Role of the degree of oligomerization in the structure and function of human surfactant protein A.
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.
Sánchez-Barbero
Fernando
F
Department of Biochemistry and Molecular Biology I, Complutense University of Madrid, 28040 Madrid, Spain.
Strassner
Jochen
J
García-Cañero
Rafael
R
Steinhilber
Wolfram
W
Casals
Cristina
C
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
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