(Received for publication, June 20, 1995)
From the
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.
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) ()is the common
precursor of several important peptide hormones, including ACTH,
-MSH, and
-endorphin. In anterior pituitary corticotropes,
major POMC-derived peptides include ACTH and
-lipotropin, whereas
in intermediate pituitary melanotropes, ACTH is further processed into
-MSH, and
-lipotropin is processed to
-endorphin(1-31) and
-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
-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-terminally shaded region represents the
prosequence; the openregion represents the
subtilisin-like protease domain and the dottedregion represents the carboxyl-terminal extension. PC1
C and the
PC1
C portion in Fur-P
PC1
C 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- and COOH-terminal domains of the PCs and highlight the
importance of enzyme activity for correct routing and storage of the
PCs.
Figure 2:
PC1C processing in AtT-20, hEK293,
and hLoVo cells. hEK293 cells, hLoVo cells (top), and
AtT-20 cells transfected with pCIS. PC1
C 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; proPC1
C, 70-kDa proform of
PC1
C.
Next, the processing of PC1C was examined in cells that express
PC1 endogenously (AtT-20 cells; Fig. 2, middleleft). In AtT-20 cells expressing PC1
C protein (Fig. 2, middleright) with a 15-min pulse
labeling, the 70-kDa proPC1
C was converted to 63-kDa PC1
C 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 PC1
C protein was secreted
more rapidly than the endogenous full-size PC1. Therefore it was
important to examine whether stimulation of AtT-20 PC1
C cells with
secretagogues would significantly increase the secretion of PC1
C
protein (Fig. 2, bottom). The results showed that the
majority of PC1
C protein never reached mature secretory granules,
potentially missing the chance to alter POMC processing.
To
determine if expression of PC1C yields a protein that is
enzymatically active in POMC processing, POMC-derived peptides in
AtT-20 cells expressing PC1
C were analyzed (Fig. 3).
Expressing PC1
C greatly accelerated the conversion of POMC to its
smaller products, namely ACTH and glycosylated ACTH (Fig. 3) and
-endorphin (not shown). In addition, the secretion of the smaller
peptides produced in cells expressing PC1
C 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 PC1C. Aliquots of samples of the nontransfected and
PC1
C-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.
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.
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
-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
-terminal ACTH antiserum; bottom, cells and
medium from a 4-h chase incubation of the PC2-D309N cells were analyzed
with the
-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.
When Fur-PPC1
C and
Fur-P
PC2 proteins were expressed in AtT-20 cells (Fig. 6A), both Fur-P
PC1
C and Fur-P
PC2
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-P
PC1
C was processed to a PC1
C-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-P
PC1
C. The Fur-P
PC2 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-P
PC2 were both secreted during a
4-h chase incubation.
Figure 6:
Biosynthetic processing of
Fur-PPC1
C and Fur-P
PC2 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-P
PC1
C 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-P
PC2 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-P
PC1
C or Fur-P
PC2. Preliminary experiments
showed that the processing of Fur-P
PC1
C 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-P
PC1
C 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-PPC1
C and
Fur-P
PC2 were transfected into hLoVo cells. As seen in Fig. 6B, Fur-P
PC1
C protein was indeed
processed in hLoVo cells, while there was no significant processing of
Fur-P
PC2 during the 3-h incubation.
The biosynthetic
processing of POMC in AtT-20 Fur-PPC1
C and Fur-P
PC2
cells was analyzed. Expression of Fur-P
PC1
C greatly
accelerated the conversion of POMC to ACTH and did not enhance the
conversion of ACTH to ACTH(1-13)NH
(Fig. 6C), as seen in AtT-20 PC1
C cells (Fig. 3), indicating that the Fur-P
PC1
C protein was
processed into PC1 protein, which was active within the secretory
pathway. There were no changes in POMC processing in AtT-20
Fur-P
PC2 cells (not shown).
AtT-20 cells expressing proregion
swapped PC1 or PC2 protein were first studied by biosynthetic labeling.
Nontransfected cells and cells expressing PC1-PPC2 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-P
PC1 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-P
PC1. As was
seen for the processing of the PC1-S382A protein, the chimeric
PC2-P
PC1 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-P
PC1 cells. In AtT-20 PC1-P
PC2 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*
PC2; 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-PPC1
cells: 15-min pulse, 2-h chase (PC1 antiserum); for PC2,
PC1-P
PC2, and PC1-P*
PC2, 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-PPC2. For PC1
C- and
Fur-P
PC1
C-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
PC1C. A, C, E, and F, PC1
immunostaining; B, D, and G, PC2
immunostaining. Cell lines: A, nontransfected; B,
wild-type PC2; C, PC2-P
PC1; D, PC1-P
PC2; E, PC1
C; F, Fur-P
PC1
C; G,
Fur-P
PC2.
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.
The bulk of
POMC cleavages occur in secretory granules, where cleavage of the
carboxyl terminus of PC1 occurs. PC1C was quite active in
increasing POMC processing when expressed in AtT-20 cells. The major
effect of PC1
C 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
-endorphin was increased. In
contrast, overexpression of PC1
C failed to increase production of
ACTH(1-13)NH
, whereas overexpression of full-size PC1
did increase production of this smaller peptide(11) . Since
newly synthesized PC1
C protein was secreted more rapidly than the
endogenous full-size PC1, and since stimulating AtT-20 PC1
C cells
with secretagogues did not significantly increase the secretion of
PC1
C protein, the majority of PC1
C 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.
The
PC1-PPC2 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) .
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. ()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.