title: Glycosylation and secretion of surfactant-associated glycoprotein A.
abstract: Synthesis of glycoprotein A, the major surfactant-associated protein, was demonstrated in Type II epithelial cells isolated from rat lung. Predominant, secreted forms migrated as glycoproteins with asparagine-linked, complex-type oligosaccharides (32,000-36,000 daltons, pI 4.2-4.8). Primary in vitro translation products of the glycoprotein migrated as five distinct proteins of approximately 26,000 daltons which were processed by pancreatic microsomal membranes in vitro to 30,000-34,000-dalton, endoglycosidase F-sensitive forms. These in vitro processed forms of glycoprotein A co-migrated with intracellular forms immunoprecipitated from [35S]methionine-labeled, Type II cells. Pulse-chase experiments with [35S]methionine-labeled cells demonstrated rapid synthesis of endoglycosidase H-sensitive precursors of 34,000 daltons, pI 4.7-4.8, which were neither secreted from Type II cells nor detected in surfactant from alveolar lavage. These high-mannose forms were slowly processed to more acidic, endoglycosidase H-resistant, neuraminidase-sensitive forms. At between 10 and 180 min, fully sialylated or other endoglycosidase H-resistant forms were a minor fraction of intracellular glycoprotein A. After 16 h, intracellular glycoproteins A were primarily present as endoglycosidase H-resistant forms. Secretion of mature, sialylated, glycoprotein A was first detected 1 h after labeling, and was also readily detected after 16-24 h chase period. Tunicamycin, which blocks N-linked protein glycosylation, resulted in synthesis of three major 26,000-dalton proteins which co-migrated with the nonglycosylated, surfactant-associated proteins A1 present in surfactant from alveolar lavage and with the major in vitro translation products of rat lung poly(A+) mRNA. Tunicamycin inhibited secretion of glycoprotein A. Swainsonine, which inhibits Golgi alpha-mannosidase II, completely inhibited synthesis of the fully sialylated molecule. Swainsonine produced forms of glycoprotein A which were both neuraminidase- and endoglycosidase H-sensitive and were readily secreted. Monensin, an ionophore that alters protein transport, markedly inhibited intracellular sialylation and secretion. These studies demonstrate that pulmonary Type II cells rapidly synthesize and process surfactant-associated glycoprotein A precursors to endoglycosidase H-sensitive forms, which are slowly sialylated prior to secretion.
authors.forename: J A
authors.initials: JA
authors.lastname: Whitsett
authors.forename: G
authors.initials: G
authors.lastname: Ross
authors.forename: T
authors.initials: T
authors.lastname: Weaver
authors.forename: W
authors.initials: W
authors.lastname: Rice
authors.forename: C
authors.initials: C
authors.lastname: Dion
authors.forename: W
authors.initials: W
authors.lastname: Hull
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002241:Carbohydrates / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000032:analysis; D006596:Hexosaminidases / Q000378:metabolism; D007526:Isoelectric Point; D008168:Lung / Q000166:cytology; D008361:Mannosidases / Q000037:antagonists & inhibitors; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D009439:Neuraminidase / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D011510:Proteolipids / Q000032:analysis* / Q000378:metabolism; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000032:analysis* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D017026:Swainsonine; D014415:Tunicamycin / Q000494:pharmacology; D043323:alpha-Mannosidase
chemical_list: D000470:Alkaloids; D002241:Carbohydrates; D009844:Oligosaccharides; D011510:Proteolipids; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D012333:RNA, Messenger; D014415:Tunicamycin; D011061:Poly A; D008985:Monensin; D006596:Hexosaminidases; D008361:Mannosidases; D009439:Neuraminidase; D043323:alpha-Mannosidase; D017026:Swainsonine