Synthesis and processing of hydrophobic surfactant protein C by isolated rat type II cells

Abstract

Surfactant protein C (SP-C) is a 3.7-kDa hydrophobic peptide isolated from organic extracts of pulmonary surfactant which is secreted by alveolar type II cells after synthesis and posttranslational processing of a 21-kDa proSP-C peptide (SP-C21). Previously characterized epitope-specific proSP-C antisera were used to study early proteolytic steps of proSP-C processing by adult rat type II cells. Western blotting and immunocytochemistry using anti-NPROSP-C (epitope = Met10-Glu23) each demonstrated marked attenuation of proSP-C protein expression by culture on plastic. Processing was therefore studied by metabolic labeling of freshly isolated type II cells maintained in suspension in serum-free media. With the use of anti-NPROSP-C, immunoprecipitation of cell lysates continuously labeled for 4 h with [35S]methionine demonstrated radiolabeled bands of M(r) 21, 16, and 10-6,000 while anti-CTERMSP-C (epitope = Ser149-Ser166) failed to detect 35S-bands of M(r) < 16,000. Pulse-chase studies demonstrated synthesis of 35S-proSP-C21 with a time-dependent dependent appearance of 16-kDa and 10- to 6-kDa forms which was blocked by addition of brefeldin A. SP-C precursors were not detected in the media. Quantitative analysis of the major bands by direct beta-counting indicated a precursor-product relationship between SP-C21 and SP-C16. These results demonstrate the utility of freshly isolated type II cells for characterization of SP-C synthetic pathways and show that early proSP-C processing events include synthesis of a 21-kDa primary translation product followed by extensive intracellular proteolysis of the proSP-C COOH-terminal in subcellular compartments of type II cells which are distal to the trans-Golgi network.

Pulmonary surfactant‐associated polypeptide C in a mixed organic solvent transforms from a monomeric α‐helical state into insoluble β‐sheet aggregates

“…Native asp-C in vivo is generated from a 2 1 -kDa precursor, proSP-CZ1 (Glasser et al, 1988), through a sequence of proteolytic events and post-translational modifications that take place in different subcellular compartments (Beers et al, 1994;Beers & Lomax, 1995). The following pathway has been suggested (see Fig.…”

Anterograde Transport of Surfactant Protein C Proprotein to Distal Processing Compartments Requires PPDY-mediated Association with Nedd4 Ubiquitin Ligases

“…1 Both authors contributed equally to this work. 2 To whom correspondence should be addressed: Pulmonary and Critical Care Division, University of Pennsylvania School of Medicine, H410F Hill Pavilion, 380 S. University Ave., Philadelphia, PA 19104-4539. Tel.…”

“…The 3.7-kDa alveolar form of SP-C (SP-C 3.7 or mature SP-C) is a 35-amino acid, valine-rich peptide that results from synthesis of a 197 amino acid precursor form (proSP-C 21 ). Within the endoplasmic reticulum (ER), proSP-C 21 exists as a type II bitopic transmembrane protein (N cyt /C lumen ), which remains integrally membrane-associated while it is extensively processed by four post-translational endoproteolytic cleavages that take place in post-Golgi compartments (2)(3)(4)(5)(6). The resulting mature protein is transferred into the lumen of lamellar bodies for secretion into the alveolar space together with surfactant phospholipid and SP-B (7).…”

“…Cell pellets were prepared for Western blotting from freshly isolated cells by detergent lysis as previously published (2,8). absence of 10 nM dexamethasone, 0.1 mM 8-Br-cAMP, and 0.1 mM isobutyl methylxanthine (DCI) to induce and maintain type 2 cell differentiation as described (31).…”

“…The NH 2 propeptide domain of wild-type (Asp ) proSP-C depicted in Fig. 1 were amplified by PCR (supplemental Table S3) and subcloned into the GST vector, pGEX-2TK (PerkinElmer Life Sciences) using the EcoR1 and BamH1 restriction enzyme sites in the vector.…”