Inhibitors Elucidate Processing of Human Surfactant Protein-B (SP-B)

Abstract 314 Poster Session IV, Tuesday, 5/4 (poster 320)

SP-B is a 79 amino acid hydrophobic protein derived from a 381 amino acid precursor by a series of cleavages of the N- and C-terminal propeptides. Complete processing of SP-B is type 2 cell specific and linked temporally to the appearance of lamellar bodies in the developing fetal lung. The subcellular localization of SP-B processing is not fully understood. We used cultured (5d) second trimester human fetal lung tissue as a model of differentiated type 2 cells to clarify the localization of events in SP-B processing. We pulse:chase labelled lung explants (n=5) with ³⁵S-met/cys (200 µCi/ml) in the presence and absence of brefeldin A (10-20 µg/ml) which causes a redistribution of the cisGolgi to the endoplasmic reticulum and prevents distal protein modifications, or monensin (2 µM), which prevents endosomal acidification and disrupts Golgi export. We found that immunoprecipitation of labelled proteins using a polyclonal antibody to 8kD human SP-B (hSP-B) in the presence of brefeldin A prevented the first processing step from 42 to 25 kD SP-B. In contrast, monensin delayed the second cleavage step from 25 to 9 kD SP-B and prevented processing of 9 kD to 8 kD SP-B. To further localize SP-B cleavage events within lung explants, we used fluorophore-conjugated epitope-specific antisera to regions within the N- and C-terminal propeptides (NFPROX Ser¹⁴⁵-Leu¹⁶⁰; NFLANK Gln¹⁸⁶-Gln²⁰⁰; CFLANK Gly²⁸⁴-Ser³⁰⁴)in double labeling with a polyclonal antibody to MG160 identified with a Cy3-conjugated secondary. In the absence of brefeldin A, staining of MG160 (a medial Golgi resident protein) colocalized with CFLANK and NFLANK but not NFPROX or hSP-B. In the presence of brefeldin A, MG160 staining condensed to the microtubule processing region of type 2 cells and no longer colocalized with the CFLANK and NFLANK antisera. CFLANK staining was dispersed, characteristic of cisGolgi residence, whereas NFLANK staining remained confined to small vesicles. We conclude that the initial aminoterminal cleavage of proSP-B to a 25 kD intermediate is a cisGolgi event whereas the cleavage of the C-terminus is a late Golgi event, resulting in the 9 kD intermediate localizing to an endosomal compartment, possibly the multivesicular body.