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• W2007697123 abstract "Native lung surfactant protein C (SP-C) is a 4.2-kDa acylpeptide that associates with alveolar surfactant phospholipids via a transmembrane α-helix. This helix contains mainly Val, although poly-Val is inefficient in helix formation, and helical SP-C can spontaneously convert to β-sheet aggregates and amyloid-like fibrils. SP-C is cleaved out from a 21-kDa integral membrane protein, proSP-C, in the alveolar type II cell. Recently several mutations localized in the endoplasmic reticulum-lumenal (C-terminal) part of proSP-C (CTproSP-C) have been associated with intracellular accumulation of toxic forms of proSP-C, low levels of mature SP-C, and development of interstitial lung disease. CTproSP-C contains a ∼100-residue Brichos domain of unknown function that is also found in other membrane proteins associated with amyloid formation, dementia, and cancer. Here we find that recombinant CTproSP-C binds lipid-associated SP-C, which is in β-strand conformation, and that this interaction results in an increased helical content. In contrast, CTproSP-C does not bind α-helical SP-C. Recombinant CTproSP-C(L188Q), a mutation associated with interstitial lung disease, shows secondary and quaternary structures similar to those of wild type CTproSP-C but is unable to bind lipid-associated β-strand SP-C. Transfection of CTproSP-C into HEK293 cells that express proSP-C(L188Q) increases the amount of proSP-C protein, whereas no effect is seen on cells expressing wild type proSP-C. These findings suggest that CTproSP-C binds nonhelical SP-C and thereby prevents β-sheet aggregation and that mutations in CTproSP-C can interfere with this function. Native lung surfactant protein C (SP-C) is a 4.2-kDa acylpeptide that associates with alveolar surfactant phospholipids via a transmembrane α-helix. This helix contains mainly Val, although poly-Val is inefficient in helix formation, and helical SP-C can spontaneously convert to β-sheet aggregates and amyloid-like fibrils. SP-C is cleaved out from a 21-kDa integral membrane protein, proSP-C, in the alveolar type II cell. Recently several mutations localized in the endoplasmic reticulum-lumenal (C-terminal) part of proSP-C (CTproSP-C) have been associated with intracellular accumulation of toxic forms of proSP-C, low levels of mature SP-C, and development of interstitial lung disease. CTproSP-C contains a ∼100-residue Brichos domain of unknown function that is also found in other membrane proteins associated with amyloid formation, dementia, and cancer. Here we find that recombinant CTproSP-C binds lipid-associated SP-C, which is in β-strand conformation, and that this interaction results in an increased helical content. In contrast, CTproSP-C does not bind α-helical SP-C. Recombinant CTproSP-C(L188Q), a mutation associated with interstitial lung disease, shows secondary and quaternary structures similar to those of wild type CTproSP-C but is unable to bind lipid-associated β-strand SP-C. Transfection of CTproSP-C into HEK293 cells that express proSP-C(L188Q) increases the amount of proSP-C protein, whereas no effect is seen on cells expressing wild type proSP-C. These findings suggest that CTproSP-C binds nonhelical SP-C and thereby prevents β-sheet aggregation and that mutations in CTproSP-C can interfere with this function. Human surfactant protein C (SP-C) 2The abbreviations used are: SP-C, surfactant protein C; CTproSP-C, C-terminal proSP-C; ER, endoplasmic reticulum; HEK, human embryonic kidney. 2The abbreviations used are: SP-C, surfactant protein C; CTproSP-C, C-terminal proSP-C; ER, endoplasmic reticulum; HEK, human embryonic kidney. is synthesized as a 197-residue proprotein (proSP-C) that is processed to a 35-residue mature transmembrane peptide corresponding to residues 24-58 of proSP-C. ProSP-C is exclusively expressed by the alveolar type II cells and is an integral membrane protein with a type II orientation in the endoplasmic reticulum (ER) membrane (C-terminal in the ER lumen) (1Conkright J.J. Bridges J.P. Na C.L. Voorhout W.F. Trapnell B. Glasser S.W. Weaver T.E. J. Biol. Chem. 2001; 276: 14658-14664Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 2Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 269: L727-L733Crossref PubMed Google Scholar, 3Beers M.F. Lomax C. Am. J. Physiol. 1995; 269: L744-L753Crossref PubMed Google Scholar, 4Russo S.J. Wang W. Lomax C.A. Beers M.F. Am. J. Physiol. 1999; 277: L1034-L1044Crossref PubMed Google Scholar, 5Wert S.E. Glasser S.W. Korfhagen T.R. Whitsett J.A. Dev. Biol. 1993; 156: 426-443Crossref PubMed Scopus (268) Google Scholar, 6Glasser S.W. Korfhagen T.R. Wert S.E. Bruno M.D. McWilliams K.M. Vorbroker D.K. Whitsett J.A. Am. J. Physiol. 1991; 261: L349-L356PubMed Google Scholar). SP-C together with SP-B and phospholipids are responsible for lowering the surface tension at the air/liquid interface and by doing so prevent alveolar collapse, (see Ref. 7Whitsett J.A. Weaver T.E. N. Engl. J. Med. 2002; 347: 2141-2148Crossref PubMed Scopus (386) Google Scholar for a review). SP-C is a highly conserved and very hydrophobic protein that lacks known structural homologues. The transmembrane part of SP-C is an α-helix made up of almost only valines. Valines are rare in α-helices and overrepresented in β-strands, and the SP-C α-helix is metastable and can convert to β-sheet aggregates and form amyloid-like fibrils (8Kallberg Y. Gustafsson M. Persson B. Thyberg J. Johansson J. J. Biol. Chem. 2001; 276: 12945-12950Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 9Szyperski T. Vandenbussche G. Curstedt T. Ruysschaert J.M. Wuthrich K. Johansson J. Protein Sci. 1998; 7: 2533-2540Crossref PubMed Scopus (69) Google Scholar, 10Gustafsson M. Thyberg J. Naslund J. Eliasson E. Johansson J. FEBS Lett. 1999; 464: 138-142Crossref PubMed Scopus (95) Google Scholar). Such fibrils of SP-C have been isolated from patients suffering from pulmonary alveolar proteinosis, a lung disease characterized by alveolar accumulation of proteinaceous material. Expression of mature SP-C in the absence of the N- and C-terminal proparts results in aggregation of SP-C in the secretory pathway and severe lung malformation during embryonic development, indicating that the proparts are necessary for correct SP-C maturation (1Conkright J.J. Bridges J.P. Na C.L. Voorhout W.F. Trapnell B. Glasser S.W. Weaver T.E. J. Biol. Chem. 2001; 276: 14658-14664Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 11Beers M.F. Lomax C.A. Russo S.J. J. Biol. Chem. 1998; 273: 15287-15293Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Mutations in the gene encoding proSP-C are associated with familial and sporadic cases of interstitial lung disease in which the mutations cause the protein to misfold and form toxic intracellular aggregates (12Nogee L.M. Dunbar III, A.E. Wert S.E. Askin F. Hamvas A. Whitsett J.A. N. Engl. J. Med. 2001; 344: 573-579Crossref PubMed Scopus (711) Google Scholar, 13Nogee L.M. Dunbar III, A.E. Wert S. Askin F. Hamvas A. Whitsett J.A. Chest. 2002; 121: 20S-21SAbstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 14Thomas A.Q. Lane K. Phillips III, J. Prince M. Markin C. Speer M. Schwartz D.A. Gaddipati R. Marney A. Johnson J. Roberts R. Haines J. Stahlman M. Loyd J.E. Am. J. Respir. Crit. Care Med. 2002; 165: 1322-1328Crossref PubMed Scopus (525) Google Scholar). The so far characterized mutations have been identified on only one allele but can result in almost complete absence of mature SP-C. This suggests that mutations in the proSP-C gene generate a dominant-negative toxic gain of function. A mutation that causes exclusion of exon 4 by alternative splicing (SP-CΔExon 4) leading to a 37-amino acid C-terminally shortened proprotein results in total absence of mature SP-C (12Nogee L.M. Dunbar III, A.E. Wert S.E. Askin F. Hamvas A. Whitsett J.A. N. Engl. J. Med. 2001; 344: 573-579Crossref PubMed Scopus (711) Google Scholar). The exchange of glutamine for leucine at position 188 gives abnormal-appearing lamellar bodies, slowed cell growth, and signs of cytotoxicity (14Thomas A.Q. Lane K. Phillips III, J. Prince M. Markin C. Speer M. Schwartz D.A. Gaddipati R. Marney A. Johnson J. Roberts R. Haines J. Stahlman M. Loyd J.E. Am. J. Respir. Crit. Care Med. 2002; 165: 1322-1328Crossref PubMed Scopus (525) Google Scholar). Expression of proSP-C with mutations in positions 66 or 73 (E66K and I73T) give rise to detectable amounts of mature SP-C (15Brasch F. Griese M. Tredano M. Johnen G. Ochs M. Rieger C. Mulugeta S. Muller K.M. Bahuau M. Beers M.F. Eur. Respir. J. 2004; 24: 30-39Crossref PubMed Scopus (121) Google Scholar, 16Stevens P.A. Pettenazzo A. Brasch F. Mulugeta S. Baritussio A. Ochs M. Morrison L. Russo S.J. Beers M.F. Pediatr. Res. 2005; 57: 89-98Crossref PubMed Scopus (69) Google Scholar). Thus it appears that the effect on proSP-C processing depends on where the mutation is located in the proprotein. The C-terminal part of proSP-C stretches from residues 59 to 197, and it has an unknown structure and function. In this region, a novel domain called Brichos (residues 94-197 in proSP-C) is located (17Sanchez-Pulido L. Devos D. Valencia A. Trends Biochem. Sci. 2002; 27: 329-332Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Most of the mutations found in the proSP-C gene are located in the Brichos part of proSP-C (18Beers M.F. Mulugeta S. Annu. Rev. Physiol. 2005; 67: 663-696Crossref PubMed Scopus (143) Google Scholar). The Brichos domain contains about 100 amino acids and is also found in other previously unrelated proteins, such as Bri associated with amyloid formation and dementia, ChM-I associated with chondrosarcoma, and CA11 associated with stomach cancer (17Sanchez-Pulido L. Devos D. Valencia A. Trends Biochem. Sci. 2002; 27: 329-332Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). All of these proteins have a transmembrane sequence in the N-terminal region, and proSP-C and Bri are posttranslationally processed by proteases. One suggested function for the Brichos domain was a chaperone-like activity, which would prevent misfolding and aggregation of the parent protein; consistent with this hypothesis is the observation that the Brichos domain structurally matches the apical domain of GroEL (17Sanchez-Pulido L. Devos D. Valencia A. Trends Biochem. Sci. 2002; 27: 329-332Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). These findings provided the incentive to investigate the function of the C-terminal part of proSP-C in the relation to folding of the mature peptide. Protein Expression and Purification—A region from nucleotide 175 (in the codon for His59) to nucleotide 991 (in the codon for Ile197) of the proSP-C cDNA sequence (i.e. coding for CTproSP-C) was amplified from FirstChoice PCR-Ready human lung cDNA (Ambion, Cambridgeshire, UK). For the PCR amplification two primers (DNA Technology AIS, Aarhus, Denmark) were used: 5′-GGTGMCCATGGCACATGAGCCAGAAACACACGGCGATGG-3′ (forward primer) and 5′-CTCTAGAGGATCCGGATCCCTAGATGTAGTAGAGCGGCACCTCC-3′ (reverse primer); the underlined sequences are BamHI and NcoI cleavage sites, respectively. The amplified cDNA fragment was digested with BamHI and NcoI and ligated into the expression vector pET-32c (Novagen, Madison, WI) coding for thioredoxin, hexahistidine, and S tags upstream of the insertion site. Thrombin and enterokinase cleavage sites allow proteolytic removal of the tags from the fusion protein. The mutant CTproSP-C(L188Q) was created from the wild type cDNA using the forward primer 5′-AGCACCCAGTGTGGCGAGGTGCCGCTCTAC-3′ and the reverse primer 5′-GCCACACTGGGTGCTCACGGCCATGCCCAA-3′; the mutation sites are underlined. For expression of wild type and mutant CTproSP-C, transformed Escherichia coli strain Origami (DE3) pLysS (Novagen, Madison, WI) were grown at 30 °C in LB medium (1 liter of LB medium contains 10 g of tryptone,5gof yeast, and 10 g of NaCl) containing 100 μg/ml ampicillin for 16 h with constant stirring. Expression was induced at an A600 around 1.2 by the addition of isopropyl β-d-thiogalactopyranoside to 0.5 mm, the temperature was lowered to 25 °C, and the bacteria were grown for another 4 h. The cells were then harvested by centrifugation at 6000 × g for 20 min at 4 °C and stored at -70 °C. The cells were lysed by lysozyme (1 mg/ml) treatment for 30 min on ice, followed by sonication for 10 min. The cell lysate was centrifuged at 6500 × g for 20 min, and the pellet was suspended in 2 m urea in buffer A (20 mm Tris, 0.5 m NaCl) containing 5 mm imidazole, pH 8, and sonicated for 5 min. After centrifugation at 6000 × g for 30 min at 4 °C, the supernatant was filtered through a 0.45-μm filter, then mixed with 5 ml of nickel-nitrilotriacetic acid-agarose (Qiagen Ltd., West Sussex, UK), and poured into a column. The column was washed with 100 ml of 2 m urea in buffer A, 5 mm imidazole, pH 8, and then with 100 ml 1 m urea in the same solution and finally with 100 ml of buffer A with 5 mm imidazole only. The fusion protein was then eluted with 300 mm imidazole in buffer A, pH 8. The eluted protein was dialyzed against 20 mm Tris, 50 mm NaCl, pH 7.4. The thioredoxin and His tags were removed by cleavage with thrombin (kind gift from Prof. Steven Olson, Chicago, IL) at an enzyme/substrate weight ratio of 0.002 for 5-16 h at 4 °C. After cleavage, the solution was reapplied to a Ni2+ column to remove the released thioredoxin-His tag fragment and uncleaved fusion protein. After elution from the Ni2+ column, the protein was applied on an anion exchange column (5 ml of HiTrap QFF from Amersham Biosciences) equilibrated with 20 mm Tris, 20 mm NaCl, pH 7.4. The protein eluted as a single peak using a linear gradient from 20 mm to 1 m NaCl and was dialyzed against buffer B (20 mm NaH2PO4,5mm NaCl), pH 7.4. The protein concentration was determined from the absorbance at 280 nm using an extinction coefficient of 1.15 μm-1 cm-1. The protein purity was checked by SDS-PAGE under nonreducing conditions and by nondenaturing PAGE. Protein-Peptide and Protein-Lipid Interactions—Phospholipids were isolated from the modified natural surfactant preparation, Curosurf, which contains 98% (w/w) phospholipids and about 1% each of SP-B and SP-C (Chiesei Farmaceutici, Parma, Italy). Curosurf paste was dissolved in MeOH/CHCl3 (4:1, v/v), and phospholipids were separated from SP-B and SP-C by reversed phase liquid chromatography using a Lipidex 5000 column (40 × 6.5 cm; Amersham Biosciences) (19Stark M. Wang Y. Danielsson O. Jornvall H. Johansson J. Anal. Biochem. 1998; 265: 97-102Crossref PubMed Scopus (24) Google Scholar). Synthetic porcine SP-C (LRIPCCPVNLKRLLVVVVVVVLVVVVIVGALLMGL) was made and purified as described (20Johansson J. Nilsson G. Stromberg R. Robertson B. Jornvall H. Curstedt T. Biochem. J. 1995; 307: 535-541Crossref PubMed Scopus (87) Google Scholar), and native SP-C was purified from pig lung homogenates as described (21Curstedt T. Jornvall H. Robertson B. Bergman T. Berggren P. Eur. J. Biochem. 1987; 168: 255-262Crossref PubMed Scopus (244) Google Scholar). Synthetic SP-C forms β-sheet aggregates that can only be dissolved in neat acids, e.g. formic acid (20Johansson J. Nilsson G. Stromberg R. Robertson B. Jornvall H. Curstedt T. Biochem. J. 1995; 307: 535-541Crossref PubMed Scopus (87) Google Scholar). For reconstitution of peptides and phospholipids, surfactant phospholipids were dissolved in MeOH/CHCl3 (1:1, v/v) and peptides, synthetic SP-C, or native SP-C dissolved in formic acid or in MeOH/CHCl3 (1:1 v/v), respectively, were added, and the solutions were incubated at 37 °C until the solvents were evaporated. Lipids and peptides were then resuspended in buffer B, pH 7.4, by vortexing and sonication, giving a final concentration of 5 mg/ml phospholipids and 20 μm peptide. Phospholipids without peptide and Curosurf paste were resuspended in the same manner. Wild type or mutant CTproSP-C was then added to the phospholipids or phospholipid/peptide mixtures to a concentration of 50 μg/ml. The mixtures were incubated at 37 °C for 1 h and then centrifuged at 40 000 × g for 30 min at 4 °C. The pellets and supernatants obtained were analyzed by SDS-PAGE using a 15% gel run under nonreducing conditions. After transfer to a nitrocellulose membrane, CTproSP-C was visualized by Western blot using a horseradish peroxidase-conjugated anti-S tag antibody (Novagen, Madison, WI). For analysis of interactions between CTproSP-C and water-soluble poly-valine peptides, the peptides KKVVVVVVVKK (K2V7K2) and KKVVVVVKK (K2V5K2) were purchased from Thermo Electron GmBH, Germany. The Lys residues at the N and C termini were introduced to make the peptides soluble in water. CTproSP-C (10 μm) and peptides (10, 50, or 100 μm) were coincubated in buffer B, pH 7.4, at 37 °C for 1 h, and thereafter the mixtures were resolved by nondenaturing PAGE using a 12% gel. CD Experiments—CD spectra in the far-UV region (190-260 nm) were recorded at 22 °C with a Jasco J-810-150S spectropolarimeter (Jasco, Tokyo, Japan) using a bandwidth of 1 nm and a response time of 2 s, and 10 data points/nm were collected. Each spectrum shown is the average of three consecutive recordings. Spectra were recorded of wild type or mutant CTproSP-C (10 μm) in (i) buffer B, pH 7.4, or in (ii) 2% (w/v) SDS micelles in buffer B and of (iii) synthetic or native SP-C (10 μm) in 2% (w/v) SDS micelles in buffer B and (iv) combinations of CTproSP-C and SP-C peptides in 2% (w/v) SDS micelles. For incorporation of peptides in SDS micelles, SDS was dissolved in MeOH, and synthetic or native SP-C (dissolved in formic acid or MeOH/CHCl3, respectively) was added, and the solutions were incubated at 37 °C until the solvents were evaporated. SDS micelles, with or without peptides, were then prepared by resuspension in buffer B, pH 7.4. For analysis of CTproSP-C in the presence of SDS micelles, the micelles were prepared first, and then the CTproSP-C was added. Estimation of secondary structure contents from the CD spectra were performed by deconvolution into four simple components (22Perczel A. Park K. Fasman G.D. Anal. Biochem. 1992; 203: 83-93Crossref PubMed Scopus (420) Google Scholar) or by using the residual molar ellipticity values for 208 and 222 nm (23Barrow C.J. Yasuda A. Kenny P.T.M. Zagorski M.G. J. Mol. Biol. 1992; 225: 1075-1093Crossref PubMed Scopus (605) Google Scholar). Recombinant cystatin A (kind gift from Prof. Ingemar Björk, Uppsala, Sweden) was used instead of CTproSP-C in control experiments. Human Embryonic Kidney (HEK) 293 Cell Experiments— HEK293 cells stably transfected with human wild type proSP-C (24Bridges J.P. Na C.-L. Wong H.R. Weaver T.E. J. Cell Biol. 2006; 172: 395-40722Crossref PubMed Scopus (100) Google Scholar), proSP-C(L188Q), or empty vector (pTRE2-Hyg; BD Biosciences) were grown in Eagle's minimum essential medium (Sigma) supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 units/ml PeSt (SVA, Uppsala, Sweden), and 75 μg/ml hygromycin B (Duchefa, Haarlem, The Netherlands), at 37 °C in 5% CO2. For testing the ability of CTproSP-C to rescue misfolded proSP-C(L188Q), 1 × 106 of each of the stably transfected cells were plated into 6-well plates and then grown in the same medium as described above, except for the omission of hygromycin B. After 24 h the cells were transfected with ∼1 μg/well of a vector (pIRES2) encoding human SP-B1-23-CT-proSP-C (SP-B1-23 encodes a signal peptide) and enhanced green fluorescent protein. For each transfection 250 μl of OPTI-MEM (Invitrogen) was mixed with 4 μl of Lipofectamine 2000 (Invitrogen), and the mixture was left at room temperature for 5 min. The vector DNA was mixed with 250 μl of OPTI-MEM and then mixed with the Lipofectamine solution. Finally, the DNA/Lipofectamine solution was added dropwise to the cells, and the cells were incubated at 37 °C with 5% CO2 for 24 h. The cells were then harvested in Laemmli buffer and sonicated for 5 min. The samples were analyzed by SDS-PAGE/Western blotting with antibodies against the N- or C-terminal proparts of proSP-C, and an antibody against enhanced green fluorescent protein. CTproSP-C Expression and Characterization—A fragment covering residues 59-197 of human proSP-C was expressed in E. coli and purified to homogeneity. This fragment starts just C-terminally of the part corresponding to the mature SP-C peptide and ends with the proSP-C C terminus. CTproSP-C was expressed as a fusion protein with thioredoxin, His, and S tags at the N-terminal end. Solubilization of the pellet obtained after cell lysis with 2 m urea followed by removal of urea, affinity chromatography, and cleavage of the thioredoxin and His tags, resulted in a yield of about 4 mg of S tag-CTproSP-C/liter of culture. The S tag was kept to allow Western blot analysis using commercially available anti-S tag antibodies. Analysis by non-reducing SDS-PAGE (Fig. 1A) shows that >90% of the purified protein corresponds to monomeric CTproSP-C. Western blot analysis (Fig. 1C) indicates that the minor bands with higher molecular mass correspond to oligomeric forms of CTproSP-C. Nondenaturing PAGE (Fig. 1B) likewise shows one major band, with a minor band migrating just anodally. Comparison of the migration of CTproSP-C upon nondenaturing PAGE with that of proteins with similar calculated sizes and isoelectric points indicate that CTproSP-C forms an oligomer, and gel filtration shows that the main form is a tetramer (data not shown). CD spectroscopy of CTproSP-C (Fig. 2) shows a broad minimum from 205 to 225 nm, indicating that the protein is folded and contains a mixture of helical and sheet structure. The rather low residual molar ellipticity suggests that CTproSP-C also contains unordered structure. From the spectrum in Fig. 2, a helical content of 20-30% and a β-sheet content of 30% is estimated. This is in reasonable agreement with the secondary structure content of CTproSP-C predicted using the ExPASy server (www.expasy.org), which yields about 30% helix and 20% β-sheet. In the presence of SDS micelles (see Fig. 5A) or phospholipid vesicles (data not shown), the amplitudes of the CTproSP-C residual molar ellipticity in the 200-230- and 190-nm regions are increased compared with the values in buffer (Fig. 2), indicating that CTproSP-C is more structurally ordered in the presence of detergents or phospholipids. The structure and stability of CTproSP-C under different conditions is currently under further investigation.FIGURE 2Far-UV CD spectra of wild type CTproSP-C and CTproSP-C(L188Q). The spectra were obtained in buffer B, pH 7.4, at a protein concentration of 10 μm. The residual molar ellipticity, θ, is expressed in kdeg × cm2 × dmol-1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5A, recorded far-UV CD spectra of wild type CTproSP-C (wt), synthetic SP-C, and the mixture thereof (denoted exp) in SDS micelles. The combination of the spectra for CTproSP-C and the synthetic SP-C is also shown (denoted calc). B, difference spectra obtained by subtraction of the experimentally measured and calculated spectra for mixtures of CTproSP-C and synthetic SP-C (solid line) and for mixtures of CTproSP-C and native SP-C (dashed line). C, corresponding spectra as in A, but with native SP-C instead of synthetic SP-C. The residual molar ellipticity (θ) is expressed in kdeg × cm2 × dmol-1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Association of CTproSP-C with Phospholipid Membranes and SP-C—In sproSP-C, the region corresponding to CTproSP-C is localized close to lipid membranes because the SP-C part forms a transmembrane region. We therefore investigated whether recombinant CTproSP-C binds to phospholipid membranes. CTproSP-C was incubated with surfactant phospholipids with and without incorporated SP-C peptides for 1 h at 37°C,andthe membrane fraction was collected by centrifugation at 40,000 × g. The CTproSP-C contents in the pellets and supernatants were analyzed by SDS-PAGE and Western blot (Fig. 3). Virtually no detectable CTproSP-C associates with the peptide-free phospholipid membranes. The presence of synthetic SP-C, which is non-helical (see below) in the phospholipid membranes, however, leads to a significant portion of membrane-associated CTproSP-C (Fig. 3, lane 9), whereas CTproSP-C does not bind to membranes containing native,α-helical, SP-C (lane 5). Incubation of phospholipid membranes containing both SP-B and SP-C resulted in detectable CTproSP-C exclusively in the supernatant after centrifugation, showing that CTproSP-C does not bind SP-B and that the presence of SP-B does not result in binding to helical SP-C. Centrifugation of CT-proSP-C in the absence of phospholipid membranes leads to no detectable pelleted protein (data not shown). These experiments show that CTproSP-C binds to phospholipid membranes harboring synthetic (non helical) SP-C. CTproSP-C Interactions with Soluble Poly-Val Peptides— The apparent interaction between CTproSP-C and membrane-bound nonhelical SP-C prompted us to investigate whether CTproSP-C can bind a poly-Val peptide in the absence of lipids. CTproSP-C and the peptide K2V7K2 were coincubated for 1 h, and then the mixture was resolved by nondenaturing PAGE (Fig. 4). This resulted in the formation of a CTproSP-C-peptide complex when the peptide was present in excess. CTproSP-C incubated with peptides containing no or shorter poly-Val segments (K2V5K2 or peptides corresponding to the N-terminal 12 or 17 residues of SP-C; see “Experimental Procedures” for the amino acid sequence of SP-C) did not result in detectable complexes. CD Experiments of CTproSP-C-Peptide Mixtures in Detergent Micelles—Experiments were carried out to investigate whether interactions between CTproSP-C and SP-C peptides with helical (native SP-C; see CD spectrum in Fig. 5C) or non-helical (synthetic SP-C; see CD spectrum in Fig. 5A) structure result in a change in conformation. The strictly hydrophobic nature of SP-C necessitated the use of SDS micelles for these experiments. The far-UV CD spectra of CTproSP-C or synthetic SP-C in SDS micelles and the mixture thereof are shown in Fig. 5A. Fig. 5A also shows the calculated spectrum of the mixture of CTproSP-C and synthetic SP-C, obtained by combining the individual spectra of the two components. Evidently the experimental and calculated spectra of CTproSP-C-synthetic SP-C mixtures differ; the difference spectrum (Fig. 5B) indicates that the protein-peptide interaction results in an increase in helical content, as judged from the double minima around 205 and 222 nm. In contrast, mixing CTproSP-C and α-helical (native) SP-C does not result in any change in conformation (Fig. 5C), and the corresponding difference spectrum is consequently close to base line (Fig. 5B). The same experiment was performed with cystatin A instead of CTproSP-C, and with K2V7K2 instead of SP-C, in both cases resulting in no change in conformation (graphs not shown). Structure and Activity of CTproSP-C(L188Q)—CTproSP-C carrying the mutation L188Q was expressed and purified in the same manner as the wild type protein, yielding about 0.5 mg of protein/liter of cell culture. CTproSP-C(L188Q) migrates mainly as a monomer upon nonreducing SDS-PAGE (Fig. 1A), shows the same response on Western blot as the wild type protein (Fig. 1C), and elutes like the wild type protein on size exclusion chromatography (not shown), and the secondary structure contents of wild type and L188Q CTproSP-C in aqueous buffer are virtually identical, as judged by CD spectroscopy (Fig. 2). Denaturation experiments under nonreducing and reducing conditions show that wild type and L188Q CTproSP-C show similar stability properties and that both proteins contain disulfide bridge(s) that increase stability. 3H. Johansson, K. Nordling, and J. Johansson, unpublished data. However, CTproSP-C(L188Q) in contrast to the wild type counterpart does not change its structure in the presence of SDS micelles (compare Figs. 2 and 6A). From this we conclude that CTproSP-C(LI88Q) can form a soluble protein with an overall secondary structure in water that is similar to that of the wild type protein but that the two proteins differ in the way they are influenced by the presence of detergent micelles. Mixing CTproSP-C(L188Q) with phospholipids containing synthetic SP-C result in significantly lower membrane binding than for the wild type protein (Fig. 3, lanes 9 and 11). Likewise, mixing CTproSP-C(L188Q) with synthetic SP-C in SDS micelles does not result in a change in conformation (Fig. 6). Like wild type CTproSP-C, the L188Q mutant does not bind pure membranes or membranes containing α-helical SP-C (Fig. 3, lanes 3 and 7), nor does mixing CTproSP-C(L188Q) with helical SP-C result in a conformational change (Fig. 6, B and C). CTproSP-C Effects on Wild Type and Mutant proSP-C Levels in HEK293 Cells—The results described above suggest that CTproSP-C can bind β-sheet SP-C and thereby prevent aggregation (see “Discussion”). To investigate whether CTproSP-C can influence proSP-C in a cell, we used HEK293 cells stably transfected with either wild type proSP-C or proSP-C(L188Q). In these cells about 60-70% of the proSP-C(L188Q) protein is rapidly degraded and only a small fraction escapes to the secretory pathway. 4T. E. Weaver, unpublished data. Transient transfection of these cells with a CTproSP-C construct containing a signal peptide results in about 70% increase in the amounts of proSP-C(L188Q) but no" @default.
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• W2007697123 date "2006-07-01" @default.
• W2007697123 modified "2023-10-09" @default.
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