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Mechanism of Lamellar Body Formation by Lung Surfactant Protein B.

ABSTRACT: 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.

SUBMITTER: Sever N

PROVIDER: S-EPMC7797001 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Mechanism of Lamellar Body Formation by Lung Surfactant Protein B.

Sever Navdar N Miličić Goran G Bodnar Nicholas O NO Wu Xudong X Rapoport Tom A TA

Molecular cell 20201125 1

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 thes ...[more]

PMID: 33242393

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Project description:BackgroundThe pulmonary surfactant that lines the air-liquid surface within alveoli is a protein-lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecular and cellular mechanisms that regulate these processes are incompletely understood. We previously identified an essential role of general control of amino acid synthesis 5 like 1 (GCN5L1) and the biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1) in surfactant system development in zebrafish. Here, we explored the role of GCN5L1 in pulmonary surfactant regulation.MethodGCN5L1 knockout cell lines were generated with the CRISPR/Cas9 system. Cell viability was analyzed by MTT assay. Released surfactant proteins were measured by ELISA. Released surfactant lipids were measured based on coupled enzymatic reactions. Gene overexpression was mediated through lentivirus. The RNA levels were detected through RNA-sequencing (RNA-seq) and quantitative reverse transcription (qRT)- polymerase chain reaction (PCR). The protein levels were detected through western blotting. The cellular localization was analyzed by immunofluorescence. Morphology of the lamellar body was analyzed through transmission electron microscopy (TEM), Lysotracker staining, and BODIPY phosphatidylcholine labeling.ResultsKnocking out GCN5L1 in MLE-12 significantly decreased the release of surfactant proteins and lipids. We detected the downregulation of some surfactant-related genes and misregulation of the ROS-Erk-Foxo1-Cebpα axis in mutant cells. Modulating the activity of the axis or reconstructing the mitochondrial expression of GCN5L1 could partially restore the expression of these surfactant-related genes. We further showed that MLE-12 cells contained many LB-like organelles that were lipid enriched and positive for multiple LB markers. These organelles were smaller in size and accumulated in the absence of GCN5L1, indicating both biogenesis and trafficking defects. Accumulated endogenous surfactant protein (SP)-B or exogenously expressed SP-B/SP-C in adenosine triphosphate-binding cassette transporterA3 (ABCA3)-positive organelles was detected in mutant cells. GCN5L1 localized to the mitochondria and LBs. Reconstruction of mitochondrial GCN5L1 expression rescued the organelle morphology but failed to restore the trafficking defect and surfactant release, indicating specific roles associated with different subcellular localizations.ConclusionsIn summary, our study identified GCN5L1 as a new regulator of pulmonary surfactant that plays a role in the biogenesis and positioning/trafficking of surfactant-containing LBs.

Project description:This study sets out to investigate the effect of the presence of small water-soluble additives on the tunability of the surfactant gel-to-liquid crystalline (Lβ-Lα) phase transition temperature (T m) for a bilayer-forming cationic surfactant and the phase behavior of such surfactant systems on dilution. This is strongly driven by the fact that this type of cationic surfactant has many interesting unanswered scientific questions and has found applications in various areas such as consumer care, the petrochemical industry, food science, etc. The underlying surfactant/additive interactions and the interfacial behavior of lamellar surfactant systems including the surfactant deposition on surfaces can provide new avenues to develop novel product formulations. We have examined dioctadecyldimethyl ammonium chloride (DODAC) in the presence of small polar additives, with respect to the phase behavior upon dilution and the deposition on silica. Differential scanning calorimetry (DSC) is used to track the transition temperature, T m, and synchrotron and laboratory-based small and wide-angle X-ray scattering (SAXS and WAXS) were used to determine the self-assembled surfactant structure below and above the T m. DSC scans showed that upon dilution the additives could be removed from the surfactant bilayer which in turn tuned the T m. A spontaneous transition from a liquid crystalline (Lα) phase to a gel (Lβ) phase on dilution was demonstrated, which indicated that additives could be taken out from the Lα phase. By means of in situ null ellipsometry, the deposition of a diluted surfactant Lβ phase upon replacement of bulk solution by deionized water was followed. This technique enables time-resolved monitoring of the deposited surfactant layer thickness and adsorbed amount, which allows us to understand the deposition on surfaces. Robust layers at least one bilayer-thick were deposited onto the surface and shown to be irreversibly adsorbed due to poor surfactant solvency in water. The thickest layer of surfactant deposited after dilution was found for mixtures with small amounts of additive since high amounts might lead to a phase-separated system.

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Mechanism of Lamellar Body Formation by Lung Surfactant Protein B.

Publications

Mechanism of Lamellar Body Formation by Lung Surfactant Protein B.

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