Recombinant Surfactant Protein C Research Articles

Exploring protein-protein interactions and oligomerization state of pulmonary surfactant protein C (SP-C) through FRET and fluorescence self-quenching.

Pulmonary surfactant (PS) is a lipid-protein complex that forms films reducing surface tension at the alveolar air-liquid interface. Surfactant protein C (SP-C) plays a key role in rearranging the lipids at the PS surface layers during breathing. The N-terminal segment of SP-C, a lipopeptide of 35 amino acids, contains two palmitoylated cysteines, which affect the stability and structure of the molecule. The C-terminal region comprises a transmembrane α-helix that contains a ALLMG motif, supposedly analogous to a well-studied dimerization motif in glycophorin A. Previous studies have demonstrated the potential interaction between SP-C molecules using approaches such as Bimolecular Complementation assays or computational simulations. In this work, the oligomerization state of SP-C in membrane systems has been studied using fluorescence spectroscopy techniques. We have performed self-quenching and FRET assays to analyze dimerization of native palmitoylated SP-C and a non-palmitoylated recombinant version of SP-C (rSP-C) using fluorescently labeled versions of either protein reconstituted in different lipid systems mimicking pulmonary surfactant environments. Our results reveal that doubly palmitoylated native SP-C remains primarily monomeric. In contrast, non-palmitoylated recombinant SP-C exhibits dimerization, potentiated at high concentrations, especially in membranes with lipid phase separation. Therefore, palmitoylation could play a crucial role in stabilizing the monomeric α-helical conformation of SP-C. Depalmitoylation, high protein densities as a consequence of membrane compartmentalization, and other factors may all lead to the formation of protein dimers and higher-order oligomers, which could have functional implications under certain pathological conditions and contribute to membrane transformations associated with surfactant metabolism and alveolar homeostasis.

Palmitoylation as a key factor to modulate SP-C–lipid interactions in lung surfactant membrane multilayers

Surfactant protein C (SP-C) has been regarded as the most specific protein linked to development of mammalian lungs, and great efforts have been done to understand its structure–function relationships. Previous evidence has outlined the importance of SP-C palmitoylation to sustain the proper dynamics of lung surfactant, but the mechanism by which this posttranslational modification aids SP-C to stabilize the interfacial surfactant film along the compression–expansion breathing cycles, is still unrevealed. In this work we have compared the structure, orientation and lipid–protein interactions of a native palmitoylated SP-C with those of a non-palmitoylated recombinant SP-C (rSP-C) form in air-exposed multilayer membrane environments, by means of ATR-FTIR spectroscopy. Palmitoylation does not affect the secondary structure of the protein, which exhibits a full α-helical conformation in partly dehydrated phospholipid multilayer films. However, differences between the Amide I band of the IR spectrum of palmitoylated and non-palmitoylated proteins suggest subtle differences affecting the environment of their helical component. These differences are accompanied by differential effects on the IR bands from phospholipid phosphates, indicating that palmitoylation modulates lipid–protein interactions at the headgroup region of phospholipid layers. On the other hand, the relative dichroic absorption of polarized IR has allowed calculating that the palmitoylated protein adopts a more tilted transmembrane orientation than the non-palmitoylated SP-C, likely contributing to more compact, dehydrated and possibly stable multilayer lipid–protein films. As a whole, the behavior of multilayer films containing palmitoylated SP-C may reflect favorable structural properties for surfactant reservoirs at the air–liquid respiratory interface.

Palmitoylation as a Key Factor to Understand Sp-C-Lipid Interactions in the Lung Surfactant System

Surfactant protein C (SP-C) has been regarded as the most specific protein linked to lung development. So far, great efforts have been done to understand the structure-function relationships of this lipopeptide, nevertheless its high hydrophobicity and tendency to aggregate forming amyloid-like structures have made its study a challenging task. Previous evidence has pointed out the importance of SP-C palmitoylation in sustaining the proper dynamics of lung surfactant, but the mechanism by which this posttranslational modification stabilizes the interfacial surfactant film under dynamic compression-expansion cycles mimicking the process of breathing, is still unrevealed. In this work we have compared the behavior of a native palmitoylated SP-C with a non-palmitoylated recombinant SP-C (rSP-C) in membrane environments by means of ATR-FTIR spectroscopy. Our results suggest that palmitoylation modulates SP-C-lipid interactions and besides, it may play a dual role in combination with electrostatic interactions with anionic phospholipids (POPG) to maintain a proper SP-C conformation in the lung surfactant context. Functional approaches will provide further insights into SP-C palmitoylation-induced effects, allowing the characterization of SP-C structure-function determinants.

A Novel Continuous Powder Aerosolizer (CPA) for Inhalative Administration of Highly Concentrated Recombinant Surfactant Protein-C (rSP-C) Surfactant to Preterm Neonates

In pulmonary medicine, aerosolization of substances for continuous inhalation is confined to different classes of nebulizers with their inherent limitations. Among the unmet medical needs is the lack of an aerosolized surfactant preparation for inhalation by preterm neonates, to avoid the risks associated with endotracheal intubation and surfactant bolus instillation. In the present report, we describe a high-concentration continuous powder aerosolization system developed for delivery of inhalable surfactant to preterm neonates. The developed device uses a technique that allows efficient aerosolization of dry surfactant powder, generating a surfactant aerosol of high concentration. In a subsequent humidification step, the heated aerosol particles are covered with a surface layer of water. The wet surfactant aerosol is then delivered to the patient interface (e.g., nasal prongs) through a tube. The performance characteristics of the system are given as mass concentration, dose rate, and size distribution of the generated aerosol. Continuous aerosol flows of about 0.84 L/min can be generated from dry recombinant surfactant protein-C surfactant, with concentrations of up to 12 g/m(3) and median particle sizes of the humidified particles in the range of 3 to 3.5 μm at the patient interface. The system has been successfully used in preclinical studies. The device with its continuous high-concentration delivery is promising for noninvasive delivery of surfactant aerosol to neonates and has the potential for becoming a versatile disperser platform closing the gap between continuously operating nebulizers and discontinuously operating dry powder inhaler devices.

The non-intubated, spontaneously breathing, continuous positive airway pressure (CPAP) ventilated pre-term lamb: A unique animal model

Neonatologists prefer non-invasive ventilation methods for pre-term neonates, who often require surfactant treatment. Therefore, a technology for non-invasive surfactant administration would be highly appreciated. We have developed a Continuous Powder Aerosolization (CPA) system for the generation of a humidified recombinant surfactant protein-C (rSP-C) surfactant aerosol for non-invasive administration to pre-term neonates via bi-nasal prongs. Before conducting clinical trials, safety testing in an adequate pre-clinical animal model is necessary. In contrast to existing pre-term lamb models, this model should use non-intubated animals to include upper airways for safety testing. Pre-term animals should have already a sufficient respiratory drive to breathe spontaneously on non-invasive continuous positive airway pressure (CPAP) support, but their lungs should still be pre-mature to be comparable with the clinical situation for the treatment of pre-term infants. The aim of this feasibility study was therefore to establish a CPAP-stable, non-intubated pre-term lamb model for the investigation of safety, efficacy, and pulmonary deposition of a humidified rSP-C surfactant aerosol. For this purpose, 19 pre-term lambs with a gestational age of 135–137 days (term: about 144 days) were delivered via Caesarean section. Four animals died before start of treatment, while the remaining animals were treated via customized bi-nasal prongs with rSP-C surfactant aerosol or humidified air as vehicle control. To determine pulmonary deposition, selected animals received rSP-C surfactant labelled with samarium oxide as non-radioactive tracer. Treatment was started at 30min of age and was continued for 1 or 2.5h. Investigations during the in-life phase included observation of clinical signs, haematology, blood gas analysis, and determination of minute volume. At 3h of age, animals were euthanized and organs removed for histopathology investigation or for determination of pulmonary deposition. Administration of humidified, aerosolized rSP-C surfactant was well tolerated, and histopathology investigation of upper airways and lungs revealed no aerosol-related changes. Mean body weight-corrected pulmonary deposition of rSP-C surfactant ranged from 1.7 to 7.7mg/kg depending on the duration of treatment and aerosolization parameters used. A trend towards reduced spontaneous minute volumes indicating reduced breathing efforts and towards reduced lung weights indicating less fluid in the lungs of surfactant-treated animals compared to animals of the vehicle control group could be seen. Taken together, a CPAP-stable, non-intubated pre-term lamb model was successfully established and the parameters for the investigation of safety, efficacy, and pulmonary deposition of aerosolized rSP-C surfactant for the subsequent main study were identified.

Dry powder aerosolization of a recombinant surfactant protein-C–based surfactant for inhalative treatment of the acutely inflamed lung*

Inhalative application of substantial amounts of pulmonary surfactant to the acutely inflamed lung represents a desirable therapeutic approach but was impossible under clinical conditions because of the technical limitations of currently available devices. We developed a new dry powder aerosolizer for administration of a recombinant surfactant protein-C-based surfactant, determined aerosol characteristics, and evaluated its use in animal models of acute lung injury. Laboratory experiment. University laboratory. Rabbits and mice. The efficacy of an aerosol application of recombinant surfactant protein-C surfactant was assessed in three animal models of acute lung injury: in rabbits with acute lung injury caused by repetitive lavage with prolonged and injurious ventilation; in rabbits at day 4 after inhalative application of bleomycin; and in bleomycin-challenged, spontaneously breathing mice. Analysis of aerosolizer characteristics revealed favorable properties making inhalative surfactant treatment in acute lung injury/acute respiratory distress syndrome possible. The generated aerosol had a mass median aerodynamic diameter of 1.6 microm, with 85% of all particles being smaller than 5 microm. The average mass of surfactant being aerosolized was approximately 800 mg/min, thus allowing delivery of large amounts of surfactant. Biochemical and biophysical surfactant properties remained unaltered after aerosolization. In both rabbit models aerosolization of approximately 500 mg recombinant surfactant protein-C surfactant resulted in a far-reaching restoration of gas exchange and compliance, with Pao2/Fio2 values approaching control values. In bleomycin-challenged, spontaneously breathing mice, surfactant aerosolization resulted in a restoration of compliance. The described dry powder aerosolizer may be applicable to surfactant therapy of acute lung injury/acute respiratory distress syndrome. This conclusion is based on four main factors. High doses comparable to those used for intratracheal instillation in humans can be generated within a relatively short time period, the device can be connected to the inspiratory limb of the ventilator circuit, the aerosolized surfactant material is biophysically fully active, and therapeutic efficacy was proven in three different animal models of acute lung injury/acute respiratory distress syndrome.

SP-C Palmitoylation is Crucial for Stabilizing Cholesterol-Containing Surfactant Films during Continuous Compression/Expansion Cycling

Cholesterol is critical to maintain a dynamic lateral structure in pulmonary surfactant membranes, including a defined fluid-ordered/fluid-disordered phase equilibrium and proper lateral sorting of surfactant proteins and lipids. However, an excess of cholesterol has been linked to impaired surface activity both in surfactant models and in surfactant from injured lungs. Surfactant protein C (SP-C), the smallest and most hydrophobic of all surfactant proteins, has been shown to interact with cholesterol and dual palmitoylation of its N-terminal segment has been shown to drive association with ordered phases in model membranes. Furthermore, it has been proposed that native palmitoylated SP-C can act in concert with surfactant protein B (SP-B) to permit cholesterol-containing surfactant films to reach very low surface tensions upon compression. In the present work, we report that palmitoylation of SP-C is important for its ability to counteract deleterious effects of cholesterol on surfactant film stability under continuous expansion/compression cycling, as evaluated in a captive bubble surfactometer (CBS) setup. Presence of 5% cholesterol impairs significantly the stability under quasi static and dynamic compression of films composed of DPPC/POPC/POPG/SP-B (50:25:15:1, w/w/w/w), which are able to reach tensions below 3 mN/m with only 20% compression and almost no hysteresis in the absence of cholesterol. Incorporation in the films of 2% native palmitoylated SP-C could alleviate these deleterious effects. However, recombinant non-palmitoylated SP-C was not able to reproduce the stabilizing effect of native SP-C, confirming that palmitoylation of SP-C at its N-terminal end is crucial for its potential function of stabilizing surfactant films during the respiratory cycles in the lung.

A Search for Subgroups of Patients With ARDS Who May Benefit From Surfactant Replacement Therapy: A Pooled Analysis of Five Studies With Recombinant Surfactant Protein-C Surfactant (Venticute)

Studies to date have shown no survival benefit for the use of exogenous surfactant to treat patients with the ARDS. To identify specific patient subgroups for future study, we performed an exploratory post hoc analysis of clinical trials of recombinant surfactant protein-C (rSP-C) surfactant (Venticute; Nycomed GmbH; Konstanz, Germany). We performed a pooled analysis of all five multicenter studies in which patients with ARDS due to various predisposing events were treated with rSP-C surfactant. Patients received either usual care (n = 266) or usual care plus up to four intratracheal doses (50 mg/kg) of rSP-C surfactant (n = 266). Factors influencing the study end points were analyzed using descriptive statistics, analysis of covariance, and logistic regression models. ARDS was most often associated with pneumonia or aspiration, sepsis, and trauma or surgery. For the overall patient population, treatment with rSP-C surfactant significantly improved oxygenation (p = 0.002) but had no effect on mortality (32.6%). Multivariate analysis showed age and acute physiology and chronic health evaluation (APACHE) II score to be the strongest predictors of mortality. In the subgroup of patients with severe ARDS due to pneumonia or aspiration, surfactant treatment was associated with markedly improved oxygenation (p = 0.0008) and improved survival (p = 0.018). rSP-C surfactant improved oxygenation in patients with ARDS irrespective of the predisposition. Post hoc evidence of reduced mortality associated with surfactant treatment was obtained in patients with severe respiratory insufficiency due to pneumonia or aspiration. Those patients are the focus of a current randomized, blinded, clinical trial with rSP-C surfactant.

Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity

Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich α helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use. We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% α-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression π–A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations.

Intratracheal application of recombinant surfactant protein-C surfactant to rabbits attenuates acute lung injury induced by intratracheal acidified infant formula.

Our aim in the current study was to determine whether recombinant surfactant protein-C (rSP-C) surfactant improves acute lung injury (ALI) induced by intratracheal acidified milk products. Twenty-eight rabbits were randomly divided into four groups. ALI was induced with intratracheal acidified infant formula (0.8 mL/kg, pH 1.8) in 3 groups. The control group received intratracheal acidified saline. Therapy groups received 1 of 2 doses of intratracheal rSP-C surfactant (0.5 or 2 SP-C mg/kg) 30 min after the acidified infant formula. The lungs were ventilated with 100% oxygen for 4 h after induction of ALI. Acidified infant formula dramatically reduced oxygenation and lung compliance, and increased resistance. Both doses of rSP-C improved the variables [mean PaO(2) (mm Hg) and compliance (mL/cm H(2)O) at 4 h: 61 and 0.4 for infant formula, 162 and 1.0 for small-dose rSP-C, and 152 and 1.2 for large-dose rSP-C, respectively; P < 0.05]. Pulmonary leukosequestration and edema, and severe morphological changes were attenuated by rSP-C treatment (ALI score: 14, 7, 7 in infant formula, small-dose rSP-C, and large-dose rSP-C; P < 0.05). The efficacy was similar for the two doses of rSP-C. These findings suggest that intratracheal administration of rSP-C ameliorates ALI induced by aspiration of acidified milk products. Small or large doses of recombinant surfactant protein-C surfactant given 30 min after intratracheal acidified infant formula attenuated physiological, biochemical, and morphological lung damage.

Resistance of different surfactant preparations to inactivation by meconium.

A disease similar to acute respiratory distress syndrome may occur in neonates after aspiration of meconium. The aim of the study was to compare the inhibitory effects of human meconium on the following surfactant preparations suspended at a concentration of 2.5 mg/mL: Curosurf, Alveofact, Survanta, Exosurf, Pumactant, rabbit natural surfactant from bronchoalveolar lavage, and two synthetic surfactants based on recombinant surfactant protein-C (Venticute) or a leucine/lysine polypeptide. Minimum surface tension, determined with a pulsating bubble surfactometer, was increased >10 mN/m at meconium concentrations >or=0.04 mg/mL for Curosurf, Alveofact, or Survanta, >or=0.32 mg/mL for recombinant surfactant protein-C, >or=1.25 mg/mL for leucine/lysine polypeptide, and >or=20 mg/mL for rabbit natural surfactant. The protein-free synthetic surfactants Exosurf and Pumactant did not reach minimum surface tension <10 mN/m even in the absence of meconium. We conclude that surfactant activity is inhibited by meconium in a dose-dependent manner. Recombinant surfactant protein-C and leucine/lysine polypeptide surfactant were more resistant to inhibition than the modified natural surfactants Curosurf, Alveofact, or Survanta but less resistant than natural lavage surfactant containing surfactant protein-A. We speculate that recombinant hydrophobic surfactant proteins or synthetic analogs of these proteins can be used for the design of new surfactant preparations that are relatively resistant to inactivation and therefore suitable for treatment of acute respiratory distress syndrome.

Dosing and delivery of a recombinant surfactant in lung-injured adult sheep.

The purpose of this study was to evaluate a surfactant based on a recombinant surfactant protein-C (rSP-C) at three different doses (25, 100, and 200 mg lipid/kg) in the saline lavage adult sheep model of acute lung injury. All three doses resulted in significant improvements in gas exchange, although the 100 and 200 mg/kg doses were superior to the 25 mg/kg dose. There were no significant differences in effect of the 100 and 200 mg/kg doses. In addition, the physiologic efficacy and lobar surfactant distribution patterns were similar when two different surfactant delivery methods were compared. This comparison involved administering the surfactant directly into each lobe under bronchoscopic guidance, versus instilling the surfactant through an endotracheal tube into the lungs. However, the former technique took significantly longer to perform (24.5 +/- 3.3 min versus 11.6 +/- 2.5 min, p < 0.05) and required a skilled bronchoscopist. In conclusion, rSP-C surfactant was effective in improving gas exchange in this model of lung injury, although higher doses were required for optimal responses. The bronchoscopic administration technique produced results similar to those of the tracheal instillation method, but had some disadvantages that may limit the widespread clinical use of this technique in patients with lung injury.

Production of surfactant protein C in the baculovirus expression system: the information required for correct folding and palmitoylation of SP-C is contained within the mature sequence

Surfactant protein C (SP-C) is synthesized in the alveolar type II cells of the lung as a 21 kDa propeptide which is proteolytically processed to a 4.2 kDa mature active form. The main function of this extremely hydrophobic protein is to enhance lipid insertion into the air/liquid interface in the lung upon inhalation. This is necessary to maintain a relatively low surface tension at this interface during breathing. In this report we describe the production of mature human SP-C in the baculovirus expression system. The recombinant protein contains a secondary structure with a high α-helical content (73%), comparable to native SP-C, as determined by circular dichroism and attenuated total reflection Fourier transform infrared analysis. The expressed protein is a mixture of dipalmitoylated (15%) and non-palmitoylated SP-C. This suggests that the information required for palmitoylation is contained within the sequence of the mature protein. The activity of the protein to insert phospholipids into a preformed monolayer of lipids at an air/liquid interface was determined with a captive bubble surfactometer. Recombinant SP-C significantly reduced the surface tension at the air/liquid interface during dynamic expansion and compression. We conclude that correctly folded, dipalmitoylated and active SP-C can be expressed in the baculovirus expression system. Our results may facilitate investigations into the relation between structure and function of SP-C and into protein palmitoylation in general.

Lung function in premature lambs and rabbits treated with a recombinant SP-C surfactant.

We evaluated the efficacy of a recombinant surfactant protein-C (rSP-C) surfactant in vitro and in vivo, using ventilated preterm lambs and rabbits. The surface tension measured with a Wilhelmy balance of the rSP-C surfactant was lower than the surface tension of natural sheep surfactant (containing SP-B and SP-C). The rSP-C and natural surfactants were equivalently sensitive to inhibition by plasma. Over a 5-h study period, lambs treated with 100 mg/kg rSP-C and natural surfactants at birth had similar PCO2, lung mechanics, and FRC values. Indicators of injury also were not different. In preterm rabbits ventilated with 3 cm H2O positive end-expiratory pressure (PEEP), similar compliance responses were measured for the rSP-C and natural surfactant. Large-aggregate surfactants recovered from alveolar washes of the lambs treated with rSP-C or natural surfactant retained their function when tested in preterm rabbits. In conclusion, the rSP-C surfactant was effective in preterm animals with surfactant deficiency.

Association of surfactant protein C with isolated alveolar type II cells.

Surfactant protein C (SP-C) is a small hydrophobic protein that is synthesized and secreted by alveolar type II cells. The mechanism of clearance of SP-C from the alveolar airspace is not well understood, although previous studies demonstrated that recombinant SP-C instilled into the lungs of spontaneously breathing anaesthetized rats was taken up by type II cells and incorporated into lamellar bodies. The current investigation was undertaken to characterize the interaction of a complex of SP-C and surfactant-like lipids with freshly isolated rat alveolar type II cells under conditions in which the extracellular milieu can be regulated. SP-C was isolated from alveolar proteinosis lavage fluid and radiolabeled with 125I-Bolton-Hunter reagent. The radiolabeled protein retained its ability to facilitate adsorption of phospholipids to an air/liquid interface. Labeled human SP-C associated with isolated type II cells in a concentration-dependent manner that was also dependent upon temperature and time. The association of labeled SP-C with isolated type II cells did not saturate up to 150 micrograms/ml. SP-A significantly enhanced the association of SP-C with isolated type II cells. Under the experimental conditions tested, SP-C was not degraded to TCA-soluble products. These results are consistent with the hypothesis that association or uptake of SP-C by type II cells may be enhanced by SP-A and that like SP-A, SP-C is recycled by type II cells.

54 CHARACTERIZATION OF SURFACTANT PROTEIN C (SP-C)

Pulmonary surfactant protein C (SP-C) is one of the pulmonary surfactant proteins. It is a small, extremely hydrophobic peptide and has a highly conservative primary structure. The protein is normally palmitoylated on two adjacent cysteine residues. One arginine and one lysine residue are positively charged. SP-C enhances the adsorption of phospholipids into the air-water interface of the alveolar space. Structure and function of SP-C were investigated. Palmitoylated and non-palmitoylated human recombinant SP-C had a high α-helix content, but the orientation of the α-helix was dependent on the presence of palmitoyl chains. The physical properties of the acylated SP-C were different from the non-palmitoylated SP-C. Two forms of canine SP-C were isolated: monomeric palmitoylated SP-C, and dimeric, non-palmitoylated SP-C. The dimerization of SP-C had no impact on the physical properties of the protein. Binding of phospholipid-vesicles to the monolayer, and insertion of phospholipids into the monolayer was dependent on the positively charged amino acids.

Effect of acylation on structure and function of surfactant protein C at the air-liquid interface.

Pulmonary surfactant protein C (SP-C) is a small hydrophobic peptide that is palmitoylated on 2 adjacent cysteine residues. SP-C enhances the adsorption of phospholipids into a monolayer. The function of the acylation is not clear yet. The experiments described in this article were carried out in order to investigate the function of SP-C acylation in (protein-catalyzed) lipid monolayer formation, and in bilayer interactions. Palmitoylated and nonpalmitoylated human recombinant SP-C were used. In addition, a nonacylated SP-C with a Cys-->Ser mutations was included in these studies. In Wilhelmy plate experiments using negatively charged, protein-containing phospholipid monolayers and negatively charged vesicles, CaCl2 was required to obtain a maximal insertion rate of lipids into the monolayer. If the negatively charged phospholipids in the monolayer were replaced by neutral phospholipids, CaCl2 was only required to show a maximal SP-C-catalyzed insertion rate (if the molecule is palmitoylated, but not if nonpalmitoylated proteins were added). In pressure area measurements, the palmitoylated protein showed a different change in pressure as a function of the surface area, as compared with the nonpalmitoylated proteins. Circular dichroism experiments showed that all three proteins had a high content of alpha-helix. All three proteins showed a preferential orientation at the air-water interface, but the palmitoylated protein has an orientation which is more parallel to the monolayer than that of the nonpalmitoylated proteins. It is concluded that acylation of SP-C alters structural and physical properties of this protein.

Surfactant protein C precursor is palmitoylated and associates with subcellular membranes

Surfactant protein C (SP-C) is a 3.7 kDa, hydrophobic protein that enhances the adsorption of phospholipids in pulmonary surfactant. SP-C is generated by protcolytic processing of a 21 kDa precursor. Murine fetal lung explant cultures and a Chinese hamster ovary cell line expressing recombinant human SP-C gene (CHO/SPC) were used to determine the subcellular location and post-translational modification(s) of proSP-C. After in vitro translation, proSP-C of M 1 = 21 000 was generated. ProSP-C was associated with canine pancreatic microsomes during co-translation and was partially protected from digestion with proteinase K, supporting the concept that proSP-C enters but does not completely traverse the membrane of the endoplasmic reticulum (ER). Association of proSP-C isoforms of 21 and 26 kDa with intracellular membranes was demonstrated by subcellular fractionation of CHO/SPC cells. Pulse/chase experiments demonstrated that the 21 kDa SP-C proprotein was synthesized first and after 15 min was modified to produce a 26 kDa isoform in CHO/SPC cells or a 24 kDa isoform in murine fetal lung. Both the 21 and 26 kDa proSP-C isoforms were detected after labelling CHO/SPC cells with [ 3H]palmitic acid. The formation of the 26 kDa proSP-C isoform in CHO/SPC cells and the 24 kDa proSP-C isoform in murine fetal lung was blocked by cerulenin, an inhibitor of fatty acid synthesis. In conclusion, proSP-C is associated with subcellular membranes. ProSP-C is palmitoylated and undergoes additional post-translational modification that is blocked by an inhibitor of fatty acid synthesis.

Lung surfactant protein SP-C from human, bovine, and canine sources contains palmityl cysteine thioester linkages

Lung surfactant is a complex mixture of lipids and proteins that coats the alveoli to reduce surface tension and prevent airspace collapse. One of the principal protein constituents, surfactant protein C (SP-C), has been characterized following isolation from human, canine, and bovine sources. In each species, this highly hydrophobic protein is composed of 33-35 amino acids, the differences being due to NH2-terminal heterogeneity. A COOH-terminal leucine is conserved throughout. The cysteines in each species were found by fast atom bombardment mass spectrometry to be present as thioesters of palmitic acid. Acylation of recombinant SP-C with palmityl coenzyme A, followed by characterization before and after release of the acyl group with 1,4-dithiothreitol, provided corroborating evidence for the native structure.