Glycosylation and Secretion of Surfactant-associated Glycoprotein

Synthesis of glycoprotein A, the major surfactantassociated protein, was demonstrated in Type I1 epithelial cells isolated from rat lung. Predominant, secreted forms migrated as glycoproteins with asparagine-linked, complex-type oligosaccharides (32,00036,000 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 Fsensitive forms. These in vitro processed forms of glycoprotein A co-migrated with intracellular forms immunoprecipitated from [3SS]methionine-labeled, Type I1 cells. Pulse-chase experiments with [3SS]methioninelabeled cells demonstrated rapid synthesis of endoglycosidase H-sensitive precursors of 34,000 daltons, PI 4.7-4.8, which were neither secreted from Type I1 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 26,000dalton proteins which co-migrated with the nonglycosylated, surfactant-associated proteins AI 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 a-mannosidase 11, completely inhibited synthesis of the fully sialylated molecule. Swainsonine produced forms of glycoprotein A which were both neuraminidaseand 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 I1 cells rapidly synthesize and process surfactant-associated glycoprotein A precursors to endoglycosidase H-sensitive forms, which are slowly sialylated prior to secretion.