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

Abstract

Lung surfactant protein B (SP-B) is required for proper surface activity of pulmonary surfactant. In model lung surfactant lipid systems composed of saturated and unsaturated lipids, the unsaturated lipids are removed from the film at high compression. It is thought that SP-B helps anchor these lipids closely to the monolayer in three-dimensional cylindrical structures termed "nanosilos" seen by atomic force microscopy imaging of deposited monolayers at high surface pressures. Here we explore the role of the SP-B NH(2) terminus in the formation and stability of these cylindrical structures, specifically the distribution of lipid stack height, width, and density with four SP-B truncation peptides: SP-B 1-25, SP-B 9-25, SP-B 11-25, and SP-B 1-25Nflex (prolines 2 and 4 substituted with alanine). The first nine amino acids, termed the insertion sequence and the interface seeking tryptophan residue 9, are shown to stabilize the formation of nanosilos while an increase in the insertion sequence flexibility (SP-B 1-25Nflex) may improve peptide functionality. This provides a functional understanding of the insertion sequence beyond anchoring the protein to the two-dimensional membrane lining the lung, as it also stabilizes formation of nanosilos, creating reversible repositories for fluid lipids at high compression. In lavaged, surfactant-deficient rats, instillation of a mixture of SP-B 1-25 (as a monomer or dimer) and synthetic lung lavage lipids quickly improved oxygenation and dynamic compliance, whereas SP-B 11-25 surfactants showed oxygenation and dynamic compliance values similar to that of lipids alone, demonstrating a positive correlation between formation of stable, but reversible, nanosilos and in vivo efficacy.

Fig. 1.

Surface pressure vs. molecular area isotherms for lung surfactant model mixtures of 7:3 dipalmitoylphosphatidylcholine:palmitoyloleoylphosphatidylglycerol (DPPC:POPG) and various lung surfactant protein B (SP-B) truncation peptides 10 wt%. Thick black line, no peptide; dashed black line, SP-B 1–25; hatched line, SP-B 9–25; dashed dotted line, SP-B 11–25; thin black line, SP-B 1–25Nflex. Inset: plateau region of monolayers with SP-B 9–25 and SP-B 11–25. All isotherms were taken on pure water at 25°C.

Fig. 2.

Fluorescence microscope images of 7:3 DPPC:POPG with 10 wt% SP-B 1–25 (A) at a surface pressure of 55 mN/m. Note the speckles of increased intensity (nanosilos) in the bright phase. B: on decompression, fluorescent speckles gradually disappear, indicating reversible respreading of the collapsed material stored in the nanosilos into the lipid monolayer.

Fig. 3.

Atomic force microscopy (AFM) topographic images of 7:3 DPPC:POPG monolayers containing 10 wt% SP-B truncation peptides transferred at 55 mN/m from a water subphase at 25°C (z-scale 5 nm). A, no peptide; B, SP-B 1–25; C, SP-B 9–25; D, SP-B 11–25; E, SP-B 1–25Nflex.

Fig. 4.

AFM topographic images of 7:3 DPPC:POPG monolayers with 10 wt% (A1 and A2) SP-B 1–25 and (B) SP-B 1–25Nflex monolayers transferred at 55 mN/m from a 40 wt% glycerol subphase at 25°C (z-scale 5 nm). All nanosilos are ∼5 nm above the height of the monolayer.

Fig. 5.

Oxygenation, expressed as arterial Po₂ (mmHg), in rats treated with porcine SP-B surfactant (positive control), lipids (negative control), and the four experimental surfactant preparations with monomeric or dimeric SP-B 1–25 and SP-B 11–25 peptides. The differences between porcine SP-B surfactant and monomeric and dimeric SP-B 1–25 surfactant and the differences between SP-B 1–25 and SP-B 11–25 surfactants or lipids alone are significant (P < 0.001).

Fig. 6.

Dynamic compliance (ml · kg⁻¹ · cmH₂O⁻¹) in rats treated with porcine SP-B surfactant (positive control), lipids (negative control), and the four experimental surfactant preparations with monomeric or dimeric SP-B 1–25 and SP-B 11–25 peptides. The differences between porcine SP-B surfactant, monomeric and dimeric SP-B 1–25 surfactant, and monomeric and dimeric SP-B 11–25 surfactants or lipids alone are significant (P < 0.005).

Fig. 7.

Cross-sectional view of bicelle structures. A, unstable structure where the edge is exposed to water; B, edge stabilized by more fluid, shorter tailed lipids; C, edge stabilized by amphiphilic peptide.

Fig. 8.

Proposed bicelle structures for SP-B 1–25 and 1–25Nflex (A); SP-B 9–25 (B). Note the NH₂-terminal insertion sequence is anchored between the 2 layers of the bilayer. C: comparison of stable bicelle nanosilo to fluid lipid vesicle.