Am. J. Respir. Cell Mol. Biol., Volume 24, Number 3, March, 2001 253-263

Post-Translational Processing of Surfactant Protein-C Proprotein
Targeting Motifs in the NH₂-Terminal Flanking Domain Are Cleaved in Late Compartments

Amy L. Johnson, Paola Braidotti, Giuseppe G. Pietra, Scott J. Russo, Albert Kabore, Wen-Jing Wang, and Michael F. Beers

Lung Epithelial Cell Biology Laboratories, Pulmonary and Critical Care Division, Department of Medicine; Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; Department of Pathology, School of Medicine, San Paolo Hospital, Milan, Italy

Rat surfactant protein (SP)-C is a 3.7-kD hydrophobic lung-specific protein generated from proteolytic processing of a 21-kD propeptide (SP-C₂₁). We have demonstrated that initial post-translational processing of SP-C₂₁ involves two cleavages of the COOH-terminus (Beers and colleagues, J. Biol. Chem. 1994;269:20318-20328). The goal of the current study was to define processing and function of the NH₂-terminal flanking domain. Epitope-specific antisera directed against spatially distinct regions of the NH₂ terminus, NPROSP-C^2-9 (epitope = D²-L⁹) and NPROSP-C^11-23 (= E¹¹-Q²³) were produced. By Western blotting, both antisera identified SP-C₂₁ in microsomes. A 6-kD form (SP-C₆), enriched in lamellar bodies (LBs), was detected only by NPROSP-C^11-23 and not extractable with NaCO₃ treatment. Immunogold staining of ultrathin lung sections with NPROSP-C^11-23 identified proSP-C in both multivesicular bodies (mvb) and LBs whereas NPROSP-C^2-9 labeled only mvb. ³⁵S-pulse chase analysis demonstrated synthesis of SP-C₂₁ and three intermediate forms (SP-C₁₆, SP-C₇, and SP-C₆). Complete processing involved four separate cleavages with a precursor- product relationship between the low molecular weight forms SP-C₇ and SP-C₆. Fluorescence microscopy of A549 cells expressing fusion proteins of enhanced green fluorescent protein (EGFP) and proSP-C NH₂-terminal deletion mutants showed targeting of EGFP/SP-C^1-194 and EGFP/SP-C^10-194 to early endosomal antigen-1-negative, CD-63-positive cytoplasmic vesicles whereas EGFP/SP-C^19-194, EGFP/SP-C^10-18, and EGFP/SP-C^24-194 were restricted to the endoplasmic reticulum (ER). We conclude that synthetic processing includes a previously unrecognized cleavage of the proximal NH₂ terminus (M¹-L⁹), which occurs after removal of COOH-flanking domains (H⁵⁹-I¹⁹⁴) but before packaging in LBs, and that the region M¹⁰-T¹⁸ is required for targeting of proSP-C to post-ER vesicular compartments in the biosynthetic pathway.

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