Biosynthesis and Post-translational Processing of the Precursor to Brain-derived Neurotrophic Factor*

S. Javad MowlaDagger §, Hooman F. FarhadiDagger , Sangeeta PareekDagger , Jasvinder K. AtwalDagger , Stephen J. MorrisDagger , Nabil G. Seidah, and Richard A. Murphy||

From the Salk Institute, La Jolla, California 92037-1099, the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-down-arrow -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 alpha 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

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1-PDX, an inhibitor of furin-like enzymes (11), was generously provided by Dr. Gary Thomas (Vollum Institute, Portland OR).

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:alpha 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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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 (alpha -BDNF), or alpha -BDNF with excess recombinant human BDNF (rhBDNF).

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.


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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.

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.


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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.

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.


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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.

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.


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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.

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.


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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.

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-down-arrow -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-down-arrow -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.

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 alpha 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 right-arrow 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.   alpha 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.

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:alpha 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:alpha 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:alpha 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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 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.

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 alpha 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.

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-down-arrow -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.

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 alpha 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 right-arrow Ala) abolished the generation of the 28-kDa form without affecting the production of mature BDNF (Fig. 8B).

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.

    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.

    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

    ABBREVIATIONS

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; alpha 1-PDX, alpha 1-antitrypsin Portland; DMEM, Dulbecco's modified Eagle's medium; rhBDNF, recombinant human BDNF.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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