Nanostructure Changes in Lung Surfactant Monolayers Induced by Interactions between Palmitoyloleoylphosphatidylglycerol and Surfactant Protein B

Abstract

Developing synthetic lung surfactants to replace animal extracts requires a fundamental understanding of the roles of the various lipids and proteins in native lung surfactant. We used Brewster angle microscopy (BAM), atomic force microscopy (AFM), and Langmuir isotherms to study the influence of palmitoyloleoylphosphatidylglycerol (POPG) in monolayers of dipalmitoylphosphatidylcholine and palmitic acid mixtures with or without dSP-B1-25, a peptide dimer based on the first 25 amino acids of surfactant protein B (SP-B). At surface pressures between 30 and 40 mN/m, only monolayers containing POPG and dSP-B1-25 showed plateaus in the isotherm similar to those in Survanta, a bovine extract replacement lung surfactant that contains native SP-B and SP-C proteins. BAM images show distinct morphological changes in the fluid phase during these plateaus, while AFM images of deposited monolayers show that multilayer structures, which we named "nanosilos", form in the fluid phase at the plateau. These nanosilos are from 50 to 300 nm in diameter and from 5 to 8 nm in height and are similar to those observed in deposited Survanta monolayers. We propose that POPG and SP-B interact to stabilize the monolayer composition by trapping POPG in three-dimensional surface-associated aggregates at high surface pressures, preventing the irreversible loss of POPG and SP-B to the subphase.

Functional importance of the NH2-terminal insertion sequence of lung surfactant protein B

“…Although much work has been done on the structure and the activity of SP-B (9,15,66), its specific interactions and molecular orientations in interfacial phospholipid films at different points of compression and expansion are not yet completely defined. Because synthesizing a proper folding mimic of this 79-residue protein is difficult, various shorter peptide models have been developed and characterized both structurally and functionally, among them, an NH 2 -terminal truncation peptide, SP-B 1-25.…”

“…Studies have shown that this SP-B analog has surface-active behavior similar to that of the full-length protein and could potentially be incorporated into synthetic surfactant formulations (23,24,40,41). This NH 2 -terminal sequence has been shown to interact specifically with fluid phase palmitoyloleoylphosphatidylglycerol (POPG) in mixed monolayers, similar to native SP-B, and leads to morphologies as seen in Survanta (a bovine lung surfactant replacement therapy) monolayers that contain native SP-B (15).…”

“…Various experimental methods have explored how SP-B helps keep fluid lipids anchored to the monolayer (14,15,37) in the form of three-dimensional reservoirs (termed "nanosilos") of lipids and proteins in the subphase. Work with the full-length SP-B protein has shown the existence of nanosilos protruding from the anionic PG-rich areas of the monolayer (37), while experiments with the dimeric form of SP-B 1-25 have demonstrated unequivocally that both the peptide and biologically relevant anionic PG lipids were needed to form the reservoirs (15).…”

“…Work with the full-length SP-B protein has shown the existence of nanosilos protruding from the anionic PG-rich areas of the monolayer (37), while experiments with the dimeric form of SP-B 1-25 have demonstrated unequivocally that both the peptide and biologically relevant anionic PG lipids were needed to form the reservoirs (15). In the present work, we further explore the role of the SP-B NH 2 nanosilos when compressed to high surface pressures; this was further compared with in vivo efficacy as a lung surfactant replacement.…”

NMR Structures of the C-Terminal Segment of Surfactant Protein B in Detergent Micelles and Hexafluoro-2-propanol

“…SP-B preferentially associates with anionic lipids in surface monolayers (16), and upon compression of the film, the SP-Banionic lipid network forms buckled structures that remain attached to the monolayer (17). Upon re-expansion of the film at a lower surface pressure, the buckled structures are rapidly reincorporated into the film to re-form a flat monolayer (18,19). SP-B is a critical component in the formation of SP-A-DPPC tubular myelin structures that are thought to be important in the transport of lipid from the aqueous subphase to the lipid monolayer (20,21).…”