Timed pregnant rats received intramuscular injections of either T3 (7 mg/kg) or placebo on days 19 and 20 of gestation and fetuses were delivered on day 21. Fetal lung SP-A, SP-B, SP-C, and AOE mRNA levels were studied by Northern analysis. AOE mRNA levels were further quantitated by solution hybridization. Total lung phospholipids (TPL) and disaturated phosphatidylcholine (DSPC) content were quantitated by a phosphorus assay. T3 significantly increased TPL and DSPC content, and significantly decreased the expression of SP-A, SP-C, CuZnSOD, and catalase genes. Because of a crucial interplay of these factors for normal lung function at the time of birth, the molecular mechanisms by which these apparently opposing changes are accomplished warrant further investigation.     

Depth profiles of pulmonary surfactant protein B in phosphatidylcholine bilayers, studied by fluorescence and electron spin resonance spectroscopy

Pulmonary surfactant-associated protein B (SP-B) has been isolated from porcine lungs and reconstituted in bilayers of dipalmitoylphosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) to characterize the extent of insertion of the protein into phospholipid bilayers. The parameters for the interaction of SP-B with DPPC or PC using different reconstitution protocols have been estimated from the changes induced in the fluorescence emission spectrum of the single protein tryptophan. All the different reconstituted SP-B-phospholipid preparations studied had similar Kd values for the binding of the protein to the lipids, on the order of a few micromolar. The depth of penetration of SP-B into phospholipid bilayers has been estimated by the parallax method, which compares the relative efficiencies of quenching of the protein fluorescence by a shallow or a deeper spin-labeled phospholipid probe. SP-B tryptophan was found to be located 10-13 A from the center of bilayers, which is consistent with a superficial location of SP-B in phosphatidylcholine membranes. Parallax experiments, as well as resonance energy transfer from SP-B tryptophan to an acceptor probe located in the center of the bilayer, indicate that there are significant differences in the extent of insertion of the protein, depending on the method of reconstitution. SP-B reconstituted from lipid/protein mixtures in organic solvents is inserted more deeply in PC or DPPC bilayers than the protein reconstituted by addition to preformed phospholipid vesicles. These differences in the extent of insertion lead to qualitative and quantitative differences in the effect of the protein on the mobility of the phospholipid acyl chains, as studied by spin-label electron spin resonance (ESR) spectroscopy, and could represent different functional stages in the surfactant cycle.     

Effect of nitroglycerin and nicorandil on regional poststenotic quantitative coronary blood flow in coronary artery disease: a combined digital quantitative angiographic and intracoronary doppler study

The biophysical activity of lung surfactant depends, to a large extent, on the presence of the hydrophobic surfactant proteins B (SP-B) and C (SP-C). The role of these proteins in lipid adsorption and lipid squeeze-out under dynamic conditions simulating breathing is not yet clear. Therefore, the aim of this study was to investigate the interaction of spread hydrophobic surfactant proteins with phospholipids in a captive-bubble surfactometer during rapid cyclic area changes (6 cycles/min). We found that SP-B and SP-C facilitated the rapid transport of lipids into the air-water interface in a concentration-dependent manner (threshold concentration > or = 0.05:0.5 mol% SP-B/SP-C). Successive rapid cyclic area changes did not affect the concentration-dependent lipid adsorption process, suggesting that SP-B and SP-C remained associated with the surface film.     

Genetics of the hydrophobic surfactant proteins

The hydrophobic surfactant proteins, SP-B and SP-C, serve important roles in surfactant function and metabolism. Both proteins are encoded by single genes, located on human chromosomes 2 and 8 respectively, which have been characterized and extensively studied. Mutations in the SP-B gene have been shown to cause severe lung disease, and polymorphisms in the SP-B gene may be associated with the development of RDS in premature infants. In contrast, mutations in the SP-C gene have not yet been identified or shown to cause lung disease, although given the apparent importance of SP-C in surfactant function, this remains a possibility.     

Site-directed mutagenesis of surfactant protein A reveals dissociation of lipid aggregation and lipid uptake by alveolar type II cells

Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195-->Gln (rE195Q), Lys201-->Ala (rK201A) and Lys203-->Ala (rK203A) for rat SP-A, Arg199-->Ala (hR199A) and Lys201-->Ala (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203-->Ala (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [125I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process.     

NT-3 regulates expression of Brn3a but not Brn3b in developing mouse trigeminal sensory neurons

To further elucidate the nature of the molecular interactions of surfactant apoprotein B (SP-B) with phospholipid (PL) membranes, we studied the binding of SP-B to PL membranes and the lipid-dependency of its subsequent effects on leakage and fusion of membranes. SP-B binding to membranes was studied by labeling the protein with the fluorophore 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) and measuring the fluorescence of the labeled protein in the presence of varying amounts of dipalmitoylphosphatidylcholine-egg phosphatidylglycerol (DPPC-eggPG; 7-3). Leakage of contents from liposomes made of DPPC and varying molar fraction of egg phosphatidylcholine (eggPC) or eggPG was assessed by measuring the fluorescence of entrapped water-soluble probes ANTS and DPX. Fusion of membranes was assessed by measuring the fluorescence of membrane-bound NBD-phosphatidylethanolamine (NBD-PE) and rhodamine-PE (RHO-PE). We found that SP-B bound to PL membranes with high affinity and appeared to irreversibly cluster at the membrane surface, leading to graded release of the vesicle contents and eventually fusion of the membranes with increasing protein-lipid ratios. All lipid mixtures tested were susceptible to the membrane disruptive effects of SP-B, but DPPC-eggPG membranes displayed a biphasic response to increasing molar fractions of eggPG, whereas increasing fractions of eggPC elicited a monotonic response.     

Palmitoylation of lung surfactant protein SP-C alters surface thermodynamics, but not protein secondary structure or orientation in 1,2-dipalmitoylphosphatidylcholine langmuir films

Pulmonary surfactant-specific protein, SP-C, isolated from porcine lung lavage, has been deacylated to investigate the role of the two thioester linked palmitoyl chains located near the N-terminus. Surface thermodynamic properties, secondary structure, and orientation of native and deacylated SP-C in 1, 2-dipalmitoylphosphatidylcholine (DPPC) monolayers has been characterized by combined surface pressure-molecular area (pi-A) isotherms and infrared reflection-absorption spectroscopy (IRRAS) measurements. The isotherms indicate that deacylation of SP-C produces more fluid monolayers at pressures less than 30 mN m-1. The helical secondary structure and tilt angle (70-80 degrees relative to the surface normal) of SP-C remained essentially unchanged upon deacylation in DPPC monolayers at a surface pressure approximately 30 mN m-1. The results are consistent with a model that acylation of SP-C may influence the rapid protein-aided spreading of a surface-associated surfactant reservoir, but not the structure of DPPC or SP-C in the monolayer at higher surface pressures.     

Mutational analysis of Arg197 of rat surfactant protein A. His197 creates specific lipid uptake defects

In previous studies, tandem mutagenesis of Glu195 and Arg197 of surfactant protein A (SP-A) has implicated both residues as critical participants in the interaction of the molecule with alveolar type II cells and phospholipids. We substituted Ala, Lys, His, Asp, and Asn mutations for Arg to evaluate the role of a basic amino acid at position 197 in SP-A action. Unexpectedly, Ala197 retained complete activity in the SP-A functions of carbohydrate binding, type II cell binding, inhibition of surfactant secretion, lipid binding, lipid aggregation, and lipid uptake by type II cells. The results unambiguously demonstrate that Arg197 is not mechanistically essential for SP-A function. The Lys197 mutation displayed all functions of the wild type protein but exhibited a 2-fold increase in lipid uptake activity. The His197 mutation displayed all SP-A functions studied except for lipid uptake. The results obtained with the His197 mutation clearly demonstrate that lipid aggregation alone by SP-A is insufficient to promote lipid uptake by type II cells. These findings indicate that specific interactions between type II cells and SP-A are involved in the phospholipid uptake processes.