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Originally published In Press as doi:10.1074/jbc.M708763200 on November 26, 2007

J. Biol. Chem., Vol. 283, Issue 4, 2373-2384, January 25, 2008
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The Regulated Cell Surface Zymogen Activation of the Proprotein Convertase PC5A Directs the Processing of Its Secretory Substrates*Formula

Gaétan Mayer{ddagger}1, Josée Hamelin{ddagger}, Marie-Claude Asselin{ddagger}, Antonella Pasquato{ddagger}, Edwidge Marcinkiewicz{ddagger}, Meiyi Tang§, Siamak Tabibzadeh§, and Nabil G. Seidah{ddagger}2

From the {ddagger}Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montréal, Quebec H2W 1R7, Canada and §Stony Brook University, Stony Brook, New York 11794

Received for publication, October 23, 2007 , and in revised form, November 14, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The proprotein convertases are synthesized as zymogens that acquire activity upon autocatalytic removal of their NH2-terminal prosegment. Based on the convertase furin, to fold properly and gain activity, the convertases PC5A, PACE4, and PC7 are presumed to undergo two sequential prosegment cleavages in the endoplasmic reticulum and then in the trans-Golgi network. However, biochemical and immunocytochemical experiments revealed that mouse PC5A is complexed to its prosegment at the plasma membrane. This labeling is lost upon treatment with heparin and is increased by overexpressing members of the syndecan family and CD44, suggesting attachment of secreted PC5A-prosegment complex to heparan sulfate proteoglycans. Following stimulation of Y1 cells with adrenocorticotropic hormone or 8-bromo-cyclic AMP, the cell surface labeling of the prosegment of PC5A is greatly diminished, whereas the signal for mature PC5A is increased. Moreover, after stimulation, the protease activity of PC5A is enhanced, as evidenced by the cleavage of the PC5A substrates Lefty, ADAMTS-4, endothelial lipase, and PCSK9. Our data suggest a novel mechanism for PC5A activation and substrate cleavage at the cell surface, through a regulated removal of its prosegment. A similar mechanism may also apply to the convertase PACE4, thereby extending our knowledge of the molecular details of the zymogen activation and functions of these heparan sulfate proteoglycan-bound convertases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian genome is predicted to express ~30,000 genes. The estimated number of primary protein products is, however, many times higher, mainly due to alternative splicing events and post-translational modifications, including irreversible proteolytic cleavage. Proteinases either degrade proteins via multiple cleavages or participate in their limited proteolysis, thereby raising the number of active products that can be generated from a single proprotein. Less frequently, a specific cleavage inactivates the substrate.

The mammalian proprotein convertases (PCs)3 form a family of nine serine proteinases that consistently modify the activation state of a wide array of bioactive proteins (1-6). Seven basic amino acid (aa)-specific PCs are related to yeast kexin: PC1 (also known as PC3), PC2, furin, PC4, PC5 (also known as PC6 or PC5/6), PACE4, and PC7. These cleave protein substrates after single or paired basic amino acids within the motif (R/K)Xn(R/K){downarrow}, where Xn represents 0, 2, 4, or 6 variable aa separating the two canonical basic residues required for cleavage recognition. Metallocarboxypeptidases then remove the carboxyl-terminal basic residues exposed by the convertases, a process shown to be necessary for the elaboration of biological activity of several polypeptides and proteins (7). Many biologically active peptides ending at G(R/K) once trimmed by a basic amino acid-specific metallocarboxypeptidase are also COOH-terminally amidated by peptidylglycine {alpha}-amidating monooxygenase (8). The other two convertases, SKI-1/S1P and PCSK9, cleave at nonbasic residues and are known for their key role in cholesterol homeostasis (3).

PC5 is indispensable for early development, since its knockout in mice is embryonic lethal (9). Ontogeny and tissue distribution analysis of PC5 expression revealed that it is dominant in somites at embryonic day 9.5, in lungs, pancreatic and adrenal primordia, intestinal tract, and kidney cortex (9). In addition, PC5 seems to be implicated in specific pathologies, such as atherosclerosis and cancer metastasis, by activating matrix metalloproteases (10) and/or by inactivating specific molecules (e.g. the neural adhesion L1) (11), respectively. The PC5 gene encodes two isoforms generated by alternative splicing, resulting in either PC5A (915 aa; 21 exons) or PC5B (1877 aa; 38 exons) (2). PC5A is sorted to both the constitutive and regulated secretory pathways, whereas PC5B traffics only within the constitutive secretory pathway (12). The membrane-bound PC5B, furin, and PC7 cycle from the cell surface back to the trans-Golgi network (TGN) through endosomes, a pathway regulated by signals in their cytosolic tails. Such PCs process precursors either in the TGN, at the cell surface, or in endosomes (4, 13-16). The secreted PC5A and PACE4, which are devoid of a transmembrane domain, contain a COOH-terminal cysteine-rich domain (CRD) that interacts with tissue inhibitors of metalloproteases (TIMPs), and allow the formation of a ternary complex with heparan sulfate proteoglycans (HSPGs) (17). Thus, like furin, PC5A and PACE4 are present at the cell surface, where they may contribute to the cleavage of HSPG-bound proteins, such as endothelial and lipoprotein lipases (18, 19).

Before they can act on protein substrates, PCs themselves must go through an ordered process of activation. PCs are initially synthesized as inactive zymogens in the endoplasmic reticulum (ER). Upon folding, they undergo an autocatalytic processing in the ER (except for PC2) downstream of their NH2-terminal prosegment, resulting in a noncovalent but tight binding complex of the inhibitory prosegment with the protease. This first zymogen cleavage is necessary to allow the PC to exit the ER for secretion, since the prosegment acts as an intramolecular chaperone allowing the proper folding of the enzyme (12, 20-24). A second autocatalytic cleavage event is then needed to liberate the protease from its prosegment, thus specifying the location within the cell where the PC is activated and can then cut substrates in trans. Although furin is activated following the dissociation of its prosegment in the TGN (22, 23, 25), the exact intracellular locations of PC5A or PACE4 activation requiring cleavage at an internal second site of their prosegment are not known.

In the present study, we provide evidence for the cell surface activation of PC5A. The complex prosegment-PC5A is anchored at the plasma membrane via HSPGs, such as syndecans and CD44. The activation of PC5A is stimulated by incubation of Y1 cells with adrenocorticotropic hormone (ACTH) or 8-Br-cAMP. Such regulated activation of PC5A triggered the cell surface endoproteolytic processing of its cognate substrates Lefty, endothelial lipase, ADAMTS-4, and PCSK9. This mode of activation is unique among the PCs and provides novel insight into the regulation and site-specific cleavages of protein substrates.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression Constructs—Mouse PC5A, mouse PC5B, human PACE4, human furin, human PCSK9, and all of their mutant cDNAs and domains were cloned, with or without a COOH-terminal V5 tag, into pIRES2-EGFP vector (Clontech) as described previously (17, 24). The cDNA coding for human Lefty-A (pcDNA3.1 vector) was reported previously (26). The cDNA encoding for human CD44 containing the variably expressed exon 3 (MGC-10468; American Type Culture Collection (ATCC)) was cloned into NheI/BamHI-digested pIRES2-EGFP. cDNAs encoding for GPI-GFP, mouse TIMP-3 (phCMV3 vector), human ADAMTS-4 and -5 (pCEP4 and pSG6 vectors, respectively), Myc-tagged human endothelial lipase (pcDNA3.1-Myc-His vector), and rat syndecan-2 and -4 (pcDNA3.1 vector) were kindly provided by Drs. Sergio Grinstein (University of Toronto), John S. Mort (McGill University), Linda Troeberg (Imperial College London), Daniel J. Rader (University of Pennsylvania), and Eok-Soo Oh (Ewha Womans University, Seoul), respectively. All cDNAs were sequenced to ensure that no errors occurred during the cloning procedures.

Cell Culture, Transfections, and Cell Treatments—COS-1 and HEK293 cell lines were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Invitrogen), whereas CHO-K1, furin-deficient CHO-FD11 (27), and heparan sulfate-deficient CHO-pgsD-677 (ATCC) (28) cells were grown in Ham's F-12/Dulbecco's modified Eagle's medium (50: 50) with 10% fetal bovine serum. Y1 adrenocortical cells (ATCC) were grown in F12K medium with 15% horse serum and 2.5% fetal bovine serum. All cells were maintained at 37 °C under 5% CO2. At about 80-90% confluence, cells were transiently transfected with Lipofectamine 2000 (Invitrogen) except for Y1 and HEK293 cells that were transfected with Effectene (Qiagen). Stable transfectants of pIRES2-EGFP-PCSK9-V5 were obtained in HEK293 cells following G418 selection.

Cells, nontransfected or 24 h post-transfection, were washed with serum-free medium and incubated either alone or in combination, as indicated in the figure legends, with 200 nM human ACTH (fragment 1-24), 5 mM 8-Br-cAMP, 10 mM ammonium chloride (NH4Cl), 20 µM recombinant human TIMP-3, 0.5 mg/ml suramin, 1 mg/ml heparin, 0.5 units/ml heparinase III, 3 units/ml phosphatidylinositol-specific phospholipase C (all obtained from Sigma), 25 µM decanoyl-RVKR-chloromethylketone (CMK; Bachem), or 25 µM GM6001 (EMD Biosciences), in serum-free medium at 37 °C for 16 h or 1 h for suramin and heparin. Following treatments, cells and conditioned media were collected for Western blot analyses, or cells were used for immunocytochemistry as described below. In some experiments, cells were also surface-biotinylated prior to lysis, immunoprecipitation, and Western blotting (see below).

Antibodies—Rabbit polyclonals against the prosegment of PC5 (Ab:pPC5) or the prosegment of furin (Ab:pfurin) were raised in house as described (24, 29). Rabbit polyclonals against PC5 recognizing both A and B isoforms (Ab:PC5) or specific for the B isoform (Ab:PC5B) were a kind gift from John Creemers (University of Leuven, Belgium). Other antibodies used were the unconjugated or horseradish peroxidase (HRP)-conjugated mouse anti-V5 tag antibodies (mAb:V5 or mAb:V5-HRP; Invitrogen), rabbit anti-syndecan-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), HRP-conjugated mouse anti-Myc tag (Invitrogen), goat anti-Lefty (R&D Systems), rabbit anti-ADAMTS-4 (Sigma), rabbit anti-TIMP-3 (rAb: TIMP-3; Chemicon), and goat anti-TIMP-3 (gAb:TIMP-3; Santa Cruz Biotechnology).

Cell Surface Biotinylation, Immunoprecipitations, and Western Blotting—For biochemical detection of PC5 and its prosegment at the plasma membrane, cells transiently transfected with FL-PC5A were scraped from plates, washed with ice-cold phosphate-buffered saline (PBS) adjusted to pH 8.0, and biotinylated with 0.2 mg of sulfo-NHS-LC-Biotin (sulfosuccinimidyl-6-(biotin-amido)hexanoate) (Pierce) per ml of cell suspension for 30 min at 4 °C. Cells were then washed with 100 mM glycine in PBS, pH 8.0, to quench the reaction. Surface biotinylation of cells transfected with TIMP-3 was done on adherent cells in culture plates and processed as described above. As a control for surface biotinylation, cells were treated with 0.05% trypsin, 0.5 mM EDTA for 20 min on ice to discard all cell surface proteins (30). Trypsin was inactivated with 10% fetal bovine serum-supplemented medium, and cells were then washed with PBS.

Cell lysis was performed in radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing a mixture of protease inhibitors (Roche Applied Science). For immunoprecipitation after surface biotinylation, cell lysates were precleared with protein A/G-agarose beads (Santa Cruz Biotechnology) and incubated overnight at 4 °C with Ab:pPC5 or Ab:PC5 (dilution 1:100). Protein A/G-agarose beads were then added to the antigen-antibody complexes, incubated for 4 h, and washed six times with cold radioimmune precipitation assay buffer. Lysates of biotinylated TIMP-3-transfected cells were incubated with streptavidin-agarose beads (Sigma) overnight at 4 °C and washed with radioimmune precipitation assay buffer. Following the addition of reducing Laemmli sample buffer, the protein samples were heated, resolved by SDS-PAGE on a 10% Tricine gel, electrotransferred onto nitrocellulose, probed with streptavidin-HRP (1:20,000; GE Healthcare) or with rAb: TIMP-3 (1:2000) and a secondary HRP-conjugated anti-rabbit IgG antibody (1:10,000), and revealed by enhanced chemiluminescence (GE Healthcare). For immunoprecipitation of ADAMTS-4, cell lysates were precleared, incubated with the anti-ADAMTS-4 antibody (1:100), and then with protein A/G-agarose beads as described above. SDS-PAGE (10% glycine gel) and Western blotting were performed with the anti-ADAMTS-4 antibody (1:2000) as described below.

Western blotting experiments were made on media or cell lysates that were reduced in Laemmli buffer, heated, and resolved either on 10% glycine SDS-polyacrylamide gels or on 10% Tricine gels for detection of the prosegment of PC5A. Separated proteins were then electrotransferred onto nitrocellulose and probed either with Ab:pPC5 (1:2000), anti-Myc-HRP (1:5000), anti-Lefty (1:2000), anti-ADAMTS-4 (1:2000), rAb-TIMP-3 (1:2000), or mAb:V5-HRP (1:5000). Bound antibodies were detected with corresponding HRP-labeled secondary antibodies, the anti-rabbit IgG-HRP (1:10,000) or the anti-goat IgG-HRP (1:100,000) (Sigma), and revealed by enhanced chemiluminescence.

Immunocytochemistry—For immunocytochemistry, cells were plated on glass bottom culture dishes (MatTek) and then either left nontransfected or transfected the following day. When indicated, cell treatments were made 24 h after transfection as described above. 48 h post-transfection, the cells were washed three times with PBS. Cell surface labelings were made under nonpermeabilizing conditions by fixation with 3.7% paraformaldehyde for 10 min at room temperature. For intracellular immunolabelings, cells were fixed and permeabilized in methanol/acetone (1:1) for 3 min at -20 °C. Cells were then washed in PBS, incubated for 5 min in 150 mM glycine, washed once in PBS, and incubated for 30 min in 1% bovine serum albumin in PBS. Cells were incubated overnight at 4 °C with Ab:pPC5, Ab:pfurin, Ab:PC5, gAb:TIMP-3 (all diluted 1:100 in PBS, 1% bovine serum albumin), Ab:PC5B (1:200), anti-syndecan-2 (1:20), or mAb:V5 (1:200) and then washed four times with PBS. Antigen-antibody complexes were revealed by incubation for 45 min at room temperature with corresponding secondary antibodies, anti-rabbit IgG-, anti-mouse IgG-, or anti-goat IgG-coupled either to Alexa-fluor-555 (red), 488 (green), or 647 (blue) (Molecular Probes, Inc., Eugene, OR) as described in the figure legends. After several washes in PBS, cells were covered with 5% 1,4-diazabicyclo(2.2.2)octane (Sigma) in PBS/glycerol 90%, and immunofluorescence analyses were performed with a Zeiss LSM-510 confocal microscope. Quantifications of the fluorescence intensity were done with the freely available ImageJ software version 1.37 (Wayne Rasband, National Institutes of Health, Bethesda, MD).

In Situ Hybridization—10-12-µm-thick cryosections were prepared from adult mice (whole body sections), fixed in 4% formaldehyde, and hybridized as previously described (31, 32) with mouse sense (negative control) and antisense cRNA probes. The latter probes corresponded to the mouse PC5 coding region (807 bases; nucleotides 262-1068; NCBI accession number L14932 [GenBank] ) and mouse Lefty-1 (365 bases; nucleotides 713-1078; NCBI accession number NM_010094 [GenBank] ). The probes were synthesized using [35S]UTP and [35S]CTP (>1000 Ci/mmol; Amersham Biosciences). For autoradiography, the sections were dipped in photographic emulsion (NTB-2; Eastman Kodak Co.), exposed for 6-12 days, and developed in D19 solution (Kodak).

In Vitro Activity Assay—Enzymatic in vitro assays were performed in 100 µl of buffer (1 mM CaCl2, 25 mM Tris-HCl, 25 mM MES buffer, pH 7.5) at 37 °C in the presence of 50 µM substrate pyroglutamic acid-RTKR-7-amido-4-methylcoumarin and 80 µl of medium from Y1 cells treated or untreated with ACTH overnight. The release of free 7-amino-4-methylcoumarin was detected with a Spectra MAX GEMINI EM microplate spectrofluorimeter (Molecular Devices) (excitation, 360 nm; emission, 460 nm).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
HSPGs Are Required for the Cell Surface Anchoring of PC5A and PACE4—Our previous observations have shown that the secreted PC5A and PACE4 can be anchored to the cell surface through their CRD (17). In order to extend these results, the molecules responsible for this plasma membrane tethering were studied. COS-1 and CHO-K1 cells were transiently transfected with cDNAs encoding either full-length PC5A (FL-PC5A) or PACE4 (FL-PACE4), their CRD-deleted versions (PC5A-{Delta}C and PACE4-{Delta}C), or the CRDs alone (PC5A-CRD and PACE4-CRD), all containing a COOH-terminal V5 tag (supplemental Fig. S1). The COOH-terminal V5 tag did not affect the immunolocalization of PC5, since it was similar to that obtained with Ab:PC5 on cells expressing PC5A without the tag (not shown). Using an anti-V5 tag monoclonal antibody (mAb:V5), the cell surface localization of PC5A and PACE4 was analyzed by immunofluorescence (Fig. 1). In both COS-1 and CHO-K1 cells, there was an intense punctate cell surface staining of PC5A or PACE4 (Fig. 1, A, E, and G). However, this labeling was completely lost when the cells were transfected with cDNA constructs encoding the CRD-deleted versions of the convertases (Fig. 1D, insets in E and G). The CRDs seem to be sufficient for plasma membrane localization of each convertase, since overexpressing their CRDs alone gave a strong cell surface signal (Fig. 1, F and H). To determine how this binding occurs, COS-1 cells expressing FL-PC5A were treated with suramin, a highly sulfated compound or with heparinase III, which cleaves at the 1,4 linkages between hexosamine and glucuronic acid residues in heparan sulfate, thereby removing cell surface heparan sulfate chains. Such treatments resulted in the release of PC5A from the surface, as shown by the significant decrease in immunofluorescence staining (Fig. 1, B and C). These results indicate that HSPGs are candidate cell surface binding proteins of secreted PCs. The critical contribution of HSPGs was analyzed directly by using the CHO-pgsD-677 cells, which specifically lack heparan sulfate but still synthesize chondroitin sulfate (28). Immunolabelings of pgsD-677 cells expressing FL-PC5A, FL-PACE4, or the CRDs alone revealed no signal at the plasma membrane, demonstrating that PC5A and PACE4 are anchored at the cell surface through binding of their CRD to HSPGs (Fig. 1, I-L).


Figure 1
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  		FIGURE 1.
Cell surface tethering of PC5A and PACE4 through binding of their CRD to HSPGs. COS-1 (A-D), CHO-K1 (E-H) (heparan sulfate-positive), or CHO-pgsD-677 (I-L) (heparan sulfate-negative) cells were transiently transfected with either full-length PC5A (FL-PC5A) (A-C, E, and I), PC5A lacking its cysteine-rich domain (PC5A-{Delta}C) (D and E, inset), the cysteine-rich domain alone (PC5A-CRD) (F and J), FL-PACE4 (G and K), PACE4-{Delta}C(G, inset), or PACE4-CRD (H and L). PC5A, PACE4, and their derivatives (all tagged with a V5 epitope) are in the pIRES2-EGFP vector. Cell surface immunofluorescence of nonpermeabilized cells was performed using the mAb:V5 (red labeling). Nuclei of transfected cells are marked by the EGFP fluorescence (green). As indicated, in some experiments, cells were treated with 0.5 mg/ml suramin (B) or 0.5 units/ml heparinase III (C). Bars, 10 µm(A-D and I-L) and 20 µm(E-H).

 


Figure 2
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  		FIGURE 2.
HSPGs syndecans and CD44 increase cell surface tethering of PC5A. A-C, cell surface immunolabeling of PC5A-V5 in COS-1 cells co-transfected with FL-PC5A along with an empty vector (A and A'), syndecan-2 (B and B'), or CD44 (C) was performed using mAb:V5 as described in the legend to Fig. 1 (red labeling). D, quantification of the fluorescence intensity for cell surface PC5A in the presence of syndecan-2, syndecan-4, and CD44. Error bars, ±S.E. E-G, cell surface immunofluorescence of COS-1 cells co-transfected with FL-PC5A and syndecan-2. Immunolabelings were performed with the mAb:V5 (red) and rabbit anti-syndecan-2 (blue). Co-localization (arrows) of FL-PC5A (E) and syndecan-2 (F) is exemplified by the pink color in the merge image (G). Bars, 10 µm.

 
The plasma membrane-anchored HSPGs include those having a transmembrane domain, such as syndecans and CD44, and those bearing a glycosylphosphatidylinositol anchor, such as glypicans (33). Cell surface tethering of PC5A through glypicans was assessed by treating COS-1 cells expressing FL-PC5A with the phosphatidylinositol-specific phospholipase C. Such treatment led to a small decrease in the intensity of staining of PC5A, pointing out that these HSPGs were not the major plasma membrane PC5A binding proteins in COS-1 cells (Fig. S2). Pursuing our quest to find HSPGs that can anchor PC5A at the membrane, COS-1 cells were co-transfected with FL-PC5A together with syndecan-2, syndecan-4, or CD44 (Fig. 2). Cell surface immunofluorescence analysis of PC5A showed an increased intensity in the presence of all three HSPGs compared with co-expression with an empty vector (Fig. 2, A-C). Confocal analysis of z-series optical sections revealed that the punctate staining was enhanced over all surface areas of the cells, with possibly some preferential accumulation of PC5A in apical versus basolateral membranes (Fig. 2, A' and B'). Quantification of the fluorescence intensity revealed an increase of cell surface PC5A over control by ~75% with syndecan-2, by ~40% with syndecan-4, and up to 100% with CD44 (Fig. 2D). Furthermore, double labeling experiments showed the co-localization of PC5A with syndecan-2 at the cell surface of transfected COS-1 cells (Fig. 2, E-G). Finally, expression of syndecan-2 in pgsD-677 cells did not result in cell surface anchoring of PC5A (data not shown), probably because of the absence of N-acetylglucosaminyltransferase and glucuronyltransferase activities in this cell line (28). This emphasizes the critical importance of heparan sulfate chains for the association of PC5A at the cell surface.

The Prosegment-PC5A Complex Is Secreted and Anchored to the Cell Surface—Although the presence of PC5A at the cell surface could be necessary to process HSPG-bound proteins, we hypothesized that HSPGs may also be required for the activation of PC5A. Previous reports showed that within the ER, the zymogen proPC5A is autocatalytically cleaved at KKRTKR116{downarrow} (Fig. 3A, site 1°) (12, 24). This first zymogen cleavage is necessary to allow the protein to exit the ER for secretion. However, the exact intracellular site of activation of PC5A that requires cleavage at the internal second site, HSR-TIKR84{downarrow} (Fig. 3A, site 2°), is not known. Accordingly, we followed the maturation of the enzyme using antibodies raised against the prosegment of PC5A (Ab:pPC5) (24) as well as the catalytic domain (Ab:PC5) and COOH-terminal V5 tag (mAb: V5) (Fig. 3A). FL-PC5A was transiently expressed in COS-1 cells, and immunofluorescence was analyzed with both Ab:pPC5 and mAb:V5. Extensive co-localization of both prosegment and full-length protein was observed in ER-like and in paranuclear Golgi-like compartments as well as at the plasma membrane (Fig. S3), indicating that at the cell surface, some prosegment has not been cleaved at site 2° and is still associated with the enzyme. Using Ab:pPC5, we further analyzed the localization of FL-PC5A and its prosegment mutants R84A and R116A of the two zymogen cleavage sites (Fig. 3, B-E). Under these conditions, an intense cell surface punctate labeling of the prosegment was clearly observed with the wild type protein and with its R84A mutant but not for the R116A mutant (Fig. 3, B, D, and E). The R116A mutant cannot be processed at site 1° and remains in the ER, as previously reported (12). This is the first evidence that the prosegment can reach the cell surface in complex with the mature enzyme and suggests that the second zymogen processing event may occur in this compartment. These results are quite different from those obtained upon COS-1 expression of FL-furin followed by cell surface immunolabeling with an in house furin prosegment antibody [Ab:pfurin]. No furin prosegment signal was observed at the cell surface (Fig. 3C), in agreement with previous studies on its zymogen processing, which demonstrated that the secondary cleavage occurs in the TGN (4, 25). Expression of the furin construct was verified by intracellular immunolabeling and Western blotting using Ab:pfurin (data not shown).


Figure 3
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  		FIGURE 3.
PC5A is secreted and anchored to the cell surface without the second zymogen cleavage of its prosegment. A, schematic diagram depicting V5-tagged proPC5A, the first (1°) and second (2°) cleavage sites of the prosegment, and specific antibodies raised against the regions underlined by red arrows. SP, signal peptide; Pro, prosegment. B-E, cell surface immunofluorescence of COS-1 cells expressing FL-PC5A (B), FL-furin (C), prosegment first cleavage site mutant PC5A-R116A (D), or prosegment second cleavage site mutant PC5A-R84A (E) using Ab:pPC5 (B, D, and E) or Ab:furin (C) (red labelings). Bars, 10 µm. F, cell lysates and conditioned media of COS-1 cells transfected either with empty pIRES2-EGFP-V5 vector (pIRES-V5), PC5A-R116A (R116A-V5), PC5A-R84A (R84A-V5), FL-PC5A (PC5A-V5), or pre-proPC5 (ppPC5, aa 1-119) were separated by SDS-PAGE on 8% glycine (left) or 10% Tricine (right) gels, Western blotted (WB) with Ab:pPC5 or mAb:V5-HRP, and revealed by chemiluminescence.

 
Western blots of cell lysates and media of COS-1 cells expressing either the vector alone (pIRES) or its recombinants PC5A-R116A, PC5A-R84A, or wild type FL-PC5A were revealed by either mAb:V5 or Ab:pPC5 (Fig. 3F). We detected a broad band of PC5A migrating with an apparent molecular mass of ~105 kDa that is recognized by both antibodies. This suggested that it represents an unresolved mixture of processed PC5A at site 1° and unprocessed ER-associated proPC5A, as reported previously (12, 24). However, only the wild type PC5A and the second prosegment cleavage site mutant PC5A-R84A were secreted (Fig. 3F, bottom left), in agreement with the retention of the unprocessed R116A mutant in the ER. The nature of the prosegment produced was analyzed, using Ab:pPC5, by Western blots of COS-1 cells overexpressing either a secretory preprosegment ppPC5 (aa 1-119) or the above constructs. In cells overexpressing ppPC5, we observed two polypeptide bands, an upper one representing the preproform still containing the signal peptide (ppPC5), and a ~9 kDa band representing the prosegment pPC5, as reported (24). A similar ~9-kDa protein was also observed in cells expressing wild type PC5A or its R84A mutant but not the R116A mutant (Fig. 3F, middle right). This demonstrates that the prosegment is not produced in the R116A mutant, as expected, since cleavage at Arg116{downarrow} is the primary processing site. In the medium, the prosegment pPC5 of PC5A-R84A was detected, whereas that of the wild type PC5A was not. This indicated that the latter has undergone the second zymogen cleavage of its prosegment, since antibody Ab:pPC5 does not recognize the internally cleaved prosegment (Fig. 3F, bottom right). Taken together, these results demonstrate that (i) cleavage of the prosegment at site 2° does not seem to be a prerequisite for cell surface binding and secretion, and (ii) wild type PC5A can reach the cell surface in a complex with its prosegment, and the secreted form in the medium is likely to be the active form.

The second zymogen processing of the prosegment leading to activation of PC5A is thought to be autocatalytic, as for the first cleavage site. However, cell surface activation by cleavage at site 2° could in theory be performed by other convertases. To test this possibility, COS-1 cells were co-transfected with FL-PC5A and either FL-furin or FL-PACE4 (Fig. S4). Immunofluorescence, using Ab:pPC5 and mAb:V5 simultaneously, showed co-localization of both the prosegment and the catalytic domain at the cell surface independent from the presence of furin or PACE4 (Fig. S4). This indicates that the prosegment of PC5A is not cleaved in trans by other convertases during its transport in the secretory pathway and that autocatalytic activation of PC5A by cleavage at site 2° probably occurs at the plasma membrane.

Y1 Cells Exhibit Endogenous PC5 Prosegment at Their Cell Surface—Our results indicate that the activation of PC5A may follow a scheme different from that of the prototype convertase furin (4). PC5A is strongly expressed in the adrenals, and it was previously found that its mRNA is expressed in mouse adrenocortical Y1 cells (9, 34). Accordingly, we chose to analyze the localization and maturation state of endogenous PC5A in Y1 cells. Immunocytochemistry of Y1 cells revealed a punctate cell surface localization of both the prosegment and catalytic domains of endogenous PC5 (Fig. 4, A and B). We also show that the Y1 cell surface prosegment immunoreactivity is abolished in the presence of heparin (Fig. 4A, lower inset). These data confirm the critical importance of HSPGs for the cell surface localization of PC5 and its prosegment and extend this observation toward the realm of an endogenous setting.


Figure 4
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  		FIGURE 4.
Endogenous PC5 and its prosegment are localized at the plasma membrane of Y1 cells. A and B, cell surface immunofluorescence of endogenous PC5 prosegment or PC5 using antibodies against the prosegment Ab:pPC5 (A) or the catalytic domain Ab:PC5 (B), respectively (green labeling) in nonpermeabilized Y1 cells. A (upper inset), immunofluorescence performed without the primary antibody is shown as a control experiment. A, lower inset, immunofluorescence using Ab:pPC5 after heparin treatment of Y1 cells. N, nucleus; PM, plasma membrane. Bars, 10 µm.

 


Figure 5
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  		FIGURE 5.
ACTH-induced activation of PC5. A-D, cell surface immunofluorescence of PC5 prosegment (Ab: pPC5, red) in untreated cells (A), after ACTH (B), after decanoyl-RVKR-chloromethylketone (CMK) (C), or after both ACTH and CMK(D)treatments of Y1 cells.A and B(insets), immunofluorescence of PC5 catalytic domain using Ab:PC5 (red) in untreated cells (A, inset) or after ACTH treatment (B, inset). Bars, 10 µm. E, quantification of the fluorescence intensity obtained for the above (A-D) immunochemical experiments. A.U., arbitrary units. Error bars, ±S.E. F, in vitro enzymatic assay of PCs in conditioned media from untreated (1) or ACTH-treated (2) Y1 cells using the fluorogenic substrate pyroglutamic acid-RTKR-7-amido-4-methylcoumarin. RFU, relative fluorescence units. G, Y1 cells transfected with FL-PC5A were treated with ACTH or both ACTH and CMK, surface-biotinylated, immunoprecipitated (IP) with Ab:pPC5 or Ab:PC5, and resolved by SDS-PAGE and analyzed by Western blotting (WB) using streptavidin-HRP (SA-HRP). Trypsin treatment was used as a control for surface biotinylation. pPC5, PC5 prosegment.

 
The Zymogen Activation of PC5A Is Induced by ACTH—It was previously shown that PC5A mRNA is up-regulated in Y1 cells treated with ACTH or cAMP and that the mRNA levels of other PCs are not modulated by ACTH (34, 35). In order to confirm and extend these data to the protein level, the cell surface localization of PC5A in Y1 cells was tested before and after 16-h treatment with either 8-Br-cAMP (Fig. S5) or ACTH (Fig. 5). The data show that although the PC5 immunoreactivity is somewhat enhanced after these treatments (Fig. 5, insets in A and B; Fig. S5), that of the prosegment is reduced by more than 60% (Fig. 5, B and E; Fig. S5). Time course experiments showed that the Y1 cell surface pPC5 immunoreactivity is significantly decreased after 3 h following stimulation with 8-Br-cAMP, whereas an increase in cell surface PC5 was observed during the same treatment time (Fig. S5). PC5A and PC5B differ only in their COOH terminus, the latter having an extended CRD, a transmembrane domain, and a cytosolic tail (36). Since they share the same prosegment and catalytic domains, it is difficult to discriminate between the two isoforms using Ab:PC5 or Ab:pPC5. However, Northern blot analyses demonstrated that adrenals and naive or ACTH-treated Y1 cells express mostly PC5A (34). Furthermore, intracellular immunofluorescence of Y1 cells, using a PC5B-specific antibody (Ab:PC5B), showed that the localization of PC5B is restricted to a Golgi-like compartment in both untreated and ACTH-treated cells. This indicates that the cell surface pPC5 signal obtained with Ab:pPC5 in untreated Y1 cells mostly represents the prosegment of PC5A, which is cleaved and released from the active enzyme upon ACTH treatment (Fig. S6A). It is also possible that ACTH treatment triggers endocytosis of the HSPG-bound prosegment-PC5A complex, resulting in the endosomal cleavage of the prosegment, thereby allowing the mature active PC5A to recycle back to the cell surface. To test the latter possibility, in the presence or absence of ACTH, Y1 cells were treated with ammonium chloride (NH4Cl), a lysosomotropic weak base that inhibits endosomal acidification. NH4Cl did not affect the immunocytochemical pPC5 labeling at the plasma membrane of Y1 cells in untreated or ACTH-treated conditions (Fig. S6B). This makes it less likely that the in trans autocatalytic prosegment cleavage at site 2° by PC5 and hence its activation requires the acidic conditions found in endosomes.


Figure 6
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  		FIGURE 6.
Regulated substrate cleavage by cell surface-activated PC5A. A, in situ hybridization of consecutive whole body sections (left) or of adrenals and uterine horns (right) from adult mice using antisense cRNA probes of mouse PC5 (top) or mouse Lefty-1 (bottom) coding regions. Tissues where PC5 and Lefty-1 co-localized are indicated by arrows. B-D, Western blotting (conditioned media) of human Lefty-A (B), endothelial lipase (EL-Myc) (C), or ADAMTS-4 (D) that were co-transfected with an empty vector (mock) or with FL-PC5A in CHO-FD11 cells (left) or transfected alone in Y1 cells (right). Cleavage of the substrates is increased after ACTH or 8-Br-cAMP treatment of Y1 cells. As indicated, Y1 cells were also treated with the convertase inhibitor CMK or heparin (Hep.). B, P, pro-Lefty; L, long form; S, short form. C, F, full-length endothelial lipase (EL). Arrowhead, cleaved EL. D, lysates of Y1 cells transfected with ADAMTS-4 were immunoprecipitated with the anti-ADAMTS-4 antibody prior to Western blotting (WB). P, pro-ADAMTS-4. Arrowhead, cleaved ADAMTS-4.

 
The ability of ACTH to induce activation of PC5A was tested by analysis of the in vitro activity toward the fluorogenic substrate pERTKR-7-amido-4-methylcoumarin (24). The data revealed that ACTH treatment significantly increased the endogenous PC-like activity found in the medium of Y1 cells, as compared with control untreated cells (Fig. 5F). To confirm these data in a biochemical setting, we analyzed the pPC5 and PC5A immunoreactivity at the cell surface of Y1 cells expressing FL-PC5A (Fig. 5G). We had to overexpress FL-PC5A, since our Ab:pPC5 is not very sensitive to detect low levels of detergent-extracted proteins. Cell surface biotinylation, immunoprecipitation with either Ab:PC5 or Ab:pPC5, and Western blotting with streptavidin-HRP revealed that although PC5 is detected in all conditions, pPC5 was seen only in naive Y1 cells and not in cells treated with ACTH (Fig. 5G). Since decanoyl-RVKR-CMK is a general PC inhibitor (37, 38), we used this compound to address the question of whether prosegment cleavage at site 2° requires an in trans activity of PC5. Accordingly, treatment of Y1 cells with 25 µM CMK resulted in an increased immunoreactivity of pPC5 in the absence (+30%) or presence (+80%) of ACTH (Fig. 5, C-E), suggesting inhibition of prosegment cleavage at site 2°. Should the CMK disrupt the prosegment-PC5A complex, we would have expected a reduction of cell surface pPC5 immunoreactivity. The fact that we observed the reverse implies that the CMK inhibits the secreted active PC5A in the medium and hence would prevent its intermolecular cleavage at site 2° of the cell surface-associated prosegment-PC5A complex. In support of this conclusion, we also observed a drastic increase in cell surface biotinylated pPC5 upon ACTH + CMK treatment but no effect on biotinylated PC5 (Fig. 5G).

The Cell Surface PC5A Processing of Lefty, Endothelial Lipase, and ADAMTS-4—We next addressed the question of which precursors would be processed in a regulated fashion by active PC5A at the cell surface. We first selected precursors known to bind HSPGs and that were reported to be processed by PC5A, such as the TGF-β-like growth factor Lefty (39), the high density lipoprotein-degrading endothelial lipase (EL) (19), and the metalloprotease ADAMTS-4 (40).

Most TGF-β-like proteins are produced as precursors, which are cleaved to release the COOH-terminal monomeric active proteins. This cleavage occurs at RXXR{downarrow} motifs by PCs (41). The mutation of both of the potential consensus sequences for PC cleavage in Lefty protein allowed these sites to be recognized as authentic cleavage sites (26). Compared with other PCs tested, PC5A has been found to be the most efficient convertase at processing Lefty (26, 39). In order to better appreciate the possible enzyme-substrate relationship between PC5 and Lefty, we first compared the distribution of their mRNAs in mouse tissues using in situ hybridization in adult whole animal sections. The tissues, such as adrenal gland, small intestine, lung, and thymus, that are rich in PC5 also express Lefty (Fig. 6A, left). Co-localization of PC5 and Lefty was also evident by in situ hybridization analysis of uterine horn and especially adrenal cortex (Fig. 6A, right). Therefore, we first co-expressed PC5A and Lefty in CHO-FD11 cells lacking endogenous furin, revealing that PC5A can completely process pro-Lefty (P) to produce both long (L) (~34-kDa) and short (S) (~28-kDa) forms (39) (Fig. 6B, left). In order to assess whether this processing can be regulated, we transfected Lefty cDNA in Y1 cells and analyzed its processing by Western blot before and after stimulation by ACTH or 8-Br-cAMP (Fig. 6B, right). In absence of stimulation, pro-Lefty was processed by an endogenous activity of Y1 cells only into the long form. However, upon stimulation, we clearly observed the generation of the short form and, especially with 8-Br-cAMP, a significant reduction in the level of pro-Lefty. This cleavage is almost completely blocked upon overnight incubation of Y1 cells with 25 mM CMK and is significantly reduced following heparin incubation of control and 8-Br-cAMP-activated cells (Fig. 6B, right). Thus, we conclude that the processing of pro-Lefty requires binding to HSPGs and that activated cell surface PC5A is critical for the generation of the ~28-kDa short form.


Figure 7
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  		FIGURE 7.
PC5A generates multiple PCSK9 cleavage products. A-D, Western blotting (WB) analyses of PCSK9-V5 and its cleavage products using the mAb:V5-HRP. A, Western blot of conditioned media from COS-1 cells co-transfected with PCSK9-V5 and either an empty vector or FL-PC5A (right) or from Y1 cells transfected with PCSK9-V5 alone (middle and right). As indicated, Y1 cells were also treated with the convertase inhibitor CMK or transfected with the PCSK9 noncleavable mutant R218S. The schematic diagram indicates the PC-cleavage site Arg218{downarrow} and the putative COOH-terminal cleavage. B, PCSK9-V5 or its mutants R218S-V5, R469W-V5, or R496W-V5 were transiently co-transfected in HEK293 cells with either an empty vector, FL-PC5A (no tag), or FL-furin. Detection of the cleavage forms was performed by Western blotting of the conditioned media. C, Western blot analysis of conditioned media from HEK293 cells stably expressing PCSK9-V5 and singly or doubly transfected with the indicated cDNA constructs. Cells were either left untreated or treated with TIMP-3, NH4Cl, or GM6001. D, Western blot of secreted PCSK9-V5 and its cleaved forms in conditioned media of Y1 cells transiently co-transfected with PCSK9 and ADAMTS-4 (TS4). The co-transfected cells were either untreated or treated with CMK or 8-Br-cAMP, and their media were analyzed by Western blotting. M, mature PCSK9. Arrowheads at ~54 kDa indicate the PC-cleaved PCSK9 at Arg218{downarrow}. The arrows point to the ~34-kDa fragment.

 
The phospholipase EL that requires binding to HSPGs to degrade the circulating high density lipoprotein (42), has been recently shown to be well cleaved, and hence inactivated, in cell lines by furin, PC5A, and PACE4 (19, 43), and such processing requires the presence of the CRD of PC5A (17). By Western blotting of Myc-tagged EL, we now show that full-length EL (F) is not processed into its inactive ~32-kDa form in either CHO-FD11 or Y1 cells (Fig. 6C). However, co-expression of PC5A and EL in CHO-FD11 cells or stimulation of Y1 cells transiently expressing EL with 8-Br-cAMP resulted in complete or partial cleavage into the ~32-kDa form (Fig. 6C).

The metalloprotease ADAMTS-4 (aggrecanase 1) is synthesized as a latent precursor protein that requires activation through removal of its prodomain before it can degrade components of the extracellular matrix (44). It has been reported that furin, PACE4, and PC5A can process pro-ADAMTS-4, resulting in its activation (40). ADAMTS-4 mRNAs are highly up-regulated upon ACTH or cAMP treatments of Y1 cells (35). Therefore, it represented a logical substrate to analyze in our system. Indeed, as shown by Western blotting of CHO-FD11 cells expressing human ADAMTS-4, the unprocessed pro-ADAMTS-4 can be completely cleaved upon co-expression with PC5A. Furthermore, cleavage of pro-ADAMTS-4 into its ~75-kDa active form can be observed when Y1 cells were stimulated with 8-Br-cAMP but abrogated if CMK is also present. All of the above data confirm that PC5A can process pro-Lefty, EL, and pro-ADAMTS-4 and that such processing by endogenous PC5A in Y1 cells depends on their prior activation by 8-Br-cAMP.

PC5A Inactivates PCSK9 via Direct and Indirect Cleavages—The proprotein convertase PCSK9 has recently been recognized as a major player in the regulation of cholesterol homeostasis via its ability to enhance the degradation of the LDLR (3). It was recently shown that furin and PC5A can process PCSK9 at Arg218{downarrow}, resulting in a truncated inactive ~54-kDa PCSK9-{Delta}N218 form missing the NH2-terminal 218 amino acids (45). We therefore used this substrate to test the importance of the cell surface localization of PC5A for this cleavage. Accordingly, Western blotting experiments showed that upon expression of PCSK9 in COS-1 cells, mature PCSK9 (M) was secreted, but its truncated PCSK9-{Delta}N218 form was only apparent upon co-expression of PC5A (Fig. 7A, left). Interestingly, we also observed a shorter novel V5 tag-positive form with an apparent molecular mass of ~34 kDa. This suggested that PC5A or an enzyme activated by PC5A can generate this fragment. We next used a similar paradigm in Y1 cells to test the possible regulation of these cleavages following transient transfection with PCSK9 cDNA. The data show that although untreated Y1 cells secrete both PCSK9 and PCSK9-{Delta}N218, stimulation with 8-Br-cAMP increased the level of PCSK9-{Delta}N218 and especially that of the ~34-kDa form (Fig. 7A, middle and right). Thus, activation of cell surface PC5A is associated with increased production of both products. When the same cells expressed the PC-resistant natural mutant form PCSK9-R218S, we noted that neither cleavage occurred in the absence or presence of 8-Br-cAMP or in the presence of CMK (Fig. 7A, middle and right). This demonstrates that the production of the ~34-kDa form requires prior cleavage of PCSK9 at Arg218{downarrow}, which was shown to result in a structurally modified PCSK9-{Delta}N218 product (45), probably exposing a new protease-sensitive site. We next showed that the production of the ~34-kDa form by PC5A was not obtained by PACE4 and that such processing requires the presence of HSPGs, since it was observed in CHO-K1 cells but not in CHO-pgsD-677 cells (Fig. S7).

Two natural mutations of PCSK9, R469W (46), and R496W (47), reported to cause hypercholesterolemia, seemed to occur at PC-like sites exhibiting basic residues at the P1- and P6-positions, namely HSGPTR469 and RSGKRR496, respectively. We thus tested them for their effects on the generation of the ~34-kDa form in HEK293 cells. However, neither of them modified the ability of PC5A to produce this product (Fig. 7B). Since these were the only possible PC candidate sites left, we hypothesized that PC5A could activate a latent cell surface protease that would then be responsible for this processing event. This is not unprecedented, since PC5A and furin were reported to activate matrix metalloproteases (48), as well as ADAM (49) and ADAMTS (44) families of metalloproteases. We thus tested the effect of the tissue inhibitor of metalloproteases TIMP-3 known to inhibit many members of the above families of metalloproteases. The addition of 20 µM TIMP-3 to the media of HEK293 cells stably expressing PCSK9 (HEK293-PCSK9) and transiently expressing PC5A completely abolished the production of the ~34-kDa form (Fig. 7C, left). Since microarray analysis of Y1 cells activated by cAMP revealed the up-regulation of PC5A and ADAMTS4 but down-regulation of TIMP-3 mRNAs (35), we focused our attention on ADAMTS-4 as a possible candidate for the generation of the ~34-kDa product. This was all the more plausible, since we already demonstrated that cell surface PC5A in Y1 cells activated pro-ADAMTS-4 (Fig. 6D). Indeed, when expressed in HEK293-PCSK9 cells, PC5A, furin, or ADAMTS-4, but not ADAMTS-5, generated this ~34-kDa product (Fig. 7C). Note the large reduction in the level of the ~54-kDa PCSK9-{Delta}N218 intermediate in the presence of ADAMTS-4, suggesting that this metalloprotease is quite adept in cleaving PCSK9-{Delta}N218 to generate the ~34-kDa product. Although some of the transfected ADAMTS-4 seems to be active in HEK293 cells, as deduced from the generation of the ~34-kDa form, co-expression of either PC5A or furin resulted in a large increase in the level of the latter form (Fig. 7C, middle). We also demonstrated that the generation of the ~34-kDa product by ADAMTS-4 occurs at the cell surface, since it is blocked in the presence of the general metalloprotease inhibitor GM6001 and does not occur in acidic compartments, since incubation with 10 mM NH4Cl had no effect (Fig. 7C, middle; Fig. S7).


Figure 8
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  		FIGURE 8.
Decreased levels of the metalloprotease inhibitor TIMP-3 and PC5 prosegment at the plasma membrane of cAMP-treated Y1 cells. A, Y1 cells transfected with TIMP-3 were left untreated or treated with 8-Br-cAMP, surface-biotinylated, immunoprecipitated (IP) with streptavidin (SA)-agarose, and analyzed by SDS-PAGE and Western blotting (WB) with rabbit Ab:TIMP-3 (lower panel). The upper panel shows the Western blot, using rabbit Ab:TIMP-3, of the corresponding whole cell lysates. The arrows point to the ~24-kDa unglycosylated TIMP-3, the arrowheads mark the ~28-kDa glycosylated form of TIMP-3, and the asterisks denote a nonspecific signal. B, cell surface immunofluorescence of PC5 prosegment (Ab:pPC5, red) and TIMP-3 (goat Ab:TIMP-3, green) in untreated (left) or 8-Br-cAMP-treated (right) Y1 cells. Co-localization areas are exemplified by arrows and by yellow color in the merged image. Bars, 10 µm.

 
Finally, we tested the effect of overexpressing ADAMTS-4 in Y1 cells in the presence or absence of 8-Br-cAMP. The results show that overexpressed ADAMTS-4 can generate the ~34-kDa polypeptide even in the absence of cAMP treatment and that such cleavage is inhibitable by CMK (Fig. 7D, left). This suggests that some of the overexpressed ADAMTS-4 can get activated by endogenous Y1 cell convertases. However, upon activation of Y1 cells by 8-Br-cAMP, we observed a dramatic increase in the level of the ~34-kDa protein, concomitant with the loss of both mature PCSK9 and PCSK9-{Delta}N218 (Fig. 7D, right), probably due to a large increase in both PC5A and ADAMTS-4 activities. Importantly, the ~34-kDa PCSK9 COOH-terminal secreted product was detected in human and mouse plasma but not in plasma derived from PCSK9 knockout mice,4 suggesting that it is not just the result of overexpressing PCSK9 in cell lines.

The substantial increase in ADAMTS-4 activity observed upon cAMP activation of Y1 cells (Fig. 7D, right) probably resulted from the combined increase in cell surface PC5A activity and reduction in the concentration of the inhibitory TIMP-3. Upon overexpression of TIMP-3 in Y1 cells, Western blot of cell lysates showed the presence a major unglycosylated ~24-kDa and minor glycosylated ~28-kDa forms, as reported (50), but that the levels of both forms did not vary in the presence of cAMP (Fig. 8A, top). However, Western blot analysis of biotinylated cell surface proteins revealed mostly the ~24-kDa TIMP-3 form and demonstrated that level of the latter is greatly diminished in the presence of cAMP (Fig. 8A, bottom), possibly due to its secretion to the extracellular matrix. We previously showed that PC5A interacts with TIMPs and that such complex is recruited to the cell surface via HSPGs (17). Therefore, we first showed by immunocytochemistry of Y1 cells that the prosegment pPC5A (hence inactive PC5A) and TIMP-3 co-localize at the cell surface (Fig. 8B, left). However, cAMP treatment provoked the loss of both proteins (Fig. 8B, right), probably rationalizing the observed activation of PC5A and ADAMTS-4.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previous work dealing with the zymogen processing of overexpressed PC5A and furin revealed that their prosegments are cleaved at the primary site (site 1°) within the neutral environment of the ER. This cleavage generates a tight binding heterodimer between the inhibitory prosegment and the enzyme, resulting in an inactive complex competent for transport to the Golgi apparatus (12, 20, 21, 23, 24). The second cleavage of the prosegment at an internal site (site 2°) is required for activation of furin and PC5A. However, mutations of site 2° had no detectable effect on furin post-TGN trafficking and recycling or on the secretion of PC5A (23, 24). In addition, although cleavage at site 2° leading to the activation of furin requires the mildly acidic and calcium-containing environment of the TGN (23, 25), the specific conditions and subcellular localization required for PC5A, PACE4, and PC7 activation remain undefined. In the present work, we extend our previous studies, include the activation of endogenous PC5A, and identify the cell surface as a likely site where secondary cleavage of the prosegment at HSRTIKR84{downarrow} occurs resulting in the activation of PC5A. The secondary cleavage requires Arg84 (17) and seems to be occurring at neutral pH, since NH4Cl had no effect (Fig. S6B). The similar behavior of PACE4 (Fig. 1) suggests that its activation may also follow the same pathway as PC5A. This goes along with the preference of PC5A (51) and PACE4 (52-54) for cleaving substrates at neutral pH and is consistent with an extracellular function for these enzymes. Notably, this puts PC5A and PACE4 in a unique regulated activation pathway. This is quite different from that of furin that is constitutively activated in the acidic environment of the TGN and can cycle between the cell surface and the TGN as an active enzyme (4).

It was previously shown that ACTH treatment of Y1 cells resulted in a substantial increase in PC5A mRNA transcripts (34). Furthermore, ACTH-induced increase of PC5A mRNA has been ascribed to the cAMP/PKA pathway (35). In the present study, we demonstrated that aside from transcriptional activation, ACTH or cAMP treatments resulted in the zymogen activation of PC5A (Figs. 5 and 6). This regulated intermolecular catalytic activation of PC5A likely occurs via a PC5A-mediated cell surface cleavage event at site 2°. The mechanism behind the cAMP activation of PC5A remains obscure but may implicate a higher level of constitutively active secreted PC5A, which would then activate in trans the cell surface enzyme (Fig. S4). Alternatively, cAMP may induce a better exposure of the processing site following binding of PC5A to phosphorylated HSPGs that may result in a conformational change and/or modification of their oligomerization state (55). A prevailing hypothesis for the different roles of cell surface HSPGs is that, like heparin, heparan sulfate chains can catalyze molecular interactions at the plasma membrane (56, 57). Indeed, upon stimulation with 8-Br-cAMP, the loss of the prosegment appears to be relatively fast, taking place in less than ~3 h (Fig. S5), suggesting that conformational changes of HSPGs result in a suitable environment favoring the activation of PC5A. Thus, although no report appeared on the regulated zymogen activation of furin or PC7, the zymogen activation of PC5A and possibly of PACE4 are regulated events.

Substrates of Cell Surface PC5A—In this work, we presented evidence for the regulated processing of four human precursor substrates of PC5A, namely Lefty, EL, ADAMTS-4, and PCSK9 (Figs. 6 and 7). In each case, the data show that activation of cell surface PC5A results in an increased processing of these proteins. Furthermore, co-localization of Lefty and PC5A expression in specific adult mouse tissues (Fig. 6A) adds further credence to our model. Therefore, we would predict that the cell surface cleavage of these proteins represents a regulatory step of their activity and hence may modulate their functions. As a corollary, perturbation of such a regulatory event may result in pathology. Our data revealed that although PC5A can activate Lefty and ADAMTS-4, it results in the inactivation of either EL or PCSK9. In the case of Lefty, we further demonstrated that activation of PC5A by cAMP results in an increased level of the short form of Lefty (Fig. 6B), which, different from the long from, can induce the MAP kinase pathway (26). ACTH or cAMP treatments of Y1 cells provoke a tremendous change in the transcriptional activity of its endogenous genes (35). Accordingly, our data exposed a cascade of events leading to complete cleavage of the ~63-kDa PCSK9 into a ~34-kDa fragment (Fig. 7). This fragment is of physiological significance, since we found a similar ~34-kDa form of PCSK9 circulating in human and mouse plasma.4

Linkage between Cell Surface HSPGs, PC5A, and TGF-β-like Substrates—The predominant role of HSPGs is to catalyze molecular encounters through a number of different mechanisms, such as modification of protein conformation and sequestration of protein ligands at the cell surface (58). The latter provides a way to restrict the function of protein ligands to a specific location (56, 58). Herein we demonstrated that PC5A can be anchored at the cell surface via HSPGs. In view of the redundancy of cleavage specificity found between basic aa-specific PCs, binding of the secreted PC5A and PACE4 to HSPGs distinguishes them as cell surface-associated convertases. The binding affinity of HSPGs for proteins is determined by their pattern of sulfation that is modeled by specific sulfotransferases and extracellular endosulfatases (33, 59). This specific sulfation pattern is recognized via linear and/or three-dimensional clusters of basic residues on the surface of proteins. Stretches of basic aa are found in the CRD of PC5A and PACE4. However, attempts to mutate a potential heparin binding motif (KRXRK690 to EEXEE690) in the CRD of PC5A failed to significantly abrogate cell surface binding, suggesting the presence of a three-dimensional concentration of basic residues forming an heparin-binding motif in the CRD.4

Recently, it became apparent that PC5A probably plays a role during embryonic development (9). During development, furin, PACE4, PC5, and PC7 are co-expressed at distinct sites together with bone morphogenic proteins and TGF-βs (60). PC5A was also shown to be critical for the activation of TGF-β-like proteins, such as BMP4 (61-64). Since HSPGs are important for the bioactivity of TGF-β-like proteins (33), it is still a matter of speculation whether cell surface PC5A is a major player in some of these processing events.

In conclusion, this study presents a novel pathway for the regulated activation/inactivation of secretory proteins through cell surface cleavage at specific sites by PC5A. This pathway adds plasticity to the repertoire of substrates of the PCs that could process precursors either at the TGN, cell surface, endosomes, or secretory granules. In view of the specific requirement of HSPGs for the activation of PC5A, the regulated cell surface processing presented in this work is distinct from that reported for membrane-bound furin (4). This mechanism adds selectivity to the substrates that would be processed by PC5A and may act in complementary and/or sequential fashion to furin cell surface processing, as would be the case of bone morphogenic proteins and Lefty. Taken together, these data enlarge our understanding of the already complex interplay between redundancy of the basic aa-specific PCs, their subcellular localization, and spatio-temporal regulation of substrate cleavage.


   FOOTNOTES
 
* This research was supported by Canadian Institute of Health Research Grant MGP-44363 and Canada Chair 201652. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S7. Back

1 Supported by postdoctoral fellowships from the Conscil de recherches en sciences naturelles et en géníe du Canada/Natural Sciences and Engineering Research Council of Canada and Institut de recherches cliniques de Montréal-Pizzagalli. Back

2 To whom correspondence should be addressed: Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Ave. W., Montreal, Quebec H2W 1R7, Canada. Tel.: 514-987-5609; Fax: 514-987-5542; E-mail: seidahn{at}ircm.qc.ca.

3 The abbreviations used are: PC, proprotein convertase; aa, amino acid(s); Ab, antibody; mAb, monoclonal antibody; CRD, cysteine-rich domain; HSPG, heparan sulfate proteoglycan; EL, endothelial lipase; TIMP, tissue inhibitor of metalloproteases; ACTH, adrenocorticotropic hormone; FL, full-length; CMK, chloromethylketone; TGN, trans-Golgi network; ER, endoplasmic reticulum; 8-Br-cAMP, 8-bromo-cyclic AMP; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; TGF, transforming growth factor; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Back

4 G. Mayer, J. Hamelin, M.-C. Asselin, A. Pasquato, E. Marcinkiewicz, M. Tang, S. Tabibzadeh, and N. G. Seidah, unpublished results. Back


   ACKNOWLEDGMENTS
 
We are grateful to Sergio Grinstein, John S. Mort, Linda Troeberg, Daniel J. Rader, Weijun Jin, and Eok-Soo Oh for cDNA plasmids and John Creemers for PC5A/B antibodies. We thank all of the members of the Seidah Laboratory for helpful discussions and Brigitte Mary for secretarial assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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