From the Salk Institute, La Jolla, California 92037-1099, the Centre for Neuronal Survival, Department of Neurology
and Neurosurgery, Montreal Neurological Institute, McGill University,
Montreal, Quebec H3A 2B4 and ¶ Laboratory of Biochemical
Neuroendocrinology, Clinical Research Institute of Montreal,
Montreal, Quebec H2W 1R7, Canada
Received for publication, September 5, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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We examined the biosynthesis and
post-translational processing of the brain-derived neurotrophic factor
precursor (pro-BDNF) in cells infected with a pro-BDNF-encoding
vaccinia virus. Metabolic labeling, immunoprecipitation, and
SDS-polyacrylamide gel electrophoresis reveal that pro-BDNF is
generated as a 32-kDa precursor that is N-glycosylated and
glycosulfated on a site, within the pro-domain. Some pro-BDNF is
released extracellularly and is biologically active as demonstrated by
its ability to mediate TrkB phosphorylation. The precursor undergoes
N-terminal cleavage within the trans-Golgi network and/or
immature secretory vesicles to generate mature BDNF (14 kDa). Small
amounts of a 28-kDa protein that is immunoprecipitated with BDNF
antibodies is also evident. This protein is generated in the
endoplasmic reticulum through N-terminal cleavage of pro-BDNF at the
Arg-Gly-Leu-Thr57- Brain-derived neurotrophic factor
(BDNF)1 along with nerve
growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) are members of the neurotrophin family of trophic factors (1).
The neurotrophins play essential roles in the development, survival,
and function of a wide range of neurons in both the peripheral and
central nervous systems.
The neurotrophins have a number of shared characteristics, including
similar molecular weights (13.2-15.9 kDa), isoelectric points (in the
range of 9-10), and ~50% identity in primary structure. They exist
in solution as noncovalently bound dimers. Six cysteine residues
conserved in the same relative positions give rise to three intra-chain
disulfide bonds (2, 3). The neurotrophins interact with two cell
surface receptors, the low affinity P75 receptor (4) and the Trk family
of high affinity tyrosine kinase receptors (5). NGF preferentially
binds TrkA, BDNF and NT4/5 bind TrkB, and NT-3 binds TrkC (and TrkA to
a lesser extent).
Sequence data predict that mature neurotrophins are generated through
the proteolytic processing of higher molecular weight precursors
(31-35 kDa), a process that has been extensively studied with respect
to the production of NGF (6, 7). Almost nothing is known, however,
about the biosynthesis and post-translational processing of the other
members of the neurotrophin family. Recent data from our laboratory
show that cells with a regulated secretory pathway, including central
nervous system neurons, release mature (i.e. fully
processed) NGF (8) and NT-3 (9) via the constitutive secretory pathway,
whereas mature BDNF is packaged in vesicles and released through the
regulated pathway (8). Furthermore, BDNF is contained in a
microvesicular fraction of lysed brain synaptosomes consistent with its
anterograde transport in large dense core vesicles (10). Differences in
the intracellular sorting of neurotrophins may arise, at least in part,
from differences in the chemistry and processing of their precursors.
Therefore, defining how neurotrophins are generated within a cell will
be key to understanding how neurotrophins are released and function within the nervous system.
In this study, we monitored the biosynthesis and post-translational
processing of the precursor to BDNF (pro-BDNF) using a vaccinia virus
(vv) expression system together with metabolic labeling,
immunoprecipitation, and SDS-PAGE. Data show that pro-BDNF is produced
as a 32-kDa precursor that undergoes N-glycosylation and
glycosulfation on residues located within the pro-domain of the
precursor. N-terminal cleavage of the precursor generates mature BDNF
as well as a minor truncated form of the precursor (28 kDa) that arises
by a different processing mechanism than mature BDNF. Site-directed
mutagenesis data suggest that 28-kDa BDNF is not an obligatory
intermediate in the formation of the mature form. Data also demonstrate
that pro-BDNF could be biologically active, as determined by its
ability to promote TrkB autophosphorylation.
Cell Culture--
Hippocampal neurons were prepared according to
the method of Brewer et al. (29). Briefly, the
hippocampus was dissected from day 18 (E18) mice (Charles River
Breeding Laboratories, Montreal, Canada), exposed to trypsin,
dissociated mechanically, and grown in 60-mm
collagen/poly-L-lysine-coated dishes. Cultures were
maintained in serum-free Neurobasal medium (Life Technologies, Inc.)
containing 0.5 mM glutamine and 1× supplemented B27 (Life
Technologies, Inc.). AtT-20, COS-7, and LoVo cells were cultured as
reported previously (7). A human glioma (U373) cell line and a variant
(U373/PDX) that stably expresses Vaccinia Virus Infections and Metabolic Labeling--
Purified
recombinant vv containing the full-length coding region of human
pro-BDNF was prepared and used to infect cells as described previously
(12). U373 and U373/PDX glial cells and AtT-20 cells were grown in
60-mm dishes and exposed to virus for 30 min or 2 h, respectively.
The cells were incubated in medium without virus overnight and either
pulsed or pulse-chase labeled at 37 °C for specified time intervals.
For pulse-chase experiments, infected cells were incubated in
cysteine/methionine-free Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal calf serum for 1 h, and then received 1.5 ml
of the same medium containing 0.2 mCi/ml [35S]Cys/Met
(PerkinElmer Life Sciences) for 30 min. For the chase, cells were
bathed for specified intervals in DMEM containing 10% fetal calf serum
plus excess (2 times) cysteine and methionine.
In experiments assessing sulfation, AtT-20 cells were labeled for
3 h with [Na235SO4 ] (0.5 mCi) (PerkinElmer Life Sciences) in
methionine/cysteine/SO4-free RPMI 1640 medium (Life
Technologies, Inc.). Sodium chlorate (1 mM) was added to
the medium in some experiments to inhibit sulfation, and in others,
tunicamycin (5 µg/ml) was added to inhibit N-linked glycosylation. In both cases, the drugs were present in the medium during the 60-min preincubation period and throughout the pulse-chase period.
Immunoprecipitation and Microsequencing--
Radiolabeled BDNF
was immunoprecipitated from cell lysates and conditioned medium as
described previously (8). We used an affinity-purified antibody to BDNF
(13, kindly supplied by Amgen) at a concentration of 0.5 µg/ml.
Samples were analyzed by 13-22% gradient SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). Gels were fixed for 1 h in 40%
methanol and 10% acetic acid, treated with ENHANCE (PerkinElmer Life
Sciences) for 1 h, washed in 10% glycerol for 1 h, and dried
for 4 h at 60 °C. Micro-sequencing was carried out on samples
from conditioned medium that were [3H]Leu-labeled and
eluted from SDS-containing gels following electrophoresis. Micro-sequencing was performed using an Applied Biosystem gas-phase sequenator model 470A, as described previously (14).
Endoglycosidase H (Endo H) and N-Glycanase
Treatment--
vv:BDNF-infected AtT-20 cells were metabolically
labeled with [35S]Cys-Met; conditioned medium was
collected and treated with antibody to BDNF, and the precipitates were
dissolved in 100 µl of reaction buffer with or without endo H (10 units; Roche Molecular Biochemicals) or N-glycanase (1.5 units; Oxford GlycoSystems). Samples were incubated overnight at
37 °C. Endo H digestions were carried out in 100 mM
sodium citrate buffer, pH 5.5, and N-glycanase digestions in
20 mM sodium phosphate buffer (pH 7.5) containing 50 mM EDTA.
Transient Transfection of R54A BDNF Mutant in COS-7
Cells--
By using the LipofectAMINE reagent (Life Technologies,
Inc.), we transfected 60-70% confluent COS-7 cells with pcDNA3
recombinants of either the wild-type or R54A mutant form of pro-BDNF.
After a 5-h incubation in serum- and antibiotic-free DMEM, the cells were incubated for another 48 h in DMEM plus 10% fetal calf
serum. Two days after transfection, cells were metabolically labeled for 6 h, and cell lysates and conditioned media were collected, immunoprecipitated, and resolved by 13-22% gradient SDS-PAGE.
TrkB Phosphorylation Assay--
For these studies, we obtained
relatively pure preparations of the BDNF precursor by coinfecting LoVo
cells, an epithelial cell line that is deficient in endogenous
furin-like enzymes (15), with vv:BDNF and vv: Antibody Specificity--
To characterize the specificity of the
BDNF antibody and to monitor its effectiveness in immunoprecipitations,
we overexpressed BDNF in AtT-20 cells using a vv encoding the
full-length precursor to hBDNF. Samples of cell lysate and conditioned
medium were divided equally and immunoprecipitated with nonimmune
serum, antibody to BDNF, or BDNF antibody with excess rhBDNF (5 ng/µl). As seen in Fig. 1, in both cell
lysate and conditioned media, the antibody to BDNF specifically
immunoprecipitated three proteins migrating at ~32, 28, and 14 kDa.
None of these proteins reacted with nonimmune serum, and none were
immunoprecipitated in the presence of excess rhBDNF. We therefore
conclude that the 32-kDa protein is unprocessed pro-BDNF, the 28-kDa
protein a truncated form of pro-BDNF, and the 14-kDa protein fully
processed mature BDNF.
Pro-BDNF Processing in vv:BDNF-infected AtT-20 Cells--
To
understand the relationship of the different forms of the BDNF
precursor to mature BDNF, we carried out pulse-chase studies using
AtT-20 cells infected with recombinant vv:BDNF, a system we have used
previously (8, 9). Fig. 2A
shows that 32-kDa BDNF precursor is apparent in cell lysates as early
as 10 min after the cells were radiolabeled and increased in intensity
through 30 min of pulse incubation. In cells labeled for 20 min, a
slightly higher molecular weight band is apparent that resolves into a doublet in cells chased for 4 h. This material likely represents differentially glycosylated and sulfated forms of the BDNF precursor (see below). Over the 8-h chase period, the 32-kDa and, to a lesser extent, the minor higher molecular weight bands decreased in intensity, whereas levels of the 14-kDa mature BDNF band increased, suggesting a
precursor-product relationship. The 28-kDa band appeared as early as 10 min pulse, and its level increased by 1 h of chase. The intensity
of the band decreased significantly thereafter. Fig. 2B
reveals that significant amounts of the 32-kDa BDNF precursor, the
28-kDa form, and mature BDNF are released into conditioned medium
during the 8-h chase period.
Pro-BDNF Is N-Glycosylated--
N-Glycanase
treatment of the 32-kDa BDNF precursor and the truncated 28-kDa form of
the precursor reduces their apparent size to around 27 and 24 kDa,
respectively, indicating that these proteins contain
N-linked complex carbohydrates (Fig.
3). N-Glycanase treatment has
no effect on the apparent molecular size of mature BDNF (14 kDa).
Treatment with endo H, which removes high mannose sugar moieties, only
partially digests the 32- and 28-kDa BDNF (Fig. 3), suggesting that the
precursor released into the conditioned medium contains a heterogeneous
mixture of complex and high mannose sugars. Note that both pro-BDNF and
28-kDa BDNF appear as doublets with the lower band being endo
H-sensitive, whereas the higher band is endo H-resistant.
To define the importance of glycosylation in the generation of BDNF
from its precursor, we infected AtT-20 cells with vv:BDNF and
metabolically labeled the cells in the presence or absence of 5 µg/ml
of tunicamycin, an inhibitor of N-glycosylation. Cells were
metabolically labeled for 30 min followed by a 2-h chase period, in the
presence or absence of tunicamycin. Fig.
4 shows that tunicamycin greatly reduced
the signal intensity of the BDNF precursors as well as mature BDNF
(compare the level of labeling in the left and right
panels of Fig. 4). In addition, the apparent molecular site
of the BDNF precursor in cell lysate and in conditioned medium was
reduced from 32 to ~27 kDa. The result suggests that glycosylation
may play an important role in stabilizing the BDNF precursor during its
processing and subcellular trafficking. Tunicamycin did not alter the
molecular size of mature BDNF (14 kDa), as expected since this form of
the protein is not N-glycosylated.
Pro-BDNF Is Glycosulfated--
Metabolic labeling of
vv:BDNF-infected AtT-20 cells with
[35SO4]Na2 (Fig.
5A) reveals that pro-BDNF as
well as the truncated 28-kDa form of the precursor are sulfated. Mature
BDNF, in contrast, is not sulfated. Furthermore, treatment of the
sulfated species with N-glycanase (Fig. 5B)
completely removes the radioactive signal, demonstrating that sulfation
occurs on carbohydrate groups.
To determine whether sulfation is essential for the processing and/or
secretion of pro-BDNF, we labeled AtT-20 cells expressing pro-BDNF with
[35S]Cys-Met for 30 min and then chased the cells for
2 h in the presence or absence of sodium chlorate (1 mM) (17). This treatment reduced by 97% the levels of
35SO4 that were incorporated into
protein immunoprecipitates measured in conditioned medium at the end of
the chase period (data not shown). The result showed that exposure to
sodium chlorate had no detectable effect on processing of pro-BDNF
or on secretion of mature BDNF (data not shown).
Generation of 28-kDa BDNF Occurs in the ER--
To determine where
in the cell the 28-kDa form of BDNF is generated, we metabolically
labeled vv:pro-BDNF infected cells with [35S]Cys-Met for
3 h in the presence or absence of brefeldin A (BFA, 5 µg/ml), a
molecule that inhibits anterograde vesicular transport from the ER
(18). The cells were analyzed immediately or after a further 2-h chase
period without BFA. Fig. 6 shows that BFA had no effect on the generation of the 28-kDa form of pro-BDNF, but it
did inhibit the generation of the 14-kDa form of mature BDNF. This
effect was reversed when the cells were chased 2 h in the absence
of BFA. These results suggest that the 28-kDa form of BDNF can be
generated in the ER, whereas the mature form of BDNF, as already shown
(8), is generated in the trans-Golgi network or a post-Golgi
compartment.
N-terminal Sequence of 28-kDa BDNF--
The data presented above
show that cell lysates and conditioned media of AtT-20 cells infected
with vv:pro-BDNF generate a truncated form of BDNF with an apparent
molecular mass of 28 kDa. We also detected this molecule in several
other cell lines as well as in primary cultures of mouse hippocampal
neurons infected with the same vv construct (Fig.
7). In a separate study, we showed that a
novel enzyme (SKI-1, subtilisin-kexin-isozyme-1) is able to increase
the level of 28-kDa BDNF when coexpressed with pro-BDNF in COS-7 cells
(19). N-terminal micro-sequencing of [3H]Leu-labeled
28-kDa BDNF revealed a unique cleavage site at
Arg54-Gly-Leu-Thr57- 28-kDa BDNF Is Not an Obligatory Intermediate in the Generation of
Mature BDNF--
In this study, we introduced a vv encoding pro-BDNF
into a cell line (U373 glial cells) that stably expresses the furin
inhibitor Pro-BDNF Is Biologically Active--
Significant amounts of
unprocessed pro-BDNF are secreted into conditioned media under our
experimental conditions, a result that led us to question whether the
precursor, if released in vivo, could be biologically
active. To test this idea, we set out to generate unprocessed pro-BDNF
by coinfecting LoVo cells, which are already deficient in furin
activity (15), with vv:BDNF along with vv:
Fig. 10C shows that medium collected from both cell types
induces robust TrkB autophosphorylation in NIH 3T3 cells that
overexpress the TrkB receptor. Medium conditioned by cells infected
with wild-type vv had no effect. We conclude from these data that once
released from a cell, the intact BDNF precursor containing small
amounts of the 28-kDa form of BDNF has the potential to be biologically active. We do not know the precise contribution of the 28-kDa form of
pro-BDNF to this activity since we were unable to obtain sufficient
amounts of the protein for testing in the absence of pro-BDNF or mature
BDNF.
Because biosynthesis of neurotrophins normally occurs at low
levels in neurons and non-neuronal cells, it is impossible to analyze
endogenous neurotrophin processing with currently available techniques.
Therefore, in this study, we used a vaccinia virus expression system to
overexpress pro-BDNF and to study its processing in a variety of cell
lines as well as in primary cultures of mouse hippocampal neurons. We
have used similar methods previously to monitor the biosynthesis and
post-translational processing of pro-NGF (7).
By using the BDNF antibody provided by Amgen, as well as a commercially
available antibody from Santa Cruz Biotechnology (data not shown), we
detected three BDNF-related products in vv:BDNF-infected AtT 20 cells
and hippocampal neurons, namely 32-, 28-, and 14-kDa forms of the
protein (Fig. 1). Results indicate that pro-BDNF is synthesized as a
32-kDa precursor that is processed within 1 h to give rise to
mature BDNF (14 kDa). We also observed a significant amount of
unprocessed pro-BDNF being released into conditioned medium by AtT-20
cells and hippocampal neurons (8), cells that can release proteins both
by the regulated and constitutive secretory pathways. In parallel
studies, we did not observe precursor release when similar methods were
used to monitor processing and release of the precursors of NGF (8) or
NT-3 (9). Indeed, previous work by others (18, 27) has shown that large
amounts of the precursors of proteins released by the regulated
secretory pathway, such as pro-opiomelanocorticotrophin and pro-renin,
are also constitutively released from AtT-20 cells. Although these
differences could simply reflect overexpression of pro-BDNF saturating
the sorting machinery in the trans-Golgi network (8),
constitutive release of the precursor could also be of biological
significance. In that regard, BDNF mRNA is present in the dendrites
of hippocampal neurons in culture (20, 21), and as yet, we know nothing
about the chemistry or fate of the BDNF protein synthesized within
dendrites. It is possible that pro-BDNF could be produced in dendrites
and released, in part, in an unprocessed form, for as yet unknown purposes.
In this study, we have shown that medium containing pro-BDNF interacts
with the TrkB receptor and activates its autophosphorylation (Fig. 10).
To eliminate the possibility of pro-BDNF being further processed by
cell surface-associated furin, the TrkB-expressing 3T3 cells
were concomitantly exposed to a medium containing a large excess of
During transit through the secretory pathway, the BDNF precursor is
glycosylated (Fig. 3), presumably at the single putative consensus
sequence for N-linked glycosylation (NX(T/S)) six
residues upstream of the cleavage site that generates mature BDNF. This glycosylation site is conserved in the same position in all
neurotrophins, suggesting a critical role for N-linked
glycosylation in neurotrophin maturation and/or trafficking. Pro-BDNF
is released into conditioned medium as a mixture of endo H-sensitive
(untrimmed) and endo H-resistant (trimmed) sugars. Both the 32- and
28-kDa forms of BDNF appear as doublets, the upper band being endo
H-resistant and the lower band is endo H-sensitive (Fig. 3). The
importance of carbohydrates in the folding of proteins has been well
documented (24). In the case of NGF, blocking
N-glycosylation with tunicamycin prevents the entry of
pro-NGF into the Golgi apparatus and its subsequent secretion (7). In
this study, blocking N-glycosylation of pro-BDNF significantly reduced the level of radiolabeling of both pro-BDNF and
mature BDNF, which may be due to incorrect folding diminishing the
half-life of newly synthesized protein. Our results also demonstrate that oligosaccharide chains attached to the pro-domain of the BDNF
precursor are sulfated (Fig. 5), as has been previously reported for
pro-NGF (7). Blocking sulfation with sodium chlorate (17) did not
affect processing and release of pro-BDNF. This result is consistent
with the recent finding of Van Kuppereld and colleagues (25) that
protein sulfation is not required for the transport, sorting, or
proteolytic processing of proteins directed to the regulated secretory pathway.
We have also identified a 28-kDa protein that is a cleavage
product of the BDNF precursor in addition to 14-kDa mature BDNF but
that is generated through a distinct processing pathway. Furthermore, its processing in U373 glial cells (Fig. 9) is not affected by Recent studies from our laboratories (19, 26) revealed that the 32-kDa
BDNF precursor is a substrate for a newly identified subtilisin/kexin-like enzyme, called SKI-1. Coexpression of pro-BDNF and SKI-1 produced sufficient 28-kDa BDNF for N-terminal
microsequencing, which revealed that cleavage occurs at the
Arg54-Gly-Leu-Thr57- Two lines of evidence suggest that the generation of mature BDNF in the
constitutive pathway does not require initial processing of pro-BDNF to
the 28-kDa form. First, in the U373-PDX cell line (a constitutive
secreting cell line expressing Much is yet to be learned about the BDNF precursor. For example, we do
not know whether the intact precursor (32 kDa) and the 28-kDa form of
the precursor, both of which can be released constitutively from cells,
could have biological roles of their own distinct from mature BDNF.
Furthermore, in cells with both the regulated and constitutive
secretory pathways, pro-BDNF is preferentially processed and released
from the regulated pathway, whereas pro-NGF (8) and pro-NT3 (9) are in
the constitutive secretory pathway. Differential targeting may well
arise because of structural differences in the pro-domains of the
neurotrophin precursors or because of differential processing. Studies
currently underway are targeted toward solving these issues.
-Ser-Leu site. Cleavage is abolished
when Arg54 is changed to Ala (R54A) by in vitro
mutagenesis. Blocking generation of 28-kDa BDNF has no effect on the
level of mature BDNF and blocking generation of mature BDNF with
1-PDX, an inhibitor of furin-like enzymes, does not lead
to accumulation of the 28-kDa form. These data suggest that 28-kDa
pro-BDNF is not an obligatory intermediate in the formation of the
14-kDa form in the constitutive secretory pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-PDX, an inhibitor of
furin-like enzymes (11), was generously provided by Dr. Gary Thomas
(Vollum Institute, Portland OR).
1-PDX (11).
The cells were incubated in virus-free medium for 10 h, followed
by a 4-h incubation in serum-free medium, which was subsequently
collected for testing. To isolate fully processed BDNF generated under
similar conditions, we coinfected LoVo cells with vv:BDNF and vv:furin,
to ensure that the precursor was cleaved, and we collected conditioned
medium 6 h later. Media collected from uninfected and wild-type
vaccinia virus-infected (vv:wild type) LoVo cells were used as
controls. To test for biological activity, we used NIH 3T3 cells that
overexpress TrkB, prepared and generously provided by Dr. David Kaplan
(Montreal Neurological Institute). The cells were bathed in conditioned
medium for 5 min, following which cell lysates were immunoprecipitated
with panTrk-203 antibody (16). The pellets were dissolved in sample buffer, fractionated by SDS-PAGE using an 8% gel, and transferred onto
a 0.2-µm nitrocellulose membrane for Western blotting. The replicas
were probed overnight at 4 °C with a monoclonal phosphotyrosine antibody (Upstate Biotechnology, Inc., Lake Placid, NY) diluted 1:10,000 in Tris-buffered saline supplemented with 0.1% Tween 20, and
for an additional 1 h with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5000). Immunoreactivity was
observed using enhanced chemiluminescence (PerkinElmer Life Sciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Immunoprecipitations with the BDNF
antibody. AtT-20 cells were infected for 1 h with vv:BDNF and
labeled with [35S]Cys-Met for 4 h. Cell lysates and
media were equally divided into three tubes and immunoprecipitated with
either nonimmune serum (NI), antibody to BDNF
( -BDNF), or
-BDNF with excess recombinant
human BDNF (rhBDNF).
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Fig. 2.
Pulse-chase labeling of AtT-20 cells infected
with vv:BDNF. Infected cells were labeled with
[35S]Cys-Met for 10, 20, and 30 min without chase or
pulsed for 30 min and then exposed to a chase medium containing excess
unlabeled cysteine and methionine for 0.5, 1, 2, 4, and 8 h. Cell
lysates (A) and conditioned media (B) were
immunoprecipitated and analyzed separately by SDS-PAGE.
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Fig. 3.
Pro-BDNF but not mature BDNF is
glycosylated. AtT-20 cells were infected with vv:BDNF, incubated
overnight in medium without virus, and labeled with
[35S]Cys-Met for 3 h. Conditioned media and cell
lysates were collected and incubated with antibody to BDNF. Following
immunoprecipitation, samples were incubated in the absence
(control) or presence of either N-glycanase or
endoglycosidase H and analyzed by SDS-PAGE.
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Fig. 4.
N-Glycosylation increases the
stability of pro-BDNF. AtT-20 cells were infected with vv:BDNF,
pulse-labeled with [35S]Cys-Met for 30 min, and chased
for 2 h in the absence ( ) or presence (+) of 5 µg/ml
tunicamycin. Immunoprecipitates from cell lysates (CL) and
conditioned media (CM) were resolved by SDS-PAGE and exposed
to film.
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Fig. 5.
Pro-BDNF is glycosulfated. A,
AtT-20 cells were infected with vv:BDNF for 2 h, incubated
overnight without virus, and then labeled with
[Na235SO4] for 3 h. Cell
lysates (CL) and conditioned media (CM) were
immunoprecipitated with antibodies to BDNF, and the precipitate was
analyzed by SDS-PAGE. Pro-BDNF (32 kDa) along with a minor (28 kDa)
form of the precursor (see below) are sulfated, but mature BDNF is not.
B, samples of conditioned media shown in A were
incubated with (+) or without ( ) N-glycanase, showing that
sulfation occurs on carbohydrate chains within the precursor.
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Fig. 6.
Generation of 28-kDa BDNF occurs in the
ER. COS-7 cells were infected with vv:BDNF for 1 h,
post-infected overnight, and metabolically labeled with
[35S]Cys-Met for 3 h in the presence (+) or absence
( ) of brefeldin A (BFA, 5 µg/ml). Cells were then chased
for 2 h in the absence of BFA.
-Ser-Leu
(shown in Fig. 8A). To
determine whether endogenous 28-kDa BDNF is also cleaved at the
same site, we mutagenized Arg 54 (which lies at the P4
position) to Ala. This residue potentially could serve as a recognition
signal for this kind of subtilase (28). Processing of the R54A pro-BDNF
mutant results in unchanged levels of mature 14-kDa BDNF with no
significant generation of the 28-kDa protein (Fig. 8B). This
result demonstrates that the endogenous protein is indeed cleaved at
the same site, and that, as seen for other PC substrates, Arg at the P4
is critical for efficient cleavage (Fig. 8B).
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Fig. 7.
Hippocampal cultures generate the 28-kDa form
of pro-BDNF. One-week-old mouse hippocampal cultures were infected
with vv:BDNF for 1 h and incubated in medium for 8 h. Cells
were then labeled with [35S]Cys-Met for 3 h. Cell
lysates (CL) and conditioned media (CM) were
collected for immunoprecipitation and SDS-PAGE (A). All of
the pro-BDNF processing products (32, 28, and 14 kDa) were detected in
cell lysates and conditioned media. In pulse-chase experiments
(B), the 28 kDa is mostly present in the conditioned media
with its signal intensity increasing with longer chase periods.
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Fig. 8.
Identification of the cleavage site within
pro-BDNF that generates 28-kDa BDNF. A, N-terminal
microsequence analysis of [3H]Leu-labeled 28-kDa BDNF.
The N-terminal sequence of the 28-kDa product in COS-7 cells infected
with vv:BDNF and vv:SKI-1 revealed a [3H]Leu at positions
2, 13, and 14. This result demonstrates that 28-kDa BDNF is generated
by a unique cleavage at Thr57 (arrow) in the
sequence
Arg54gly-Leu-Thr57- -Ser-LeuAla-Asp-Thr-Phe-Glu-His-Val-Ile-Glu-Glu-Leu-Leu-Asp
(top panel). B, transient expression of the
wild-type and R54A mutant form of pro-BDNF in COS-7 cells. COS-7 cells
were transfected with expression constructs of the wild type
(WT) or the Arg54 to Ala mutant (R54A) form of
pro-BDNF. Two days after transfection, cells were metabolically labeled
with [35S]Cys-Met for 6 h, and cell lysates
(CL) and conditioned media (CM) were collected,
immunoprecipitated with a BDNF-specific antiserum, and resolved by
SDS-PAGE.
1-PDX (10). Fig.
9 shows that inhibiting furin-like
enzymes abolishes the formation of mature BDNF but has no effect on the generation of the 28-kDa protein. Also, as shown in Fig. 8B,
transient expression of the Arg54
Ala mutant in COS-7
cells abolishes the generation of 28-kDa BDNF without affecting the
level of mature BDNF. Taken together, these results strongly suggest
that the 28-kDa species does not constitute an obligatory intermediate
in the normal processing of the BDNF precursor in the constitutive
secretory pathway.
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Fig. 9.
1-PDX does not
inhibit the generation of the 28-kDa form of BDNF. U373 and
U373-PDX cell lines were infected with vv:BDNF for 30 min, incubated
overnight without virus, and labeled with [35S]Cys-Met
for 3 h. Cell lysates (CL) and conditioned media
(CM) were immunoprecipitated and resolved by SDS-PAGE.
1-PDX. By
blocking the activity of all furin-like enzymes in the cell, we were
able to obtain conditioned medium containing pro-BDNF and the 28-kDa
BDNF without detectable amounts of mature BDNF (Fig.
10A). As a control for this
study, we collected medium from LoVo cells coinfected with vv:BDNF and
vv encoding furin (vv:furin) (7), conditions that favor the processing of pro-BDNF to mature BDNF (Fig. 10B). Conditioned medium
from these cells contained small amounts of unprocessed pro-BDNF.
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Fig. 10.
BDNF- and pro-BDNF-stimulated TrkB
autophosphorylation. A and B, metabolic
labeling of LoVo cells coinfected with vv:BDNF: 1-PDX
(A) or vv:pro-BDNF/furin (B). The cells were
labeled for 4 h, and cell lysates (CL) and conditioned
media (CM) were immunoprecipitated and analyzed by SDS-PAGE.
C, Western blot analysis of TrkB phosphorylation levels in
NIH 3T3-TrkB cells exposed for 5 min to either of the following media.
Conditioned media from uninfected LoVo cells (control), DMEM
with 100 µg/ml recombinant human BDNF (rhBDNF),
conditioned medium from LoVo cells infected with wild-type vv
(vv:WT), and conditioned medium from LoVo cells coinfected
with vv:BDNF/vv:
1-PDX (pro-BDNF,
B), or vv:BDNF and vv:furin (BDNF, C). The cell
lysates were immunoprecipitated with the panTrk-203 antibody and
analyzed by SDS-PAGE. Levels of phosphorylated TrkB were analyzed on
Western blot replicas with a phosphotyrosine antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-PDX. This ensures the blockade of furin activity at
the cell surface and has recently been shown to be effective for the
cytomegalovirus (17). Previous work (22) has shown that a BDNF mutant
containing an extension of 19 amino acids upstream of the cleavage site
of mature BDNF (25-kDa BDNF) is biologically active. Also, Edwards and
colleagues (23) have reported that pro-NGF is biologically active but
at a level 10-20-fold below that of mature NGF. Taken together, these
findings suggest that complete processing of pro-neurotophins may not
be an absolute requirement for biological activity.
1-PDX, an inhibitor of the furin-like enzymes that
likely generate mature BDNF from pro-BDNF in cells that contain a
constitutive but not regulated secretory pathway. These results
strongly suggest that the 28-kDa molecule is not processed by the known
prohormone convertases but rather by some other processing system
within the cell.
-Ser-Leu site (Fig. 8A). To determine whether the 28-kDa BDNF we
detected is generated at the same cleavage site, we mutagenized
Arg54, which lies at the P4 position relative to the
cleavage site and is potentially important for recognition by this kind
of enzyme (28). Processing of the R54A pro-BDNF mutant did not yield
significant amounts of 28-kDa BDNF, suggesting that the 28-kDa
precursor is cleaved at the same site (Fig. 8B). Thus,
although the 28-kDa form of pro-BDNF is clearly evident in our samples
including hippocampal neurons, we do not know whether the protein
is biologically important.
1-PDX), generation of
14-kDa BDNF is abolished, but there is no accumulation of 28-kDa BDNF,
as would be expected if the latter were an intermediate product (Fig.
9). Second, Ala substitution of the P4 Arg (Arg54
Ala)
abolished the generation of the 28-kDa form without affecting the
production of mature BDNF (Fig. 8B).
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ACKNOWLEDGEMENTS |
---|
We thank Amgen for providing the antibody against brain-derived neurotrophic factor. We thank Suzanne Benjannet and Claude Lazure for carrying out pro-BDNF microsequencing.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Medical Research Council of Canada (to R. A. M. and N. G. S.).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.
§ Supported by a studentship from the Iranian Ministry of Culture and Higher Education.
To whom correspondence should be addressed. Tel.:
858-453-2430; Fax: 858-546-0838; E-mail: Murphy@salk.edu.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M008104200
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ABBREVIATIONS |
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The abbreviations used are:
BDNF, brain-derived
neurotrophic factor;
NGF, nerve growth factor;
NT-3, neurotrophin-3;
NT-4/5, neurotrophin-4/5;
PAGE, polyacrylamide gel electrophoresis;
vv, vaccinia virus;
endo H, endoglycosidase H;
ER, endoplasmic reticulum;
1-PDX,
1-antitrypsin Portland;
DMEM, Dulbecco's modified Eagle's medium;
rhBDNF, recombinant human
BDNF.
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REFERENCES |
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---|
1. | Snider, D. W. (1994) Cell 77, 625-638[Medline] [Order article via Infotrieve] |
2. | Maisonpierre, P. C., Belluscio, L., Squito, S., Ip, N. Y., Furth, M. E., Lindsay, R. M., and Yancoupoulos, G. D. (1990) Science 247, 1373-1520 |
3. | Maisonpierre, P. C., Le Beau, M. M., Espinosa, R., III, Ip, N. Y., Belluscia, L., De La Monte, S. M., Squinto, S., Furth, M. E., and Yancopoulos, G. D. (1991) Genomics 10, 558-568[Medline] [Order article via Infotrieve] |
4. | Barker, P. A. (1998) Cell Death Differ. 5, 346-356[CrossRef][Medline] [Order article via Infotrieve] |
5. | Kaplan, D. R., Hemstead, B. L., Martin-Zanca, D., Chao, M. V., and Parada, L. F. (1991) Science 252, 545-558 |
6. | Edwards, R. H., Selby, M. J., Mobley, W. C., Weinrich, S. L., Hruby, D. E., and Rutter, W. J. (1988) Mol. Cell. Biol. 8, 2456-2464[Medline] [Order article via Infotrieve] |
7. | Seidah, N. G., Benjannet, S., Pareek, S., Savaria, D., Hamlin, J., Goulet, B., Laliberte, J., Lazure, C., Chretien, M., and Murphy, R. A. (1996) Biochem. J. 314, 951-960[Medline] [Order article via Infotrieve] |
8. |
Mowla, S. J.,
Pareek, S.,
Farhadi, H. F.,
Petrecca, K.,
Fawcett, J. P.,
Seidah, N. G.,
Morris, S. J.,
Sossin, W. S.,
and Murphy, R. A.
(1999)
J. Neurosci.
19,
2069-2080 |
9. |
Farhadi, H. F.,
Mowla, S. J.,
Petrecca, K.,
Morris, S. J.,
Seidah, N. G.,
and Murphy, R. A.
(2000)
J. Neurosci.
20,
4059-4068 |
10. |
Fawcett, J. P.,
Aloyz, R.,
McClean, J. H.,
Pareek, S.,
Miller, F. D.,
McPherson, P. S.,
and Murphy, R. A.
(1997)
J. Biol. Chem.
272,
8837-8840 |
11. |
Anderson, E. D.,
Thomas, L.,
Hayflick, J. S.,
and Thomas, G.
(1993)
J. Biol. Chem.
268,
24887-24891 |
12. | Seidah, N. G., Benjannet, S., Pareek, S., Chretien, M., and Murphy, R. A. (1996) FEBS Lett. 379, 248-250 |
13. | Yan, Q., Rosenfeld, R. D., Matheson, C. R., Hawkins, N., Lopez, O. T., Bennett, L., and Welcher, A. A. (1997) Neuroscience 78, 431-448[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Benjannet, S.,
Reudelhuber, T.,
Mercure, C.,
Rondeau, N.,
Chretien, M.,
and Seidah, N. G.
(1992)
J. Biol. Chem.
267,
11417-11423 |
15. | Takahashi, S., Kasal, K., Hatsuzawa, K., Kitamura, N., Misumi, Y., Ikehara, Y., Murakami, K., and Nakayama, K. (1993) Biochem. Biophys. Res. Commun. 195, 1019-1026[CrossRef][Medline] [Order article via Infotrieve] |
16. | Hempstead, B. L., Rabin, S. J., Kaplan, L., Reid, S., Parada, L. F., and Kaplan, D. R. (1992) Neuron 9, 883-896[Medline] [Order article via Infotrieve] |
17. | Baeuerle, P. A., and Huttner, W. B. (1986) Biochem. Biophys Res. Commun. 141, 870-877[Medline] [Order article via Infotrieve] |
18. |
Fernandez, C. J.,
Haugwitz, M.,
Baton, B.,
and Moore, H. P.
(1997)
Mol. Biol. Cell
8,
2171-2185 |
19. |
Seidah, N. G.,
Mowla, S. J.,
Hamelin, J.,
Mamarbachi, A. M.,
Benjannet, S.,
Toure, B. B.,
Basak, A.,
Munzer, J. S.,
Marcinkiewicz, J.,
Zhong, M.,
Barale, J. C.,
Lazure, C.,
Murphy, R. A.,
Chretien, M.,
and Marcinkiewicz, M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
1321-1326 |
20. | Crino, P. B., and Eberwine, J. (1996) Neuron 17, 1173-1187[Medline] [Order article via Infotrieve] |
21. |
Tongiori, E.,
Righi, M.,
and Catteneo, A.
(1997)
J. Neurosci.
17,
9492-9505 |
22. | Kolbeck, R., Jungbluth, S., and Barde, Y. A. (1994) Eur. J. Biochem. 225, 995-1003[Abstract] |
23. |
Edwards, R. H.,
Selby, M. J.,
Garcia, P. D.,
and Rutter, W. J.
(1988)
J. Biol. Chem.
263,
6810-6815 |
24. | Kornfeld, R., and Kornfeld, S. (1985) Annu. Rev. Biochem. 54, 5543-5551 |
25. | Van Kupperfeld, F. J., Van Horssen, A. M., and Martens, G. J. (1997) Mol. Cell. Endocrinol. 136, 29-35[CrossRef][Medline] [Order article via Infotrieve] |
26. | Mowla, S. J., Zhong, M., Mamarbachi, A. M., Seidah, N. G., and Murphy, R. A. (1999) Soc. Neurosci. Abstr. 709, 6 |
27. |
Brechler, V.,
Chu, W. N.,
Baxter, J. D.,
Thibault, G.,
and Reudelhuber, T. L.
(1996)
J. Biol. Chem.
271,
20636-20640 |
28. | Seidah, N. G., and Chretien, M. (1999) Brain Res. 848, 45-62[CrossRef][Medline] [Order article via Infotrieve] |
29. | Brewer, G. J., Torricelli, J. R., Evege, E. K., and Price, P. J. (1993) J. Neurosci. Res. 35, 567-576[Medline] [Order article via Infotrieve] |
30. |
Jean, F.,
Thomas, L.,
Molloy, S. S.,
Liu, G.,
Jarvis, M. A.,
Nelson, J. A.,
and Thomas, G.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2864-2869 |