Binding of rat and human surfactant proteins A and D to Aspergillus fumigatus conidia.
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Surfactant proteins A (SP-A) and D (SP-D) are thought to play important roles in pulmonary host defense. We investigated the interactions of rat and human SP-A and SP-D with Aspergillus fumigatus conidia. Rat SP-D but not rat SP-A bound the conidia, and the binding was inhibited by EDTA, mannose, glucose, maltose, and inositol. Binding studies using a mutant recombinant rat SP-D with altered carbohydrate recognition but normal structural organization clearly established a role for the carbohydrate recognition domain in binding to conidia. However, neither rat SP-A nor SP-D increased the association of fluorescein isothiocyanate-labeled conidia with rat alveolar macrophages as determined by flow cytometry. Both human SP-A (isolated from normal and alveolar proteinosis lungs) and SP-D (recombinant protein and protein isolated from alveolar proteinosis lungs) bound the conidia. These data indicate that important differences exist between rat and human SP-A in binding to certain fungi. Human SP-A and SP-D binding to conidia was also examined in the presence of hydrophobic surfactant components (HSC), containing both the phospholipid and hydrophobic proteins of surfactant. We found that HSC inhibited but did not eliminate human SP-A binding to Aspergillus conidia. In contrast, the SP-D binding to conidia was unaffected by HSC. These findings indicate that SP-D plays a major role in the recognition of Aspergillus conidia in alveolar fluid. (+info)
Regulation of protein phosphorylation and pathogen phagocytosis by surfactant protein A.
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Surfactant protein A (SP-A), a pulmonary member of the collectin family of proteins, facilitates the rapid clearance of pathogens by upregulating immune cell functions in the lungs. SP-A binds to bacteria and targets them for rapid phagocytosis by alveolar macrophages, but the mechanism by which this stimulation occurs is not clear. To characterize the intracellular events that may be involved, we examined the roles of protein phosphorylation and cytoskeletal polymerization in SP-A-stimulated phagocytosis. In rat alveolar macrophages, SP-A stimulated rapid tyrosine phosphorylation of specific proteins in a dose- and time-dependent manner. The pattern of proteins that were phosphorylated in response to SP-A, as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar to that observed for immunoglobulin G (IgG)-stimulated macrophages. Both SP-A and IgG stimulated increases in phagocytosis of Streptococcus pneumoniae above levels in the absence of added protein by 394% +/- 81% and 200% +/- 25%, respectively. Phagocytosis in both cases was dependent on tyrosine kinases, protein kinase C, and actin polymerization but not on microtubule activity. These studies show that SP-A utilizes pathways similar to those used by IgG to increase macrophage phagocytosis of bacteria. (+info)
Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development.
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In the mature mouse lung, the proximal-distal (P-D) axis is delineated by two distinct epithelial subpopulations: the proximal bronchiolar epithelium and the distal respiratory epithelium. Little is known about the signaling molecules that pattern the lung along the P-D axis. One candidate is Bone Morphogenetic Protein 4 (Bmp4), which is expressed in a dynamic pattern in the epithelial cells in the tips of growing lung buds. Previous studies in which Bmp4 was overexpressed in the lung endoderm (Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B. L. M. (1996) Development 122, 1693-1702) suggested that this factor plays an important role in lung morphogenesis. To further investigate this question, two complementary approaches were utilized to inhibit Bmp signaling in vivo. The Bmp antagonist Xnoggin and, independently, a dominant negative Bmp receptor (dnAlk6), were overexpressed using the surfactant protein C (Sp-C) promoter/enhancer. Inhibiting Bmp signaling results in a severe reduction in distal epithelial cell types and a concurrent increase in proximal cell types, as indicated by morphology and expression of marker genes, including the proximally expressed hepatocyte nuclear factor/forkhead homologue 4 (Hfh4) and Clara cell marker CC10, and the distal marker Sp-C. In addition, electron microscopy demonstrates the presence of ciliated cells, a proximal cell type, in the most peripheral regions of the transgenic lungs. We propose a model in which Bmp4 is a component of an apical signaling center controlling P-D patterning. Endodermal cells at the periphery of the lung, which are exposed to high levels of Bmp4, maintain or adopt a distal character, while cells receiving little or no Bmp4 signal initiate a proximal differentiation program. (+info)
Ultrastructural and protein analysis of surfactant in the Australian lungfish Neoceratodus forsteri: evidence for conservation of composition for 300 million years.
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The Australian lungfish Neoceratodus forsteri is the most primitive member of the lungfish family, with a surfactant lipid composition similar to the actinopterygiian fishes, which evolved 400 million years ago. We have analysed the proteins associated with surfactant isolated from lung lavage of this species, and used electron microscopy and immunohistochemistry to examine the surfactant structures and the subcellular localisation of these proteins. The epithelial lining of the gas-exchange region of the lungfish lung consists of one basic cell type, which has characteristics of both mammalian alveolar type I and type II cells and may be the common ancestor of both. It has long cytoplasmic plates containing microvilli, large osmiophilic bodies resembling mammalian lamellar bodies and a cytoplasm rich in metabolic organelles. Extracellular structures reminiscent of mammalian surfactant forms, but not including tubular myelin, were observed in the airspaces. Immunochemical analysis of the lungfish surfactant and lung tissue, using antibodies to human SP-A and SP-B, showed a similar staining pattern to human surfactant, indicating that SP-A- and SP-B-like proteins are present. Immunohistochemistry revealed that both SP-A and SP-B reactivity was present in the secretory cell osmiophilic bodies. In conclusion, our results suggest that, despite the great diversity in present day lung structures, a common cellular mechanism may have evolved to overcome fundamental problems associated with air-breathing. (+info)
The proteolipid of the A(1)A(0) ATP synthase from Methanococcus jannaschii has six predicted transmembrane helices but only two proton-translocating carboxyl groups.
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The proteolipid, a hydrophobic ATPase subunit essential for ion translocation, was purified from membranes of Methanococcus jannaschii by chloroform/methanol extraction and gel chromatography and was studied using molecular and biochemical techniques. Its apparent molecular mass as determined in SDS-polyacrylamide gel electrophoresis varied considerably with the conditions applied. The N-terminal sequence analysis made it possible to define the open reading frame and revealed that the gene is a triplication of the gene present in bacteria. In some of the proteolipids, the N-terminal methionine is excised. Consequently, two forms with molecular masses of 21,316 and 21,183 Da were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The molecular and biochemical data gave clear evidence that the mature proteolipid from M. jannaschii is a triplication of the 8-kDa proteolipid present in bacterial F(1)F(0) ATPases and most archaeal A(1)A(0) ATPases. Moreover, the triplicated form lacks a proton-translocating carboxyl group in the first of three pairs of transmembrane helices. This finding puts in question the current view of the evolution of H(+) ATPases and has important mechanistic consequences for the structure and function of H(+) ATPases in general. (+info)
Helical interactions and membrane disposition of the 16-kDa proteolipid subunit of the vacuolar H(+)-ATPase analyzed by cysteine replacement mutagenesis.
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Theoretical mechanisms of proton translocation by the vacuolar H(+)-ATPase require that a transmembrane acidic residue of the multicopy 16-kDa proteolipid subunit be exposed at the exterior surface of the membrane sector of the enzyme, contacting the lipid phase. However, structural support for this theoretical mechanism is lacking. To address this, we have used cysteine mutagenesis to produce a molecular model of the 16-kDa proteolipid complex. Transmembrane helical contacts were determined using oxidative cysteine cross-linking, and accessibility of cysteines to the lipid phase was determined by their reactivity to the lipid-soluble probe N-(1-pyrenyl)maleimide. A single model for organization of the four helices of each monomeric proteolipid was the best fit to the experimental data, with helix 1 lining a central pore and helix 2 and helix 3 immediately external to it and forming the principal intermolecular contacts. Helix 4, containing the crucial acidic residue, is peripheral to the complex. The model is consistent not only with theoretical proton transport mechanisms, but has structural similarity to the dodecameric ring complex formed by the related 8-kDa proteolipid of the F(1)F(0)-ATPase. This suggests some commonality between the proton translocating mechanisms of the vacuolar and F(1)F(0)-ATPases. (+info)
Interactions of pulmonary surfactant protein A with phospholipid monolayers change with pH.
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The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the pi-A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at the edges of condensed-expanded phase domains, and distributed evenly at lower concentration into the fluid phase with increasing pressure. At pH 4.5, TR-SP-A expanded DPPC monolayers to a slightly lower extent than at pH 7.4. It interacted primarily at the phase boundaries but it did not distribute into the fluid phase with increasing pressure. Films of DPPC/dipalmitoylphosphatidylglycerol (DPPG) 7:3 mol/mol were somewhat expanded by TR-SP-A at pH 7.4. The protein was distributed in aggregates only at the condensed-expanded phase boundaries at all surface pressures. At pH 4.5 TR-SP-A caused no expansion of the pi-A isotherm of DPPC/DPPG, but its fluorescence was relatively homogeneously distributed throughout the expanded phase at all pressures studied. These observations can be explained by a combination of factors including the preference for SP-A aggregates to enter monolayers at packing dislocations and their disaggregation in the presence of lipid under increasing pressure, together with the influence of pH on the aggregation state of SP-A and the interaction of SP-A with zwitterionic and acidic lipid. (+info)
Dissociation of surfactant protein B from canine surfactant large aggregates during formation of small surfactant aggregates by in vitro surface area cycling.
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Pulmonary surfactant isolated by lavage can be separated into large aggregates (LA) and small aggregates (SA). Pulse labeling experiments have shown that the LA subtype is the precursor of the SA subtype. Conversion of LA to SA can be demonstrated in vitro using the technique of surface area cycling. The precise mechanisms of surfactant subtype conversion remain unknown. We have previously reported a decline in surfactant-associated protein B (SP-B) during in vitro subtype conversion of canine surfactant. This led to the hypothesis that SP-B may be degraded by a serine protease 'convertase' during cycling. The current studies used a quantitative slot-blot assay to investigate the fates of SP-A and SP-B during in vitro cycling. These studies confirmed some SP-A is present in SA, but SP-B is confirmed to LA. Conversion leads to an apparent loss of SP-B during cycling. However, SP-B can be recovered from the walls of polypropylene and Teflon tubes by washing with chloroform:methanol. Recovered SP-B migrated on non-reducing tricine gels as a single band with an apparent molecular weight of 17 kDa, corresponding to intact SP-B dimer. Reconstitution studies demonstrated that the recovered SP-B retained its surface active properties as determined on a pulsating bubble surfactometer. We conclude in vitro surface area cycling of canine LA results in the dissociation of SP-B from surfactant lipids resulting in an apparent decline in SP-B levels. (+info)