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)