From the Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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A unique feature of the eukaryotic
subtilisin-like proprotein convertases (SPCs) is the presence of an
additional highly conserved sequence of approximately 150 residues (P
domain) located immediately downstream of the catalytic domain. To
study the function of this region, which is required for the production
of enzymatically active convertases, we have expressed and
characterized various P domain-related mutants and chimeras in HEK293
cells and -TC1-6 cells. In a series of C-terminal truncations of
PC3 (also known as PC1 or SPC3), PC3-Thr594 was
identified as the shortest active form, thereby defining the functional
C-terminal boundary of the P domain. Substitutions at
Thr594 and nearby sites indicated that residues 592-594
are crucial for activity. Chimeric SPC proteins with interchanged P
domains demonstrated dramatic changes in several properties. Compared with truncated wild-type PC3 (PC3-Asp616), both PC3/PC2Pd
and PC3/FurPd had elevated activity on several synthetic substrates as
well as reduced calcium ion dependence, whereas Fur/PC2Pd was only
slightly decreased in activity as compared with truncated furin
(Fur-Glu583). Of the three active SPC chimeras tested, all
had more alkaline pH optima. When PC3/PC2Pd was expressed in
-TC1-6
cells, it accelerated the processing of proglucagon into glicentin and
major proglucagon fragment and cleaved major proglucagon fragment to
release GLP-1 and tGLP-1, similar to wild-type PC3. Thus, P domain
exchanges generated fully active chimeric proteases in several
instances but not in all (e.g. PC2/PC3Pd was inactive). The
observed property changes indicate a role for the P domain in
regulating the stability, calcium dependence, and pH dependence of the
convertases.
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INTRODUCTION |
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Recently, a new family of serine proteases that process a wide variety of proprotein substrates has been identified in eukaryotic cells. Based on the similarity of their catalytic domain to the subtilisins, these proteases have been named subtilisin-like proprotein convertases (SPCs)1 (Refs. 1-4; for terminology see Ref. 5). To date, in addition to kexin, a yeast homologue (6, 7) and a number of SPCs found in lower species, seven SPCs have been identified in mammals as follows: furin2 (PACE or SPC1), PC3 (SPC3 or PC1), PC2, PC4, PC5/PC6, PC7/PC8 (or LPC), and PACE4 (1, 8, 9).
Each convertase has a distinct but overlapping substrate specificity and a distinctive tissue distribution, subcellular location, and maturation process, consistent with its unique role in some aspect of proprotein processing. Well characterized examples regarding these aspects are furin (expressed ubiquitously in almost all tissues), PC3, and PC2 (both restrictedly distributed in neuroendocrine tissues). We have only a limited understanding of the structural determinants which differentiate the various SPCs from each other in their function and properties. Their basic domain structure includes (Fig. 1) a signal peptide, a partially conserved propeptide, a highly conserved catalytic domain (40-50% identity among the SPCs and 25-30% to the subtilisins) followed by a relatively well conserved region called the P, homoB, or "middle" domain. Studies in recent years have clarified the role of propeptide cleavage in furin activation and the function of the propeptide as an intramolecular inhibitor which prevents early activation (10). After the P domain, various C-terminal extensions occur; these extensions seem to contain mainly routing/trafficking determinants. For instance, furin is primarily located in the trans-Golgi network, anchored by its transmembrane domain, and mutations in its cytoplasmic domain result in dramatic changes in its subcellular location (11, 12). Attachment of the P domain and C-terminal region of PC2 to the catalytic domain of furin resulted in a chimera which behaved similarly to PC2 in that it was now targeted to regulated pathway vesicles (13), whereas a truncated furin without a transmembrane domain was secreted via a non-regulated (constitutive or basal) secretory pathway. Deletion of residues from the C terminus of PC3 gave a mutant (terminating at Asp616) that was secreted in increased amounts via the constitutive pathway, whereas lesser amounts were routed into regulated secretory granules in AtT-20 cells (14). The C-terminal region after Asp616 of PC3 may also function as an inhibitor (15).
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Little is known about the function of the P domain. Due to functional differences between the subtilisins (degradative) and kexin (proteolytic processing), the conserved region after the catalytic domain of kexin was named the "P domain" when it became evident that it was required for processing activity (16). Although there are a few examples of bacterial subtilisins with extensions after the catalytic domain, a search of the protein data bank does not reveal any significant sequence similarity of the P domain with other known proteins. For both kexin and furin, a partial C-terminal deletion of the P domain, even of just a few amino acids, abolishes enzymatic activity completely (17, 18). Lovo cells (a human colon carcinoma cell line) express furin mRNA yet lack furin activity (19). Genetic analysis demonstrated that the lack of activity was due to two mutations within the P domain coding region of the furin gene, one resulting in a frameshift (20) and the other in a replacement of a conserved tryptophan with arginine (21). Recently, a defect in the PC3 gene has been found in a human patient. In this case, a glycine within the P domain region is replaced by an arginine (22). There has been no reported systematic study to elucidate the functional role(s) of the P domain and examine its structural relation to the catalytic domain.
In the present study, using PC3 as the primary model convertase, we have investigated the properties of various P domain-related mutants and chimeras expressed in endocrine and/or non-endocrine mammalian cell lines. Our findings provide evidence that in addition to stabilizing the catalytic domain, the P domain participates in the regulation of the pH and calcium dependence, as well as the substrate specificity of these enzymes.
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MATERIALS AND METHODS |
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Construction of Vectors Encoding Mutant SPCs
cDNA templates for rat PC3 (PC1), PC2, and
PC3-Asp616 (PC1C) (14) were kindly provided by Dr.
Richard Mains at the Johns Hopkins University; human furin template was
from Dr. Kazuhisa Nakayama at the University of Tsukuba, Japan.
pCMV6b/6c (b and c: identical except in the orientation of the
polylinker region) vectors were from Dr. Graeme Bell at the University
of Chicago. Unless stated otherwise, all mutants were generated by
creating mutation-containing fragments by polymerase chain reaction and
subcloning the polymerase chain reaction fragments into expression
vectors by restriction digestion and ligation. All polymerase chain
reaction-generated fragments were verified by sequencing. The
orientation of subcloned fragments was verified by restriction
digestion. Fig. 1 illustrates a schematic summary of the mutants used
in this study.
Truncation and Substitution Mutants-- Three truncation mutants, PC3-His592, PC3-Gly593, and PC3-Thr594, terminating at amino acid His592, Gly593, and Thr594, were created by adding a stop code and a restriction site immediately after the desired amino acid, respectively. In the substitution study, Thr594 in mutant PC3-Thr594 was replaced with serine, aspartic acid, and asparagine, respectively (mutants PC3-T594S/T594D/T594N). Mutant PC3-H592T was generated by replacing His592 in PC3-His592 with threonine.
P Domain-swapping Chimeric Proteins-- Three chimeric proteins, PC3/PC2Pd, PC2/PC3Pd, and PC3/FurPd, were generated by using the "gene splicing by overlap extension" (SOE) technique (23). PC3/PC2Pd was created by fusing amino acids 1-453 of PC3 with amino acids 456-638 of PC2. PC2/PC3Pd was a fusion protein with amino acids 1-455 of PC2 and amino acids 454-616 of PC3. Mutant PC3/FurPd consists of amino acids 1-453 of PC3 and amino acids 440-583 of furin. Mutant Fur/PC2Pd (18) was a kind gift from Dr. John Creemers (Katholieke Universiteit, Leuven, Belgium).
A truncated PC3 (Ref. 14; PC1Transfection and Cell Culture
HEK293 cells (human embryonic kidney tumor cells) and -TC1-6
cells (mouse pancreatic alpha cells) were routinely maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum (Life Technologies, Inc.). SPC mutant vectors were transiently
transfected into HEK293 cells using the calcium phosphate method
(Profection kit, Promega). When stable transfection was desired, as
indicated under "Results," cells were transfected using the
Lipofectin method. Plasmid pSV2neo was co-transfected with the SPC
mutant-carrying vectors to provide drug resistance. Transfected cells
were then cultured in G418-containing medium (0.5 mg/ml);
G418-resistant colonies were screened for the expression of interested
SPC protein by biosynthetic labeling/immunoprecipitation, Western
blotting, or immunofluorescence staining.
Metabolic Labeling and Analysis of SPC Proteins
Processing of transfected SPC protein was characterized by biosynthetic labeling. For transiently transfected cells, the labeling was performed 48 h after transfection. Briefly, cells were first incubated in methionine-deficient media for 30 min and then pulse-labeled with [35S]methionine (1 mCi/ml, 1000 Ci/mmol, Amersham Pharmacia Biotech) with or without a subsequent chase incubation in nonradioactive complete media. Upon termination of incubation, media were collected, and cellular proteins were extracted with a TES buffer containing 20 mM TES, pH 7.4, 10 mM mannitol, and 1% Triton X-100 (24). For immunoprecipitation, samples were diluted with an immunoprecipitation buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 0.5% Nonidet P-40, 0.02% NaN3) (25). Appropriate antibodies (as specified under "Results") and protein A (for polyclonal antibodies) or protein G (for monoclonal antibodies) (Pierce) were added. Protease inhibitors (26) were present through the sample collection and immunoprecipitation procedures. Immunoprecipitated SPC proteins were fractionated on SDS-PAGE slab gels (7.5% or 10%) and detected by fluorography.
Analysis of Enzymatic Activity on Synthetic Substrates
HEK293 cells expressing various SPCs were incubated in complete serum-free Dulbecco's modified Eagle's medium for 15-20 h. Collected medium was concentrated 20-30-fold using a 30K Ultrafree concentrating unit (Millipore Corp.). Enzymatic activity assays followed the protocol described by Vindrola and Lindberg (27) using pGlu-Arg-Thr-Lys-Arg-MCA as the substrate. In the substrate specificity assay, Boc-Arg-Val-Arg-Arg-MCA, Boc-Leu-Ser-Thr-Arg-MCA and Boc-Ala-Gly-Pro-Arg-MCA (where Boc is t-butoxycarbonyl) were the test substrates used (Peninsula Laboratories).
In experiments aimed at comparing the activity levels of various SPC
chimeras, cells were cultured in duplicate plates with equal cell
numbers. One plate was used to collect medium and analyze its activity.
Cells in the second plate were pulse-labeled with [35S]methionine for 1 h and then chase incubated in
non-radioactive complete serum-free medium for 4-6 h. Media from the
second plates of various cell lines were collected, immunoprecipitated
with appropriate SPC antibodies, and subjected to SDS-PAGE slab gel analysis. Upon loading the SDS-PAGE gel, each sample was mixed with
non-radioactive rainbow molecular weight markers. Fractionated samples
were electrically blotted onto Immobolin-P membrane (Millipore); blot
areas corresponding to the appropriate molecular weight range of the
processed SPCs were excised, treated with a stripping buffer (62.5 mM Tris, 2% SDS, 100 mM -mercaptoethanol,
pH 6.7) at 50 °C for 30 min, followed by scintillation counting.
Preliminary tests established that loaded radioactivity could be
quantitatively recovered by this procedure. Based on the radioactivity
recovered from each immunoprecipitation/SDS-PAGE, after correction for
the number of methionine residues in each SPC protein, the amount of
medium collected from the duplicate plate used for assay of activity
was adjusted so that equimolar amounts of SPC were used in each
activity assay.
Analysis of Proglucagon-related Peptides in -TC1-6 Cells
Wild-type or transfected -TC1-6 cells were labeled with
either [35S]methionine, as described above, or
[3H]leucine (0.4 mCi/ml; Amersham Pharmacia Biotech).
Cellular protein extraction and immunoprecipitation were performed as
described previously (28). After immunoprecipitation with the
appropriate antibodies (see "Results"),
[35S]methionine-labeled samples were fractionated on a
gradient SDS-PAGE slab gel; [3H]leucine-labeled samples
were analyzed by SDS-PAGE tube gels. Tube gels were sliced and eluted
(29), followed by scintillation counting. Non-radioactive rainbow
molecular weight markers were added with samples as internal
standards.
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RESULTS |
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The Shortest Active Form of PC3 Terminates at Thr594
Among the known mammalian SPCs, PC3-Asp616, which migrated as a 67-kDa species in the present study (Fig. 2), is the shortest active form. It is normally produced in neuroendocrine cells after the autocatalytic removal of the propeptide in the endoplasmic reticulum and the subsequent deletion of a C-terminal fragment (amino acids 617-736) in the secretory granules (14, 30). Non-endocrine cells usually do not endogenously express PC3. Its relative simplicity, selective distribution, and our greater understanding of its maturation process and function make this truncated form of PC3 an ideal model convertase for studies on the P domain.
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Of the mammalian P domains, the last two conserved amino acids in the C-terminal region, presumably marking their junction with the C-terminal extensions, are Gly593 and Thr594 (numbers are for rat PC3). PC3-Asp616 contains an additional 22 amino acids beyond this conserved region. To define more precisely the shortest sequence needed for activity, three truncation mutants ending at or near these conserved amino acids were expressed in HEK293 cells. Their propeptide cleavage activity and secretion were examined by a pulse-chase metabolic labeling paradigm (Fig. 2).
PC3-Asp616 undergoes very efficient autocatalytic cleavage to remove its propeptide. During a 30-min pulse-labeling period, the majority of 75-kDa pro-PC3-Asp616 was converted to 67-kDa mature PC3, and most of the latter was secreted into medium during the subsequent 3-h chase incubation. For truncation mutant PC3-Thr594 (Fig. 2, right), the dominant form during the 30-min pulse was a molecule slightly smaller than 75 kDa. Later, it was processed into a 65-kDa protein, an expected size for this truncated PC3 after removal of its propeptide. The conversion rate of pro-PC3-Thr594 to PC3-Thr594 was not as efficient as that of pro-PC3-Asp616 to PC3-Asp616, but after 3 h of chase incubation, a significant amount of the processed PC3-Thr594 was secreted into the medium. In contrast, neither PC3-Gly593 (Fig. 2, middle) nor PC3-His592 (data not shown) was processed or secreted.
To determine whether the lack of propeptide cleavage in PC3-Gly593 and PC3-His592 was due to the absence of threonine at the C terminus of the P domain, Thr594 in PC3-Thr594 was replaced with serine, aspartic acid, or asparagine, respectively. Fig. 3 shows a biosynthetic labeling experiment on HEK293 cells expressing PC3-T594S, T594D, and T594N. After a 30-min pulse and a 3-h chase incubation, a small amount of mature 65-kDa protein was seen in the media of cells expressing PC3-T594S and PC3-T594N, whereas it was not visible with mutant PC3-T594D. For mutant PC3-H592T, in which His592 in the PC3-His592 mutant was replaced with threonine, there was no detectable propeptide cleavage activity.
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These results thus demonstrated that the shortest form of PC3 capable of cleaving its propeptide is PC3-Thr594. The few conserved amino acids at the C terminus of the P domain region (His592, Gly593, and Thr594) also seem to play a crucial role in sustaining the autocatalytic activity of PC3. This also confirmed that the P domain of PC3 has a C-terminal well defined boundary.
Biosynthesis and Processing of Chimeric SPCs with Foreign P Domains
The relatively high level of conservation of the P domains among various SPCs suggests that these domains must play some essential role. We therefore reasoned that interchanges of P domains might provide information on these possible function(s). Accordingly, P domain-swapped SPCs were prepared and transfected into HEK293 cells (Fig. 1). The autocatalytic activation of each chimeric form was studied by pulse-chase metabolic labeling, and its processing and secretion were compared with that of the parental proteases.
PC3/PC2Pd was initially synthesized as a 79-kDa protein, which was then converted into a 69-kDa protein through an intermediate (Fig. 4, top panel). The conversion rate was slower than that of the propeptide processing of PC3-Asp616. The secretion of 69-kDa PC3/PC2Pd protein, however, was as efficient as the secretion of PC3-Asp616. The counterpart mutant, PC2/PC3Pd, did not show any propeptide cleavage activity (Fig. 4, bottom panel), even when 7B2 (a unique PC2 helper protein) (31-33) was co-expressed and the chase incubation was extended to 8 h (data not shown).
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Mutant PC3/FurPd also displayed propeptide cleavage activity, although it was much less efficient than that of PC3-Asp616, and the secretion of the processed form was relatively slow (Fig. 4, middle panel). The presence of two forms in the cells after a 3-hour chase presumably represents differences in glycosylation status. During the course of this study, Creemers et al. (13) reported that, in their study on the sorting mechanisms of SPCs, a chimeric protein with the catalytic domain of furin and the P domain of PC2 underwent efficient propeptide cleavage and secretion via the regulated secretory pathway in AtT-20 cells. When we expressed this mutant in HEK293 cells, it also showed efficient propeptide cleavage and secretion, similar to that of Fur-Glu583 (data not shown).
Activities of Chimeric SPCs
The successful propeptide cleavage and secretion of the chimeric SPCs suggested that they were able to fold and be transported in HEK293 cells. Next we attempted to determine whether these chimeric enzymes were active in cleaving substrates. Our preliminary experiments established that wild-type HEK293 cells in our culture system secrete negligible amounts of endoproteolytic activity toward MCA-linked synthetic substrates, as tested over a range of pH values and varying calcium ion concentrations. Collected media samples from HEK293 cells expressing various SPC mutants and wild-type SPCs were analyzed for their activities against fluorogenic substrates. PC3/PC2Pd, PC3/FurPd, and Fur/PC2Pd were all active (see below).
Three major enzymatic properties that distinguish eukaryotic SPCs from bacterial subtilisins are their dependence on calcium for activation and activity, their more acidic pH optima, and their requirement of two or more basic amino acids at the substrate cleavage site. Efforts were made to address each of these properties in the mutants.
Ca2+ Dependence-- We first tested the calcium dependence of the active chimeras. Between 0 and 20 mM calcium ion concentration, PC3-Asp616 showed a gradual increase of activity. The activity level plateaued above 20 mM calcium ion concentration. Omitting calcium and adding 2 mM EGTA or 2 mM EDTA to the assay completely suppressed the activity of PC3-Asp616 (Fig. 5, top panel). Quite strikingly, PC3/PC2Pd retained 40-55% of its activity in the presence of EGTA or EDTA, and its activity increased only moderately over the range from 0 to 40 mM calcium ion concentration. PC3/FurPd (Fig. 5, top panel), on the other hand, had little activity in the presence of EGTA or EDTA. It gained most of its activity in the lower range of calcium concentration (0-5 mM). The activity of Fur/PC2Pd responded to the changes in calcium ion concentration in a pattern similar to that of Fur-Glu583 (Fig. 5, bottom panel), i.e. no activity in the presence of EGTA or EDTA and gaining most of the activity in the low range of calcium ion concentrations.
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pH Dependence-- Full-size PC3 and C-terminally processed PC3 have different acidic pH optima (34, 35). Similar to other published results, PC3-Asp616 secreted from HEK293 cells showed maximal activity at pH 6.0 within a narrow range (Fig. 6, top panel). Fur-Glu583 had a broader optimum pH, ranging between pH 7 and pH 8 (Fig. 6, bottom panel). The optimum pH for PC3/PC2Pd was shifted to between pH 7.0 and pH 8.0 (Fig. 6, top panel). The optimum pH for PC3/FurPd was pH 7.0 (Fig. 6, top panel), in between that of Fur-Glu583 and PC3-Asp616. Fur/PC2Pd demonstrated a more alkaline and narrower optimum pH (pH 8.0) than was observed for Fur-Glu583 (Fig. 6, bottom panel). Taken together, all three chimeric SPC proteins had more alkaline pH optima than their parental wild types.
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Relative Activity Levels and Substrate Specificity-- The activity levels of various SPC chimeras with fluorogenic substrates were compared at their optimum pH and with 10 mM calcium (Table I, top). The specific activities of PC3/PC2Pd and PC3/FurPd were 3-4-fold higher than the activity of PC3-Asp616, respectively, whereas the activity of Fur/PC2Pd was slightly lower than the activity of Fur-Glu583. PC3-Thr594 had a level of activity similar to that of PC3-Asp616 (data not shown); PC3-T594S and PC3-T594N, although secreted in minor amounts, did not show any detectable enzymatic activity (data not shown).
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PC3/PC2Pd Activity in Vivo--
To examine the activity of the
chimeric SPCs with a natural preprotein substrate, we created
-TC1-6 cell lines stably expressing either PC3/PC2Pd or
PC3-Asp616 (as a control).
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DISCUSSION |
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The P domains in the proprotein convertases are unique sequences in that they are well conserved and their appearance in evolution parallels the acquisition of the specificity for multiple basic residues and other important properties that distinguish these proteases from the subtilisins. In this study, we describe the creation and expression in mammalian cells of several active chimeric SPC proteins, which have allowed us to address the functional role(s) of the P domain in greater detail.
We first investigated the C-terminal region of PC3-Asp616. A truncation study clearly defined the C-terminal boundary of the P domain of PC3 to be at Thr594. Similar findings have been reported for yeast kexin; a kexin protein terminating at Glu593, which aligns with Thr594 of PC3, was active but also exhibited a relatively slower rate of maturation and secretion (17). In PC3, Thr594 could not be replaced with other amino acids bearing a similar charge or side chain size, nor could this threonine be reserved but placed at another position (PC3-H592T). The decisive role played by Thr594 suggests that the C-terminal end of the P domain of PC3 interacts with other regions in the PC3 protein or is required for its structural integrity. A human PC3 gene mutation has been reported recently in which Gly593 was mutated to arginine, resulting in an inactive PC3 protein (22). This finding is in complete agreement with our results and supports the conclusion that this region of the P domain of PC3 is crucial for the generation of an active enzyme.
The C termini of the chimeric SPCs used in this study did not end precisely at the last conserved amino acids (Thr594 in PC3, Thr594 in PC2, and Thr573 in furin). Instead, mutant PC3/PC2Pd included the entire C-terminal region of PC2, and PC3/FurPd was extended to Glu583 of furin. Creemers et al. (13) reported that a PC2 protein truncated after Thr594 was inactive. The shortest active form of PC2 is not known at this time. The shortest active form of furin as tested in vitro was a truncated form ending at Glu583 (18); another truncated form, ending at amino acid 576, has also been reported but without data on its secretion or activity (39). More recently, it has been demonstrated that a furin protein ending at Thr573 is active when expressed in Pk15 cells (13). The foregoing findings imply that these last two conserved amino acids (Gly and Thr) do not precisely define a commonly shared C-terminal boundary for the P domains in all SPCs. Additional sequence or other structural elements might be needed in a molecule-specific manner.
The regulatory role of calcium on convertase activity is well
documented (34, 35, 40-47). On the other hand, although calcium plays
a role in stabilizing subtilisin, the enzymatic activity of subtilisin
is not regulated by calcium and may not be sensitive to chelators
(48-50). Calcium-binding sites in a number of subtilisin-related proteases are known from crystallographic studies (49, 51), whereas the
structural basis for the calcium dependence in the SPCs is unknown. In
thermitase, Ca1 (calcium-binding site 1, strong, Kd
108 M) was fully occupied without added
medium calcium ions, whereas Ca2 (calcium-binding site 2, medium
strength) exhibited various degrees of occupancy at 0, 5, and 100 mM calcium) (51). Sites homologous to Ca1 and Ca2 have been
predicted to be present in the catalytic domain of furin (52). Sequence
alignment illustrates the conservation of several amino acids located
in these predicted calcium-binding sites in other SPCs as well (4).
Other than these, no sequences resembling known calcium-binding motifs
(e.g. EF-hands, etc.) have been identified in the
convertases. If the calcium-binding sites in the convertase catalytic
domains are functionally similar to those in subtilisin or thermitase,
then at the calcium concentrations that prevail along the secretory pathway in vivo, one of the sites (Ca1) should be fully
occupied, whereas the second site (Ca2) might be playing regulatory
roles. It should be mentioned that there are no disulfide bonds in the subtilisins, whereas two disulfide bonds have been predicted in the
catalytic domain of the SPCs (52, 53). It is reasonable to postulate
that bound calcium may have a dual functional role in the convertases,
stabilizing the structure and regulating the activity.
The changes in calcium dependence that we have observed in the chimeric SPCs clearly indicate the importance of the P domain in regulation of activity. Since chimeric SPC proteins with identical catalytic domains but different P domains showed altered patterns of calcium dependence, it is likely that additional calcium-binding site(s) may either occur within the P domain or at new sites created by the intersection of the P and catalytic domains. All evidence taken together suggests that multiple calcium-binding sites may be involved in regulating convertase activity.
In vitro analyses indicate that PC3, PC2, and furin have different pH optima within the acidic to neutral range. PC2 has the lowest (pH 5.0), that of PC3 is also more acidic (pH 5.5-6.0), whereas that of furin is nearer neutrality (34, 35, 40-44, 54). It is thus very surprising that the chimeras PC3/PC2Pd, PC3/FurPd, and Fur/PC2Pd all demonstrated more alkaline pH optima than the corresponding wild-type SPCs, values ranging from pH 7 to 9. The subtilisins in general have similar neutral to alkaline pH optima. Our results thus strongly suggest that in wild-type SPC, the authentic P domain somehow enables the convertase catalytic domain to function optimally in an acidic environment. An alternative explanation could be that the chimeric enzymes were destabilized at more acidic pH values, possibly due to changes in surface charge in the Glu- and Asp-rich substrate-binding region. In either case, however, these results indicate that structural interactions between the P and catalytic domains are needed to maintain function at a neutral or acidic pH.
In an in vivo system (proglucagon processing in -TC1-6
cells), PC3/PC2Pd was as active as PC3-Asp616. It
maintained a similar cleavage site selectivity to that of PC3-Asp616, and both enzymes seemed to be able to cleave at
the single arginine site (residue Arg77 of proglucagon)
required for the production of tGLP-1 and tMPGF. The increased activity
of the PC3 chimeras toward synthetic substrates in vitro was
also surprising, as was the altered activity of PC3/PC2Pd versus PC3-Asp616 toward certain synthetic
substrates (pGlu-Arg-Thr-Lys-Arg-MCA versus
Arg-Val-Arg-Arg-MCA). Such findings, however, are not without precedent
in other serine proteases, i.e. that alteration in the region following the subtilisin-like catalytic domain would change the
substrate specificity. One such example is the Lactococcus lactis proteinase (55). L. lactis proteinase has two
isoforms, which differ in only 44 out of 1902 amino acids. Both
isoforms have C-terminal extensions (>1200 amino acids) downstream of
the catalytic domain. Fragment exchanges between the two isoforms within the C-terminal region resulted in a different pattern of casein
cleavage.
The structural relationship between the catalytic domain and the P
domain at present is not known; there has been no published crystal
structure of any SPC to date. By using predictive methods and
computer-assisted modeling, we have found that the P domains are likely
to have an eight-stranded -barrel motif as their core structure.
This independently folded domain, in turn, is likely to interact with
the catalytic domain in the SPCs through a set of hydrophobic contacts
in a defined patch that exists on the surface of the catalytic domain
opposite the active site in the SPCs but not in the
subtilisins.3 Presumably, it
is through conformational changes in this latter region that these
regulatory effects are exerted. An overall conserved intrinsic
structure of the P domain would explain why P domain swapping gives
rise to functional chimeras. However, the dramatic property changes we
have observed in these chimeras are more difficult to explain. In
preliminary thermostability experiments and limited endoprotease
digestion experiments, no significant differences between
PC3-Asp616 and PC3/PC2Pd3 were observed,
implying that domain swapping did not result in significant
conformational destabilization. Crystallographic studies and further
mutagenesis to map more precisely important sequence determinants in
the P domain may shed light on these questions.
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ACKNOWLEDGEMENTS |
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We thank Dr. John Creemers for providing Fur/PC2Pd construct; Drs. Stephen Duguay and Joseph Bass for helpful discussions; Paul Gardner and Jeffrey Stein for synthesis of oligonucleotides; Margaret Milewski for assistance in cell culture; and Rosie Ricks for help in preparing this manuscript.
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FOOTNOTES |
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* This study was supported by the Howard Hughes Medical Institute and in part by National Institutes of Health Grant DK-13914.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.
To whom correspondence should be addressed: Howard Hughes Medical
Institute, Dept. of Biochemistry and Molecular Biology, the University
of Chicago, 5841 S. Maryland Ave., MC 1028, N101, Chicago, IL 60637. Tel.: 773-702-1334; Fax: 773-702-4292; E-mail: dfsteine{at}midway.uchicago.edu.
1 The abbreviations used are: SPC, subtilisin-like prohormone convertase; peptide-MCA, peptide-methylcoumarin amide; PAGE, polyacrylamide gel electrophoresis; Bis-Tris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)-propane-1,3-diol; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MPGF, major proglucagon fragment; GLP-I, glucagon-like peptide I. t, truncated; pGlu, pyroglutamic acid.
2 For simplicity and consistence, we refer to SPC1 as furin, SPC2 as PC2, and SPC3 as PC3 (also known as PC1) in this article.
3 G. Lipkind, A. Zhou, and D. F. Steiner, unpublished results.
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REFERENCES |
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