Native pulmonary-surfactant-associated lipopolypeptide SP-C, its chemically depalmitoylated form and several synthetic analogues lacking the palmitoylcysteine residues were analysed for secondary structure in phospholipid micelles and for biophysical activity in 1,2-dipalmitoyl-sn-glycero-3- phosphocholine/phosphatidylglycerol/palmitic acid (68:22:9, by wt.). Compared with the native molecule, with the entire poly-valyl part in a known alpha-helical conformation, depalmitoylated SP-C was found to be still mainly alpha-helical, but with an approx. 20% decrease in the helical content. A synthetic hybrid polypeptide where the entire poly-valyl alpha-helical part of native SP-C had been replaced with the amino acid sequence of a transmembrane helix of bacteriorhodopsin is also predominantly alpha-helical. In contrast, synthetic SP-C analogues lacking only the palmitoyl groups, by replacement of the palmitoylcysteine residues with cysteine, phenylalanine or serine, or lacking the positively charged amino acids by replacement with alanine, are considerably less alpha-helical than both native and depalmitoylated SP-C. The data indicate that the SP-C palmitoyl groups are important for maintenance of the alpha-helical conformation in parts of the polypeptide, and that the poly-valyl alpha-helical conformation is not fully formed in synthetic SP-C polypeptides. Furthermore, the helical structure of both native and depalmitoylated SP-C in dodecylphosphocholine micelles is very resistant to thermal denaturation, exhibiting ordered structure at 90 degrees C. The alpha-helical content grossly parallels the peptide-induced acceleration of the spreading of phospholipids at an air/water interface and the increase of surface pressure. The data suggest that the alpha-helical conformation itself, rather than just the covalent structure, is of prime importance for the biological function of synthetic pulmonary-surfactant peptides.

Pulmonary surfactant contains two hydrophobic proteins, SP-B and SP-C. With the aim of identifying synthetic SP-B and SP-C substitutes for replacement therapy of respiratory distress syndromes, we have studied two transmembrane peptides and two amphipathic peptides that are located in the plane of a phospholipid bilayer. One amphipathic peptide was designed by changing the amino acid sequence, but not the composition or size, of the 21-residue peptide KL 4 . This peptide, designated KL 2.3 from its spacing of nonpolar and polar residues, exhibited similar A-helical content as KL 4 but was oriented along a phospholipid bilayer plane, in contrast to the transmembrane orientation of KL 4 in the same environment. The second amphipathic peptide analyzed was succinyl-LLEKLLEWLK-amide (WMAP10). KL 4 more efficiently accelerated the spreading of a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (Pam 2 -GroPCho)/phosphatidylglycerol (PtdGro)/palmitic acid (PamOH), 68:22:9 (by mass), at an air/water interface than did any of the amphipathic peptides. Similarly, KL 4 , but not KL 2.3 , when present in an interfacial monolayer composed of Pam 2GroPCho/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, 7: 3 (by mass), increased lipid insertion from subphase vesicles.An SP-C analogue, SP-C(Leu), with all helical valyl residues in native SP-C replaced with Leu and the palmitoylcysteines at positions 5 and 6 replaced with Ser, but otherwise with essentially the same primary structure as the native peptide, was analyzed. SP-C(Leu) exhibited similar A-helical content as native SP-C and a transmembrane orientation and, in contrast to poly-valyl-containing synthetic peptides, it folds into a helical conformation after acid-induced denaturation. SP-C(Leu) accelerated the spreading of Pam 2 GroPCho/PtdGro/PamOH, 68:22:9 (by mass), almost identically to native SP-C, and lowered the surface tension during rapid cyclic film compressions in a pulsating bubble surfactometer to near zero and 43 mN/m at minimum and maximum bubble size, respectively. Airway instillation of 2% (by mass) SP-C(Leu) combined with Pam 2 GroPCho/PtdGro/PamOH in preterm rabbit fetuses improved dynamic lung compliance by about 30% compared with untreated controls.Keywords : pulmonary surfactant; surfactant protein B ; surfactant protein C ; transmembrane peptide; amphipathic peptide.

The low-molecular-mass surfactant protein fraction, soluble in chloroform/methanol, contains at least two separate polypeptide chains. The 8-kDa form (type I) was isolated, ['4C]carboxymethylated after reduction, and submitted to structural analysis. Its highly hydrophobic nature complicated purification, proteolytic cleavages, and sequence analysis. Acid hydrolysis in 6 M HCI for 7 days was necessary for release of branched-chain residues in full yield. Pepsin was the only enzyme found to cleave the surfactant protein and was used to complement peptide generation by chemical cleavage with CNBr. The primary structure deduced consists of 79 residues with 8 half-cystine residues, and a total of 39% branched-chain hydrophobic residues. However, 11 residues are charged at physiological pH, and all properties of the primary structure are not entirely outstanding in relation to those of other proteins. Hydrophobic segments, coupled with a presumably tight folding from the presence of disulfide bridges, probably explain the unusual properties and the solubility in organic solvents.Pulmonary surfactant is a phospholipid/protein complex essential for normal respiration by reducing surface tension at the air-liquid interface of the alveoli [l]. Different surfactantspecific proteins have been detected. One group, composed of comparatively large glycoproteins with molecular masses varying over 28-36 kDa depending on the degree of glycosylation [2, 31, is soluble in water and the primary structure of the forms from canine and human lung has been determined [4 -61. In the presence of calcium ions this protein type apparently participates in the formation of surface-active tubular myelin from secreted lamellar bodies [7] and increases the rate of adsorption of surfactant phospholipids [8]. Although this protein is probably part of the endogenous surfactant, synthesized in the alveolar epithelial type I1 cells, it does not seem to be essential for the physiological activity of exogenous surfactant preparations designed for replacement therapy [9 -1 I].A second group of surfactant-specific proteins is composed of forms with low molecular masses (< 14 kDa) [2, 9,12 -211. These proteins are highly hydrophobic and include different polypeptides which may be soluble [9] or insoluble [2, 16, 171 in ether/ethanol. We have recently isolated and characterized two of these hydrophobic proteins from pig surfactant [21]. Both proteins require organic solvents for solubilization and purification, and are heterogenous by multiple start positions in the N-terminal regions. Recombination of either of these proteins with synthetic phospholipids yields a surfactant preparation with physical and biological properties which in many respects are similar to those of natural pulmonary surfactant [21].The two low-molecular-mass proteins have unrelated structures and sizes; the smaller form is not a fragment of the larger [21]. Recently, cDNA segments of the longer form [22] have been described from dog, but exact structures, sizes, or further prop...

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