(Received for publication, May 30, 1995; and in revised form, August 11, 1995)
From the
The proteinase mPC1, a neuroendocrine member of the mammalian
family of subtilisin-like enzymes, has previously been shown to be
converted to a carboxyl-terminally truncated 66-kDa form during
transport through the secretory pathway. The cleavage site and the
function of this carboxyl-terminal truncation event are unknown. We
have performed site-directed mutagenesis of two paired basic sites in
the mPC1 carboxyl-terminal tail and expressed these constructs in PC12
cells, a rat pheochromocytoma known to lack endogenous PC1. We found
that the most likely site for the truncation event was at
Arg-Arg
since mutation of this site to
Lys-His prevented processing of 87-kDa PC1. A PC1 mutant
carboxyl-terminally truncated at this site and expressed in PC12 cells
was efficiently routed to the secretory pathway and stored in secretory
granules, indicating that the carboxyl-terminal extension is not
required for sorting of this enzyme. The function of the various PC1
constructs was assessed by analyzing proneurotensin cleavage to various
forms. The carboxyl-terminally truncated PC1 mutant was found to
perform most of the cleavages of this precursor as well as wild-type
PC1; however, the blockade mutant processed proneurotensin much less
efficiently. Differences between the site preferences of the various
enzymes were noted. Our results support the notion that
carboxyl-terminal processing of PC1 serves to regulate PC1 activity.
We have shown previously that in AtT-20 cells, constitutively released PC1 is present mostly in the 87-kDa form, while PC1 (also known as PC3) released through stimulation predominantly consists of a carboxyl-terminally truncated, 66-kDa protein (Vindrola and Lindberg, 1992). This difference indicates that carboxyl-terminal processing of 87-kDa PC1 probably largely occurs within regulated secretory vesicles. Recent studies employing temperature block, brefeldin A, and determination of oligosaccharide maturation have supported the idea that PC1 is carboxyl-terminally cleaved within the post-trans-Golgi network compartments in the regulated secretory pathway (Benjannet et al., 1992; Lindberg, 1994; Milgram and Mains, 1994; Zhou and Mains, 1994a). Through purification and characterization of recombinant PC1, we have demonstrated that both the 87-and the 74/66-kDa forms of PC1 are enzymatically active (Zhou and Lindberg, 1994). The conversion from 87-kDa PC1 to the 74/66-kDa forms not only increases specific activity while decreasing overall stability, it also narrows the pH optimum, increases calcium-dependence, and alters susceptibility to certain proteinase inhibitors (Zhou and Lindberg, 1994). Thus, we speculate that proteolytic processing of PC1 may play an important role in the regulation of PC1 enzymatic activity in vivo. The carboxyl-terminal segment may also be involved in the targeting and sorting of PC1 to secretory vesicles. In the experiments described here, we have used site-directed mutagenesis to study the biosynthetic processing, sorting, and function of the various domains of PC1 in PC12 cells.
The mutation at Arg-Arg
of mPC1 was carried out using the Muta-Gene(TM) in vitro mutagenesis kit (Bio-Rad). A mutagenesis primer, 5`-CTT TTC CAC
TCC GTG CTT GTC ATT CTG GAC TG-3`, was synthesized by LSUMC Core
Laboratories. Single-strand DNA was prepared as described by Ausubel et al.(1987). The mutation in mPC1591BL/CMV was confirmed by
DNA sequencing using a DNA Sequenase(TM) version 2.0 kit (U. S.
Biochemical Corp.). The mutations at Arg
-Arg
and Gly
were performed using a PCR mutagenesis
method. Primers XBAI-3 (5`-CAC AAC AAC TCT AGA CCC AGG AAC-3`) and
BSTXI-5 (5`-TAA ATG CCA AAG CTC TGG TGG-3`) were synthesized to match
the sequences of mPC1 cDNA at bp 1405-1426 or 2392-2415
(with a mutation switching CG to TA at 2404 bp to introduce an XbaI cleavage site). Two pairs of mutagenesis primers were
synthesized to introduce mutations at Arg
-Arg
or at Gly
. These primers were 628BL-5
(5`-GCA-ATG-TGG-AGG-GTA-AAG CGG ATG AGC AGG TAC-3`), 628BL-3 (5`-GTA
CCTGCT CAT CCG CTT TAC CCT CCA CAT TGC-3`), 591ST-5 (5`CCA GAA TGA CAG
GAG ATA AGT GGA AAA GAT GGT G-3`), and 591ST-3 (5`-CAC CAT CTT TTC CAC
TTA TCT CCT GTC ATT CTG G-3`). The mPC1/CMV and mPC1591BL/CMV vectors
were transfected into JM101 Escherichia coli, then were
amplified and purified using a Wizard(TM) Miniprep DNA purification
system (Promega, Madison, WI). About 2 µg of mPC1/CMV DNA were
digested with 1.5 µl of XbaI (30 units, New England
Biolabs) in a 20-µl volume at 37 °C for 1.5 h; after heating at
70 °C for 10 min, 15 µl of this reaction mixture was further
digested with 1.5 µl of BstXI (15 units, New England
Biolabs) in a 30-µl volume at 55 °C for 1.5 h. The reaction
products were separated on 1% agarose; a 6800-bp cDNA fragment was
recovered and extracted from the agarose gel using a Geneclean II kit
(Bio 101, Inc.). This cDNA was used as the vector in the following
ligation reaction. Using 0.5 µg of mPC1/CMV or mPC1591BL/CMV DNA as
template, three separate PCR reactions were carried out. The BSTXI-5
and 591ST-3 (or 628 BL-3) were used as PCR primers in the first
reaction, while 591ST-5 (or 628BL-5) and XBAI-3 were used in the second
reaction. After 10 cycles of amplification, the products were separated
from template and primers on 1.5% agarose gel; the major DNA product of
each PCR reaction was recovered and extracted from an agarose gel using
a Geneclean II kit. In the third PCR reaction, the major products of
the first two reactions were mixed and annealed to generate a template,
and BSTXI-5 and XBAI-3 were used as PCR primers. The amplification was
performed for 15 cycles, and the product (1005 bp) was separated from
template and primers on a 1% agarose gel and DNA recovered using a
Geneclean II kit. The product of the third PCR was digested using BstXI and XbaI as described above; the largest
product was separated from small DNA segments using the Geneclean II
kit. In order to ligate the PCR products into the digested vector (6800
bp), 60 ng of vector DNA was mixed with half of the PCR product and 1
µl (400 units) T
DNA ligase (New England Biolabs) in a
20-µl final volume for 45 min at room temperature. The ligation
mixture was then transformed into Ultracomp(TM) INV
F` cells
(Invitrogen). To confirm the mutations and accuracy of PCR
amplification, the 1005-bp PCR product region was sequenced using a DNA
Sequenase(TM) version 2.0 kit with three different sequencing
primers. Plasmids of correctly mutated mPC1SB/CMV
(Arg
-Arg
Lys-Ala), mPC1DB/CMV
(Arg
-Arg
Lys-His and
Arg
-Arg
Lys-Ala), and mPC1ST/CMV
(truncation at Gly
) were then amplified in E. coli (XL1 Blue; Stratagene) and purified using a Qiagen plasmid kit.
Figure 2: Western blotting of recombinant mPC1 proteins synthesized in PC12 cells. PC12 cells, transfected with mPC1, mPC1SB, mPC1DB, or mPC1ST, were homogenized in Laemmli sample buffer; the cell extracts were analyzed on 8.8% SDS-PAGE, followed by Western blotting using PC1 amino-terminal antiserum. ST, mPC1ST; WT, wild-type mPC1; SB, mPC1SB; DB, mPC1DB.
Figure 5: Analysis of proneurotensin processing in PC12 cells expressing various forms of PC1. Products of proneurotensin processing were detected by radioimmunoassay using specific radioimmunoassays directed against the epitopes indicated. A schematic diagram of proneurotensin is shown in panel a. Within each cell line, the percentage of each particular processing product is shown in panel b as a percentage of total proneurotensin. Numbers in parentheses refer to dibasic sites detected by each assay. Quantitation of total proneurotensin in each cell extract was determined by assaying iK6L following tryptic digestion (CTiK6L); the amounts of CTiK6L in PC12 Ctr (control), mPC1DB/PC12, mPC1/PC12, and mPC1ST/PC12 cells were 37.4 ± 4.9, 10.1 ± 0.9, 11.6 ± 2.5, and 34.5 ± 1.9 pmol/mg protein, respectively (mean ± S.E., n = 4).
The rat proneurotensin precursor (Kislaukis et al., 1988)
is depicted in Fig. 5a. It consists of a 169-residue
polypeptide, which begins with a NH-terminal signal
peptide(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) .
Neurotensin is located near the COOH terminus of the precursor and is
flanked by two Lys-Arg sequences at positions 148-149 and
163-164. Neurotensin is preceded by a neuromedin N sequence
located between Lys
-Arg
and
Lys
-Arg
. A fourth Lys-Arg sequence occurs
near the middle of the precursor at position 85-86. This doublet
and the one at the NH
terminus of neuromedin N delimit a
53-residue peptide, which starts with a neuromedin N-like sequence
(Lys-Leu-Pro-Leu-Val-Leu, designated K6L) and ends with an acidic
sequence (Glu-Lys-Glu-Glu-Val-Ile, designated E6I).
The
specificities of the neurotensin, neuromedin N, E6I, and K6L antisera
used here have been described previously in detail (Bidard et
al., 1993; Rovere et al., 1993). Briefly, the neurotensin
and E6I antisera react with the free COOH termini, while the neuromedin
N and K6L antisera recognize the free NH termini, of their
respective antigens. These antisera cross-react poorly (<1%) with
antigenic sequences that are internal to proneurotensin or
proneurotensin fragments. Thus, the neurotensin antiserum will detect
all precursor products with a COOH-terminal neurotensin sequence
(including authentic neurotensin). Similarly, the E6I antiserum will
measure all the precursor forms ending with the E6I sequence, while the
neuromedin N and K6L antiserum will assay the precursor products
bearing NH
-terminal neuromedin N and K6L sequences,
respectively. The radioimmunoassay and reverse-phase HPLC procedures
employed here to quantitate the various proneurotensin-derived peptides
have been fully described elsewhere (Bidard et al., 1993;
Rovere et al., 1993).
All cell extracts were directly
assayed for their content in immunoreactive neurotensin (iNT), E6I
(iE6I), and K6L (iK6L). Because of the above-described antisera
specificity, the iNT, iE6I, and iK6L assays measure the amounts of
precursor products that are processed at the
Lys-Arg
,
Lys
-Arg
, and Lys
-Arg
sequences, respectively. Portions of the cell extracts were
submitted to Arg-directed tryptic digestion (Bidard et al.,
1993; Rovere et al., 1993) and then assayed for immunoreactive
K6L. The value of CTiK6L thus obtained provides an index of the total
amount of proneurotensin (either processed or unprocessed) that was
synthesized and stored in the cells during the induction period. The
remainder of the trypsin-treated samples was applied to reverse-phase
HPLC, and the fractions were assayed for their immunoreactive
neuromedin N (iNN) content. Previous studies have shown that
trypsin-generated iNN can be resolved by HPLC into two peaks, one
comigrating with synthetic neuromedin N and the other with neuromedin N
bearing a COOH-terminal Lys-Arg extension (Bidard et al.,
1993; Rovere et al., 1993). The latter peptide is produced by
Arg-directed tryptic digestion of precursor forms in which the
neuromedin N sequence is internal, whereas the former is generated by
cleavage of peptides that end with a COOH-terminal neuromedin N
sequence. Thus, the post-HPLC assay of trypsin-generated neuromedin N
provides a measurement of all the precursor products that are processed
at the Lys
-Arg
sequence (including
authentic neuromedin N). The results were normalized for the amount of
protein in each extract. The percentages of cleavage at the
Lys
-Arg
,
Lys
-Arg
,
Lys
-Arg
, and Lys
-Arg
sequences were calculated by dividing, respectively, iNT,
trypsin-generated neuromedin N, iE6I, and iK6L by CTiK6L and by
multiplying these ratio values by 100. Duplicate independent samples
were analyzed on two separate occasions.
Figure 1: Diagram of site-directed mutants of mPC1.
Figure 3: Western blotting of recombinant mPC1 proteins secreted from PC12 cells. PC12 cells transfected with different forms of mPC1 were incubated with or without 50 mM KCl for 40 min. The conditioned media were concentrated and analyzed on 8.8% SDS-PAGE, followed by Western blotting using PC1 amino-terminal antiserum. ST, mPC1ST; WT, wild-type mPC1; SB, mPC1SB; DB, mPC1DB.
In
pulse-chase labeling experiments, we found that amino-terminal
conversions of pro-mPC1DB and pro-mPC1ST were completed within the
first 20 min of synthesis, similar to wild-type pro-mPC1 (data not
shown). This result indicates that substitution of
Arg-Arg
and Arg
-Arg
or deletion of the PC1 carboxyl-terminal region (residues
592-726) apparently had little effect on proPC1 conversion.
During the later stages of biosynthesis, intracellular 87-kDa mPC1DB
remained intact after a 4-h chase period, while wild-type PC1 was
converted to the 66-kDa form (Fig. 4). Constitutive secretion of
the 87-kDa form of both mPC1DB and wild-type PC1 occurred within 1 h
after the pulse period (Fig. 4). Similar studies of the mPC1ST
mutant demonstrated efficient synthesis of a 66-kDa form of PC1 and
constitutive secretion into the medium over the same time period
(results not shown).
Figure 4:
Biosynthesis of wild-type mPC1and mPC1DB
in PC12 cells. PC12 cells transfected with either wild-type mPC1 or
mPC1DB were labeled with [S]Met for 20 min, then
chased in methionine-containing medium with 2% dialyzed fetal bovine
serum for various periods of time as indicated. The conditioned media
and cell extracts were immunoprecipitated using PC1 amino-terminal
antiserum. The immunoprecipitates were separated using SDS-PAGE and
subjected to fluorography.
The amount of proneurotensin (processed and unprocessed) stored in the various cell lines during the induction period ranged between approximately 10 and 30 pmol/mg protein (CTiK6L values are given in the legend to Fig. 5). Similarly to wild-type mPC1, mPC1DB and mPC1ST both possessed the ability to process proneurotensin (Fig. 5).
All
forms of PC1 markedly increased proneurotensin cleavage at the
Lys-Arg
and Lys
-Arg
dibasic sites as compared to the control (Fig. 5b). They also cleaved, though less efficiently,
the Lys
-Arg
site. This cleavage event was not
observed in control PC12 cells. Only mPC1 and mPC1ST were able to
increase processing at the Lys
-Arg
site
above the level seen in the control, whereas mPC1DB appeared inactive
in that respect. In general, and especially given the fact that it had
the highest level of expression, mPC1DB was much less efficient in
processing proneurotensin than mPC1 and mPC1ST. Interestingly, mPC1ST,
the enzyme expressed at the lowest level, was the most active in
processing the Lys
-Arg
site, in contrast to
mPC1DB which apparently did not cleave this dibasic despite its high
level of expression. Thus, there appear to be certain differences in
proneurotensin processing efficiency and site usage between the 66- and
87-kDa PC1 forms.
PC1 is known to be cleaved within its carboxyl-terminal
region at a late stage of its biosynthesis (Vindrola and Lindberg,
1992). Previous work has suggested that an autocatalytic mechanism may
be involved in this process (Zhou and Lindberg, 1994); however, the
site of this carboxyl-terminal cleavage event has not yet been
identified. In this work, we have assumed that this cleavage occurs at
a paired basic site, since these are known to represent consensus
sequences for PC1 cleavage. Four paired basic residues are located in
the mPC1 carboxyl-terminal region; cleavage at these sites can generate
products with estimated molecular masses between 62 and 73 kDa.
However, among these sites, only Arg-Arg
and Arg
-Arg
are conserved among the
PC1 sequences of human, rat, mouse, and anglerfish (Seidah et
al., 1991, 1992; Smeekens et al., 1991; Bloomquist et
al., 1991; Roth et al., 1993); thus, these two sites were
thought to represent likely candidate sites for PC1 carboxyl-terminal
cleavage. By performing site-directed mutagenesis at these two sites,
we found that mutation of Arg
-Arg
alone had
little effect on the generation of the 66-kDa form, while mutations of
both Arg
-Arg
and
Arg
-Arg
were able to block the conversion
of 87-kDa PC1 to the 66-kDa form. Furthermore, mPC1ST (truncation at
Gly
) exhibited a molecular mass on SDS-PAGE identical to
that of endogenous 66-kDa PC1 converted from the 87-kDa wild-type mPC1.
Subsequent to the generation of our mutants, the sequence of Aplysia PC1 was published (Chun et al., 1994); PC1a
from this species contains the first of these dibasics, but not the
second. Taken together, these data strongly suggest that
Arg
-Arg
is the major cleavage site for the
generation of 66-kDa PC1 in vivo. Since wild-type PC12 cells
possess a regulated secretory pathway, but lack the ability to process
prohormones at paired basic residues, PC1 cleavage at
Arg
-Arg
within PC12 cells is likely to be
attributable to an autocatalytic mechanism. This interpretation is
supported by our in vitro work (Zhou and Lindberg, 1994) and in vivo results obtained in AtT-20 cells, which indicate that
overexpression of PC1 results in increased COOH-terminal proteolytic
processing (Zhou and Mains, 1994a).
The presence of an intermediate
form of PC1 of approximately 74 kDa has been observed in in vitro studies (Zhou and Lindberg, 1994); however, little 74-kDa mPC1 is
found in PC12 cells (this study) or in AtT-20 cells (Vindrola and
Lindberg, 1992; Milgram and Mains, 1994). Taken together with the
finding of lesser effects of the mutation of
Arg-Arg
in PC12 cells, these results
suggest that 74-kDa PC1 may represent a minor product during the
carboxyl-terminal processing of PC1 in vivo. A possible
explanation for these differences is that the cleavage site generating
the 74-kDa form is blocked in vivo, possibly due to an
association of PC1 carboxyl-terminal region with membrane or with other
proteins. The association of PC1 with membranes and association with
other granule proteins have both been reported (Vindrola and Lindberg,
1992; Palmer and Christie, 1992).
Although mutation at
Arg-Arg
and Arg
-Arg
substantially blocked PC1 carboxyl-terminal conversion, a small
portion of 87-kDa mPC1DB was still cleaved. The product was slightly
larger than wild-type 66-kDa PC1 on SDS-PAGE, suggesting that an
alternative cleavage site is involved. Through limited digestion using
chymotrypsin, trypsin, and subtilisin, which possess different
substrate specificities, we found that all three proteinases were able
to convert 87-kDa recombinant PC1 to 66- and 74-kDa-like products in
its carboxyl-terminal region (Zhou and Lindberg, 1994). These results
suggest that the cleavage site in the PC1 carboxyl terminus is located
in an exposed region which can readily be attacked. Therefore, the
alternative cleavage site usage in PC12 cells may be due to the action
of other proteinases located in the regulated secretory pathway.
Alternatively, PC1 itself may also act at alternative cleavage sites.
This idea is supported by our in vitro studies that
demonstrate spontaneous carboxyl-terminal cleavage of 87-kDa mPC1DB
purified from Chinese hamster ovary cells amplified for the production
of this protein (results not shown). A possible alternative cleavage
site for transfected mPC1 may be Lys
-Arg
,
although this site is present only in mouse PC1. Cleavage at the
Lys
-Arg
site can generate a product 12
amino acids longer than the product cleaved at
Arg
-Arg
(66-kDa PC1); this molecular mass
is also consistent with our observed molecular masses on SDS-PAGE.
Construction of a PC1 vector encoding a further mutation at
Lys
-Arg
will be required to investigate
this possibility.
Carboxyl-terminal conversion of PC1 occurs mainly in regulated secretory granules, as evidenced by previous studies in AtT-20 and PC12 cells (Vindrola and Lindberg, 1992; Benjannet et al., 1992; Lindberg et al., 1994; Lindberg, 1994; Milgram and Mains, 1994; Zhou and Mains, 1994a). However, the functional significance of this conversion event is not clear. The activation of proPC1 occurs within the endoplasmic reticulum (Lindberg, 1994; Milgram and Mains, 1994; Goodman and Gorman, 1994), while peptide hormone precursors are thought to be cleaved within the later stages of the secretory pathway (reviewed by Loh et al.(1992)). It may thus be necessary for the cell to regulate PC1 function during intracellular transport. The decreasing pH gradient from the endoplasmic reticulum to the secretory granules may represent one important aspect of this regulation. The pH within the trans-Golgi network and regulated secretory granules corresponds well with the optimal pH of PC1 activity, between 5.0 and 6.5 (Zhou and Lindberg, 1993; Jean et al., 1993; Rufaut et al., 1993). On the other hand, since the timing and location of PC1 carboxyl-terminal processing coincide with the timing and location of prohormone processing, truncation of PC1 may also play a role in the regulation of enzyme activity. In vitro studies have shown that that carboxyl-terminal cleavage of PC1 dramatically increases PC1 activity against peptide and prohormone substrates (Zhou and Lindberg, 1994), suggesting that carboxyl-terminal cleavage of PC1 could potentially possess physiological significance.
In order to determine the function of the PC1 carboxyl-terminal
region in vivo, we compared the processing of proneurotensin
in PC12 cells stably transfected with wild-type mPC1, mutated mPC1DB,
or mPC1ST. In contrast to AtT-20 cells, PC12 cells do not express
prohormone convertases; thus, neurotensin is stored mainly in precursor
form (Carraway et al., 1993; Rovere et al., 1993).
When the varying expression levels are taken into account, the 66-kDa
form of PC1 was found to be the most active against proneurotensin,
especially relative to the 87-kDa blockade mutant, which was expressed
at much higher levels. The comparatively low activity of mPC1DB against
proneurotensin confirms our previous in vitro results, which
indicate that the 87-kDa PC1 represents only a partially active PC1
form (Zhou and Lindberg, 1994); removal of the carboxyl-terminal region
appears to be required to fully activate PC1. The finding that the
various forms of PC1 are differentially active against proneurotensin
supports our in vitro results showing that the 87- and
74/66-kDa recombinant PC1s exhibit differing specific activities (Zhou
and Lindberg, 1994). A recent study has also demonstrated that
expression of carboxyl-terminally truncated PC1 (Stop)
in AtT-20 cells increases the rate of conversion of proopiomelanocortin
(Zhou and Mains, 1994b). Based on these in vivo and in
vitro observations, we speculate that carboxyl-terminal processing
of PC1 during transport through the secretory pathway may control the
amount of PC1 activity available for the processing of prohormones.
In conclusion, we have demonstrated that PC1 carboxyl-terminal
conversion largely occurs at Arg-Arg
site
through a possible autocatalytic mechanism; the PC1 carboxyl-terminal
domain (Gly
to Asn
) is required neither for
activation nor for intracellular transport of PC1 to secretory
granules. However, removal of this domain appears to increase total PC1
activity and alters cleavage site preference. In line with our previous in vitro data, these results support the idea that
carboxyl-terminal processing is important for the regulation of PC1
function.