Post-translational processing of surfactant protein-C proprotein: targeting motifs in the NH(2)-terminal flanking domain are cleaved in late compartments.
Rat surfactant protein (SP)-C is a 3.7-kD hydrophobic lung-specific protein generated from proteolytic processing of a 21-kD propeptide (SP-C(21)). We have demonstrated that initial post-translational processing of SP-C(21) involves two cleavages of the COOH-terminus (Beers and colleagues, J. Biol. Chem. 1994;269:20,318--20,328). The goal of the current study was to define processing and function of the NH(2)-terminal flanking domain. Epitope-specific antisera directed against spatially distinct regions of the NH(2) terminus, NPROSP-C(2-9) (epitope = D(2)-L(9)) and NPROSP-C(11-23) (= E(11)-Q(23)) were produced. By Western blotting, both antisera identified SP-C(21) in microsomes. A 6-kD form (SP-C(6)), enriched in lamellar bodies (LBs), was detected only by NPROSP-C(11-23) and not extractable with NaCO(3) 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. (35)S-pulse chase analysis demonstrated synthesis of SP-C(21) and three intermediate forms (SP-C(16), SP-C(7), and SP-C(6)). Complete processing involved four separate cleavages with a precursor- product relationship between the low molecular weight forms SP-C(7) and SP-C(6). Fluorescence microscopy of A549 cells expressing fusion proteins of enhanced green fluorescent protein (EGFP) and proSP-C NH(2)-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(Delta 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 NH(2) terminus (M(1)-L(9)), which occurs after removal of COOH-flanking domains (H(59)-I(194)) but before packaging in LBs, and that the region M(10)-T(18) is required for targeting of proSP-C to post-ER vesicular compartments in the biosynthetic pathway.
Lung Epithelial Cell Biology Laboratories, Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6061, USA.
A L
AL
Johnson
P
P
Braidotti
G G
GG
Pietra
S J
SJ
Russo
A
A
Kabore
W J
WJ
Wang
M F
MF
Beers
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D000939:Epitopes; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008168:Lung / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D016253:Microscopy, Immunoelectron; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D037721:Pulmonary Surfactant-Associated Protein C; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D017931:DNA Primers; D000939:Epitopes; D008164:Luminescent Proteins; D010455:Peptides; D011510:Proteolipids; D037721:Pulmonary Surfactant-Associated Protein C; D011663:Pulmonary Surfactants; D011993:Recombinant Fusion Proteins; C489679:SFTPC protein, human; C077063:Sftpc protein, rat; D049452:Green Fluorescent Proteins