Structural Elements of PC2 Required for Interaction with Its Helper Protein 7B2*

Xiaorong ZhuDagger , Laurent Muller, Richard E. Mains§, and Iris Lindberg

From the Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112 and the § Departments of Neuroscience and Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The structures of the eukaryotic subtilisin protease family members can be divided into four distinct domains as follows: the proregion, the catalytic domain, the P domain, and the carboxyl-terminal region. Although these enzymes are evolutionarily related, only prohormone convertase 2 (PC2) requires 7B2 for activation. To examine the potential contribution of each domain of PC2 to PC2-7B2 interactions, we performed sequential deletions, site-directed mutagenesis, and domain swapping to replace individual domains or particular amino acids of pro-PC2 with the corresponding segments/amino acids of pro-PC1. These chimeras and mutant enzyme molecules were then expressed in AtT-20 cells and analyzed for 7B2 binding, maturation ability, and enzymatic activity. The results revealed that 1) the PC2 proregion is required but is not sufficient to confer 7B2 binding; 2) the P domain is required for the stabilization of PC2 structure and is not exchangeable with the P domain of PC1; and 3) the carboxyl-terminal domain is not involved in 7B2 binding. Site-directed mutagenesis of pro-PC2 further showed that a single residue replacement in the catalytic domain, Tyr-194 right-arrow Asp, prevented pro-PC2 from binding 7B2 and blocked activation. This residue is present within a loop rich in aromatic amino acids which appears to be on the surface of the molecule as extrapolated from the crystal structure of subtilisin. This loop may represent the primary recognition site for 7B2 within the catalytic domain.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The processing of many peptide hormones and other protein precursors is mediated by a family of subtilisin-like serine endoproteases that act through cleavage at paired or multiple basic residues. Two members of this family, prohormone convertase 1 (PC1,1 also known as PC3) and 2 (PC2), are the major proteinases involved in the cleavage of many hormone precursors (reviewed in Ref. 1). These enzymes have been found to function sequentially in the biosynthesis of neuropeptides; PC1, which is activated in the endoplasmic reticulum, is believed to act early in the processing pathway, whereas PC2, which is activated only in the trans-Golgi network/immature secretory granules, is thought to be involved in the later stages of prohormone proteolysis (2, 3). Like other members of this protease family these two enzymes exhibit four distinct structural domains (Fig. 1) as follows: a poorly conserved amino-terminal proregion, a highly conserved subtilisin catalytic domain, a less well-conserved middle or P domain, and a non-conserved carboxyl terminus (1, 4-6). The domain boundaries are distinguishable by comparison of enzyme sequences from different species (7).

A unique feature of PC2 as opposed to other members of this family is that another protein, 7B2, is involved in its biosynthesis (8, 9) and is required for the expression of enzymatic activity (10). The neuroendocrine protein 7B2, whose expression is restricted to the central nervous system and to endocrine tissues (11-13), is a bifunctional molecule. Its amino-terminal domain is responsible for the facilitation of maturation of pro-PC2 (10), and its carboxyl-terminal peptide is a potent inhibitor of PC2 (14-16). We have previously reported on the structural features of 7B2 required for its interaction with pro-PC2/PC2 and provided evidence that the proline-rich region in the interior of this molecule (residues 88-95) along with a flanking putative alpha -helix are pivotal in assisting pro-PC2 maturation (17).

PC1 and PC2 are closely evolutionarily related, yet PC1 does not require 7B2 for its maturation and activation. To determine what structural element(s) unique to PC2 are involved in the PC2-7B2 interaction, we compared the sequences of all known PC2s, including that of Drosophila PC2,2 with those of PC1 and furin. These efforts identified several regions unique to PC2s. To examine the potential contribution of these PC2-specific sequences to 7B2 binding (as well as the contributions of the proregion, the catalytic region, the P domain, and the carboxyl-terminal tail of pro-PC2), we used sequential deletion and domain swapping as well as site-directed mutagenesis to replace individual domains or particular amino acids of pro-PC2 with the corresponding segments or individual amino acids of pro-PC1. These chimeras and mutant enzyme molecules were then expressed in neuroendocrine cells and analyzed for 7B2 binding, for maturation, and for enzymatic activity. Except for the carboxyl-terminal region, all of the other PC2 domains, i.e. the proregion, the catalytic domain, and the P domain of PC2, were implicated in successful folding and/or PC2-7B2 interactions. We further identified a single amino acid in the catalytic domain, Tyr-194, as particularly critical to PC2-7B2 binding.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Construction of PC2 Mutants-- pRc/CMV (Invitrogen) was used in constructing all PC2-derived mutants. The serial deletions, domain swapping, and point-directed mutation constructs were created by PCR-mediated procedures as described earlier (10). The PC2 construct lacking the P and carboxyl-terminal domains and the construct lacking the PC2 carboxyl-terminal domain only (hereafter referred to as PC2Delta PC and PC2Delta C, respectively (Fig. 1)) were made using a common amino-terminal primer (PC2HindIII), 5'-GGGCCAAGCTTCACTCCCAAAGAAGGATGG-3'; the carboxyl-terminal primer used in PC2Delta PC (amino acids 1-452) was 5'-CGCGCTCTAGACTACACCATGGCACCTGCATC-3'; and for PC2Delta C (amino acids 1-593), 5'-CGCGCTCTAGACTATGTGCCGTGAAGCATCAG-3'. The PCR-generated fragments were inserted into pRc/CMV between HindIII and EcoRI sites. For domain-swapped mutants, the amino-terminal primer 5'-GGGCCGAATTCTTTCACTCCCAAAGAAGG-3' (mPC2A-EcoRI), and the carboxyl-terminal primer 5'-GGGCGCTCTAGATTAATTCTCCTCATTTAGGAT-3' (mPC1-XbaI end) were used in making PC2[PC1-PC] and PC2[PC1-C] (Fig. 1). To make the chimera PC2[PC1-PC], which has the P and C domains of PC2 replaced with those of PC1, two rounds of PCR were performed. Two independent PCRs were involved in the first round. In one reaction, the template was mouse PC2, and the primers were mPC2A-EcoRI and mPC2PC1-P2 (5'-GCCAAATCCAAATCGGCTATTAAATTCCAGGCCAAC-3'. In another reaction, the template was mPC1 and the primers were mPC1-XbaI and mPC2PC1-P1 (5'-GTTGGCCTGGAATTTAATAGCCGATTTGGATTTGGC-3'). The PCR products of both reactions were purified as described previously (10). These two fragments were then pooled together and used as template for the second round of PCR, in which primers PC2A-EcoRI and PC1-XbaI-end were used. The construction of PC2[PC1-C], in which the PC2 carboxyl-terminal domain was replaced with that of PC1, followed the same procedure, except that two different primers were used as follows: PC2PC1-C2 (5'-GTGCTCTGGTTGAGAAGATGTGCCGTGAAGCATCAG-3') and PC2PC1-C1 (5'-CTGATGCTTCACGGCACATCTTCTCAACCAGAGCAC-3') were used in the place of PC2PC1-P2 and PC2PC1-P1, respectively. The fragments were cloned into pRc/CMV between the EcoRI and XbaI sites. The four site-directed mutants were generated as described previously (10). All constructs used the same amino- and carboxyl-end primers, PC2HindIII and PC2XbaI (5'-CGCGCTCTAGAGTGAAGGCGGAAGCGTGGCC-3'), respectively. The middle primers were as follows: for construct PC2-(189-194), 5'-ATCATGATCATTATCGTTGAAGTCTAACTTGCATC-3' and AACGATAATGATCATGATCCATACCCTCGATACACA-3'; for PC2-(201-206), 5'-TTTATTTTCATTTGTGAGTGTGTATCGAGGGTATGG-3' and 5'-CTCACAAATGAAAATAAACATGGAACTAGGTGTGCA-3'; for PC2-S190D, 5'-GGGGTCATTATCGCTGAAGTCATAACTTGC-3' and 5'-TTCAGCGATAATGACCCCTACCCATAC-3'; for PC2-Y194D, 5'-AGGGTATGGATCGGGGTCATTGCTGCTGAA-3' and 5'-GACCCCGATCCATACCCTCGATACACA-3'. Expand High Fidelity PCR System (Boehringer Mannheim) was used in all PCR reactions. The PCR-generated fragments were cloned into pRc/CMV between HindIII and XbaI. The fidelity of PCR-generated fragments was verified by DNA sequencing.

Cell Culture, Transfection, and Selection-- PC2-P:PC1, Fur-P:PC2, and oxyanion hole-mutated PC2 (PC2-D309N) represent stable AtT-20 cell line derivatives which have been described previously (18). These cell lines were supertransfected with 21-kDa 7B2 in the pCEP4 expression vector (Invitrogen) (10). An AtT-20 cell line stably transfected with 21-kDa 7B2 in pCEP4 (10) was used for transfections of the PC2 constructs described above. Transfection and isolation of mutant PC2-expressing clones was performed following the procedures described previously (10). Two or three clones from each transfection were selected and analyzed to minimize potential clonal variation.

Antisera-- Antiserum LSU13 (against residues 23-39 of vertebrate 7B2) and antiserum LSU18 (against the carboxyl terminus of PC2) have been described (10). LSU3 was raised against residues 714-724 of mouse PC1 (the carboxyl terminus) (19). LSU7 was raised against the first 13 residues of mature mouse PC2.3 All antisera were raised in rabbits against peptides conjugated to hemocyanin.

Metabolic Labeling and Immunoprecipitation-- Metabolic labeling was performed in a 6-well tissue culture plate. In all cases 5 × 105 cells were seeded and labeled 2 days later in Met-Cys- medium containing 0.5 mCi of 35S-labeled Pro-Mix (Amersham Corp.) in 1 ml for 20 min. For co-immunoprecipitation using antisera LSU3 and LSU18, cells were extracted immediately after labeling. Alternatively, for 7B2 immunoprecipitations using LSU13 antiserum, cells were labeled and then chased for 20 min with Met/Cys-containing Dulbecco's modified Eagle's medium (with 2% fetal bovine serum). Cells were extracted with 1% Triton buffer (0.1 M NaCl, 25 mM Tris-HCl, pH 7.4, 10 mM iodoacetamide, 5 mM EDTA, and 1% Triton (20)). The samples were then diluted with an equal volume of extraction buffer (lacking Triton). Phenylmethanesulfonyl fluoride (30 µl of 100 mM stock) and p-chloromercuriphenylsulfonic acid (30 µl of 10 mM stock) were then added, and the samples were clarified by centrifugation and subjected to immunoprecipitation. Pulse-chase experiments were carried out as described previously (10). SDS-polyacrylamide gel electrophoresis (8.8% acrylamide for pulse-chase samples, 15% for co-immunoprecipitated samples) was also performed as described previously (21). The gels were treated with Amplify (Amersham Corp.) following the manufacturer's recommendations prior to fluorography. PhosphorImager analysis was used in some cases (as indicated in the figure legends). All experiments were repeated at least twice and usually three times.

Collection of Conditioned Medium and Enzyme Assay-- For assay of PC2 enzymatic activity, 500,000 cells of each clone were plated per 35-mm well (two independent clones were used per experiment). Two days after plating, the wells were rinsed twice with 3 ml of phosphate-buffered saline, and 1 ml of serum-free medium (Opti-MEM; Life Technologies) containing 100 µg/ml aprotinin was placed on the cells for 16 h. The conditioned medium was removed, centrifuged briefly to remove cells and debris, and stored frozen prior to analysis for PC2 enzymatic activity and radioimmunoassay. The cells were washed with phosphate-buffered saline, extracted with 0.5 ml of acid extraction solution (0.1% beta -mercaptoethanol, 1.0 M acetic acid, 20 mM HCl), and the acid solution subjected to protein assay to correct for variations in cell growth. 7B2 radioimmunoassay (10) was performed on 50-µl triplicates of the conditioned medium. PC2 enzymatic activity was assayed using 35 µl of conditioned cell culture medium in duplicate for a 4-h incubation period, in the presence and absence of the PC2 inhibitor CT peptide; 200 µM pGlu-Arg-Thr-Lys-Arg-AMC (Peptides International, Lexington, KY) was used as a substrate (10). It should be noted that varying expression levels cannot account for the differences observed in enzymatic assays of conditioned media, as pulse-labeling experiments indicated roughly comparable expression levels of the various mutant PC2s and wtPC2.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The PC2 Proregion Is Required but Is Not Sufficient for 7B2 Binding-- Similar to the other members of the kex2-like protease family, pro-PC2 can be divided into four distinct domains. To determine whether a particular domain might confer 7B2 binding, we constructed chimeric and truncated pro-PC2 molecules (as depicted in Fig. 1) for stable transfection into AtT-20/21-kDa 7B2 cells.


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Fig. 1.   Diagram of PC2 and chimeric constructs. The open areas depict domains derived from PC2. The shaded and filled regions represent PC1 and furin-derived domains, respectively. The fur-P:PC2 chimera contains the furin propeptide and cleavage site, with the PC2 catalytic, P and carboxyl-terminal domains (18). The PC2-P:PC1 chimera contains the PC2 propeptide and cleavage site, with the PC1 catalytic, P and carboxyl-terminal domains (18). The PC2[PC1-P] construct contains the first 438 residues of mPC2 (including the signal sequence). The PC2[PC1-C] contains the first 593 residues of PC2 and residues 595-753 of mPC1. PC2Delta C terminates after residue 593, and PC2 Delta PC terminates after residue 452.

We first examined the role of the PC2 proregion in the binding of 7B2 by a co-immunoprecipitation study using Fur-P:PC2 (in which the PC2 proregion was replaced with the furin proregion) and PC2-P:PC1 (in which the PC1 proregion was replaced with that of PC2) (18). AtT-20/PC2/21-kDa 7B2, AtT-20/Fur-P:PC2/21-kDa 7B2, and AtT-20/PC2-P:PC1/21-kDa 7B2 cells were labeled with [35S]Met and -Cys ProMix for 20 min, and cell extracts were immunoprecipitated under non-denaturing conditions using antisera LSU18 (for PC2 and Fur-P:PC2) and LSU3 (for PC2-P:PC1) to examine potential co-immunoprecipitation of PC2 domains with 7B2. The results, presented in Fig. 2, showed no 7B2 binding to the chimera containing the furin rather than the PC2 proregion (Fur-P:PC2). It should be noted that both the Fur-P:PC2 and PC2-P:PC1 proteins are not efficiently processed and secreted (18) and therefore may not represent well folded proteins. However, if folding of these chimeras is assumed, then it can be concluded that the PC2 proregion alone cannot confer ability to co-immunoprecipitate 7B2 since, even after prolonged exposure of the gel to film, no 7B2 bands were visible in co-immunoprecipitations (not shown).


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Fig. 2.   The proregion of PC2 is required but is not sufficient for 7B2 binding. AtT-20/PC2-7B2, AtT-20/PC2-P:PC1-7B2, and AtT-20/Fur-P:PC2-7B2 expressing cells were pulsed for 20 min prior to extraction under native conditions and co-immunoprecipitation. The PC2 antiserum LSU18 (for AtT-20/PC2-7B2 and AtT-20/Fur-P:PC2) and the PC1 antiserum LSU3 (for AtT-20/PC2-P:PC1-7B2) were used.

The PC2 Carboxyl-terminal Region Is Not Involved in the Binding of 7B2; the P Domain Is Required for the Stabilization of PC2-- The carboxyl-terminal domains are not conserved between the various PCs, suggesting PC-specific roles such as 7B2 binding; however, carboxyl-terminal domains from PC2s of various species do not exhibit remarkable homology, potentially ruling out a role for this domain in binding of 7B2. To investigate the possibility of the involvement of the carboxyl domain with respect to 7B2 binding, we constructed a truncated PC2 mutant, PC2Delta C. In addition, a mutant with both the P and carboxyl-terminal domains deleted (PC2Delta PC) was also created. Both constructs were transfected into AtT-20/21-kDa 7B2 cells. The results, presented in Fig. 3a, showed that PC2Delta C was still capable of binding 7B2. These data indicate that the carboxyl-terminal domain is not essential for binding of 7B2. However, there was no mature form of PC2Delta C detectable inside the cell, although it was found in the medium (Fig. 3b). Interestingly, this truncated form of PC2 was still active (Table I). Further deletion of the P domain (PC2Delta PC) destabilized pro-PC2; this protein may represent an unfolded form which was presumably degraded since it was neither released into the medium nor retained intracellularly (Fig. 3b).


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Fig. 3.   Pro-PC2 containing a carboxyl-terminal deletion is still able to bind to 7B2. a, co-immunoprecipitation of the two PC2 deletion mutants with 7B2. AtT-20/PC2-7B2, AtT-20/PC2Delta C-7B2, and AtT-20/PC2Delta PC-7B2 cells were pulsed for 20 min and chased for 20 min to enable newly synthesized 7B2 to bind to pro-PC2 and its mutants (30). Anti-7B2 antiserum LSU13 was used to immunoprecipitate 7B2·PC2 complexes under native conditions. b, Pulse-chase analysis of PC2 deletion mutants. AtT-20/PC2-7B2, AtT-20/PC2Delta C-7B2 (two independent clones), and AtT-20/PC2Delta PC-7B2 cells were pulsed for 20 min and then either lysed or chased for 2 h in Met/Cys-containing medium prior to immunoprecipitation with PC2 antiserum LSU18. Phosphorimage analysis is shown.

                              
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Table I
Enzymatic activity of PC2 deletion mutants and chimeras

The P Domain Is Not Interchangeable between PC2 and PC1-- Since the truncated pro-PC2 construct lacking the P domain was not stable, and with the knowledge that the P domains of PC2 and PC1 are relatively well conserved, we exchanged both the PC2 P and carboxyl-terminal domains with those of PC1 (PC2[PC1-P]). Analysis of expression confirmed that the resulting PC2[PC1-P] protein was well expressed in AtT-20/21-kDa 7B2 cells; however, it was unable to bind 7B2 (Fig. 4a). Furthermore, this chimera appeared to remain in the cell for an unusually long time (Fig. 4b); it was neither processed nor released, indicating potential problems in proper folding. We conclude that the PC1 and PC2 P domains are not interchangeable with respect to either folding and/or 7B2 binding.


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Fig. 4.   The carboxyl-terminal domain-swapped PC2 binds to 7B2, whereas the P + carboxyl-terminal domain-swapped PC2 does not. a, co-immunoprecipitation of PC2[PC1-C], containing the carboxyl-terminal domain of PC1 instead of PC2, with 7B2. AtT-20/PC2-7B2, AtT-20 PC2[PC1-C]-7B2, and AtT-20/PC2[PC1-P]-7B2 cells were pulsed for 20 min and then either lysed or chased for 2 h in Met/Cys-containing medium before co-immunoprecipitation using PC1 antiserum LSU3 (for AtT-20 PC2[PC1-C]-7B2 and AtT-20/PC2[PC1-P]-7B2) and PC2 antiserum LSU18 (AtT-20/PC2-7B2). b, pulse-chase analysis of domain-swapped PC2 mutants. Cells were pulsed for 20 min and then either lysed or chased for 2 h. Antiserum LSU7 was used for the immunoprecipitation due to the absence of the LSU18 epitope.

In contrast, the PC2 chimera containing only the PC1 carboxyl-terminal domain (PC2[PC1-C]), but not the PC1 P domain, was capable of binding 7B2 (Fig. 4a). Interestingly, the mature form of PC2[PC1-C] was found only in the medium but not inside the cell (Fig. 4b). This construct also exhibited enzymatic activity (Table I), indicating successful traverse of the secretory pathway. These results support the lack of importance of the carboxyl-terminal domain in 7B2 binding and further demonstrate that despite proper 7B2 binding, PC2 constructs containing a PC1 carboxyl terminus do not exhibit correct cellular routing.

Mutation of the Oxyanion Hole of PC2 Curtails 7B2 Binding and PC2 Activity-- One striking difference between PC2 and all other known PCs is that a catalytically important asparagine residue is replaced by an aspartate (Asp-309). The asparagine, which is involved in the formation of the oxyanion hole (22), is conserved in all other members of the subtilisin family except PC2. Seidah and colleagues (23) have suggested a critical role for this residue in the interaction between PC2 and 7B2. We subjected the AtT-20/PC2-D309N cell line, in which Asp-309 was intentionally mutated to asparagine (18), to supertransfection with 21-kDa 7B2. In the new 7B2-expressing cell line, the PC2-D309N protein was well expressed and released (Fig. 5). PC2-D309N was still able to bind 7B2, although in three separate experiments 7B2 was bound to a lesser degree than to wild-type PC2 (Fig. 5a). This mutant form of PC2 was processed more slowly than wild-type PC2 (Fig. 5b) and was also slightly less active, although it also exhibited lowered 7B2 expression which could account for this decrease in activity (Table II).


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Fig. 5.   The PC2 oxyanion hole mutant is capable of binding 7B2. a, PC2-D309N co-immunoprecipitation with 7B2. AtT-20/PC2-D309N-7B2 and AtT-20/PC2-7B2 cells were labeled for 20 min followed by co-immunoprecipitation of extracts using antiserum LSU18. Phosphorimage analysis is shown; the sensitivity of the bottom panel was set approximately five times higher than that of the top panel. b, pulse-chase analysis of PC2-D309N. AtT-20/PC2-D309N-7B2 and AtT-20/PC2-7B2 cells were pulsed for 20 min and chased for 2 h. Phosphorimage analysis is shown.

                              
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Table II
Enzymatic activity of residue-switched PC2 mutants

Replacement of a Six Amino Acid Sequence (Residues 189-194) in the Catalytic Domain of PC2 with the Corresponding PC1 Stretch Abolishes both 7B2 Binding and PC2 Activity-- Our previous studies have shown that a putative polyproline helix formed by the proline-rich region in 7B2 is involved in the interaction between pro-PC2 and 7B2. It has been suggested that elongated patches of aromatic residues represent likely candidates for binding of the relatively hydrophobic polyproline helix (24, 25). A close inspection of the PC2 amino acid sequence revealed that a relatively aromatic residue-rich region lies between residues 186 and 204, a region well conserved among all known PC2s (Fig. 8b). Seven aromatic residues are contained within this stretch of 19 amino acids; only four of these are present in the homologous sequence of PC1 and two within that of furin. To determine whether this region could be involved in 7B2 binding, two PC2 mutants were made in this region, each containing six amino acid stretches replaced with the homologous sequences of PC1. This strategy resulted in the replacement of the three non-conserved aromatic residues of PC2. In the PC2-(189-194) construct, the SSNDPY sequence (residues 189-194) was replaced with the corresponding NDNDHD (residues 190-195) sequence present in PC1; for the PC2-(201-206) construct, the DDWFNS sequence (residues 201-206) was mutated to the LTNENK sequence (residues 202-207) found in PC1. The expression of both PC2-(189-194) and PC2-(201-206) was similar to those of wild-type PC2 (Fig. 6), and both proteins were released into the medium, indicating successful traverse of the secretory pathway. Co-immunoprecipitation results, presented in Fig. 6a, demonstrated that PC2-(189-194) was completely unable to bind to 7B2; furthermore, this mutant exhibited no PC2 activity (Table II). On the other hand, PC2-(201-206) was still capable of binding 7B2 and was enzymatically active, although to a lesser extent than wild-type PC2. The processing of pro-PC2-(201-206) was also impaired compared with that of wild-type pro-PC2 (Fig. 6b). Unexpectedly, however, even in the absence of 7B2 binding, the proteolytic processing of pro-PC2-(189-194) was more rapid than that of wild-type PC2, as judged from the disappearance of the proform (Fig. 6b).


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Fig. 6.   The substitution of a six amino acid sequence (amino acids 189-194) of PC2 with corresponding PC1 sequence abolishes PC2-7B2 binding. a, PC2-(189-194) cannot co-immunoprecipitate 7B2. AtT-20/PC2-7B2, AtT-20/PC2-(189-194)-7B2 and AtT-20/PC2-(201-206)-7B2 cells were pulsed for 20 min, followed by co-immunoprecipitation with antiserum LSU18. b, PC2-(189-194) can undergo proregion removal. The data presented in Table II indicate that processed PC2-(189-194) is inactive.

The Tyr-194 Residue Is Pivotal for 7B2 Binding and for Enzymatic Activity-- A comparison of residues 189-194 of PC2, SSNDPY, with the corresponding PC1 and furin sequences (NDNDHD and NDQDPD, respectively) suggested that the two residues that were most likely to be critical for 7B2 binding were Ser-190 and Tyr-194. Based on this observation, the PC2 point mutants PC2-S190D and PC2-Y194D, which replaced Ser-190 and Tyr-194 with the corresponding PC1 residues Asp-190 and Asp-194, respectively, were made. These two mutants were transfected into AtT-20/21-kDa 7B2 cells. Both constructs were expressed (Fig. 7) and were efficiently released (not shown). The co-immunoprecipitation results revealed that 7B2 still bound to PC2-S190D, whereas no detectable 7B2 co-immunoprecipitated with PC2-Y194D (Fig. 7a). Both constructs were, however, enzymatically inactive (Table II). It is interesting that binding of 7B2 was unable to facilitate the intracellular processing of pro-PC2-S190D, which remained as the proform, whereas the processing of pro-PC2-Y194D, which was unable to bind to 7B2, was actually more rapid than that of wild-type PC2 (Fig. 7b).


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Fig. 7.   Tyr-194 of PC2 is critical to the PC2-7B2 interaction. a, PC2-Y194D cannot co-immunoprecipitate 7B2. AtT-20/PC2-7B2, AtT-20/PC2-S190D-7B2, and AtT-20/PC2-Y194D-7B2 cells were pulsed for 20 min before they were subjected to co-immunoprecipitation. b, enhanced processing of PC2-Y194D. The data presented in Table II indicate that processed PC2-Y194D is inactive.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study we have attempted to define the regions of pro-PC2 important for interaction with its helper protein 7B2. We first investigated the role of each individual domain of PC2 by replacement with the respective PC1 or furin domains. It has been demonstrated that the proregion is required for the proper folding of subtilisin (26) and appears to be required for other members of this protease family (18, 27-29). The interchangeability of proregions between these members of this family, however, is not clear. It has been shown that a furin chimera, in which the furin proregion was replaced with the PC2 proregion, was unprocessed and inactive (27). However, this study did not employ exact proregion and catalytic boundary domains, and it has been argued that preservation of exact boundaries is essential for proPC2 cleavage. Using chimeras constructed with precise junctions, Zhou et al. (18) found that the PC1 proregion and furin proregion were interchangeable, whereas those of PC1 and PC2 were not. They further showed that PC2 containing a furin proregion (PC2-P:fur) was not active, although it underwent very limited proteolytic processing (18). Using these same constructs, our studies suggest that with regard to 7B2 binding, the proregion of PC2 cannot be replaced with the proregion of furin. However, this may simply be due to the inability of this chimera to fold properly, since we have recently shown that proper folding of pro-PC2 precedes, and is required for, 7B2 binding (30). If the PC2-P:fur mutant is assumed to be properly folded, our results indicate that the PC2 proregion alone cannot confer the ability to bind 7B2.

With regard to the role of the middle or P domain, this domain has already been shown to be essential for the activation of Kex2, a yeast member of the subtilisin family. Any disruption of this region totally blocks the cleavage of the proregion (37). In agreement with the Kex2 experiments, we found that P domain-deleted PC2 is synthesized but is degraded rapidly. Some interchanges of this domain with other family members are apparently possible; Creemers et al. (28) showed that a furin chimera containing the P and carboxyl-terminal domains of PC2 matured normally and was released. In the case of the PC2 chimera containing PC1 P and carboxyl-terminal domains, the protein was synthesized and was not degraded, but it was also neither processed nor released, again leading to questions as to proper folding. We observed no detectable 7B2 binding to any P domain-deleted or swapped pro-PC2 mutant. However, since the P domain truncation mutant and the chimera were not able to traverse the secretory pathway, it is possible that they were misfolded, and we therefore cannot come to a firm conclusion about the role of the P domain in 7B2 binding based on co-immunoprecipitation data. Our data only provide evidence that the PC2 P domain is indispensible for the activation of pro-PC2. These results indicate that for PC2, the P domain is not exchangeable with the homologous domain from other members of this protease family.

The carboxyl termini of PC1 and PC2 are predicted to form an amphipathic alpha -helical segment (31) and are believed to be involved in sorting and/or storage (18, 28). Carboxyl-terminal truncated PC1 (PC1Delta C) matured normally and was enzymatically active (18, 32, 33). However, overexpression of PC1Delta C did not enhance POMC processing, and PC1Delta C was not as well stored as full-length PC1 (18). These results support a role for the COOH-terminal domain in routing. In the case of furin, Creemers et al. (34) showed that although furin containing only the pro, catalytic, and P domains was enzymatically active, this truncated furin was not stored. However, the PC2 carboxyl-terminal domain could reinstate intracellular retention when attached to truncated furin (28), implying that the carboxyl-terminal region of other PCs can serve to support intracellular storage of furin. We have shown above that the carboxyl-terminal domain of PC2 is not involved in the PC2-7B2 interaction, as 7B2 could still be co-immunoprecipitated with carboxyl-terminal region-deleted or swapped pro-PC2. Our pulse-chase data further demonstrated that there were no intracellular processed forms of either of these carboxyl-terminal mutant PC2s; however, they were found in the medium, suggesting that they were released soon after the completion of processing. These observations support the notion that the PC2 carboxyl-terminal domain is in fact required for proper storage and that for this function it is not replaceable with that of another family member.

One interesting difference between PC2 and other members of the subtilisin family is that an asparagine residue in the oxyanion hole (35, 36) is replaced by an aspartate residue. By using the vaccinia virus expression system, Benjannet et al. (23) reported that D309N PC2, in which this Asp residue was replaced by an Asn, exhibited a significant reduction in its capacity to produce beta -endorphin from POMC. They further reported that the D309N protein no longer bound to pro-7B2, suggesting that Asp-309 is important for the binding of pro-7B2 to pro-PC2. Other experiments, however, showed that the enzymatic activity of D309N PC2 on POMC was similar to that of wild-type PC2 (18). By using 21-kDa 7B2 supertransfected AtT-20/PC2-D309N cells, in three experiments we indeed observed some reduction in 7B2 binding (as compared with wild-type PC2) in 7B2-expressing AtT-20 cells. However, the efficient cleavage of POMC observed in stably transfected D309N AtT-20 cells (18) supports the efficacy of intracellular PC2 in accomplishing the required conversion events.

Based on the three-dimensional structural model proposed for furin (37), the aromatic residue-rich region of the catalytic domain is exposed on the surface in a distinct loop (Fig. 8a), potentially making this region accessible to the other proteins (assuming it is not covered by the P and carboxyl-terminal domains, which have not been modeled since the crystal structure of eukaryotic subtilisin-like enzymes is not yet available). Of the two PC2 mutants with 6 residue exchanges with corresponding PC1 sequences, one mutant, PC2-(201-206), exhibited minimal alteration in 7B2 binding. Although the rate of processing of this mutant pro-PC2 was reduced, it was still quite enzymatically active. The PC2-(189-194) aromatic amino acid loop mutant, however, totally lost both its ability to bind to 7B2 and any expression of enzymatic activity. Surprisingly, with this mutant, the processing of pro-PC2 to mature, although inactive, PC2 was enhanced. Similar results were obtained using the point mutant PC2-Y194D. We speculate that the proteolytic processing of PC2-(189-194) and PC2-Y194D is "unproductive," i.e. in these mutants, the cleavage of proregion (although resulting in a molecule with a molecular mass indistinguishable from that of mature PC2) does not yield active enzyme but instead a misfolded species (or one unable to release cleaved propeptide). Binding of 7B2 can potentially prevent such unproductive cleavage of propeptide; for example, pro-PC2-S190D, which binds 7B2 (but is not active), is processed much more slowly than either wild-type pro-PC2, non-7B2 binding pro-PC2-(189-194), or pro-PC2-Y194D. It is not clear whether this enhanced processing of PC2 mutants is autocatalytic or whether other proteases, such as furin, are involved in proregion cleavage which results in inactive enzyme. It has been proposed that like the other members of this family of enzymes, the processing of native pro-PC2 to mature, active PC2 is an autocatalytic reaction (38).4 Autocatalytic cleavage of propeptide by an otherwise inactive enzyme has been observed by Li and Inouye (39) with the active site serine-mutated thiol subtilisin.


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Fig. 8.   a, model of the structure of the prohormone convertase 2 catalytic domain. This representation is based upon the model of furin proposed by Siezen and colleagues (37). In this model, the white space-fill residues identify the catalytic triad His/Asn/Ser of furin. The gray space-fill amino acid corresponds to Tyr-194. The gray backbone identifies the furin substrate. b, comparison of the PC2 aromatic residue-rich region with PC1 and furin. Alignment of amino acids 186-206 of PC2 and the corresponding PC1 and furin sequences. Aromatic residues are in bold. The line indicates the six amino acid stretch that was mutated in PC2-(189-194). The arrowhead depicts the crucial Tyr-194.

In summary, in this paper we have attempted to define which sequences within pro-PC2 are involved in binding 7B2. Due to difficulty in establishing whether the pro and P domain chimeras are indeed properly folded, and the knowledge that binding of 7B2 follows folding of pro-PC2 (30), we cannot unequivocally assign the presence of 7B2 binding determinants to the pro and P domains at this time. However, our data rule out a significant role for the carboxyl-terminal domain and positively identify the catalytic domain as important to 7B2 binding. Within the catalytic domain residue Tyr-194 appears to play an especially critical role for binding of 7B2. We propose that wild-type PC2 and mutant PC2s can undergo three distinct biochemical pathways following intracellular encounter with 7B2: 1) normal binding of 7B2, productive proregion cleavage, and generation of enzymatically active enzyme (such as wild-type PC2 and PC2-(201-206)); 2) normal or slightly diminished binding of 7B2 (which may, however, be ineffectual), unproductive proregion cleavage, and no generation of enzyme activity (such as PC2-S190D); and 3) no binding of 7B2, unproductive proregion cleavage, which may even be enhanced with regard to rate, but which also results in an inactive enzyme species (such as PC2-(189-194) and PC2-Y194D). These data have implications for the molecular mechanism of action of 7B2 in pro-PC2 activation. The potential internal cleavage of the proregion and its dissociation from the proenzyme are also likely to play a role in the activation process, as recently demonstrated for furin (29). The cell lines containing the mutant PC2s described above should provide valuable tools for the further study of the activation of the pro-PC2·7B2 complex.

    ACKNOWLEDGEMENTS

We thank Elizabeth Guerra for help with radioimmunoassays and protein assays; Joelle Finley for invaluable assistance with cell culture; An Zhou for creating several of the initial cell lines; Nabil Seidah for mouse PC1 and PC2 cDNAs; and Jack Dixon for 7B2 cDNA. We thank Daria Siekhaus and Bob Fuller for providing the sequence of Drosophila PC2 in advance of publication.

    FOOTNOTES

* This work was funded in part by National Institutes of Health Grants DA 05084, DK 49703 (to I. L.), and DA 00266 (to R. E. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by National Institutes of Health Grant DA 05700.

Supported by National Institutes of Health Grant DA 00204. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, LSUMC, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504-568-4799; Fax: 504-568-3370; E-mail: ilindb{at}lsumc.edu.

1 The abbreviations used are: PC1 and PC2, prohormone convertase 1 and 2; AMC, aminomethylcoumarin; POMC, proopiomelanocortin; PCR, polymerase chain reaction; m, mouse; wt, wild type; 7B2 CT-peptide, human 7B2155-185; CMV, cytomegalovirus.

2 D. Siekhaus and R. S. Fuller, manuscript in preparation.

3 I. Lindberg, unpublished observations.

4 N.S. Lamango and I. Lindberg, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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