Journal Article
Characterization of alpha-soluble N-ethylmaleimide-sensitive fusion attachment protein in alveolar type II cells: implications in lung surfactant secretion
Abonyo, BO; Wang, P; Narasaraju, TA; Rowan, WH; Mcmillan, DH; Zimmerman, UJ; Liu, L
American Journal of Respiratory Cell and Molecular Biology
ISSN: 1044-1549
EISSN: 1535-4989
Am J Respir Cell Mol Biol.
N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (alpha-SNAP) are thought to be soluble factors that transiently bind and disassemble SNAP receptor complex during exocytosis in neuronal and endocrine cells. Lung surfactant is secreted via exocytosis of lamellar bodies from alveolar epithelial type II cells. However, the secretion of lung surfactant is a relatively slow process, and involvement of SNAP receptor and its cofactors (NSF and alpha-SNAP) in this process has not been demonstrated. In this study, we investigated a possible role of alpha-SNAP in surfactant secretion. alpha-SNAP was predominantly associated with the membranes in alveolar type II cells as determined by Western blot and immunocytochemical analysis using confocal microscope. Membrane-associated alpha-SNAP was not released from the membrane fraction when the cells were lyzed in the presence of Ca2+ or Mg2+ATP. The alkaline condition (0.1 M Na2CO3, pH 12), known to extract peripheral membrane proteins also failed to release it from the membrane. Phase separation using Triton X-114 showed that alpha-SNAP partitioned into both aqueous and detergent phases. NSF had membrane-bound characteristics similar to alpha-SNAP in type II cells. Permeabilization of type II cells with beta-escin resulted in a partial loss of alpha-SNAP from the cells, but cellular NSF was relatively unchanged. Addition of exogenous alpha-SNAP to the permeabilized cells increased surfactant secretion in a dose-dependent manner, whereas exogenous NSF has much less effects. An alpha-SNAP antisense oligonucleotide decreased its protein level and inhibited surfactant secretion. Our results suggest a role of alpha-SNAP in lung surfactant secretion.
Adenosine Triphosphate/chemistry/metabolism; Animals; Blotting, Western; Calcium/metabolism; Carrier Proteins/chemistry; Cell Membrane/metabolism; Cytosol/metabolism; Detergents/pharmacology; Dose-Response Relationship, Drug; Enzyme Inhibitors/pharmacology; Escin/metabolism; Ethylmaleimide/pharmacology; Formaldehyde/pharmacology; Hydrogen-Ion Concentration; Immunohistochemistry; Magnesium/metabolism; Membrane Proteins/chemistry; Microscopy, Confocal; Microscopy, Fluorescence; Neurons/metabolism; Oligonucleotides, Antisense/pharmacology; Polyethylene Glycols/pharmacology; Polymers/pharmacology; Protein Binding; Pulmonary Alveoli/cytology; Rats, Sprague-Dawley; Recombinant Fusion Proteins/metabolism; Recombinant Proteins/metabolism; Sodium Dodecyl Sulfate/pharmacology; Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; Sulfhydryl Reagents/pharmacology; Surface-Active Agents/metabolism; Vesicular Transport Proteins; 1HG84L3525; 368GB5141J; 3WJQ0SDW1A; 6805-41-0; 8L70Q75FXE; 9036-19-5; I38ZP9992A; O3C74ACM9V; SY7Q814VUP; Y19UC83H8E