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)) ≃ 29,000-33,000 were detected in the transfected cells, whereas glycosylated forms with apparent M(r) ≃ 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. When COS-1 cells were transfected with an expression plasmid containing the SP-A cDNA lacking coding sequences for seven NH2-terminal amino acids, the expressed truncated protein was localized predominantly in the cytosol and did not appear to be posttranslationally modified or secreted, suggesting the role of this region as part of a signal sequence.
|Original language||English (US)|
|Journal||American Journal of Physiology - Lung Cellular and Molecular Physiology|
|Issue number||4 6-4|
|Publication status||Published - 1992|
ASJC Scopus subject areas
- Cell Biology
- Pulmonary and Respiratory Medicine