INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pulmonary surfactant is a mixture of lipids and specific proteins that is secreted into the alveolar fluid by alveolar type II cells. Its main function is to reduce the surface tension at the air/liquid interface in the lung. This is achieved by forming a surface film that consists of a monolayer that is highly enriched in dipalmitoylphosphatidylcholine (DPPC) and bilayer lipid/protein structures closely attached to it. This surface film reduces the work of breathing and prevents alveolar collapse upon end expiration. The in vivo existence of such a film has recently been confirmed by electron microscopy (Schürch et al., 1995).

The highly hydrophobic surfactant-specific proteins B and C (SP-B and SP-C) are required for a rapid insertion of new material from the lipid bilayer structures to the monolayer upon inspiration. In addition to reducing the work required for breathing, this prevents serum components from entering the surface film. These serum components, of which albumin is the most abundant, disturb surfactant function, and an increase in these serum components in the bronchoalveolar lavage is often observed in several diseases related to surfactant dysfunction (Byrne et al., 1992; Hallman et al., 1989; Jorens et al., 1992; Matsumoto et al., 1992; Nakos et al., 1997). SP-B and SP-C are also likely involved in the DPPC enrichment of the monolayer (for a review see Pérez-Gil and Keough, 1998). A monolayer enrichment of DPPC is required to create stable surface films with near-zero surface tensions.

SP-B is a ~17 kDa homodimer. Secondary structure analysis and homology with related proteins, especially members of the saposin-like family (Patthy, 1991), predict an SP-B structure that contains four amphipathic -helices (Andersson et al., 1995). These helices are thought to interact with the surfactant lipids, with a specific interaction between anionic lipids and the positive charges of SP-B (Baatz et al., 1990; Longo et al., 1993). The structure of SP-C, a 4.2-kDa protein containing 35 amino acids, has been completely resolved in solution and micelles by NMR (Johansson et al., 1994). Its main characteristics are a polyvalyl -helix that can exactly span a lipid bilayer and a tail that contains two palmitoylated cysteines.

The importance of SP-B in surfactant is apparent from the lethal respiratory distress that is caused by SP-B deficiency in humans (Nogee et al., 1994) and in SP-B knock-out mice (Tokieda et al., 1997). A lowered or total lack of all surfactant components, due to immaturity of the lungs in premature infants or inflammation or trauma in adults, leads to respiratory distress syndrome (RDS). Administration of exogenous surfactant has been proven a successful strategy for treating the neonatal form of RDS, and improvements are observed in ARDS as well (for a review see Robertson and Halliday, 1998). However, human surfactant is scarce, and therefore replacement surfactants are derived from animals. The disadvantage of this is that immunological responses and viral infections of the treated patient might occur. A completely synthetic surfactant might overcome these problems, and a major research area has evolved in this direction.

The development of a purely synthetic surfactant requires a thorough understanding of the role of the protein and lipid components in pulmonary surfactant. Several peptides based on either SP-B or SP-C have been tested (Bruni et al., 1991, 1998; Johansson et al., 1995; Nilsson et al., 1998; Takei et al., 1996). SP-B_1-25 is a peptide based on the N-terminal segment of human SP-B (hSP-B) and has been shown to have an amphipathic helical structure (Gordon et al., 1996). It has a high affinity for phospholipid monolayers and increases the collapse pressure of palmitic acid (PA) monolayers (Lipp et al., 1996; Waring et al., 1989). The peptide also permits resistance to the inhibitory effect of plasma constituents that inhibit surfactant and partly restores lung compliance in two animal models (Bruni et al., 1998). All of these activities are known characteristics of native SP-B. To mimic the dimeric state of native SP-B we synthesized the new peptide dSP-B_1-25. The monomers of this dimer differ from the previously mentioned SP-B_1-25 in that a cysteine at position 11 was substituted by alanine, leaving only one cysteine in the peptide. A disulfide linkage between two cysteines of the monomers results in a dimer of the SP-B_1-25 peptide (Fig. 1). The activity of both peptides was tested in vitro, using the captive bubble surfactometer (CBS) and lipid mixing experiments. Both peptides show good activity in several aspects of surface film formation and dynamics, with a clear positive effect of the dimerization on the respreadability of lipids under dynamic conditions.

View larger version (15K): [in this window] [in a new window]   FIGURE 1   The sequence of SP-B1-25 and dSP-B1-25 in one-letter code.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG) were obtained from Avanti Polar Lipids (Alabaster, AL); 1-palmitoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (pyrene-PC) was from Molecular Probes; HEPES was from Life Technologies (Paisley, Scotland); EDTA, calcium chloride (CaCl₂), chloroform (CHCl₃), and methanol (MeOH) were from Baker Chemicals B.V. (Deventer, the Netherlands). Organic solvents were distilled before use. hSP-B and hSP-C were isolated from amniotic fluid, using protocols normally used for lung lavage, as described previously (Oosterlaken Dijksterhuis et al., 1991).

Peptide synthesis

Molecular design rational

View larger version (9K): [in this window] [in a new window]   FIGURE 2   The amino acid sequence and specific secondary structure of SP-B1-25 in surfactant lipid (Gordon et al., 2000; Protein Data Bank ID: 1DFW). Residue specific secondary assignments are as follows: h, helix; e, extended; s, bend; _, disordered.

Solid-phase peptide synthesis, purification, and characterization of SP-B_1-25 and dSP-B_1-25

Vesicle preparation

Small unilamellar vesicles (SUVs) for lipid mixing and captive bubble surfactometer experiments were prepared as follows. Lipids from stock solutions in CHCl₃/MeOH with or without peptides in CHCl₃/MeOH were dried under a continuous stream of nitrogen at room temperature. The resulting lipid/peptide film was rehydrated by adding the appropriate buffer, vortexing, and incubating at 55°C for 15 min. The multilamellar vesicles thus formed were sonicated for 2 × 1 min at 55°C with a 10-s interval. The vesicles were cooled to 37°C and used immediately.

Lipid mixing

Lipid mixing experiments were performed as described by Oosterlaken-Dijksterhuis (Oosterlaken-Dijksterhuis et al., 1992). Phospholipid SUVs (300 nmol of lipid) with or without peptides were mixed with pyrene-PC-labeled SUVs (15 nmol of lipid containing 10% pyrene-PC) in a HEPES buffer (25 mM, pH 7.0) supplemented with 0.2 mM EGTA at a final volume of 2 ml. A standard concentration of 0.4 mol% was used for the two peptides and hSP-B, and an additional experiment with a twofold concentration for SP-B_1-25 was performed. Fluorescence emission spectra were recorded before and after the addition of 30 µl 0.3M CaCl₂ on a Perkin-Elmer LS 50 fluorescence spectrophotometer. The excitation wavelength was 343 nm, and the emission between 360 and 550 nm was recorded. The excimer/monomer (E/M) ratio, indicative of the amount of lipid mixing, was calculated by dividing the intensity at 475 nm (excimer fluorescence) by the intensity at 377 nm (monomer fluorescence).

Captive bubble surfactometry

The activity of the synthetic SP-B analogs in inserting lipids into the air/liquid interface was determined using a pressure-driven captive bubble surfactometer (Putz et al., 1994). A bubble (0.5 cm²) was formed in subphase buffer (140 mM NaCl, 10 mM HEPES, 0.5 mM EDTA, 2.5 mM CaCl₂, pH 6.9) by injecting air (28.5 µl) into the sample chamber at 1.0 bar and 37°C. Two methods of surface film formation were used, i.e., film spreading and adsorption from the subphase.

Film spreading

Film adsorption

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lipid mixing

The peptides SP-B_1-25 and dSP-B_1-25 were tested for their activity to induce lipid mixing. When donor vesicles containing pyrene-PC are mixed with acceptor vesicles (initiated by the addition of calcium ions), the fluorescent lipid probe becomes diluted, leading to more monomeric fluorescence and less excimeric fluorescence. The results are depicted in Fig. 3; they show that both SP-B-based peptides have an activity comparable to that of hSP-B. SP-B_1-25 has a slightly lower activity based on molar concentrations, but doubling the amount of SP-B_1-25 (to achieve the same amount of protein by weight compared to dSP-B_1-25) diminishes this difference.

View larger version (18K): [in this window] [in a new window]   FIGURE 3   Effect of SP-B analogs on lipid mixing. Black bars represent the E/M ratio before and white bars the E/M ratio after calcium addition. 2× SP-B1-25 has a twofold increased peptide concentration (0.8%) compared to dSP-B1-25 and hSP-B. Values shown are the average of at least four separate experiments ± SEM.

Captive bubble surfactometry

Adsorption

Cycling of adsorbed films
DPPC:POPG vesicles

View larger version (19K): [in this window] [in a new window]   FIGURE 4   Maximum and minimum surface tensions during cycling of adsorbed films from DPPC:POPG (8:2) SUV. Two hundred micrograms of SUVs was injected into the subphase and allowed to adsorb to the interface for 15 min. Then cycling was performed by varying the pressure between 0.5 and 2.8 bar. The values shown are the average of at least three separate experiments ± SEM.

DPPC:POPG:PA vesicles

View larger version (21K): [in this window] [in a new window]   FIGURE 5   Maximum and minimum surface tensions during cycling of adsorbed films from DPPC:POPG:PA (7:2:1) SUV. Two hundred micrograms of SUVs was injected into the subphase and allowed to adsorb to the interface for 15 min. Then cycling was performed by varying the pressure between 0.5 and 2.8 bar. The values shown are the average of at least three separate experiments ± SEM.

Spread films

View larger version (17K): [in this window] [in a new window]   FIGURE 6   Maximum and minimum surface tensions during cycling of spread films of DPPC:POPG (8:2). Peptide/lipid mixtures were dissolved in CHCl3/MeOH. Lipids (0.25 nmol) in 0.05 µl were spread at the interface. The subphase was washed, and vesicles without peptides were injected into the subphase. Then cycling was performed by varying the pressure between 0.5 and 2.8 bar. The values shown are the average of at least three separate experiments ± SEM.

Refinement of the surface film

View larger version (18K): [in this window] [in a new window]   FIGURE 7   Representative surface tension-area isotherms of adsorbed films of 1% dSP-B1-25 (A) and 0.02% hSP-B (B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this paper the in vitro surface activity of two synthetic SP-B analogs, based on the N-terminal segment of human SP-B, was tested. The monomeric SP-B_1-25 shows a clear activity compared to nonprotein samples. dSP-B_1-25, the dimeric peptide, shows an even better activity in some aspects of surface film dynamics, especially respreadability of lipids, which resembles the native protein activity.

The lipid mixing activities of dSP-B_1-25 and SP-B_1-25 were found to be similar to native SP-B (Fig. 3). The fusogenic properties are thought to be important in surfactant function in several aspects. They likely play a role in forming contact points between the lipid monolayer at the air/liquid interface and the bilayer structures that are attached to it. This enables the lung to insert new material into the interface when the surface is enlarged (during inspiration) to maintain a relatively low surface tension. Fusogenic events are also thought to be important in the formation of tubular myelin, which contains highly curved structures at the edges of the tubules where lipid bilayers merge.

The adsorption of lipids to a clean air/liquid interface from vesicles in the subphase was clearly stimulated by the presence of SP-B_1-25 or dSP-B_1-25. However, the values obtained were, in all of the experiments performed, ~15 mN/m or more above the observed equilibrium surface tension of 23 mN/m (Table 1). This indicates that the peptides cannot penetrate an existing, relatively tightly packed monolayer with a surface tension of 35 mN/m and fuse with it. These results are in line with results that were obtained for SP-B_1-25 with a Langmuir-Wilhelmy balance (Bruni et al., 1991). In those experiments it was observed that if lipid films were spread to an initial surface pressure of ~42 mN/m (surface tension of 30 mN/m), no insertion of peptide could occur. However, in our experiments it was observed that "extra" adsorption can occur despite the existing monolayer after the first cycling series. Somehow, because of the cycling procedure, new material from peptide-containing vesicles was now capable of inserting into the interface. A possible explanation for this phenomenon is that cycling has induced a lipid reservoir, probably consisting of lipid bilayer structures in close contact with the monolayer at the interface. The contact points between the monolayer and the reservoir would then allow an easy adsorption of new lipids into the interface, despite the relatively low surface tension of ~40 mN/m, which prevents adsorption from "normal" subphase vesicles.

The cycling experiments with spread films show that dSP-B_1-25 films but not SP-B_1-25 give rise to stable, near-zero _min during the whole cycling procedure. This shows that the material that is squeezed out during compression stays attached to the monolayer and is directly available for reinsertion upon the next expansion. Insertion of new lipids from the subphase vesicles occurs at a much slower rate and will therefore only play a minor role during the dynamic cycling. The easiest explanation for the observed difference is that dSP-B_1-25 can form a bridge between the monolayer and the squeezed-out subphase lipids by attaching one monomer part to each membrane structure. SP-B_1-25 does not offer this possibility, and loss of material is observed through rising _max and _min values upon cycling. The major role of the dimerization of native SP-B is thought to be this bridging function. The results obtained in this study strongly support this theory.

Recently, the requirement of SP-B dimerization for optimal activity was demonstrated in a mouse animal model (Beck et al., 2000). These mice expressed a monomeric SP-B mutant in an SP-B / background. Large aggregate surfactant from bronchoalveolar lavage and purified SP-B monomer or dimer, reconstituted with lipids, was tested and revealed slower adsorption kinetics and higher _min values during cycling experiments. Although we did not find improved adsorption for dSP-B_1-25 compared to SP-B_1-25, these results strongly support our findings.

Contrary to the results presented here, the monomeric SP-B_1-25 has been shown in monolayer studies to be able to create a reservoir of squeezed-out lipids that can reenter upon expansion (Lipp et al., 1997, 1998). This discrepancy is probably due to the dynamic system that was used in the current experiments. The differences observed for the protein-free sample with former captive bubble studies (Putz et al., 1999; Veldhuizen et al., 1999) in which the protein-free sample also creates low _min values during the whole cycling procedure are probably due to the lower lipid subphase concentration in our current experiments. Similar rising _min values are observed when no lipid vesicles are injected into the subphase (unpublished results).

A dramatic effect of PA on the peptides' activity was observed (Fig. 4 and 5). In contrast to the results with DPPC:POPG vesicles, near-zero surface tensions upon the first cycle were observed when PA was included in the vesicles. In other words, PA somehow enhances the creation of a DPPC-enriched monolayer upon compression. We propose that the presence of PA induces a specific squeeze-out of non-DPPC lipids during compression. DPPC-poor domains within the monolayer could be more easily formed, which are then squeezed out upon compression of the bubble, leading to an almost pure DPPC monolayer at the interface. Following this theory, the adsorbed films from DPPC:POPG vesicles would contain enough DPPC to reach very low surface tensions, but fail to do so because DPPC is squeezed out together with POPG when the bubble is compressed. Interestingly, the advantageous effect of PA was not observed when hSP-B was used in adsorbed films (results not shown). The effect could therefore be specific to the combination with the peptides, or the advantageous effect of PA could be more subtle with hSP-B and only show up at lower concentrations of SP-B or in a different experimental setup.

The surface tension-area isotherms show that upon cycling increasingly less surface area reduction is needed to reach a very low surface tension (Fig. 7). This reflects an enrichment in DPPC of the monolayer. The area reduction required to reach very low surface tensions decreased significantly for both peptides upon cycling. A pure DPPC monolayer requires 15% area reduction to reach this value from equilibrium surface tension (Schürch et al., 1989, 1992), which is close to the value obtained for the SP-B_1-25 and dSP-B_1-25 films after a few cycles. The hSP-B sample showed a smaller effect in these decreasing values but started already with a relatively small required area reduction upon the first cycle. This implies that the monolayer was already enriched in DPPC during the initial adsorption period. This has been observed before, using the natural surfactant BLES (Schürch et al., 1992). In these experiments a fast adsorption of the material was always accompanied by lower area reductions needed to reach near-zero surface tensions. In other words, a fast adsorption results in a specific DPPC enrichment of the monolayer. The adsorption of SP-B_1-25 and dSP-B_1-25 in our experiments was slow (and that of hSP-B fast), and following this line of thought, no specific DPPC adsorption has occurred. This indicates that, with the synthetic peptides, the surface film is mostly enriched in DPPC because of selective squeeze-out of the non-DPPC lipids.

In summary, two peptides based on the N-terminal segment of SP-B have been tested. Both peptides contain many characteristics of the native protein in lipid mixing and captive bubble surfactometer experiments. The dimeric peptide has a clearly increased activity compared to the monomeric peptide in reservoir formation upon cycling. We presume that dimerization is required to create a lipid reservoir in close contact with the lipid monolayer at the interface and that dimerization of native SP-B has a similar function.