The lung lectin surfactant protein A aggregates phospholipid vesicles via a novel mechanism.
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ABSTRACT:
SUBMITTER: Haagsman HP
PROVIDER: S-EPMC1150045 | biostudies-other | 1991 Apr
REPOSITORIES: biostudies-other
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Project description:Breathing depends on pulmonary surfactant, a mixture of phospholipids and proteins, secreted by alveolar type II cells. Surfactant requires lamellar bodies (LBs), organelles containing densely packed concentric membrane layers, for storage and secretion. LB biogenesis remains mysterious but requires surfactant protein B (SP-B), which is synthesized as a precursor (pre-proSP-B) that is cleaved during trafficking into three related proteins. Here, we elucidate the functions and cooperation of these proteins in LB formation. We show that the N-terminal domain of proSP-B is a phospholipid-binding and -transfer protein whose activities are required for proSP-B export from the endoplasmic reticulum (ER) and sorting to LBs, the conversion of proSP-B into lipoprotein particles, and neonatal viability in mice. The C-terminal domain facilitates ER export of proSP-B. The mature middle domain, generated after proteolytic cleavage of proSP-B, generates the striking membrane layers characteristic of LBs. Together, our results lead to a mechanistic model of LB biogenesis.
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Project description:The fluorescence characteristics of surfactant protein A (SP-A) from porcine and human bronchoalveolar lavage were determined in the presence and absence of lipids. After excitation at either 275 or 295 nm, the fluorescence emission spectrum of both proteins was characterized by two maxima at about 326 and 337 nm, indicating heterogeneity in the emission of the two tryptophan residues of SP-A, and also revealing a partially buried character for these fluorophores. Interaction of both human and porcine SP-A with various phospholipid vesicles resulted in an increase in the fluorescence emission of tryptophan without any shift in the emission wavelength maxima. This change in intrinsic fluorescence was found to be more pronounced in the presence of dipalmitoyl phosphatidylcholine (DPPC) than with dipalmitoyl phosphatidylglycerol (DPPG), DPPC/DPPG (7:3, w/w) and 1-palmitoyl-sn-glycerol-3-phosphocholine (LPC). Intrinsic fluorescence of SP-A was almost completely unaffected in the presence of egg phosphatidylcholine (egg-PC). In addition, we demonstrated a shielding of the tryptophan fluorescence from quenching by acrylamide on interaction of porcine SP-A with DPPC, DPPG or LPC. This shielding was most pronounced in the presence of DPPC. In the case of human SP-A, shielding was only observed on interaction with DPPC. From the intrinsic fluorescence measurements as well as from the quenching experiments, we concluded that the interaction of some phospholipid vesicles with SP-A produces a conformational change on the protein molecule and that the interaction of SP-A with DPPC is stronger than with other phospholipids. This interaction appeared to be independent of Ca2+ ions. Physiological ionic strength was found to be required for the interaction of SP-A with negatively charged vesicles of either DPPG or DPPC/DPPG (7:3, w/w). Intrinsic fluorescence of SP-A was sensitive to the physical state of the DPPC vesicles. The increase in intrinsic fluorescence of SP-A in the presence of DPPC vesicles was much stronger when the vesicles were in the gel state than when they were in the liquid-crystalline state. The effect produced by SP-A on the lipid vesicles was also dependent on temperature. The aggregation of DPPC, DPPC/DPPG (7:3, w/w) or dimyristoyl phosphatidylglycerol (DMPG) was many times higher below the phase-transition temperature of the corresponding phospholipids. These results strongly indicate that the interaction of SP-A with phospholipid vesicles requires the lipids to be in the gel phase.
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Project description:Pulmonary surfactant protein B (SP-B) facilitates the rapid transfer of phospholipids from bilayer stores into air-liquid interfacial films along the breathing cycle, and contributes to the formation of a surface-associated multilayer reservoir of surfactant to optimize the stability of the respiratory interface. To obtain more insights into the mechanisms underlying this transfer and multilayer formation, we established a simple model system that captures different features of SP-B action. We monitored the formation of supported planar bilayers from the collapse of intact phospholipid vesicles on a silica surface using a technique called quartz crystal microbalance with dissipation, which provides information on changes in membrane thickness and viscosity. At physiologically relevant concentrations, SP-B dramatically alters vesicle collapse. This manifests itself as a reduced buildup of intact vesicles on the surface before collapse, and allows the stepwise buildup of multilayered deposits. Accumulation of lipids in these multilayer deposits requires the presence of SP-B in both the receptor and the arriving membranes, surrounded by a comparable phospholipid charge. Thus, the quartz crystal microbalance with dissipation system provides a useful, simplified way to mimic the effect of surfactant protein on vesicle dynamics and permits a detailed characterization of the parameters governing reorganization of surfactant layers.
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Project description:Surfactant protein D (SP-D) is a soluble C-type lectin, belonging to the collectin (collagen-containing calcium-dependent lectin) family, which acts as an innate immune pattern recognition molecule in the lungs at other mucosal surfaces. Immune regulation and surfactant homeostasis are salient functions of SP-D. SP-D can bind to a range of viral, bacterial, and fungal pathogens and trigger clearance mechanisms. SP-D binds to gp120, the envelope protein expressed on HIV-1, through its C-type lectin or carbohydrate recognition domain. This is of importance since SP-D is secreted by human mucosal epithelial cells and is present in the female reproductive tract, including vagina. Another C-type lectin, dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), present on the surface of the DCs, also binds to HIV-1 gp120 and facilitates viral transfer to the lymphoid tissues. DCs are also present at the site of HIV-1 entry, embedded in vaginal or rectal mucosa. In the present study, we report a direct protein-protein interaction between recombinant forms of SP-D (rfhSP-D) and DC-SIGN via their C-type lectin domains. Both SP-D and DC-SIGN competed for binding to immobilized HIV-1 gp120. Pre-incubation of human embryonic kidney cells expressing surface DC-SIGN with rfhSP-D significantly inhibited the HIV-1 transfer to activated peripheral blood mononuclear cells. In silico analysis revealed that SP-D and gp120 may occupy same sites on DC-SIGN, which may explain the reduced transfer of HIV-1. In summary, we demonstrate, for the first time, that DC-SIGN is a novel binding partner of SP-D, and this interaction can modulate HIV-1 capture and transfer to CD4+ T cells. In addition, the present study also reveals a novel and distinct mechanism of host defense by SP-D against HIV-1.
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Project description:In this letter, we report a facile method to prepare robust phospholipid vesicles using commonly available phospholipids that are stabilized via the formation of an interpenetrating, acid-labile, cross-linked polymer network that imparts a site for controlled polymer destabilization and subsequent vesicle degradation. The polymer network was formed in the inner lamella of the phospholipid bilayer using 2,2-di(methacryloyloxy-1-ethoxy)propane (DMOEP) and butyl methacrylate (BMA). Upon exposure to acidic conditions, the highly cross-linked polymer network was partially converted to smaller linear polymers, resulting in substantially reduced vesicle stability upon exposure to chemical and physical insults. Isolated polymers had pH-dependent-solubility in THF. Transmission electron microscopy and dynamic light scattering revealed time-dependent enhanced vesicle stability in high concentrations of surfactant and vacuum conditions at elevated pH, whereas exposure to acidic pH rapidly decreased the vesicle stability, with complete destabilization observed in less than 24 h. The resultant transiently stabilized vesicles may prove useful for enhanced drug delivery and chemical sensing applications and allow for improved physiological clearance.
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Project description:Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL(4) is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL(4) is helical in a lipid bilayer environment, but its exact secondary structure and orientation within the bilayer remain controversial. To investigate the partitioning and dynamics of KL(4) in phospholipid bilayers, we introduced CD(3)-enriched leucines at four positions along the peptide to serve as probes of side chain dynamics via (2)H solid-state NMR. The chosen labels allow distinction between models of helical secondary structure as well as between a transmembrane orientation or partitioning in the plane of the lipid leaflets. Leucine side chains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL(4) in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL(4) lying in the plane of the bilayers and adopting an unusual helical structure which confers amphipathicity and allows partitioning into the lipid hydrophobic interior. At physiologic temperatures, the partitioning depth and dynamics of the peptide are dependent on the degree of saturation present in the lipids. The deeper partitioning of KL(4) relative to antimicrobial amphipathic alpha-helices leads to negative membrane curvature strain as evidenced by the formation of hexagonal phase structures in a POPE/POPG phospholipid mixture on addition of KL(4). The unusual secondary structure of KL(4) and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.
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