Hydrophobic surfactant-associated polypeptides: SP-C is a lipopeptide with two palmitoylated cysteine residues, whereas SP-B lacks covalently linked fatty acyl groups.

Abstract

Pulmonary surfactant contains two hydrophobic polypeptides, SP-B and SP-C, with known amino acid sequences and with truncated subforms lacking the N-terminal residues. Treatment of SP-C with KOH releases fatty acids (palmitic acid to more than 85%) in molar ratios of 1.8-2.0 relative to the polypeptide. Furthermore, plasma-desorption mass spectrometry shows native SP-C of both the intact and truncated types to be monomers with masses about 500 units higher than those expected for the polypeptide chains. After treatment with KOH, trimethylamine, or dithioerythritol, the polypeptide masses are obtained. These results prove that native SP-C is a lipopeptide with two palmitoyl groups covalently linked to the polypeptide chain. The deacylation conditions, the presence of two cysteine residues in the polypeptide, and the absence of other possible attachment sites establish that the palmitoyl groups are thioester-linked to the two adjacent cysteine residues. In contrast, the major form of porcine SP-B is a dimer without fatty acid components. That SP-C is a true lipopeptide with covalently bound palmitoyl groups suggests possibilities for functional interactions. It gives a direct physical link between SP-C and surfactant phospholipid components. Long-chain acylation may constitute a means for association of proteins with membranes and could conceivably modulate the stability and biological activity of surfactant fims.polypeptides in different subforms constituting N-terminally truncated molecules (20,21).Nevertheless, the primary structures of the polypeptides have been determined (18)(19)(20), showing SP-B to be a 79-residue polypeptide with a high content of cysteine residues, and SP-C to be a 35-residue polypeptide with two adjacent cysteine residues in a "palindromic" sequence (-Ile-Pro- Surfactant is essential for pulmonary function. It reduces the alveolar surface tension, preventing alveolar collapse, and is a prerequisite for normal respiration (1). The capacity for surfactant synthesis is limited in premature infants, and low amounts of surfactant are associated with neonatal respiratory distress syndrome. This serious disease can be treated effectively by replacement therapy through instillation of surfactant in the airways (2-7). Natural surfactant is composed of phospholipids and small amounts of specific polypeptides. In particular, two highly hydrophobic, low molecular weight polypeptides named SP-B and SP-C (8) appear to be essential components of surfactant preparations for replacement therapy (2, 3, 5-7). Both are derived from larger precursor polypeptides and have been structurally analyzed at the cDNA (9-16) and protein (17)(18)(19)(20) levels. Protein studies are essential for characterization ofthe mature polypeptides but are complicated by the extreme peptide hydrophobicities. SP-C in particular has essentially no aqueous solubility and is largely inert to conventional hydrolysis and other analytical steps. Furthermore, native preparations are heterogeneous, with the presence of the two M...

The 21‐residue surfactant peptide (LysLeu4)4Lys(KL4) is a transmembrane α‐helix with a mixed nonpolar/polar surface

“…Native SP-C (LRIPCCPVNLKRLLVVVVVVVLVVVVIVGALL-MGL with both cysteines palmitoylated [6]) was purified from porcine lungs [21].…”

“…SP-B, a homodimer of two 79-residue polypeptide chains [6,7], has an a-helical content of about 45% as shown by Fourier transform infrared (FTIR) [8] and circular dichroism (CD) spectroscopy [9]. SP-B has been proposed to contain four amphipathic helices interacting preferentially with superficial parts of a phospholipid bilayer [9].…”

“…SP-B has been proposed to contain four amphipathic helices interacting preferentially with superficial parts of a phospholipid bilayer [9]. SP-C is a 35-residue polypeptide [10] with palmitoyl groups thioester-linked to the cysteine residues in the N-terminal part of the molecule [6]. FTIR spectroscopy revealed that SP-C adopts mainly an ahelical conformation with its a-helix oriented parallel to the lipid acyl chains [11,12].…”

Antibodies against synthetic amphipathic helical sequences of surfactant protein SP‐B detect a conformational change in the native protein

“…In particular antipeptide antibodies offer a sensitive approach for the detection of conformational changes in surface accessible sequences of proteins [16]. This approach is especially useful in the study of the native SP-B protein domains because, with the possible exception of disulfide dependent oligomerization, SP-B is not known to undergo post translational modification such as phosphorylation or glycosylation [5]. In this report we describe the binding characteristics of two antibodies raised against synthetic peptides containing surface seeking segments of surfactant protein B.…”

Molecular Structures and Interactions of Pulmonary Surfactant Components

“…The lack of exact agreement between the molecular mass calculated from the amino acid sequence (8.7 kDa for the monomer and 17.4 kDa for the dimer) and the masses observed by gel electrophoresis could be due to abnormal binding of SDS by the hydrophobic SP-B polypeptide, the fact that SP-B retains a folded structure in SDS and/or additional factors. Covalent linkage of non-protein constituents to SP-B have been ruled out by mass spectrometry (Curstedt et al, 1990;Voss et al, 1992). Each polypeptide chain of the dimer contains three disulphide bridges: Cys8-Cys77, Cysll -Cys71 and Cys35-Cys46.…”