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First published online 23 December 2002
doi: 10.1242/jcs.00267

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Deletion of exon 4 from human surfactant protein C results in aggresome formation and generation of a dominant negative

Lung Epithelial Cell Biology Laboratories, Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

View larger version (26K): [in a new window]   Fig. 1. Constructs used for analysis of human proSP-C trafficking. SP-C inserts containing wild-type or mutant human SP-C were generated by PCR and subcloned into either the EGFPC1 or pcDNA3 vectors as described in Materials and Methods. From top to bottom: hSP-C1-197 construct inserted into pcDNA3 containing full-length human SP-C shown with putative intrachain disulfide bonding between cys189 and cys120/121; EGFP/SP-C1-197 created by ligation of PCR generated hSP-C1-197 insert into the pEGFPC1 vector; EGFP/hSP-CExon4 is a deletional construct produced by removal of amino acids 110-144 encoded by Exon 4, which includes cys121/122; EGFP/hSP-CC120/121G is a double cysteine folding mutant containing Gly for Cys substitutions at residues 120 and 121 in the COOH flanking propeptide; EGFP/hSP-CC120/121/189G contains substitutions of Gly for Cys at codons 120, 121 and 189; HA/hSP-C1-197 is a full-length human SP-C cDNA containing the HA sequence peptide linked to the N-terminus and subcloned into pcDNA3. Amino acid nomenclature is based on published human SP-C sequence.

View larger version (77K): [in a new window]   Fig. 2. Expression of EGFP/hSP-C, pcDNA3/hSP-C and HA/hSP-C in transfected A549 cells. Plasmids containing cDNA for EGFP/hSP-C (A), pcDNA3/hSP-C (B), and HA/hSP-C (C) were introduced into A549 cells by CaPO4 precipitation. 48 hours after transfection, cells were fixed and/or stained with polyclonal -NPROSP-C and Texas-Red-conjugated goat anti-rabbit serum or monoclonal anti-HA plus Texas-Red-conjugated goat anti-mouse IgG. The fluorescence images were acquired using High Q FITC filter package for EGFP/hSP-C (A) or a High Q Texas Red filter package for NPROSP-C (B) and HA staining (C). Corresponding phase images appear below each fluorescence panel.

View larger version (37K): [in a new window]   Fig. 3. EGFP/hSP-C1-197 chimeric protein is directed to CD63 positive cytoplasmic vesicles. A549 cells grown on glass coverslips at 50-60% confluence were transfected with 10 µg of EGFP-C1/SP-C1-197 using CaPO4 as described in Materials and Methods. At 48 hours after transfection, cells were fixed, permeabilized and stained with primary monoclonal CD-63 antiserum and IgG-specific secondary Texas-Red-labeled antisera. Images were acquired by video fluorescence microscopy as in Fig. 2 and are representative of six separate experiments and >50 cells for each construct. The majority of EGFP/hSP-C1-197 (A) was associated with CD-63 staining (B). Phase image is shown in C.

View larger version (64K): [in a new window]   Fig. 4. Unprocessed EGFP/hSP-CExon4 accumulates in juxtanuclear inclusions. (A-D) Immunofluorescence of A549 cells grown on coverslips were transfected with either EGFP/hSP-C1-197 (A) or EGFP/hSP-CExon4 (C). Forty-eight hours after introduction of plasmid DNA, cells were fixed and fluorescence images acquired. Corresponding phase images are presented in panels B and D; (E) Western blotting for detection of EGFP proteins. Nuclear-free lysates were prepared as detailed in Materials and Methods using cell pellets from dishes transfected identically to those in panels A-D. Fifty percent of each lysate was subjected to 12% SDS-PAGE. Separated proteins were transferred to nitrocellulose and immunoblotted with primary rabbit polyclonal anti-GFP. Bands were visualized using enhanced chemiluminescence. EGFPC1 was expressed as a major product with Mr 27,000 (lane 1). Analysis of EGFP/hSP-C1-197 fusion protein expression (lane 2) demonstrated two doublets bands with relative molecular weight (Mr) of 48-50,000 and 33-38,000, consistent with a primary translation product/palmitoylated form and two processing intermediates. In contrast, EGFP/hSP-CExon4 was expressed as a single smaller band (hatched arrow, lane 3).

View larger version (57K): [in a new window]   Fig. 5. Ubiquitinated EGFP/hSP-CExon4 is associated with the MOTC. A549 cells grown on coverslips were transfected with EGFP/hSP-CExon4 using CaPO4. Forty-eight hours after introduction of plasmid DNA, cells were fixed and staining was performed using monoclonal antiserum against either ubiquitin (A-C) or -tubulin (1:100) (D-F) and visualized with secondary goat antimouse-IgG—Texas-Red. Two channel fluorescence images were acquired for EGFP (A,D) and Texas Red (B,E).

View larger version (56K): [in a new window]   Fig. 6. Mutation of Cys120/Cys121 in the COOH flanking propeptide mimics EGFP/hSP-CExon4 expression. A549 cells were transiently transfected with EGFP/hSP-C1-197 (A,B) or the COOH cysteine mutant EGFP/hSP-CC120/121G (C,D) using CaPO4. Images for EGFP expression were acquired 48 hours after transfection by using fluorescence microscopy with a FITC filter package. As for Exon4, the cysteine mutant was restricted to a juxtanuclear region showing aggregation. (E) Western blotting for detection of EGFP proteins. Nuclear-free lysates were prepared from cell pellets obtained from dishes identically transfected with EGFP/hSP-CC120/121G, EGFP/hSP-CC120/121/189G, EGFP/hSP-CC189G, EGFP/hSP-C1-197, or EGFP/hSP-CExon4 as detailed in Materials and Methods. Immunoblotting was performed with primary rabbit polyclonal anti-GFP and bands visualized using enhanced chemiluminescence. EGFPC1 was expressed as a major product with Mr 27,000 (not shown). Analysis of EGFP/hSP-C1-197 fusion protein expression (lane 5) demonstrated a primary translation bands with relative molecular weight (Mr) of 50-48,000 and processed forms of 37-33,000. EGFP/hSP-CC120/121G (lane 3), EGFP/hSP-CC120/121/189G (lane 4), and EGFP/hSP-CC189G (lane 1) were each expressed as a single band with Mr 48,000. Mock transfected cells (lane 2) yield no immunoreactive bands.

View larger version (92K): [in a new window]   Fig. 7. hSP-CExon4 alters trafficking of wild-type proSP-C. A549 cells were co-transfected with 5 µg of HA/hSP-C1-197 plasmid plus 5 µg of either EGFP/hSP-C1-197 (A-C) or EGFP/hSP-CExon4 (D-F) plasmids using CaPO4. Cotransfected cells fixed 48 hours after transfection, permeabilized and stained with anti-HA using Texas-Red-conjugated secondary antibody were subjected to fluorescence microscopy for EGFP (A,D) and -HA (Texas Red) staining (B,E). EGFP- and HA-tagged hSP-C1-197 colocalize in the same cytoplasmic vesicles. In contrast, EGFP/hSP-CExon4 induced aggregation of HA/hSP-C1-197 in perinuclear compartments.

View larger version (12K): [in a new window]   Fig. 8. 4-Phenylbutyric acid blocks accumulation of EGFP/hSP-CExon4 in aggresomes. A549 cells grown on coverslips were transfected with 5 µg EGFP/hSP-CExon4 using CaPO4. At the time of transfection, cells were treated with either 0, 1 mM, or 5 mM Na 4-phenylbutyrate as indicated. Forty-eight hours after introduction of plasmid DNA, cells were fixed and fluorescence images for EGFP expression were acquired.

View larger version (29K): [in a new window]   Fig. 9. Model of heterozygous expression of mutant SP-C. Schematic presentation of possible fates of synthesized proSP-C. (A) Expression of wild-type SP-C (hSP-C1-197) results in homomeric association, sorting and direction to CD63-positive vesicles (lamellar bodies). (B) In contrast, COOH folding mutants such as Exon 4 are retrotranslocated from the ER and directed preferentially to degradative pathways. Aggresomes form if protein expression exceeds degradation. (C) In heterozygous expression in vivo or co-transfection in vitro, via heteromeric association and retrotranslocation prior to sorting in the Golgi, folding mutants interact with wild-type SP-C to alter trafficking producing dominant negatives.