Lung surfactant is a mixture of phospholipids, neutral lipids, and surfactant protein A (SP-A) 1 SP-B, SP-C, and SP-D, which are secreted into the air spaces by alveolar type II cells and Clara cells of the distal pulmonary epithelium (1). Although the primary function of surfactant is to reduce surface tension, the contribution of each molecular component to surface activity is not completely understood. Surfactant phospholipids form a film at the air-liquid interface that maintains air space patency by resisting compression as the alveolar radius decreases during expiration. Data from in vitro experiments, gene-targeted animals, and naturally occurring mutations in humans indicate that the hydrophobic surfactant proteins, SP-B and SP-C, participate in the assembly and biophysical properties of the surfactant film (2). The hydrophilic surfactant proteins, SP-A and SP-D, have a complex functional profile. The recognition that SP-A and SP-D are structurally homologous to mannosebinding protein has identified them as members of the collectin family of innate opsonins and directed attention to their host defense properties (3). Like mannose-binding protein, SP-A and SP-D bind to a wide range of microorganisms and enhance microbial phagocytosis and killing by alveolar macrophages. These in vitro activities appear to be physiologically relevant, since gene-targeted SP-A Ϫ/Ϫ and SP-D Ϫ/Ϫ mice clear microbial infections less effectively than pulmonary collectin-sufficient mice (4 -7). However, SP-A Ϫ/Ϫ and SP-D Ϫ/Ϫ mice also exhibit abnormalities of surfactant structure, metabolism and function (8 -10). Surfactant isolated from SP-A Ϫ/Ϫ mice does not contain the large aggregate tubular myelin and has impaired surface activity in the presence of plasma inhibitors (11). SP-D Ϫ/Ϫ mice develop progressive alveolar phospholipidosis and air space dilation (9, 10), associated with increased macrophage production of metalloproteinases and oxidant species (12). All of these defects are corrected by lung-specific expression of the cognate collectin in the SP-A Ϫ/Ϫ and SP-D Ϫ/Ϫ mice (13, 14). The structural basis of SP-A and SP-D surfactant functions has been explored by mutagenesis using in vitro and in vivo analyses. The primary structure of both proteins includes an N-terminal segment containing interchain linkages formed by Cys residues, a collagen-like region of Gly-X-Y repeats, a hydrophobic "neck" domain, and a carbohydrate recognition domain (CRD) (15,16). Trimeric association of subunits occurs by the folding of the collagen-like domains into triple helices (17) and coiled-coil bundling of ␣-helices in the neck (18). In the fully assembled molecules, the N-terminal sequences and di- ϩ/ϩ mice disrupted oligomeric assembly of the endogenous SP-D and produced air space dilation and foamy macrophage formation without phospholipidosis (24). These data suggested that the in vivo activity of SP-D is dependent on its oligomeric structure. The purpose of this study was to examine the role of the N-terminal segment-dependent olig...
We have reported that Gram-negative organisms decorated with rough lipopolysaccharide (LPS) are particularly susceptible to the direct antimicrobial actions of the pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D). In this study, we examined the lipid and LPS components required for the permeabilizing effects of the collectins on model bacterial membranes. Liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), with or without rough Escherichia coli LPS (J5), smooth E. coli LPS (B5), or cholesterol, were loaded with self-quenching probes and exposed to native or oxidatively modified SP-A. Fluorescence that resulted from permeabilization of liposomes and diffusion of dyes was assessed by microscopy or fluorimetry. Human SP-A and melittin increased the permeability of J5 LPS/POPE liposomes, but not B5 LPS/POPE liposomes or control (POPE only) liposomes. At a human SP-A concentration of 100 microg/mL, the permeability of the J5 LPS/POPE membranes increased 4.4-fold (p < 0.02) compared to the control with no added SP-A. Rat SP-A and SP-D also permeabilized the J5-containing liposomes. Incorporation of cholesterol into J5 LPS/POPE liposomes at a POPE:cholesterol molar ratio of 1:0.15 blocked human SP-A or melittin-induced permeability (p < 0.05) compared to cholesterol-free liposomes. Exposure of human SP-A to surfactant lipid peroxidation blocked the permeabilizing activity of the protein. We conclude that SP-A permeabilizes phospholipid membranes in an LPS-dependent and rough LPS-specific manner, that the effect is neither SP-A- nor species-specific, and that oxidative damage to SP-A abolishes its membrane destabilizing properties. Incorporation of cholesterol into the membrane enhances resistance to permeabilization by SP-A, most likely by increasing the packing density and membrane rigidity.
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