From the Institute for Environmental Medicine,
University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104-6068 and the § Pulmonary and Critical
Care Division, Department of Medicine, Hospital of the University
of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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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 a 35S-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.
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INTRODUCTION |
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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) (1). Organic extracts of isolated surfactant have been shown to contain two small lipophilic proteins (SP's), SP-B1 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 (2). SP-C, an extremely hydrophobic 3.7-kDa peptide, is the exclusive product of the alveolar type II cell (3-5) and is a component of most clinical surfactant preparations (3, 6, 7).
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) (8). In vitro translation of lung RNA produces SP-C primary translation products of Mr 21,000-22,000 (8). Similar sized products have been detected in freshly isolated rat type II cells (9, 10) and produced in cultured Chinese hamster ovary cells transfected with a human SP-C cDNA (11). 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 (12, 13). 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 (3, 7, 14). 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 (9, 10, 15-17). 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 (3, 7, 14).
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 (9-11, 15-17). 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) (9, 15, 16). A similar processing pattern has been confirmed by others utilizing pulse-chase analysis of type II cells and immunoprecipitation with different polyclonal antisera (10). The proteolysis of proSP-C21 can be blocked either by the use of brefeldin A (9, 16) or by low temperature incubation (20 °C) (10), 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 (18, 19), 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 (15).
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 (20, 21), 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.
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EXPERIMENTAL PROCEDURES |
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Materials
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.
ProSP-C Antisera
Monospecific polyclonal rat proSP-C antisera were produced from synthetic peptide immunogens and have been previously characterized (16, 17). 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.
Cell Lines
A549 Cells-- The lung epithelial cell line A549 (22) 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 Cells-- 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. (23). The preparation obtained after panning on IgG coated plates (i.e. fresh type II cells) contained approximately 80-85% type II cells.
SP-C cDNA Expression Constructs
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, -lactamase and neomycin
resistance genes, as well as T7 and SP6 promoters for sense/antisense
in vitro transcription.
All procedures involving oligonucleotide and cDNA manipulations were performed essentially as described by Ausbel et al. (24). 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.
Wild Type SP-C
A full-length rat SP-C cDNA (816 base pairs) insert was
prepared by EcoRI digestion of a previously characterized
prokaryotic SP-C expression vector, PGEM4Z-SP-C (8+) (8). Purified SP-C insert was ligated into pcDNA3 polylinker at the EcoRI
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.
COOH-terminal Truncations-- 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 Table I. Amplification reactions containing 0.2 µM primers, 1.25 µM dNTP mixture, 1.5 µM MgCl2, 10 ng of template, and 2.5 units of Taq 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 KpnI and XhoI.
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Transfection
SP-C constructs were transiently transfected into A549 cells using calcium phosphate precipitation (0.18 ml of 0.25 M CaCl2 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) (24).
Immunocytochemistry
Immunocytochemical localization of expressed proSP-C proteins was performed on transfected epithelial cell lines fixed and stained as described previously (17). 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 (25). Fluorescent images were captured using a 12-bit CCD camera and processed using IMAGE 1 software (Universal Imaging Corporation, West Chester, PA).
Metabolic Labeling and Immunoprecipitation
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 (9).
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 (9, 15). Captured proteins were separated by SDS-PAGE and visualized by autoradiography as described below.
Analytical Methods
Polyacrylamide Gel Electrophoresis and Immunoblotting-- One-dimensional SDS-PAGE was performed in 16.5% polyacrylamide gels using a Tris-Tricine buffer system (26) as modified in our laboratory for surfactant proteins (9, 15, 16). Immunoblotting of transferred samples was done using proSP-C antisera and bands were visualized by enhanced chemiluminescence using the ECL kit (Amersham, Inc.).
Protein Determination-- Total protein was quantified by the Bradford (27) method using bovine immunoglobulin as standard.
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RESULTS |
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pcDNA3-rSP-C Constructs-- 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).
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Expression of Wild Type ProSP-C Protein by Transfected A549
Cells--
A549 cells transfected with pcDNA3-SP-Cwt
cells stained with anti-NPROSP-C consistently demonstrated expression
of proSP-C within cytoplasmic vesicles (Fig.
3A). The specificity of the
immunohistochemical staining was confirmed by the substitution of
preimmune serum for primary anti-NPROSP-C (Fig. 3B). 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. 3C). 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).
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COOH-terminal SP-C Deletional Mutants Are Translated but Not Proteolytically Processed-- A549 cells transiently transfected with SP-C1-185, SP-C1-175, SP-C1-147, or SP-C1-72 each produced a 35S-labeled primary translation product of predicted molecular weight (Fig. 6). However, compared with the SP-Cwt (Fig. 4B), there were no lower molecular weight forms indicative of proteolytic processing. ProSP-C forms were not detected in the media (not shown).
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Deletion of the COOH Terminus of ProSP-C21 Interrupts Intracellular Targeting-- Immunofluorescence micrographs of A549 cells transfected with pcDNA3-SP-Cwt showed consistent expression of proSP-C within cytoplasmic vesicles (Fig. 7A). Expression of pcDNA3-SP-C1-175 was markedly different with proSP-C staining manifested as a juxtanuclear, reticular pattern without focal accumulation in cytoplasmic vesicles (Fig. 7B). This pattern was repeated with SP-C1-72 (Fig. 7C) and SP-C1-185 (Fig. 8C).
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DISCUSSION |
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The primary sequence of the 3.7-kDa alveolar form of SP-C contains
a transmembrane spanning -helical region and is one of the most
hydrophobic amino acid domains known (3). The complete synthesis of
SP-C by the type II lung epithelium requires that a series of discrete
processing steps occur within the secretory pathway: 1) translation,
folding, and translocation of apoproprotein across the ER membrane; 2)
post-translational addition of covalent palmitic acid; 3) sorting and
exit of the lipoproprotein from the Golgi followed by cleavage of
flanking domains; 4) assembly of mature SP-C with surfactant
phospholipid and other proteins in the lamellar body prior to secretion
into the alveolus (9, 10, 15-17). Previous studies from our laboratory
have shown that early events in the synthetic processing of SP-C
involve cleavages of COOH-terminal flanking domains of the propeptide
which can only occur after its successful export from the Golgi. We now show that deletion of as little as 10 amino acids from the distal COOH
terminus results in retention of proSP-C protein in ER/Golgi compartments of transfected A549 cells. Truncated mutants are translated but not proteolytically cleaved providing evidence that the
C terminus is required for sorting of proSP-C to distal compartments
for processing.
The A549 cell line was chosen as the experimental model because it represents a transformed cell line of type II cell origin (22). Isolated alveolar type II cells are inadequate for use in prolonged transfection studies because when placed in primary culture on most matrices, they have been characterized by marked phenotypic instability which includes a loss of mRNA for all major surfactant proteins (21) and down-regulation of SP-C proprotein expression within 24-48 h of plating (9). It appears that the regulated secretory pathway of alveolar type II cells is also altered by culture. Studies using adenovirus-mediated transfection of SP-B in cultured type II cells have shown that the translated recombinant SP-B proprotein is neither sorted nor proteolytically processed by these primary cultures (20). In contrast, when SP-Cwt was transfected into A549 cells, we observed synthesis and proteolytic processing of 35S-proSP-C21 in a manner identical to that demonstrated in fresh type II cells in which two intermediates (16 and 6 kDa) result from cleavage of COOH-terminal propeptide domains (9, 15). The kinetics of the initial proteolytic events leading to SP-C16 and SP-C6 were similar in A549 cells and type II cells (Fig. 5), however, differences in the rate of disappearance of SP-C6 were observed such that type II cells appeared to have a slower rate of clearance of this intermediate. This could represent differences in the fate of SP-C6 in the distal synthetic pathway of the two systems: i.e. a regulated, rate-limiting conversion of SP-C6 to SP-C3.7 by type II cells versus degradation of SP-C6 in A549 cells. We have shown that the appearance of SP-C6 in the lysates of type II cells is followed by conversion to mature SP-C3.7 (15), but have not yet, for technical reasons, been able to reliably detect its production in transfected A549 cells using similar methods.2
Prior to transfection, the fidelity of the open reading frame of each mutant SP-C construct and the ability of epitope-specific antisera to recognize translated protein was demonstrated. The pattern of 35S-doublets captured by immunoprecipitation of translated SP-Cwt and truncated mutants (Fig. 2) had been observed by us during in vitro transcription/translation SP-Cwt cDNA in a PGEM-4Z vector and is due to the use of an alternative start site (codon 10) by the TNT reticulocyte lysate kit (16).
When transfected into A549 cells, constructs containing deletions of the proSP-C COOH terminus ranging from 10 to 134 amino acids consistently showed synthesis of a primary translation product of predicted length without demonstrable proteolytic processing (Fig. 6). The low level of expression of SP-C1-72 as well as the presence of oligomeric forms suggests that in the absence of substantial amounts of flanking propeptide, mutant protein could be less efficiently translocated into the ER and/or rapidly degraded. Similar findings were obtained with SP-C1-59 (not shown) and have been reported for the translation of a similarly truncated proSP-B construct by transfected Chinese hamster ovary cells (28).
The failure to process truncated proSP-C was associated with an inability of the A549 cells to direct the export of the mutated proteins from early synthetic compartments into cytoplasmic vesicles (Figs. 7 and 8). Co-localization of SP-C1-185 with ConA indicated that an intact distal COOH terminus (Cys186-Ile194) is necessary for targeting. This data in A549 cells extends observations obtained with a mutated human SP-C construct reported by Keller et al. (29) using transfected Chinese hamster ovary fibroblasts. By fluorescence immunocytochemistry, full-length proSP-C tagged with an epitope of c-myc oncogene was directed to a population of cytoplasmic vesicles which co-localized with a lysosomal marker. Deletion of 25 amino acids from the proSP-C COOH terminus resulted in restriction of tagged proSP-C to the Golgi. The effect of smaller deletions on either targeting or processing were not examined; however, when combined with the present study, indicate that the COOH terminus of proSP-C is necessary for proper targeting in both epithelial and non-epithelial cells. The mechanism by which this occurs is not fully defined but the domain Cys186-Ile194 could act as a functional signal peptide, a previously unrecognized sorting signal, or a structural domain required for proper propeptide folding.
Using empiric criteria (30), sequence analysis of proSP-C indicates an absence of a consensus signal peptide motif (8, 14). Nevertheless, constructs containing even small deletions of the COOH terminus are translated (Fig. 6) and appear in ER/Golgi compartments (Figs. 7 and 8). The failure to process proSP-C is more consistent with a failure to sort and export mutant proprotein to later compartments and suggests another region of proSP-C, either the NH2-terminal flanking domain or mature SP-C itself, serves to enable import into the ER. Previous in vitro studies using deletional human SP-C mutants have provided some indication that the mature SP-C sequence (Phe24-Leu58) could serve this function (31). Since all SP-C constructs in this study contain NH2-terminal and mature SP-C sequences, additional mutagenesis will be needed to further localize a functional signal sequence.
A comparison of proSP-C protein sequences from 4 species indicates a high degree of conserved amino acids in the COOH terminus including the terminal residue Ile194 (Fig. 9). BLAST analysis demonstrates no apparent conserved homology with other proteins or conformation to any consensus targeting sequences. However, site-directed mutagenesis of the COOH-terminal Ile of the bitopic lysosome-associated membrane protein, LAMP-2, affects its steady state subcellular distribution (32). This raises the possibility that Ile194 could provide a similar motif for direction of proSP-C to the lamellar body, which contains the lysosomal glycoprotein markers, CD63 (33) and LAMP-1 (see Ref. 36).3
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Misfolding of the natural conformation of proteins has been associated with aggregation and retention in the early stages of the secretory pathway. The role of intrachain disulfide linkages in propeptide folding is well known. Mutagenesis of residues Cys8 and/or Cys20 in the propeptide NH2 terminus of proopiomelanocortin disrupts targeting of proopiomelanocortin to the regulated secretory pathway of Neuro-2a cells (34). For proSP-C, cysteine residues in the COOH terminus (at positions 121 and 186) are conserved across species but Cys186 is deleted during truncation of the distal COOH terminus. Because of the extreme hydrophobicity and membrane avidity of mature SP-C (Phe24-Leu58), the restriction of mutant proSP-C to ConA compartments could be explained by the disruption of propeptide folding and aggregation. Altered post-translational processing secondary to misfolding has been implicated as the mechanism for the absence of SP-B in surfactant observed in cases of congenital SP-B deficiency associated with the R236C mutation (35). While SP-C deficiency has not been reported to date, our data suggests that similar small changes in the distal proSP-C COOH terminus, while not altering mRNA stability, could result in aberrant SP-C processing leading to a deficiency of SP-C3.7.
In summary, like SP-B and many peptide hormones, zymogens, and secreted proteins, SP-C is expressed as a proprotein. Detailed analysis of many of these proteins has failed to identify any conserved amino acid sequences that mediate selective targeting to distal compartments of the exocytic pathway. The results of the present study using A549 lung epithelial cells indicate that the COOH terminus of the SP-C propeptide is critical for its intracellular transport and processing. The transfected A549 cell model will enable further study of other deletional mutants as well as analysis of heterologous chimeric proteins containing the propeptide COOH terminus to further define both the motifs and mechanisms of proSP-C processing.
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ACKNOWLEDGEMENTS |
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We thank Kathy Notarfrancesco for assistance with immunocytochemistry and Drs. Michael Koval, Harry Ischiropoulos, and Aron B. Fisher for helpful suggestions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL-02869, HL-19737, and P50-HL56401 (to M. F. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a Clinician-Scientist Award from the American Heart Association. To whom correspondence should be addressed: Institute for Environmental Medicine, University of Pennsylvania School of Medicine, 1 John Morgan Bldg., 36th and Hamilton Walk, Philadelphia, PA 19104-6068. Tel.: 215-898-9100; Fax: 215-898-0868; E-mail: mfbeers{at}mail.med.upenn.edu.
1 The 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.
2 M. F. Beers, unpublished observations.
3 H. Shuman, unpublished data.
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REFERENCES |
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