Synthesis of surfactant-associated glycoprotein A by rat type II epithelial cells. Primary translation products and post-translational modification.
Surfactant-associated glycoproteins A, 38 (A3), 32 (A2) and 26 (A1) kDa, pI (4.2-4.8), were identified as related proteins present in surfactant isolated from rat lung lavage fluid. Differences in size and charge among surfactant-associated glycoproteins A were related to differences in glycosylation as determined by reduction of the larger forms (38 and 32 kDa) to 26 kDa by endoglycosidase F and by increased isoelectric points of the glycosylated forms after treatment with neuraminidase. Synthesis and secretion of surfactant-associated glycoproteins A and precursors were demonstrated in purified rat Type II epithelial cells by immunoprecipitation of [35S]methionine-labelled proteins with anti-surfactant-associated glycoprotein A antisera. In pulse-chase experiments, labelled proteins 26-34 kDa, appeared within 10 min and smaller forms co-migrated with surfactant-associated glycoprotein A from alveolar lavage. The relative abundance of the larger molecular mass forms (30-34 kDa, pI 4.8) increased at later times up to 3 h. More acidic mature forms, which co-migrated with surfactant-associated glycoproteins A2 and A3 in surfactant (38 and 32 kDa), were readily detectable in the media, but were not abundant forms in lysates of labelled Type II cells after 1-3 h of incubation. Primary translation products of surfactant-associated glycoprotein A were immunoprecipitated with monospecific anti-surfactant-associated glycoprotein A antiserum after in vitro translation of poly(A)+ mRNA isolated from adult rat lung. The immunoprecipitated translation product migrated at 26 kDa, pI 4.8, and migrated slightly faster than surfactant-associated glycoprotein A1 from surfactant. Treatment of surfactant-associated glycoprotein A with bacterial collagenase resulted in proteolytic fragments 23-20 kDa, pI 4.2-4.8, which no longer underwent sulfhydryl-dependent cross-linking, suggesting that the collagen-like domain was required for the sulfhydryl-dependent oligomerization. Surfactant-associated glycoproteins A are synthesized by rat Type II epithelial cells as pre-proteins, 26-34 kDa. Larger forms result primarily from N-linked glycosylation of the 26 kDa primary translation product. Mature, more acidic forms result from further addition of sialic acid.
Whitsett
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JA
Weaver
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Hull
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Ross
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Dion
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D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D006026:Glycoside Hydrolases / Q000378:metabolism; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008715:Methionine / Q000378:metabolism; D003012:Microbial Collagenase / Q000378:metabolism; D008970:Molecular Weight; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011650:Pulmonary Alveoli / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D006023:Glycoproteins; D012333:RNA, Messenger; D011061:Poly A; D008715:Methionine; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D003012:Microbial Collagenase