Effect of subtransition in 1,2-dipalmitoylphosphatidylcholine model membrane on the spin probe motion

The effect of subgel----gel phase transition (subtransition) on conventional electron spin resonance spectra of cholestane, fatty acid and alkylammonium-type spin probes has been studied in aqueous 1,2-dipalmitoyl-sn-phosphatidylcholine dispersions. The cooperative onset of the cholestane spin probe rotation about its long axis, with an effective correlation time of 2-3 ns, has been detected at a temperature coinciding with the calorimetric substransition, indicating onset of the host lipid rotational motion. The lipid rotation results in dissolution of the spin probe clusters in the host lipid. In the gel phase, the lateral distribution of impurity molecules is more isotropic than in the subgel phase.     

Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography.

X-ray interferometry/holography was applied to meridional x-ray diffraction data to determine uniquely the profile structures of a single monolayer of an integral membrane protein and a peripheral membrane protein, each tethered to the surface of a solid inorganic substrate. Bifunctional, organic self-assembled monolayers (SAMs) were utilized to tether the proteins to the surface of Ge/Si multilayer substrates, fabricated by molecular beam epitaxy, to facilitate the interferometric/holographic x-ray structure determination. The peripheral membrane protein yeast cytochrome c was covalently tethered to the surface of a sulfhydryl-terminated 11-siloxyundecanethiol SAM via a disulfide linkage with residue 102. The detergent-solubilized, photosynthetic reaction center integral membrane protein was electrostatically tethered to the surface of an analogous amine-terminated SAM. Optical absorption measurements performed on these two tethered protein monolayer systems were consistent with the x-ray diffraction results indicating the reversible formation of densely packed single monolayers of each fully functional membrane protein on the surface of the respective SAM. The importance of utilizing the organic self-assembled monolayers (as opposed to Langmuir-Blodgett) lies in their ability to tether specifically both soluble peripheral membrane proteins and detergent-solubilized integral membrane proteins. The vectorial orientations of the cytochrome c and the reaction center molecules were readily distinguishable in the profile structure of each monolayer at a spatial resolution of 7 A.     

Surfactant protein A regulates complement activation.

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.     

Interaction of amphiphilic chlorin-based photosensitizers with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monolayers

The drawbacks of the presently used photosensitizers include their relatively low selectivity toward cancer cells, and long-lasting accumulation in healthy tissues. Our recent results indicate that conjugating a photosensitizer with folic acid both enhances the active uptake by cells, and decreases the accumulation in healthy tissue. Here, the interaction between 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers used as model membranes, and three different photosensitizers were studied; the derivatives were the non-conjugated meta-tetrahydroxyphenylchlorin (m-THPC, CHL1) and tris(3-hydroxyphenyl)-4-carboxyphenylchlorin (CHL2), as well as a folic acid-conjugated m-THPC-like molecule (CHL3). The results obtained indicate that the folate moiety present in the conjugated derivative CHL3 is involved in the interaction with the phospholipid polar heads. This interaction may be responsible for a better miscibility of CHL3 with the DPPC films compared to CHL1 and CHL2, while elimination of CHL3 from the tissue may be due rather to specific, biological processes and not to its polarity.     

2D crystal forms of annexin IV on lipid monolayers.

Two-dimensional crystalline arrays of annexin IV were generated by interaction of the purified protein with a phospholipid monolayer. Image analysis of electron micrographs of the protein crystals, which diffracted to 3.5 nm respectively, revealed p6 and p3 symmetry. Annexin IV gave two crystal forms with unit cells of 18 x 18 nm and 28 x 28 nm. The former unit cell was similar to a previously described form of annexin VI. The implications of these observations are discussed.     

Surfactant protein A mediates pulmonary clearance of Staphylococcus aureus

Surfactant protein A has been shown to enhance opsonization and clearance of Staphylococcus aureus in vitro. Here, the phagocytosis of alveolar S. aureus was investigated in vivo using intravital microscopy. Fluorescence labelled S. aureus Newman cells were intratracheally administered to anesthetized mice and the alveolar surface was observed for fifteen minutes. Confirming previously reported in vitro data, surfactant protein A-deficient mice showed a significantly reduced uptake of bacteria compared to wild-type mice.     

Surfactant protein A regulates IgG-mediated phagocytosis in inflammatory neutrophils

Surfactant proteins (SP)-A and SP-D have been shown to affect the functions of a variety of innate immune cells and to interact with various immune proteins such as complement and immunoglobulins. The goal of the current study is to test the hypothesis that SP-A regulates IgG-mediated phagocytosis by neutrophils, which are major effector cells of the innate immune response that remove invading pathogens by phagocytosis and by extracellular killing mediated by reactive oxygen and nitrogen. We have previously shown that SP-A stimulates chemotaxis by inflammatory, but not peripheral, neutrophils. To evaluate the ability of SP-A to modulate IgG-mediated phagocytosis, polystyrene beads were coated with BSA and treated with anti-BSA IgG. SP-A significantly and specifically enhanced IgG-mediated phagocytosis by inflammatory neutrophils, but it had no effect on beads not treated with IgG. SP-A bound to IgG-coated beads and enhanced their uptake via direct interactions with the beads as well as direct interactions with the neutrophils. SP-A did not affect reactive oxygen production or binding of IgG to neutrophils and had modest effects on polymerization of actin. These data suggest that SP-A plays an important role in mediating the phagocytic response of neutrophils to IgG-opsonized particles.     

Methylation effects on the microdomain structures of phosphatidylethanolamine monolayers.

Increasing methylation of the headgroup in DPPE results in an increase of minimum area per molecule in highly compressed monolayers at the air-water interface. The shape of solid domains, as observed by epifluorescence microscopy, also exhibits marked changes upon increasing headgroup methylation. Branching domains are observed in DPPE and DP(Me)PE, whereas U-shaped or round domains are observed in DP(Me)2PE and DPPC under our experimental conditions. The domain shape is determined more by the headgroup methylatin than by the corresponding shift in critical temperatures, as shown by the study of PCs of different acyl chain moieties. In mixed lipid monolayers, PC (phosphatidylcholine) and PE (phosphatidylethanolamine) do not mix ideally, as indicated by the non-linear variation of the average area per molecule with composition, and by distinct domain shapes in LE/LC (liquid expanded/liquid condensed) coexisting phases representing PE-enriched or PC-enriched domains in those mixed monolayers.     

The effects of deimination of myelin basic protein on structures formed by its interaction with phosphoinositide-containing lipid monolayers

The recombinant 18.5-kDa charge isoform of murine myelin basic protein (rmMBP) is unmodified posttranslationally and was used to study the effects of deimination, i.e., the conversion of arginyl to citrullinyl residues, on the protein's interactions with itself and with lipids. The unmodified species rmMBP-Cit(0) (i.e., containing no citrullinyl residues) interacted with binary monolayers containing acidic (phosphatidylinositol) and nickel-chelating lipids to form paracrystalline arrays with 4.8-nm spacing. A sample of protein was deiminated to an average of 9 citrullinyl residues per molecule of protein, yielding rmMBP-Cit(9). Under both low- and high-salt conditions, this species formed better-ordered domains than rmMBP-Cit(0), viz., planar crystalline assemblies. Thus, deimination of MBP resulted in a significant alteration of its lipid-organizing and self-interaction properties that might be operative in myelin in vivo, especially in progression of the autoimmune disease multiple sclerosis. Comparisons of amino acid sequences indicated significant similarities of MBP with filaggrin, a protein that is deiminated in another autoimmune disease, rheumatoid arthritis, suggesting that comparable epitopes could be targeted in both pathologies. In contrast, binary lipid monolayers consisting of phosphatidylinositol-4-phosphate (or phosphatidylinositol-4,5-bisphosphate) and a nickel-chelating lipid formed helical tubular vesicular structures, which appeared to be induced and/or stabilized by rmMBP, especially in its deiminated form. Sequence comparisons with other actin- and phosphoinositide-binding proteins (vinculin, ActA, MARCKS) suggested that the carboxyl-terminal segment of MBP could form an amphipathic alpha helix and was the phosphoinositide binding site.     

Efficiency of detergents at maintaining membrane protein structures in their biologically relevant forms

High-resolution structural analysis of membrane proteins by X-ray crystallography or solution NMR spectroscopy often requires their solubilization in the membrane-mimetic environments of detergents. Yet the choice of a detergent suitable for a given study remains largely empirical. In the present work, we considered the micelle-crystallized structures of lactose permease (LacY), the sodium/galactose symporter (vSGLT), the vitamin B(12) transporter (BtuCD), and the arginine/agmatine antiporter (AdiC). Representative transmembrane (TM) segments were selected from these proteins based on their relative contact(s) with water, lipid, and/or within the protein, and were synthesized as Lys-tagged peptides. Each peptide was studied by circular dichroism and fluorescence spectroscopy in water, and in the presence of the detergents sodium dodecylsulfate (SDS, anionic); n-dodecyl phosphatidylcholine (DPC, zwitterionic); n-dodecyl-β-d-maltoside (DDM, neutral); and n-octyl-β-d-glucoside (OG, neutral, varying acyl tail length). We found that (i) the secondary structures of the TM segments were statistically indistinguishable in the four detergents studied; and (ii) a strong correlation exists between the extent of helical structure of each individual TM segment in detergents with its helicity level as it exists in the full-length protein, indicating that helix adoption is fundamentally the same in both environments. The denaturing properties of so-called 'harsh' detergents may thus largely be due to their interactions with non-membranous regions of proteins. Given the consistency of structural features observed for each TM segment in a variety of micellar media, the overall results suggest that the structure likely corresponds to its relevant biological form in the intact protein in its native lipid bilayer environment.     

Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex

Protein glutaminase, which converts a protein glutamine residue to a glutamate residue, is expected to be useful as a new food-processing enzyme. The crystal structures of the mature and pro forms of the enzyme were refined at 1.15 and 1.73 ? resolution, respectively. The overall structure of the mature enzyme has a weak homology to the core domain of human transglutaminase-2. The catalytic triad (Cys-His-Asp) common to transglutaminases and cysteine proteases is located in the bottom of the active site pocket. The structure of the recombinant pro form shows that a short loop between S2 and S3 in the proregion covers and interacts with the active site of the mature region, mimicking the protein substrate of the enzyme. Ala-47 is located just above the pocket of the active site. Two mutant structures (A47Q-1 and A47Q-2) refined at 1.5 ? resolution were found to correspond to the enzyme-substrate complex and an S-acyl intermediate. Based on these structures, the catalytic mechanism of protein glutaminase is proposed.     

Crystal structures of hydrogenase maturation protein HypE in the Apo and ATP-bound forms

The hydrogenase maturation protein HypE serves an essential function in the biosynthesis of the nitrile group, which is subsequently coordinated to Fe as CN(-) ligands in [Ni-Fe] hydrogenase. Here, we present the crystal structures of HypE from Desulfovibrio vulgaris Hildenborough in the presence and in the absence of ATP at a resolution of 2.0 A and 2.6 A, respectively. Comparison of the apo structure with the ATP-bound structure reveals that binding ATP causes an induced-fit movement of the N-terminal portion, but does not entail an overall structural change. The residue Cys341 at the C terminus, whose thiol group is supposed to be carbamoylated before the nitrile group synthesis, is completely buried within the protein and is located in the vicinity of the gamma-phosphate group of the bound ATP. This suggests that the catalytic reaction occurs in this configuration but that a conformational change is required for the carbamoylation of Cys341. A glutamate residue is found close to the thiol group as well, which is suggestive of deprotonation of the carbamoyl group at the beginning of the reactions.     

Phase transitions in 1,2-and 1,3-diacyl-sn-glycerol-3-phosphocholine monolayers at the oil/water interface

Moving the phosphatidylcholine group from the 3-to the 2-position in monolayers of $\text{distearoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ at the oil/water interface expands the surface pressure-area isotherm and markedly increases the surface pressure at which phase separation occurs with only a slight change in the monolayer surface density at the onset of the transition. This is interpreted in terms of a change in an ordering parameter in the solid-condensed state.     

Surfactant protein A differentially regulates peripheral and inflammatory neutrophil chemotaxis

Surfactant protein A (SP-A), a pulmonary lectin, plays an important role in regulating innate immune cell function. Besides accelerating pathogen clearance by pulmonary phagocytes, SP-A also stimulates alveolar macrophage chemotaxis and directed actin polymerization. We hypothesized that SP-A would also stimulate neutrophil chemotaxis. With the use of a Boyden chamber assay, we found that SP-A (0.5-25 microg/ml) did not stimulate chemotaxis of rat peripheral neutrophils or inflammatory bronchoalveolar lavage (BAL) neutrophils isolated from LPS-treated lungs. However, SP-A affected neutrophil chemotaxis toward the bacterial peptide formyl-met-leu-phe (fMLP). Surprisingly, the effect was different for the two neutrophil populations: SP-A reduced peripheral neutrophil chemotaxis toward fMLP (49 +/- 5% fMLP alone) and enhanced inflammatory BAL neutrophil chemotaxis (277 +/- 48% fMLP alone). This differential effect was not seen for the homologous proteins mannose binding lectin and complement protein 1q but was recapitulated by type IV collagen. SP-A bound both neutrophil populations comparably and did not alter formyl peptide binding. These data support a role for SP-A in regulating neutrophil migration in pulmonary tissue.     

Surfactant protein A enhances alveolar macrophage phagocytosis of apoptotic neutrophils

Surfactant protein A (SP-A) is an innate immune molecule that binds foreign organisms that invade the lungs and targets them for phagocytic clearance by the resident pulmonary phagocyte, the alveolar macrophage (AM). We hypothesized that SP-A binds to and enhances macrophage uptake of other nonself particles, specifically apoptotic polymorphonuclear neutrophils (PMNs). PMNs are recruited into the lungs during inflammation, but as inflammation is resolved, PMNs undergo apoptosis and are phagocytosed by AMs. We determined that SP-A increases AM phagocytosis of apoptotic PMNs 280 +/- 62% above the no protein control value. The increase is dose dependent, and heat-treated SP-A still enhanced uptake, whereas deglycosylated SP-A had significantly diminished ability to enhance phagocytosis. Surfactant protein D also increased phagocytosis of apoptotic PMNs by approximately 125%. However, other proteins that are structurally homologous to SP-A, mannose-binding lectin and complement protein 1q, did not. SP-A enhances phagocytosis via an opsonization-dependent mechanism and binds apoptotic PMNs approximately 4-fold more than viable PMNs. Also, binding of SP-A to apoptotic PMNs does not appear to involve SP-A's lectin domain. These data suggest that the pulmonary collectins SP-A and SP-D facilitate the resolution of inflammation by accelerating apoptotic PMN clearance.     

Surfactant protein A prevents silica-mediated toxicity to rat alveolar macrophages

Silicosis is a serious occupational lung disease associated with irreversible pulmonary fibrosis. The interaction between inhaled crystalline silica and the alveolar macrophage (AM) is thought to be a key event in the development of silicosis and fibrosis. Silica can cause direct injury to AMs and can induce AMs to release various inflammatory mediators. Acute silicosis is also characterized by a marked elevation in surfactant apoprotein A (SP-A); however, the role of SP-A in silicosis is unknown. We investigated whether SP-A directly affects the response of AMs to silica. In this study, the degree of silica toxicity to cultured rat AMs as assessed by a (51)Cr cytotoxicity assay was shown to be dependent on the time of exposure and the concentration and size of the silica particles. Silica directly injured rat AMs as evidenced by a cytotoxic index of 32.9 +/- 2.5, whereas the addition of rat SP-A (5 microg/ml) significantly reduced the cytotoxic index to 16.6 +/- 1.2 (P < 0. 001). This effect was reversed when SP-A was incubated with either polyclonal rabbit anti-rat SP-A antibody or D-mannose. These data indicate that SP-A mitigates the effect of silica on AM viability, and this effect may involve the carbohydrate recognition domain of SP-A. The elevation of SP-A in acute silicosis may serve as a normal host response to prevent lung cell injury after exposure to silica.     

Surfactant protein A is defective in abrogating inflammation in asthma

Surfactant protein A (SP-A) regulates a variety of immune cell functions. We determined the ability of SP-A derived from normal and asthmatic subjects to modulate the inflammatory response elicited by Mycoplasma pneumoniae, a pathogen known to exacerbate asthma. Fourteen asthmatic and 10 normal control subjects underwent bronchoscopy with airway brushing and bronchoalveolar lavage (BAL). Total SP-A was extracted from BAL. The ratio of SP-A1 to total SP-A (SP-A1/SP-A) and the binding of total SP-A to M. pneumoniae membranes were determined. Airway epithelial cells from subjects were exposed to either normal or asthmatic SP-A before exposure to M. pneumoniae. IL-8 protein and MUC5AC mRNA were measured. Total BAL SP-A concentration did not differ between groups, but the percentage SP-A1 was significantly increased in BAL of asthmatic compared with normal subjects. SP-A1/SP-A significantly correlated with maximum binding of total SP-A to M. pneumoniae, but only in asthma. SP-A derived from asthmatic subjects did not significantly attenuate IL-8 and MUC5AC in the setting of M. pneumoniae infection compared with SP-A derived from normal subjects. We conclude that SP-A derived from asthmatic subjects does not abrogate inflammation effectively, and this dysfunction may be modulated by SP-A1/SP-A.