Secretion of Surfactant Protein C, an Integral Membrane
Protein, Requires the N-terminal Propeptide*
Juliana Johnson
Conkright
,
James P.
Bridges
,
Cheng-Lun
Na
,
Wim F.
Voorhout§,
Bruce
Trapnell
,
Stephan W.
Glasser
, and
Timothy E.
Weaver
¶
From the
Division of Pulmonary Biology, Children's
Hospital Medical Center, Cincinnati, Ohio 45229-3039 and the
§ Department of Functional Morphology, University of
Utrecht, 3508 TD Utrecht, The Netherlands
Received for publication, December 28, 2000, and in revised form, January 25, 2001
 |
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.
 |
INTRODUCTION |
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 (1, 2). 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.2 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 (3-5).
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 (6). The topology of the SP-C proprotein in
the membrane is not clear, with reports of both type II (7) and type
III (8) 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 (8, 9).
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 (8, 10). 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 (11-13). 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 (14, 15),
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.
 |
MATERIALS AND METHODS |
DNA Constructs and Transfection--
Full-length human SP-C
cDNA was cloned into pcDNA3 (Invitrogen, San Diego, CA). To
generate SP-C deletion constructs (Fig. 2A), 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 Cell Culture, Metabolic Labeling, and
Immunoprecipitation--
PC12 cells (a gift from D. Cutler, University
College London) were cultured as described previously (16). 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. (17) with 5 µl of an
antibody directed against the SP-C N-terminal propeptide (18).
SDS-PAGE and autoradiography were performed as previously
described (17).
Immunostaining and Confocal Microscopy--
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 (18)
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 (18) or C-terminal peptide domain (9), 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 Cell Isolation and Transfection--
Type II cells were
prepared from 6-week old female C57B/6 mice as described by Corti
et al. (19) 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 (19) 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.
Generation of Adenoviral Constructs--
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 (20) and sequenced bidirectionally to confirm
the integrity of the reading frame. Recombination and virus production
were performed as described previously (20).
Analysis of Lung Tissue and Surfactant from Adenovirus-infected
Mice--
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 (21) 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 (9). 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 (22). Lamellar bodies were isolated
from pooled lungs of three injected mice as previously described by
Osanai et al. (23). Equal amounts of protein, determined by
bicinchoninic acid protein assay (24), 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).
 |
RESULTS |
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.
1A). 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.
1B, upper panel). These results,
coupled with previous reports that SP-C is an integral membrane protein
(7, 25), 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. 1B). The N-terminal propeptide was
detected only after cell permeabilization, confirming that this peptide
domain is located in the cytoplasm (Fig. 1B). 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.

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Fig. 1.
SP-C1-197 is a type II membrane
protein. A, PC12 cells were transiently transfected
with SP-C1-197, labeled with
[35S]methionine/cysteine, and immunoprecipitated for
SP-C. SP-C proprotein (Mr = 21,000) was detected
in cell lysates (lanes 1 and 3) but
not processed to the active mature peptide (Mr = 4000) or secreted into the media (lanes 2 and 4). Molecular weight markers (× 103) are
indicated to the right. B, PC12 cells were
transiently transfected with SP-C1-197; stained with an
antibody directed against the N-terminal or C-terminal propeptide,
chromogranin A, or wheat germ agglutinin lectin; and examined by
confocal microscopy. SP-C1-197 proprotein colocalized with
chromogranin A in dense core granule in permeabilized cells stained
with antibody directed against the N-terminal propeptide
(upper panels). The N-terminal antibody did not
detect SP-C in nonpermeabilized cells (middle
panels); in contrast, the C-terminal antibody detected SP-C,
which colocalized with wheat germ agglutinin lectin at the cell surface
in nonpermeabilized cells (lower panels).
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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. 2A)
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.
2B); 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). SPC24-58
contains 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
SS
and SP-CCC
AA) in the context of the proprotein (Fig.
2A). 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).

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Fig. 2.
SP-C mature peptide (SP-C24-58)
is sorted to the regulated secretory pathway in PC12 cells.
A, a series of constructs were generated in which the
N-terminal propeptide (residues 1-23) was replaced with GFP and the
C-terminal peptide domain (residues 59-197) was successively deleted.
GFP/SP-C24-53 lacks a dileucine motif at positions 54 and
55 of the proprotein. Additional constructs were generated in which
cysteines ( ) at positions 28 and 29 were mutated to serine
(SP-C1-197(CC SS)) or alanine
(SP-C1-197(CC AA)). Each construct was transiently
transfected into PC12 cells and analyzed by confocal microscopy for
colocalization with chromogranin A in dense core granules.
B, PC12 cells were transiently transfected with SP-C mature
peptide (GFP/SP-C24-58). GFP fluorescence
(upper left panel) colocalized with
chromogranin A (Chr. A, upper right
panel) staining in the merged image (lower
left panel). Colocalization of
GFP/SP-C24-58 and chromogranin A was detected for each of
the constructs in A.
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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 (26), 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.

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Fig. 3.
GFP/SP-C24-58 is sorted to
lamellar bodies/multivesicular bodies in transfected mouse type II
cells. Isolated mouse type II cells were plated on collagen-coated
coverslips and transfected with gold particles coated with
GFP/SP-C24-58 plasmid using a hand-held gene gun. Gold
particles (arrows) were detected in the DIC image
(lower right panel). Cells were
immunostained with an antibody to mature SP-B (upper
right panel), a marker for lamellar
bodies/multivesicular bodies, and imaged by confocal microscopy.
GFP/SP-C24-58 (upper left
panel) colocalized with SP-B staining in the merged image
(lower left panel).
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To determine if SP-C mature peptide could be sorted to lamellar bodies
in 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 and
C). Within multivesicular bodies, HA labeling was detected
on the limiting membrane and the inner vesicles (Fig. 4B)
similar to the distribution of SP-C proprotein in wild type (data not
shown) and SP-B null mice (Fig. 4A). Localization of SP-C on
the inner vesicles of multivesicular bodies suggested that
SP-C24-58HA could be secreted.

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Fig. 4.
SP-C24-58HA is detected in
multivesicular and lamellar bodies in type II cells in
vivo. An adenoviral construct encoding the SP-C mature
peptide with a hemagglutinin tag (SP-C24-58HA) was
generated for intratracheal injection into adult mice. Four days after
infection, the subcellular distribution of SP-C24-58HA was
analyzed by immunogold labeling of fixed, cryoprotected frozen lung
sections. A, SP-C proprotein in internal vesicles of
multivesicular bodies in type II cells from an SP-B ( / ) mouse.
B and C, HA immunoreactivity was detected in
multivesicular bodies (MVB) and lamellar bodies
(LB) in type II cells of infected wild type mice.
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|
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.
5A). 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. 5B). 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.

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Fig. 5.
SP-C24-58HA is secreted by wild
type mice. Four days after intratracheal injection with
SP-C24-58HA adenovirus, mice were lavaged and surfactant
was isolated by centrifugation of bronchoalveolar lavage fluid.
A, 4% of the total surfactant pellet was immunoblotted with
a polyclonal anti-HA antibody. A duplicate blot containing 2% of the
total surfactant pellet was probed with an antibody directed against
the SP-C mature peptide. SP-C24-58HA and the endogenous
SP-C mature peptide were detected in all infected mice.
(n = 27 mice). B, BAL from infected mice was
fractionated into large (LA) and small (SA)
aggregates. 4% of each fraction was Western blotted with a polyclonal
anti-HA antibody. A duplicate blot containing 0.5% of each fraction
was blotted with an antibody directed against SP-B mature peptide.
SP-C24-58HA sedimented with wild type SP-B in the large
aggregate fraction (shown in duplicate) (n = 7 experiments of three animals each). Molecular weight markers (× 103) are indicated to the right.
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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.
6A). 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. 6B). 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.

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Fig. 6.
SP-C24-58HA is not secreted by
SP-C ( / ) mice.
Wild type and SP-C ( / ) mice were intratracheally injected with
SP- C24-58HA adenovirus. A, 4 days after
injection, mice were lavaged, large (LA) and small
(SA) aggregate surfactant fractions were isolated, and 4 or
0.5% of each fraction was analyzed by Western blotting with anti-HA
antibody or an antibody directed against the SP-B mature peptide,
respectively. SP-C24-58HA was detected in the large
aggregate fraction of wild type but not SP-C ( / ) mice. SP-B mature
peptide was detected in large aggregate fraction of both wild type and
SP-C ( / ) mice. (n = 3 experiments of three animals
each) (B). In addition to large aggregate fractions
(LA) from lavage fluid, lamellar body fractions
(LB) were isolated from lung homogenates and Western blotted
with HA antibody or antibody directed against mature SP-B.
SP-C24-58HA was detected in lamellar bodies isolated from
wild type mice and comigrated with SP-C24-58HA in large
aggregate surfactant (LA); in contrast,
SP-C24-58HA was not detected in lamellar bodies isolated
from SP-C ( / ) mice, although SP-B was readily detected
(n = 3 experiments of four animals each). Molecular
weight markers (× 103) are indicated to the
right.
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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.

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Fig. 7.
SP-C1-58HA is secreted by SP-C
( / ) mice. An
adenoviral construct encoding the SP-C N-terminal propeptide and the
mature peptide (SP-C1-58HA) was generated for
intratracheal injection into SP-C ( / ) mice. Three days after
infection, mice were lavaged, and large aggregate surfactant fractions
were isolated. HA immunoreactivity detected in large aggregate
surfactant of SP-C ( / ) mice comigrated with
SP-C24-58HA from wild type mice and was smaller than
SP-C1-58HA expressed in HEK 293 cells (control),
consistent with cleavage of the N-terminal propeptide. The two
immunoreactive bands detected in 293 cells probably represent
palmitoylated and nonpalmitoylated forms of SP-C1-58HA
(n = 6 experiments of three animals each). Molecular
weight markers (× 103) are indicated to the
right.
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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.

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Fig. 8.
SPC1-33PDGFR531-553HA is sorted to
lamellar bodies and secreted by SP-C
( / ) mice. An
adenoviral construct containing the SP-C cytosolic domain and the PDGFR
transmembrane domain was intratracheally injected into SP-C ( / )
mice. Three days after infection, mice were lavaged, and large
aggregate surfactant was isolated; in addition, lamellar bodies were
extracted from lung tissue.
SPC1-33PDGFR531-553HA was detected in
lamellar body and large aggregate fractions of infected mice
(n = 3 experiments of three animals each). Molecular
weight markers (× 103) are indicated to the
right.
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 |
DISCUSSION |
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 (14, 15). Previous studies have identified the SP-C
proprotein as a type III integral membrane protein (8), in which the
C-terminal propeptide is located in the cytoplasm, or a type II
membrane integral membrane protein (7), 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. (7). 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 (27,
28). 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 (8, 9).
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 (25, 29). 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 (25, 29, 30).
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 (31-35). 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 (16, 17). 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 (8, 10, 26); 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 (36-38). 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 (39, 40), 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 (41, 42).
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.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants POI-HL6164 (to T. E. W.) and ROI-HL50046 (to S. W. G.).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.
¶
To whom correspondence should be addressed: Children's
Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Tel.: 513-636-7223; Fax: 513-636-7868; E-mail: Tim.Weaver@chmcc.org.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011770200
2
S. Glasser, submitted for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
SP-B, surfactant
protein B;
SP-C, surfactant protein C;
PDGFR, platelet-derived growth
factor receptor;
PBS, phosphate-buffered saline;
GFP, green fluorescent
protein;
HA, hemagglutinin.
 |
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