SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2058448098> ?p ?o ?g. }

Showing items 1 to 100 of ±115 with 100 items per page.

W2058448098 endingPage "14664" @default.
W2058448098 startingPage "14658" @default.
W2058448098 abstract "Proteolytic processing of surfactant protein C (SP-C) proprotein in multivesicular bodies of alveolar type II cells results in a 35-residue mature peptide, consisting of a transmembrane domain and a 10-residue extramembrane domain. SP-C mature peptide is stored in lamellar bodies (a lysosomal-like organelle) and secreted with surfactant phopholipids into the alveolar space. This study was designed to identify the peptide domain of SP-C required for sorting and secretion of this integral membrane peptide. Deletion analyses in transiently transfected PC12 cells and isolated mouse type II cells suggested the extramembrane domain of mature SP-C was cytosolic and sufficient for sorting to the regulated secretory pathway. Intratracheal injection of adenovirus encoding SP-C mature peptide resulted in secretion into the alveolar space of wild type mice but not SP-C (−/−) mice. SP-C secretion in null mice was restored by the addition of the N-terminal propeptide. The cytosolic domain, consisting of the N- terminal propeptide and extramembrane domain of mature SP-C peptide, supported secretion of the transmembrane domain of platelet-derived growth factor receptor. Collectively, these studies indicate that the N-terminal propeptide of SP-C is required for intracellular sorting and secretion of SP-C. Proteolytic processing of surfactant protein C (SP-C) proprotein in multivesicular bodies of alveolar type II cells results in a 35-residue mature peptide, consisting of a transmembrane domain and a 10-residue extramembrane domain. SP-C mature peptide is stored in lamellar bodies (a lysosomal-like organelle) and secreted with surfactant phopholipids into the alveolar space. This study was designed to identify the peptide domain of SP-C required for sorting and secretion of this integral membrane peptide. Deletion analyses in transiently transfected PC12 cells and isolated mouse type II cells suggested the extramembrane domain of mature SP-C was cytosolic and sufficient for sorting to the regulated secretory pathway. Intratracheal injection of adenovirus encoding SP-C mature peptide resulted in secretion into the alveolar space of wild type mice but not SP-C (−/−) mice. SP-C secretion in null mice was restored by the addition of the N-terminal propeptide. The cytosolic domain, consisting of the N- terminal propeptide and extramembrane domain of mature SP-C peptide, supported secretion of the transmembrane domain of platelet-derived growth factor receptor. Collectively, these studies indicate that the N-terminal propeptide of SP-C is required for intracellular sorting and secretion of SP-C. surfactant protein B surfactant protein C platelet-derived growth factor receptor phosphate-buffered saline green fluorescent protein hemagglutinin Type II epithelial cells synthesize and secrete pulmonary surfactant, a complex mixture of phospholipids and proteins that reduces surface tension along the alveolar air-liquid interface at end respiration. The peptide components of surfactant, in particular surfactant protein B (SP-B1) and SP-C, are critical for surfactant film formation and function. Newborn infants and mice lacking SP-B have dramatically reduced pulmonary compliance and develop lethal, respiratory distress syndrome shortly after birth (1Whitsett J.A. Nogee L.M. Weaver T.E. Horowitz A.D. Physiol. Rev. 1995; 75: 749-757Crossref PubMed Scopus (161) Google Scholar, 2Nogee L.M. BBA Mol. Basis Dis. 1998; 1408: 323-333Crossref PubMed Scopus (51) Google Scholar). Disruption of the SP-B locus results in incomplete processing of pro-SP-C to its mature peptide, leading to deficiency of both SP-C and SP-B. SP-C null mice have normal levels of SP-B and survive with subtle changes in lung function but normal lung structure and surfactant pool sizes. 2S. Glasser, submitted for publication.2S. Glasser, submitted for publication. The importance of SP-C for normal lung function is inferred from experiments in which intratracheal administration of surfactant containing SP-C as the sole protein component to preterm animals restored lung function to values comparable with animals treated with native surfactant (3Hafner D. Beume R. Kilian U. Krasznai G. Lachmann B. Br. J. Pharmacol. 1995; 115: 451-458Crossref PubMed Scopus (55) Google Scholar, 4Hawgood S. Ogawa A. Yukitake K. Schlueter M. Brown C. White T. Buckley D. Lesikar D. Benson B. Am. J. Respir. Crit. Care Med. 1996; 154: 484-490Crossref PubMed Scopus (57) Google Scholar, 5Davis A.J. Jobe A.H. Häfner D. Ikegami M. Am. J. Respir. Crit. Care Med. 1998; 157: 553-559Crossref PubMed Scopus (102) Google Scholar). Collectively, these results suggest that SP-C and SP-B are functionally interchangeable with respect to biophysical activity. SP-C is synthesized by the alveolar type II epithelial cell as a 197-amino acid proprotein in which the mature peptide (residues 24–58) is flanked by N-terminal (residues 1–23) and C-terminal (residues 59–197) peptide domains. Unlike SP-B, SP-C is an integral membrane protein, which contains a single membrane-spanning domain located within the mature peptide (6Johansson J. Szyperski T. Curstedt T. Wuthrich K. Biochemistry. 1994; 33: 6015-6023Crossref PubMed Scopus (176) Google Scholar). The topology of the SP-C proprotein in the membrane is not clear, with reports of both type II (7Keller A. Eistetter H.R. Voss T. Schäfer K.P. Biochem. J. 1991; 277: 493-499Crossref PubMed Scopus (51) Google Scholar) and type III (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar) orientations. SP-C proprotein is detected in endoplasmic reticulum, Golgi, and multivesicular bodies but not in lamellar bodies, the intracellular storage compartment for pulmonary surfactant (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar, 9Voorhout W.F. Weaver T.E. Haagsman H.P. Geuze H.J. van Golde L.M.J. Microsc. Res. Technique. 1993; 26: 366-373Crossref PubMed Scopus (67) Google Scholar). Processing of the proprotein results in cleavage of the propeptides and generation of the 35-amino acid mature peptide that is detected only in the multivesicular body and lamellar body (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar, 10Beers M.F. Lomax C. Am. J. Physiol. 1995; 13: L744-L753Google Scholar). Colocalization of both proprotein and mature peptide in the multivesicular body strongly suggests that processing of the SP-C precursor to the biologically active peptide occurs within this compartment. In order to promote absorption and spreading of surfactant lipids at the alveolar air-liquid interface, mature SP-C peptide must be secreted by the type II cell. The mature peptide consists of an extremely hydrophobic transmembrane domain and a 10–12-amino acid extramembrane domain that contains palmitoylated cysteines at positions 5 and 6 (residues 28 and 29 of the proprotein) in most species (11Curstedt T. Johansson J. Persson P. Eklund A. Robertson B. Löwenadler B. Jornvall H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2985-2989Crossref PubMed Scopus (181) Google Scholar, 12Johansson J. Persson P. Löwenadler B. Robertson B. Jornvall H. Curstedt T. FEBS Lett. 1991; 281: 119-122Crossref PubMed Scopus (44) Google Scholar, 13Stults J.T. Griffin P.R. Lesikar D.D. Naidu A. Moffat B. Benson B.J. Am. J. Physiol. 1991; 261: L118-L125PubMed Google Scholar). The mechanism underlying secretion of this integral membrane peptide is not clear but probably involves two discrete steps. The proprotein is first sorted to the multivesicular body that ultimately fuses with the lamellar body, a lysosome-related organelle. Sorting of integral membrane proteins to lysosomes and secretory granules is dependent upon information encoded in the cytosolic domain of the protein (14Hunziker W. Geuze H.J. Bioessays. 1996; 18: 379-389Crossref PubMed Scopus (237) Google Scholar, 15Marks M.S. Ohno H. Kirchhausen T. Bonifacino S.J. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar), suggesting that either the N-terminal or C-terminal peptide domain of the proprotein plays an important role in sorting SP-C to lamellar bodies. Since SP-C mature peptide is detected only in the lumen of the lamellar body, a second step is required to relocate SP-C from the limiting membrane to the lumen of the multivesicular/lamellar body. It is likely that the N-terminal or C-terminal peptide domain also facilitates luminal internalization of SP-C. This study was designed to identify the compartment in which SP-C internalization occurs and the peptide domains required for sorting and secretion of SP-C into the airspace. Full-length human SP-C cDNA was cloned into pcDNA3 (Invitrogen, San Diego, CA). To generate SP-C deletion constructs (Fig. 2 A), SP-C fragments were amplified by polymerase chain reaction using specific primers to human SP-C containing 5′ SacI and 3′ SacII cleavage sites. Polymerase chain reaction fragments were subcloned into rEGFP vector (CLONTECH, San Jose, CA) and subjected to bidirectional sequencing to verify the fidelity of the polymerase chain reaction product throughout the SP-C coding sequence and the green fluorescent protein (GFP)/SP-C junction. PC12 cells (a gift from D. Cutler, University College London) were cultured as described previously (16Lin 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). Cells were grown in T25 flasks until 80% confluent and transiently transfected with 6 μg of plasmid DNA and 60 μl of Fugene 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. After 48 h of culture, PC12 cells were labeled with 0.5 mCi of [35S]methionine/cysteine (Amersham Pharmacia Biotech) for 4 h. Cell lysates and media were immunoprecipitated exactly as described previously by Lin et al. (17Lin S. Phillips K.S. Wilder M.R. Weaver T.E. BBA Mol. Cell Res. 1996; 1312: 177-185Crossref PubMed Scopus (70) Google Scholar) with 5 μl of an antibody directed against the SP-C N-terminal propeptide (18Vorbroker D.K. Profitt S.A. Nogee L.M. Whitsett J.A. Am. J. Physiol. 1995; 268: L647-L656Crossref PubMed Google Scholar). SDS-PAGE and autoradiography were performed as previously described (17Lin S. Phillips K.S. Wilder M.R. Weaver T.E. BBA Mol. Cell Res. 1996; 1312: 177-185Crossref PubMed Scopus (70) Google Scholar). 1.5 × 106 PC12 cells were plated on laminin/polylysine-coated coverslips. After 24 h of culture, PC12 cells were transiently transfected with 2 μg of plasmid DNA and 20 μl of Fugene 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. 48 h post-transfection, PC12 cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% saponin, and stained with antibodies directed against the SP-C N-terminal propeptide (18Vorbroker D.K. Profitt S.A. Nogee L.M. Whitsett J.A. Am. J. Physiol. 1995; 268: L647-L656Crossref PubMed Google Scholar) and chromogranin A (1:100) (ICN Biomedical, Costa Mesa, CA) for 1 h at room temperature. Cells were washed and incubated with anti-rabbit Texas Red (1:100) and anti-mouse Cy5 secondary antibodies (1:400) (Jackson Immunoresearch, West Grove, PA) for 1 h at room temperature. After cooling to 4 °C, nonpermeabilized cells were incubated for 30 min at 4 °C with antibody directed against the SP-C N-terminal (18Vorbroker D.K. Profitt S.A. Nogee L.M. Whitsett J.A. Am. J. Physiol. 1995; 268: L647-L656Crossref PubMed Google Scholar) or C-terminal peptide domain (9Voorhout W.F. Weaver T.E. Haagsman H.P. Geuze H.J. van Golde L.M.J. Microsc. Res. Technique. 1993; 26: 366-373Crossref PubMed Scopus (67) Google Scholar), biotinylated wheat germ agglutinin lectin (1:100) (Vector Laboratories, Burlingame, CA), and TOPRO3 to assess membrane integrity. Cells were washed with cold PBS and fixed with cold 4% paraformaldehyde in PBS at 4 °C. After 20 min, cells were washed and incubated with anti-rabbit Texas Red (1:100) and anti-biotin Cy5 secondary antibodies (1:400) (Jackson Immunoresearch) for 1 h at room temperature. All cells were washed with PBS and then double distilled H2O, mounted on slides with Vectashield mounting medium (Vector Laboratories), and sealed with nail polish. Fluorescence was imaged with a Leica confocal microscope and analyzed using Metamorph Imaging Software (Universal Imaging Corp., West Chester, PA). Type II cells were prepared from 6-week old female C57B/6 mice as described by Cortiet al. (19Corti M. Brody A.R. Harrison J.H. Am. J. Respir. Cell Mol. Biol. 1996; 14: 309-315Crossref PubMed Scopus (275) Google Scholar) with the following modifications. A crude cell suspension was prepared by rapid instillation of 3 ml of Dispase into the lung followed immediately by 0.5 ml of 1% low melting temperature agarose, warmed to 45 °C. Single cell suspensions were subsequently isolated as previously described (19Corti M. Brody A.R. Harrison J.H. Am. J. Respir. Cell Mol. Biol. 1996; 14: 309-315Crossref PubMed Scopus (275) Google Scholar) and added to 100-mm culture dishes coated with 42 μg of CD45 and 16 μg of CD32 antibodies (Pharmingen, San Diego, CA) in 6 ml of PBS followed by incubation for 1 h at 37 °C. Plates were gently “panned” to free settled type II cells and pelleted at 130 × g for 7 min at 4 °C. Type II cells were resuspended in culture media (Dulbecco's modified Eagle's medium, 25 mm HEPES, 10% fetal bovine serum, and 1% penicillin/streptomycin), plated at a density of 3 × 106 cells/well, and allowed to attach to collagen-coated 22 × 22 coverslips for 4 h. Cells were washed with calcium-free Hanks' balanced salt buffer (Life Technologies, Inc.) and transfected with a hand-held gene gun (Bio-Rad) at 100 p.s.i. with GFP/SP-C24–58 plasmid-coated 0.6 μm gold particles. Culture medium was replaced, and cells were cultured for 24 h followed by immunostaining with antibody directed against mature SP-C as described above. The sequence encoding amino acids 24–58 or 1–58 was amplified from cDNA generated by reverse transcriptase-polymerase chain reaction of type II cell RNA isolated from C57B/6 mice using specific primers that included a Kozak consensus sequence in the 5′ primer and a hemagglutinin tag (YPYDVPDYA) in the 3′ primer. The SP-C1–33PDGFR531–553HA adenoviral construct was generated by using overlapping primers to include the SP-C cytosolic domain (residues 1–33 of the proprotein), the PDGFR transmembrane domain (residues 531–53), and an HA tag. These polymerase chain reaction fragments were cloned into the Adv2 adenoviral shuttle vector (20Davis A.R. Meyers K. Wilson J.M. Gene Ther. 1998; 5: 1148-1152Crossref PubMed Scopus (41) Google Scholar) and sequenced bidirectionally to confirm the integrity of the reading frame. Recombination and virus production were performed as described previously (20Davis A.R. Meyers K. Wilson J.M. Gene Ther. 1998; 5: 1148-1152Crossref PubMed Scopus (41) Google Scholar). Purified adenoviral particles were intratracheally injected into wild type Swiss Black and SP-C (−/−) mice (Stephan Glasser, Cincinnati, OH). Mice were anesthetized and injected with 2 × 109 plaque-forming units of adenovirus/mouse in Hanks' balanced salt buffer containing 12 mm EGTA (21Wang G. Zabner J. Deering C. Launspach J. Shao J. Bodner M. Jolly D.J. Davidson B.L. McCray P. Am. J. Respir. Cell Mol. Biol. 2000; 22: 129-138Crossref PubMed Scopus (127) Google Scholar) in a final volume of 100 μl. Three or four days after infection, lung tissue was fixed with 2% paraformaldehyde and 0.5% gluteraldehyde, and the fixed, cryoprotected frozen tissue was processed for immunogold labeling with HA antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as described previously (9Voorhout W.F. Weaver T.E. Haagsman H.P. Geuze H.J. van Golde L.M.J. Microsc. Res. Technique. 1993; 26: 366-373Crossref PubMed Scopus (67) Google Scholar). Injected mice were lavaged with saline (5 × 1 ml), and surfactant was isolated by centrifugation at 18,000 × g for 30 min at 4 °C. Bronchoalveolar lavage from three injected mice was pooled, and large and small aggregate fractions were isolated on a 0.8 m sucrose gradient as described previously (22Ikegami M. Ueda T. Hull W. Whitsett J.A. Mulligan R.C. Dranoff G. Jobe A.H. Am. J. Physiol. 1996; 14: L650-L658Google Scholar). Lamellar bodies were isolated from pooled lungs of three injected mice as previously described by Osanai et al. (23Osanai K. Mason R.J. Voelker D.R. Am. J. Respir. Cell Mol. Biol. 1998; 19: 929-935Crossref PubMed Scopus (50) Google Scholar). Equal amounts of protein, determined by bicinchoninic acid protein assay (24Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18497) Google Scholar), from large and small aggregate fractions were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with an antibody directed against the HA tag (Santa Cruz Biotechnology). This study was designed to identify the peptide domain(s) that directs sorting and secretion of SP-C. Because isolated type II cells are very difficult to transfect, initial sorting studies were performed in transiently transfected PC12 cells. PC12 cells were transfected with wild type SP-C1–197 and labeled with [35S]cysteine/methionine, and cell lysates and media were immunoprecipitated. SP-C proprotein (Mr = 21,000) was detected in cell lysates but was not processed to the active peptide (Mr = 4000) or secreted into the media (Fig.1 A). To determine if wild type SP-C1–197 was sorted to the regulated secretory pathway, PC12 cells were transfected with SP-C1–197 and stained with antibodies directed against the SP-C proprotein and chromogranin A, a marker for dense core granules. Wild type SP-C1–197 proprotein colocalized with chromogranin A consistent with sorting to the regulated secretory pathway (Fig.1 B, upper panel). These results, coupled with previous reports that SP-C is an integral membrane protein (7Keller A. Eistetter H.R. Voss T. Schäfer K.P. Biochem. J. 1991; 277: 493-499Crossref PubMed Scopus (51) Google Scholar, 25Keller A. Stienhilber W. Schäfer K.P. Voss T. Am. J. Respir. Cell Mol. Biol. 1992; 6: 601-608Crossref PubMed Scopus (32) Google Scholar), suggested that the proprotein was located in the limiting membrane of the dense core granule and that exocytosis would lead to localization of SP-C on the cell surface. To test this hypothesis, PC12 cells were transiently transfected with wild type SP-C1–197 and stained with an antibody directed against the N-terminal or C-terminal peptide domains and wheat germ agglutinin lectin to define the cell surface. In nonpermeabilized cells, SP-C immunoreactivity was detected with antibody directed against the C-terminal peptide, consistent with an extracellular location for this peptide domain (Fig. 1 B). The N-terminal propeptide was detected only after cell permeabilization, confirming that this peptide domain is located in the cytoplasm (Fig. 1 B). Collectively, these data suggest that SP-C is a type II integral membrane protein and that sorting to the regulated secretory pathway occurs independently of proprotein processing. To determine the minimal peptide sequence required for sorting of the SP-C proprotein to the regulated secretory pathway, a series of deletion constructs (Fig. 2 A) were cloned in frame with GFP, transfected into PC12 cells, and analyzed for colocalization with chromogranin A. All deletion constructs, including a construct consisting of only the mature SP-C peptide (SPC24–58), colocalized with chromogranin A (Fig.2 B); further, SP-C was sorted to the regulated secretory pathway irrespective of the position of GFP at the N- or C terminus of the mature SP-C peptide (data not shown). SPC24–58contains two candidate-sorting motifs, a dileucine motif at positions 31 and 32 (residues 54 and 55 of the proprotein) and two palmitoylated cysteines at positions 5 and 6 (residues 28 and 29 of the proprotein). The sorting activity of the dileucine motif was tested by deleting the last 5 residues of the mature peptide (SP-C24–53); the sorting activity of the palmitoylated cysteines was tested by mutating both cysteine residues to serine or alanine (SP-CCC→SSand SP-CCC→AA) in the context of the proprotein (Fig.2 A). The proteins encoded by all of these constructs colocalized with chromogranin A following transfection of PC12 cells, indicating that neither motif is involved in SP-C sorting (data not shown). We conclude from these experiments that the N- and C-terminal peptide domains of SP-C are not required for sorting to the regulated secretory pathway of PC12 cells and that a novel sorting motif is located in the 10-residue extramembrane, cytosolic domain of the mature peptide (residues 24 and 33 of the proprotein). The results in PC12 cells predicted that the mature SP-C peptide (SP-C24–58) would be sorted to lamellar bodies in type II cells. This hypothesis was tested by transfecting freshly isolated mouse type II cells with a construct encoding the mature SP-C peptide (GFP/SP-C24–58). The plasmid was precipitated onto gold particles and propelled into type II cells with a hand-held gene gun (Bio-Rad). Following 24 h of culture, GFP fluorescence was detected in vesicular structures that also stained positively for the mature SP-B peptide (Fig. 3). Since SP-B mature peptide is only detected in multivesicular bodies and lamellar bodies of type II cells (26Voorhout W.F. Veenendaal T. Haagsman H.P. Weaver T.E. Whitsett J.A. van Golde L.M.G. Geuze H.J. Am. J. Physiol. 1992; 263: L479-L486PubMed Google Scholar), these results confirm that the mature SP-C peptide is sorted to the secretory pathway in the absence of the flanking N- and C-terminal peptide domains. To determine if SP-C mature peptide could be sorted to lamellar bodiesin vivo, an adenoviral construct encoding SP-C mature peptide with a hemagglutinin tag (SP-C24–58HA) was generated for intratracheal injection into adult mice. The subcellular distribution of SP-C24–58HA in infected mice was analyzed by immunogold labeling of fixed, cryoprotected frozen lung sections. Gold particles were detected throughout the regulated secretory pathway including the endoplasmic reticulum, Golgi, multivesicular bodies, and lamellar bodies, but not in the nucleus, mitochondria, or plasma membrane (Fig. 4, B andC). Within multivesicular bodies, HA labeling was detected on the limiting membrane and the inner vesicles (Fig. 4 B) similar to the distribution of SP-C proprotein in wild type (data not shown) and SP-B null mice (Fig. 4 A). Localization of SP-C on the inner vesicles of multivesicular bodies suggested that SP-C24–58HA could be secreted. To determine if the cytosolic domain of the mature SP-C peptide (residues 1–10) was sufficient to direct secretion of SP-C, mice were intratracheally injected with SP-C24–58HA adenovirus. Four days postinfection, mice were lavaged, and the surfactant pellet was isolated. SP-C24–58HA was detected in the surfactant pellet of all seven infected mice, consistent with secretion of the peptide into the airway (Fig.5 A). Fractionation of BAL into large and small aggregates revealed that SP-C24–58HA co-sedimented with wild type mature SP-B in the surface-active, large aggregate fraction (Fig. 5 B). Immunohistochemical analyses of the lungs from injected mice detected HA staining in epithelial cells and in the alveolar spaces without any evidence of cytotoxicity; further, when mice were infected with adenovirus encoding luciferase, reporter activity was not detected in BAL (data not shown). These data indicate that SP-C mature peptide is secreted into the alveolar space and associates with the large aggregate surfactant fraction. To determine if SP-C24–58HA could be secreted in the absence of endogenous SP-C, SP-C knockout mice were intratracheally injected with SP-C24–58HA adenovirus. Four days after infection, mice were lavaged, and large and small aggregate fractions were isolated. SP-C24–58HA was detected in the large aggregate fraction of wild type but not SP-C null mice (Fig.6 A). To determine if SP-C24–58HA was sorted to lamellar bodies in the absence of endogenous SP-C, lamellar bodies from lung homogenates were isolated from infected wild type and SP-C knockout mice. SP-C24–58HA was detected in lamellar body fractions of wild type mice but not SP-C (−/−) (Fig. 6 B). We conclude from these experiments that, in the absence of endogenous SP-C, the cytosolic domain of the mature peptide is not sufficient to sort SP-C to the regulated secretory pathway in type II cells. To test the hypothesis that the N-terminal propeptide of SP-C could direct sorting and secretion of SP-C24–58 in SP-C (−/−) mice, an adenovirus encoding the SP-C N-terminal propeptide and mature peptide (SP-C1–58HA) was generated. Three days after infection, SP-C (−/−) mice were lavaged, and the large aggregate fraction was isolated. Western analysis detected SP-C1–58HA in the large aggregate surfactant fraction that also contained mature SP-B peptide (Fig.7). SP-C1–58HA comigrated with SP-C24–58HA in wild type mice, consistent with cleavage of the N-terminal propeptide. The results of these experiments indicate that the N-terminal propeptide is required for secretion of SP-C. To determine if the cytosolic domain of SP-C could direct sorting and secretion of a heterologous transmembrane domain, an adenoviral construct consisting of the SP-C cytosolic domain (residues 1–33), the platelet-derived growth factor receptor (PDGFR) transmembrane domain (residues 531–553), and the 9-amino acid HA tag (SPC1–33PDGFR531–553HA) was generated. Three days after infection, SP-C (−/−) mice were lavaged, and the large aggregate fraction isolated; in addition, lamellar body fractions were isolated from lung tissue. SPC1–33PDGFR531–553HA was detected in both large aggregate and lamellar body fractions (Fig.8); however, SPC1–33PDGFR531–553HA did not comigrate with SP-C1–58HA, suggesting that the N-terminal propeptide was not cleaved from the chimeric protein. These results demonstrate that the cytosolic domain of the SP-C proprotein can direct the sorting and secretion of a heterologous transmembrane domain but is not sufficient to direct processing of the propeptide. SP-C is an integral membrane protein that is sorted to the lamellar body and subsequently secreted with surfactant lipids into the alveolar space. The current study was undertaken to identify the peptide domain(s) involved in the sorting of SP-C to the regulated secretory pathway in type II cells and secretion of this integral membrane protein. In the presence of endogenous SP-C, the mature peptide was sorted to the regulated secretory pathway and was secreted into the airspace. However, in the absence of endogenous SP-C, the SP-C N-terminal propeptide was required for secretion of the mature peptide. The importance of the cytosolic domain for secretion of SP-C was confirmed by demonstrating that residues 1–33 of the proprotein was sufficient to direct secretion of a truncated PDGFR receptor. Integral membrane proteins are sorted to specific subcellular compartments based on information encoded in the cytosolic domain of the protein (14Hunziker W. Geuze H.J. Bioessays. 1996; 18: 379-389Crossref PubMed Scopus (237) Google Scholar, 15Marks M.S. Ohno H. Kirchhausen T. Bonifacino S.J. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar). Previous studies have identified the SP-C proprotein as a type III integral membrane protein (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar), in which the C-terminal propeptide is located in the cytoplasm, or a type II membrane integral membrane protein (7Keller A. Eistetter H.R. Voss T. Schäfer K.P. Biochem. J. 1991; 277: 493-499Crossref PubMed Scopus (51) Google Scholar), in which the N-terminal propeptide is cytosolic. The results of the current study in PC12 cells suggest a type II membrane orientation consistent with the results reported by Keller et al. (7Keller A. Eistetter H.R. Voss T. Schäfer K.P. Biochem. J. 1991; 277: 493-499Crossref PubMed Scopus (51) Google Scholar). This conclusion is also consistent with the presence of a putative stop-transfer sequence (amino acids 34 and 35 of the proprotein) and the palmitoylation of adjacent cysteine residues (amino acids 28 and 29 of the proprotein), motifs that frequently occur on the cytosolic side of the membrane (27Dunphy J.T. Linder M.E. Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (315) Google Scholar,28Sipos L. von Heijne G. Eur. J. Biochem. 1993; 213: 1333-1340Crossref PubMed Scopus (252) Google Scholar). Further evidence comes from immunogold labeling studies that detected the N-terminal propeptide inside the internal vesicles of multivesicular bodies, consistent with a type II orientation (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar, 9Voorhout W.F. Weaver T.E. Haagsman H.P. Geuze H.J. van Golde L.M.J. Microsc. Res. Technique. 1993; 26: 366-373Crossref PubMed Scopus (67) Google Scholar). Taken together, these results suggest that the cytosolic domain of SP-C is composed of the 23-amino acid N-terminal propeptide and the first 10 amino acids of the mature peptide. Consistent with a cytosolic location for the N-terminal propeptide, deletion of the entire 139-amino acid C-terminal propeptide of the SP-C proprotein did not perturb sorting to the regulated secretory pathway in PC12 cells. Moreover, removal of the last 5 amino acids of the mature peptide (residues 54–58 of the proprotein), which contains a dileucine motif at positions 54 and 55, did not affect SP-C sorting. SP-C was also sorted following the deletion of the 23-amino acid N-terminal propeptide, suggesting that the 10-amino acid cytosolic domain of the mature peptide (residues 24–33 of the proprotein) is sufficient to direct SP-C to the regulated secretory pathway. This outcome is difficult to reconcile with results of previous studies suggesting that the C-terminal propeptide of the proprotein was critical for intracellular trafficking of SP-C (25Keller A. Stienhilber W. Schäfer K.P. Voss T. Am. J. Respir. Cell Mol. Biol. 1992; 6: 601-608Crossref PubMed Scopus (32) Google Scholar, 29Beers M.F. Lomax C.A. Russo S.J. J. Biol. Chem. 1998; 273: 15287-15293Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). It is possible that selected mutations/deletions within the C-terminal peptide resulted in misfolding and degradation of SP-C in a cytosolic compartment; for example, in previous studies, a potential intramolecular sulfhydryl bridge was disrupted by deletion of cysteine 189 and/or cysteine 121, residues that are strictly conserved in the SP-C proprotein of all seven species analyzed to date (25Keller A. Stienhilber W. Schäfer K.P. Voss T. Am. J. Respir. Cell Mol. Biol. 1992; 6: 601-608Crossref PubMed Scopus (32) Google Scholar, 29Beers M.F. Lomax C.A. Russo S.J. J. Biol. Chem. 1998; 273: 15287-15293Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 30Russo S.J. Wang W.J. Lomax C.A. Beers M.F. Am. J. Physiol. 1999; 277: L1034-L1044Crossref PubMed Google Scholar). Interpretation of the latter studies is further complicated by expression of SP-C deletion constructs in cell types that lack a typical regulated secretory pathway. PC12 cells used in this study have a well characterized regulated secretory pathway and have been used extensively for sorting and secretion studies (31Stahlman M.T. Gray M.E. Ross G.F. Hull W.M. Wikenheiser K. Dingle S. Zelenskilow K.R. Whitsett J.A. Pediatr. Res. 1992; 31: 364-371Crossref PubMed Scopus (22) Google Scholar, 32Cool D.R. Louie D.Y. Loh Y.P. Endocrinology. 1996; 137: 5441-5446Crossref PubMed Scopus (11) Google Scholar, 33Schweitzer E.S. Jeng C.J. TaoCheng J.H. J. Neurosci. Res. 1996; 46: 519-530Crossref PubMed Scopus (5) Google Scholar, 34Blagoveshchenskaya A.D. Norcott J.P. Cutler D.F. J. Biol. Chem. 1998; 273: 2729-2737Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 35Chuman Y. Bergman A.C. Ueno T. Saito S. Sakaguchi K. Alaiya A.A. Franzen B. Bergman T. Arnott D. Auer G. Appella E. Jornvall H. Linder S. FEBS Lett. 1999; 462: 129-134Crossref PubMed Scopus (100) Google Scholar). We have previously used PC12 cells to identify a sorting determinant in the SP-B proprotein and confirmed the identity of the sorting signal in transgenic mice (16Lin 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, 17Lin S. Phillips K.S. Wilder M.R. Weaver T.E. BBA Mol. Cell Res. 1996; 1312: 177-185Crossref PubMed Scopus (70) Google Scholar). In the current study, the results of experiments in PC12 cells were confirmed by transfecting isolated type II cells and by intratracheal injection of wild type mice with adenovirus encoding the mature peptide. In both cases, SP-C mature peptide (SP-C24–58HA) was detected in lamellar bodies, consistent with sorting of the mature peptide to this compartment in the absence of the N- and C-terminal peptide domains. Interestingly, SP-C was not detected in lamellar bodies or BAL of SP-C (−/−) mice infected with adenovirus encoding SP-C24–58HA. This outcome suggests that in wild type mice endogenous SP-C associated with transfected mature SP-C peptide to facilitate its trafficking to lamellar bodies; SP-C mature peptide may similarly interact with an endogenous protein(s) in PC12 cells, leading to localization in dense core secretory granules. The sorting deficit in SP-C (−/−) mice was corrected by the addition of the 23-amino acid N-terminal propeptide to SP-C24–58. SP-C1–58HA was processed to the mature peptide consistent with trafficking through the multivesicular body, a compartment previously shown to be involved in processing of the SP-C and SP-B proproteins (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar, 10Beers M.F. Lomax C. Am. J. Physiol. 1995; 13: L744-L753Google Scholar, 26Voorhout W.F. Veenendaal T. Haagsman H.P. Weaver T.E. Whitsett J.A. van Golde L.M.G. Geuze H.J. Am. J. Physiol. 1992; 263: L479-L486PubMed Google Scholar); further, the processed peptide was detected in the extracellular, surface-active, large aggregate fraction of surfactant. Collectively, these studies suggest that the N-terminal propeptide is required for efficient sorting of SP-C to the distal secretory pathway in type II cells and that sorting occurs independently of the C-terminal peptide. Furthermore, the cytosolic domain of SP-C was able to direct both the sorting (to lamellar bodies) and secretion of the transmembrane domain of PDGFR. However, SPC1–33PDGFR531–553HA was not efficiently processed to a smaller form, suggesting that the SP-C transmembrane domain may contribute to the formation of an optimal cleavage site between the propeptide and mature peptide. Sorting of SP-C to the distal secretory pathway is necessary but not sufficient for secretion of SP-C into the alveolar space. In transfected PC12 cells, SP-C was detected in the limiting membrane of dense core granules and was transferred to the plasma membrane during exocytosis; however, in type II epithelial cells, SP-C was detected in the lumen of the lamellar body and was secreted with surfactant phospholipids. These observations suggest that the SP-C proprotein is relocated from the limiting membrane of a transport or storage vesicle to the vesicle lumen during transit in the secretory pathway of the type II cell. Ultrastructural analyses detected SP-C proprotein in the limiting membrane and luminal vesicles of multivesicular bodies, suggesting a key role for this compartment in SP-C secretion. Relocation of SP-C within the multivesicular body is probably the result of inward vesiculation of the limiting membrane, similar to the process leading to internalization and subsequent degradation of the epidermal growth factor receptor in lysosomes (36Felder S. Miller K. Moehren G. Ullrich A. Schlessinger J. Hopkins C.R. Cell. 1990; 61: 623-634Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 37Futter C.E. Pearse A. Hewlett L.J. Hopkins C.R. J. Cell Biol. 1996; 132: 1011-1023Crossref PubMed Scopus (433) Google Scholar, 38Kornilova E. Sorkina T. Beguinot L. Sorkin A. J. Biol. Chem. 1996; 271: 30340-30346Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Inward vesiculation of the multivesicular body-limiting membrane is probably dependent on the interaction of specific residues in the cytosolic domain of the SP-C proprotein with unidentified cytoplasmic proteins. Not all proteins on the limiting membrane of the multivesicular body are included in the inner vesicles (39Denzer K. Weber B. HilleRehfeld A. vonFigura K. Pohlmann R. Biochem. J. 1997; 326: 497-505Crossref PubMed Scopus (32) Google Scholar, 40Escola J.M. Kleijmeer M.J. Stoorvogel W. Griffith J.M. Yoshie O. Geuze H.J. J. Biol. Chem. 1998; 273: 20121-20127Abstract Full Text Full Text PDF PubMed Scopus (883) Google Scholar), suggesting that SP-C is selectively sorted to the inner vesicle. The finding that SP-C1–58HA is secreted following infection of SP-C (−/−) mice suggests that residues 1–34 of the proprotein contain one of more motifs that direct sorting to the secretory pathway and internalization of SP-C into the luminal vesicles of the multivesicular bodies. Following internal vesicle formation, the multivesicular body fuses with the lamellar body, resulting in the incorporation of the internal vesicles of the multivesicular body into the internal membranes of the lamellar body and, ultimately, secretion of SP-C with surfactant phospholipids (41Stahlman M.T. Gray M.P. Falconieri M.W. Whitsett J.A. Weaver T.E. Lab. Invest. 2000; 80: 395-403Crossref PubMed Scopus (87) Google Scholar, 42Sorokin S.P. J. Histochem. Cytochem. 1967; 14: 884-897Crossref Scopus (186) Google Scholar). In summary, the mature SP-C peptide is sorted to the regulated secretory pathway of PC12 cells, isolated mouse type II cells, and adenovirus-infected wild type mice; however, mature SP-C peptide was not sorted to lamellar bodies or secreted into the airway of adenovirus-infected SP-C (−/−) mice. The addition of the SP-C N-terminal propeptide to the mature peptide (SP-C1–58HA) restored sorting, processing, and secretion of SP-C as part of a functional surfactant complex; further, the cytosolic domain of SP-C was sufficient to direct the sorting and secretion of a heterologous transmembrane domain. These studies demonstrate that the N-terminal propeptide of SP-C is critical for the intracellular trafficking and secretion of SP-C in type II cells." @default.
W2058448098 created "2016-06-24" @default.
W2058448098 creator A5027381383 @default.
W2058448098 creator A5028739100 @default.
W2058448098 creator A5050169529 @default.
W2058448098 creator A5055786810 @default.
W2058448098 creator A5068993756 @default.
W2058448098 creator A5069592417 @default.
W2058448098 creator A5083579246 @default.
W2058448098 date "2001-01-01" @default.
W2058448098 modified "2023-09-30" @default.
W2058448098 title "Secretion of Surfactant Protein C, an Integral Membrane Protein, Requires the N-terminal Propeptide" @default.
W2058448098 cites W1807798123 @default.
W2058448098 cites W1965412830 @default.
W2058448098 cites W1966079789 @default.
W2058448098 cites W1967985864 @default.
W2058448098 cites W1978689611 @default.
W2058448098 cites W1980565826 @default.
W2058448098 cites W1983464528 @default.
W2058448098 cites W1989728523 @default.
W2058448098 cites W1995574514 @default.
W2058448098 cites W1996751084 @default.
W2058448098 cites W1996819436 @default.
W2058448098 cites W1996895913 @default.
W2058448098 cites W2010459343 @default.
W2058448098 cites W2011225090 @default.
W2058448098 cites W2013951841 @default.
W2058448098 cites W2017384797 @default.
W2058448098 cites W2028248159 @default.
W2058448098 cites W2029074080 @default.
W2058448098 cites W2041611018 @default.
W2058448098 cites W2057188635 @default.
W2058448098 cites W2059618188 @default.
W2058448098 cites W2066129384 @default.
W2058448098 cites W2068808582 @default.
W2058448098 cites W2078334493 @default.
W2058448098 cites W2080934311 @default.
W2058448098 cites W2082543204 @default.
W2058448098 cites W2087849748 @default.
W2058448098 cites W2098856298 @default.
W2058448098 cites W2106223472 @default.
W2058448098 cites W2165853056 @default.
W2058448098 cites W2179225990 @default.
W2058448098 cites W2318698174 @default.
W2058448098 cites W2342135985 @default.
W2058448098 cites W2347145398 @default.
W2058448098 cites W4232534952 @default.
W2058448098 doi "https://doi.org/10.1074/jbc.m011770200" @default.
W2058448098 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11278984" @default.
W2058448098 hasPublicationYear "2001" @default.
W2058448098 type Work @default.
W2058448098 sameAs 2058448098 @default.
W2058448098 citedByCount "60" @default.
W2058448098 countsByYear W20584480982012 @default.
W2058448098 countsByYear W20584480982014 @default.
W2058448098 countsByYear W20584480982015 @default.
W2058448098 countsByYear W20584480982017 @default.
W2058448098 countsByYear W20584480982019 @default.
W2058448098 countsByYear W20584480982021 @default.
W2058448098 crossrefType "journal-article" @default.
W2058448098 hasAuthorship W2058448098A5027381383 @default.
W2058448098 hasAuthorship W2058448098A5028739100 @default.
W2058448098 hasAuthorship W2058448098A5050169529 @default.
W2058448098 hasAuthorship W2058448098A5055786810 @default.
W2058448098 hasAuthorship W2058448098A5068993756 @default.
W2058448098 hasAuthorship W2058448098A5069592417 @default.
W2058448098 hasAuthorship W2058448098A5083579246 @default.
W2058448098 hasBestOaLocation W20584480981 @default.
W2058448098 hasConcept C104317684 @default.
W2058448098 hasConcept C144647389 @default.
W2058448098 hasConcept C156399914 @default.
W2058448098 hasConcept C161200384 @default.
W2058448098 hasConcept C185592680 @default.
W2058448098 hasConcept C2779664074 @default.
W2058448098 hasConcept C41008148 @default.
W2058448098 hasConcept C41625074 @default.
W2058448098 hasConcept C49039625 @default.
W2058448098 hasConcept C55493867 @default.
W2058448098 hasConcept C58226133 @default.
W2058448098 hasConcept C76155785 @default.
W2058448098 hasConcept C7860815 @default.
W2058448098 hasConceptScore W2058448098C104317684 @default.
W2058448098 hasConceptScore W2058448098C144647389 @default.
W2058448098 hasConceptScore W2058448098C156399914 @default.
W2058448098 hasConceptScore W2058448098C161200384 @default.
W2058448098 hasConceptScore W2058448098C185592680 @default.
W2058448098 hasConceptScore W2058448098C2779664074 @default.
W2058448098 hasConceptScore W2058448098C41008148 @default.
W2058448098 hasConceptScore W2058448098C41625074 @default.
W2058448098 hasConceptScore W2058448098C49039625 @default.
W2058448098 hasConceptScore W2058448098C55493867 @default.
W2058448098 hasConceptScore W2058448098C58226133 @default.
W2058448098 hasConceptScore W2058448098C76155785 @default.
W2058448098 hasConceptScore W2058448098C7860815 @default.
W2058448098 hasIssue "18" @default.
W2058448098 hasLocation W20584480981 @default.
W2058448098 hasOpenAccess W2058448098 @default.
W2058448098 hasPrimaryLocation W20584480981 @default.