title: Alanine mutagenesis of surfactant protein A reveals that lipid binding and pH-dependent liposome aggregation are mediated by the carbohydrate recognition domain.
abstract: The carbohydrate recognition domain (CRD) of surfactant protein A (SP-A) is critical for the modulation of surfactant secretion from isolated type II cells and for the Ca(2+)-dependent aggregation of surfactant liposomes, but the domains of SP-A that mediate lipid binding have not been precisely mapped. To determine the role of the CRD in lipid interactions and other functions, the conserved amino acids of the putative Ca2+ and carbohydrate binding site (Glu195, Glu202, Asn214, Asp215) were substituted with alanine. The wild-type recombinant protein, SP-Ahyp, and mutant SP-As SP-Ahyp,E195A, SP-Ahyp,E202A, SP-Ahyp,N214A, and SP-Ahyp,D215A, were expressed in insect cells using baculovirus vectors and compared functionally. The Ca(2+)-dependent binding and aggregation of liposomes at pH 7.0 by SP-Ahyp,N214A were comparable to SP-Ahyp, but these activities were blocked in SP-Ahyp,E195A, SP-Ahyp,E202A, and SP-Ahyp,D215A. In contrast, the SP-Ahyp,D215A but not the other mutant proteins induced the Ca(2+)-independent aggregation of phospholipid liposomes at pH 4.0. The mutant recombinant proteins did not compete with 125I-labeled rat SP-A for high-affinity receptor occupancy on isolated type II cells and were much less potent than SP-Ahyp as regulators of surfactant secretion and uptake from type II cells. We conclude that (1) lipid binding and pH-dependent liposome aggregation are mediated by the CRD of SP-A, (2) distinct but overlapping domains within the CRD are required for pH- and Ca(2+)-dependent liposome aggregation, and (3) conserved acidic and polar residues of the carbohydrate binding site of SP-A are essential for interactions with type II cells.
authors.affiliation: Division of Pulmonary/Critical Care Medicine, University of Cincinnati College of Medicine, Ohio, USA. frank.mccormack@uc.edu
authors.forename: F X
authors.initials: FX
authors.lastname: McCormack
authors.forename: J
authors.initials: J
authors.lastname: Stewart
authors.forename: D R
authors.initials: DR
authors.lastname: Voelker
authors.forename: M
authors.initials: M
authors.lastname: Damodarasamy
mesh_terms: D000409:Alanine / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D002241:Carbohydrates / Q000502:physiology*; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism*; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D002241:Carbohydrates; D006023:Glycoproteins; D008081:Liposomes; D046911:Macromolecular Substances; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C067774:surfactant protein A receptor; D000409:Alanine; D002118:Calcium