(Received for publication, September 26, 1994; and in revised form, February 6, 1995)
From the
Carboxypeptidase E (CPE) is involved with the biosynthesis of
numerous peptide hormones and neurotransmitters. Several forms of CPE
have been previously detected in neuroendocrine cells, including a form
which is soluble at pH 5.5 (S-CPE), a form which can be extracted from
membranes with 1 M NaCl at pH 5.5 (M1-CPE), and a form which
requires both 1% Triton X-100 and 1 M NaCl for extraction from
membranes at pH 5.5 (M2-CPE). Like other peptide processing enzymes,
CPE is known to be sorted into peptide-containing secretory vesicles of
the regulated pathway. One mechanism that has been proposed to be
important for sorting of regulated pathway proteins is Ca and pH-induced aggregation. CPE purified from bovine pituitary
membranes aggregates at pH 5.5 when the concentration of CPE is 0.3
µg/µl or higher, but not when the concentration is 0.01
µg/µl. Aggregation of CPE is pH-dependent, with very little
aggregation occurring at pH 6 or above. At pH 5.0-5.5, the M2
form of CPE shows a greater tendency to aggregate than the other two
forms. At pH 6, Ca
concentrations from 1-30
mM increase the aggregation of M1- and M2-CPE, but not S-CPE.
The aggregation of M2-CPE does not explain the apparent membrane
binding of this protein since the aggregate is solubilized by 1% Triton
X-100 at pH 5.5 or by pH 6.0, whereas M2-CPE is not extracted from
membranes under these conditions. Taken together, these results are
consistent with a model in which the decreasing pH and increasing
Ca
levels in the trans Golgi network induce
the aggregation of CPE, which contributes to the sorting of this
protein into regulated pathway secretory vesicles.
Many peptide hormones and neurotransmitters are produced by the
selective proteolysis of prohormone precursors (Steiner, 1991).
Initially, endopeptidases cleave the precursor at basic amino acid
consensus sites (Lindberg and Hutton, 1991; Devi, 1991), and then a
carboxypeptidase removes these basic residues from the peptide
intermediates (Fricker, 1988a, 1991). Several mammalian
peptide-processing endopeptidase have been reported, including
prohormone convertase 1 (PC1, also known as PC3), PC2, PACE 4, PC4, PC5
(also known as PC6), and furin (Smeekens and Steiner, 1990; Seidah et al., 1990, 1991; Smeekens et al., 1991; Kiefer et al., 1991; Nakayama et al., 1992, 1993). In
contrast to the multitude of endopeptidases, only a single
carboxypeptidase is thought to be involved with intracellular peptide
processing (Fricker, 1988a, 1991). Carboxypeptidase E (CPE) ()is present in all neuroendocrine tissues, and in several
tissues this enzyme has been localized to the peptide-containing
secretory vesicles (Fricker, 1988a, 1991).
Peptide processing is
thought to begin in the trans Golgi network and continue in
the secretory vesicle (Orci et al., 1987; Schnabel et
al., 1989). Thus, it is essential that the peptide processing
enzymes and the substrates are co-segregated into secretory vesicles.
Two distinct mechanisms of sorting have been proposed; one mechanism
requires a specific protein which binds to and sorts the proteins
destined for the secretory vesicles (Kelly, 1985). Although a
prohormone-specific sorting protein was reported (Chung et
al., 1989), this protein has been identified as chymotrypsinogen
(Gorr et al., 1992), and it is unlikely that this protein
participates in the sorting of most secretory proteins. The second
mechanism proposed for the sorting of secretory proteins is the
selective aggregation hypothesis (Chanat and Huttner, 1991; Reaves and
Dannies, 1991). In this model, proteins sort themselves by forming
aggregates in the environment of the trans Golgi network
(Bauerfeind and Huttner, 1993). This compartment is slightly acidic
(Anderson and Pathak, 1985), and has been estimated to contain mM concentrations of Ca (Chanat and Huttner, 1991),
based on the Ca
concentrations in other organelles of
the secretory pathway (Bulenda and Gratzl, 1985; Roos, 1988; Sambrook,
1990). Evidence for this aggregation model of protein sorting is
provided by the finding that several secretory granule proteins undergo
aggregation at slightly acidic pH and in the presence of mM concentrations of Ca
(Gorr et al.,
1989; Yoo and Albanesi, 1990; Chanat and Huttner, 1991; Shennan et
al., 1994). For example, chromogranin A and B and secretogranin II
form aggregates in the presence of 10-20 mM Ca
at pH 5-6, but not at neutral pH
(Chanat and Huttner, 1991). Recently PC2 has been found to undergo pH-
and Ca
-induced aggregation (Shennan et al.,
1994).
The sorting of CPE into secretory vesicles was hypothesized
to proceed by the binding of a C-terminal amphipathic helix to
membranes at acidic, but not neutral pH values (Fricker et
al., 1990). Evidence that this C-terminal region may contribute to
the sorting of CPE was provided by studies using fusion proteins;
albumin with the C-terminal region of CPE is sorted into the regulated
pathway, although with a low efficiency compared to the sorting of
native CPE (Mitra et al., 1994). This result raised the
possibility that additional regions of CPE are involved in the
efficient sorting of this protein. CPE has homology to the
Ca-binding region of the bacterial metalloenzyme
carboxypeptidase T, and CPE has recently been found to bind
Ca
(Nalamachu et al., 1994). Since bacteria
and mammals diverged approximately 1.8 billion years ago, this homology
between the Ca
-binding region of carboxypeptidase T
and CPE implies that the binding of Ca
to CPE is
important. However, Ca
does not substantially alter
CPE activity, and has only a small effect on CPE stability (Nalamachu et al., 1994). The possibility that Ca
influences the aggregation of CPE was previously investigated
using low concentrations (0.01-0.1 µg/µl) of protein at
pH 5.5 (Nalamachu et al., 1994). Although no evidence for
aggregation was found, these concentrations are much lower than the
protein concentrations typically used to study aggregation (Gorr et
al., 1989; Yoo and Albanesi, 1990; Chanat and Huttner, 1991). In
the present study, we find that CPE does aggregate at pH 5.5 when the
concentration is 0.3 µg/µl or higher. This aggregation is
dependent on pH and is augmented by 1-30 mM Ca
, although not all forms of CPE show the same
dependence on pH or Ca
. Since these concentrations of
CPE, Ca
, and H
are thought to be
physiological, these findings raise the possibility that the
aggregation of CPE may participate in the sorting of this protein into
the regulated pathway.
For experiments where the amount of protein was analyzed only by gel electrophoresis, the supernatant was removed using microcapillary pipette tips (Marsh) and diluted with 10 volumes of SDS gel loading buffer. A similar volume of SDS gel loading buffer was added to the pellet fraction, the tubes were mixed, and aliquots were taken for gel electrophoresis. Samples were heated at 95 °C for 5 min and analyzed on a denaturing 10% polyacrylamide gel. Following electrophoresis, the protein was detected using the silver staining procedure (Morrissey, 1981). Quantitation of the silver-stained gels was performed using an image analysis system consisting of a Cohu video camera, a PC visions frame grabber, and Jandel Scientific's JAVA software.
For experiments where the
amount of CPE was quantitated by enzyme and protein assays in addition
to the gel electrophoresis, the supernatant from the 100,000 g centrifugation step was diluted 1:10 into 5 mM Tris-Cl, pH 8.0, containing 0.01% Triton X-100. An aliquot was
further diluted 1:200 in 100 mM NaAc buffer at pH 5.5
containing 0.01% Triton X-100, and then 20 µl were used to measure
CPE activity using the substrate dansyl-Phe-Ala-Arg as described by
Fricker(1994). The amount of protein in the supernatant was determined
using the Bradford assay with 20 µl of the 1:10 diluted
supernatant. An aliquot of the 1:10 diluted supernatant was also
analyzed on denaturing polyacrylamide gels, along with an aliquot of
the pellet fraction after extraction with SDS gel loading buffer, as
described above. All CPE activity and protein measurements were
performed in triplicate; variation was typically less than 5%. Each
experiment was repeated a minimum of two times with comparable results.
Extraction of CPE from Bovine Pituitary Membranes and Western Blot
Analysis-Bovine pituitary glands (Pel-Freez) were homogenized
(Polytron, Brinkman) and centrifuged as described above for the
purification of CPE. Pellets after the extraction of S-CPE and M1-CPE
were homogenized in various buffers, as indicated in Fig. 5. The
homogenate/buffer mixture was mixed and then centrifuged for 30 min at
50,000 g at 4 °C. The supernatant was removed, and
a 100-µl aliquot analyzed on 10% denaturing polyacrylamide gels.
Following electrophoresis, the protein was transferred to
nitrocellulose and Western blots were performed as described previously
(Fricker et al., 1990). CPE was detected using a 1:1000
dilution of an antiserum raised against a peptide corresponding to the
N-terminal region of CPE (Fricker et al., 1990), using
I-protein A to detect the primary antisera. An aliquot of
the supernatant was diluted with 100 mM NaAc, pH 5.5, buffer
and used for CPE activity determinations, as described above.
Figure 5:
Effect of different buffers on the
extraction of M2-CPE from bovine pituitary membranes. Bovine pituitary
glands were homogenized in 5 volumes of 20 mM NaAc buffer
containing 1 M NaCl and centrifuged at 50,000 g for 30 min. This step was repeated one time, and then the pellets
were resuspended in 5 volumes of 20 mM NaAc buffer,
homogenized, and centrifuged again. The pellets were combined with
various buffers: 100 mM NaAc at pH 5.5 or 6.0; 100 mM Tris-Cl at pH 7.0, 8.0, or 9.0; and in the presence (+) or
absence(-) of 1 M NaCl or 1% Triton X-100. The mixture
was homogenized, and then centrifuged at 50,000
g for
30 min. An aliquot of the supernatant was analyzed on a denaturing 10%
polyacrylamide gel followed by Western blot analysis as described under
``Materials and Methods.'' The positions and molecular masses
of prestained molecular weight standards (Bio-Rad) are
indicated.
When purified M2-CPE was incubated at a concentration of 0.01
µg/µl for 2 h at 37 °C at pH 5.5 and then centrifuged at
100,000 g for 30 min, nearly all of the protein was
recovered in the supernatant (Fig. 1). This finding is
consistent with a previous study which found no evidence for the
aggregation of CPE under these conditions (Nalamachu et al. 1994). However, when the concentration of the protein was increase
to 0.3 µg/µl, a large amount of the M2-CPE was recovered in the
pellet (Fig. 1). For this analysis, the samples were diluted
after centrifugation so that equal amounts of protein could be loaded
onto the gel. Concentrations of M2-CPE from 1 to 3 µg/µl showed
slightly more protein in the pellet, compared with the 0.3
µg/µl sample (Fig. 1). These results indicate that CPE
aggregates at high protein concentrations at pH 5.5 in the absence of
added Ca
.
Figure 1:
Effect of CPE concentration on the
aggregation of M2-CPE. Purified M2-CPE at the indicated concentration
was incubated for 2 h at 37 °C in 25 mM NaAc buffer, pH
5.5, and then centrifuged at 100,000 g for 30 min, as
described under ``Materials and Methods.'' The entire
supernatant (S) and the pellet (P) fractions from the
0.01 µg/µl group were used without dilution. The sets
containing higher amounts of CPE were diluted according to the initial
amount of protein so that each set would contain equal amounts of
protein for the gel. The positions and molecular masses of protein
markers (Bio-Rad) are shown (M).
The aggregation of CPE was not found to be substantially different when samples were incubated at 37 or at 4 °C (data not shown), and so the physiologically relevant 37 °C was used in subsequent experiments. Samples which were not incubated before centrifugation showed no aggregate, with the maximal amount of aggregate observed after 1-2 h of incubation (data not shown). To test the effect of pH on the aggregation of CPE, 1.5 µg/µl purified M2-CPE was incubated for 2 h, at pH values between 5 and 8, and then centrifuged and analyzed on a denaturing polyacrylamide gel (Fig. 2, top). At pH 5.0, nearly all of the M2-CPE was recovered in the pellet fraction (Fig. 2, top). Approximately 50% of the M2-CPE was particulate at pH 5.5, and very little was particulate at pH values of 6.0 or higher (Fig. 2, top). The amounts of CPE in the supernatant and pellet fractions were quantitated by three different methods; the silver stained polyacrylamide gels were quantitated using an image analysis system, the amount of protein in the supernatant was determined using the Bradford protein assay, and the amount of CPE activity in the supernatant was determined using an enzyme assay (after diluting the sample into pH 5.5 buffer). All three of these methods gave comparable results, and the enzyme assay was found to be the most convenient and sensitive. Using the enzyme assay to quantitate the amount of CPE in the supernatant, the different forms of CPE were compared for the pH-dependent aggregation (Fig. 2, bottom). All forms of CPE showed pH-dependent aggregation, although S-CPE and M1-CPE did not produce as much aggregate as M2-CPE (Fig. 2, bottom). At pH 5.0, approximately 30-40% of the S- and M1-CPE was recovered in the soluble faction. These observations were verified by polyacrylamide gel electrophoresis of the soluble and pellet fractions of each form of CPE (data not shown).
Figure 2:
Effect of pH on the aggregation of CPE. Top, polyacrylamide gel analysis of the supernatant and pellet
fractions after incubation of 1.5 µg/µl M2-CPE at the indicated
pH for 2 h at 37 °C and then centrifugation at 100,000 g for 30 min, as described under ``Materials and
Methods.'' NaAc buffers were used for the samples at pH
5-6.5, and Tris-Cl buffers were used for the pH 7 and 8 groups.
The positions and molecular masses of protein markers (Bio-Rad) are
shown. Bottom, quantitation of the amount of CPE activity in
the supernatant after incubation at the indicated pH and
centrifugation. Samples were diluted 1:200 into 100 mM NaAc,
pH 5.5, prior to CPE activity analysis. CPE activities are shown in
relative units, which were adjusted to 100 for the pH 7.0 data since
the polyacrylamide gels revealed that virtually all of the CPE was
soluble at this pH. Error bars show range of data from two separate
determinations. Points without error bars had range values smaller than
the symbol size.
To test the Ca dependence of the aggregation of CPE, 1.5 µg/µl CPE was
incubated with 0.3 to 30 mM CaCl
at pH 6.0, and
then centrifuged and analyzed by gel electrophoresis (Fig. 3, top) or by enzyme assay after dilution with pH 5.5 buffer (Fig. 3, bottom). As found in Fig. 2, very
little M2-CPE was detected in the pellet when tested at pH 6.0 in the
absence of Ca
(Fig. 3, top). However,
when CaCl
is included in the incubation mixture, a moderate
amount of M2-CPE was detected in the pellet fraction (Fig. 3, top). Quantitation of the amount of CPE in the supernatant
fraction showed that 10-30 mM CaCl
reduced
this amount by approximately 30%, while 1 mM CaCl
reduced this amount by approximately 20% (Fig. 3, bottom). Similar results were obtained using M1-CPE, whereas
the amount of S-CPE in the supernatant did not change by more than 10%
for any concentration of CaCl
(Fig. 3, bottom).
Figure 3:
Effect of CaCl on the
aggregation of CPE. Top, polyacrylamide gel electrophoresis of
the supernatant and pellet fractions after incubation of M2-CPE with
the indicated concentration of CaCl
and centrifugation, as
described under ``Materials and Methods.'' The positions and
molecular masses of protein markers (Bio-Rad) are shown. Bottom, quantitation of the amount of CPE activity in the
supernatant after incubation of M2-CPE with the indicated amount of
CaCl
and centrifugation. Samples were diluted 1:200 into
100 mM NaAc, pH 5.5, prior to the enzyme assay. CPE activities
are shown in relative units, which were adjusted to 100 for the samples
incubated in the absence of CaCl
since the polyacrylamide
gel analysis revealed that nearly all of the CPE was soluble under this
condition. Error bars show standard error: n = 3 for
S-CPE and M1-CPE, and n = 5 for M2-CPE. Points without
error bars had standard error smaller than the symbol
size.
The finding that aggregation of S-, M1-, and
M2-CPE differ in their pH and/or Ca dependence ( Fig. 2and Fig. 3, bottom) raises the possibility
that aggregation, and not membrane binding, may account for the
presence of CPE in the particulate fraction of cell extracts. For
example, the M2-form of CPE might not represent a membrane-bound form,
as hypothesized, but instead might be due to an insoluble aggregate. To
test this, we compared the ability of various buffers to solubilize
aggregates of M2-CPE. The aggregates were produced by incubating 1.5
µg/µl M2-CPE at pH 5.0 for 2 h at 37 °C, and then the
aggregates were collected by centrifugation. When the aggregates were
mixed with sodium acetate buffer at pH 5.5 and then re-centrifuged, the
majority of CPE was present in the pellet (Fig. 4). Addition of
1 M NaCl to the buffer did not substantially alter the amount
of CPE which could be extracted from the aggregate, whereas 1% Triton
X-100 was very effective in solubilizing the aggregate (Fig. 4).
The combination of 1 M NaCl and 1% Triton X-100 was slightly
more effective than Triton X-100 alone (Fig. 4). As found for
the pH-dependence of the formation of the aggregate, the dissolution of
the aggregate was also pH-dependent, with nearly all of the aggregate
extracted at pH 6.0 (Fig. 4). Higher pH buffers (7.0 and 8.0)
were similar to pH 6 buffers in extracting CPE from the aggregate (data
not shown).
Figure 4:
Effect
of different buffers on the extraction of CPE from aggregates of
M2-CPE. The aggregates of M2-CPE were obtained by incubating 1.5
µg/µl M2-CPE for 2 h at 37 °C in 25 mM NaAc
buffer, pH 5.0, containing 10 mM CaCl. The
aggregates were collected by centrifugation at 100,000
g for 30 min. The pellets were combined with 100 mM NaAc
buffer at the indicated pH, either containing (+) or not
containing(-) 1 M NaCl and/or 1% Triton X-100, as
indicated. The mixture was sonicated 20 s and then centrifuged again at
100,000
g for 30 min at 4 °C. Both supernatant and
pellet were diluted and analyzed on a denaturing polyacrylamide gel, as
described under ``Materials and Methods.'' The positions and
molecular masses of protein markers (Bio-Rad) are
shown.
The conditions which extract M2-CPE from the aggregate are distinct from the conditions previously reported to extract CPE activity from the membranes (Supattapone et al., 1984; Fricker, 1988b; Fricker et al., 1990). Specifically, very little CPE activity is extracted by pH 6.0 or by 1% Triton X-100 alone at pH 5.5; efficient extraction of membrane-bound CPE requires either a combination of NaCl and Triton X-100 at pH 5.5 or pH values in the neutral to basic range (Fricker, 1988b; Fricker et al., 1990). However, these previous studies measured enzyme activity and not the extraction of CPE protein. To test whether the extraction of CPE protein from membranes is similar to that previously reported for the enzyme activity (and distinct from the conditions that solubilize the M2-CPE aggregate), bovine pituitary membranes were extracted with various buffers and the CPE protein identified using both enzyme assays and Western blot analysis. In this procedure, the bovine pituitary membranes were first washed with 1 M NaCl to remove the S-CPE and M1-CPE, so that the analysis would detect only the M2-CPE form. When membranes were extracted with pH 5.5 buffer alone, virtually no CPE could be detected in the extract (Fig. 5). Either 1 M NaCl or 1% Triton X-100 alone at pH 5.5 only extracted a small amount of M2-CPE, whereas the combination of salt and detergent extracted a large amount of M2-CPE (Fig. 5). Importantly, very little CPE could be extracted from the membranes at pH 6.0, although large amounts were extracted at pH 7.0 or higher (Fig. 5). The amounts of CPE protein in the various extracts, as detected by Western blotting, were comparable to the amount of CPE activity detected in the extracts (data not shown). These results indicate that the conditions for the extraction of M2-CPE from membranes is distinct from the conditions which extract M2-CPE from aggregates.
The major finding of this study is that CPE can aggregate
under conditions that resemble those found in the regulated secretory
pathway. It is difficult to assign exact values for the pH and
Ca concentration of the trans Golgi
apparatus, and it has been estimated that the pH is slightly acidic
(6.2-6.5) and Ca
concentrations are around 10
mM (Chanat and Huttner, 1991). Mature secretory vesicles are
even more acidic, with an internal pH of 5.0-5.5 and
Ca
concentrations of 20 mM or higher
(Bulenda and Gratzl, 1985; Roos, 1988). The concentration of CPE in the
secretory pathway is difficult to calculate; if secretory vesicles and
the trans Golgi network constitute 0.5-5% of the total
volume of the pituitary, then the concentration of CPE would be
approximately 0.5-5 µg/µl (based on the typical recovery
of 50 µg of CPE per pituitary used for the purification of CPE).
Thus, it is likely that the conditions used for the present study
resemble those inside the secretory pathway of a cell, and that CPE
aggregates in vivo. Support for this hypothesis was provided
by immunocytochemical studies of rat islet cells; both immunoreactive
CPE and insulin were found to be co-localized in discreet patches on
the outside of cells (Aguilar-Diosdado et al., 1994). The
presence of cell surface insulin, and presumably CPE, is thought to
reflect the secretion and incomplete dissolution of a dense-core
aggregate (AguilarDiosdado et al., 1994).
If aggregation of
CPE occurs in the trans Golgi network, this could be a
mechanism which contributes to the sorting of CPE into the regulated
pathway. The combination of low pH and high Ca is
likely to be important for CPE to aggregate in vivo in the trans Golgi network since little aggregation of CPE occurs in
the absence of Ca
at pH 6. The combination of low pH
and high Ca
is thought to be involved with
aggregation of other proteins found in the regulated secretory pathway.
Several studies have found that chromogranin A and B, and secretogranin
II aggregate at acidic pH values in the presence of Ca
(Gorr et al., 1989; Yoo and Albanesi, 1990; Chanat and
Huttner, 1991). For example, Chanat and Huttner(1991) reported that
aggregation of secretogranin II was stimulated by 1-10 mM Ca
at pH 6.4, whereas little aggregation
occurred even in the presence of Ca
at pH 6.9 or 7.4.
Similar to our findings with CPE, the aggregation of these other
secretory granule proteins is not complete at pH 6-7, even with
elevated levels of Ca
(Gorr et al., 1989;
Chanat and Huttner, 1991). It will be important to determine whether
CPE and the other secretory granule proteins can form co-aggregates.
One study has reported that CPE is present in a complex with
glycoprotein III in bovine adrenal chromaffin granules (Palmer and
Christie, 1992). However, this complex is not likely to be an insoluble
aggregate since it was soluble upon extraction of the membranes at pH
7.0 with 2% octylglucoside and centrifugation at 100,000
g (Palmer and Christie, 1992).
According to the selective aggregation hypothesis, once the protein aggregate forms in the trans Golgi network it must somehow bind to membranes (Bauerfeind and Huttner, 1993). In the case of chromogranin A and B, both soluble and membrane bound forms have been detected in neuroendocrine cells (Settleman et al., 1985; Pimplikar and Huttner, 1992). CPE is also found in soluble and membrane-bound forms (Fricker and Snyder, 1982; Supattapone et al., 1984). The membrane binding domain of CPE is thought to reside in the C-terminal region, possibly due to an amphiphilic helix (Fricker et al., 1990). This membrane binding of CPE is pH-dependent, with substantial binding observed at pH 5-6, and much less binding observed at pH values of 7 or higher (Fricker, 1988b; Fricker et al., 1990). It is likely that the combination of pH-dependent aggregation and membrane binding both contribute to the efficient sorting of CPE into the regulated pathway. This model would explain why a fusion protein containing only the C-terminal region of CPE was sorted into the regulated pathway with low efficiency (Mitra et al., 1994).
The different aggregation properties of the various forms of CPE
were unexpected. All forms of CPE bind Ca (Nalamachu et al., 1994), although the affinity and number of
Ca
binding sites has not been determined for the
various forms. It is possible that the highly acidic sequence near the
C terminus of CPE (ERKEEEKEE) binds Ca
; since this
region is thought to be missing from S-CPE (Fricker et al.,
1990), this could be responsible for the different effect of
Ca
on S-CPE, compared with M1- and M2-CPE. The
different pH sensitivity of M2-CPE, compared to S- and M1-CPE is not
due to differences in the isoelectric points of these proteins because
isoelectric focusing of S-, M1-, and M2-CPE showed a similar pattern;
all three samples contained a broad distribution of proteins with pI
values between 4.8 and 5.2 (data not shown). This is consistent with
previous reports that soluble and membrane forms of CPE are each
composed of multiple proteins with pI values around 5 (Laslop et
al., 1986; Christie and Palmer, 1990; Palmer and Christie, 1992).
The molecular basis for the different forms of CPE upon isoelectric
focusing is not known. Further studies examining the various forms of
CPE, and the interaction of these forms with Ca
are
in progress.