title: Assembly and disulfide rearrangement of recombinant surfactant protein A in vitro.
abstract: The surfactant-associated protein, protein A, produced by transgenic Chinese hamster ovary cells exhibits a heterogeneous population of structures. Electron microscopy reveals lollipop-shaped monomers consisting of a collagenous triple helix and a globular domain as well as oligomers in which two, three or more protomers are connected by their collagenous stalks. Each protomer consists of three alpha-chains (36 kDa) but under non-reducing conditions few free alpha-chains are observed by SDS/PAGE. Instead gamma-components (three chains), gamma 2 (six chains) and higher components are observed which are derived from intra- and inter-protomer disulfide cross-linking. Complete reduction at low temperature dissociates the oligomers, but preserves the intact structure of monomers as demonstrated by electron microscopy and trypsin digestion. Circular dichroism revealed an unfolding of the collagen triple helices of fully reduced protein A at 26 degrees C and of the unreduced protein A around 41.5 degrees C. Reoxidation of the fully reduced protein A re-established mainly the disulfide bonds within the triple helix but not between monomers. Very few higher assembly forms were reformed even at high protein A concentrations. Cellular in vivo systems must possess an efficient assembly mechanism which cannot be simulated by an in vitro system.
authors.affiliation: Abteilung für Biophysikalische Chemie, Biozentrum der Universität Basel, Switzerland.
authors.forename: C
authors.initials: C
authors.lastname: Haas
authors.forename: T
authors.initials: T
authors.lastname: Voss
authors.forename: J
authors.initials: J
authors.lastname: Engel
mesh_terms: D004220:Disulfides; D004355:Drug Stability; D010084:Oxidation-Reduction; D011510:Proteolipids / Q000737:chemistry*; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D011510:Proteolipids; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins