From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several recently discovered members of the
carboxypeptidase E (CPE) gene family lack critical active site residues
that are conserved in other family members. For example, three CPE-like proteins contain a Tyr in place of Glu300 (equivalent
to Glu270 of carboxypeptidase A and B). To investigate the
importance of this position, Glu300 of rat CPE was
converted into Gln, Lys, or Tyr, and the proteins expressed in
Sf9 cells using the baculovirus system. All three mutants were
secreted from the cells, but the media showed no enzyme activity above
background levels. Wild-type CPE and the Gln300 point
mutant bound to a p-aminobenzoyl-Arg-Sepharose affinity resin, and this binding was competed by an active site-directed inhibitor, guanidinoethylmercaptosuccinic acid. The affinity purified mutant CPE protein showed no detectable enzyme activity (<0.004% of
wild-type CPE) toward dansyl-Phe-Ala-Arg. Expression of the Gln300 and Lys300 mutant CPE proteins in the
NIT3 mouse pancreatic beta-cell line showed that these mutants are
routed into secretory vesicles and secreted via the regulated pathway.
Taken together, these results indicate that Glu300 of CPE
is essential for enzyme activity, but not required for substrate
binding or for routing into the regulated secretory pathway.
Metallocarboxypeptidases perform many physiological functions.
Pancreatic carboxypeptidases A and B
(CPA1 and CPB) are primarily
involved with the digestion of food, whereas CPE and related family
members perform more selective cleavages and are involved in the
biosynthesis of peptides and proteins (1). Altogether, there are 13 members of this gene family, which is subdivided into two groups based
on homology and size. Each group has 30-70% amino acid sequence
identity with other members of the same group, but only 15-25% amino
acid sequence identity with members of the other group. One group
includes pancreatic CPA1, CPA2, CPB, mast cell CPA, and
carboxypeptidase U (also known as plasma CPB) (2-6). These family
members are all enzymatically active, and are 30-40-kDa proteins. The
other group includes CPE, carboxypeptidase M (CPM), carboxypeptidase N
(CPN), carboxypeptidase D (CPD), carboxypeptidase Z (CPZ), and three
proteins designated CPX1, CPX2, and AEBP1 (7-17). Except for CPD,
which is 180 kDa and contains 3 distinct carboxypeptidase-like domains,
the members of this family contain a single carboxypeptidase domain of
approximately 400 amino acids. Only CPE, CPM, CPN, CPZ, and the first
two domains of CPD have been demonstrated to have enzymatic activity;
the third domain of CPD and the other proteins in this group do not appear to cleave the standard carboxypeptidase substrates (13-15, 18).
The observation that CPX2, AEBP1, and the third domain of CPD are not
active toward standard substrates is consistent with a comparison of
active site residues of the various members of the family. CPX2, AEBP1,
and the third domain of CPD lack a Glu in the position equivalent to
Glu270 of CPA and CPB. Instead, these novel members of the
gene family contain Tyr in this position (Fig.
1). Glu270 in CPA/B is a
critical catalytic residue that is thought to contribute a proton to
the leaving group following hydrolysis of the amide bond (19).
Theoretically, it is possible that a Tyr in this position could also
function as a proton donor, but the catalytic activity would be
predicted to be greatly reduced. However, substrate binding may not be
greatly affected by the absence of a Glu in this position. These
observations led to the prediction that the novel members of the
metallocarboxypeptidase gene family that lack the Glu270
equivalent residue are binding proteins, rather than active
enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Fig. 1.
Comparison of the amino acid sequences of CPE
and other mammalian metallocarboxypeptidases. The region
surrounding Glu300 of CPE (top) and the
numbering system of CPA/CPB (bottom) are shown. Residues
conserved among all members of the metallocarboxypeptidase family are
shown in bold. D1, D2, and D3, the first, second, and third
domains of CPD; MCA, mast cell CPA; #, residue corresponding to
Thr268 of CPB and CPA1, which is present in the side chain
binding cavity (46).
In the present study, we tested the importance of Glu300 in
rat CPE. In addition to testing the affect on enzyme activity and binding to a substrate affinity resin, we also tested whether the
mutant CPE could be correctly sorted into the regulated secretory pathway in a CPE-deficient pancreatic beta cell line. Recently, CPE has
been proposed to be involved in the sorting of neuroendocrine peptides
into the regulated secretory pathway (20). Thus, we were interested in
testing whether mutations that inactivate CPE had an influence on the
sorting of CPE in a neuroendocrine cell line. For this analysis, we
used the NIT3 cell line (21), which was derived from
Cpefat/Cpefat mice and does not
contain active CPE due to a point mutation of Ser202 to Pro
in the coding region of the CPE gene (22). This cell line is an ideal
test system to examine the sorting of CPE because it contains a
regulated secretory pathway without endogenous CPE (the
Pro202 mutant CPE is degraded before entry into the Golgi)
(21). Our findings that mutations of Glu300 eliminate CPE
activity but not binding to a substrate affinity column supports the
hypothesis that the novel family members lacking a Glu in the
equivalent position are binding proteins rather than active enzymes.
Furthermore, our finding that the CPE proteins with mutations of
Glu300 are sorted into the regulated pathway in the NIT3
cells indicates that neither enzyme activity or substrate binding are
required for this sorting.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of Constructs-- To generate point mutations, Altered Sites II in vitro mutagenesis system (Promega) was used. Briefly, full-length rat CPE cDNA was subcloned into the vector pAlter-1. Oligonucleotides with the desired codon changes were used to generate single amino acid substitutions. The cDNA fragments with point mutations were subcloned into the BamHI and EcoRI sites of the baculovirus transfer vector pVL-1393. Dideoxynucleotide sequencing was performed to confirm the sequences of the mutated region of CPE.
For expression in NIT3 cells, a SacI/EcoRI fragment of the CPE cDNA (which contains the point mutant) was combined with a BamHI/SacI fragment from a construct containing the hemagglutinin (HA) epitope tag between the pro region of CPE and the mature form of the protein (23). These fragments were subcloned into the pcDNA3 vector (Invitrogen). The resulting proteins contain the N-terminal HA epitope tag and the various mutations of Glu300.
Prodynorphin cDNA in the pcDNAI/Amp expression vector was obtained from Dr. Lakshmi Devi (Dept. of Pharmacology, New York University).
Expression of Proteins in the Baculovirus System-- CPE proteins were expressed using the recombinant baculovirus expression system (Pharmingen) as described previously (24). For large scale protein expression, Sf9 cells were grown to 2 × 106/ml in shaker flasks and infected with the baculovirus. Cells were harvested by centrifugation at 10,000 × g for 30 min. Cell pellets were homogenized (Polytron, Brinkman Instruments) in 0.1 M NaAc, pH 5.5, buffer containing 1 M NaCl and 1% Triton X-100 and centrifuged at 50,000 × g for 1 h. Aliquots of media and cell extracts were assayed for carboxypeptidase activity as described below.
Western Blot Analysis-- Aliquots of expression cell extracts and media were resolved on a 10% denaturing polyacrylamide gel and electrophoretically transferred onto nitrocellulose membrane. The blot was probed with an antiserum to the CPE C-terminal region (24), followed by horseradish peroxidase-labeled goat anti-rabbit antisera. The enhanced chemiluminescence method (Amersham Pharmacia Biotech) was used to detect the bound antiserum.
Enzyme Activity Assays-- In a typical assay, 25-µl aliquots of media or cell extracts were combined with 225 µl of 0.1 M NaAc, pH 5.5, 0.01% Triton X-100, and 0.22 mM dansyl-Phe-Ala-Arg. After incubation at 37 °C for 1 h, product was detected by measuring fluorescence after extraction into chloroform from an acidified aqueous phase (25). A similar assay was used for the affinity purified proteins.
Affinity Chromatography-- To test the binding ability of the CPE mutants, 50 ml of baculovirus-infected Sf9 media or cell extracts was adjusted to pH 5.5 and 0.15 M NaCl and applied to a 0.5-ml column of p-aminobenzoyl-Arg-Sepharose (25). The columns were washed with 50 mM NaAc, pH 5.5, buffer containing 0.5 M NaCl and 0.5%Triton X-100. In some experiments, bound proteins were eluted with 5 ml of 50 mM Tris-Cl buffer, pH 8.0, containing 100 mM NaCl and 0.01% Triton X-100. Following this treatment, the columns were then eluted with 5 ml of 25 mM Arg in the same buffer. In other experiments, bound proteins were competitively eluted with increasing concentration of guanidinoethylmercaptosuccinic acid (GEMSA) in 0.6 ml of washing buffer. The GEMSA solution was applied to the column, and the mixture was recycled 2 times over a 30-min period of time at 4 °C. Following this step, the remaining CPE was eluted with 0.6 ml of 50 mM Tris-Cl, pH 8.0, buffer with 100 mM NaCl. The various column fractions were analyzed on denaturing polyacrylamide gels. For quantitation of the amount of CPE recovered by the various concentrations of GEMSA, the protein was detected using the silver staining method (26). The protein gel was densitized using the Java Image analysis program (Jandel Scientific). The percentage of the bound CPE eluted by GEMSA was determined by calculating the amount of CPE protein in the GEMSA elute versus the total amount of CPE recovered from the column (i.e. the combined amount of the GEMSA and pH 8.0 elutes).
Expression of CPE in NIT3 Cells-- For the studies investigating whether different mutants of CPE co-localize with prodynorphin, NIT3 cells were transiently transfected with CPE cDNA and prodynorphin cDNA in expression vectors, and co-stained for the HA-tagged CPE and prodynorphin. Briefly, cells were co-transfected using the standard calcium phosphate procedure (27) with equal amounts of the two constructs. Two days after transfection, the cells were replated on glass growth-supporting coverslips (Fisher Scientific), cultured for one more day, and then fixed in 4% paraformaldehyde for 10 min. The cells were rinsed in phosphate-buffered saline (PBS), permeabilized in 0.1% Triton X-100 in PBS for 15 min, and then blocked in 5% bovine serum albumin for 1 h. After blocking, the cells were immunostained for 1 h with 1:2,500 dilution of monoclonal antibodies to the HA tag (a gift of Dr. Jonathan Backer, Molecular Pharmacology, Albert Einstein College of Medicine), and 1:1,000 dilution of polyclonal antiserum "13S" raised against dynorphin B-13 (a gift of Dr. Lakshmi Devi). This antiserum to dynorphin recognizes both prodynorphin and processed peptides (28). Following the incubation with primary antisera, fluorescein-labeled goat anti-rabbit IgG and rhodamine-labeled goat anti-mouse IgG (1:200 dilutions) were added, and the cells were incubated in the dark for 1 h. The coverslips were extensively washed, mounted on glass slides in 50% glycerol in PBS and examined using a Bio-Rad confocal microscope. The images represent a single plane of focus. The transfections and analysis were performed twice with similar results.
Secretion of CPE from NIT3 Cells--
To examine whether
different CPE mutants undergo regulated secretion, NIT3 cells were
transfected as described above using CPE cDNA in the pcDNA3
expression vector. Stably transfected cells were selected using 0.8 mg/ml of geneticin (G418). Typically, 2-3 separate clones expressing
different CPE mutants were examined for the regulated secretion. Cells
were cultured in 6-well plates. Medium was removed, and the cells were
washed three times with PBS and then incubated in Krebs Ringer buffer
containing no addition or 45 mM KCl (ionic strength was
maintained by reducing the NaCl concentration). After 40 min of
incubation, the medium was removed and subjected to Western blot
analysis using antibodies to the HA epitope-tag (1:1,000 dilution).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Wild type CPE has previously been expressed in high levels in
Sf9 cells using the baculovirus system (24). Mutation of the Glu300 into Lys, Gln, or Tyr does not affect the relative
levels of CPE protein expression in the baculovirus system (Fig.
2). High levels of CPE protein are
detected in both cells and media for all three point mutants. However,
enzymatic activity toward dansyl-Phe-Ala-Arg is detectable only for the
wild-type CPE (Table I). The level of
enzyme activity of the wild-type CPE is similar to that determined in
previous studies (13). The three point mutants show only background
activity that is comparable with controls with nonexpressing virus
(Table I). Similar results were observed in three separate experiments.
Because of the presence of low levels of endogenous activities in the
Sf9 cells, studies on crude cells or media cannot rule out the
possibility that the mutant proteins have low levels of enzyme
activity. To investigate this further, purification of the proteins
were attempted on a substrate affinity resin.
|
|
When applied to a Sepharose column containing
p-aminobenzoyl-Arg, the majority of the wild-type CPE is
bound and only trace levels are detected in the flow through fraction
(Fig. 3, F). Raising the pH to
8.0 causes most of the bound CPE to elute from the column (Fig. 3,
E1). Similar analysis of the three point mutants showed that
only the Gln300 form of CPE was able to bind to the
affinity resin, and subsequently elute with high pH buffer (Fig. 3).
Both the Lys300 and the Tyr300 mutants were
unable to bind to the affinity resin. To test whether a small amount of
protein bound to the column but could not be eluted by the high pH, the
columns were also eluted with a high concentration of Arg after the
high pH treatment. This treatment, which elutes other carboxypeptidases
such as CPD, did not elute any detectable amount of Lys300
or Tyr300 CPE (Fig. 3, E2). Thus, these two
mutants do not appear able to bind to the substrate affinity resin,
whereas the Gln300 mutant is able to bind.
|
To ensure that the binding of the Gln300 form of CPE to the
p-aminobenzoyl-Arg-Sepharose represents binding to the
active site of the protein, we tested whether an active site-directed
inhibitor could compete for this binding. GEMSA is a competitive
inhibitor of CPE with Ki around 10 nM
(29). When affinity column-bound wild-type or Gln300 CPE
was incubated with 0.1 mM GEMSA, nearly all of the bound protein was eluted. To quantitate this, the experiment was performed with various concentrations of GEMSA. Approximately 50% of the wild-type CPE is eluted from the p-aminobenzoyl-Arg column
with 6 µM GEMSA (Fig. 4).
Similar analysis of the Gln300 form of CPE shows that 2 µM GEMSA elutes 50% of the bound protein (Fig. 4). Thus,
both wild-type and Gln300 CPE bind to the affinity column
through their active sites, and this binding is competed by generally
similar concentrations of an active site-directed inhibitor.
|
Following purification of the Gln300 form of CPE on the affinity columns, the activity toward the standard carboxypeptidase substrate dansyl-Phe-Ala-Arg was tested. For this analysis, the affinity column-bound protein was eluted by raising the pH to 8.0 (as done in Fig. 3) rather than with GEMSA (as done in Fig. 4) because GEMSA would interfere with the enzyme activity measurement. Wild type CPE showed a Vmax of 20 µmol/min/mg protein, whereas no activity could be detected for the Gln300 form of CPE. Based on the sensitivity of the assay and the amount of purified protein tested, the Gln300 CPE has less than 0.004% of the activity of wild-type CPE.
To test whether CPE activity or substrate binding are required for
sorting of CPE into the regulated pathway, we expressed the proteins in
the NIT3 pancreatic cell line. For this analysis, the HA epitope was
added to the N terminus of the CPE so that the expressed protein could
be detected above the background of endogenous CPE, which is only
detected in the endoplasmic reticulum of these cells due to the
naturally occurring point mutation (21). The cells were co-transfected
with a plasmid expressing rat prodynorphin so that the distribution of
the CPE could be compared with that of a prohormone. Prodynorphin has
been previously found to be routed into the secretory pathway of
neuroendocrine cells (30, 31). When
expressed in NIT3 cells, the epitope
tagged wild-type CPE (Glu300 showed a punctate pattern
throughout the cytoplasm) (Fig. 5). Co-staining of the cells with a rabbit polyclonal antiserum to prodynorphin showed a similar pattern of distribution (Fig. 5). This
pattern is consistent with localization of the CPE to secretory vesicles in this cell type. The distribution of the Lys300
and Gln300 forms of CPE also showed overlap to the
prodynorphin (Fig. 5), suggesting that they were also routed to the
secretory vesicles.
|
|
To directly examine if the expressed CPE was routed to the regulated
secretory pathway, the media of the cells were examined following
stimulation with 45 mM KCl. In the absence of stimulation, extremely low levels of CPE protein are detected in the media (Fig.
6). Following stimulation with KCl,
there is a substantial increase in the amount of CPE detected in the
media (Fig. 6). Quantitation of the results from three experiments
shows the secretion of the Gln300 and Lys300
forms of CPE to be stimulated by KCl to a similar degree as the epitope-tagged wild-type Glu300 form of CPE. Thus, enzyme
activity or substrate binding do not appear to be required for entry of
CPE into the regulated secretory pathway.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A major finding of this study is that Glu300 of CPE is
essential for enzyme activity but not for substrate binding. The
critical nature of this residue was predicted from previous studies on the structure and function of CPA and CPB, but due to the relatively low conservation of amino acids between CPE and the CPA/B subfamily, it
was important to test this directly. In CPA and CPB, the Glu in a
comparable position (Glu270 is thought to be responsible
for the protonation of the leaving group following hydrolysis of the
amide bond (19). In other hydrolases, such as -lactamases, some
members of the gene family contain a Glu in a position thought to
function as a general base, whereas other family members contain a Tyr
in a comparable position (32). Thus, Tyr is potentially able to
function in this capacity in the novel members of the CPE gene family.
In this study, replacement of the Glu300 of CPE with Tyr resulted in an inactive enzyme that did not bind to the substrate affinity column. It is possible that the bulky side chain of the Tyr caused changes in the active site that precluded substrate binding. The substitution of Gln for Glu is relatively minor, changing only the -COOH into a -CONH2. This smaller change preserved binding to the substrate affinity column, indicating that binding does not require Glu300. Even though the Tyr300 and Lys300 forms of CPE were not able to bind to the affinity resin, these forms were secreted from both the Sf9 cells, and the Lys300 CPE was secreted from NIT3 cells (Tyr300 CPE was not examined in NIT3 cells). In contrast, forms of CPE containing deletions of 33 or more C-terminal residues, or point mutation of Ser202 into Pro are not secreted from either cell type and are degraded before protein transport into the Golgi (24, 33). The failure of these other mutations of CPE to be secreted presumably reflects the improper folding of these proteins. Because the Tyr300 and Lys300 forms of CPE were secreted in levels comparable with wild-type CPE, these point mutants are unlikely to be completely misfolded.
The regulated secretion of Gln300 and Lys300 CPE in the NIT3 cells indicates that neither enzyme activity or substrate binding are required for entry into the regulated pathway. Recently, Y. P. Loh and colleagues (20, 34) have proposed that CPE is a hormone-sorting receptor. This proposal is based on studies examining the sorting of prohormones in the fat/fat mouse, and in cell lines that have a reduced level of CPE protein due to the expression of antisense RNA. However, proinsulin sorting is not dependent on CPE, based on studies of primary cultures of pancreatic beta cells (35) or the NIT3 beta cell line (21). The sorting of proinsulin and insulin into the regulated pathway of the beta cells proceeds in the complete absence of CPE from this pathway. Also, previous studies have found that prodynorphin is routed to the regulated secretory pathway in the NIT3 cell line.2 Thus, CPE is not essential for the sorting of prohormones in pancreatic beta cells, although it is possible that CPE contributes to this process in other cell types. The mechanism by which CPE is sorted into the regulated pathway has not been elucidated. The C-terminal region of CPE appears to contain an element that contributes to the sorting into the regulated pathway; attachment of the C-terminal 50 residues to albumin causes a portion of the fusion protein to be secreted via the regulated pathway (33). This region is distinct from the membrane-binding motif also present in the C-terminal region of CPE (36). CPE aggregates at slightly acidic pH values, and this aggregation is enhanced by Ca2+ (37, 38); it is possible that this aggregation contributes to the routing of CPE. Models of co-aggregation of the secretory vesicle constituents have been proposed to account for the routing of the proteins into the regulated pathway (39, 40). Because the mutants of CPE that do not bind to the substrate affinity column are correctly routed in the NIT3 cells, the mechanism of CPE sorting presumably does not involve substrate binding to the active site.
The finding that Glu300 is not essential for binding of CPE to the affinity column supports the hypothesis that the new members of the CPE gene family that lack a Glu in the comparable position function as binding proteins rather than active enzymes. These three proteins (CPX2, AEBP1, and the third CP-like domain of CPD) have not been found to have enzyme activity upon expression in Sf9 cells using the baculovirus system (13, 14, 18). Although AEBP1 was reported to have detectable activity upon expression in bacteria (16), this activity was extremely low (0.02 absorption units at 256 nm with the substrate hippuryl-Arg after 10 min with 2 µg of enzyme). Furthermore, attempts to repeat the experiment using the identical constructs, expression system, and assay were unsuccessful.3 However, it remains possible that these proteins are active enzymes. The substitution of the Tyr for Glu300 of CPE was created with the hope that the protein would still bind to the affinity column and thus be able to be purified and tested with substrate in the absence of other enzymes present in the Sf9 cells and media. This was done for the Gln300 mutant, with the finding that this variant had less than 0.004% of the activity of wild-type CPE. However, as discussed above, the bulky side chain of the Tyr may have altered the active site and prevented binding to the affinity resin. Although alternative purification strategies could have been used to isolate the enzyme, the failure to bind to the substrate affinity resin suggests that the protein would not bind substrates and would therefore be unlikely to possess enzyme activity. The lack of enzyme activity in the media or cell extracts of the Sf9 cells infected with Tyr300 CPE-expressing baculovirus indicates that this mutant has less than 0.6% of the wild-type CPE activity.
In summary, Glu300 of CPE is essential for enzyme activity
but not substrate binding. The recent identification of three members of the CPE gene family that lack a Glu in a comparable position highlights the importance of this residue. Interestingly, several other
enzyme families contain members with substitutions of critical active
site residues that affect enzyme activity but not substrate binding.
For example, aspartyl protease-like proteins that lack the critical Asp
have been found in bovine and sheep placenta (41, 42), and
disintegrin-like proteins that lack the consensus site for metal
binding have been found in human brain, testis, and other tissues (43,
44). In addition to these inactive members of protease gene families,
several phosphatase-like proteins that lack one or more residues
critical for catalytic activity have been discovered (45). Taken
together with the present study, these results suggest that the
evolution of proteins that lack enzyme activity but not substrate
binding is a widespread mechanism for the generation of binding proteins.
![]() |
ACKNOWLEDGEMENTS |
---|
Confocal microscopy was performed in the Analytical Imaging Facility of the Albert Einstein College of Medicine.
![]() |
FOOTNOTES |
---|
* This work was supported primarily by National Institutes of Health Grants R01 DA-04494 and Research Scientist Development Award DA-00194 (to L. D. F.). The DNA sequencing facility of the Albert Einstein College of Medicine is supported in part by Cancer Center grant CA13330.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: Dept. of Molecular
Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park
Ave., Bronx, NY 10461. Tel.: 718-430-4225; Fax: 718-430-8954; E-mail:
fricker{at}aecom.yu.edu.
2 L. Devi, personal communication.
3 Y. Qian, O. Varlamov, and L. D. Fricker, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CPA, carboxypeptidase A; CPB, carboxypeptidase B; CPD, carboxypeptidase D; HA, hemagglutinin; GEMSA, guanidinoethylmercaptosuccinic acid; PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|