Expression and transport of rabbit surfactant protein A in COS-1 cells.
SV40-transformed green monkey kidney (COS-1) cells were transfected with expression plasmids that contained either the structural gene or cDNA for surfactant protein A (SP-A), a major protein of rabbit lung surfactant. The transfected COS-1 cells synthesized several isoforms of SP-A that were found to be less acidic than those produced in rabbit lung tissue. SP-A species with apparent molecular weight (M(r)) approximately equal to 29,000-33,000 were detected in the transfected cells, whereas glycosylated forms with apparent M(r) approximately equal to 33,000-38,000 were detectable only in the culture medium. Analysis of transfected cells by indirect immunofluorescence revealed that SP-A was localized in punctate bodies throughout the cytoplasm. Expressed SP-A was not detectable on the cell surface nor was there evidence that secreted SP-A was endocytosed by COS-1 cells. After subcellular fractionation of the transfected COS-1 cells, SP-A was found to be localized predominantly in the 5,000- and 18,000-g pellet fractions; little or no immunoreactive SP-A was detectable in cytosolic fractions. Treatment of transfected cells with the glycosylation inhibitor tunicamycin prevented secretion of SP-A into the medium, suggesting a role of glycosylation in secretion of SP-A. On the other hand, treatment of transfected cells with inhibitors of proline hydroxylation, which may cause destabilization of the collagen-like domain of SP-A, reduced but did not prevent secretion of SP-A into the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Alcorn
J L
JL
Department of Biochemistry, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas 75235.
Chen
Q
Q
Boggaram
V
V
Mendelson
C R
CR
D000818:Animals; D001692:Biological Transport; D002461:Cell Line, Transformed; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000378:metabolism; D006900:Hydroxylation; D015151:Immunoblotting; D011392:Proline / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism*; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000378:metabolism*; D011817:Rabbits; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution; D014415:Tunicamycin / Q000494:pharmacology
D006023:Glycoproteins; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D014415:Tunicamycin; D011392:Proline