Interaction of SP-A (surfactant protein A) with bacterial rough lipopolysaccharide (Re-LPS), and effects of SP-A on the binding of Re-LPS to CD14 and LPS-binding protein.
SP-A (surfactant protein A) is a lipid-binding collectin primarily involved in innate lung immunity. SP-A interacts with the bacterial rough LPS (lipopolysaccharide) Re-LPS (Re595 mutant of LPS from Salmonella minnesota), but not with smooth LPS. In the present study, we first examined the characteristics of the interaction of human SP-A with Re-LPS. Fluorescence intensity and anisotropy measurements of FITC-labelled Re-LPS in the presence and absence of SP-A indicated that SP-A bound to Re-LPS in solution in a Ca2+-independent manner, with a dissociation constant of 2.8x10(-8) M. In the presence of calcium, a high-mobility complex of SP-A and [3H]Rb-LPS (Rb mutant of LPS from Escherichia coli strain LCD 25) micelles was formed, as detected by sucrose density gradients. Re-LPS aggregation induced by SP-A was further characterized by light scattering. On the other hand, human SP-A inhibited TNF-alpha (tumour necrosis factor-alpha) secretion by human macrophage-like U937 cells stimulated with either Re-LPS or smooth LPS. We further examined the effects of human SP-A on the binding of Re-LPS to LBP (LPS-binding protein) and CD14. SP-A decreased the binding of Re-LPS to CD14, but not to LBP, as detected by cross-linking experiments with 125I-ASD-Re-LPS [125I-labelled sulphosuccinimidyl-2-(p-azidosalicylamido)-1,3-dithiopropionate derivative of Re-LPS] and fluorescence analysis with FITC-Re-LPS. When SP-A, LBP and CD14 were incubated together, SP-A reduced the ability of LBP to transfer 125I-ASD-Re-LPS to CD14. These SP-A effects were not due to the ability of SP-A to aggregate Re-LPS in the presence of calcium, since they were observed in both the absence and the presence of calcium. These studies suggest that SP-A could contribute to modulate Re-LPS responses by altering the competence of the LBP-CD14 receptor complex.
García-Verdugo
Ignacio
I
Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University of Madrid, 28040-Madrid, Spain.
Sánchez-Barbero
Fernando
F
Soldau
Katrin
K
Tobias
Peter S
PS
Casals
Cristina
C
D000209:Acute-Phase Proteins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008823:Micelles; D009154:Mutation; D011485:Protein Binding / Q000187:drug effects; D037662:Pulmonary Surfactant-Associated Protein A / Q000378:metabolism* / Q000494:pharmacology; D012475:Salmonella / Q000378:metabolism; D020298:U937 Cells; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D002118:Calcium; D002352:Carrier Proteins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008823:Micelles; D037662:Pulmonary Surfactant-Associated Protein A; D000099231:Lipopolysaccharide-Binding Protein