7B2 Prevents Unfolding and Aggregation of ProhormoneConvertase 2
Sang-Nam Lee and Iris Lindberg
Department of Biochemistry and Molecular Biology (S.-N.L., I.L.), Louisiana State University Health SciencesCenter/Research Institute for Children, New Orleans, Louisiana 70118; and Department of Anatomy and Neurobiology(I.L.), University of Maryland Medical School, Baltimore, Maryland 21201
Prohormone convertase 2 (PC2) requires interaction with theneuroendocrine protein 7B2 for the production of an activat-able zymogen; the mechanism for this effect is unknown. 7B2could act proactively to generate an activation-competentform of pro-PC2 during synthesis, or block spontaneous gen-eration of activation-incompetent forms. We here demon-strate that addition of exogenous recombinant 7B2 to CHOcells expressing pro-PC2 prevented the unfolding and aggre-gation of secreted PC2 forms in a dose-dependent manner,as assessed by aggregation assays, activity assays, cross-linking experiments, and sucrose density gradients. Intra-cellular pro-PC2 was also found to exist in part as higher-order oligomers that were reduced in the presence ofcoexpressed 7B2. 7B2 addition did not result in the acqui-sition of enzymatic competence unless added before or veryrapidly after pro-PC2 secretion, indicating that an activa-
tion-competent structure cannot be maintained in the ab-sence of 7B2. Velocity sedimentation experiments showedthat addition of extracellular 7B2 solubilized three differ-ent PC2 species from a precipitable aggregate: two activat-able pro-PC2 species, the intact zymogen and a zymogenwith a partially cleaved propeptide, and an inactive 66-kDaform. Our results suggest that 7B2 possesses chaperone ac-tivity that blocks partially unfolded pro-PC2 forms fromlosing catalytic competence and then aggregating. The lossof the catalytically competent conformer appears to repre-sent the earliest indicator of pro-PC2 unfolding and is fol-lowed on a slower time scale by the appearance of aggre-gates. Because 7B2 expression is not confined to areasexpressing pro-PC2, 7B2 may represent a general intracel-lular and extracellular secretory chaperone. (Endocrinology149: 4116–4127, 2008)
MOST SMALL PEPTIDES and proteins, including manypeptide hormones and neuropeptide neurotransmit-
ters, are initially synthesized as larger precursors (1). Pro-hormone convertases (PCs), proteases belonging to the eu-karyotic subtilisin family, are responsible for the enzymaticmaturation of these protein precursors (1, 2). These enzymesare themselves synthesized as precursors; the propeptides offurin (3), PACE4 (4), PC1 (5, 6), and PC5/6 (7) contain twocleavage sites and are processed at the primary cleavage sitein the endoplasmic reticulum (ER). For all convertases exceptfor pro-PC2, this process is a prerequisite for exit from the ER.In the more acidic trans-Golgi network (TGN)/secretorygranule compartment, the cleaved (but still associated)propeptides are processed at an internal cleavage site, re-sulting in propeptide dissociation and release (8–11).
The propeptides of all proprotein convertases studied thusfar have been found to act as intramolecular chaperones(IMC) essential to the correct folding of their cognate catalyticdomains (12, 13). IMC-mediated folding has been well stud-ied in the bacterial serine endoproteases �-lytic protease andsubtilisin (14, 15). These IMC propeptides fold first and thencatalyze folding of their cognate protease domains (13). Al-
though the cleaved propeptide remains tightly bound toconvertase, the propeptide primary site cleavage event isparticularly important to convertase maturation, becauseblockade of this cleavage results in ER retention of furin (9)and PC1 (reviewed in Ref. 16). Primary-site cleavage appar-ently results in an alteration of conformation recognized byER quality control mechanisms.
Unlike these other convertases, cleavage of the PC2propeptide at the primary site occurs in the TGN/secretorygranules (6, 17). Pro-PC2 also differs in requiring a bindingprotein, 7B2, for production of an active enzyme species.Pro-PC2 forms released from CHO/PC2 cells not expressing7B2 (CHO/PC2 cells) are neither active nor activatable (18),whereas coexpression of 7B2 in CHO/PC2 (CHO/PC2/7B2cells) results in the secretion of activation-competent pro-PC2(19) (reviewed in Ref. 16). Proteolytic maturation of 27-kDa7B2 to its 21-kDa form is mediated by furin or a furin-likeconvertase within the TGN (20); this domain is sufficient forthe production of activation-competent pro-PC2 (reviewedin Ref. 16). However, the mechanism for this effect is not yetunderstood. The autocatalytic activation of pro-PC2 does notappear to depend on the presence of 7B2 and occurs spon-taneously when the pH is lowered to 5.0 (21).
We here demonstrate using chemical cross-linking andsedimentation velocity analysis coupled with activity assaysthat oligomerization and aggregation of pro-PC2 occur nat-urally, both intracellularly and extracellularly. We find thatextracellular recombinant 7B2 has a postsynthesis chaper-one-like activity in the prevention of pro-PC2 unfolding (asassessed by inability to activate) and on enzyme aggregation.
First Published Online May 8, 2008Abbreviations: DSP, Dithiobis[sulfosuccinimidylpropionate]; ER,
endoplasmic reticulum; IMC, intramolecular chaperone; Mr, relativemolecular mass; PC, prohormone convertase; TBS, Tris-buffered saline;TGN, trans-Golgi network.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.
We have previously reported the construction of two dihydrofolatereductase-coupled amplified CHO cell lines, CHO/PC2 cells (18) andCHO/PC2/7B2 cells (19), which highly overexpress recombinant mousepro-PC2. All cells were cultured at 37 C in 5% CO2. CHO/PC2 cells weregrown in �-MEM (Invitrogen, Gaithersburg, MD) containing 10% dia-lyzed fetal bovine serum (Invitrogen, Gaithersburg, MD) and 50 �mmethotrexate as described previously (18). CHO/PC2/7B2 cells werecultured in the same medium except for the appropriate selection agents,50 �g/ml hygromycin and 800 �g/ml 50% active G418 (Invitrogen). Forexperimentation, CHO/PC2 and CHO/PC2/7B2 cells were plated intosix-well plates with 1–3 � 106 cells per well. After 24 h, each well waswashed with Opti-MEM and incubated with 1 ml Opti-MEM containing100 �g/ml aprotinin in the presence or absence of recombinant 21-kDarat 7B2 (22) for 16 h. Recombinant 7B2 proteins were prepared byprokaryotic expression as described previously (23). The DNAs codingfor the deleted 7B2s were generated by PCR using the following primers:a common carboxyl-terminal primer (5�-GCGGCAAGCTTCTACTGTC-CTCCCTTCATCTT-3�) and amino-terminal primers for the 7B230–150
construct, 5�-CGCGGATCCCCACGTGTGGAGTACCCA-3�, and for the7B268–150 construct, 5�-GCCGGATCCATCGTGGCAGAGTTG-3�. Theconditioned medium was collected and briefly centrifuged at low speedto remove any floating cells and then used for aggregation assays,chemical cross-linking, Western blotting, and the determination of PC2activity.
In vitro aggregation assays
Conditioned medium samples were kept on ice for 30 min and thencentrifuged for 15 min at 20,800 � g at 4 C. Similar proportions of thesupernatant and the pellet were subjected to electrophoresis on nonre-ducing 4–20% SDS-PAGE gels (Bio-Rad, Hercules, CA), followed byWestern blotting.
Chemical cross-linking experiments
For in vitro cross-linking with glutaraldehyde, 30 �l of each condi-tioned medium sample was treated with 0.05% glutaraldehyde for 30min at room temperature and then quenched by the addition of 200 mmethanolamine. After electrophoresis on nonreducing 4–20% SDS-PAGEgels (Bio-Rad), the cross-linked products were analyzed by Westernblotting.
For in vivo cross-linking experiments, CHO/PC2 and CHO/PC2/7B2cells were plated into six-well plates with 1–3 � 106 cells per well 1 dbefore labeling. The cells were washed twice with PBS and incubatedwith 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 (pH7.5) and incubation for 15 min. The cells were washed with PBS andresuspended in sample buffer without 2-mercaptoethanol and boiled for5 min. Cell lysates were analyzed by Western blotting.
Sucrose gradients
Each conditioned medium sample of 100 �l was loaded on top of a2.1-ml 10–40% (wt/vol) linear sucrose gradient in HEPES buffer [10 mmHEPES (pH 7.5), 150 mm NaCl, 2 mm CaCl2, 0.4 mm n-dodecyl-�-d-maltoside]. Gradients were centrifuged for 10 h at 54,000 rpm in a TLS-55rotor at 4 C, and 150-�l fractions were collected from the top of thegradient. Pellets were resuspended in 40 �l 2� sample buffer. Aliquotsof the fractions were analyzed using 4–20% SDS-PAGE gels, followedby Western blotting.
For radiolabeling immunoprecipitation experiments, cells were la-beled with 0.5 mCi/ml [35S]methionine and cysteine (Met/Cys) Promix(Amersham Corp., Arlington Heights, IL) for 20 min and then lysed in1 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, and1 mm EDTA] containing 10 �l 100 mm phenylmethylsulfonyl fluorideand 10 �l 10 mm parachloromercuriphenyl sulfonate. Samples wereincubated for 5 min on ice and used for immunoprecipitation. Cellextracts were preincubated with 0.1 ml 20% protein A-Sepharose CL-4B
(Pharmacia, Uppsala, Sweden) hydrated, washed with RIPA buffer at 4C for 1 h, and then centrifuged. Five microliters of antiserum against PC2were then added to the supernatant, along with 0.5 mm phenylmeth-ylsulfonyl fluoride and 0.5 mm parachloromercuriphenyl sulfonate.Samples were incubated overnight at 4 C with agitation. One hundredmicroliters of 20% protein A-Sepharose were then added and the sam-ples rocked at 4 C for 1 h. The beads were washed five times with RIPAbuffer. Immunoprecipitates were resuspended in sample buffer without2-mercaptoethanol and analyzed using nonreducing 4–20% SDS-PAGEgels. The dried gels were exposed to PhosphorImager screens and an-alyzed with a Typhoon 9410 variable-mode imager and ImageQuantsoftware (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The mi-gration of labeled material was compared with that of unlabeled stan-dard proteins analyzed on the same gel. The same samples were sub-jected to nonreducing SDS-PAGE (4–20%), and the gel slice containinglabeled material migrating at the position of the positive signal exhibitedon the screen was then excised. The gel slice was incubated in 2� samplebuffer containing 5% �-mercaptoethanol at 37 C for 30 min, placed onreduced 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 thePC2 propeptide (LSU26) was raised against residues His58-Asp80 ofmouse pro-PC2 (8). The antiserum against rat 7B2 (LSU13) was directedagainst residues 23–39 (22). Samples were subjected to electrophoresison either reducing (8%) or nonreducing (4–20%) SDS-PAGE gels (Bio-Rad, Hercules, CA), followed by Western blotting using the appropriateantisera. Proteins were transferred from gels to nitrocellulose mem-branes, and the membranes were preincubated in 5% nonfat milk inTris-buffered saline (TBS) for 30 min at room temperature before incu-bation overnight at 4 C with antiserum diluted 1:1000 in milk. Mem-branes were washed three times with TBS containing 0.05% Tweenfollowed by incubation at room temperature for 1 h with secondaryantibody (goat antirabbit IgG coupled to alkaline phosphatase). Mem-branes were then washed once with TBS containing 0.05% Tween andtwice 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 microtiterplates using 5 or 25 �l of each conditioned medium sample in a totalvolume of 50 �l containing 200 �m fluorogenic substrate, pyr-Glu-Arg-Thr-Lys-Arg-methylcoumarinamide as a substrate and 100 mm sodiumacetate (pH 5.0), 5 mm CaCl2, and 0.1% Brij 35 in the presence of aprotease inhibitormixture composed of 1 �m pepstatin, 0.28 mm tosyl-phenylalanyl chloromethyl ketone (TPCK), 1 �m trans-epoxysuccinicacid (E-64), and 0.14 mm tosyllysyl chloromethyl ketone (TLCK). In someexperiments, the activity was also measured in the presence of 1 �m 7B2CT 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 nmand an emission wavelength of 460 nm for 1 h at 37 C. Enzyme activitywas measured in triplicate and is given in fluorescence units (FU) perminute in which one FU corresponds to 5.33 pmol methylcoumarinamide.
ResultsAddition of recombinant 21-kDa 7B2 reverses pro-PC2/PC2aggregation and restores enzymatic competence
Our previous attempts to purify PC2 forms from the con-ditioned medium of CHO/PC2 cells using anion-exchangechromatography showed that eluted PC2 proteins were dis-tributed across the entire salt gradient, indicating that se-creted PC2 species are highly heterogeneous by charge (datanot shown). We first assessed the molecular weight patternof PC2-containing species present in the overnight condi-tioned medium of CHO/PC2 cells (i.e. no added 7B2) using
Lee and Lindberg • 7B2 Blocks Pro-PC2 Aggregation Endocrinology, August 2008, 149(8):4116–4127 4117
standard reducing SDS-PAGE. Western blotting of dena-tured medium reveals that these cells secrete three PC2species with molecular masses of 66 kDa (no propeptide), 71kDa (intermediate zymogen with a cleavage at an internalpropeptide site), and 75 kDa (complete zymogen), respec-tively (Fig. 1A, top panel); none are enzymatically active. Thethree potential cleavage sites in the mouse PC2 propeptideare shown in Fig. 1A (bottom panel).
A considerable amount of the PC2 species in this mediumwas precipitable after centrifugation. We subjected aliquotsof centrifuged medium and the corresponding pellets to de-naturing but nonreducing gel analysis. Under these condi-tions, PC2 forms consisted mainly of large aggregates thatcould not enter gels as well as many other multimers (Fig.1B). These may represent mixed disulfide-bonded proteins,
because they were stable to the addition of sodium dodecylsulfate but not to �-mercaptoethanol (see Fig. 1A).
Prior addition of recombinant 7B2 to the overnight con-ditioned medium of CHO/PC2 cells changed the molecularmass pattern of the recovered PC2 species; 7B2 increased theamount of the low-molecular-weight PC2 form in the su-pernatant [with a relative molecular mass (Mr) consistentwith a monomer] in a concentration-dependent manner (Fig.1B, 0.1–5 �m 7B2) and decreased the amount of this form inthe pellet (Fig. 1B). These data show that added 7B2 preventsPC2 forms from aggregating into precipitable forms.
To gain further information on PC2-containing complexespresent in CHO/PC2 cell conditioned media in the presenceand absence of 7B2, the formation of soluble oligomericforms was examined after cross-linking with 0.05% glutar-
FIG. 1. Addition of recombinant 7B2 to CHO/PC2 cells facilitates the formation of soluble, activatable PC2 species. CHO/PC2 cells werecultured with the indicated concentrations of recombinant 7B2 for 16 h, and the conditioned medium was collected. Samples were subjectedto either reducing (8%) (A) or nonreducing (B and C) SDS-PAGE (4 –20%) and analyzed by Western blotting with PC2 antiserum (LSU18,antiserum against a carboxyl-terminal peptide of mature PC2). A, Bottom, Sequence of the PC2 propeptide, with three potential cleavagesites within the pro-PC2 propeptide shown in bold and predicted solvent-accessible residues in the propeptide hydrophobic patch byhomology with the PC1 propeptide highlighted in gray (43); B, aggregation assay using the conditioned medium; C, cross-linking of PC2with 0.05% glutaraldehyde; D, PC2 activity assay. FU, Fluorescence units.
4118 Endocrinology, August 2008, 149(8):4116–4127 Lee and Lindberg • 7B2 Blocks Pro-PC2 Aggregation
aldehyde. Western blots of cross-linked samples preparedfrom conditioned medium with no added 7B2 contained onlyminor amounts of monomeric PC2 with a molecular mass ofabout 70 kDa (Fig. 1C, 0 �m 7B2). The majority of secretedPC2 species in cross-linked, non-7B2-containing mediumwas unable to enter the gel, indicating a high proportion ofaggregated material. However, cross-linked samples pre-pared from conditioned medium with added 7B2 containedan increased proportion of lower-molecular-weight PC2 spe-cies and a reduced amount of aggregated protein at the topof the gel (Fig. 1C, compare 0 and 5 �m 7B2). These cross-linking data support the idea that the presence of 7B2 reducesthe amounts of cross-linkable, self-associated PC2 species.
Surprisingly, the addition of extracellular 7B2 also resultedin the robust production of active PC2 from otherwise totallyinactive medium (Fig. 1D). The acquisition of enzyme activ-ity was strongly correlated with 7B2 concentration. We in-terpret this result to indicate that extracellular 7B2 blocks theunfolding events that result in enzymatic incompetenceand/or assists in the correct refolding of partially unfoldedpro-PC2 forms, or both; the end result is the formation of anactivation-competent zymogen.
In summary, these results show that exogenously added7B2 blocks the formation of aggregated PC2 forms and in-creases the amounts of soluble, smaller oligomeric complex-
es; this effect is associated with the ability of secreted pro-PC2to 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 ag-gregation events that we observed in the conditioned me-dium also occur intracellularly. We used cell-permeablecross-linking reagents coupled with Western blotting to de-termine whether intracellular PC2 forms are also present asmultimeric forms. The majority of intracellular PC2-contain-ing species in cross-linked cell extracts consisted of largeaggregates and other oligomers with lower Mr (Fig. 2A). Themolecular masses of the total PC2 species within CHO/PC2/7B2 cells were somewhat different from those of CHO/PC2cells (Fig. 2A, Western blotting), most likely due to the for-mation of 7B2-containing complexes. Whereas Fig. 2A showsthe steady state of PC2-containing species, Fig. 2B shows themolecular masses of newly synthesized 35S-labeled PC2-con-taining molecules. Most of these newly synthesized PC2forms were present in high-molecular-mass multimersand large aggregates (over 500 kDa, Fig. 2B, left panel). Inaddition to the large aggregates, 35S-labeled PC2 appeared asa strongly labeled band of smaller Mr in both cell lines (Fig.
FIG. 2. Intracellular PC2 exists as multimeric forms and aslarge aggregates. A, Both CHO/PC2 and CHO/PC2/7B2cells were cross-linked with the reducible cross-linker DSPand immunoblotted with PC2 antiserum. B, Both CHO/PC2and CHO/PC2/7B2 cells were labeled with [35S]Met/Cys for20 min followed by incubation in the presence and absenceof 1 mM DSP for 30 min at room temperature. Cell extractswere immunoprecipitated with PC2 antiserum. The immu-noprecipitates were subjected to nonreducing SDS-PAGE(4–20%). Gel slices exhibiting immunoreactivity were in-cubated in 2� sample buffer containing 5% �-mercapto-ethanol at 37 C for 30 min, subjected to reducing SDS-PAGE, and analyzed with phosphorimaging.
Lee and Lindberg • 7B2 Blocks Pro-PC2 Aggregation Endocrinology, August 2008, 149(8):4116–4127 4119
2B). The presence of 7B2 in this complex in the CHO/PC2/7B2 sample was experimentally confirmed by reducing thecross-linker with �-mercaptoethanol followed by electro-phoresis on reducing SDS-PAGE. As expected, we observeda 7B2-sized product only in the samples obtained fromCHO/PC2/7B2 cells (Fig. 2B, right panel).
We conclude from these data that a high proportion of theintracellular pro-PC2/PC2-containing species in CHO/PC2/7B2 cells can be assembled into higher-order complex-es; if 7B2 is present, these complexes will contain 7B2.
7B2 must be added before or immediately after secretion ofpro-PC2 for activatable species to be generated
Interestingly, when we added recombinant 7B2 to aliquotsof conditioned medium collected from CHO/PC2 cell cul-tures, 7B2 addition (0–5 �m) had no effect on PC2 enzymeactivity (Fig. 3A) but still inhibited PC2 aggregation (Fig. 3B).Because no activity could be rescued by this later addition of7B2, it is clear that secreted pro-PC2/PC2 forms must veryrapidly lose enzymatic competence in the absence of 7B2 andthat 7B2-induced solubilization (Fig. 3B) alone is not suffi-cient to restore enzymatic competence. These results high-light the extreme lability of the activation-competent form.
If the ability of exogenous 7B2 to confer enzymatic com-petence to released PC2 species is labile, we would expectthat increased enzyme activity would be strongly correlatedwith the time that secreted pro-PC2 spends in the presenceof 7B2. We therefore cultured CHO/PC2 cells in a six-wellplate overnight and then added recombinant 7B2 to each wellat different time points (at 2-h intervals) during the succeed-ing 10 h. Indeed, although the total secretion of PC2 in allwells after 10 h in culture was identical (results not shown),PC2 activity increased in direct proportion to the time inculture 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 pre-sumed unfolding reaction, we performed a time-dependentactivity assay with added 7B2 (Fig. 4). We cultured CHO/PC2 cells in two 12-well plates overnight; the next day, themedium in one plate was substituted with medium contain-ing recombinant 7B2, whereas medium without 7B2 wasadded to the other plate. Conditioned medium was collectedfrom each well at different time points. For samples from theplate lacking 7B2, recombinant 7B2 was added to each sam-ple immediately after collection so that final concentrationsof 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 PC2was exposed to 7B2. We then measured enzymatic activity(corresponding to total PC2-containing forms secreted ateach time point); the amount of PC2 secreted in both con-ditions was identical.
We observed that all medium samples cleaved substrate ata similar rate during the first 5 min or so. After that first timeperiod, only media samples with initially added 7B2 exhib-ited increased PC2 activity in a time-dependent manner (Fig.4, Œ); after 5 min without 7B2, increased activity was notpresent in samples with delayed addition of 7B2, showingthat in vitro rescue of PC2 activity with 7B2 was ineffectiveafter this time (Fig. 4, F). Our interpretation of these data isthat in the absence of immediate interaction with extracel-lular 7B2, released pro-PC2 undergoes an irreversible con-formational change that results in loss of catalytic compe-tence. Therefore, PC2 forms secreted at time points after 5min are not able to contribute to the final activity, leading tothe 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/PC2species in conditioned medium exist as large aggregates, butthe addition of extracellular 7B2 increases the quantity ofsoluble forms. To furnish detail on the apparent sizes of thecomplexes in the presence or absence of 7B2, we analyzed theconditioned medium obtained from CHO/PC2 cells using
FIG. 4. Secreted PC2 activity is extremely unstable in the absence ofimmediate interaction with 7B2. CHO/PC2 cells were cultured in two12-well plates overnight. One plate was changed to medium contain-ing recombinant 7B2 (1 �M), and the conditioned medium was col-lected at the time points shown (Œ). The other plate was changed tomedium lacking 7B2, and medium samples were collected at the sametime points; recombinant 7B2 was added to each sample immediatelyafter collection (F). Medium samples were then assayed for PC2activity.
FIG. 3. 7B2 addition to the conditioned medium collected from CHO/PC2 cell cultures can prevent PC2 aggregation but does not renderPC2 competent for activation. Conditioned medium was collectedfrom CHO/PC2 cells, and recombinant 7B2 was added to the mediumat the indicated concentrations. A, Western blotting and PC2 activity;B, aggregation assay using conditioned medium.
4120 Endocrinology, August 2008, 149(8):4116–4127 Lee and Lindberg • 7B2 Blocks Pro-PC2 Aggregation
velocity centrifugation on 10–40% sucrose gradients. Paral-lel gradients containing BSA monomer and dimers (66 and132 kDa) or urease monomers and dimers (272 and 545 kDa)were used as molecular standards.
In conditioned medium obtained from CHO/PC2 cellswith no added 7B2, the majority of the 66-kDa PC2 wasdetected in the pellet and in fractions 6 and 7, which containproteins of Mr from 60–70 kDa (Fig. 5A), suggesting thatsecreted 66-kDa is present both as a monomer and in pre-cipitable aggregates. A trace amount of the intermediatezymogen (71 kDa) was detected in fractions 6 and 7, but the71-kDa form was not present in the pellet (Fig. 5A), indicatingthat this PC2 species does not exhibit a propensity to aggre-gate. By contrast, the majority of the intact 75-kDa zymogenwas located in the pellet, supporting the idea that secretedpro-PC2 is mostly highly aggregated (Fig. 5A).
In contrast, when exogenous 7B2 was added to CHO/PC2cells at 1–5 �m concentrations, increased amounts of mono-meric 66-kDa PC2 were detected in all lower Mr-containingfractions, whereas the proportion of 66-kDa PC2 in the pelletwas reduced (Fig. 5, C and D); these data indicate that ex-ogenous 7B2 is able to solubilize large 66-kDa PC2-contain-ing aggregates into smaller forms (although these were en-zymatically inactive; see below). Significantly increasedamounts of both soluble zymogen and intermediate formswere detected in fractions 6–10, with Mr corresponding topotential monomer, dimer, and trimer forms (Fig. 5, B–D).
Composition of higher-order pro-PC2–7B2 complexes in themedium
To gain information on the molecular composition of pro-PC2 complexes, we performed gel filtration of recombinantPC2 forms partially purified from conditioned medium; afterenzymatic assay, we cross-linked fractions with glutaralde-hyde and performed Western blotting using 7B2 and PC2antisera (supplemental Fig. 1, published as supplementaldata on The Endocrine Society’s Journals Online web site athttp://endo.endojournals.org). These data show the pres-ence of multiple oligomeric forms, most of which also contain7B2. These data were confirmed by velocity sedimentationand Western blotting of cross-linked conditioned medium(not shown).
7B2 addition protects pro-PC2 from cleavage at theprimary site
Although within CHO/PC2 cells the majority of intracel-lular PC2 is present as the proform (Fig. 2A), secreted PC2species consisted largely of the two zymogen forms togetherwith the 66-kDa processed form (Fig. 5A). These data indicatethat processing of two zymogen forms to the 66-kDa form islikely to occur extracellularly, possibly by extraneous pro-teases on the cell membrane or in the overnight-conditionedculture medium. (This event can be distinguished from au-tocatalytic propeptide cleavage, which requires an acidicpH.) Adding 7B2 to the medium increased the amounts ofboth soluble zymogen forms (Fig. 5, B–D). The pronouncedincrease in the amounts of intact and intermediate zymogenforms in the presence of 7B2 indicates that binding of exog-
enous 7B2 protects the primary cleavage site from extraneouscleavage, potentially by preventing unfolding and propep-tide exposure to solvent.
Addition of recombinant 7B2 to cells already expressing7B2 (CHO/PC2/7B2 cells) increases both the amount ofsoluble PC2 forms as well as enzyme activity
CHO/PC2/7B2 cells already express large quantities ofactive PC2 and lesser quantities of 7B2 (due to the methodof construction of the cells; PC2 was expressed using thepowerful dihydrofolate reductase-coupled amplificationmethod, whereas 7B2 expression was coupled only to twoantibiotic resistance plasmids). If 7B2 expression is limit-ing for the expression of PC2 activity in a postsynthesismanner, the addition of recombinant 7B2 should also in-crease the quantity of active PC2 forms secreted fromCHO/PC2/7B2 cells. This was indeed found to be the case(Fig. 6A). Western blot analysis using cross-linked me-dium with extracellular 7B2 showed considerably in-creased quantities of soluble PC2 forms with molecularmasses from 70 – 400 kDa, corresponding to mono-, di-,tri-, and higher oligomeric forms (Fig. 6B). Comparison ofvelocity centrifugation experiments using the conditionedmedium with added 7B2 and medium without 7B2 (Fig.7A) shows that 7B2 addition resulted in highly increasedamounts of both soluble pro-PC2 and the intermediateform and that this was associated with significantly in-creased enzyme activity (�4-fold; Figs. 6B and 7B).
We conclude that even in CHO/PC2/7B2 cells, a largeportion of the PC2 species is released without achievingintracellular interaction with 7B2, resulting in an enzymat-ically incompetent, potentially unfolded form; however, thisparticular unfolding event must be reversible, because thezymogen can still be rendered competent for activation byexogenously added 7B2. As with cells not expressing any7B2, addition of recombinant protein solubilized PC2 speciesfrom precipitable aggregates.
Structure-function analysis of 7B2 effects on aggregation
We next performed structure-function studies to test therole of the amino-terminal segments of 7B2 in the preventionof PC2/pro-PC2 aggregation. We constructed two amino-terminally deleted 7B2 mutants, 7B230–150 and 7B268–150, toidentify the determinant within 7B2 that inhibits pro-PC2aggregation (Fig. 8A). As found previously (27), the condi-tioned medium obtained from CHO/PC2 cells with addedrecombinant 7B268–150 or 7B230–150 protein showed a con-centration-dependent increase in PC2 activity (Fig. 8B). Inaddition, two truncated 7B2 proteins were able to inhibitPC2/pro-PC2 aggregation; however, the anti-aggregant ac-tivity of 7B268–150 was not as efficient as that of recombinant21-kDa 7B2 and 7B230–150 (Fig. 8C). We conclude that 7B2segments containing the 36-residue helix previously shownto be important in the acquisition of enzyme activity bypro-PC2 (27) are also effective in our extracellular 7B2 acti-vation system.
Lee and Lindberg • 7B2 Blocks Pro-PC2 Aggregation Endocrinology, August 2008, 149(8):4116–4127 4121
FIG. 5. 7B2 addition to PC2-expressing cells increasesthe amounts of both soluble zymogen and intermediatePC2 forms: velocity sedimentation and cross-linkingstudies. Conditioned medium obtained from CHO/PC2cells with either no added 7B2 (A) or added 7B2 (B–D)was separated on a 10–40% sucrose gradient. Fifteenmicroliters of each fraction (150 �l) and 15 �l of pelletsresuspended in 40 �l 2� sample buffer were subjected toreducing SDS-PAGE and analyzed by Western blottingusing antisera against either PC2 (A–D) or 7B2 (D, bot-tom). The fractions were also assayed for PC2 activity(B–D). The positions of molecular weight standards areindicated. Conditioned medium was cross-linked with0.05% glutaraldehyde and then separated on a 10–40%sucrose gradient. P, Pellet.
4122 Endocrinology, August 2008, 149(8):4116–4127 Lee and Lindberg • 7B2 Blocks Pro-PC2 Aggregation
7B2 addition to SK-N-MC cells also results in theacquisition of PC2 activity in the conditioned medium
The human neuroblastoma cell line SK-N-MC expressesPC2 but not 7B2 (28); peptide precursors are not processedin this cell line (29), indicating that SK-N-MC cell-expressedPC2 forms are enzymatically incompetent. We testedwhether exogenous 7B2 addition could also result in theacquisition of PC2 enzyme activity in SK-N-MC cells; thiswas indeed found to be the case (Fig. 9). The activity wasidentified as PC2 by virtue of its complete inhibition with the7B2 CT peptide (23, 24). We conclude that the ability of 7B2to act as an extracellular chaperone for pro-PC2 is not aconsequence of pro-PC2 overexpression.
Discussion
The observation that 7B2 is a required binding protein forthe manifestation of PC2 activity is now 13 yr old and hasbeen confirmed by several groups (22, 28, 30), but the mech-anism of this interaction is still not understood. Although weinitially believed that 7B2 would be a required protein duringthe autocatalytic activation process, our previous data show-ing that 7B2 can be removed by antibody affinity chroma-tography from purified recombinant pro-PC2 without effecton enzyme activity (21) indicated that this was not a likelymechanism. We similarly ruled out an effect of 7B2 duringinitial folding of pro-PC2 (12). Although we and others haveobtained structure-function information on the 7B2/pro-PC2interaction both for 7B2 as well as for pro-PC2 (reviewed inRef. 16), exactly how 7B2 is able to facilitate the acquisitionof activation-competent forms of pro-PC2 has been enig-matic. We have here approached this question by examiningeffects 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 experi-ments both demonstrate that majority of the PC2 speciesreleased from CHO/PC2 cells is highly aggregated. How-ever, in the presence of extracellular 7B2, large amounts ofthe pro-PC2 aggregates become solubilized, forming mono-,di, tri, and smaller oligomers, and this effect is clearly pro-portional to the 7B2 concentration. Exposed hydrophobicsurfaces, presumably due to partial unfolding, may generatethe phenomenon of aggregation and precipitation of PC2-containing complexes; we speculate that 7B2 binds to thesehydrophobic surfaces. Molecular chaperones are known tointeract with unstructured, aggregation-prone folding inter-mediates that expose a significant amount of hydrophobicsurface to solvent, thereby preventing an irreversible path-way toward misfolding and aggregation (31). The cytoplas-mic small heat-shock proteins (32) and clusterin, an extra-cellular mammalian chaperone protein (33), are found in avariety of disease states and in stress conditions. These pro-teins preferentially recognize hydrophobic sequences ofpartly folded protein intermediates that are slowly aggre-gating and inhibit stress-induced precipitation of many dif-ferent proteins in an ATP-independent manner (34, 35). Wehave previously shown that the N-terminal 21-kDa domainof 7B2 interacts in part with hydrophobic residues in the PC2catalytic 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 domainthat is involved in the binding of a propeptide hydrophobicpatch, implying that the N-terminal domain of 7B2 may alsointeract with the propeptide (37). Indeed, early immunopre-cipitation experiments showed that 7B2 does bind the pro-PC2 propeptide (38). We speculate that 7B2-mediated inhi-bition of PC2 aggregation results from binding of 7B2 to alarge hydrophobic surface of pro-PC2 that includes both thisTyr-194 domain and the propeptide sequence. Co-crystal-lography of 7B2 with pro-PC2 will be required to definitivelyanswer the question of where 7B2 binds pro-PC2; such ex-periments are ongoing.
FIG. 6. 7B2 addition to 7B2-expressing CHO/PC2 cells further in-creases the amount of soluble PC2 species and the amount of PC2activity: cross-linking studies. CHO/PC2/7B2 cells were cultured withthe indicated concentration of 7B2 for 16 h, and the conditionedmedium was collected. A, Conditioned medium was analyzed by West-ern blotting with PC2 antiserum and also assayed for PC2 activity;B, cross-linking of PC2 with 0.05% glutaraldehyde.
Lee and Lindberg • 7B2 Blocks Pro-PC2 Aggregation Endocrinology, August 2008, 149(8):4116–4127 4123
Exogenous 7B2 protects pro-PC2 from extraneous processingat the primary cleavage site
Within the CHO/PC2 cell, PC2 species are largelypresent as intact zymogen, whereas secreted PC2 speciesconsist mainly of soluble and insoluble 66-kDa forms to-gether with a large amount of insoluble (precipitable) zy-mogen and a small amount of soluble intermediate zy-mogen. In the absence of 7B2, these two secreted zymogenforms could not undergo autocatalytic processing even ata pH low enough to autoactivate pro-PC2 in vitro, dem-onstrating their catalytic incompetence. However, the me-dium still contained significant quantities of inactive,cleaved 66-kDa PC2. These results strongly suggest thatthese zymogen forms are not properly folded and that theprimary cleavage site of this secreted pro-PC2 is exposedto solvent, possibly due to partial unfolding. Cleavage byextraneous proteases may then result; although the endresult is a protein with the molecular mass of mature PC2,this unfolded enzyme is irreversibly inactivated. The factthat recombinant 7B2 added to CHO/PC2 cells increasedthe amounts of both soluble intact and intermediate zy-mogens demonstrates that extracellular 7B2 can protectpro-PC2 from this primary-site cleavage event, possibly byblocking the initial unfolding event and thereby stabiliz-ing the activation-competent zymogen conformer. Wehave previously reported that 7B2 can stabilize the enzyme
activity of recombinant PC2 during heat denaturation (19),and others have reported that 7B2 addition increases thecleavage of proopiomelanocortin by released PC2 (39). Thesame protective effect is observed when conditioned me-dium from CHO/PC2 is heated; binding of 7B2 protectssecreted forms from heat denaturation (Lee, S. N., and I.Lindberg, results not shown). Thus, in several differentsystems, 7B2 has been shown to protect PC2 species fromunfolding events, supporting the notion that it representsan authentic postsynthesis chaperone.
Unlike the primary site, internal cleavage of the PC2propeptide (see Fig. 1A) was not protected by 7B2 addi-tion, suggesting that this site is accessible to proteaseswhether or not 7B2 is present. In recent experiments, wehave observed that the PC2 propeptide can be cleaved atthe internal cleavage site by furin in vitro (Lee, S. N., andI. Lindberg, unpublished results), indicating that unlikethe furin propeptide (40), the internal cleavage site of thePC2 propeptide is always solvent accessible.
In summary, these data support the idea that 7B2interacts with residues in the catalytic and propeptidedomains flanking the primary site, resulting in protec-tion from primary site (but not internal site) process-ing, conferring enhanced stability to unfolding events,and preventing enzyme aggregation leading to precipi-tation.
FIG. 7. Addition of 7B2 to PC2- and 7B2-expressing cellsresults in increased amounts of both soluble pro-PC2 andthe intermediate zymogen form: velocity sedimentation.The conditioned medium collected from CHO/PC2/7B2cells with either no added 7B2 (A) or added 7B2 (B) wasanalyzed by velocity sedimentation using a 10–40% su-crose gradient. The fractions were analyzed by Westernblotting or assayed for PC2 activity.
4124 Endocrinology, August 2008, 149(8):4116–4127 Lee and Lindberg • 7B2 Blocks Pro-PC2 Aggregation
Pro-PC2 aggregation
Oligomerization and aggregation of pro-PC2 are not con-fined to the extracellular medium. Our work here has clearlyshown that intracellular forms of pro-PC2 are present as
higher-order oligomers; other groups have also shown thataggregation and membrane association of pro-PC2 occur inthe TGN/immature secretory granules (26, 41). In AtT-20/PC2 cells, late secretory compartment pro-PC2 aggregateswere shown to interact with lipid rafts via either the PC2propeptide (41) or the PC2 C terminus (26). This was pos-tulated to lead to release of soluble mature PC2 from themembrane at the neutral pH of the external environment (41).However, the data presented here clearly show that releasedPC2 forms are also highly aggregated. Thus, aggregationappears to occur both at the acidic pH within neuroendocrinecells as well as at the neutral pH of culture medium.
7B2 binding prevents both enzyme unfolding andaggregation
Figure 10 depicts our current hypothesis as to the mech-anism 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 absenceof 7B2 that an irreversible unfolding event occurs that ismanifested first by the loss of activation competence and,on a longer time scale, further oligomerization of the zy-mogen, leading to irreversible protein aggregation intoinsoluble precipitates (path A). If 7B2 is added later, i.e.after an initial rapid irreversible unfolding event, pre-cipitation is prevented, but the zymogen cannot refoldto recover activation competence (path B). Finally, if pro-PC2 immediately encounters 7B2 after secretion the zy-mogen will be stabilized and will be activation competent(path C).
In summary, we have shown using a postsynthesis se-cretory system that 7B2 can reverse and prevent the oc-currence of pro-PC2 aggregation. Although rapid expo-sure of pro-PC2 to 7B2 restores enzymatic competence toa presumably partially unfolded form, later exposure to7B2 does not. These postsynthesis chaperone-like activitiesof 7B2 are likely to occur via binding to an exposed hy-drophobic surface of pro-PC2 molecules, which appear tohave a natural tendency to aggregate. Although the mostsensitive indicator of 7B2 efficacy on PC2 folding events isthe prevention of the loss of activation competence, theeffects of this small neuroendocrine protein on enzymeaggregation are quantitatively quite remarkable. In fact,there is historical precedent for an antiaggregant activityof 7B2; 7B2 has been reported to assist in the formation ofcorrectly folded active, monomeric human IGF-I via dis-solution of incorrectly folded, aggregated multimers
FIG. 8. 7B2 structure-function analysis: prevention of pro-PC2 ag-gregation. A, Schematic representation of 7B2 amino-terminal dele-tions. The gray box corresponds to the major determinant of thebinding and activation of pro-PC2 in vivo (27). The first residue of each7B2 construct is indicated. CHO/PC2 cells were cultured with addedrecombinant 21-kDa 7B2, 7B230–150, or 7B268–150 protein for 16 h, andthe conditioned medium was collected. B, Conditioned medium wasassayed for PC2 activity. C, Aggregation assay using the conditionedmedium. Cell numbers were similar, as shown by equal amounts of�-actin.
FIG. 9. Addition of recombinant 7B2 to the neuroblas-toma cell line SK-N-MC results in the acquisition of PC2activity in the conditioned medium. SK-N-MC cells werecultured with the indicated concentrations of 7B2 for 16 h,and the conditioned medium was collected and assayed forPC2 activity in the presence or absence of 1 �M 7B2 CTpeptide, a specific PC2 inhibitor.
Lee and Lindberg • 7B2 Blocks Pro-PC2 Aggregation Endocrinology, August 2008, 149(8):4116–4127 4125
in vitro (42). Because 7B2 exhibits a pan-neuronal andendocrine distribution much broader than that of PC2, wespeculate that 7B2’s antiaggregant action may apply toother as yet unknown neural and endocrine substrates.
Acknowledgments
Received January 15, 2008. Accepted April 25, 2008.Address all correspondence and requests for reprints to: Dr. Iris
Lindberg, Department of Anatomy and Neurobiology, University ofMaryland Medical School, 20 Penn Street, Baltimore, Maryland 21201.E-mail: [email protected].
This work was supported by National Institutes of Health GrantDK049703 (to I.L.).
Present address for S.-N.L.: The Airway Mucus Institute, YonseiUniversity College of Medicine, Seoul, Korea 120-752.
Disclosure Statement: The authors have nothing to disclose.
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