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

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

Analysis of the CD2 and spliceosomal Sm B/B' polyproline-arginine motifs defined by a monoclonal antibody using a phage-displayed random peptide library

The cytoplasmic region of the CD2 receptor of lymphocytes contains proline-rich motifs, which are involved in T cell activation and interleukin-2 production. An intracellular CD2 binding protein, CD2BP2, interacts with two tandem PPPPGHR segments of the CD2 tail. CD2BP2 contains a GYF (glycine-tyrosine-phenylalanine) domain that confers binding to these proline-rich sequences. Monoclonal antibody 3E10 that was previously raised against a peptide containing the CD2 PPPPGHR segment reacts with the native CD2 molecule and spliceosomal Sm B/B' proteins. To identify the exact epitope on the CD2 peptide recognized by 3E10, a phage-displayed combinatorial peptide library was used. Analysis of the selected clones revealed that the mAb 3E10 binds preferentially to the motif PxxPPGxR. Experiments using amino acid substitutions with synthetic peptides confirmed the reactivity of mAb 3E10 with this motif. In addition, we show that several similarities exist between this motif and the CD2BP2-GFY recognition motif PPGxR/K. Binding of antibody 3E10 indicates some degree of degeneracy, which is consistent with its ability to recognize structurally related polyproline-arginine motifs found in intracellular proteins including Sm B/B' proteins and other RNA binding proteins. Thus, mAb 3E10 can be used to specifically identify a sub-class of proline-rich motifs, and as such can be used to study the potential role of these proline-rich sequences in mediating protein-protein interactions.     

Orientation and depth of surfactant protein B C-terminal helix in lung surfactant bilayers

SP-B(CTERM) is a cationic amphipathic helical peptide and functional fragment composed of residues 63 to 78 of surfactant protein B (SP-B). Static oriented and magic angle spinning solid state NMR, along with molecular dynamics simulation was used to investigate its structure, orientation, and depth in lipid bilayers of several compositions, namely POPC, DPPC, DPPC/POPC/POPG, and bovine lung surfactant extract (BLES). In all lipid environments the peptide was oriented parallel to the membrane surface. While maintaining this approximately planar orientation, SP-B(CTERM) exhibited a flexible topology controlled by subtle variations in lipid composition. SP-B(CTERM)-induced lipid realignment and/or conformational changes at the level of the head group were observed using (31)P solid-state NMR spectroscopy. Measurements of the depth of SP-B(CTERM) indicated the peptide center positions ~8? more deeply than the phosphate headgroups, a topology that may allow the peptide to promote functional lipid structures without causing micellization upon compression.     

Conformation and molecular topography of the N-terminal segment of surfactant protein B in structure-promoting environments.

Although the effects of surfactant protein B (SP-B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure-activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N-terminal SP-B peptides in lipids and structure-promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP-B1-25 (residues 1-25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP-B1-25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP-B1-25 sequence. Residues Cys-8-Gly-25 are largely alpha-helix in methanol, whereas the N-terminal segment Phe-1-Cys-8 had turn and helical propensities. Addition of SP-B1-25 spin-labeled at the N-terminal Phe (i.e., SP-B1-25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP-B1-25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP-B1-25 argues that the reporter on Phe-1 resides in the headgroup of the lipid dispersions. The blue-shift in the endogenous fluorescence of Trp-9 near the N-terminus of SP-B1-25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP-B1-25 interacting with lipids.     

Structure of mini-B, a functional fragment of surfactant protein B, in detergent micelles

Surfactant protein B (SP-B) is essential for normal lung surfactant function, which is in itself essential to life. However, the molecular basis for SP-B's activity is not understood and a high-resolution structure for SP-B has not been determined. Mini-B is a 34-residue peptide with internal disulfide linkages that is composed of the N- and C-terminal helical regions of SP-B. It has been shown to retain similar activity to full-length SP-B in certain in vitro and in vivo studies. We have used solution NMR to determine the structure of Mini-B in the presence of micelles composed of the anionic detergent sodium dodecyl sulfate (SDS). Under these conditions, Mini-B forms two alpha-helices connected by an unstructured loop. Mini-B possesses a strikingly amphipathic surface with a large positively charged patch on one face of the peptide and a large hydrophobic patch on the opposite face. A tryptophan side chain extends outward from the peptide in a position to interact with lipids at the polar/apolar interface. Interhelix interactions are stabilized by both disulfide bonds and by interleaving of hydrophobic side chains from the two helices.     

Dimeric N-terminal segment of human surfactant protein B (dSP-B(1-25)) has enhanced surface properties compared to monomeric SP-B(1-25)

Surfactant protein B (SP-B) is a 17-kDa dimeric protein produced by alveolar type II cells. Its main function is to lower the surface tension by inserting lipids into the air/liquid interface of the lung. SP-B's function can be mimicked by a 25-amino acid peptide, SP-B(1-25), which is based on the N-terminal sequence of SP-B. We synthesized a dimeric version of this peptide, dSP-B(1-25), and the two peptides were tested for their surface activity. Both SP-B(1-25) and dSP-B(1-25) showed good lipid mixing and adsorption activities. The dimeric peptide showed activity comparable to that of native SP-B in the pressure-driven captive bubble surfactometer. Spread surface films led to stable near-zero minimum surface tensions during cycling while protein free, and films containing SP-B(1-25) lost material from the interface during compression. We propose that dimerization of the peptide is required to create a lipid reservoir attached to the monolayer from which new material can enter the surface film upon expansion of the air/liquid interface. The dimeric state of SP-B can fulfill the same function in vivo.     

Structure and properties of phospholipid-peptide monolayers containing monomeric SP-B(1-25) II. Peptide conformation by infrared spectroscopy

The conformation and orientation of synthetic monomeric human sequence SP-B(1-25) (mSP-B(1-25)) was studied in films with phospholipids at the air-water (A/W) interface by polarization modulation infrared reflectance absorption spectroscopy (PM-IRRAS). Modified two-dimensional infrared (2D IR) correlation analysis was applied to PM-IRRAS spectra to define changes in the secondary structure and rates of reorientation of mSP-B(1-25) in the monolayer during compression. PM-IRRAS spectra and 2D IR correlation analysis showed that, in pure films, mSP-B(1-25) had a major alpha-helical conformation plus regions of beta-sheet structure. These alpha-helical regions reoriented later during film compression than beta structural regions, and became oriented normal to the A/W interface as surface pressure increased. In mixed films with 4:1 mol:mol acyl chain perdeuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPPC-d(62):DOPG), the IR spectra of mSP-B(1-25) showed that a significant, concentration-dependent conformational change occurred when mSP-B(1-25) was incorporated into a DPPC-d(62):DOPG monolayer. At an mSP-B(1-25) concentration of 10 wt.%, the peptide assumed a predominantly beta-sheet conformation with no contribution from alpha-helical structures. At lower, more physiological peptide concentrations, 2D IR correlation analysis showed that the propensity of mSP-B(1-25) to form alpha-helical structures was increased. In phospholipid films containing 5 wt.% mSP-B(1-25), a substantial alpha-helical peptide structural component was observed, but regions of alpha and beta structure reoriented together rather than independently during compression. In films containing 1 wt.% mSP-B(1-25), peptide conformation was predominantly alpha-helical and the helical regions reoriented later during compression than the remaining beta structural components. The increased alpha-helical structure of mSP-B(1-25) demonstrated here by PM-IRRAS and 2D IR correlation analysis in monolayers of 4:1 DPPC:DOPG containing 1 wt.% (and, to a lesser extent, 5 wt.%) peptide may be relevant for the formation of the intermediate order 'dendritic' surface phase observed in similar surface films by epi-fluorescence.     

Location of structural transitions in an isotopically labeled lung surfactant SP-B peptide by IRRAS

Pulmonary surfactant, a lipid/protein complex that lines the air/water interface in the mammalian lung, functions to reduce the work of breathing. Surfactant protein B (SP-B) is a small, hydrophobic protein that is an essential component of this mixture. Structure-function relationships of SP-B are currently under investigation as the protein and its peptide analogs are being incorporated into surfactant replacement therapies. Knowledge of the structure of SP-B and its related peptides in bulk and monolayer phases will facilitate the design of later generation therapeutic agents. Prior infrared reflection-absorption spectroscopic studies reported notable, reversible surface pressure-induced antiparallel beta-sheet formation in a synthetic peptide derived from human SP-B, residues 9-36 (SP-B(9-36)). In the current work, infrared reflection-absorption spectroscopy is applied in conjunction with isotopic labeling to detect the site and pressure dependence of the conformational change. SP-B(9-36), synthesized with (13)C=O-labeled Ala residues in positions 26, 28, 30, and 32, shifted the beta-sheet marker band to approximately 1600 cm(-1) and thus immediately identified this structural element within the labeled region. Surface pressure-induced alterations in the relative intensities of Amide I band constituents are interpreted using a semiempirical transition dipole coupling model. In addition, electron micrographs reveal the formation of tubular myelin structures from in vitro preparations using SP-B(9-36) in place of porcine SP-B indicating that the peptide has the potential to mimic this property of the native protein.     

Membrane activity of (Cys48Ser) lung surfactant protein B increases with dimerisation

One of the possible functions of lung surfactant protein B (SP-B), an hydrophobic membrane-associated saposin-like protein, is to reduce the alveolar surface tension by promoting insertion of phospholipids into the air/liquid interface of the lung. SP-B is a covalent homodimer; Cys48 of two polypeptides form an intermolecular disulphide bond. In order to test whether dimerisation of SP-B is important for surfactant function, transgenic mice which express (Cys48Ser) human SP-B in a mouse SP-B null background were generated. In previous studies (Cys48Ser)SP-B showed a concentration-dependent in vitro activity, suggesting that it may form non-covalent dimers. Here (Cys48Ser)SP-B isolated from bronchoalveolar lavage of transgenic mice was studied at different concentrations by circular dichroism (CD) spectroscopy, pulsating bubble surfactometry, mass spectrometry and reversed-phase HPLC. The results indicate that (Cys48Ser)SP-B, both in a phospholipid environment and in organic solvents, is largely monomeric and exhibits low activity at concentrations lower than 1 -2 microM, while at higher concentrations it forms non-covalent dimers, which are nearly functionally equivalent to native SP-B in vitro. Furthermore, electrospray mass spectrometry showed that more dimers were found relative to the monomer when the polarity of the solvent was decreased, and when the concentration of SP-B increased. (Cys48Ser)SP-B also eluted earlier than native SP-B in reversed-phase HPLC. Taken together, these results indicate that a polar surface is buried upon dimerisation, thereby promoting formation of interchain ion pairs between Glu51-Arg52' and Glu51'-Arg52.     

Effect of hydrophobic surfactant proteins SP-B and SP-C on binary phospholipid monolayers: II. Infrared external reflectance-absorption spectroscopy

In situ external reflection infrared spectroscopy at the air-water interface was used to study the influence on phospholipid structure of an endogenous mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, which are thought to play pivotal roles in the adsorption and function of pulmonary surfactant. Mixtures studied were 1:1, 2:1, and 7:1 (mol:mol) DPPC-d(62):DPPG, and 7:1 DPPC-d(62):DOPG, alone and in the presence of 0.5-10 wt % mixed SP-B/C purified chromatographically from calf lung surfactant extract. Perdeuteration of DPPC produced a shift in vibrational frequencies so that it could be differentiated spectroscopically from the phosphoglycerol component in the surface monolayer. CH(2) antisymmetric and symmetric stretching bands ( approximately 2920 and 2852 cm(-1)) along with the analogous CD(2) stretching bands ( approximately 2194 and 2089 cm(-1)) were analyzed, and band heights and peak wavenumber positions were assessed as a function of monolayer surface pressure. Small, near-physiological contents of 1-2 wt % SP-B/C typically produced the maximum observed spectroscopic effects, which were abolished at high protein contents of 10 wt %. Analysis of CH(2) and CD(2) stretching bands and C-H/C-D band height ratios indicated that SP-B/C affected PC and PG lipids differently within the surface monolayer. SP-B/C had preferential interactions with DPPG in 1:1, 2:1, and 7:1 DPPC-d(62):DPPG films that increased its acyl chain order. SP-B/C also interacted specifically with DOPG in 7:1 DPPC-d(62):DOPG monolayers, but in this case an increase in CH(2) band heights and peak wavenumber positions indicated a further disordering of the already fluid DOPG acyl chains. CD(2) band height and peak wavenumber analysis indicated that SP-B/C had no significant effect on the structure of DPPC-d(62) chains in 7:1 films with DPPG or DOPG, and had only a slight tendency to increase the acyl chain order in 1:1 films of DPPC-d(62):DPPG. SP-B/C had no significant effect on DPPC-d(62) structure in films with DOPG. Infrared results also indicated that interactions involving SP-B/C and lipids led to exclusion of PC and PG lipids from the compressed interfacial monolayer, in agreement with our previous report on the phase morphology of lipid monolayers containing 1 wt % SP-B/C.     

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

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

Surfactant peptides stimulate uptake of phosphatidylcholine by isolated cells

To determine whether small hydrophobic surfactant peptides (SP-B and SP-C) participate in recycling of pulmonary surfactant phospholipid, we determined the effect of these peptides on transfer of 3H- or 14C-labelled phosphatidylcholine from liposomes to isolated rat alveolar Type II cells and Chinese hamster lung fibroblasts. Both natural and synthetic SP-B and SP-C markedly stimulated phosphatidylcholine transfer to alveolar Type II cells and Chinese hamster lung fibroblasts in a dose- and time-dependent fashion. Effects of the peptides on phospholipid uptake were dose-dependent, but not saturable and occurred at both 4 and 37 degrees C. Uptake of labelled phospholipid into a lamellar body fraction prepared from Type II cells was augmented in the presence of SP-B. Neither SP-B nor SP-C augmented exchange of labelled plasma membrane phosphatidylcholine from isolated Type II cells or enhanced the release of surfactant phospholipid when compared to liposomes without SP-B or SP-C. Addition of native bovine SP-B and SP-C to the phospholipid vesicles perturbed the size and structure of the vesicles as determined by electron microscopy. To determine the structural elements responsible for the effect of the peptides on phospholipid uptake, fragments of SP-B were synthesized by solid-phase protein synthesis and their effects on phospholipid uptake assessed in Type II epithelial cells. SP-B (1-60) stimulated phospholipid uptake 7-fold. A smaller fragment of SP-B (15-60) was less active and the SP-B peptide (40-60) failed to augment phospholipid uptake significantly. Like SP-B and SP-C, surfactant-associated protein (SP-A) enhanced phospholipid uptake by Type II cells. However, SP-A failed to significantly stimulate phosphatidylcholine uptake by Chinese hamster lung fibroblasts. These studies demonstrate the independent activity of surfactant proteins SP-B and SP-C on the uptake of phospholipid by Type II epithelial cells and Chinese hamster lung fibroblasts in vitro.     

Antimicrobial activity of native and synthetic surfactant protein B peptides

Surfactant protein B (SP-B) is secreted into the airspaces with surfactant phospholipids where it reduces surface tension and prevents alveolar collapse at end expiration. SP-B is a member of the saposin-like family of proteins, several of which have antimicrobial properties. SP-B lyses negatively charged liposomes and was previously reported to inhibit the growth of Escherichia coli in vitro; however, a separate study indicated that elevated levels of SP-B in the airspaces of transgenic mice did not confer resistance to infection. The goal of this study was to assess the antimicrobial properties of native SP-B and synthetic peptides derived from the native peptide. Native SP-B aggregated and killed clinical isolates of Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and group B streptococcus by increasing membrane permeability; however, SP-B also lysed RBC, indicating that the membranolytic activity was not selective for bacteria. Both the antimicrobial and hemolytic activities of native SP-B were inhibited by surfactant phospholipids, suggesting that endogenous SP-B may not play a significant role in alveolar host defense. Synthetic peptides derived from native SP-B were effective at killing both Gram-positive and Gram-negative bacteria at low peptide concentrations (0.15-5.0 microM). The SP-B derivatives selectively lysed bacterial membranes and were more resistant to inhibition by phospholipids; furthermore, helix 1 (residues 7-22) retained significant antimicrobial activity in the presence of native surfactant. These results suggest that the role of endogenous SP-B in host defense may be limited; however, synthetic peptides derived from SP-B may be useful in the treatment of bacterial pneumonias.     

Secondary structure and orientation of the surfactant protein SP-B in a lipid environment. A Fourier transform infrared spectroscopy study.

Attenuated total reflection Fourier transform infrared spectroscopy was used to investigate the secondary structure of the surfactant protein SP-B. Nearly half of the polypeptide chain is folded in an alpha-helical conformation. No significant change of the secondary structure content was observed when the protein is associated to a lipid bilayer of dipalmitoylphosphatidylcholine (DPPC)/phosphatidylglycerol (PG) or of dipalmitoylphosphatidylglycerol (DPPG). The parameters related to the gamma w(CH2) vibration of the saturated acyl chains reveal no modification of the conformation or orientation of the lipids in the presence of SP-B. A model of orientation of the protein at the lipid/water interface is proposed. In this model, electrostatic interactions between charged residues of SP-B and polar headgroups of PG, and the presence of small hydrophobic alpha-helical peptide stretches slightly inside the bilayers, would maintain SP-B at the membrane surface.     

Mapping and analysis of the lytic and fusogenic domains of surfactant protein B

Surfactant protein B (SP-B) is a hydrophobic, 79 amino acid peptide that regulates the structure and function of surfactant phospholipid membranes in the airspaces of the lung. Addition of SP-B to liposomes composed of DPPC/PG (7:3) leads to membrane binding, destabilization, and fusion, ultimately resulting in rearrangement of membrane structure. The goal of this study was to map the fusogenic and lytic domains of SP-B and assess the effects of altered fusion and lysis on surface activity. Synthetic peptides were generated to predicted helices and/or interhelical loops of SP-B and tested for fusion, lytic, and surface activities. The N-terminal half of SP-B (residues 1-37), which includes the nonhelical N-terminal amino acids in addition to helices 1 and 2, promoted rapid liposome fusion whereas shorter peptides were significantly less effective. The requirements for optimal surface tension reduction were similar to those for fusion; in contrast, helix 1 (residues 7-22) alone was sufficient for liposome lysis. The C-terminal half of SP-B (residues 43-79), which includes helices 3, 4, and 5, exhibited significantly lower levels of fusogenic, lytic, and surface tension reducing activities compared to the N-terminal region. These results indicate that SP-B fusion, lytic and surface activities map predominantly to the N-terminal half of SP-B. Amino acid substitutions in synthetic peptides corresponding to the N-terminal half of SP-B indicated that, in general, decreased fusion or lytic activities were associated with altered surface tension reducing properties of the peptide. However, the presence of fusion and lytic activities alone could not account for the surface tension reducing property of SP-B. We propose a model in which association of helix 1 with lipids leads to membrane permeabilization but not aggregation; helix 2 mediates membrane cross-linking (aggregation), which, in turn, facilitates lipid mixing, membrane fusion, and interfacial adsorption/surface tension reduction.     

The surfactant peptide KL4 in lipid monolayers: phase behavior, topography, and chemical distribution

Studies of different fragments and mutants of SP-B suggest that the function related structural and compositional characteristics in SP-B are its positive charges with intermittent hydrophobic domains. KL4 ([lysine-(leucine)4]4-lysine) is a synthetic peptide based on SP-B structure and is the major constituent of Surfaxin, a potential therapeutic agent for respiratory distress syndrome in premature infants. There is, however, no clear understanding about the possible lipid-KL4 interactions behind its function, which is an inevitable knowledge to design improved therapeutic agents. To examine the phase behavior, topography, and lipid specificity of KL4/lipid systems, we aimed to study different surfactant model systems containing KL4, neutral dipalmitoylphosphatidylcholine (DPPC) and/or negatively charged dipalmitoylphosphatidylglycerol (DPPG) in the presence of Ca2+ ions. Surface pressure-area isotherms, fluorescence microscopic images, scanning force microscopy as well as time-of-flight secondary ion mass spectrometry suggest (i) that KL4 is not miscible with DPPC and therefore forms peptide aggregates in DPPC/KL4 mixtures; (ii) that KL4 specifically interacts with DPPG via electrostatic interactions and induces percolation of DPPG-rich phases; (iii) that existing DPPG-Ca2+ interactions are too strong to be overcome by KL4, the reason why the peptide remains excluded from condensed DPPG domains and passively colocalizes with DPPC in a demixed fluid phase; and (iv) that the presence of negatively charged lipid is necessary for the formation of bilayer protrusions. These results indicate that the capability of the peptide to induce the formation of a defined surface-confined reservoir depends on the lipid environment, especially on the presence of anionic lipids.     

Processing of pulmonary surfactant protein B by napsin and cathepsin H

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2-terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2-terminal propeptide between Leu178 and Pro179, 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.     

NMR structures of the C-terminal segment of surfactant protein B in detergent micelles and hexafluoro-2-propanol

Although the membrane-associated surfactant protein B (SP-B) is an essential component of lung surfactant, which is itself essential for life, the molecular basis for its activity is not understood. SP-B's biophysical functions can be partially mimicked by subfragments of the protein, including the C-terminus. We have used NMR to determine the structure of a C-terminal fragment of human SP-B that includes residues 63-78. Structure determination was performed both in the fluorinated alcohol hexafluoro-2-propanol (HFIP) and in sodium dodecyl sulfate (SDS) micelles. In both solvents, residues 68-78 take on an amphipathic helical structure, in agreement with predictions made by comparison to homologous saposin family proteins. In HFIP, the five N-terminal residues of the peptide are largely unstructured, while in SDS micelles, these residues take on a well-defined compact conformation. Differences in helical residue side chain positioning between the two solvents were also found, with better agreement between the structures for the hydrophobic face than the hydrophilic face. A paramagnetic probe was used to investigate the position of the peptide within the SDS micelles and indicated that the peptide is located at the water interface with the hydrophobic face of the helix oriented inward, the hydrophilic face of the helix oriented outward, and the N-terminal residues even farther from the micelle center than those on the hydrophilic face of the alpha-helix. Interactions of basic residues of SP-B with anionic lipid headgroups are known to have an impact on function, and these studies demonstrate structural ramifications of such interactions via the differences observed between the peptide structures determined in HFIP and SDS.     

Content-dependent activity of lung surfactant protein B in mixtures with lipids

The content-dependent activity of surfactant protein (SP)-B was studied in mixtures with dipalmitoyl phosphatidylcholine (DPPC), synthetic lipids (SL), and purified phospholipids (PPL) from calf lung surfactant extract (CLSE). At fixed SP-B content, adsorption and dynamic surface tension lowering were ordered as PPL/SP-B approximately SL/SP-B > DPPC/SP-B. All mixtures were similar in having increased surface activity as SP-B content was incrementally raised from 0.05 to 0.75% by weight. SP-B had small but measurable effects on interfacial properties even at very low levels < or =0.1% by weight. PPL/SP-B (0.75%) had the highest adsorption and dynamic surface activity, approaching the behavior of CLSE. All mixtures containing 0.75% SP-B reached minimum surface tensions <1 mN/m in pulsating bubble studies at low phospholipid concentration (1 mg/ml). Mixtures of PPL or SL with SP-B (0.5%) also had minimum surface tensions <1 mN/m at 1 mg/ml, whereas DPPC/SP-B (0.5%) reached <1 mN/m at 2.5 mg/ml. Physiological activity also was strongly dependent on SP-B content. The ability of instilled SL/SP-B mixtures to improve surfactant-deficient pressure-volume mechanics in excised lavaged rat lungs increased as SP-B content was raised from 0.1 to 0.75% by weight. This study emphasizes the crucial functional activity of SP-B in lung surfactants. Significant differences in SP-B content between exogenous surfactants used to treat respiratory disease could be associated with substantial activity variations.