title: Role of the degree of oligomerization in the structure and function of human surfactant protein A.
abstract: The role of the degree of oligomerization in the structure and function of human surfactant protein A (SP-A) was investigated using a human SP-A1 mutant (SP-A1(DeltaAVC,C6S)), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys(6) and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys(6) mutant lacked the NH(2)-terminal Ala(-3)-Val(-2)-Cys(-1) (DeltaAVC) extension present in some SP-A1 isoforms. SP-A1(DeltaAVC,C6S) was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1(DeltaAVC,C6S) was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated from human lung lavages. SP-A1(DeltaAVC,C6S) showed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin degradation. The T(m) was 32.7 degrees C for SP-A1(DeltaAVC,C6S) and 44.5 degrees C for SP-A1. Although SP-A1(DeltaAVC,C6S) was capable of binding to calcium, rough lipopolysaccharide, and phospholipid vesicles, this mutant was unable to induce rough lipopolysaccharide and phospholipid vesicle aggregation, to enhance the interfacial adsorption of SP-B/SP-C-surfactant membranes, and to undergo self-association in the presence of Ca(2+). On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1(DeltaAVC,C6S) to inhibit the production of tumor necrosis factor-alpha by macrophage-like U937 cells stimulated with either smooth or rough lipopolysaccharide. We conclude that supratrimeric assembly of human SP-A is essential for collagen triple helix stability at physiological temperatures, protection against proteases, protein self-association, and SP-A-induced ligand aggregation. The supratrimeric assembly is not essential for the binding of SP-A to ligands and anti-inflammatory effects of SP-A.
authors.affiliation: Department of Biochemistry and Molecular Biology I, Complutense University of Madrid, 28040 Madrid, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Sánchez-Barbero
authors.forename: Jochen
authors.initials: J
authors.lastname: Strassner
authors.forename: Rafael
authors.initials: R
authors.lastname: García-Cañero
authors.forename: Wolfram
authors.initials: W
authors.lastname: Steinhilber
authors.forename: Cristina
authors.initials: C
authors.lastname: Casals
mesh_terms: D000327:Adsorption; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D018893:Bronchoalveolar Lavage; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007249:Inflammation; D007700:Kinetics; D008024:Ligands; D008070:Lipopolysaccharides / Q000737:chemistry; D008264:Macrophages / Q000378:metabolism; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D037662:Pulmonary Surfactant-Associated Protein A / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D020298:U937 Cells
chemical_list: D000893:Anti-Inflammatory Agents; D008024:Ligands; D008070:Lipopolysaccharides; D010743:Phospholipids; D020033:Protein Isoforms; D037662:Pulmonary Surfactant-Associated Protein A; D011994:Recombinant Proteins; D014357:Trypsin; D003545:Cysteine; D002118:Calcium