FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2029074080> ?p ?o ?g. }

• W2029074080 endingPage "15293" @default.
• W2029074080 startingPage "15287" @default.
• W2029074080 abstract "Surfactant protein C (SP-C) is synthesized by alveolar type II cells as a 21-kDa propeptide (proSP-C21) which is proteolytically processed in subcellular compartments distal to the trans-Golgi network to yield a 35-residue mature form. Initial synthetic processing events for SP-C include post-translational cleavages of the COOH terminus of proSP-C21 yielding two intermediates (16 and 6 kDa). To test the role of specific COOH-terminal domains in intracellular targeting and proteolysis of proSP-C21, synthesis and processing of SP-C was evaluated using a lung epithelial cell line (A549) transfected with a eukaryotic expression vector containing either the full-length cDNA for rat SP-C (SP-Cwt) or one of six polymerase chain reaction (PCR)-generated COOH terminally truncated forms (SP-C1–185, SP-C1–175, SP-C1–147, SP-C1–120, SP-C1–72, and SP-C1–59). Using in vitro transcription/translation, each of the seven constructs produced a35S-labeled product of appropriate length which could be immunoprecipitated by epitope specific proSP-C antisera. Immunoprecipitation of 35S-labeled A549 cell lysates from SP-Cwt transfectants demonstrated rapid synthesis of [35S]proSP-C21 with processing to SP-C16 and SP-C6 intermediates via cleavages of the COOH-terminal propeptide. Both the intermediates as well as the kinetics of processing in A549 cells were similar to that observed in rat type II cells. In contrast, constructs SP-C1–185, SP-C1–175, SP-C1–147, SP-C1–120, SP-C1–72, and SP-C1–59 were each translated but degraded without evidence of proteolytic processing. Fluorescence immunocytochemistry identified proSP-Cwt in cytoplasmic vesicles of A549 cells while all COOH-terminal deletional mutants were restricted to an endoplasmic reticulum/Golgi compartment identified by co-localization with fluorescein isothiocyanate-concanavalin A. We conclude that SP-Cwt expressed in A549 cells is directed to cytoplasmic vesicles where it is proteolytically processed in a manner similar to native type II cells and that amino acids Cys186-Ile194 located at the COOH terminus of proSP-C21 are necessary for correct intracellular targeting and subsequent cleavage events. Surfactant protein C (SP-C) is synthesized by alveolar type II cells as a 21-kDa propeptide (proSP-C21) which is proteolytically processed in subcellular compartments distal to the trans-Golgi network to yield a 35-residue mature form. Initial synthetic processing events for SP-C include post-translational cleavages of the COOH terminus of proSP-C21 yielding two intermediates (16 and 6 kDa). To test the role of specific COOH-terminal domains in intracellular targeting and proteolysis of proSP-C21, synthesis and processing of SP-C was evaluated using a lung epithelial cell line (A549) transfected with a eukaryotic expression vector containing either the full-length cDNA for rat SP-C (SP-Cwt) or one of six polymerase chain reaction (PCR)-generated COOH terminally truncated forms (SP-C1–185, SP-C1–175, SP-C1–147, SP-C1–120, SP-C1–72, and SP-C1–59). Using in vitro transcription/translation, each of the seven constructs produced a35S-labeled product of appropriate length which could be immunoprecipitated by epitope specific proSP-C antisera. Immunoprecipitation of 35S-labeled A549 cell lysates from SP-Cwt transfectants demonstrated rapid synthesis of [35S]proSP-C21 with processing to SP-C16 and SP-C6 intermediates via cleavages of the COOH-terminal propeptide. Both the intermediates as well as the kinetics of processing in A549 cells were similar to that observed in rat type II cells. In contrast, constructs SP-C1–185, SP-C1–175, SP-C1–147, SP-C1–120, SP-C1–72, and SP-C1–59 were each translated but degraded without evidence of proteolytic processing. Fluorescence immunocytochemistry identified proSP-Cwt in cytoplasmic vesicles of A549 cells while all COOH-terminal deletional mutants were restricted to an endoplasmic reticulum/Golgi compartment identified by co-localization with fluorescein isothiocyanate-concanavalin A. We conclude that SP-Cwt expressed in A549 cells is directed to cytoplasmic vesicles where it is proteolytically processed in a manner similar to native type II cells and that amino acids Cys186-Ile194 located at the COOH terminus of proSP-C21 are necessary for correct intracellular targeting and subsequent cleavage events. The alveolar epithelium synthesizes pulmonary surfactant, a surface active lining film consisting of a biochemically complex mixture of lipids and proteins, which serves to reduce surface tension at the alveolar surface, thereby allowing for maintenance of alveolar stability at low lung volumes (end-expiration) (1King R.J. J. Appl. Physiol. 1982; 53: 1-8Crossref PubMed Scopus (112) Google Scholar). Organic extracts of isolated surfactant have been shown to contain two small lipophilic proteins (SP's), SP-B 1The abbreviations used are: SP-B, pulmonary surfactant protein B (9 kDa); SP-C, pulmonary surfactant protein C (3.7 kDa); Tricine,N -[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; ConA, concanavalin A; PCR, polymerase chain reaction; ER, endoplasmic reticulum. 1The abbreviations used are: SP-B, pulmonary surfactant protein B (9 kDa); SP-C, pulmonary surfactant protein C (3.7 kDa); Tricine,N -[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; ConA, concanavalin A; PCR, polymerase chain reaction; ER, endoplasmic reticulum. and SP-C, which alone or in combination are sufficient to confer properties of rapid surface adsorption and surface tension lowering to reconstituted mixtures of synthetic phospholipids (2Mathalgian N. Possmayer F. Biochim. Biophys. Acta. 1990; 1045: 121-127Crossref PubMed Scopus (24) Google Scholar). SP-C, an extremely hydrophobic 3.7-kDa peptide, is the exclusive product of the alveolar type II cell (3Beers M.F. Fisher A.B. Am J. Physiol. 1992; 263: L151-L160PubMed Google Scholar, 4Kalina M. Mason R.J. Shannon J.M. Am. J. Respir. Cell. Mol. Biol. 1992; 6: 594-600Crossref PubMed Scopus (130) Google Scholar, 5Phelps D.S. Floros J. Exp. Lung Res. 1991; 17: 985-995Crossref PubMed Scopus (104) Google Scholar) and is a component of most clinical surfactant preparations (3Beers M.F. Fisher A.B. Am J. Physiol. 1992; 263: L151-L160PubMed Google Scholar, 6Boncuk-Dayanikli P. Taeusch H.W. Robertson B. Taeusch H.W. Surfactant Therapy for Lung Disease. Marcel-Dekker, Inc., New York1995: 217-238Google Scholar, 7Rooney S.A. Young S.L. Mendelson C.R. FASEB J. 1994; 8: 957-967Crossref PubMed Scopus (299) Google Scholar). The full-length rat SP-C mRNA (0.9 kilobases) is produced by splicing of multiple exons and yields a primary translation product 194 amino acids in length (proSP-C21) (8Fisher J.H. Shannon J.M. Hofmann T. Mason R.J. Biochim. Biophys. Acta. 1989; 995: 225-230Crossref PubMed Scopus (73) Google Scholar). In vitro translation of lung RNA produces SP-C primary translation products ofM r 21,000–22,000 (8Fisher J.H. Shannon J.M. Hofmann T. Mason R.J. Biochim. Biophys. Acta. 1989; 995: 225-230Crossref PubMed Scopus (73) Google Scholar). Similar sized products have been detected in freshly isolated rat type II cells (9Beers M.F. Lomax C. Am. J. Physiol. 1995; 269: L744-L753Crossref PubMed Google Scholar, 10Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 269: L727-L733Crossref PubMed Google Scholar) and produced in cultured Chinese hamster ovary cells transfected with a human SP-C cDNA (11Vorbroker D.K. Dey C. Weaver T.E. Whitsett J.A. Biochim. Biophys. Acta. 1992; 1105: 161-169Crossref PubMed Scopus (46) Google Scholar). The predominant form of SP-C isolated from extracellular surfactant (“mature SP-C”) is a 35-amino acid monomer which also contains 1–2 covalently linked palmitic acid residues (12Curstedt T. Johansson J. Persson P. Eklund A. Robertson B. Lowenadler B. Jornvall H. Proc. Nat. Acad. Sci. U. S. A. 1990; 87: 2985-2989Crossref PubMed Scopus (181) Google Scholar,13Stults J.T. Green P.R. Leskiar D.D. Naidu A. Moffat B. Benson B.J. Am. J. Physiol. 1991; 261: L118-L125PubMed Google Scholar). Mature SP-C3.7 is contained within the larger precursor proprotein, encompassing residues 24–58 of the proSP-C21 sequence. Unlike other surfactant-associated proteins, the NH2 terminus of the primary translation product does not contain a classic “signal” sequence and there are no sites for asparagine-linked glycosylation (3Beers M.F. Fisher A.B. Am J. Physiol. 1992; 263: L151-L160PubMed Google Scholar, 7Rooney S.A. Young S.L. Mendelson C.R. FASEB J. 1994; 8: 957-967Crossref PubMed Scopus (299) Google Scholar, 14Hawgood S. Am. J. Physiol. 1989; 263: L13-L22Google Scholar). Nonetheless, proSP-C21 must be translocated across the ER membrane and routed to the distal secretory pathway where it has been shown to undergo synthetic processing leading to production of the 3.7-kDa alveolar form (9Beers M.F. Lomax C. Am. J. Physiol. 1995; 269: L744-L753Crossref PubMed Google Scholar, 10Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 269: L727-L733Crossref PubMed Google Scholar, 15Beers M.F. J. Biol. Chem. 1996; 271: 14361-14370Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 16Beers M.F. Kim C.Y. Dodia C. Fisher A.B. J. Biol. Chem. 1994; 269: 20318-20328Abstract Full Text PDF PubMed Google Scholar, 17Beers M.F. Wali A. Eckenhoff M.E.F. Feinstein S. Fisher J.H. Fisher A.B. Am J. Respir. Cell. Mol. Biol. 1992; 7: 368-378Crossref PubMed Scopus (39) Google Scholar). The processing events triggered by delivery of proSP-C21 from the ER include post-translational addition of covalent palmitic acid residues and intracellular proteolysis involving cleavage of 23 residues of NH2- and 136 residues of COOH-terminal flanking domains of the precursor molecule (3Beers M.F. Fisher A.B. Am J. Physiol. 1992; 263: L151-L160PubMed Google Scholar, 7Rooney S.A. Young S.L. Mendelson C.R. FASEB J. 1994; 8: 957-967Crossref PubMed Scopus (299) Google Scholar, 14Hawgood S. Am. J. Physiol. 1989; 263: L13-L22Google Scholar). Using several different in vitro models, the processing events which lead to the appearance of SP-C3.7 in alveolar surfactant have been partially characterized (9Beers M.F. Lomax C. Am. J. Physiol. 1995; 269: L744-L753Crossref PubMed Google Scholar, 10Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 269: L727-L733Crossref PubMed Google Scholar, 11Vorbroker D.K. Dey C. Weaver T.E. Whitsett J.A. Biochim. Biophys. Acta. 1992; 1105: 161-169Crossref PubMed Scopus (46) Google Scholar, 15Beers M.F. J. Biol. Chem. 1996; 271: 14361-14370Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 16Beers M.F. Kim C.Y. Dodia C. Fisher A.B. J. Biol. Chem. 1994; 269: 20318-20328Abstract Full Text PDF PubMed Google Scholar, 17Beers M.F. Wali A. Eckenhoff M.E.F. Feinstein S. Fisher J.H. Fisher A.B. Am J. Respir. Cell. Mol. Biol. 1992; 7: 368-378Crossref PubMed Scopus (39) Google Scholar). In both a perfused rat lung preparation and freshly isolated rat type II cells, we have demonstrated processing of proSP-C21 through 16- and 6-kDa intermediate forms (proSP-C16, proSP-C6) (9Beers M.F. Lomax C. Am. J. Physiol. 1995; 269: L744-L753Crossref PubMed Google Scholar, 15Beers M.F. J. Biol. Chem. 1996; 271: 14361-14370Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 16Beers M.F. Kim C.Y. Dodia C. Fisher A.B. J. Biol. Chem. 1994; 269: 20318-20328Abstract Full Text PDF PubMed Google Scholar). A similar processing pattern has been confirmed by others utilizing pulse-chase analysis of type II cells and immunoprecipitation with different polyclonal antisera (10Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 269: L727-L733Crossref PubMed Google Scholar). The proteolysis of proSP-C21 can be blocked either by the use of brefeldin A (9Beers M.F. Lomax C. Am. J. Physiol. 1995; 269: L744-L753Crossref PubMed Google Scholar, 16Beers M.F. Kim C.Y. Dodia C. Fisher A.B. J. Biol. Chem. 1994; 269: 20318-20328Abstract Full Text PDF PubMed Google Scholar) or by low temperature incubation (20 °C) (10Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 269: L727-L733Crossref PubMed Google Scholar), indicating that intracellular processing of proSP-C is occurring in subcellular compartments located distal to the trans-Golgi. SP-C3.7 has also been recovered from the isolated lamellar body, a phospholipid storage organelle found within type II cells (18Oosterlaken-Dijksterhuis M.A. van Eijk M. van Beul B.L.M. van Golde L.M.G. Haagsman H.P. Biochem. J. 1991; 274: 115-119Crossref PubMed Scopus (88) Google Scholar,19Phizackerly P.J. Town R.M.H. Newman G.E. Biochem. J. 1979; 183: 731-736Crossref PubMed Scopus (83) Google Scholar), which indicates that all proteolysis of proSP-C21 and proSP-C intermediates occurs intracellularly prior to secretion of the mature peptide into the alveolus. Furthermore, the use of inhibitors of organellar acidification has further elucidated that these intracellular proteolytic events are taking place within acidic subcellular compartments of the exocytic pathway (15Beers M.F. J. Biol. Chem. 1996; 271: 14361-14370Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Despite what is known of the major cleavage events, their localization, and cellular factors important for the regulation of SP-C synthetic processing, the role of specific peptide domains contained within the proSP-C sequence in the direction of its post-translational processing and/or of its subcellular targeting has not been forthcoming due to limitations imposed by previous experimental models. Additional insights have been hampered because primary alveolar type II cells in culture are phenotypically unstable and not easily transfected (20Lin S. Akinbi H.T. Breslin J.S. Weaver T.E. J. Biol. Chem. 1996; 271: 19689-19695Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar,21Shannon J.M. Emrie P.A. Fisher J.H. Kuroki Y. Jennings S.D. Mason R.J. Am. J. Respir. Cell. Mol. Biol. 1990; 2: 183-192Crossref PubMed Scopus (83) Google Scholar), and a relevant experimental lung epithelial cell line capable of demonstrating synthesis, targeting, and post-translational proteolysis of transfected proSP-C cDNA constructs has not been characterized. The present study was undertaken to identify peptide domains that facilitate intracellular transport and processing of proSP-C. Initially, synthetic processing of wild type SP-C was defined using the A549 lung epithelial cell line transfected with a eukaryotic expression vector containing a full-length rat SP-C cDNA under the control of a strong viral promoter. Results obtained using this system show that the patterns of expression and processing of recombinant SP-C were similar to that observed for endogenous SP-C in native rat type II cells and demonstrate the feasibility of this model for use in studies aimed at evaluating functional domains contained within the proSP-C primary sequence. COOH terminally truncated forms of proSP-C were generated using PCR-based mutagenesis. Transfection of these mutant constructs into A549 cells demonstrated that deletion of as little as 10 amino acids from the COOH terminus of the proSP-C molecule causes mistargeting of the translated protein resulting in disruption of post-translational proteolytic events showing that an intact COOH-terminal peptide of proSP-C is necessary for proper post-translational processing. Trans35S-label (70% l-methionine 15%-l-cysteine; 1100 mCi/ml as methionine) was purchased from ICN/Flow, Inc., Irvine, CA. Protein A-agarose was obtained from Bethesda Research Labs, Gaithersburg, MD. FITC-concanavalin A was obtained from Sigma. Except where noted, other reagents were electrophoretic grade and were purchased from Bio-Rad or Sigma. Monospecific polyclonal rat proSP-C antisera were produced from synthetic peptide immunogens and have been previously characterized (16Beers M.F. Kim C.Y. Dodia C. Fisher A.B. J. Biol. Chem. 1994; 269: 20318-20328Abstract Full Text PDF PubMed Google Scholar, 17Beers M.F. Wali A. Eckenhoff M.E.F. Feinstein S. Fisher J.H. Fisher A.B. Am J. Respir. Cell. Mol. Biol. 1992; 7: 368-378Crossref PubMed Scopus (39) Google Scholar). Anti-NPROSP-C (Met10-Glu23), anti-hCPROSP-C (His59-Ser72), and anti-CTERMSP-C (Ser149-Ser166) each recognize spatially distinct regions of the linearized proSP-C molecule but do not recognize mature SP-C. The lung epithelial cell line A549 (22Giard D.J. Aaronson S.A. Tordaro G.J. Arnstein P. Kersey J.H. Dosik H. Parks W.P. J. Natl. Cancer Inst. 1973; 51: 1417-1423Crossref PubMed Scopus (1821) Google Scholar) utilized in all transfection studies were originally obtained through the American Type Culture Collection (Rockville, MD) and made available as a gift of Dr. S. I. Feinstein. A549 cells were grown at 37 °C, 5% CO2 in minimal essential medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Type II pneumocytes were isolated using elastase digestion of lungs from adult Sprague-Dawley rats (age 2–3 months) by the method of Dobbs et al. (23Dobbs L.G. Gonzalez R. Williams M.C. Biochim. Biophys. Acta. 1982; 713: 118-127Crossref PubMed Scopus (77) Google Scholar). The preparation obtained after panning on IgG coated plates (i.e. fresh type II cells) contained approximately 80–85% type II cells. The expression vector chosen as the backbone for transfection of epithelial cells in culture is the pcDNA3 eukaryotic expression plasmid (Invitrogen, Inc., San Diego, CA) which contains the human cytomegalovirus promoter (early promoter and enhancer region), bovine growth hormone polyadenylation sequence, β-lactamase and neomycin resistance genes, as well as T7 and SP6 promoters for sense/antisensein vitro transcription. All procedures involving oligonucleotide and cDNA manipulations were performed essentially as described by Ausbel et al. (24Ausbel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley, Inc., New York1987Google Scholar). The wild type rat SP-C (Met1-Ile194) and six mutant construct inserts containing progressively larger truncations of the proSP-C COOH terminus are schematically illustrated in Fig. 1. A full-length rat SP-C cDNA (816 base pairs) insert was prepared by Eco RI digestion of a previously characterized prokaryotic SP-C expression vector, PGEM4Z-SP-C (8+) (8Fisher J.H. Shannon J.M. Hofmann T. Mason R.J. Biochim. Biophys. Acta. 1989; 995: 225-230Crossref PubMed Scopus (73) Google Scholar). Purified SP-C insert was ligated into pcDNA3 polylinker at the Eco RI site. Restriction analysis of amplified subclones of pcDNA3-SP-Cwt confirmed successful insertion of a full-length cDNA in the sense orientation. A subclone containing the antisense (−) orientation was also identified and used as a control in transfection studies. Mutant cDNA deletion constructs containing progressively larger truncations of the COOH flanking region of proSP-C (His59-Ile194) (Fig.1) were generated by the PCR. PcDNA3-SP-Cwt was used as template and the oligonucleotide primers are listed in TableI. Amplification reactions containing 0.2 μm primers, 1.25 μm dNTP mixture, 1.5 μm MgCl2, 10 ng of template, and 2.5 units ofTaq polymerase (Perkin-Elmer, Inc., Foster City, CA) consisted of 30 cycles of: denaturation at 95 °C for 30 s, primer annealing at 50–55 °C for 30 s, and primer extension at 72 °C for 15 s. After the last cycle, the mixture was incubated at 72 °C for 7 min. Purified inserts were ligated into pcDNA3 sequentially digested with Kpn I and Xho I.Table ITemplates and primers used in PCR reactions for generation of rat SP-C constructsSP-C construct nameSP-C insert (amino acids)PCR primers1-aNumbers correspond to nucleic acids in the rat SP-C cDNA (Ref. 8).Template5′ (forward)3′ (reverse)pcDNA3-SPCwtMet1-Ile194None1-bSP-C was subcloned from pGEM4Z-SPC8+ (Ref. 8) into the Eco RI site of pcDNA3.None1-bSP-C was subcloned from pGEM4Z-SPC8+ (Ref. 8) into the Eco RI site of pcDNA3.pGEM4Z-SPC8+pcDNA3-SPC1–194Met1-Ile194pcDNA3Kpn I1-cPrimer pcDNA3Kpn I is located in the pcDNA3 polylinker (5′-GACCCAAGCTTGGTACCGAG-3′).pcDNA3XhoI1-dPrimer pcDNA3Xho I is located in the pcDNA3 polylinker (5′-TCTAGATGCATGCTCGAGCG-3′).pcDNA3-SPC3+pcDNA3-SPC1–185Met1-Leu185pcDNA3Kpn I1-cPrimer pcDNA3Kpn I is located in the pcDNA3 polylinker (5′-GACCCAAGCTTGGTACCGAG-3′).[551–567]1-eA stop codon (ATT ) and an Xho I site were added to the 5′ end of each reverse primer (3′-[ ]-ATTGTGAGCTCCC-5′).pcDNA3-SPC3+pcDNA3-SPC1–175Met1-Leu175pcDNA3Kpn I1-cPrimer pcDNA3Kpn I is located in the pcDNA3 polylinker (5′-GACCCAAGCTTGGTACCGAG-3′).[521–537]1-eA stop codon (ATT ) and an Xho I site were added to the 5′ end of each reverse primer (3′-[ ]-ATTGTGAGCTCCC-5′).pcDNA3-SPC3+pcDNA3-SPC1–147Met1-Asp147pcDNA3Kpn I1-cPrimer pcDNA3Kpn I is located in the pcDNA3 polylinker (5′-GACCCAAGCTTGGTACCGAG-3′).[437–453]1-eA stop codon (ATT ) and an Xho I site were added to the 5′ end of each reverse primer (3′-[ ]-ATTGTGAGCTCCC-5′).pcDNA3-SPC3+pcDNA3-SPC1–120Met1-Thr120pcDNA3Kpn I1-eA stop codon (ATT ) and an Xho I site were added to the 5′ end of each reverse primer (3′-[ ]-ATTGTGAGCTCCC-5′).[346–362]1-eA stop codon (ATT ) and an Xho I site were added to the 5′ end of each reverse primer (3′-[ ]-ATTGTGAGCTCCC-5′).pcDNA3-SPC3+pcDNA3-SPC1–72Met1-Ser72pcDNA3Kpn I1-cPrimer pcDNA3Kpn I is located in the pcDNA3 polylinker (5′-GACCCAAGCTTGGTACCGAG-3′).[212–228]1-eA stop codon (ATT ) and an Xho I site were added to the 5′ end of each reverse primer (3′-[ ]-ATTGTGAGCTCCC-5′).pcDNA3-SPC3+pcDNA3-SPC1–59Met1-His59pcDNA3Kpn I1-cPrimer pcDNA3Kpn I is located in the pcDNA3 polylinker (5′-GACCCAAGCTTGGTACCGAG-3′).[173–189]1-eA stop codon (ATT ) and an Xho I site were added to the 5′ end of each reverse primer (3′-[ ]-ATTGTGAGCTCCC-5′).pcDNA3-SPC3+1-a Numbers correspond to nucleic acids in the rat SP-C cDNA (Ref. 8Fisher J.H. Shannon J.M. Hofmann T. Mason R.J. Biochim. Biophys. Acta. 1989; 995: 225-230Crossref PubMed Scopus (73) Google Scholar).1-b SP-C was subcloned from pGEM4Z-SPC8+ (Ref. 8Fisher J.H. Shannon J.M. Hofmann T. Mason R.J. Biochim. Biophys. Acta. 1989; 995: 225-230Crossref PubMed Scopus (73) Google Scholar) into the Eco RI site of pcDNA3.1-c Primer pcDNA3Kpn I is located in the pcDNA3 polylinker (5′-GACCCAAGCTTGGTACCGAG-3′).1-d Primer pcDNA3Xho I is located in the pcDNA3 polylinker (5′-TCTAGATGCATGCTCGAGCG-3′).1-e A stop codon (ATT ) and an Xho I site were added to the 5′ end of each reverse primer (3′-[ ]-ATTGTGAGCTCCC-5′). Open table in a new tab Automated DNA sequencing in both directions performed at the Core Facility in the Department of Genetics at the University of Pennsylvania detected no nucleotide mutations in the coding region of SP-Cwt or any deletional constructs. For SP-Cwt, a single deviation from the published rat SP-C cDNA (8Fisher J.H. Shannon J.M. Hofmann T. Mason R.J. Biochim. Biophys. Acta. 1989; 995: 225-230Crossref PubMed Scopus (73) Google Scholar) sequence occurred at nucleotides 612–613 located within the 3′-untranslated region (GC for CG). The open reading frame of each construct was characterized by production of 35S-labeled protein using sequential in vitro transcription/translation of SP-C cDNAs with Trans35S-label and the TNT T7 reticulocyte lysate system (Promega, Inc., Madison, WI) as described by the manufacturer. SP-C constructs were transiently transfected into A549 cells using calcium phosphate precipitation (0.18 ml of 0.25 mCaCl2 was added dropwise to 0.18 ml of plasmid DNA dissolved in 2 × HEPES-buffered saline (50 mm HEPES, 280 mm NaCl, 1.5 mm NaPO4, pH 7.1) (24Ausbel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley, Inc., New York1987Google Scholar). Immunocytochemical localization of expressed proSP-C proteins was performed on transfected epithelial cell lines fixed and stained as described previously (17Beers M.F. Wali A. Eckenhoff M.E.F. Feinstein S. Fisher J.H. Fisher A.B. Am J. Respir. Cell. Mol. Biol. 1992; 7: 368-378Crossref PubMed Scopus (39) Google Scholar). ProSP-C staining was visualized using primary anti-NPROSP-C (1:200) and secondary goat anti-rabbit IgG-Texas red. Concanavlin A conjugated to fluorescein (FITC-ConA) was used for staining ER and Golgi (25Tartakoff A.M. Vassalli P. J. Cell Biol. 1983; 97: 1243-1248Crossref PubMed Scopus (148) Google Scholar). Fluorescent images were captured using a 12-bit CCD camera and processed using IMAGE 1 software (Universal Imaging Corporation, West Chester, PA). Sixty hours following the introduction of plasmid DNA, transiently transfected cell line monolayers (80–90% confluence) equilibrated in serum-free Dulbecco's modified essential medium-Cys/Met were labeled for 30 min with 100 μCi/ml Trans55S-label, then chased in Met/Cys replete minimal essential medium for up to 4 h. Labeled cells were harvested by scraping and pelleted by centrifugation at 130 × g for 10 min. Fresh type II cells were metabolically labeled with Trans35S-label in serum-free, Met/Cys-free Dulbecco's modified Eagle's medium, using suspension cultures (3–5 × 106 cells/ml) as described previously (9Beers M.F. Lomax C. Am. J. Physiol. 1995; 269: L744-L753Crossref PubMed Google Scholar). All radiolabeled cell pellets were solubilized in buffer containing 150 mm NaCl, 50 mm Tris-HCl, pH 7.40, 1 mm phenylmethylsulfonyl fluoride, 1% (v/v) Triton X-100, 5 mm EDTA, and 5 μg/ml each of aprotinin, leupeptin, and pepstatin and immunoprecipitated using proSP-C antiserum as previously published (9Beers M.F. Lomax C. Am. J. Physiol. 1995; 269: L744-L753Crossref PubMed Google Scholar, 15Beers M.F. J. Biol. Chem. 1996; 271: 14361-14370Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Captured proteins were separated by SDS-PAGE and visualized by autoradiography as described below. One-dimensional SDS-PAGE was performed in 16.5% polyacrylamide gels using a Tris-Tricine buffer system (26Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10407) Google Scholar) as modified in our laboratory for surfactant proteins (9Beers M.F. Lomax C. Am. J. Physiol. 1995; 269: L744-L753Crossref PubMed Google Scholar, 15Beers M.F. J. Biol. Chem. 1996; 271: 14361-14370Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 16Beers M.F. Kim C.Y. Dodia C. Fisher A.B. J. Biol. Chem. 1994; 269: 20318-20328Abstract Full Text PDF PubMed Google Scholar). Immunoblotting of transferred samples was done using proSP-C antisera and bands were visualized by enhanced chemiluminescence using the ECL kit (Amersham, Inc.). Total protein was quantified by the Bradford (27Bradford M.M. Anal. Biochemistry. 1976; 72: 248-254Crossref PubMed Scopus (211805) Google Scholar) method using bovine immunoglobulin as standard. Each pcDNA-SP-C construct (Fig. 1) generated in vitro translation products which were immunoprecipitated by epitope-specific proSP-C antisera (Fig. 2). SDS-PAGE of a TNT rabbit reticulocyte lysate reaction containing full-length or truncated plasmid DNA identified 35S-labeled bands of predicted molecular weight which were not seen in reactions omitting plasmid DNA (not shown). The three proSP-C antisera specifically recognized the appropriate in vitro translation product in a pattern restricted by epitope specificity. Similar patterns were obtained using translation products from SP-C1–147, SP-C1–120, and SP-C1–59 (not shown). A549 cells transfected with pcDNA3-SP-Cwtcells stained with anti-NPROSP-C consistently demonstrated expression of proSP-C within cytoplasmic vesicles (Fig.3 A ). The specificity of the immunohistochemical staining was confirmed by the substitution of preimmune serum for primary anti-NPROSP-C (Fig. 3 B ). Control experiments using an antisense construct, pcDNA3-SP-C (−), transfected under identical conditions showed a complete absence of proSP-C staining by the A549 cell line (Fig. 3 C ). Western blots of transient transfections of A549 cells with pcDNA3-SP-Cwt confirmed detectable expression of proSP-C proteins at 48 and 72 h following introduction of DNA (not shown). The processing profile of SP-Cwt expressed in A549 cells was compared with native type II cells. Pulse-chase analysis demonstrates synthesis and proteolytic processing of35S-proSP-C21 by type II cells (Fig.4 A ). Using anti-NPROSP-C (which recognizes all major proSP-C forms),35S-proSP-C21 appeared by completion of the pulse (30 min) followed by a time-dependent appearance of a 16-kDa intermediate and low molecular weight proSP-C forms during the chase. 35S-Lysates from SP-Cwt transfected A549 cells immunoprecipitated under identical conditions yielded the same intermediates (Fig. 4 B ). Immunoprecipitation of SP-Cwt transfected A549 cells with anti-CTERMSP-C demonstrated that initial processing is due to cleavage of COOH-terminal regions (Fig. 4 C ) as had been previously shown for rat type II cells (16Beers M.F. Kim C.Y. Dodia C. Fisher A.B. J. Biol. Chem. 1994; 269: 20318-20328Abstract Full Text PDF PubMed Google Scholar). Immunoprecipitation of the media failed to detect the presence of proSP-C21 or smaller intermediates at chase periods of up to 4 h (not shown). The kinetics of early proSP-C processing by A549 cells was nearly identical to that of rat type II cells (Fig.5). In both models, at the conclusion of the pulse period, almost 95% of the total counts were in SP-C21. A quantitative precursor-product relationship between SP-C21 and SP-C16 can be seen. The appearance of low molecular weight forms immediately followed the appearance of SP-C16. A549 cells transiently transfecte" @default.
• W2029074080 created "2016-06-24" @default.
• W2029074080 creator A5025905469 @default.
• W2029074080 creator A5040694955 @default.
• W2029074080 creator A5061586074 @default.
• W2029074080 date "1998-06-01" @default.
• W2029074080 modified "2023-09-30" @default.
• W2029074080 title "Synthetic Processing of Surfactant Protein C by Alevolar Epithelial Cells" @default.
• W2029074080 cites W1501290006 @default.
• W2029074080 cites W1608454061 @default.
• W2029074080 cites W1858962045 @default.
• W2029074080 cites W1975085160 @default.
• W2029074080 cites W1993446768 @default.
• W2029074080 cites W1996751084 @default.
• W2029074080 cites W1996895913 @default.
• W2029074080 cites W2003558962 @default.
• W2029074080 cites W2011225090 @default.
• W2029074080 cites W2017241568 @default.
• W2029074080 cites W2037385260 @default.
• W2029074080 cites W2050171843 @default.
• W2029074080 cites W2052518317 @default.
• W2029074080 cites W2062790216 @default.
• W2029074080 cites W2068808582 @default.
• W2029074080 cites W2076945592 @default.
• W2029074080 cites W2089658522 @default.
• W2029074080 cites W2102838769 @default.
• W2029074080 cites W2117951701 @default.
• W2029074080 cites W2129224957 @default.
• W2029074080 cites W2135766746 @default.
• W2029074080 cites W2138879032 @default.
• W2029074080 cites W2162315767 @default.
• W2029074080 cites W2289289126 @default.
• W2029074080 cites W2347145398 @default.
• W2029074080 cites W2462018642 @default.
• W2029074080 cites W4236508265 @default.
• W2029074080 cites W4293247451 @default.
• W2029074080 doi "https://doi.org/10.1074/jbc.273.24.15287" @default.
• W2029074080 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9614145" @default.
• W2029074080 hasPublicationYear "1998" @default.
• W2029074080 type Work @default.
• W2029074080 sameAs 2029074080 @default.
• W2029074080 citedByCount "55" @default.
• W2029074080 countsByYear W20290740802012 @default.
• W2029074080 countsByYear W20290740802013 @default.
• W2029074080 countsByYear W20290740802014 @default.
• W2029074080 countsByYear W20290740802016 @default.
• W2029074080 countsByYear W20290740802017 @default.
• W2029074080 countsByYear W20290740802018 @default.
• W2029074080 countsByYear W20290740802019 @default.
• W2029074080 countsByYear W20290740802020 @default.
• W2029074080 countsByYear W20290740802021 @default.
• W2029074080 crossrefType "journal-article" @default.
• W2029074080 hasAuthorship W2029074080A5025905469 @default.
• W2029074080 hasAuthorship W2029074080A5040694955 @default.
• W2029074080 hasAuthorship W2029074080A5061586074 @default.
• W2029074080 hasBestOaLocation W20290740801 @default.
• W2029074080 hasConcept C12554922 @default.
• W2029074080 hasConcept C185592680 @default.
• W2029074080 hasConcept C55493867 @default.
• W2029074080 hasConcept C58226133 @default.
• W2029074080 hasConcept C86803240 @default.
• W2029074080 hasConcept C95444343 @default.
• W2029074080 hasConceptScore W2029074080C12554922 @default.
• W2029074080 hasConceptScore W2029074080C185592680 @default.
• W2029074080 hasConceptScore W2029074080C55493867 @default.
• W2029074080 hasConceptScore W2029074080C58226133 @default.
• W2029074080 hasConceptScore W2029074080C86803240 @default.
• W2029074080 hasConceptScore W2029074080C95444343 @default.
• W2029074080 hasIssue "24" @default.
• W2029074080 hasLocation W20290740801 @default.
• W2029074080 hasOpenAccess W2029074080 @default.
• W2029074080 hasPrimaryLocation W20290740801 @default.
• W2029074080 hasRelatedWork W1531601525 @default.
• W2029074080 hasRelatedWork W2048107595 @default.
• W2029074080 hasRelatedWork W2076441233 @default.
• W2029074080 hasRelatedWork W2606230654 @default.
• W2029074080 hasRelatedWork W2607424097 @default.
• W2029074080 hasRelatedWork W2748952813 @default.
• W2029074080 hasRelatedWork W2868263345 @default.
• W2029074080 hasRelatedWork W2899084033 @default.
• W2029074080 hasRelatedWork W2948807893 @default.
• W2029074080 hasRelatedWork W2778153218 @default.
• W2029074080 hasVolume "273" @default.
• W2029074080 isParatext "false" @default.
• W2029074080 isRetracted "false" @default.
• W2029074080 magId "2029074080" @default.
• W2029074080 workType "article" @default.