Membrane activity of (Cys48Ser) lung surfactant protein B increases with dimerisation.
One of the possible functions of lung surfactant protein B (SP-B), an hydrophobic 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 polypeptides form an intermolecular disulphide bond. In order to test whether dimerisation of SP-B is important for surfactant function, transgenic mice which express (Cys48Ser) human SP-B in a mouse SP-B null background were generated. In previous studies (Cys48Ser)SP-B showed a concentration-dependent in vitro activity, suggesting that it may form non-covalent dimers. Here (Cys48Ser)SP-B isolated from bronchoalveolar lavage of transgenic mice was studied at different concentrations by circular dichroism (CD) spectroscopy, pulsating bubble surfactometry, mass spectrometry and reversed-phase HPLC. The results indicate that (Cys48Ser)SP-B, both in a phospholipid environment and in organic solvents, is largely monomeric and exhibits low activity at concentrations lower than 1 -2 microM, 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 dimers were found relative to the monomer when the polarity of the solvent was 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' and Glu51'-Arg52.
Zaltash
S
S
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Griffiths
W J
WJ
Beck
D
D
Duan
C X
CX
Weaver
T E
TE
Johansson
J
J
D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects*; D002942:Circular Dichroism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D008567:Membranes, Artificial; D051379:Mice; D008822:Mice, Transgenic; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D013500:Surface Tension / Q000187:drug effects
D008567:Membranes, Artificial; D011510:Proteolipids; D011663:Pulmonary Surfactants