STRUCTURAL REQUIREMENTS FOR TARGETING OF SURFACTANT PROTEIN-B (SP-B) TO SECRETORY GRANULES IN-VITRO AND IN-VIVO

Human surfactant protein B (SP-B) is synthesized by type II cells as a 381-residue preproprotein which is proteolytically processed to a 79- residue mature peptide and targeted to lamellar bodies for secretion. To identify secretory granule targeting determinants, constructs encod ing the SP-B preproprotein (SP-B), COOH-terminally deleted SP-B (SP-B- Delta C), the NH2-terminal propeptide (SP-B-N), and a chimeric molecul e consisting of albumin and the mature peptide (ALB/SP-B-M) were trans fected into AtT-20 and PC12 cells. Pulse-chase studies demonstrated th at 10-30% of SP-B and SP-B-Delta C remained in cells in an endoglycosi dase H-resistant form. Secretion of stored SP-B was stimulated by fors kolin/12-O-tetradecanoylphorbol-13-acetate and intracellular SP-B was localized to secretory granules by immunoelectron microscopy. In contr ast, SP-B-N and ALB/SP-B-M were constitutively secreted and not detect ed in secretory granules. Specific processing of SP-B was not detected in either AtT-20 or PC12 cells. Expression of SP-B-Delta C in transge nic mice resulted in secretion of fully processed mature SP-B, indicat ing correct processing and targeting of this construct in vivo. We con clude that 1) SP-B processing occurs in a cell-specific manner, 2) the proprotein contains secretory granule targeting determinants that are not cell-specific, 3) the NH2-terminal propeptide and the mature pept ide are required for targeting SP-B to lamellar body, and 4) the COOH- terminal propeptide is not required for processing or sorting of SP-B.