Requirements for Trafficking in the N-terminal Propeptide of Human Surfactant Protein B (SP-B)

Abstract 299 Poster Session IV, Tuesday, 5/4 (poster 321)

The 79 amino acid mature human SP-B protein is highly hydrophobic and initially synthesized as a large 381 amino acid precursor that undergoes extensive post-translational processing before entering the lamellar body. Previous work by Lin et al (J. Biol. Chem. 271:19689, 1996; Biochim. Biophys. Acta 1312:177, 1996) suggested that the N-terminus is required in its entirety for appropriate folding and trafficking of SP-B to the lamellar body. We have recently described a short segment of the N-terminus (NFlank) which is retained during processing of SP-B until the last cleavage step (AJPLung, 275(19):L559, 1998). To begin to examine the role of this retained peptide, we engineered plasmid DNA expressing portions of the proSP-B N-terminus as fusion peptides with green fluorescent protein (pEGFP). pNT-EGFP contains the entire aminoterminus and the first 5 amino acids of mature SP-B (Met¹-Leu²⁰⁵). pNP-EGFP is deleted of the NFlank and mature SP-B residues (Met¹-Pro¹⁸²). We used A549 cells and type 2 cells isolated from second trimester human fetal lung explants after 4 d of culture in the presence of glucocorticoid and cAMP in transfections using FuGene6. Under these culture conditions, type 2 cells retained SP-A, -B, and -C mRNAs through the study period. Cells were transfected with 5 µg of plasmid dNA/35 mm dish overnight and photographed using fluorescence microscopy at 24 and 48h (n=3 with duplicate dishes per plasmid). The unmodified pEGFP plasmid was used as a positive control and to assess transfection efficiency which was estimated at ∼5% for both A549 and type 2 cells. A549 and type 2 cells exhibited homogeneous cytoplasmic EGFP, which does not contain a signal peptide. In A549 cells, which do not possess a regulated secretory pathway, NT-EGFP was localized to the endoplasmic reticulum and Golgi, whereas NP-EGFP was localized only to the endoplasmic reticulum. In isolated type 2 cells by 48 h, NT-EGFP was found in vesicles in addition to endoplasmic reticulum and Golgi, whereas NP-EGFP was only found in endoplasmic reticulum. We conclude that a novel trafficking sequence contained within amino acids Pro¹⁸²-Leu²⁰⁵ of proSP-B may be required for targeting SP-B toward lamellar bodies.