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J Biol Chem, Vol. 275, Issue 12, 8672-8679, March 24, 2000
From the Division of Neonatology, University of Pennsylvania School
of Medicine, Department of Pediatrics, Children's Hospital of
Philadelphia, Philadelphia, Pennsylvania 19104
Surfactant protein B (SP-B) is essential to the
function of pulmonary surfactant and to alveolar type 2 cell phenotype.
Human SP-B is the 79-amino acid product of extensive post-translational processing of a 381-amino acid preproprotein. Processing involves modification of the primary translation product from 39 to 42 kDa and
at least 3 subsequent proteolytic cleavages to produce the mature 8-kDa
SP-B. To examine the intracellular sites of SP-B processing, we carried
out immunofluorescence cytochemistry and inhibitor studies on human
fetal lung in explant culture and isolated type 2 cells in monolayer
culture using polyclonal antibodies to human SP-B8
(Phe201-Met279) and specific epitopes within
the N- (NFProx, Ser145-Leu160; NFlank
Gln186-Gln200) and C-terminal (CFlank,
Gly284-Ser304) propeptides of pro-SP-B.
Fluorescence immunocytochemistry using epitope-specific antisera showed
colocalization of pro-SP-B with the endoplasmic reticulum resident
protein BiP. The 25-kDa intermediate was partially endo H-sensitive,
colocalized with the medial Golgi resident protein MG160, and shifted
into the endoplasmic reticulum in the presence of brefeldin A, which
interferes with anterograde transport from endoplasmic reticulum to
Golgi. The 9-kDa intermediate colocalized in part with MG160 but not
with Lamp-1, a transmembrane protein resident in late endosomes and
lamellar bodies. Brefeldin A induced a loss of colocalization between
MG160 and NFlank, shifting NFlank immunostaining to a juxtanuclear
tubular array. In pulse-chase studies, brefeldin A blocked all
processing of 42-kDa pro-SP-B whereas similar studies using monensin
blocked the final N-terminal processing event of 9 to 8 kDa SP-B. We
conclude that: 1) the first enzymatic cleavage of pro-SP-B to the
25-kDa intermediate is in the brefeldin A-sensitive, medial Golgi; 2)
cleavage of the 25-kDa intermediate to a 9-kDa form is a trans-Golgi
event that is slowed but not blocked by monensin; 3) the final cleavage of 9 to 8 kDa SP-B is a monensin-sensitive, post-Golgi event occurring prior to transfer of SP-B to lamellar bodies.
Surfactant protein B is a 79-amino acid hydrophobic protein that
is essential to the function of pulmonary surfactant, as illustrated by
lethal SP-B deficiency in humans and the transgenic homozygous SP-B
knock-out mouse (reviewed in Ref. 1). The 8-kDa protein is the result
of extensive post-translational processing of a large 381-amino acid
precursor within alveolar type 2 cells. Previous studies in cell lines,
isolated rat type 2 cells, and human fetal lung (2-7) indicated that
processing to the mature 8-kDa protein involves signal peptide cleavage
and glycosylation of the C terminus, followed by cleavage of the N
terminus and C terminus in succession. We have recently shown that
cleavage of the N terminus occurs in two steps, leaving an
approximately 10-amino acid remnant flanking mature SP-B which is
removed in a final processing step that releases mature SP-B (8). The subcellular location of these processing events and the enzymes necessary for processing SP-B are poorly understood. Previous work by
Voorhout and colleagues (9) utilizing immunoelectron microscopy with
antisera to mature SP-B and a synthetic pro-SP-B showed pro-SP-B in the
endoplasmic reticulum and mature SP-B in lamellar bodies of adult human
type 2 cells. Analysis of grain density over other organelles showed
intermediate grain densities over multivesicular bodies and Golgi,
indicating the involvement of these organelles in SP-B transport and/or processing.
The extensive post-translational processing of SP-B is similar to the
post-translational processing of the other hydrophobic surfactant
protein, SP-C (10-13). The 21-kDa pro-SP-C undergoes sequential
enzymatic cleavages resulting in a 3.7-kDa mature protein. Pro-SP-C is
detected in endoplasmic reticulum and a 6-kDa intermediate is enriched
in lamellar bodies. Inhibitors of intracellular trafficking and
acidification in vitro disrupt all processing beyond the
16-kDa SP-C intermediate. Processing of SP-B and SP-C are linked, since in alveolar type 2 cells of patients with inherited SP-B deficiency SP-C is not processed beyond the 6-kDa intermediate (14, 15).
In this report, we use epitope-specific antisera and pulse-chase
labeling studies with inhibitors of protein processing to show that
most human pro-SP-B processing is in post-endoplasmic reticulum but
pre-lamellar body compartments. Our data extend previous observations
of pro-SP-B trafficking and processing to show that early N-terminal
propeptide and C-terminal propeptide processing events occur within the
Golgi apparatus with processing of the small vestigial N-terminal
propeptide domain as a post-Golgi event. We speculate that the
N-terminal remnant is involved in trafficking SP-B toward the lamellar
body. Previous reports of these data have appeared elsewhere in
abstract form (16, 17).
Reagents--
Express Protein Labeling Mix was obtained from NEN
Life Science Products Inc. (Boston, MA). Protein A-agarose was obtained from Life Technologies, Inc. (Gaithersburg, MD). Dexamethasone, isobutylmethylxanthine, and 8-Br-cAMP were obtained from Sigma. Endoglycosidase H (endo H) and PNGase F were obtained from New England
Biolabs (Beverly, MA). All other reagents were electrophoretic grade
and were purchased from either Bio-Rad or Novex (San Diego, CA).
Culture media were produced by the Cell Center Facility, University of Pennsylvania.
The polyclonal antibody to BiP was supplied by StressGen
Biotechnologies Corp. (Victoria, British Columbia, Canada). The MG160 polyclonal antiserum was the generous gift of N. Gonatas, Division of
Neuropathology, University of Pennsylvania. The Lamp-1 (H4A3) monoclonal antibody developed by J. T. August and J. E. K. Hildreth was obtained from the Developmental Studies Hybridoma Bank
developed under the auspices of the NICHD, National Institutes of
Health, and maintained by The University of Iowa, Department of
Biological Sciences, Iowa City, IA.
Explant and Cell Culture--
Human fetal lung was obtained from
second trimester therapeutic abortions (20-23-week estimated
gestational age) under protocols approved by the Committee for Human
Research, Children's Hospital of Philadelphia. Fetal lung parenchyma
was dissected free of large airways, chopped into 1-mm3
explants, and cultured in Waymouth's media on a rocking platform as
described previously (18). After overnight culture, hormones (10 nM dexamethasone, 0.1 mM 8-Br-cAMP, and 0.1 mM isobutylmethylxanthine (DCI)) were added to the media
for the remainder of the culture period. Media were changed daily and
tissues were studied on day 5 of culture. Type 2 cells were isolated
from human fetal lung explants after 4 days in culture with DCI using
collagenase-trypsin digestion and differential adhesion to remove
fibroblasts and plated on coverslips coated with extracellular matrix
of Madin-Darby canine kidney cells (19, 20). Fibroblast contamination
of the final culture varied between 5 and 10% of cells. Cells were cultured in Waymouth's media supplemented with DCI in 35-mm dishes for
up to 4 days. Under these conditions, type 2 alveolar cells maintain
expression of SP-A, -B, and -C mRNA, process SP-B and -C
proproteins and incorporate choline into surfactant phospholipids for
at least 4 days in culture (20).
Human SP-B Antisera and
FITC1 Labeling--
Based on
the antigenicity index of prepro-SP-B, peptide sequences were chosen
for production of synthetic peptides and antiserum preparation (NFProx,
Ser145-Leu160, NFlank,
Gln186-Gln200, and CFlank,
Gly284-Ser304) as described previously (8) and
illustrated in Fig. 1. Human SP-B
antiserum was prepared using purified human SP-B8 isolated from patients with pulmonary alveolar proteinosis as described previously for anti-bovine SP-B antibody (21). Antisera were screened
for reactivity against the immunizing peptide by immunodot blot assay.
The IgG fractions of NFlank, CFlank, NFProx, and hSP-B antisera were
isolated using the Serum IgG Purification Kit (Bio-Rad) and conjugated
to FITC using Fluoreporter Protein labeling Kit (Molecular Probes,
Eugene, OR).
Immunofluorescence Cytochemistry--
For single labeling
studies, cells were cultured overnight before immunofluorescent
labeling. In double labeling studies, isolated type 2 cells were
harvested on culture day 4 with no further treatment (control) or after
treatment with brefeldin A (10 µg/ml) for 30 min. Cells were washed
free of media in PBS and fixed in 1% paraformaldehyde in PBS followed
by washes in PBS with 5 mM NH4Cl. Coverslips
were incubated in freshly prepared 0.1% sodium borohydride to reduce
autofluorescence followed by 5% bovine serum albumin, 10% normal goat
serum (Vector Laboratories, Burlingame, CA) in PBS for 30 min at room
temperature to reduce nonspecific binding. The cells were permeabilized
using 0.3% Triton X-100 in PBS. Specific antisera were diluted in PBS
containing 0.3% Triton X-100 + 5% bovine serum albumin and 10%
normal goat serum. The unlabeled primary antibody was applied as
follows: polyclonal MG160, 1:500, overnight at room temperature; BiP,
1:1500 overnight at 4 °C; Lamp-1, 1:100, overnight at room
temperature. After washing in PBS + 0.3% Triton X-100, Cy-3-conjugated
secondary goat anti-rabbit IgG (Zymed Laboratories
Inc., San Francisco, CA) was diluted at 1:300 in the blocking
solution described above and incubated for 1 h at room
temperature. After additional washes in PBS + 0.3% Triton X-100,
FITC-conjugated SP-B antisera were applied at 1:100 dilution and
incubated overnight at room temperature. Coverslips were washed in PBS + 0.3% Triton X-100, air dried, and mounted using Vectashield (Vector
Labs, Burlingame, CA) to reduce fading. All immunostaining experiments
were done in triplicate.
Confocal Microscopy--
Confocal microscopic images were
obtained using a computer-interfaced, laser-scanning microscope (Leica
TCS 4D), of the Confocal Core Facility, Children's Hospital of
Philadelphia. Immunolabeled slides (n = 3-4
representative fields per slide), were sectioned optically at 0.5-µm
intervals through the cell monolayer to obtain the appropriate focal
depth. The representative 0.5-µm image chosen contained nucleus and
the relevent organelle of interest (ER, Golgi, and/or lamellar body).
Simultaneous wavelength scanning allowed superimposition of fluorescent
labeling with FITC and Cy3 fluorophores. Laser power was fixed at 75%
for all image acquisition. Image output was at 1024 × 1024 pixels
and photomicrographs were later embossed with a 20-µm bar unless
otherwise indicated.
Pulse-Chase Labeling of Human Fetal Lung Explants--
Culture
media was replaced with Met-Cys-free Dulbecco's modified Eagle's
medium (2 ml/60-mm plate) with or without inhibitors for 2 h while
incubating in 95% air, 5% CO2 on a rocking platform. The
inhibitors brefeldin A (10 µg/ml) or monensin (2 µM)
were added at the beginning of the starvation period and maintained throughout the pulse and chase periods. Met-Cys-free Dulbecco's modified Eagle's medium ± inhibitors was then replaced with
Met-Cys-free Dulbecco's modified Eagle's medium ± inhibitors
supplemented with 200 µCi/ml 35S-Express Protein Labeling
Mix (2 ml/60-mm plate) which is composed of 70% methionine and 15%
cysteine (NEN Life Science Products Inc.). After a 1-h pulse, the media
was changed to complete Waymouth's media with DCI ± inhibitors.
To ensure that inhibitor concentrations remained constant, media was
changed at each subsequent time point through the 8-h time point of the
24-h chase. Samples were harvested immediately after the
35S labeling and at regular intervals through 8 h
post-labeling. Samples were washed in PBS with protease inhibitors (10 mM N-ethylmaleimide, 2 mM
benzamidine HCl, and 80 mM phenylmethylsulfonyl fluoride) and then sonicated in 500 µl of 1% SDS with protease inhibitors.
Immunoprecipitation--
Radiolabeled lung homogenates were
immunoprecipitated by modification of our previous method (8).
Immunoprecipitations were performed on samples containing
106 trichloroacetic acid precipitable counts/min unless
otherwise specified, using 3 µl of anti-human SP-B antibody (hSP-B)
or preimmune rabbit serum. After the first immunoprecipitation, protein
A-agarose beads were washed and the immunoprecipitated proteins were
solubilized in 40 µl of gel sample buffer (62.5 mM
Tris-HCl, pH 6.8, 2% SDS, 0.72 M 2-mercaptoethanol, 10%
glycerol, 0.0075% bromphenol blue). A 5-µl aliquot was taken for
scintillation counting and a 30-µl aliquot was subjected to SDS-PAGE
in 16.5% polyacrylamide gels using a Tris-Tricine buffer system as
described previously (8). Electrophoresed samples were
transferred to polyvinylidene difluoride (Bio-Rad) at 20 mA/cm2
for 13-16 h. After transfer to membranes, blots were visualized using
the Storm PhosphorImager system (Molecular Dynamics, Sunnyvale, CA),
analyzed using Imagequant software and later subjected to autoradiography.
Endoglycosidase H and PNGase F--
After immunoprecipitation
from hormone-treated human fetal lung pulse-chase labeled for 4 h,
triplicate samples still complexed to protein A-agarose beads were
treated with either endo H or PNGase F. Control samples were incubated
in wash buffer. Endo H-treated samples were resuspended in 1 × G5
buffer with 2000 units of endo H while PNGase F-treated samples were
resuspended in 1 × G7 buffer, 1% Nonidet P-40 with 4000 units of
PNGase F. All samples were incubated at 37 °C for 2 h followed
by a final wash before solubilizing the beads in NuPAGE SDS sample
buffer with DTT as the reducing agent. For superior band resolution, these samples were separated using a 10% NuPAGE Bis-Tris gel with MES
SDS Running Buffer as per the manufacturer's protocol (Novex, San
Diego, CA), including transfer to Duralose membrane (Stratagene, La
Jolla, CA) for PhosphorImager analysis.
Subcellular Localization of Pro-SP-B Peptides Using
Immunofluorescence with Epitope-specific Antisera--
To localize
SP-B precursor and intermediate forms within type 2 cells of human
fetal lung, we used epitope-specific antisera developed against
antigenic sequences within the N- and C-terminal propeptides.
Fluorescence cytochemistry of type 2 cells isolated from cultured,
hormone-treated human fetal lung showed distinct immunostaining
patterns for each of the antisera (Fig.
2). The NFProx antiserum immunostaining
occupied a perinuclear pattern. The CFlank antiserum exhibited a
vesicular pattern of fluorescence throughout the cell. The NFlank
antiserum also showed a vesicular pattern but in close approximation to
lamellar bodies which have a perinuclear distribution by phase-contrast
microscopy. By comparison, the antiserum to mature SP-B (hSP-B)
intensely labeled lamellar bodies and no other structures presumably
due to the high concentration of SP-B8 within lamellar
bodies. The specificity of each antiserum was confirmed by the ability
of synthetic peptide to block the signal in immunohistochemistry (data
not shown).
Double immunofluorescence labeling was used to identify the subcellular
location of staining with the epitope-specific antisera using known
markers of subcellular organelles in type 2 cells. Contaminating
fibroblasts were occasionally present but were recognizable by absence
of staining with the FITC-labeled epitope-specific antisera despite
positive staining for subcellular organelles. Confocal images were
obtained using the following laser parameters for FITC-labeled primary
SP-B antisera: NFProx voltage mean 808 (range 781-840), offset
Fig. 3 illustrates the results of double
immunofluorescence cytochemistry of isolated type 2 cells using
monoclonal antibody to BiP (as a marker of endoplasmic reticulum and
identified by Cy3-labeled secondary antibody) and SP-B epitope-specific
antisera directly conjugated to FITC. Colocalization of NFProx
immunostaining with BiP in the endoplasmic reticulum was evident in
both merged (yellow; Fig. 3a) and unmerged images
(Fig. 3b). There was minor colocalization of CFlank and
NFlank antisera with BiP, whereas the steady-state pool of pro-SP-B in
the endoplasmic reticulum was preferentially identified by NFProx.
There was minimal colocalization of BiP with hSP-B antiserum.
Fig. 4 illustrates double
immunofluorescence photomicrographs of isolated type 2 cells using
polyclonal antibody to MG160, identified by Cy3-labeled secondary
antibody, and FITC-conjugated epitope-specific antisera. MG160 is a
medial Golgi resident transmembrane sialoglycoprotein which is found as
a component of the Golgi apparatus of most cells (22, 23). By confocal
fluorescence microscopy, NFProx did not colocalize with MG160. CFlank
and NFlank immunostaining colocalized intensely with MG160 in a tubular
network lying in close proximity to the nucleus which is characteristic
of the medial Golgi. Although this region was also close to lamellar bodies, there was no colocalization of hSP-B with MG160.
Fig. 5 shows double immunofluorescence
images of isolated type 2 cells labeled using Lamp-1 monoclonal
antibody, identified by Cy3-labeled secondary antibody, and
FITC-conjugated epitope-specific antisera. Lamp-1 is a transmembrane
protein which localizes to late endosomes, lysosomes and, in alveolar
type 2 cells, lamellar bodies (24). Neither NFProx nor CFlank antisera
colocalized with Lamp-1. NFlank immunostaining highlighted vesicles
adjacent to lamellar bodies (as seen in Fig. 2) which were
Lamp-1-negative. By contrast, the hSP-B antiserum localized to dense
regions within Lamp-1-positive lamellar bodies (Fig. 5,
inset). Taken together, these immunofluorescence data
suggest that at steady state pro-SP-B is endoplasmic reticulum resident
and the 25-kDa intermediate, which is identified by both CFlank and
NFlank, is distributed within the Golgi. In contrast, mature SP-B is
concentrated within the lamellar body.
Initial Cleavage of the N Terminus of Pro-SP-B Occurs in the Medial
Golgi--
To examine ER to Golgi transport of SP-B precursors, we
carried out pulse-chase labeling of cultured human fetal lung in the presence or absence of brefeldin A, an inhibitor of anterograde trafficking between endoplasmic reticulum and Golgi (25). We showed
previously that processing from pro-SP-B through 25- and 9-kDa
intermediates to mature 8-kDa SP-B occurs within 1-2 h postlabeling in
hormone-treated human fetal lung explants (8) (Fig.
6). In the presence of brefeldin A,
processing of pro-SP-B was blocked with no accumulation of 25 kDa or
more distal intermediates over 8 h of chase (Fig. 6). This
suggests that the initial N-terminal cleavage of pro-SP-B occurs in a
brefeldin A-sensitive cis or medial Golgi compartment.
We also performed endoglycosidase digestions of SP-B intermediates
isolated by immunoprecipitation after a 4-h pulse-chase. The 9- and
8-kDa SP-B proteins are not glycosylated and do not shift apparent
Mr after treatment with either PNGase F or endo H (Fig. 7). PNGase F, which cleaves all
carbohydrates, reduces the 42-kDa pro-SP-B to ~39 kDa and the 25-kDa
intermediate to ~21 kDa. After endo H treatment, both pro-SP-B and
the 25-kDa intermediate appear to be partially endo H-sensitive (39- and 21-kDa bands, respectively) and endo H-resistant (42- and 25-kDa bands, respectively). The majority of pro-SP-B is endo H-sensitive but
a small fraction are endo H-resistant. Conversely, a small amount of
the 25-kDa intermediate is endo H-sensitive and the bulk of this
intermediate is endo H-resistant. Together with the brefeldin A
studies, these data place the initial N-terminal cleavage of pro-SP-B
to 25-kDa intermediate in the medial Golgi.
C-terminal Propeptide Cleavage of the 25-kDa SP-B Intermediate
Occurs in the Trans-Golgi--
To discriminate the location of the
first N-terminal cleavage from the subsequent cleavage of the C
terminus, we examined the steady state distribution of SP-B
intermediates in the presence of brefeldin A using double
immunofluorescence staining of isolated type 2 cells. In Fig.
8, brefeldin A-treated cells were fixed and double stained using antibodies to BiP (Fig. 8A) or
MG160 (Fig. 8B) in combination with the FITC-conjugated
epitope-specific antisera. Control cells (not shown) mimicked the
immunostaining patterns illustrated in Fig. 4. BiP, NFProx, and hSP-B
immunostaining patterns were unaffected by brefeldin A treatment.
CFlank immunostaining dispersed after exposure to brefeldin A, as did
the distribution of MG160, with both antisera now colocalizing with
BiP. CFlank immunostaining identifies a pool of SP-B intermediate
within a brefeldin A-sensitive, cis/medial Golgi compartment similar to MG160. By contrast, NFlank identified an intermediate that at steady
state was predominantly distal to the brefeldin A-sensitive Golgi
regions with the majority of NFlank immunostaining collapsing into
perinuclear tubular arrays characteristic of trans-Golgi proteins
exposed to brefeldin.
Cleavage of the Vestigial N Terminus Occurs in a Post-Golgi but
Prelamellar Body Compartment--
To localize late processing events,
we carried out pulse-chase labeling of lung explants in the presence
and absence of monensin. Monensin, an ionophore mediating monovalent
cation exchange across cellular membranes, reversibly slows the rate of
intracellular transport of newly synthesized proteins, especially
interfering with transfer across Golgi compartments and compromising
secretion from the trans-Golgi (26). Although pro-SP-B processing was slowed, monensin treatment did not prevent the appearance of 25- and
9-kDa SP-B intermediates (Fig. 9).
However, processing to 8-kDa mature SP-B was not demonstrated up to
8 h after labeling, suggesting that the final N-terminal cleavage
of te 9-kDa intermediate is a pH-dependent, post-Golgi
event. Combined with the observation that NFlank immunostaining is not
found within lamellar bodies, this places the terminal cleavage event
in a post-Golgi but pre-lamellar body compartment.
The processing of pro-SP-B to mature SP-B in alveolar type 2 cells
requires a series of post-translational modifications and proteolytic
cleavages. Through the course of SP-B processing, the mature protein
must be transported to the lamellar body where it is concentrated with
SP-C and surfactant-specific phospholipids. The mechanisms controlling
the process of lamellar body formation and the aggregation of these
diverse surfactant components are poorly understood. To elucidate these
events, it became important to examine the intracellular localization
of SP-B processing. Previous studies by others using immunoelectron
microscopy localized the primary translation product, pro-SP-B, to the
endoplasmic reticulum with mature SP-B concentrated in lamellar bodies.
The present study extends these observations with new data
demonstrating that the initial proteolytic cleavage of the N-terminal
propeptide is in the brefeldin A-sensitive, medial Golgi with a
subsequent C-terminal cleavage in the trans-Golgi and a final
N-terminal cleavage event in a post-Golgi but pre-lamellar body
compartment as depicted in Fig. 10.
We used type 2 cells isolated from hormone-treated human fetal lung
explants for immunofluorescence studies. Recent advances in type 2 cell
culture allow maintenance of type 2 cell phenotype for extended periods
(19, 20). Although isolated type 2 cells lose their basal-apical
orientation, the lamellar bodies have a characteristic perinuclear
distribution which permits detailed examination of subcellular
structures in close proximity to lamellar bodies. We have recently used
these cells in pulse-chase labeling studies and have found no
significant differences in SP-B processing over 4 h compared with
similar studies in human fetal lung
explants.2 This culture
technique provides a useful system for studies characterizing lamellar
body genesis and secretion as well as in the surfactant protein
processing. In the present study the combination of type 2 cell
immunofluorescence cytochemistry with pulse-chase studies of human
fetal lung explants facilitated correlation of steady state pools of
SP-B intermediates with the effects of inhibitors and endoglycosidases
on dynamic SP-B processing.
Pro-SP-B has a complex tertiary structure inferred from its amino acid
sequence homology to NK-lysin and prosaposin (27, 28). Given the
proposed structure of pro-SP-B, our epitope-specific antisera were
designed to recognize peptides within intervening segments between
tight Our previous studies also showed that the N-terminal propeptide is
cleaved in two steps, exposing first a small vestigial propeptide that
is later cleaved in the final event liberating SP-B (8). We have now
shown that the initial N-terminal cleavage is a medial Golgi event.
Brefeldin A, a small hydrophobic molecule that disrupts budding
vesicles and induces collapse of the cis/medial Golgi which
redistribute to the endoplasmic reticulum (recently reviewed in Ref.
25), prevents all proteolytic processing of pro-SP-B. Monensin, which
acts primarily on later Golgi compartments (26), does not prevent this
first proteolytic cleavage event. In addition, brefeldin shifted CFlank
immunostaining out of a Golgi pattern to colocalize with the
ER-resident BiP. This method has been used by others to examine the
trafficking of proteins to regions of the Golgi (31, 32), including
MG160 which characteristically disperses after brefeldin exposure (33).
Finally, the only explanation for an endo H sensitive pool of both
42-kDa pro-SP-B and the 25-kDa intermediate is that the first
N-terminal cleavage occurs in the medial Golgi while the
oligosaccharide modification is still endo H-sensitive. Mannosidase II,
which removes mannose residues from the oligosaccharide rendering the
N-acetylglucosamine residues endo H-resistant, typically
localizes to the cis/medial Golgi depending on cell type although
overlap into the trans-Golgi has been described (34-36). Thus, endo
H-sensitive pro-SP-B is rapidly transported from the ER to the medial
Golgi where it becomes endo H-resistant concomitantly with initial
N-terminal cleavage. The failure of the NFProx antiserum to colocalize
with MG160 at steady state indicates that the relative size of the ER
pool of pro-SP-B is much greater than the pool passing through the Golgi.
The next step in pro-SP-B processing is C-terminal propeptide cleavage.
Our results indicate that this occurs in a late Golgi compartment, most
likely the trans-Golgi. This cleavage was not inhibited by monensin.
The immunostaining results for CFlank and NFlank in the presence of
brefeldin show that the steady state pools of the 25-kDa and 9-kDa
intermediates are in separate compartments. Although both CFlank and
NFlank antisera identify the 25-kDa intermediate, our previous
immunoblotting studies showed that CFlank does not identify a
C-terminal propeptide fragment which would potentially confound
interpretation of these studies (8). Furthermore, NFlank, but not
CFlank, antiserum identifies the 9-kDa intermediate. As mentioned
above, CFlank immunostaining showed a shift to endoplasmic reticulum
and colocalization with BiP after brefeldin while the change in NFlank
immunostaining was more characteristic of other trans-Golgi resident
proteins. Thus the CFlank antiserum identifies primarily the 25-kDa
intermediate moving within cis and medial Golgi compartments while the
NFlank antiserum identifies a trans-Golgi pool of 9-kDa SP-B
intermediate, with the cleavage event occurring in the trans-Golgi.
The precise location of the final N-terminal cleavage event remains
unclear. This step appears to be inhibited by monensin, placing it in a
pH-sensitive, post-Golgi compartment. NFlank immunostaining was not
seen within lamellar bodies, nor did it colocalize in Lamp-1-positive
vesicles as with the hSP-B antiserum. Instead, NFlank-positive small
vesicles were found in close proximity to Lamp-1-positive vesicles.
Lamp-1 has been identified in the membranes of the small vesicles
within multivesicular bodies of type 2 cells (24). Immunoelectron
microscopy studies will be required to determine whether NFlank and
Lamp-1 antisera identify distinct populations of vesicles within
multivesicular bodies.
The complexity of pro-SP-B processing is reminiscent of prohormone
processing, in which inactive prohormones are sequentially modified and
cleaved to release active forms at their site of action (reviewed in
Ref. 37), and of post-translational processing of surfactant protein C
(38). SP-C is also synthesized as a larger proprotein that is
sequentially processed to a hydrophobic mature protein. Inhibitor
studies of surfactant protein C processing showed that both brefeldin
and monensin blocked most pro-SP-C processing, indicating that
post-Golgi compartments (i.e. multivesicular body and/or
lamellar body) are major loci for SP-C processing (11, 13). By
contrast, our inhibitor studies indicated that most pro-SP-B processing
occurs in a pre-lamellar body compartment.
The biologic role of the complexity of SP-B processing has been
elusive. The structural and functional parallels between SP-B and
saposins have been pointed out by others (27, 39). Both SP-B and
saposins A, B, C, and D arise from large precursors. All 4 saposins
arise from a single precursor protein (prosaposin ABCD) and are
liberated through a complex series of enzymatic cleavages (40). Each of
the monosaposins has a unique function in sphingolipid hydrolysis.
Despite sequence homology between the monosaposins and the N- and
C-terminal propeptides of pro-SP-B, to date no unique cellular
functions have been attributed to the cleaved SP-B propeptides.
Subcellular localization studies have shown the saposins only in
lysosomes with processing kinetics that are more rapid than we have
observed for SP-B (41, 42). Recently, the dermatologic manifestations
of the prosaposin knock-out mouse have been characterized and show
interesting functional parallels with the homozygous SP-B The many parallels between saposin and SP-B suggest post-translational
processing complexity is necessary to maintain these proteins in an
inactive state until reaching their sites of action. In addition, it is
possible that processing uncovers trafficking motifs that facilitate
the movement of these proteins to their final destination. There are no
known trafficking motifs within the amino acid sequences of either
prosaposin or pro-SP-B. However, the small N-terminal peptide cleaved
in the final step of SP-B processing is a potential candidate. Studies
in transgenic homozygous SP-B We acknowledge the technical assistance of
Sree Angampalli in culturing lung explants and preparation of isolated
type 2 cells, as well as the editorial assistance of Philip Ballard,
Michael Beers, and Michael Koval in the preparation of this manuscript.
*
This work was supported by the Gisela and Dennis Alter
endowed chair in Pediatrics and National Institutes of Health Grants 5 P30 HD-28815, 1 P50 HL-56401, and 1 RO1 HL-59959.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.
2
L. W. Gonzales and S. H. Guttentag,
unpublished data.
The abbreviations used are:
FITC, fluorescein
isothiocyanate;
PBS, phosphate-buffered saline;
ER, endoplasmic
reticulum;
PAGE, polyacrylamide gel electrophoresis;
Tricine, N-[1-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
Bis-Tris, 2-[bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-propane-1,3-diol.
Intracellular Localization of Processing Events in Human
Surfactant Protein B Biosynthesis*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Antigenic epitopes used to produce SP-B
epitope-specific antisera. Synthetic peptides were produced using
antigenic sequences of pro-SP-B as described previously (8). The
antisera are designated: NFProx, Ser145-Leu160;
NFlank, Gln186-Gln200; CFlank,
Gly284-Ser304. The hSP-B antiserum was
developed using 8 kDa SP-B isolated from human alveolar proteinosis
fluid (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Immunostaining using SP-B epitope-specific
antisera identifies unique human fetal type 2 cell subcellular
compartments. Human fetal type 2 cells isolated and cultured
overnight on plastic were fixed and immunostained using the
epitope-specific polyclonal antisera NFProx, CFlank, NFlank, and the
polyclonal antiserum to mature human SP-B. Corresponding phase-contrast
photomicrographs appear adjacent to fluorescence photomicrographs.
Exposure time was corrected for all epitope-specific antisera but is
1/10 for the hSP-B photomicrograph due to the intensity of the lamellar
body immunostaining. Each epitope-specific antiserum identifies unique
subcellular structures that do not correspond to lamellar bodies
identified by the hSP-B antiserum (arrow). Images are
representative of duplicate experiments; immunostaining patterns shown
were characteristic of ~90% of cells per slide. Bar, 20 µm.
15 to
17; CFlank voltage mean 844 (range 812-877), offset 7 to
26; NFlank voltage mean 775 (range 754-789), offset 19 to 24;
hSP-B voltage mean 726 (range 705-740), offset 16 to 19. By
comparison, the parameters for Cy3-labeled secondary IgG were voltage
mean 807 (range 691-891), offset 4 to 22. Pinhole settings, which
were identical for both FITC and Cy3 images due to simultaneous
scanning, were: NFProx/Cy3 mean 169 (range 157-181), CFlank/Cy3
mean 179 (range 146-207), NFlank/Cy3 mean 173 (range 141-191), hSP-B/Cy3 mean 163 (range 129-194). Overall, the
hSP-B antiserum yielded more intense staining as reflected by the lower voltage and pinhole settings required for optimal image acquisition. Conversely, the epitope-specific antisera required higher acquisition settings to achieve equivalent image quality.
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Fig. 3.
Colocalization of SP-B epitope-specific
antisera with BiP. Isolated alveolar type 2 cells were fixed and
immunostained for BiP (red) and SP-B intermediates using the
epitope-specific antisera as indicated (green). Confocal
microscopy showed intense colocalization of immunostaining
(yellow) by BiP and NFProx and to a lesser extent for CFlank
and NFlank (panel A, merged images; panel B,
unmerged images for NFProx and BiP). There was no colocalization
between BiP and hSP-B. Images are representative of triplicate
experiments; immunostaining patterns shown were characteristic of
~75% of cells per slide. Bar, 20 µm.
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Fig. 4.
Colocalization of SP-B epitope-specific
antisera with MG160. Isolated alveolar type 2 cells were fixed and
immunostained for MG160 (red) and SP-B intermediates using
the epitope-specific antisera as indicated (green). Confocal
microscopy showed intense colocalization of immunostaining
(yellow) occurred with MG160 and CFlank and NFlank in a
juxtanuclear tubular network. There was no colocalization of MG160 with
either NFProx or hSP-B antisera. Images are representative of
triplicate experiments; immunostaining patterns shown were
characteristic of ~75% of cells per slide. Bar, 20 µm.
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Fig. 5.
Colocalization of SP-B epitope-specific
antisera with Lamp-1. Type 2 cells were fixed and immunostained
for Lamp-1 (red) and SP-B intermediates using the
epitope-specific antisera as indicated (green). Lamp-1
stained the limiting membrane of lamellar bodies but did not colocalize
with hSP-B staining. The hSP-B antiserum stained intensely as discrete
foci within Lamp-1 demarcated lamellar bodies and the NFlank
antiserum-stained Lamp-1-negative vesicles in close proximity to
lamellar bodies. The inserted frames illustrate lamellar body detail.
Images are representative of triplicate experiments; immunostaining
patterns shown were characteristic of ~75% of cells per slide.
Bar, 20 µm.
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Fig. 6.
Brefeldin A blocks the first proteolytic
cleavage of pro-SP-B to a 25-kDa intermediate. Fetal lung explants
were pulse-labeled with [35S]Met-Cys for 1 h and
chased in cold complete medium for up to 8 h in the presence or
absence of brefeldin A (10 µg/ml). Samples collected at the indicated
time points were immunoprecipitated with the hSP-B8
antiserum and analyzed by Tris-Tricine SDS-PAGE. The PhosphorImager
results of untreated control samples indicated full processing to 8 kDa
by 1-2 h post-pulse whereas brefeldin A treatment blocked processing
beyond 42 kDa pro-SP-B.
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Fig. 7.
Pro-SP-B and the 25-kDa SP-B intermediate
reside in endo H-sensitive and endo H-resistant pools. Triplicate
samples from a 4-h pulse-chase of fetal lung explants were
immunoprecipitated with the hSP-B antiserum. Samples were treated with
or without endo H or PNGase F before 10% Bis-Tris SDS-PAGE. Control
samples showed processing of 42-kDa pro-SP-B through 25- and 9-kDa
intermediates to 8-kDa mature SP-B by 2 h. Endo H and PNGase F did
not alter migration of the 9- and 8-kDa proteins. PNGase F reduced
pro-SP-B from 42 to 39 kDa and the 25-kDa intermediate to 21 kDa. Endo
H reduced pro-SP-B to 39 kDa with a endo H-resistant minor pool at 42 kDa. The 25-kDa intermediate also appeared in 2 pools: a minor 21-kDa
endo H-sensitive pool and a major 25-kDa endo H-sensitive pool.
14C-Labeled molecular weight markers (molecular mass: 46, 30, 21.5, 14.3, and 6.5 kDa) and non-immune serum immunoprecipitation
(NIS) lanes are also shown.
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Fig. 8.
Brefeldin A induces a redistribution of
CFlank immunostaining. Isolated alveolar type 2 cells were
incubated with or without brefeldin A (10 µg/ml) for 30 min prior to
fixation and immunostaining for SP-B intermediates using the SP-B
epitope-specific antisera as indicated (green) and BiP
(red, panel A), or MG160 (red, panel B).
Immunostaining of control cells was no different from Figs. 3 and 4. In
the presence of brefeldin A, there was no change in the immunostaining
patterns of hSP-B, or NFProx and BiP, which both continue to colocalize
(yellow). CFlank and MG160 lost the tubular appearance seen
in control cells. CFlank colocalized with BiP and MG160 colocalized
with NFProx after brefeldin A treatment. NFlank immunostaining shifted
after brefeldin A but had a juxtanuclear tubular pattern, losing all
colocalization with MG160. Images are representative of triplicate
experiments; immunostaining patterns shown were characteristic of
~75% of cells per slide. Bar, 20 µm.
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Fig. 9.
Monensin slows pro-SP-B processing and
prevents the final N-terminal processing event. Cultured
hormone-treated human fetal lung explants were pulse-labeled with
[35S]Met-Cys for 1 h and chased in cold complete
medium for up to 8 h in the presence or absence of monensin (2 mM). Samples collected at the indicated time points were
immunoprecipitated with the hSP-B8 antiserum and analyzed
by Tris-Tricine SDS-PAGE. A, PhosphorImager results of
untreated control samples showed the 8-kDa mature SP-B by 1-2 h
post-pulse whereas monensin treatment resulted in delayed appearance of
25- and 9-kDa intermediates and no 8-kDa SP-B at 8 h.
B, control samples from 4 and 8 h and monensin-treated
sample from 8 h were immunoprecipitated and electrophoresed
together on the same gel to illustrate the lack of processing beyond 9 kDa in the presence of monensin (C, control; M,
monensin).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
Model of intracellular localization of SP-B
Processing. Prepro-SP-B (40 kDa) is modified by glycosylation and
signal peptide cleavage resulting in 42-kDa pro-SP-B. These events
occur within the endoplasmic reticulum with prepro-SP-B and pro-SP-B,
as indicated by NFProx immunostaining, colocalizing with BiP. The
initial proteolytic cleavage of the N terminus occurs within the medial
Golgi since brefeldin A prevents all pro-SP-B processing and shifts
CFlank immunostaining. These findings along with partial sensitivity to
endo H indicate that both N terminus cleavage and oligosaccharide
modification by mannosidase II occur in medial Golgi compartments.
Cleavage of the C terminus, which is not monensin-sensitive, occurs in
the trans-Golgi leaving a steady state pool of 9-kDa intermediate in
the trans-Golgi and/or a post-Golgi compartment. In the presence of
brefeldin, this pool loses colocalization with MG160 and takes on a
juxtanuclear tubular appearance characteristic of trans-Golgi proteins.
The final N-terminal cleavage occurs in a monensin-sensitive post-Golgi
compartment, possibly the multivesicular body, resulting in only the
mature form of SP-B in the lamellar body.
-helical saposin-consensus regions. Our immunofluorescence
studies relied on the ability of these antisera to discriminate SP-B
intermediates. We have shown previously by Western immunoblotting that
these antisera appropriately recognize the relevant SP-B intermediates
containing the immunizing peptide sequences and are successfully
competed by preincubating each antiserum with its immunizing peptide
(8). In using the epitope-specific antisera for immunolocalization
studies, we assumed that all epitopes would be equally exposed and
available for antibody recognition based upon our prior Western
blotting experiments. Based upon this assumption, all of the SP-B
antisera would recognize pro-SP-B in the ER and continue to identify
intermediates until the epitope was lost in post-translational
processing events. Instead, our immunofluorescence images showed
variations in the intensity of fluorescence with each antiserum, as
reflected in both the images and the laser settings used to generate
the images. NFProx antiserum, which recognizes both pro-SP-B and the
excised N terminus by Western blotting, colocalized predominantly with
the lumenal endoplasmic reticulum marker BiP (29, 30). This places
pro-SP-B predominantly in the endoplasmic reticulum, which is in
agreement with previous work by Voorhout and colleagues (9). However,
CFlank, NFlank, and hSP-B antisera, which identify pro-SP-B by
immunoblotting, showed little colocalization with BiP. These
observations can be explained by variation in the affinities of the
antisera for their epitopes, in the FITC labeling of the primary
antisera, in the relative concentration of SP-B proteins within the
organelles, or by altered accessibility of the epitopes within the SP-B
proteins. Immunostaining procedures were optimized for each antiserum
to attempt to control for variability in antiserum affinity and FITC labeling and our methodologies could not evaluate whether epitopes were
accessible. However, the relative amounts of pro-SP-B, intermediates and mature SP-B protein are not constants between organelles and more
likely explains some of the immunostaining variability. Previously, we
showed that pro-SP-B is rapidly processed to the 25-kDa intermediate in
human fetal lung explants with differentiated type 2 cells (8). The
accumulation of mature SP-B protein occurs more slowly. Therefore at
steady state, there is relatively less 42-kDa pro-SP-B within the type
2 cell than 25-kDa intermediate. Immunostaining using the CFlank and
NFlank antisera would favor detection of the larger pool of 25-kDa
intermediate over the smaller pool of pro-SP-B. Likewise, mature SP-B
accumulates and is concentrated within lamellar bodies, achieving a
much higher steady state pool size than any of the intermediates. As a
result, immunostaining using the hSP-B antiserum favors detection of
the lamellar body pool of mature SP-B over the smaller intermediate pools.
/
knock-out mouse. Keratinocytes, like alveolar type 2 cells, develop
lamellar bodies which are extruded into the extracellular space where
the monosaposins modify ceramides to form a water-tight barrier. The
homozygous prosaposin knock-out mice have a thinned epidermis that on
electron microscopy appears disordered. The keratinocytes have abnormal
lamellar bodies reminiscent of the abnormal lamellar bodies of alveolar
type 2 cells of the homozygous SP-B knock-out mouse and human infants with inherited SP-B deficiency.
/ mice involving the knocking-in of
various SP-B constructs have shown that the N terminus is essential for
successful rescue of this lethal phenotype (5, 6, 43). In these studies the complete N terminus, including the vestigial N-terminal epitope retained after the initial pro-SP-B cleavage, was eliminated. Our data
show that this epitope was retained through a post-Golgi, pre-lamellar
body compartment which suggests a role for this epitope in trafficking
SP-B toward lamellar bodies. Additional studies will be required to
determine whether this peptide is both necessary and sufficient for
trafficking SP-B to similar compartments within type 2 cells and other
cell types with specialized secretory functions.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Div. of Neonatology,
Children's Hospital of Philadelphia, 416G Abramson Research Bldg.,
34th St. and Civic Center Blvd., Philadelphia, PA 19104. Tel.:
215-590-2806; Fax: 215-590-4267; E-mail:
guttentag@email.chop.edu.
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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