title: Site-directed mutagenesis of surfactant protein A reveals dissociation of lipid aggregation and lipid uptake by alveolar type II cells.
abstract: Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195-->Gln (rE195Q), Lys201-->Ala (rK201A) and Lys203-->Ala (rK203A) for rat SP-A, Arg199-->Ala (hR199A) and Lys201-->Ala (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203-->Ala (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [125I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process.
authors.affiliation: Department of Biochemistry, Sapporo Medical University School of Medicine, South-1 West-17, Chuo-ku, Sapporo 060, Japan.
authors.forename: W
authors.initials: W
authors.lastname: Tsunezawa
authors.forename: H
authors.initials: H
authors.lastname: Sano
authors.forename: H
authors.initials: H
authors.lastname: Sohma
authors.forename: F X
authors.initials: FX
authors.lastname: McCormack
authors.forename: D R
authors.initials: DR
authors.lastname: Voelker
authors.forename: Y
authors.initials: Y
authors.lastname: Kuroki
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D006801:Humans; D008055:Lipids / Q000493:pharmacokinetics*; D008081:Liposomes / Q000493:pharmacokinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010715:Phosphatidylglycerols / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011510:Proteolipids / Q000737:chemistry* / Q000494:pharmacology; D011650:Pulmonary Alveoli / Q000378:metabolism*; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000737:chemistry* / Q000494:pharmacology; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D012697:Serine Endopeptidases / Q000378:metabolism
chemical_list: D008055:Lipids; D008081:Liposomes; D010715:Phosphatidylglycerols; 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; D015060:1,2-Dipalmitoylphosphatidylcholine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase