Pulmonary surfactant proteins B and C : molecular organisation and involvement in respiratory disease

Sammanfattning: Lung surfactant is a complex mixture of phospholipids and proteins with the main function to reduce the surface tension at the alveolar air/liquid interface. Surfactant protein B (SP-B) and C (SP-C) are hydrophobic but unrelated in structure, and probably have unique functional roles in the formation of the surface-active monolayer. SP-B deficiency causes lethal respiratory failure, but SP-C null mice show no respiratory dysfunction. This thesis is focused on the molecular architecture of SP-B and its proform, and the presence of native and aggregated forms of SP-C associated with surfactant abnormalities. Circular dichroism (CD) spectroscopy of recombinant proSP-B showed that it is composed of about 35% [alpha]-helical structure, similarly to the approximately 45% helix found in SP-B. Limited proteolysis of rproSP- B occurs predominantly between three tandem saposin-like domains previously proposed from amino acid sequence comparisons, supporting that proSP-B contains, in addition to SP-B, two further saposin domains. A model of dimeric SP-B based on the available NMR-structure of monomeric NK-lysin was generated. This suggests that SP-B is an elongated molecule with clusters of positive charges located at both poles, separated by a predominantly nonpolar region. The model is compatible with a function of SP-B in lipid crosslinking and fusion. Intriguingly, polymyxin B (which cross-links lipid vesicles but is structurally unrelated to SP-B) exhibits in vitro surface activity similar to SP-B. This suggests an avenue for identification of SP-B analogues that can be used in synthetic surfactants for treatment of RDS. (Cys48Ser)human SP-B expressed in transgenic mice deficient in mouse SP-B was isolated and studied by CD spectroscopy, pulsating bubble surfactometry, mass spectrometry and reversed-phase HPLC. (Cys48Ser)SP- B, both in a phospholipid environment and in organic solvents, is largely monomeric and exhibits low activity at concentrations < ~ 2 mM, while at higher concentrations it forms non-covalent dimers. (Cys48Ser)SP-B elutes earlier than native SP-B upon reversed-phase HPLC, and mass spectrometry revealed more dimers relative to the monomer when the polarity of the solvent was decreased. These results are compatible with the involvement of Glu51-Arg52 ion pair dimerisation, as suggested from the SP-B model. Respiratory distress syndrome (RDS) of unknown origin has been reported in Belgian White and Blue (BWB) calves. The SP-B and SP-C contents were compared in pulmonary surfactant from 7 healthy and 14 RDS BWB calves. This revealed a very low concentration of SP-C in RDS calves, while no significant difference was detected concerning the SP-B level. This is the first time that an isolated SP-C deficiency in RDS is reported. Addition of SP-C to surfactant from sick calves improved but did not completely restore, surface activity. This suggest that low SP-C levels contribute to the respiratory distress, but that also other factors are important. SP-C amyloid fibrils were found in bronchoalveolar lavage fluid (BAL) from mice deficient in either SP-D or GM-CSF. SDS-PAGE and electron microscopy revealed 10-100 fold higher amounts of fibrils in the SP-D (-/- ) mice. This could be related to the much elevated production of hydrogen peroxide in macrophages from SP-D (-/-) compared to GM-CSF (-/-) mice. In vitro experiments showed that SP-C also forms fibrils in the presence of about 500-fold molar excess of phospholipids, and to a higher extent at low temperature. This indicates that SP-C constitutively fomrs amyloid fibrils under physiological conditions, but the formation is increased under certain conditions.