title: Intermolecular cross-links mediate aggregation of phospholipid vesicles by pulmonary surfactant protein SP-A.
abstract: As the most abundant glycoprotein component of pulmonary surfactant, SP-A (Mr = 30,000-36,000) plays a central role in the organization of phospholipid bilayers in the alveolar air space. SP-A, isolated from lung lavage, exists in oligomeric forms (N = 6, 12, 18, ...), mediated by collagen-like triple helices and intermolecular disulfide bonds. These protein-protein interactions, involving the amino-terminal domain of SP-A, are hypothesized to facilitate the alignment of surfactant lipid bilayers into unique tubular myelin structures. SP-A reorganization of surfactant lipid was assessed in vitro by quantitating the calcium-dependent light scattering properties of lipid vesicle suspensions induced by SP-A. Accelerated aggregation of unilamellar vesicles required SP-A and at least 3 mM free calcium. The initial rate of aggregation was proportional to the concentration of canine SP-A over lipid:protein molar ratios ranging from 200:1 to 5000:1. Digestion with bacterial collagenase or incubation with dithiothreitol (DTT) completely blocked lipid aggregation activity. Both treatments decreased the binding of SP-A to phospholipids. The conditions used in the DTT experiments (10 mM DTT, nondenaturing Tris buffer, 37 degrees C) resulted in the selective reduction and 14C-alkylation of the intermolecular disulfide bond involving residue 9Cys, whereas the four cysteines found in the noncollagenous domain of SP-A were inefficiently alkylated with [14C]-iodoacetate. HPLC analysis of tryptic SP-A peptides revealed that these four cysteine residues participate in intramolecular disulfide bond formation (138Cys-229Cys and 207Cys-221Cys). Our data demonstrate the importance of the quaternary structure (triple helix and intermolecular disulfide bond) of SP-A for the aggregation of unilamellar phospholipid vesicles.
authors.affiliation: Department of Pediatrics, College of Medicine, University of Cincinnati, Ohio 45267-0541.
authors.forename: G F
authors.initials: GF
authors.lastname: Ross
authors.forename: J
authors.initials: J
authors.lastname: Sawyer
authors.forename: T
authors.initials: T
authors.lastname: O'Connor
authors.forename: J A
authors.initials: JA
authors.lastname: Whitsett
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D004285:Dogs; D006023:Glycoproteins / Q000494:pharmacology*; D008051:Lipid Bilayers / Q000378:metabolism*; D008168:Lung / Q000737:chemistry; D008970:Molecular Weight; D010743:Phospholipids / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011510:Proteolipids / Q000494:pharmacology*; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000494:pharmacology*
chemical_list: D000596:Amino Acids; D003432:Cross-Linking Reagents; D006023:Glycoproteins; D008051:Lipid Bilayers; D010743:Phospholipids; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D004229:Dithiothreitol