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Endocrinology. Aug 2008; 149(8): 4116–4127.
Published online May 8, 2008. doi:  10.1210/en.2008-0064
PMCID: PMC2488232

7B2 Prevents Unfolding and Aggregation of Prohormone Convertase 2

Abstract

Prohormone convertase 2 (PC2) requires interaction with the neuroendocrine protein 7B2 for the production of an activatable zymogen; the mechanism for this effect is unknown. 7B2 could act proactively to generate an activation-competent form of pro-PC2 during synthesis, or block spontaneous generation of activation-incompetent forms. We here demonstrate that addition of exogenous recombinant 7B2 to CHO cells expressing pro-PC2 prevented the unfolding and aggregation of secreted PC2 forms in a dose-dependent manner, as assessed by aggregation assays, activity assays, cross-linking experiments, and sucrose density gradients. Intracellular pro-PC2 was also found to exist in part as higher-order oligomers that were reduced in the presence of coexpressed 7B2. 7B2 addition did not result in the acquisition of enzymatic competence unless added before or very rapidly after pro-PC2 secretion, indicating that an activation-competent structure cannot be maintained in the absence of 7B2. Velocity sedimentation experiments showed that addition of extracellular 7B2 solubilized three different PC2 species from a precipitable aggregate: two activatable pro-PC2 species, the intact zymogen and a zymogen with a partially cleaved propeptide, and an inactive 66-kDa form. Our results suggest that 7B2 possesses chaperone activity that blocks partially unfolded pro-PC2 forms from losing catalytic competence and then aggregating. The loss of the catalytically competent conformer appears to represent the earliest indicator of pro-PC2 unfolding and is followed on a slower time scale by the appearance of aggregates. Because 7B2 expression is not confined to areas expressing pro-PC2, 7B2 may represent a general intracellular and extracellular secretory chaperone.

MOST SMALL PEPTIDES and proteins, including many peptide hormones and neuropeptide neurotransmitters, are initially synthesized as larger precursors (1). Prohormone convertases (PCs), proteases belonging to the eukaryotic subtilisin family, are responsible for the enzymatic maturation of these protein precursors (1,2). These enzymes are themselves synthesized as precursors; the propeptides of furin (3), PACE4 (4), PC1 (5,6), and PC5/6 (7) contain two cleavage sites and are processed at the primary cleavage site in the endoplasmic reticulum (ER). For all convertases except for pro-PC2, this process is a prerequisite for exit from the ER. In the more acidic trans-Golgi network (TGN)/secretory granule compartment, the cleaved (but still associated) propeptides are processed at an internal cleavage site, resulting in propeptide dissociation and release (8,9,10,11).

The propeptides of all proprotein convertases studied thus far have been found to act as intramolecular chaperones (IMC) essential to the correct folding of their cognate catalytic domains (12,13). IMC-mediated folding has been well studied in the bacterial serine endoproteases α-lytic protease and subtilisin (14,15). These IMC propeptides fold first and then catalyze folding of their cognate protease domains (13). Although the cleaved propeptide remains tightly bound to convertase, the propeptide primary site cleavage event is particularly important to convertase maturation, because blockade of this cleavage results in ER retention of furin (9) and PC1 (reviewed in Ref. 16). Primary-site cleavage apparently results in an alteration of conformation recognized by ER quality control mechanisms.

Unlike these other convertases, cleavage of the PC2 propeptide at the primary site occurs in the TGN/secretory granules (6,17). Pro-PC2 also differs in requiring a binding protein, 7B2, for production of an active enzyme species. Pro-PC2 forms released from CHO/PC2 cells not expressing 7B2 (CHO/PC2 cells) are neither active nor activatable (18), whereas coexpression of 7B2 in CHO/PC2 (CHO/PC2/7B2 cells) results in the secretion of activation-competent pro-PC2 (19) (reviewed in Ref. 16). Proteolytic maturation of 27-kDa 7B2 to its 21-kDa form is mediated by furin or a furin-like convertase within the TGN (20); this domain is sufficient for the production of activation-competent pro-PC2 (reviewed in Ref. 16). However, the mechanism for this effect is not yet understood. The autocatalytic activation of pro-PC2 does not appear to depend on the presence of 7B2 and occurs spontaneously when the pH is lowered to 5.0 (21).

We here demonstrate using chemical cross-linking and sedimentation velocity analysis coupled with activity assays that oligomerization and aggregation of pro-PC2 occur naturally, both intracellularly and extracellularly. We find that extracellular recombinant 7B2 has a postsynthesis chaperone-like activity in the prevention of pro-PC2 unfolding (as assessed by inability to activate) and on enzyme aggregation.

Materials and Methods

Cell culture

We have previously reported the construction of two dihydrofolate reductase-coupled amplified CHO cell lines, CHO/PC2 cells (18) and CHO/PC2/7B2 cells (19), which highly overexpress recombinant mouse pro-PC2. All cells were cultured at 37 C in 5% CO2. CHO/PC2 cells were grown in α-MEM (Invitrogen, Gaithersburg, MD) containing 10% dialyzed fetal bovine serum (Invitrogen, Gaithersburg, MD) and 50 μm methotrexate as described previously (18). CHO/PC2/7B2 cells were cultured in the same medium except for the appropriate selection agents, 50 μg/ml hygromycin and 800 μg/ml 50% active G418 (Invitrogen). For experimentation, CHO/PC2 and CHO/PC2/7B2 cells were plated into six-well plates with 1–3 × 106 cells per well. After 24 h, each well was washed with Opti-MEM and incubated with 1 ml Opti-MEM containing 100 μg/ml aprotinin in the presence or absence of recombinant 21-kDa rat 7B2 (22) for 16 h. Recombinant 7B2 proteins were prepared by prokaryotic expression as described previously (23). The DNAs coding for the deleted 7B2s were generated by PCR using the following primers: a common carboxyl-terminal primer (5′-GCGGCAAGCTTCTACTGTCCTCCCTTCATCTT-3′) and amino-terminal primers for the 7B230–150 construct, 5′-CGCGGATCCCCACGTGTGGAGTACCCA-3′, and for the 7B268–150 construct, 5′-GCCGGATCCATCGTGGCAGAGTTG-3′. The conditioned medium was collected and briefly centrifuged at low speed to remove any floating cells and then used for aggregation assays, chemical cross-linking, Western blotting, and the determination of PC2 activity.

In vitro aggregation assays

Conditioned medium samples were kept on ice for 30 min and then centrifuged for 15 min at 20,800 × g at 4 C. Similar proportions of the supernatant and the pellet were subjected to electrophoresis on nonreducing 4–20% SDS-PAGE gels (Bio-Rad, Hercules, CA), followed by Western blotting.

Chemical cross-linking experiments

For in vitro cross-linking with glutaraldehyde, 30 μl of each conditioned medium sample was treated with 0.05% glutaraldehyde for 30 min at room temperature and then quenched by the addition of 200 mm ethanolamine. After electrophoresis on nonreducing 4–20% SDS-PAGE gels (Bio-Rad), the cross-linked products were analyzed by Western blotting.

For in vivo cross-linking experiments, CHO/PC2 and CHO/PC2/7B2 cells were plated into six-well plates with 1–3 × 106 cells per well 1 d before labeling. The cells were washed twice with PBS and incubated with the reducible cross-linker dithiobis[sulfosuccinimidylpropionate] (DSP) (Pierce, Rockford, IL) at room temperature for 30 min. The cross-linking reaction was quenched with the addition of 20 mm Tris-HCl (pH 7.5) and incubation for 15 min. The cells were washed with PBS and resuspended in sample buffer without 2-mercaptoethanol and boiled for 5 min. Cell lysates were analyzed by Western blotting.

Sucrose gradients

Each conditioned medium sample of 100 μl was loaded on top of a 2.1-ml 10–40% (wt/vol) linear sucrose gradient in HEPES buffer [10 mm HEPES (pH 7.5), 150 mm NaCl, 2 mm CaCl2, 0.4 mm n-dodecyl-β-d-maltoside]. Gradients were centrifuged for 10 h at 54,000 rpm in a TLS-55 rotor at 4 C, and 150-μl fractions were collected from the top of the gradient. Pellets were resuspended in 40 μl 2× sample buffer. Aliquots of the fractions were analyzed using 4–20% SDS-PAGE gels, followed by Western blotting.

For radiolabeling immunoprecipitation experiments, cells were labeled with 0.5 mCi/ml [35S]methionine and cysteine (Met/Cys) Promix (Amersham Corp., Arlington Heights, IL) for 20 min and then lysed in 1 ml ice-cold RIPA buffer [50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate, and 1 mm EDTA] containing 10 μl 100 mm phenylmethylsulfonyl fluoride and 10 μl 10 mm parachloromercuriphenyl sulfonate. Samples were incubated for 5 min on ice and used for immunoprecipitation. Cell extracts were preincubated with 0.1 ml 20% protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden) hydrated, washed with RIPA buffer at 4 C for 1 h, and then centrifuged. Five microliters of antiserum against PC2 were then added to the supernatant, along with 0.5 mm phenylmethylsulfonyl fluoride and 0.5 mm parachloromercuriphenyl sulfonate. Samples were incubated overnight at 4 C with agitation. One hundred microliters of 20% protein A-Sepharose were then added and the samples rocked at 4 C for 1 h. The beads were washed five times with RIPA buffer. Immunoprecipitates were resuspended in sample buffer without 2-mercaptoethanol and analyzed using nonreducing 4–20% SDS-PAGE gels. The dried gels were exposed to PhosphorImager screens and analyzed with a Typhoon 9410 variable-mode imager and ImageQuant software (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The migration of labeled material was compared with that of unlabeled standard proteins analyzed on the same gel. The same samples were subjected to nonreducing SDS-PAGE (4–20%), and the gel slice containing labeled material migrating at the position of the positive signal exhibited on the screen was then excised. The gel slice was incubated in 2× sample buffer containing 5% β-mercaptoethanol at 37 C for 30 min, placed on reduced 4–20% SDS-PAGE, and analyzed with a Typhoon 9410 variable-mode imager.

Western blotting

The antiserum against PC2 (LSU18) was directed against a COOH-terminal peptide of mature mouse PC2 (18). The antiserum against the PC2 propeptide (LSU26) was raised against residues His58-Asp80 of mouse pro-PC2 (8). The antiserum against rat 7B2 (LSU13) was directed against residues 23–39 (22). Samples were subjected to electrophoresis on either reducing (8%) or nonreducing (4–20%) SDS-PAGE gels (Bio-Rad, Hercules, CA), followed by Western blotting using the appropriate antisera. Proteins were transferred from gels to nitrocellulose membranes, and the membranes were preincubated in 5% nonfat milk in Tris-buffered saline (TBS) for 30 min at room temperature before incubation overnight at 4 C with antiserum diluted 1:1000 in milk. Membranes were washed three times with TBS containing 0.05% Tween followed by incubation at room temperature for 1 h with secondary antibody (goat antirabbit IgG coupled to alkaline phosphatase). Membranes were then washed once with TBS containing 0.05% Tween and twice with TBS alone and then developed with 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt/p-nitroblue tetrazolium chloride.

Enzyme assays

The assay for PC2 was carried out in 96-well polypropylene microtiter plates using 5 or 25 μl of each conditioned medium sample in a total volume of 50 μl containing 200 μm fluorogenic substrate, pyr-Glu-Arg-Thr-Lys-Arg-methylcoumarinamide as a substrate and 100 mm sodium acetate (pH 5.0), 5 mm CaCl2, and 0.1% Brij 35 in the presence of a protease inhibitormixture composed of 1 μm pepstatin, 0.28 mm tosylphenylalanyl chloromethyl ketone (TPCK), 1 μm trans-epoxysuccinic acid (E-64), and 0.14 mm tosyllysyl chloromethyl ketone (TLCK). In some experiments, the activity was also measured in the presence of 1 μm 7B2 CT peptide, a specific inhibitor of PC2 activity (23,24). Released 7-amino-4-methylcoumarin was measured with a Fluoroscan Ascent fluorometer (LabSystems, Waltham, MA) using an excitation wavelength of 380 nm and an emission wavelength of 460 nm for 1 h at 37 C. Enzyme activity was measured in triplicate and is given in fluorescence units (FU) per minute in which one FU corresponds to 5.33 pmol methylcoumarinamide.

Results

Addition of recombinant 21-kDa 7B2 reverses pro-PC2/PC2 aggregation and restores enzymatic competence

Our previous attempts to purify PC2 forms from the conditioned medium of CHO/PC2 cells using anion-exchange chromatography showed that eluted PC2 proteins were distributed across the entire salt gradient, indicating that secreted PC2 species are highly heterogeneous by charge (data not shown). We first assessed the molecular weight pattern of PC2-containing species present in the overnight conditioned medium of CHO/PC2 cells (i.e. no added 7B2) using standard reducing SDS-PAGE. Western blotting of denatured medium reveals that these cells secrete three PC2 species with molecular masses of 66 kDa (no propeptide), 71 kDa (intermediate zymogen with a cleavage at an internal propeptide site), and 75 kDa (complete zymogen), respectively (Fig. 1A1A,, top panel); none are enzymatically active. The three potential cleavage sites in the mouse PC2 propeptide are shown in Fig. 1A1A (bottom panel).

Figure 1
Addition of recombinant 7B2 to CHO/PC2 cells facilitates the formation of soluble, activatable PC2 species. CHO/PC2 cells were cultured with the indicated concentrations of recombinant 7B2 for 16 h, and the conditioned medium was collected. Samples were ...

A considerable amount of the PC2 species in this medium was precipitable after centrifugation. We subjected aliquots of centrifuged medium and the corresponding pellets to denaturing but nonreducing gel analysis. Under these conditions, PC2 forms consisted mainly of large aggregates that could not enter gels as well as many other multimers (Fig. 1B1B).). These may represent mixed disulfide-bonded proteins, because they were stable to the addition of sodium dodecyl sulfate but not to β-mercaptoethanol (see Fig. 1A1A).

Prior addition of recombinant 7B2 to the overnight conditioned medium of CHO/PC2 cells changed the molecular mass pattern of the recovered PC2 species; 7B2 increased the amount of the low-molecular-weight PC2 form in the supernatant [with a relative molecular mass (Mr) consistent with a monomer] in a concentration-dependent manner (Fig. 1B1B,, 0.1–5 μm 7B2) and decreased the amount of this form in the pellet (Fig. 1B1B).). These data show that added 7B2 prevents PC2 forms from aggregating into precipitable forms.

To gain further information on PC2-containing complexes present in CHO/PC2 cell conditioned media in the presence and absence of 7B2, the formation of soluble oligomeric forms was examined after cross-linking with 0.05% glutaraldehyde. Western blots of cross-linked samples prepared from conditioned medium with no added 7B2 contained only minor amounts of monomeric PC2 with a molecular mass of about 70 kDa (Fig. 1C1C,, 0 μm 7B2). The majority of secreted PC2 species in cross-linked, non-7B2-containing medium was unable to enter the gel, indicating a high proportion of aggregated material. However, cross-linked samples prepared from conditioned medium with added 7B2 contained an increased proportion of lower-molecular-weight PC2 species and a reduced amount of aggregated protein at the top of the gel (Fig. 1C1C,, compare 0 and 5 μm 7B2). These cross-linking data support the idea that the presence of 7B2 reduces the amounts of cross-linkable, self-associated PC2 species.

Surprisingly, the addition of extracellular 7B2 also resulted in the robust production of active PC2 from otherwise totally inactive medium (Fig. 1D1D).). The acquisition of enzyme activity was strongly correlated with 7B2 concentration. We interpret this result to indicate that extracellular 7B2 blocks the unfolding events that result in enzymatic incompetence and/or assists in the correct refolding of partially unfolded pro-PC2 forms, or both; the end result is the formation of an activation-competent zymogen.

In summary, these results show that exogenously added 7B2 blocks the formation of aggregated PC2 forms and increases the amounts of soluble, smaller oligomeric complexes; this effect is associated with the ability of secreted pro-PC2 to retain enzymatic competence.

PC2 forms also exist in large aggregates within cells

Because pro-PC2 has been shown to aggregate in a pH-dependent manner (25,26), we wondered whether the aggregation events that we observed in the conditioned medium also occur intracellularly. We used cell-permeable cross-linking reagents coupled with Western blotting to determine whether intracellular PC2 forms are also present as multimeric forms. The majority of intracellular PC2-containing species in cross-linked cell extracts consisted of large aggregates and other oligomers with lower Mr (Fig. 2A2A).). The molecular masses of the total PC2 species within CHO/PC2/7B2 cells were somewhat different from those of CHO/PC2 cells (Fig. 2A2A,, Western blotting), most likely due to the formation of 7B2-containing complexes. Whereas Fig. 2A2A shows the steady state of PC2-containing species, Fig. 2B2B shows the molecular masses of newly synthesized 35S-labeled PC2-containing molecules. Most of these newly synthesized PC2 forms were present in high-molecular-mass multimers and large aggregates (over 500 kDa, Fig. 2B2B,, left panel). In addition to the large aggregates, 35S-labeled PC2 appeared as a strongly labeled band of smaller Mr in both cell lines (Fig. 2B2B).). The presence of 7B2 in this complex in the CHO/PC2/7B2 sample was experimentally confirmed by reducing the cross-linker with β-mercaptoethanol followed by electrophoresis on reducing SDS-PAGE. As expected, we observed a 7B2-sized product only in the samples obtained from CHO/PC2/7B2 cells (Fig. 2B2B,, right panel).

Figure 2
Intracellular PC2 exists as multimeric forms and as large aggregates. A, Both CHO/PC2 and CHO/PC2/7B2 cells were cross-linked with the reducible cross-linker DSP and immunoblotted with PC2 antiserum. B, Both CHO/PC2 and CHO/PC2/7B2 cells were labeled ...

We conclude from these data that a high proportion of the intracellular pro-PC2/PC2-containing species in CHO/PC2/7B2 cells can be assembled into higher-order complexes; if 7B2 is present, these complexes will contain 7B2.

7B2 must be added before or immediately after secretion of pro-PC2 for activatable species to be generated

Interestingly, when we added recombinant 7B2 to aliquots of conditioned medium collected from CHO/PC2 cell cultures, 7B2 addition (0–5 μm) had no effect on PC2 enzyme activity (Fig. 3A3A)) but still inhibited PC2 aggregation (Fig. 3B3B).). Because no activity could be rescued by this later addition of 7B2, it is clear that secreted pro-PC2/PC2 forms must very rapidly lose enzymatic competence in the absence of 7B2 and that 7B2-induced solubilization (Fig. 3B3B)) alone is not sufficient to restore enzymatic competence. These results highlight the extreme lability of the activation-competent form.

Figure 3
7B2 addition to the conditioned medium collected from CHO/PC2 cell cultures can prevent PC2 aggregation but does not render PC2 competent for activation. Conditioned medium was collected from CHO/PC2 cells, and recombinant 7B2 was added to the medium ...

If the ability of exogenous 7B2 to confer enzymatic competence to released PC2 species is labile, we would expect that increased enzyme activity would be strongly correlated with the time that secreted pro-PC2 spends in the presence of 7B2. We therefore cultured CHO/PC2 cells in a six-well plate overnight and then added recombinant 7B2 to each well at different time points (at 2-h intervals) during the succeeding 10 h. Indeed, although the total secretion of PC2 in all wells after 10 h in culture was identical (results not shown), PC2 activity increased in direct proportion to the time in culture spent in the presence of 7B2 (results not shown).

To provide more direct evidence for the requirement of immediate interaction of pro-PC2 with 7B2 to prevent a presumed unfolding reaction, we performed a time-dependent activity assay with added 7B2 (Fig. 44).). We cultured CHO/PC2 cells in two 12-well plates overnight; the next day, the medium in one plate was substituted with medium containing recombinant 7B2, whereas medium without 7B2 was added to the other plate. Conditioned medium was collected from each well at different time points. For samples from the plate lacking 7B2, recombinant 7B2 was added to each sample immediately after collection so that final concentrations of 7B2 in all samples were equal; however, in the one case, the secreted PC2 species were continuously exposed to 7B2, and in the other, 2–60 min elapsed before the secreted PC2 was exposed to 7B2. We then measured enzymatic activity (corresponding to total PC2-containing forms secreted at each time point); the amount of PC2 secreted in both conditions was identical.

Figure 4
Secreted PC2 activity is extremely unstable in the absence of immediate interaction with 7B2. CHO/PC2 cells were cultured in two 12-well plates overnight. One plate was changed to medium containing recombinant 7B2 (1 μm), and the conditioned medium ...

We observed that all medium samples cleaved substrate at a similar rate during the first 5 min or so. After that first time period, only media samples with initially added 7B2 exhibited increased PC2 activity in a time-dependent manner (Fig. 44,, [filled triangle]); after 5 min without 7B2, increased activity was not present in samples with delayed addition of 7B2, showing that in vitro rescue of PC2 activity with 7B2 was ineffective after this time (Fig. 44,, •). Our interpretation of these data is that in the absence of immediate interaction with extracellular 7B2, released pro-PC2 undergoes an irreversible conformational change that results in loss of catalytic competence. Therefore, PC2 forms secreted at time points after 5 min are not able to contribute to the final activity, leading to the observed plateau.

Extracellular 7B2 increases the proportion of PC2-containing species that migrate as monomers and dimers: sucrose density gradient centrifugation

As described above, most of the secreted pro-PC2/PC2 species in conditioned medium exist as large aggregates, but the addition of extracellular 7B2 increases the quantity of soluble forms. To furnish detail on the apparent sizes of the complexes in the presence or absence of 7B2, we analyzed the conditioned medium obtained from CHO/PC2 cells using velocity centrifugation on 10–40% sucrose gradients. Parallel gradients containing BSA monomer and dimers (66 and 132 kDa) or urease monomers and dimers (272 and 545 kDa) were used as molecular standards.

In conditioned medium obtained from CHO/PC2 cells with no added 7B2, the majority of the 66-kDa PC2 was detected in the pellet and in fractions 6 and 7, which contain proteins of Mr from 60–70 kDa (Fig. 5A5A),), suggesting that secreted 66-kDa is present both as a monomer and in precipitable aggregates. A trace amount of the intermediate zymogen (71 kDa) was detected in fractions 6 and 7, but the 71-kDa form was not present in the pellet (Fig. 5A5A),), indicating that this PC2 species does not exhibit a propensity to aggregate. By contrast, the majority of the intact 75-kDa zymogen was located in the pellet, supporting the idea that secreted pro-PC2 is mostly highly aggregated (Fig. 5A5A).

Figure 5
7B2 addition to PC2-expressing cells increases the amounts of both soluble zymogen and intermediate PC2 forms: velocity sedimentation and cross-linking studies. Conditioned medium obtained from CHO/PC2 cells with either no added 7B2 (A) or added 7B2 (B–D) ...

In contrast, when exogenous 7B2 was added to CHO/PC2 cells at 1–5 μm concentrations, increased amounts of monomeric 66-kDa PC2 were detected in all lower Mr-containing fractions, whereas the proportion of 66-kDa PC2 in the pellet was reduced (Fig. 55,, C and D); these data indicate that exogenous 7B2 is able to solubilize large 66-kDa PC2-containing aggregates into smaller forms (although these were enzymatically inactive; see below). Significantly increased amounts of both soluble zymogen and intermediate forms were detected in fractions 6–10, with Mr corresponding to potential monomer, dimer, and trimer forms (Fig. 55,, B–D).

Composition of higher-order pro-PC2–7B2 complexes in the medium

To gain information on the molecular composition of pro-PC2 complexes, we performed gel filtration of recombinant PC2 forms partially purified from conditioned medium; after enzymatic assay, we cross-linked fractions with glutaraldehyde and performed Western blotting using 7B2 and PC2 antisera (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). These data show the presence of multiple oligomeric forms, most of which also contain 7B2. These data were confirmed by velocity sedimentation and Western blotting of cross-linked conditioned medium (not shown).

7B2 addition protects pro-PC2 from cleavage at the primary site

Although within CHO/PC2 cells the majority of intracellular PC2 is present as the proform (Fig. 2A2A),), secreted PC2 species consisted largely of the two zymogen forms together with the 66-kDa processed form (Fig. 5A5A).). These data indicate that processing of two zymogen forms to the 66-kDa form is likely to occur extracellularly, possibly by extraneous proteases on the cell membrane or in the overnight-conditioned culture medium. (This event can be distinguished from autocatalytic propeptide cleavage, which requires an acidic pH.) Adding 7B2 to the medium increased the amounts of both soluble zymogen forms (Fig. 55,, B–D). The pronounced increase in the amounts of intact and intermediate zymogen forms in the presence of 7B2 indicates that binding of exogenous 7B2 protects the primary cleavage site from extraneous cleavage, potentially by preventing unfolding and propeptide exposure to solvent.

Addition of recombinant 7B2 to cells already expressing 7B2 (CHO/PC2/7B2 cells) increases both the amount of soluble PC2 forms as well as enzyme activity

CHO/PC2/7B2 cells already express large quantities of active PC2 and lesser quantities of 7B2 (due to the method of construction of the cells; PC2 was expressed using the powerful dihydrofolate reductase-coupled amplification method, whereas 7B2 expression was coupled only to two antibiotic resistance plasmids). If 7B2 expression is limiting for the expression of PC2 activity in a postsynthesis manner, the addition of recombinant 7B2 should also increase the quantity of active PC2 forms secreted from CHO/PC2/7B2 cells. This was indeed found to be the case (Fig. 6A6A).). Western blot analysis using cross-linked medium with extracellular 7B2 showed considerably increased quantities of soluble PC2 forms with molecular masses from 70–400 kDa, corresponding to mono-, di-, tri-, and higher oligomeric forms (Fig. 6B6B).). Comparison of velocity centrifugation experiments using the conditioned medium with added 7B2 and medium without 7B2 (Fig. 7A7A)) shows that 7B2 addition resulted in highly increased amounts of both soluble pro-PC2 and the intermediate form and that this was associated with significantly increased enzyme activity (~4-fold; Figs. 6B6B and 7B7B).

Figure 6
7B2 addition to 7B2-expressing CHO/PC2 cells further increases the amount of soluble PC2 species and the amount of PC2 activity: cross-linking studies. CHO/PC2/7B2 cells were cultured with the indicated concentration of 7B2 for 16 h, and the conditioned ...
Figure 7
Addition of 7B2 to PC2- and 7B2-expressing cells results in increased amounts of both soluble pro-PC2 and the intermediate zymogen form: velocity sedimentation. The conditioned medium collected from CHO/PC2/7B2 cells with either no added 7B2 (A) or added ...

We conclude that even in CHO/PC2/7B2 cells, a large portion of the PC2 species is released without achieving intracellular interaction with 7B2, resulting in an enzymatically incompetent, potentially unfolded form; however, this particular unfolding event must be reversible, because the zymogen can still be rendered competent for activation by exogenously added 7B2. As with cells not expressing any 7B2, addition of recombinant protein solubilized PC2 species from precipitable aggregates.

Structure-function analysis of 7B2 effects on aggregation

We next performed structure-function studies to test the role of the amino-terminal segments of 7B2 in the prevention of PC2/pro-PC2 aggregation. We constructed two amino-terminally deleted 7B2 mutants, 7B230–150 and 7B268–150, to identify the determinant within 7B2 that inhibits pro-PC2 aggregation (Fig. 8A8A).). As found previously (27), the conditioned medium obtained from CHO/PC2 cells with added recombinant 7B268–150 or 7B230–150 protein showed a concentration-dependent increase in PC2 activity (Fig. 8B8B).). In addition, two truncated 7B2 proteins were able to inhibit PC2/pro-PC2 aggregation; however, the anti-aggregant activity of 7B268–150 was not as efficient as that of recombinant 21-kDa 7B2 and 7B230–150 (Fig. 8C8C).). We conclude that 7B2 segments containing the 36-residue helix previously shown to be important in the acquisition of enzyme activity by pro-PC2 (27) are also effective in our extracellular 7B2 activation system.

Figure 8
7B2 structure-function analysis: prevention of pro-PC2 aggregation. A, Schematic representation of 7B2 amino-terminal deletions. The gray box corresponds to the major determinant of the binding and activation of pro-PC2 in vivo (27). The first residue ...

7B2 addition to SK-N-MC cells also results in the acquisition of PC2 activity in the conditioned medium

The human neuroblastoma cell line SK-N-MC expresses PC2 but not 7B2 (28); peptide precursors are not processed in this cell line (29), indicating that SK-N-MC cell-expressed PC2 forms are enzymatically incompetent. We tested whether exogenous 7B2 addition could also result in the acquisition of PC2 enzyme activity in SK-N-MC cells; this was indeed found to be the case (Fig. 99).). The activity was identified as PC2 by virtue of its complete inhibition with the 7B2 CT peptide (23,24). We conclude that the ability of 7B2 to act as an extracellular chaperone for pro-PC2 is not a consequence of pro-PC2 overexpression.

Figure 9
Addition of recombinant 7B2 to the neuroblastoma cell line SK-N-MC results in the acquisition of PC2 activity in the conditioned medium. SK-N-MC cells were cultured with the indicated concentrations of 7B2 for 16 h, and the conditioned medium was collected ...

Discussion

The observation that 7B2 is a required binding protein for the manifestation of PC2 activity is now 13 yr old and has been confirmed by several groups (22,28,30), but the mechanism of this interaction is still not understood. Although we initially believed that 7B2 would be a required protein during the autocatalytic activation process, our previous data showing that 7B2 can be removed by antibody affinity chromatography from purified recombinant pro-PC2 without effect on enzyme activity (21) indicated that this was not a likely mechanism. We similarly ruled out an effect of 7B2 during initial folding of pro-PC2 (12). Although we and others have obtained structure-function information on the 7B2/pro-PC2 interaction both for 7B2 as well as for pro-PC2 (reviewed in Ref. 16), exactly how 7B2 is able to facilitate the acquisition of activation-competent forms of pro-PC2 has been enigmatic. We have here approached this question by examining effects of extracellular 7B2 on the structure, oligomerization, and enzymatic activity of secreted pro-PC2.

7B2 can act as an extracellular chaperone

In vitro cross-linking and velocity sedimentation experiments both demonstrate that majority of the PC2 species released from CHO/PC2 cells is highly aggregated. However, in the presence of extracellular 7B2, large amounts of the pro-PC2 aggregates become solubilized, forming mono-, di, tri, and smaller oligomers, and this effect is clearly proportional to the 7B2 concentration. Exposed hydrophobic surfaces, presumably due to partial unfolding, may generate the phenomenon of aggregation and precipitation of PC2-containing complexes; we speculate that 7B2 binds to these hydrophobic surfaces. Molecular chaperones are known to interact with unstructured, aggregation-prone folding intermediates that expose a significant amount of hydrophobic surface to solvent, thereby preventing an irreversible pathway toward misfolding and aggregation (31). The cytoplasmic small heat-shock proteins (32) and clusterin, an extracellular mammalian chaperone protein (33), are found in a variety of disease states and in stress conditions. These proteins preferentially recognize hydrophobic sequences of partly folded protein intermediates that are slowly aggregating and inhibit stress-induced precipitation of many different proteins in an ATP-independent manner (34,35). We have previously shown that the N-terminal 21-kDa domain of 7B2 interacts in part with hydrophobic residues in the PC2 catalytic domain in a surface-exposed loop containing Tyr-194 (36). Based on the pro-PC1-furin chimera structure (37), this loop is adjacent to a surface patch of the catalytic domain that is involved in the binding of a propeptide hydrophobic patch, implying that the N-terminal domain of 7B2 may also interact with the propeptide (37). Indeed, early immunoprecipitation experiments showed that 7B2 does bind the pro-PC2 propeptide (38). We speculate that 7B2-mediated inhibition of PC2 aggregation results from binding of 7B2 to a large hydrophobic surface of pro-PC2 that includes both this Tyr-194 domain and the propeptide sequence. Co-crystallography of 7B2 with pro-PC2 will be required to definitively answer the question of where 7B2 binds pro-PC2; such experiments are ongoing.

Exogenous 7B2 protects pro-PC2 from extraneous processing at the primary cleavage site

Within the CHO/PC2 cell, PC2 species are largely present as intact zymogen, whereas secreted PC2 species consist mainly of soluble and insoluble 66-kDa forms together with a large amount of insoluble (precipitable) zymogen and a small amount of soluble intermediate zymogen. In the absence of 7B2, these two secreted zymogen forms could not undergo autocatalytic processing even at a pH low enough to autoactivate pro-PC2 in vitro, demonstrating their catalytic incompetence. However, the medium still contained significant quantities of inactive, cleaved 66-kDa PC2. These results strongly suggest that these zymogen forms are not properly folded and that the primary cleavage site of this secreted pro-PC2 is exposed to solvent, possibly due to partial unfolding. Cleavage by extraneous proteases may then result; although the end result is a protein with the molecular mass of mature PC2, this unfolded enzyme is irreversibly inactivated. The fact that recombinant 7B2 added to CHO/PC2 cells increased the amounts of both soluble intact and intermediate zymogens demonstrates that extracellular 7B2 can protect pro-PC2 from this primary-site cleavage event, possibly by blocking the initial unfolding event and thereby stabilizing the activation-competent zymogen conformer. We have previously reported that 7B2 can stabilize the enzyme activity of recombinant PC2 during heat denaturation (19), and others have reported that 7B2 addition increases the cleavage of proopiomelanocortin by released PC2 (39). The same protective effect is observed when conditioned medium from CHO/PC2 is heated; binding of 7B2 protects secreted forms from heat denaturation (Lee, S. N., and I. Lindberg, results not shown). Thus, in several different systems, 7B2 has been shown to protect PC2 species from unfolding events, supporting the notion that it represents an authentic postsynthesis chaperone.

Unlike the primary site, internal cleavage of the PC2 propeptide (see Fig. 1A1A)) was not protected by 7B2 addition, suggesting that this site is accessible to proteases whether or not 7B2 is present. In recent experiments, we have observed that the PC2 propeptide can be cleaved at the internal cleavage site by furin in vitro (Lee, S. N., and I. Lindberg, unpublished results), indicating that unlike the furin propeptide (40), the internal cleavage site of the PC2 propeptide is always solvent accessible.

In summary, these data support the idea that 7B2 interacts with residues in the catalytic and propeptide domains flanking the primary site, resulting in protection from primary site (but not internal site) processing, conferring enhanced stability to unfolding events, and preventing enzyme aggregation leading to precipitation.

Pro-PC2 aggregation

Oligomerization and aggregation of pro-PC2 are not confined to the extracellular medium. Our work here has clearly shown that intracellular forms of pro-PC2 are present as higher-order oligomers; other groups have also shown that aggregation and membrane association of pro-PC2 occur in the TGN/immature secretory granules (26,41). In AtT-20/PC2 cells, late secretory compartment pro-PC2 aggregates were shown to interact with lipid rafts via either the PC2 propeptide (41) or the PC2 C terminus (26). This was postulated to lead to release of soluble mature PC2 from the membrane at the neutral pH of the external environment (41). However, the data presented here clearly show that released PC2 forms are also highly aggregated. Thus, aggregation appears to occur both at the acidic pH within neuroendocrine cells as well as at the neutral pH of culture medium.

7B2 binding prevents both enzyme unfolding and aggregation

Figure 1010 depicts our current hypothesis as to the mechanism of action of 7B2. We postulate that within the cell, pro-PC2 forms already exist as higher-order oligomers; released pro-PC2 appears to be so unstable in the absence of 7B2 that an irreversible unfolding event occurs that is manifested first by the loss of activation competence and, on a longer time scale, further oligomerization of the zymogen, leading to irreversible protein aggregation into insoluble precipitates (path A). If 7B2 is added later, i.e. after an initial rapid irreversible unfolding event, precipitation is prevented, but the zymogen cannot refold to recover activation competence (path B). Finally, if pro-PC2 immediately encounters 7B2 after secretion the zymogen will be stabilized and will be activation competent (path C).

Figure 10
Schematic representation of a possible mechanism for the chaperone-like activity of extracellular 7B2 in CHO/PC2 cells. Partially unfolded PC2 aggregates released from CHO/PC2 cells become slowly further aggregated (A). Exogenous 7B2 blocks further aggregation ...

In summary, we have shown using a postsynthesis secretory system that 7B2 can reverse and prevent the occurrence of pro-PC2 aggregation. Although rapid exposure of pro-PC2 to 7B2 restores enzymatic competence to a presumably partially unfolded form, later exposure to 7B2 does not. These postsynthesis chaperone-like activities of 7B2 are likely to occur via binding to an exposed hydrophobic surface of pro-PC2 molecules, which appear to have a natural tendency to aggregate. Although the most sensitive indicator of 7B2 efficacy on PC2 folding events is the prevention of the loss of activation competence, the effects of this small neuroendocrine protein on enzyme aggregation are quantitatively quite remarkable. In fact, there is historical precedent for an antiaggregant activity of 7B2; 7B2 has been reported to assist in the formation of correctly folded active, monomeric human IGF-I via dissolution of incorrectly folded, aggregated multimers in vitro (42). Because 7B2 exhibits a pan-neuronal and endocrine distribution much broader than that of PC2, we speculate that 7B2’s antiaggregant action may apply to other as yet unknown neural and endocrine substrates.

Supplementary Material

[Supplemental Data]

Footnotes

This work was supported by National Institutes of Health Grant DK049703 (to I.L.).

Present address for S.-N.L.: The Airway Mucus Institute, Yonsei University College of Medicine, Seoul, Korea 120-752.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 8, 2008

Abbreviations: DSP, Dithiobis[sulfosuccinimidylpropionate]; ER, endoplasmic reticulum; IMC, intramolecular chaperone; Mr, relative molecular mass; PC, prohormone convertase; TBS, Tris-buffered saline; TGN, trans-Golgi network.

References

  • Rouille Y, Duguay SJ, Lund K, Furuta M, Gong Q, Lipkind G, Oliva AAJ, Chan SJ, Steiner DF 1995 Proteolytic processing mechanisms in the biosynthesis of neuroendocrine peptides: the subtilisin-like proprotein convertases. Front Neuroendocrinol 16:322–361 [PubMed]
  • Seidah NG, Chretien M 1997 Eukaryotic protein processing: endoproteolysis of precursor proteins. Curr Opin Biotechnol 8:602–607 [PubMed]
  • Creemers JW, Vey M, Schafer W, Ayoubi TA, Roebroek AJ, Klenk HD, Garten W, Van de Ven WJ 1995 Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum. J Biol Chem 270:2695–2702 [PubMed]
  • Mains RE, Berard CA, Denault JB, Zhou A, Johnson RC, Leduc R 1997 PACE4: a subtilisin-like endoprotease with unique properties. Biochem J 321(Pt 3):587–593 [PMC free article] [PubMed]
  • Lindberg I 1994 Evidence for cleavage of the PC1/PC3 pro-segment in the endoplasmic reticulum. Mol Cell Neurosci 5:263–268 [PubMed]
  • Zhou A, Mains RE 1994 Endoproteolytic processing of proopiomelanocortin and prohormone convertases 1 and 2 in neuroendocrine cells overexpressing prohormone convertases 1 or 2. J Biol Chem 269:17440–17447 [PubMed]
  • De Bie I, Marcinkiewicz M, Malide D, Lazure C, Nakayama K, Bendayan M, Seidah NG 1996 The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments. J Cell Biol 135:1261–1275 [PMC free article] [PubMed]
  • Muller L, Cameron A, Fortenberry Y, Apletalina EV, Lindberg I 2000 Processing and sorting of the prohormone convertase 2 propeptide. J Biol Chem 275:39213–39222 [PubMed]
  • Anderson ED, Molloy SS, Jean F, Fei H, Shimamura S, Thomas G 2002 The ordered and compartment-specific autoproteolytic removal of the furin intramolecular chaperone is required for enzyme activation. J Biol Chem 277:12879–12890 [PMC free article] [PubMed]
  • Molloy SS, Thomas L, VanSlyke JK, Stenberg PE, Thomas G 1994 Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. EMBO J 13:18–33 [PMC free article] [PubMed]
  • Vey M, Schafer W, Berghofer S, Klenk HD, Garten W 1994 Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation. J Cell Biol 127:1829–1842 [PMC free article] [PubMed]
  • Muller L, Zhu X, Lindberg I 1997 Mechanism of the facilitation of PC2 maturation by 7B2: involvement in Pro-PC2 transport and activation but not folding. J Cell Biol 139:625–638 [PMC free article] [PubMed]
  • Shinde U, Fu X, Inouye M 1999 A pathway for conformational diversity in proteins mediated by intramolecular chaperones. J Biol Chem 274:15615–15621 [PubMed]
  • Zhu X, Ohta Y, Jordan F, Inouye M 1989 Prosequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature 339:483–484 [PubMed]
  • Eder J, Rheinnecker M, Fersht AR 1993 Folding of subtilisin BPN′: characterization of a folding intermediate. Biochemistry 32:18–26 [PubMed]
  • Muller L, Lindberg I 1999 The cell biology of the prohormone convertases PC1 and PC2. Prog Nucleic Acids Res 63:69–108 [PubMed]
  • Benjannet S, Rondeau N, Paquet L, Boudreault A, Lazure C, Chretien M, Seidah NG 1993 Comparative biosynthesis, covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2. Biochem J 294(Pt 3):735–743 [PMC free article] [PubMed]
  • Shen FS, Seidah NG, Lindberg I 1993 Biosynthesis of the prohormone convertase PC2 in Chinese hamster ovary cells and in rat insulinoma cells. J Biol Chem 268:24910–24915 [PubMed]
  • Lamango NS, Zhu X, Lindberg I 1996 Purification and enzymatic characterization of recombinant prohormone convertase 2: stabilization of activity by 21 kDa 7B2. Arch Biochem Biophys 330:238–250 [PubMed]
  • Paquet L, Bergeron F, Boudreault A, Seidah NG, Chretien M, Mbikay M, Lazure C 1994 The neuroendocrine precursor 7B2 is a sulfated protein proteolytically processed by a ubiquitous furin-like convertase. J Biol Chem 269:19279–19285 [PubMed]
  • Lamango NS, Apletalina E, Liu J, Lindberg I 1999 The proteolytic maturation of prohormone convertase 2 (PC2) is a pH-driven process. Arch Biochem Biophys 362:275–282 [PubMed]
  • Zhu X, Lindberg I 1995 7B2 facilitates the maturation of pro-PC2 in neuroendocrine cells and is required for the expression of enzymatic activity. J Cell Biol 129:1641–1650 [PMC free article] [PubMed]
  • Martens GJ, Braks JA, Eib DW, Zhou Y, Lindberg I 1994 The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2. Proc Natl Acad Sci USA 91:5784–5787 [PMC free article] [PubMed]
  • Lindberg I, van den Hurk WH, Bui C, Batie CJ 1995 Enzymatic characterization of immunopurified prohormone convertase 2: potent inhibition by a 7B2 peptide fragment. Biochemistry 34:5486–5493 [PubMed]
  • Shennan KIJ, Taylor NA, Docherty K 1994 Calcium- and pH-dependent aggregation and membrane association of the precursor of the prohormone convertase PC2. J Biol Chem 269:18646–18650 [PubMed]
  • Assadi M, Sharpe JC, Snell C, Loh YP 2004 The C-terminus of prohormone convertase 2 is sufficient and necessary for raft association and sorting to the regulated secretory pathway. Biochemistry 43:7798–7807 [PubMed]
  • Muller L, Zhu P, Juliano MA, Juliano L, Lindberg I 1999 A 36-residue peptide contains all of the information required for 7B2-mediated activation of prohormone convertase 2. J Biol Chem 274:21471–21477 [PubMed]
  • Seidel B, Dong W, Savaria D, Zheng M, Pintar JE, Day R 1998 Neuroendocrine protein 7B2 is essential for proteolytic conversion and activation of proprotein convertase 2 in vivo. DNA Cell Biol 17:1017–1029 [PubMed]
  • Lindberg I, Shaw E 1992 Posttranslational processing of proenkephalin in SK-N-MC cells: evidence for phosphorylation. J Neurochem 58:448–453 [PubMed]
  • Benjannet S, Mamarbachi AM, Hamelin J, Savaria D, Munzer JS, Chretien M, Seidah NS 1998 Residues unique to the prohormone convertase PC2 modulate its autoactivation, binding to 7B2, and enzymatic activity. FEBS Lett 428:37–42 [PubMed]
  • Fink AL 1999 Chaperone-mediated protein folding. Physiol Rev 79:425–449 [PubMed]
  • Haslbeck M, Walke S, Stromer T, Ehrnsperger M, White HE, Chen S, Saibil HR, Buchner J 1999 Hsp26: a temperature-regulated chaperone. EMBO J 18:6744–6751 [PMC free article] [PubMed]
  • Poon S, Rybchyn MS, Easterbrook-Smith SB, Carver JA, Pankhurst GJ, Wilson MR 2002 Mildly acidic pH activates the extracellular molecular chaperone clusterin. J Biol Chem 277:39532–39540 [PubMed]
  • Jakob U, Gaestel M, Engel K, Buchner J 1993 Small heat shock proteins are molecular chaperones. J Biol Chem 268:1517–1520 [PubMed]
  • Poon S, Easterbrook-Smith SB, Rybchyn MS, Carver JA, Wilson MR 2000 Clusterin is an ATP-independent chaperone with very broad substrate specificity that stabilizes stressed proteins in a folding-competent state. Biochemistry 39:15953–15960 [PubMed]
  • Zhu X, Muller L, Mains RE, Lindberg I 1998 Structural elements of PC2 required for interaction with its helper protein 7B2. J Biol Chem 273: 1158–1164 [PubMed]
  • Henrich S, Lindberg I, Bode W, Than ME 2005 Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity. J Mol Biol 345:211–227 [PubMed]
  • Benjannet S, Savaria D, Chretien M, Seidah NG 1995 7B2 is a specific intracellular binding protein of the prohormone convertase PC2. J Neurochem 64:2303–2311 [PubMed]
  • Braks JAM, Martens GJM 1995 The neuroendocrine chaperone 7B2 can enhance in vitro POMC cleavage by prohormone convertase PC2. FEBS Lett 371:154–158 [PubMed]
  • Bhattacharjya S, Xu P, Xiang H, Chretien M, Seidah NG, Ni F 2001 pH-induced conformational transitions of a molten-globule-like state of the inhibitory prodomain of furin: implications for zymogen activation. Protein Sci 10:934–942 [PMC free article] [PubMed]
  • Blazquez M, Thiele C, Huttner WB, Docherty K, Shennan KI 2000 Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway. Biochem J 349:843–852 [PMC free article] [PubMed]
  • Chaudhuri B, Stephen C, Huijbregts RP, Martens GJ 1995 The neuroendocrine protein 7B2 acts as a molecular chaperone in the in vitro folding of human insulin-like growth factor-1 secreted from yeast. Biochem Biophys Res Commun 211:417–425 [PubMed]
  • Tangrea MA, Bryan PN, Sari N, Orban J 2002 Solution structure of the pro-hormone convertase 1 pro-domain from Mus musculus. J Mol Biol 320:801–812 [PubMed]

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