Palmitoylation and amyloid fibril formation of lung surfactant protein C

Sammanfattning: Lung surfactant is a mixture of lipids and a few proteins, of which surfactant proteins (SP)-B and SP-C are lipophilic. Surfactant is essential for the reduction of surface tension at the alveolar air/liquid interface. The extremely hydrophobic SP-C is a 35-residue transmembraneous [alpha]-helical peptide containing a poly-Val stretch and two palmitoylated Cys residues. In this thesis the structural and functional importance of the SP-C palmitoyl groups and the poly-Val helix have been studied An HPLC system with high resolution was developed to purify very unpolar polypeptides and maintain their secondary structure. Thereby, a tripalmitoylated SP-C isoform, with the third palmitoyl group linked to the side-chain of Lys- 11 was detected, and it was possible to distinguish between helical and non-helical SP-C from their elution profiles. 5% of natural SP-C was identified as an elongated SP-C isoform with a C-terminal extension of Met-36-His-37 using electro-spray mass spectrometry. ProSP-C is believed to be inserted into a lipid bilayer before processing to SP-C takes place. The isoform detected suggests that proteolysis is not very specific and that membrane shielding influences the C-terminal end of SP-C. The surface properties of two peptide analogues with the same amino acid composition but different helical surfaces, KL2,3 and KL4, were compared. KL2,3 was determined to be an amphipathic helix oriented parallel to the lipid surface while KL4 presents a mixed polar/unpolar surface and, like SP-C, is transmembraneous. KL2,3 is inferior to KL4 in reducing the surface tension of an air/water interface when mixed with DPPC/PG/PA, 68:22:9 (w/w) and it was concluded that a transmembraneous helix is superior in this respect. Natural and synthetic SP-C, as well as SP-C analogues with a conserved poly-Val part aggregate. An analogue with the poly-Val stretch substituted for poly-Leu was designed. This peptide is almost identical to SP-C in secondary structure and hydrophobicity, but does not aggregate. A lipid mixture containing this peptide exhibits excellent surface properties in vitro and increases lung-thorax compliance with 30% in premature rabbits. The poly-Leu substituted analogue was palmitoylated and mixed with DPPC/PG/PA, 68:22:9 (w/w). The palmitoyl groups were important for lipid respreading of cycled films, surface stability at low surface tensions, and for association of subphase-lipids to the interfacial monolayer. Amyloid fibrils were detected after SP-C was incubated in organic solvents containing a small portion of water, and depalmitoylation of SP-C increased the fibrillation rate. In lavage fluid from the lungs of a patient with pulmonary alveolar proteinosis, large amounts of fibrils were found. These fibrils were composed of SP-C and were shown to be, at least partly, depalmitoylated. Using mass spectrometry it was shown that [alpha]-helical SP-C transforms into a non-helical form following first order kinetics. The half-life of SP-C was 70 h while the halflife of non- and mono-palmitoylated SP-C was 35 h. Formation of oligomeric/aggregated intermediates with a half-life of >300 h precedes formation of elongated fibrils. Hydrogen/deuterium exchange experiments revealed that the SP-C helix is extremely rigid and does not "breathe", but once started, unfolding of the helix is a highly cooperative process.