ABSTRACT 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^sup 2^/dimer molecule for SP-B and 1.15 nm^sup 2^/molecule for SP-C in [alpha]-helical conformation and 1.05 nm^sup 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 [arrow right] [beta]-sheet conversion of SP-C occurs simply by an increase of the summary protein/lipid concentration in solution.
INTRODUCTION
Pulmonary surfactant contains four specific proteins: SP-A, SP-B, SP-C, and SP-D. SP-A and SP-D are hydrophilic proteins and as members of the collectin family of proteins contribute to host defense against invading pathogens (Haagsmann and Diemel, 2001; Crouch and Wright, 2001). SP-B and SP-C are extremely Hydrophobic and play important roles in regulating surface tension in the lungs, thereby preventing alveoli collapse (Weaver, 1998; Perez-Gil and Keough, 1998).
Ovine SP-B consists of 79 amino acids and has a molecular weight of 8690 Da. The content of hydrophobic amino acids in SP-B is 40.5% (11 Val, 13.9%; 15 Leu, 19%; and 6 Ile, 7.6%). Mature SP-B is commonly a homodimer in many species, with two monomeric units linked by disulphide bounds at Cys 48. Bovine SP-B occurs also as covalent trimer (Haagsmann and Diemel, 2001; Hawgood et al., 1998), and oligomeric forms of ovine SP-B have been described (Bunger et al, 2001). SP-B is remarkably thermally stable, and its [alpha]-helical domains are not much influenced by reduction of the disulfide bounds (Hawgood et al., 1998). SP-B is essential for lung function and its absence is lethal (Nogee, 1998). Despite the functional importance of SP-B, little is known about the biophysical activity of the different oligomeric forms.
Ovine SP-C consists of 35 amino acids and has a molecular weight of 4200 Da. The content of hydrophobic amino acids in SP-C is 65.7% (12 Val, 34.3%; 7 Leu, 20%; and 4 Ile, 11.4%). Despite its low molecular weight, SP-C has several structural features. The [alpha]-helical valyl-rich domain consists of amino acids in positions 11-34 and is 25 amino acids long.
Palmitoylation of the cysteines at positions 5 and 6 increases hydrophobicity of SP-C (Johansson et al., 1994; Johansson, 1998; Weaver, 1998). Dimeric SP-C that has almost exclusively [beta]-sheet structure and is not acylated enhances the surface tension lowering properties of surfactant (Baatz et al., 1992). The [beta]-sheet structure of SP-C develops from an [alpha]-helix located at the amino acids in position 11-34 upon incubation in solution (Kallberg et al., 2001). Removal of the palmitoyl groups accelerates [alpha]-helix [arrow right] [beta]-sheet conversion and fibril formation. Amyloidlike fibrils of SP-C were found in lung washings from patients with alveolar proteinosis (Gustafsson et al., 2001). The specific functional role of SP-C for breathing and the consequences of [alpha]-helix [arrow right] [beta]-sheet conversion are unknown.
The experimental determination of the conformation and the secondary structure of a protein provides understanding of the protein function. The conformation of proteins can be changed during each single step of pretreatment, isolation, and purification procedures, as well as sample preparation for the different methods used for the spectroscopic measurements (Lohner et al., 1997; Heremans and Smeller, 1998).
To date, there are only a few studies about the effect of the isolation method on surfactant protein conformation and the influence of protein conformation on the interfacial behavior. Therefore, the first objective of this study is to investigate the structure of hydrophobic surfactant proteins obtained by different isolation and purification procedures using circular dichroism (CD), Fourier transform infrared (FTIR) and matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF) spectroscopy. A second objective of this study is to compare the interfacial behavior of SP-B and SP-C with their structural data and to determine the molecular cross section of these proteins. Surface behavior of SP-B and SP-C monolayers was evaluated in a captive bubble surfactometer using axisymmetric drop shape analysis (ADSA).
MATERIALS AND METHODS
Materials
Chloroform (Ultra-Resi analyzed) was obtained from J.T. Baker (Griesheim, Germany) and methanol (LiChrosolv, gradient grade) from Merck (Darmstadt, Germany). Water was purified by means of a Milli-Q Plus Water System (Millipore, Eschborn, Germany) and had a surface tension of 72.4 + or - 0.2 mN/m at 23[degrees]C as determined by using the axisymmetric drop shape analysis for captive bubbles (ADSA-CB) (Prokop et al., 1998). All glass vessels used for this study and the measuring cell were cleaned in KOH-saturated isopropanol.
Isolation of SP-B and SP-C
The pulmonary surfactant was obtained from cell-free sheep lung lavage fluid after 2-h centrifugation at 53,000 g. The pellet was homogenized in 1.64 N NaBr for density gradient centrifugation at 100,000 g overnight. (Hawgood et al., 1985; Pison et al., 1989). The pellicle was removed, washed, and homogenized in 4 mL water and the hydrophobic surfactant components were extracted into either 1-butanol (Bunger et al., 2000) or chloroform/methanol (Folch et al., 1957). The hydrophilic components of pulmonary sheep surfactant were discharged and the remaining solvents containing the hydrophobic surfactant components were evaporated in vacuum at 40[degrees]C and residues were weighed.
The first residue contained the hydrophobic components of pulmonary surfactant from butanol extraction (lipids, SP-B-1, and SP-C-1). It was weighted and resolved in 5 mL acidified (5% 0.1 M HCl) chloroform/methanol (1:1, v/v). Surfactant proteins were isolated using gel exclusion chromatography on LH-60 column (100 x 2.6 cm ID) (Pharmacia, Upsala, Sweden) with 5% 0.1 M HCl acidified chloroform/methanol (1:1, v/v) solvent as the mobile phase. SP-B-1 and SP-C-1 fractions were collected (50 ml of each protein) and aliquots of the solutions (1.5 ml) with final concentration of 0.04 to 0.06 mg/ml were stored at -20[degrees]C. The final concentration was determined using the HPLC method as described elsewhere (Bunger et al., 2000).
The second residue contained the hydrophobic components of pulmonary surfactant from chloroform/methanol extraction (lipids, SP-B-2 and SP-C-2). It was resolved in 5 mL acidified (5% 0.1 N trifluoroacetic acid) chloroform/methanol (1:1, v/v). Surfactant proteins were isolated using a semipreparative HPLC column (250 x 10 mm ID) with Vydac C4, a butyl silica gel (Bunger et al., 2000). SP-B-2 and SP-C-2 fractions were collected, the solvent was evaporated and the purified proteins weighted and then redisolved in chloroform/methanol (1:1, v/v) to give a final concentration of ~0.8 -1.0 mg/ml. Aliquots were stored at -20[degrees]C.
The third residue contained the hydrophobic components of pulmonary surfactant from chloroform/methanol extraction (lipids, SP-B-3 and SP-C-3). It was resolved in 1.5 mL acidified (5% 0.1 N trifluoroacetic acid) chloroform/methanol (1:1, v/v) but then treated as the second residue. It should be emphasized that the only difference between residues 2 and 3 is the concentration of their protein/lipid extracts entering the HPLC column.
On SDS polyacrylamide gel electrophoresis (16% gels) under nonreducing conditions, SP-B-1 showed a single wide band centered at ~23-24 kDa, and SP-B-2 and SP-B-3, at ~29 kDa. SP-C-1, SP-C-2, and SP-C-3 showed only one band at ~5 kDa (Fig. 1). Table 1 summarizes the three isolation and purification procedures that were used to obtain SP-B and SP-C.
Mass spectrometry
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the protein samples was performed as previously described (Plasencia et al., 2001) on a BIFLEX time-of-flight instrument (Bruker-Franzen Analytik, Bremen, Germany) operated in the positive mode. A saturated solution of sinapinic acid in acetonitrile:water (1:2) with 0.1% trifluoroacetic acid was used as the matrix. Equal volumes of the matrix and the sample
air cushion of at least 0.1 [mu]l after filling of the syringe needle with the spreading solution. Standard syringes were used with a sharp, 90[degrees] cut needle tip (pst3), which guarantees proper contact of the needle with the bubble surface. The spreading solution was slowly and gently injected on to the surface of the bubble under continuous video monitoring. After optimization of all parameters it was possible to spread even 0.4 [mu]l solution without an problems caused by solvent vapors in the bubble. Ten min after solvent evaporation and equilibration of the surface film, protein monolayers were continuously compressed. To evaluate the spreading quantity of protein, each spreading experiment was repeated 5-10 times.
The surface pressure-area isotherms were plotted using nm^sup 2^/molecule calculated by using a molecular weight for SP-B of 17,380 (dimer, 158 amino acid residues) and 4200 for SP-C (dipalmitoylated form, 35 amino acid residues).
It is not possible to determine the point where a monolayer is compressed to its complete coverage without auxiliary experiments. This critical point corresponds to the minimal area demand per molecule or molecule cross section. During further compression of protein monolayer, the formation of bi- or multilayers begins. This point can be determined using dynamic monolayer cycling, i.e., repeated fast monolayer compression/dilatation. The compression of the monolayer to molecular areas smaller than the minimal area demand per molecule yields a gradual shift of the whole isotherm to the left, i.e., to smaller molecular areas.
Therefore, after [pi]-A isotherm measurements, dynamic cycling of the monolayer was carried out by compressing and expanding the bubble surface. For cycling experiments the bubble volume was continuously changed at 10 cycles per min.
RESULTS
Mass spectrometry
Molecular masses of SP-B and SP-C were determined using MALDI-TOF MS. The main peak of all SP-B samples was caused by the [M + H]^sup +^ ion of the dimeric protein (Table 2).
There were no crucial differences between SP-B-1, SP-B-2, and SP-B-3 concerning their mass spectra if the measurements were carried out using a protein concentration of 0.05-0.1 mg/ml. If the concentration of the protein increased to 0.8-1.0 mg/ml as for SP-B-3, the molar masses were consistent with dimeric and oligomeric forms of SP-B-3 (Bunger et al., 2001). Additionally, the HPLC chromatogram of SP-B-1 showed two peaks corresponding to the dimeric and oligomeric forms of protein, whereas only one peak corresponding to the oligomeric form of SP-B-2 and SP-B-3 was found (unpublished).
The differences between the theoretical molecular mass of ovine SP-B (17380) and the average masses of the major forms of SP-B-1 (17558), SP-B-2 (17424), and SP-B-3 (17423) are shown in Table 2. They are consistent with possible protein modification by the solvents (Taneva et al., 1998), i.e., butanol in the case of SP-B-1 and methanol in the case of SP-B-2 and SP-B-3.
Common mass spectra were obtained for SP-C samples (Fig. 2). The SP-C-1 showed a main component with an [M + H]^sup +^ ion at m/z 4217; SP-C-2 and SP-C-3, however, showed a main component with an [M + H]^sup +^ ion at m/z 4201, indicating that monomeric SP-C is predominantly dipalmitoylated (Table 2). The difference between the theoretical molecular mass of ovine SP-C (4201) and the average masses of the major forms of SP-C-1 (4217) is consistent with possible protein modification by the solvent used for the second step of the isolation procedure (methanol) or by oxidation of Met33 (Griffiths et al., 1998).
started to increase at a surface coverage of 23 nm^sup 2^/molecule. This particular point, the so-called liftoff point, was similar for all investigated SP-B samples. The isotherm for SP-B-1 had one plateau region between 20 and 25 mN/m. In contrast, the SP-B-2 isotherm had a plateau region at a much higher surface pressure, i.e., 42 to 46 mN/m, whereas SP-B-3 had a corresponding plateau at 44 to 50 mN/m. The starting point of plateaus for all SP-B samples corresponds to a critical area of 12.5-12.8 nm^sup 2^ per molecule. Molecular cross sections were almost identical for all three SP-B samples, i.e., ~6 nm^sup 2^/molecule or 0.038 nm^sup 2^/amino acid respectively.
The minimal area demands on molecule cross sections for all proteins were determined using dynamic cycling experiments. An example of the first and the tenth compression/dilatation cycle for monolayer of one of SP-C samples is given in Fig. 7.
The [pi]-A isotherms for monolayer compression of SP-C samples are shown in Fig. 8. The SP-C-1 and SP-C-2 isotherms consist of two separate curves (points and line) taken from two experiments, one of which was carried out in the lower surface pressure region and the other in the upper region. The surface pressure for each of the three SP-C samples lifted off at different molecular areas, which are given in Table 1. The SP-C-1 isotherm had at least three plateau regions, the first plateau at [pi]~15 mN/m, the second at [pi]~30 mN/m, and a third at [pi]~37-43 mN/m. Furthermore, there was a slightly pronounced inflection point at 25 mN/m. The SP-C-2 isotherm had a distinct plateau at 22-25 mN/m and a plateau at [pi]~37-4-3 mN/m. Both plateaus of the SP-C-2 isotherm had the appropriated pendant in the SP-C-1 curves. The isotherm of SP-C-3 had an absolutely unusual shape. The surface pressure increased very quickly during compression to ~60 mN/m after collapse of the monolayer. The starting points of plateaus are given in Table 1. The starting point of the last SP-C-1 and SP-C-2 plateau is a critical area of 2.3-2.4 nm^sup 2^ per molecule. The molecule cross sections of SP-C-1 and SP-C-2 were similar, ~1.15 nm^sup 2^/molecule or 0.034 nm^sup 2^/amino acid. The molecule cross section of SP-C3 was different: 1.05 nm^sup 2^/ molecule or 0.03 nm^sup 2^/amino acid. Fig. 9 represents the [pi]-A isotherms for monolayers of all proteins recalculated for area per one amino acid residue of each protein. The parameters which characterize surface behavior and which were drawn from the isotherms of SP-B and SP-C samples are summarized in Table 1.
DISCUSSION
In the present study we evaluate the conformation of native hydrophobic surfactant proteins SP-B and SP-C isolated from sheep lung washings by either butanol or chloroform/methanol lipid extraction using gel exclusion or high performance liquid chromatography. SDS gel electrophoresis data and the structural data obtained from CD and FTIR spectroscopy are compared with the interfacial behavior of the pure proteins.
From examination of SDS gel electrophoresis data under nonreducing conditions it appears that the SP-B-1 sample extracted using butanol had a higher molecular weight than porcine SP-B (Fig. 1 A), but a lower molecular weight than SP-B-2 and SP-B-3 samples extracted in chloroform/methanol. These data suggest that the SP-B-1 sample consists mostly of dimers and probably trimers, whereas the SP-B-2 and SP-B-3 samples are mostly trimers. The existence of dimer/oligomer mixture in the SP-B-1 sample was additionally confirmed by the appearance of two peaks in HPLC chromatograms. By contrast, the chromatograms of the other samples showed only one peak. Therefore, it was concluded that the use of butanol instead of chloroform/methanol may decrease the oligomerization of SP-B.
The appearance of SP-B trimers in our preparation is consistent with a report by Baatz et al. (2001), who described trimers of SP-B in bovine lung surfactant. Oligomerization of modified human SP-B (Cys48Ser) expressed in transgenic mice was studied by Zaltash et al. (2001), who found more noncovalent dimers of SP-B (Cys48Ser) if the polarity of the solvent was decreased and/or if the concentration of SP-B increased. In chloroform/methanol solutions multimeric forms of the protein were observed as well. These results are in full agreement with our own observations that changes in the protein purification procedure, such as protein/lipid concentration and/or solvent polarity, obviously change the oligomerization state of protein. The oligomerization state of SP-B might be important for in vivo function as suggested by Beck et al. (2000).
The secondary structure of SP-B and SP-C was evaluated using CD and FTIR spectra. The CD and FTIR spectra of all three SP-B samples were consistent with the secondary structure of [alpha]-helix, as were the CD and FTIR spectra of SP-C-1. The CD spectrum and FTIR data of SP-C-2 showed that this sample consists of a mixture of two protein conformations: [alpha]-helix and [beta]-sheet. In contrast, the CD and FTIR spectra of SP-C-3 strongly corresponded to secondary structure of antiparallel [beta]-sheet.
Comparing the purification protocols of SP-C-1 and SP-C-2, we assumed that the [alpha]-/[beta]-conversion of protein could be caused either by utilization of chloroform/methanol instead of butanol in the extraction step or by the utilization of HPLC instead of gel exclusion chromatography. Comparing the purification protocols of SP-C-2 and SP-C-3, it is essential to note that the both chloroform/methanol extracted SP-C samples differed only in their protein/lipid concentration after extraction (Table 1); all other parameters of the isolation procedure were identical. We concluded that the formation of [beta]-sheets in SP-C-3 sample occurred after resolving of protein/lipid extracts in an extremely small amount of solvent but before the separation of proteins from lipids. This [beta]-sheet formation probably took place by exceeding the summary critical micelle concentration of the protein/ lipid mixture in the organic solvent, and not as a result of separation.
The phospholipid concentration of the SP-C-2 containing extracts was 70 mg/ml (0.1 M, considering all lipids as DPPC) and the total protein concentrations was 1.5 mg/ml. The lipid concentration of SP-C-3 containing extract, however, was 330 mg/ml (0.45 M, considering all lipids as DPPC) and the total protein concentration was 6 mg/ml. For example, the critical micelle concentration of DPPC in methanol is 1 x 10^sup -2^ M (Smith and Tanford, 1972). It was also found (Datta et al., 1992) that DPPC exists as reverse micelles in chloroform solutions at concentrations beyond 6 x 10^sup 3^ M. This means that the driving force of [alpha]-/[beta]-conversion was definitively the extremely high concentration of protein/lipid extracts of the SP-C-3 sample.
Our assumption that the protein/lipid concentration influences the secondary structure of SP-C is supported by several other studies. Blondelle and co-workers (Blondelle et al., 1997) used different concentrations of monomeric sodium dodecyl sulfate (SDS) to mimic a lipid environment
[alpha]-helix oriented vertically to the interface plane in a highly compressed monolayer. The cross section of two palmitoyl groups is ~0.4 nm^sup 2^ , yielding a cross section for the apolar [alpha]-helix of ~0.75 nm^sup 2^. This size corresponds to the size of a single [alpha]-helix as deduced above for SP-B and reported for transmembrane helices (Bowie, 1997).
Finally, from the results described in this paper, we would like to comment on the biological consequences of extensive alpha-to-beta transformation in SP-C. Several human diseases of different etiology are characterized by the extracellular deposition of amyloidlike fibrils, a process that is initiated by the transition of [alpha]-helices into [beta]-sheets of amyloid-forming proteins (Guijarro et al., 1998; Baskakov et al., 2001). Our results suggest that [alpha]-helix to [beta]-sheet transition could originate as a simple consequence of increase of the summary protein/lipid concentration in the alveolar or cellular environment, similar to the [alpha]-helix [arrow right] [beta]-sheet conversion occurring in the SP-C-3 sample. The mechanism may be common in the development of several human diseases of different etiology characterized by the extracellular deposition of amyloid (Soto, 1999) and may influence lung diseases such as alveolar proteinosis, acute respiratory distress syndrome, and lung fibrosis.
The authors are grateful to L. Kaufner and H. Bunger for protein isolation and determination of protein concentration; G. Brezesinski for technical support at the CD experiments; U. Bentrup for kind assistance with the FTIR experiments; and I. Plasencia and A. Prieto, for technical support with PAGE and mass spectrometry analyses.
Financial support by the Deutsche Forschungsgemeinschaft (Grant Pi 165/ 7) and Spanish Direccion General de Investigacion Cientifica y Tecnica (BIO2000-0929) is gratefully acknowledged.
[Sidebar]
Biophysical Journal Volume 84 March 2003 1940-1949
[Reference]
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[Author Affiliation]
N. Wustneck,* R. Wustneck,[dagger] J. Perez-Gil,[double dagger] and U. Pison*
* Humboldt-Universitat Berlin, Charite Campus Virchow-Klinikum, Anaesthesiologie, Berlin, Germany;
[double dagger] Departamento Bioquimica y Biologia Molecular I, Facultad Biologia, Universidad Complutense, Madrid, Spain; and
[dagger] Universitat Potsdam, Institut fur Physik, Potsdam, Germany
[Author Affiliation]
Submitted June 21, 2002, and accepted for publication November 20, 2002.
Address reprint requests to N. Wustneck, Humboldt-Universitat Berlin, Charite Campus Virchow-Klinikum, Anaesthesiologie, Augustenburger Platz 1, D-13344 Berlin, Germany. E-mail: wustneck@charite.de.
(C) 2003 by the Biophysical Society
0006-3495/03/03/1940/10 $2.00
