Processing, stability and interactions of lung surfactant protein C

Abstract: Mature SP-C is a 4.2 kDa transmembrane protein which is uniquely expressed in the alveolar type II cell. Human SP-C is generated via multistep proteolytic cleavage of both the C-terminal and Nterminal regions of proSP-C. The function of SP-C in vivo remains unclear, but effects of SP-C on the adsorption, spreading, and stability of lipid films at an air/water interface have been documented in a number of in vitro studies. Infants with inherited deficiency of SP-B and SP-B knock-out mice show neonatal respiratory distress syndrome. SP-B deficiency is associated with accumulation of a processing intermediate, SP-Ci, which was characterized in paper I. The molecular weight of SP-Ci, determined by mass spectrometry, is 5458 Da. Determination of the SP-Ci covalent structure revealed a 12-residue N-terminal peptide segment, followed by a 35residue segment that is identical to mature SP-C. Unlike SP-C, SP-Ci exhibits a very poor ability to promote phospholipid adsorption, gives high surface tension during cyclic film compression, and does not bind lipopolysaccharide (LPS) in vitro. SP-C is composed of a flexible N-terminal end and an a-helical C-terminal part. In paper II, the secondary structure and the stability of SP-Ci was investigated. The circular dichroism (CD) spec~ of SP-Ci shows that the dominant structure is a-helical. Unlike SP-C, SP-Ci helix does not unfold or aggregate during several weeks of incubation in aqueous organic solvents. Hydrogen/deuterium exchange experiments showed that 15 amide hydrogen atoms in SP-Ci are protected from exchange for at least several weeks. This number is in agreement with the number of residues spanning the poly-valine part. This leads to the conclusion that the polyvaline part of SP-Ci is locked in a helical conformation for weeks. However, if SP-Ci is incubated in an acidic environment, SP-C unfolds and aggregates into amyloid fibrils like SP-C. The reasons for the reduced function and more stable a-helix of SP-Ci were studied in paper III. The NMR structure of an analogue of SP-Ci, SP-Ci(1 -31), shows that the a-helix is extended Nterminally compared to mature SP-C. CD spectroscopy of SP-Ci(1 -31) in lipids shows a mixture of helical and extended conformation at pH 6, and a shift to more unordered structure at pH 5. Addition in trans of a synthetic dodecapeptide corresponding to the propeptide part of SP-Ci to SPC results in slower aggregation kinetics, altered amyloid fibril formation, and reduced surface activity of phospholipid-bound SP-C. These data suggest that the propeptide part of SP-Ci prevents helix unfolding by locking the conformation of the N-terminal part of the helix, and that acidic pH results in structural disordering of the region that is proteolytically cleaved to generate SP-C. SP-C can recognize LPS. In paper IV using synthetic SP-C analogues, it was shown that the capacity of SP-C to bind LPS requires both the hydrophilic N-terminal region and the C-terminal hydrophobic region of the peptide. Using chemically modified LPS and synthetic lipid A analogs, we established that the phosphate residue, when present in aconfiguration, is required for the interaction. Finally, more efficient binding between SP-C and LPS was observed in a neutral lipid environment.