©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Elements That Direct Specific Processing of Different Mammalian Subtilisin-like Prohormone Convertases (*)

(Received for publication, June 20, 1995)

An Zhou Luc Paquet Richard E. Mains (§)

From the Departments of Neuroscience and Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

PC1 and PC2 are two important subtilisin-like prohormone convertases (PC) that undergo differential endoproteolytic processing steps and sequentially mediate proopiomelanocortin (POMC) processing. To investigate the structural elements directing the processing of different PCs, we constructed a series of mutant and chimeric PC proteins and expressed them in cell lines with different patterns of expression of endogenous PCs: AtT-20, hEK293, and hLoVo cells. The COOH-terminally truncated PC1 underwent efficient proregion cleavage and rapid secretion in all three cell lines, while proregion cleavage and secretion were completely blocked in an active-site mutant of PC1. The truncated PC1 produced dramatic changes in POMC processing in AtT-20 cells. PC2 with the potential oxyanion hole Asp residue changed to Asn was processed and altered several aspects of POMC processing in a manner similar to that of wild-type PC2. PC1 protein with its proregion substituted with that of furin was cleaved after its proregion, producing active PC1 enzyme. A similar furin/PC2 fusion protein underwent proregion cleavage at low efficiency. By contrast, when the proregions of PC1 and PC2 were substituted with one another, both fusion proteins failed to cleave the foreign prosequences, were unable to undergo oligosaccharide maturation, and remained in the ER. Although inactive PC mutants could theoretically function as dominant negatives, none interfered with the processing of endogenous active PCs or with POMC processing. We conclude that the COOH-terminal of PC1 plays an important role in the routing or storage of PC1, the proregions of these PC proteins are replaceable in a molecule-specific manner, removal of proregion is essential for routing and for endoproteolytic activity, and the role of the potential oxyanion hole in PC2 is still unclear.


INTRODUCTION

Virtually all known mammalian bioactive peptides are synthesized first as precursor proteins in which product peptides are flanked by cleavage sites, most often paired basic amino acids. In different neuroendocrine tissues, depending on the availability of different endoproteases, precursors are cleaved into tissue-specific sets of final products. Proopiomelanocortin (POMC) (^1)is the common precursor of several important peptide hormones, including ACTH, alpha-MSH, and beta-endorphin. In anterior pituitary corticotropes, major POMC-derived peptides include ACTH and beta-lipotropin, whereas in intermediate pituitary melanotropes, ACTH is further processed into alpha-MSH, and beta-lipotropin is processed to beta-endorphin(1-31) and beta-endorphin(1-27)(1, 2) .

In the last few years, a group of subtilisin-like serine endoproteases that cleave specifically at sequences containing paired basic amino acids have been identified in mammalian tissues(3, 4, 5, 6, 7, 8, 9) . The group includes PC1 (prohormone convertase 1, also called PC3), PC2, PC4, PC5/6, furin, and PACE4. PC1 and PC2 are predominantly detected in neuroendocrine tissues. Using as a model the AtT-20 mouse corticotrope tumor cell line, which has very high levels of endogenous PC1 and barely detectable PC2, we and others have established the specific and sequential roles of PC1 and PC2 in POMC processing(8, 9, 10, 11, 12, 13, 14, 15) .

Subtilisin-like PCs share a common domain structure: a heterogeneous 10-kDa NH(2)-terminal proregion, a highly conserved 55-kDa subtilisin-like catalytic domain featuring a serine-histidine-aspartic acid catalytic triad, and a carboxyl-terminal domain that varies greatly in length and sequence among the different PCs (3) (Fig. 1). For example, PC1 has a much longer carboxyl-terminal domain than PC2, and the carboxyl-terminal domain of furin contains transmembrane and cytoplasmic regions (Fig. 1). Like many other proteases, subtilisin-like PCs are initially synthesized as inactive proenzymes. The proregion is generally believed to function as an intramolecular chaperone, guiding the correct folding of the protease domain and preventing it from being active until the proregion is removed in the appropriate compartment(16, 17) .


Figure 1: Domain structures of native and mutated prohormone convertases. For each protein, the NH(2)-terminally shaded region represents the prosequence; the openregion represents the subtilisin-like protease domain and the dottedregion represents the carboxyl-terminal extension. PC1DeltaC and the PC1DeltaC portion in Fur-PbulletPC1DeltaC end at amino acid D of PC1.



In AtT-20 cells, PC1 undergoes very rapid proregion cleavage in the ER and a later cleavage to remove the carboxyl-terminal domain in immature granules(11, 18, 19) . When PC2 is expressed in AtT-20 cells by transfection, cleavage of its proregion does not take place until the protein reaches immature granules(11, 20, 21) . The oligosaccharide chains of the two PCs also mature at very different rates, with PC2 oligosaccharide maturation much slower than PC1. No detailed studies have addressed the structural elements that distinguish the maturation of PC1 and PC2.

In the present study, we investigated the importance of the carboxyl-terminal domain of PC1 by deleting it. We investigated the specificities of the proregions of PC1 and PC2 by substituting them with foreign proregions. We also examined the importance of an unique Asp in PC2 located in the predicted oxyanion hole, which in all other PCs is Asn. Mutated and chimeric PCs (Fig. 1) were transfected into three different cells; AtT-20 cells have furin and PC1 with a regulated secretory pathway, hEK293 (human embryonic kidney) cells have furin but no regulated secretory pathway, hLoVo (human colon carcinoma) cells have no active furin or PACE4 and no regulated secretory pathway(22, 23) . The biosynthetic processing of the PCs and their effects on POMC processing in AtT-20 cells were characterized. The results demonstrate unique roles for the NH(2)- and COOH-terminal domains of the PCs and highlight the importance of enzyme activity for correct routing and storage of the PCs.


MATERIALS AND METHODS

Construction of Vectors Encoding Mutant PC Proteins

Carboxyl-terminally Truncated PC1 (PC1DeltaC), Active-site Mutated PC1 (PC1-S382A), and Oxyanion Hole Mutated PC2 (PC2-D309N)

The in vivo removal of the carboxyl-terminal domain of PC1 presumably takes place at the carboxyl-terminal side of -Asp-Arg-Arg, followed by removal of basic residues by carboxypeptidase H(24) . To delete nucleotides encoding amino acids 617-752 and the 3`-untranslated region of rat PC1, a 725-base pair fragment of pBS.rPC1 (8) was prepared by amplification with polymerase chain reaction (PCR), using a sense primer CAGGCACCTCAGCTTCTG (PC1 nucleotides 1292-1309) and an antisense primer GGGACTCGAGCTAGTCATTCTGGACAGTAT (PC1 nucleotides 2004-1988, extended by the underlined region, which generated a stop codon (bold) and a XhoI site). The fragment was digested with StyI and XhoI and ligated into pBS.rPC1 digested with the same restriction enzymes, resulting in plasmid pBS.rPC1DeltaC (ending at Asp of PC1). The active-site mutant of PC1 (PC1-S382A) and the oxyanion hole mutant of PC2 (PC2-D309N) were created by two-step sequential PCR(25) .

Proregion-substituted Chimeric Proteins

To create proregion-substituted chimeric proteins, the gene splicing by overlap extension (SOE) technique was employed(26) . Fragments were amplified by PCR using as templates the pBluescript plasmids carrying the cDNAs of the desired PCs, cloned into pBluescript and verified by sequencing. Table 1delineates the amino acid sequences at the proregion cleavage site for the five chimeric proteins generated in this study. In each construct, the proregion (bold) was preceded by its authentic signal peptide and ended with the amino acids of its authentic cleavage site. The mature enzyme started immediately after the cleavage site of the foreign proregion, except for the PC1-P*bulletPC2 construct, in which the cleavage site followed the PC2 sequence. For expression in mammalian cells, all recombinant cDNAs were inserted into the pCIS.2CXXNH vector(27) . For all mutants created in this study, the sequences of PCR-amplified regions were verified by sequencing.



Tissue Culture of AtT-20, hEK-293, and hLoVo Cells and Stable Transfection

AtT-20 cells and hEK-293 cells were cultured as described previously(10) . hLoVo cells were purchased from American Type Culture Collection (ATCC) and carried in Dulbecco's modified Eagle's medium/Ham's F-12 supplemented with 10% NuSerum and 10% fetal clone serum (HyClone Laboratories, Logan, UT). The pCIS plasmids carrying various cDNAs were cotransfected with a neomycin resistance plasmid (10) into AtT-20 or hEK-293 cells using lipofection, followed by drug selection with G418 (0.5 mg/ml)(10) . G418-resistant cell lines were screened by either Western blot or immunostaining using antibodies directed against amino acids 359-373 of PC1 or amino acids 626-638 of PC2, respectively(10) . hLoVo cells were transiently transfected by lipofection. Cells were plated 1 day prior to transfection, and first incubated in complete serum-free medium (CSFM) for 1 h. Meanwhile, 10 µg of cDNAs of interest were suspended in 10 µl of CSFM and gently mixed with 10 µl of Lipofectin. The mixtures were incubated at room temperature for 20 min and then diluted to 10 µg/ml with CSFM. Cells were incubated with the Lipofectin-DNA mixture for 6 h, followed by another 18-h incubation in growth medium before analysis. The immunostaining procedure followed the protocol described by Milgram et al.(27) .

Biosynthetic Labeling and Analyses of PC1 and PC2 Proteins and POMC Peptides

Processing of the transfected PC proteins in various cell lines, as well as POMC processing in AtT-20 cells expressing the various PCs, were characterized by biosynthetic labeling as described previously (11) . Briefly, cells were incubated in methionine/cysteine-deficient media for 5 min to deplete the corresponding intracellular amino acids pools, then pulse-incubated with [S]methionine/cysteine (1 mCi/ml, 1000 Ci/mmol; in vitro cell labeling mix, Amersham Corp.) for 15-30 min with or without subsequent chase incubations in nonradioactive complete media. Cellular proteins were extracted with a preheated buffer (95 °C) containing 50 mM sodium phosphate, pH 7.4, 1% SDS, 50 mM beta-mercaptoethanol, 2 mM EDTA plus protease inhibitors(19) . S-Labeled wild-type, mutant, and chimeric PC1 and PC2 proteins and POMC-derived peptides were immunoprecipitated with specific antisera(11) . For PC proteins, immunoprecipitated samples were fractionated on 10% SDS-PAGE slab gels and analyzed by fluorography. POMC-related peptides were fractionated on 12% tube gels using a borate-acetate buffer and quantitated by liquid scintillation counting(10) .


RESULTS

Processing and Function of Carboxyl-terminal Truncated PC1 (PC1DeltaC)

The endoproteolytic processing and secretion of rat PC1DeltaC in hEK293 cells and hLoVo cells were examined using a pulse/chase metabolic labeling paradigm (Fig. 2). First, when expressed in cells that do not express PC1 endogenously (hEK293 cells; Fig. 2, topleft), the 70-kDa PC1DeltaC protein showed rapid intracellular conversion to the 63-kDa PC1DeltaC, followed by rapid secretion of the 63-kDa form. To determine whether the processing of PC1DeltaC protein observed in hEK293 cells might be mediated by the endogenous furin, PC1DeltaC was expressed into hLoVo cells (Fig. 2, top right), a cell line that lacks endogenous furin or PACE4 activity(22, 23) . Cells were biosynthetically labeled, and PC1 protein was immunoprecipitated. Even in hLoVo cells, the PC1DeltaC protein was efficiently cleaved.


Figure 2: PC1DeltaC processing in AtT-20, hEK293, and hLoVo cells. hEK293 cells, hLoVo cells (top), and AtT-20 cells transfected with pCIS. PC1DeltaC and nontransfected (wt) AtT-20 cells (middle) were labeled with Met/Cys for 15 min (P) and then incubated in non-radioactive medium for 1 h. For hLoVo cells, an extended chase incubation in medium was performed. Cell extracts (c) and media (m) from pulse and chase incubations were collected and extracted. Samples were immunoprecipitated with antiserum against a peptide within the catalytic domain of PC1 and analyzed by SDS-PAGE followed by fluorography. Similar results were obtained from another two independent experiments. Bottom, AtT-20 cells. Cells were labeled with [S]Met/Cys and chased in nonradioactive medium as above, followed by an additional 30-min incubation in nonradioactive medium with or without (±) 50 nM PMA. proPC1, 87-kDa proform of PC1; proPC1DeltaC, 70-kDa proform of PC1DeltaC.



Next, the processing of PC1DeltaC was examined in cells that express PC1 endogenously (AtT-20 cells; Fig. 2, middleleft). In AtT-20 cells expressing PC1DeltaC protein (Fig. 2, middleright) with a 15-min pulse labeling, the 70-kDa proPC1DeltaC was converted to 63-kDa PC1DeltaC to an extent identical to the conversion of the endogenous full-size PC1 (87-kDa PC1 to 81-kDa PC1); the endogenous 81-kDa PC1 intermediate was later cleaved at the carboxyl terminus to the 63-kDa PC1 (a step barely started during the 1-h chase). During the subsequent 1-h chase incubation, about half of the truncated PC1 protein was secreted into the medium, while the majority of the endogenous PC1 was still retained in the cells. Thus, newly synthesized PC1DeltaC protein was secreted more rapidly than the endogenous full-size PC1. Therefore it was important to examine whether stimulation of AtT-20 PC1DeltaC cells with secretagogues would significantly increase the secretion of PC1DeltaC protein (Fig. 2, bottom). The results showed that the majority of PC1DeltaC protein never reached mature secretory granules, potentially missing the chance to alter POMC processing.

To determine if expression of PC1DeltaC yields a protein that is enzymatically active in POMC processing, POMC-derived peptides in AtT-20 cells expressing PC1DeltaC were analyzed (Fig. 3). Expressing PC1DeltaC greatly accelerated the conversion of POMC to its smaller products, namely ACTH and glycosylated ACTH (Fig. 3) and beta-endorphin (not shown). In addition, the secretion of the smaller peptides produced in cells expressing PC1DeltaC was stimulatable by secretagogues (not shown). Therefore, some of the truncated PC1 protein is active within the regulated secretory pathway, as seen previously for cells overexpressing full-sized PC1(11) .


Figure 3: Processing of POMC in AtT-20 cells expressing PC1DeltaC. Aliquots of samples of the nontransfected and PC1DeltaC-expressing AtT-20 cells collected from the biosynthetic labeling experiment described in Fig. 2were immunoprecipitated with antiserum against ACTH and analyzed by SDS-PAGE(10) . ABI, adrenocorticotropin biosynthetic intermediate; gACTH, glycosylated ACTH. Data from a 1-h chase incubation for cells (A) and medium (B) are shown. Similar experiments were performed another three times with identical results.



Processing of Active-site Mutated PC1

To examine whether a mutant form of proPC1 that should not yield active enzyme could have its proregion cleaved, the serine residue in the catalytic triad of PC1 was mutated to alanine (S382A). When this PC1-S382A was expressed in hEK293 cells (Fig. 4, bottomright), which do not express endogenous PC1 but do express furin, an accumulation of 87-kDa PC1 protein inside the cells was observed. Even 2 h after the pulse, there was no detectable production of 81-kDa PC1 protein; neither intact precursor nor processed form was seen in the medium. To examine whether endogenous PC1 might activate the proPC1-S382A, or perhaps that proPC1-S382A might interfere with the cleavage of the proregion of the endogenous PC1, AtT-20 cells expressing proPC1-S382A were studied. Despite the simultaneous production of a high level of endogenous PC1, the biosynthetic processing of PC1-S382A showed a similar pattern as in hEK293 cells. In the biosynthetic labeling experiment shown in Fig. 4, the cells were first pulse-labeled with [S]Met/Cys for 15 min and then incubated in nonradioactive medium for another 1 and 4 h. PC1 immunoprecipitable proteins were analyzed. The top band in Fig. 4(fourthlane from right) represents the sum of the endogenous proPC1 and transfected proPC1-S382A mutant. At a time when the endogenous proPC1 was completely processed to PC1, as demonstrated by the identical labeling experiment on nontransfected cells (Fig. 4, topleft), the mutated PC1 remained unprocessed. This proPC1-sized band was never seen in the medium. To examine whether the mutant protein ever reached the medial Golgi apparatus and acquired mature oligosaccharide chains, cell extracts from the 4-h chase incubation were subjected to endoglycosidase H treatment (Fig. 4, bottomleft). The mutated proform-sized PC1 present after a 4-h chase was still sensitive to endoglycosidase H, while the endogenous 63-kDa PC1 became resistant to endoglycosidase H. The pattern of POMC processing in PC1-S382A cells did not show any changes, as compared with nontransfected cells (not shown).


Figure 4: Maturation of PC1-S382A in AtT-20 cells and hEK293 cells. hEK293 cells stably expressing PC1-S382A protein were biosynthetically labeled with [S]Met/Cys for 15 min followed by a 2-h incubation in non-radioactive medium. AtT-20 cells were similarly labeled with 1- and 4-h chase incubations, respectively. Samples were immunoprecipitated with PC1 antiserum. For nontransfected (wt) and PC1-S382A-expressing AtT-20 cells, immunoprecipitated PC1 proteins from the 4-h chase incubation were also subjected to endoglycosidase H digestion (1 milliunit/100 µl of reaction mixture, 17 h at 37 °C) before analysis by SDS-PAGE (lowerleft). The arrowhead indicates the 87-kDa proPC1-S382A protein.



Oxyanion Hole Mutated PC2 (PC2-D309N)

In subtilisin, the formation of the oxyanion hole involves the side chain of Asn(28) . This Asn is conserved in the corresponding position in Kex2 and in all known PCs with the exception of PC2, in which the corresponding residue (amino acid 309) is an Asp. To examine if this Asp may contribute to the slowness of proregion cleavage of PC2 and have an impact on the specificity of PC2, we replaced it with an Asn and stably transfected the mutant into AtT-20 cells. As shown in Fig. 5A, the 75-kDa proPC2-D309N protein was processed into 64-kDa mature PC2 at a rate indistinguishable from wild-type PC2. The mutated PC2 also altered POMC processing with similar kinetics and final products as that of wild-type PC2 (Fig. 5B; (10) ). PC2-D309N stimulated the cleavage of ACTH(1-39) to ACTH(1-13)NH(2) and the cleavage of the Lys-Arg in beta-lipotropin to produce beta-endorphin(1-31) (Fig. 5B). In addition, PC2-D309N stimulated the cleavage of the Lys-Lys pair in beta-endorphin(1-31) to produce beta-endorphin(1-27), and the cleavage of the Arg-Lys in the NH(2)-terminal of POMC to produce (3)MSH (not shown; analyses as in (10) ). In addition, the release of PC2-D309N and PC2 was stimulatable in response to secretagogues to a similar extent (Fig. 5A), and release of the new peptides produced in response to PC2-D309N was also stimulatable (Fig. 5B, middle). Since the results with PC2-D309N were identical to those with the wild-type PC2(10, 11) , the exogenous PC2 mRNA in the PC2-D309N cells was subjected to reverse transcription and DNA sequencing, which verified that the PC2-D309N cells were indeed expressing the desired mutant form of PC2.


Figure 5: Processing of oxyanion-hole mutant PC2 protein and its effect on POMC processing. AtT-20 cells stably transfected with PC2-D309N cDNA were labeled with [S]Met/Cys for 30 min and then incubated in nonradioactive medium as indicated. A, PC2 immunoprecipitation. Top, to examine PC2 processing, 1- and 4-h chase incubations were performed; bottom, to examine stimulation of secretion after a 2-h chase, cells were incubated an additional hour with or without 50 nM PMA. B, ACTH and beta-endorphin immunoprecipitations. Top, cell extracts of nontransfected cells and PC2-D309N cells were examined after a 3-h chase incubation, focusing on smaller peptides; middle, medium from the PMA stimulation of PC2-D309N cells in panelA above were analyzed with the NH(2)-terminal ACTH antiserum; bottom, cells and medium from a 4-h chase incubation of the PC2-D309N cells were analyzed with the beta-endorphin antiserum. The pattern is identical to that of wild-type PC2-expressing cells (not shown; (10) ). Similar results were obtained in two additional experiments.



Furin-proregion Substituted PC1 and PC2 Proteins

Proregions of furin, PC1 and PC2 are nearly identical in length. Furin has more homology to PC1 and to PC2 than PC1 and PC2 have to each other(5) . All known mammalian subtilisin-like PCs share a similar amino acid sequence at the proregion cleavage sites (RXKR), raising the possibility that some proregions could be interchangeable among PCs.

When Fur-PbulletPC1DeltaC and Fur-PbulletPC2 proteins were expressed in AtT-20 cells (Fig. 6A), both Fur-PbulletPC1DeltaC and Fur-PbulletPC2 underwent substantial proregion cleavage, although at a slower rate than their natural counterparts possessing the native proregions. During a 15-min pulse labeling period, a significant fraction of the Fur-PbulletPC1DeltaC was processed to a PC1DeltaC-sized form (63 kDa) and this conversion went to completion within 1 h. There was no significant acceleration of secretion, however, of 63-kDa PC1 processed from Fur-PbulletPC1DeltaC. The Fur-PbulletPC2 protein was also slowly cleaved, yielding a 71-kDa form, instead of the 64-kDa PC2 protein seen in the wild-type PC2 processing (Fig. 6A). The proform-sized and processed Fur-PbulletPC2 were both secreted during a 4-h chase incubation.


Figure 6: Biosynthetic processing of Fur-PbulletPC1DeltaC and Fur-PbulletPC2 proteins in AtT-20 cells and hLoVo cells. Cells were labeled with [S]Met/Cys and then incubated in non-radioactive medium for the indicated periods of time. Samples were immunoprecipitated with appropriate antisera, fractionated by SDS-PAGE, and visualized by fluorography. A, nontransfected AtT-20 cells and AtT-20 cells stably expressing Fur-PbulletPC1DeltaC were labeled for short times (5 and 15 min) to examine processing at the very early stage of biosynthesis. AtT-20 cells stably expressing Fur-PbulletPC2 were labeled for 30 min and then incubated for 1 or 4 h in non-radioactive medium. For comparison, a similar labeling experiment was carried in AtT-20 cells expressing wild-type PC2. B, hLoVo cells were transiently transfected with Fur-PbulletPC1DeltaC or Fur-PbulletPC2. Preliminary experiments showed that the processing of Fur-PbulletPC1DeltaC protein in hLoVo cells was relatively slower than in AtT-20 cells, so a 30-min pulse labeling period was employed. Ctr, samples from mock transfection. C, AtT-20 cells expressing Fur-PbulletPC1DeltaC were labeled with [S]Met/Cys for 15 min and then incubated in non-radioactive medium for 1 h. Media were immunoprecipitated with antiserum against ACTH and analyzed by SDS-PAGE tube gels. Similar results were obtained in another two independent experiments.



To evaluate processing of chimeric PC proteins in a cell line lacking functional furin, both Fur-PbulletPC1DeltaC and Fur-PbulletPC2 were transfected into hLoVo cells. As seen in Fig. 6B, Fur-PbulletPC1DeltaC protein was indeed processed in hLoVo cells, while there was no significant processing of Fur-PbulletPC2 during the 3-h incubation.

The biosynthetic processing of POMC in AtT-20 Fur-PbulletPC1DeltaC and Fur-PbulletPC2 cells was analyzed. Expression of Fur-PbulletPC1DeltaC greatly accelerated the conversion of POMC to ACTH and did not enhance the conversion of ACTH to ACTH(1-13)NH(2) (Fig. 6C), as seen in AtT-20 PC1DeltaC cells (Fig. 3), indicating that the Fur-PbulletPC1DeltaC protein was processed into PC1 protein, which was active within the secretory pathway. There were no changes in POMC processing in AtT-20 Fur-PbulletPC2 cells (not shown).

Proregion-swapped PC1 and PC2 Chimeric Proteins

We have previously shown that, when stably expressed in AtT-20 cells, PC2 undergoes slow but substantial proregion cleavage(11) . For both PC1 and PC2, the processed forms are secreted into the medium under basal conditions, and release of 63-kDa PC1 and 64-kDa PC2 can be stimulated with secretagogues.

AtT-20 cells expressing proregion swapped PC1 or PC2 protein were first studied by biosynthetic labeling. Nontransfected cells and cells expressing PC1-PbulletPC2 were pulse-labeled for 15 min with [S]Met/Cys and then incubated in nonradioactive medium for another 2 h. Since the prosequence of PC2 is almost identical in length to the prosequence of PC1, PC2-PbulletPC1 and wild-type proPC1 cannot be resolved. The most intense 87-kDa band in Fig. 7(top; fourthlane from left) observed during the 15-min pulse represents the sum of the endogenous proPC1 and PC2-PbulletPC1. As was seen for the processing of the PC1-S382A protein, the chimeric PC2-PbulletPC1 protein was not processed to any detectable extent and was easily detectable after the 2-h chase. There was no difference in the production of 81- or 63-kDa PC1 between nontransfected cells and PC2-PbulletPC1 cells. In AtT-20 PC1-PbulletPC2 cells (Fig. 7, middle), without interference from endogenous PC2, it was clear that the chimeric protein stayed at the size of the proform and was not seen in the medium. Since cleavage of the proregion might only occur at a site whose sequence is tied to the specificity of the enzymatic region, an additional construct was expressed, with the PC1 proregion but with the cleavage site as well as the enzyme from PC2 (PC1-P*bulletPC2; Fig. 7, bottom); however, this hybrid molecule was also not processed in AtT-20 cells.


Figure 7: Biosynthetic processing of proregion-swapped PC1 and PC2 proteins in AtT-20 cells. AtT-20 cell lines expressing wild-type PC2, proregion-swapped PC1 or PC2 proteins and nontransfected cells were labeled and analyzed as described in Fig. 1. For nontransfected (wt) and PC2-PbulletPC1 cells: 15-min pulse, 2-h chase (PC1 antiserum); for PC2, PC1-PbulletPC2, and PC1-P*bulletPC2, 30-min pulse, 3-h chase (PC2 antiserum). Similar results were seen in another two experiments.



Fig. 8illustrates immunostaining of PC1, PC2 and mutant PC1 or PC2 proteins expressed in AtT-20 cells. The endogenous PC1 and the transfected natural form of PC2 were clearly seen in the processes where secretory granules reside(26, 28) . There was significant PC2 immunofluorescence in the processes, as well as over cell bodies of cells expressing Fur-PbulletPC2. For PC1DeltaC- and Fur-PbulletPC1DeltaC-expressing cells, increased PC1 immunofluorescence was distributed over cell bodies and processes. Both proregion-swapped PC1 and PC2 proteins were distributed diffusely over cell bodies, and they were never seen in the processes, no matter how high the expression level.


Figure 8: Immunostaining of PC1 and PC2 proteins in AtT-20 cells expressing wild-type or mutated PC1 and PC2. All exposures were for the same times and prints were processed identically; with longer exposure times, the pattern for endogenous PC1 in nontransfected AtT-20 cells (52) is the same as shown for cells expressing PC1DeltaC. A, C, E, and F, PC1 immunostaining; B, D, and G, PC2 immunostaining. Cell lines: A, nontransfected; B, wild-type PC2; C, PC2-PbulletPC1; D, PC1-PbulletPC2; E, PC1DeltaC; F, Fur-PbulletPC1DeltaC; G, Fur-PbulletPC2.




DISCUSSION

The subset of mammalian subtilisin-like enzymes involved in prohormone processing clearly includes PC1 and PC2. In contrast, furin, another member of this family of enzymes, does not appear to be involved in secretory granule-associated processing events. PC1 and PC2 differ from each other in very important functional ways. PC1 is activated by pro-sequence cleavage in the ER within minutes of its synthesis, and PC1 initiates prohormone processing early in peptide biosynthesis in the trans-Golgi network. By contrast, PC2 is only activated in the trans-Golgi network or immature secretory granules and mediates relatively late steps in biosynthetic endoproteolysis. This work establishes that there are important roles played by the amino- and carboxyl termini of PC1 and PC2 in routing and storage, and that the enzymatic activity of the PCs can also be crucial to routing and storage. Although PC1 and PC2 both function in secretory granules, the proregions of PC1 and PC2 are not interchangeable, while the proregion of the Golgi enzyme furin can replace the proregions of PC1 and PC2.

Role of the COOH Terminus

The subtilisin catalytic core is a 35-kDa domain. Eukaryotic subtilisin-like prohormone convertases have more complicated carboxyl-terminal domains than bacterial subtilisins, which suggests they subserve important functions in regulating the properties of PCs. Eukaryotic PCs function intracellularly within specific regions of the secretory pathway, whereas the bacterial enzymes function only after secretion. For example, a COOH-terminally shortened PC1 has pH and Ca optima consistent with an ability to function in more mature granules (30) . In all three cell lines tested, the PC1DeltaC protein underwent proregion cleavage as did the wild-type enzyme; the simplest interpretation is that proregion cleavage of PC1 is an intramolecular autocatalytic event that does not require the carboxyl-terminal domain or another identified enzyme.

The bulk of POMC cleavages occur in secretory granules, where cleavage of the carboxyl terminus of PC1 occurs. PC1DeltaC was quite active in increasing POMC processing when expressed in AtT-20 cells. The major effect of PC1DeltaC expression on POMC processing was identical to one of the effects of full-sized PC1 overexpressed in AtT-20 cells; the conversion of POMC to ACTH and beta-endorphin was increased. In contrast, overexpression of PC1DeltaC failed to increase production of ACTH(1-13)NH(2), whereas overexpression of full-size PC1 did increase production of this smaller peptide(11) . Since newly synthesized PC1DeltaC protein was secreted more rapidly than the endogenous full-size PC1, and since stimulating AtT-20 PC1DeltaC cells with secretagogues did not significantly increase the secretion of PC1DeltaC protein, the majority of PC1DeltaC protein never reached mature secretory granules, therefore missing the chance to mediate further POMC processing. Thus, the carboxyl-terminal domain of full-size PC1 appears to enhance delivery or storage of PC1 in secretory granules.

Inactive PC Mutants Do Not Interfere with the Processing of the Endogenous Active PC Proteins or POMC Processing

We had hypothesized that the PC1-S382A mutant might function as a dominant negative mutant in AtT-20 cells, blocking maturation or function of the endogenous native PC1. However, when expressed in AtT-20 cells, the PC1-S382A did not interfere with the processing of the endogenous PC1 or with the processing of POMC, nor was PC1-S382A cleaved by the endogenous PC1. The result that PC1-S382A was incapable of proregion cleavage is in agreement with similar studies on active-site mutated furin(31) , PC2(32, 33) , and Kex2 endoprotease(34, 35) , although the serine-to-cysteine mutant of bacterial subtilisin was able to undergo processing of the proregion in vivo(36) . In another study (37) , some PC1-S382A was cleaved at an abnormal site within the proregion when expressed in hEK293 cells, and secretion of both cleaved and unprocessed PC1-S382A was reported. In this work, we extracted cells in a boiling SDS buffer and included EDTA to block possible PC enzyme activity (including endogenous furin), since previous studies established that milder buffers do not prevent cleavages during subsequent analyses(19) .

The PC1-PbulletPC2 chimera expressed in AtT-20 cells did not undergo proregion cleavage. Endogenous PC1 was apparently unable to rescue the chimeric protein. Likewise, maturation of the endogenous PC1 protein was unaltered by coexpression of the unprocessed chimera. Similar results have been obtained in studies on furin; cotransfection of inactive furin mutants with active furin did not result in processing of the mutants(38, 39) . In contrast, inactive prosubtilisin-like enzymes are sometimes activated by active enzymes in the same cells (33, 40) , and separately expressed proregions can actually guide the correct folding of proregion-deleted mutants(41, 42) .

Replacing PC2 Oxyanion Hole Asp with Asn Resulted in a Fully Active PC2 Protein

The role of the oxyanion-hole Asn of subtilisin-like endoproteases has been studied only in subtilisin and Kex2 protease (43, 44) . Based on the assumption that Asp of PC2 can only function after it is protonated in an acidic environment, it has been hypothesized that this residue plays a crucial role in the secretory granule-associated activation and function of PC2(3) . Thus, one might predict that PC2-D309N could be activated earlier than normal in the regulated secretory pathway, that the mutant protein would be nonfunctional in secretory granules, or that the mutant enzyme might never become activated at all. In fact, the processing pattern of the PC2-D309N protein and the several changes in POMC processing were indistinguishable from AtT-20 cells expressing wild-type PC2(10) . Because this result was quite unexpected, the exogenous PC2 mRNA was rescued from the stably transfected AtT-20 cells and sequenced, to verify the presence of the mutation. Replacing the oxyanion hole Asn with Asp in Kex2 did not alter cleavage of the proregion but did reduce the k of the enzyme for a synthetic substrate substantially(44) . One possible interpretation is that PC2 has a different oxyanion binding site, or it may function without the oxyanion site(44) . A substantial loss in PC2 activity might still produce major changes in POMC processing, given the high levels of PC2 expression achieved.

Proregions of PCs Are Replaceable in a Molecule-specific Manner

The role of the proregion of bacterial subtilisins as an intramolecular chaperone is well established(17) . Rehemtulla et al.(45) replaced the proregion of furin with the proregion of PC2, creating a chimera that was unprocessed and inactive, and suggested that the proregions of subtilisin-like PCs are not interchangeable. However, the boundaries of the signal peptide, proregion, and active enzyme were not precisely maintained in this PC2-PbulletFur construct(45) . In this work, several chimeric proteins were generated to address the role of the proregion in PC activation, being careful to retain the accepted boundaries of domains (Fig. 1), as in the corresponding studies with bacterial enzymes(40, 41) . These results demonstrated that in both AtT-20 cells and hLoVo cells, the Fur-PbulletPC1 chimeric protein was processed into an enzymatically active PC1 protein. Despite the fact that PC1 and PC2 function in secretory granule-associated processing, their proregions are not interchangeable. Our results with PC1-P*bulletPC2 demonstrated that preserving the authentic cleavage site of PC2 in a chimeric protein was not adequate to enable the cleavage of the proregion to occur. Therefore, PC2 is unique among PCs in that its proregion is cleaved extremely slowly(11) , the neuroendocrine-specific protein 7B2 is a potential inhibitor or activator(46, 47, 48) , and it has Asn instead of Asp in the oxyanion hole(8, 49, 50) .

Within the proregion, there are several components that could influence whether or not a particular prosegment could guide the folding of the downstream endoprotease domain into an active enzyme: the sequence of the whole prosegment, its secondary and tertiary structure, the amino acids at the cleavage site, plus interactions with other proteins. Fig. 9illustrates the sequence alignment of proregions of PC1-6, PACE4, and furin from many species(51) . For all mammalian subtilisin-like prohormone proteases, the proregions and catalytic domains are separated by the RXKR motif (positions -4 to -1 from the cleavage site). Interestingly, all PCs except PC2 commonly have a basic residue at the -6 position, while in PC2, this position is either phenylalanine or tyrosine. The catalytic domain of PC2 may not tolerate a positively charged residue at the -6 position of the cleavage site, whereas other PCs require the positive charge, so that chimeric proteins involving PC2 will not be efficiently processed. In support of this assumption, the PACE4 catalytic domain preceded by the proregion of PC1 underwent proregion removal when expressed in AtT-20 cells. (^2)In fact, the corresponding substrate binding pocket S6 in PC2 has fewer negatively charged amino acids. Chimeric proteins with altered cleavage site residues and/or changes in substrate binding sites may be necessary to clarify this question.


Figure 9: Sequence comparisons. Sequences listed corresponding to amino acids -1 to -50 from proregion cleavage site (indicated by the arrow). Amino acids that appear solely in PC2 are illustrated in the toprow; amino acids present in all other PCs but not in PC2 are listed in the middlerow; the bottomrow lists amino acids unique to PC1. S1 through S6 stand for predicted substrate binding pockets(51) ; P-1 to P-6 indicate the positions of amino acids from the proregion cleavage site.




FOOTNOTES

*
This study was supported by National Institutes of Drug Abuse Grant DA-00266 (to R. E. M.) and a grant from the Medical Research Council of Canada (to L. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should addressed: Dept. of Neuroscience, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-6938; Fax: 410-955-0681; dick.mains{at}qmail.bs.jhu.edu

(^1)
The abbreviations used are: POMC, proopiomelanocortin; PC, prohormone convertase; ER, endoplasmic reticulum; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; ACTH, adrenocorticotropic hormone; alpha-MSH, alpha-melanocyte-stimulating hormone; PAGE, polyacrylamide gel electrophoresis; CSFM, complete serum-free medium.

(^2)
A. Zhou, C. A. Berard, and R. E. Mains, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Betty Eipper and Martin Schiller for many helpful discussions, Rich Johnson for help constructing expression vectors, Carla Berard for help with cell culture, Andrew Quon and Marie Bell for general lab assistance, and Zina Garrett for laboratory administrative management.


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