RESUMEN
Lung surfactant is a mixture of lipids and proteins and is essential for air breathing in mammals. The hydrophobic surfactant proteins B and C (SP-B and SP-C) assist in reducing surface tension in the lung alveoli by organizing the surfactant lipids. SP-B deficiency is life-threatening, and a lack of SP-C can lead to progressive interstitial lung disease. B-YL (41 amino acids) is a highly surface-active, sulfur-free peptide mimic of SP-B (79 amino acids) in which the four cysteine residues are replaced by tyrosine. Mammalian SP-C (35 amino acids) contains two cysteine-linked palmitoyl groups at positions 5 and 6 in the N-terminal region that override the ß-sheet propensities of the native sequence. Canine SP-C (34 amino acids) is exceptional because it has only one palmitoylated cysteine residue at position 4 and a phenylalanine at position 5. We developed canine SP-C constructs in which the palmitoylated cysteine residue at position 4 is replaced by phenylalanine (SP-Cff) or serine (SP-Csf) and a glutamic acid-lysine ion-lock was placed at sequence positions 20-24 of the hydrophobic helical domain to enhance its alpha helical propensity. AI modeling, molecular dynamics, circular dichroism spectroscopy, Fourier Transform InfraRed spectroscopy, and electron spin resonance studies showed that the secondary structure of canine SP-Cff ion-lock peptide was like that of native SP-C, suggesting that substitution of phenylalanine for cysteine has no apparent effect on the secondary structure of the peptide. Captive bubble surfactometry demonstrated higher surface activity for canine SP-Cff ion-lock peptide in combination with B-YL in surfactant lipids than with canine SP-Csf ion-lock peptide. These studies demonstrate the potential of canine SP-Cff ion-lock peptide to enhance the functionality of the SP-B peptide mimic B-YL in synthetic surfactant lipids.
RESUMEN
Background: Animal-derived surfactants containing surfactant proteins B (SP-B) and C (SP-C) are used to treat respiratory distress syndrome (RDS) in preterm infants. SP-B (79 residues) plays a pivotal role in lung function and the design of synthetic lung surfactant. Super Mini-B (SMB), a 41-residue peptide based on the N- and C-domains of SP-B covalently joined with a turn and two disulfides, folds as an α-helix hairpin mimicking the properties of these domains in SP-B. Here, we studied 'B-YL', a 41-residue SMB variant that has its four cysteine and two methionine residues replaced by tyrosine and leucine, respectively, to test whether these hydrophobic substitutions produce a surface-active, α-helix hairpin. Methods: Structure and function of B-YL and SMB in surfactant lipids were compared with CD and FTIR spectroscopy, and surface activity with captive bubble surfactometry and in lavaged, surfactant-deficient adult rabbits. Results: CD and FTIR spectroscopy of B-YL in surfactant lipids showed secondary structures compatible with peptide folding as an α-helix hairpin, similar to SMB in lipids. B-YL in surfactant lipids demonstrated excellent in vitro surface activity and good oxygenation and dynamic compliance in lavaged, surfactant-deficient adult rabbits, suggesting that the four tyrosine substitutions are an effective replacement for the disulfide-reinforced helix-turn of SMB. Here, the B-YL fold may be stabilized by a core of clustered tyrosines linking the N- and C-helices through non-covalent interactions involving aromatic rings. Conclusions: 'Sulfur-free' B-YL forms an amphipathic helix-hairpin in surfactant liposomes with high surface activity and is functionally similar to SMB and native SP-B. The removal of the cysteines makes B-YL more feasible to scale up production for clinical application. B-YL's possible resistance against free oxygen radical damage to methionines by substitutions with leucine provides an extra edge over SMB in the treatment of respiratory failure in preterm infants with RDS.