Membrane activity of (Cys48Ser) lung surfactant protein B increases with dimerisation

One of the possible functions of lung surfactant protein B (SP-B), an hydro phobic membrane-associated saposin-like protein, is to reduce the alveolar surface tension by promoting insertion of phospholipids into the air/liquid interface of the lung. SP-B is a covalent homodimer; Cys48 of two polypept ides form an intermolecular disulphide bond. In order to test whether dimer isation of SP-B is important for surfactant function, transgenic mice which express (Cys48Ser) human SP-B in a mouse SP-B null background were generat ed. In previous studies (Cys48Ser)SP-B showed a concentration-dependent in vitro activity, suggesting that it may form non-covalent dimers. Here (Cys4 8Ser)SP-B isolated from bronchoalveolar lavage of transgenic mice was studi ed at different concentrations by circular dichroism (CD) spectroscopy, pul sating bubble surfactometry, mass spectrometry and reversed-phase HPLC. The results indicate that (Cys48Ser)SP-B, both in a phospholipid environment a nd in organic solvents, is largely monomeric and exhibits low activity at c oncentrations lower than 1 -2 muM, while at higher concentrations it forms non-covalent dimers, which are nearly functionally equivalent to native SP- B in vitro. Furthermore, electrospray mass spectrometry showed that more di nners were found relative to the monomer when the polarity of the solvent w as decreased, and when the concentration of SP-B increased. (Cys48Ser)SP-B also eluted earlier than native SP-B in reversed-phase HPLC. Taken together , these results indicate that a polar surface is buried upon dimerisation, thereby promoting formation of interchain ion pairs between Glu51-Arg52' an d Glu51'-Arg52.