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 encoding the SP-B preproprotein (SP-B), COOH-terminally deleted SP-B (SP-BDeltaC), the NH2-terminal propeptide (SP-BN), and a chimeric molecule consisting of albumin and the mature peptide (ALB/SP-BM) were transfected into AtT-20 and PC12 cells. Pulse-chase studies demonstrated that 10-30% of SP-B and SP-BDeltaC remained in cells in an endoglycosidase H-resistant form. Secretion of stored SP-B was stimulated by forskolin/12-O-tetradecanoylphorbol-13-acetate and intracellular SP-B was localized to secretory granules by immunoelectron microscopy. In contrast, SP-BN and ALB/SP-BM were constitutively secreted and not detected in secretory granules. Specific processing of SP-B was not detected in either AtT-20 or PC12 cells. Expression of SP-BDeltaC in transgenic mice resulted in secretion of fully processed mature SP-B, indicating correct processing and targeting of this construct in vivo. We conclude 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 peptide 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.
Lin
S
S
Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039, USA.
Akinbi
H T
HT
Breslin
J S
JS
Weaver
T E
TE
D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016716:PC12 Cells; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011510:Proteolipids / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000378:metabolism / Q000648:ultrastructure; D011663:Pulmonary Surfactants / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D017931:DNA Primers; D011498:Protein Precursors; D011510:Proteolipids; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins