(Received for publication, June 28, 1995; and in revised form, August 3, 1995)
From the
Carboxypeptidase E (CPE) is involved in the biosynthesis of most
neuropeptides and peptide hormones. Until recently, CPE was the only
intracellular carboxypeptidase thought to be involved in neuroendocrine
peptide processing. However, the finding that fat/fat mice,
which have a mutation within the CPE gene that inactivates the enzyme,
are capable of a reduced amount of insulin processing suggests that
another carboxypeptidase is present within the secretory pathway. We
have detected a CPE-like enzyme, designated CPD, which has many
properties in common with those of CPE. Like CPE, CPD is a
metallocarboxypeptidase that has a pH optimum of 5.5-6. The K and K
values for
a series of short peptide substrates show only minor differences
between CPD and CPE. Several active site-directed inhibitors also show
generally similar potency toward the two enzymes, although
guanidinoethylmercaptosuccinic acid is approximately 10-fold more
potent, and hippuryl-Arg is approximately 100-fold more potent as an
inhibitor of CPD than of CPE. A major difference between the two
enzymes is the molecular masses; CPE is 50,000-56,000, whereas
CPD is approximately 180,000. Also, CPD does not elute from a substrate
affinity column when the pH is raised to 8, which elutes CPE, although
CPD can subsequently be eluted by arginine. Both CPE and CPD are
present in purified bovine anterior pituitary secretory vesicles, but
the tissue distribution of CPD is more uniform than that of CPE.
Antisera to the N- and C-terminal regions of CPE do not recognize CPD.
The partial N-terminal amino acid sequence of bovine CPD shows
30-40% homology with an N-terminal region of bovine and rat CPE
and 70% homology with a duck protein known as gp180, a hepatitis B
virus particle binding protein that shows 47% homology to CPE. Taken
together, these results suggest that CPD is a novel secretory pathway
enzyme that may be the bovine homologue of gp180.
Most peptide hormones are initially produced as precursors
(Docherty and Steiner, 1982; Eipper et al., 1986; Steiner,
1991). The conversion of prohormones into bioactive peptides requires
the action of an endopeptidase which cleaves at pairs of basic amino
acids and then a carboxypeptidase which removes the basic amino acids
from the C terminus of the intermediate peptides. Endopeptidases
responsible for peptide biosynthesis include furin, prohormone
convertase 3 (PC3, also known as PC1), and PC2 (Smeekens and Steiner,
1990; Seidah et al., 1990, 1991; Smeekens et al.,
1991; Hosaka et al., 1991; Molloy et al., 1992;
Hatsuzawa et al., 1992; Day et al., 1992). Both PC2
and -3 appear to be required for proper processing of a variety of
peptides, such as insulin (Davidson et al., 1988; Bennett et al., 1992; Smeekens et al., 1992; Steiner et
al., 1992). Until recently, CPE ()was the only
secretory pathway carboxypeptidase thought to be involved with the
removal of Arg and/or Lys residues from the C terminus of the peptide
intermediates (Fricker, 1988a, 1991). However, recent studies on the fat/fat mouse suggest that CPE is not the only
peptide-processing carboxypeptidase (discussed below).
CPE was initially discovered as an enzyme associated with the biosynthesis of enkephalin in the bovine adrenal medulla (Fricker and Snyder, 1982) and is thought to be involved in the processing of many peptide hormones and neurotransmitters (Fricker, 1988a, 1991). CPE has been designated EC 3.4.17.10 and is alternatively known as CPH and enkephalin convertase (Fricker and Snyder, 1982; Webb, 1986). CPE activity and protein are highest in pancreatic islets and pituitary, with intermediate levels in brain, and low but detectable levels in many neuroendocrine tissues (Fricker, 1988a, 1991; Schafer et al., 1994; Zheng et al., 1994). The amino acid sequence of CPE is highly conserved among species, with 80% amino acid identity among fish and human and greater than 94% amino acid identity among the various mammalian species (Fricker et al., 1986, 1989; Manser et al., 1990; Roth et al., 1991). Although multiple forms of CPE protein are detected in all tissues that express CPE, only a single gene has been identified (Jung et al., 1991). The various forms of CPE protein arise from a single precursor, proCPE, through post-translational processing, and not through alternative RNA splicing (Jung et al., 1991; Fricker and Devi, 1993).
CPE is a member of the carboxypeptidase B (CPB) gene family. Altogether, there are 7-8 known mammalian members of the metallocarboxypeptidase gene family, depending on the species (i.e. rat has two forms of CPA) (Bradshaw et al., 1969; Titani et al., 1975; Tan et al., 1989; Reynolds et al., 1989; Gebhard et al., 1989; Eaton et al., 1991; Clauser et al., 1988). Of these previously described enzymes, CPE is unique in having an acidic pH optimum in the 5-6 range (Greene et al., 1992). The acidic pH optimum of CPE presumably reflects the environment of the secretory vesicles in which CPE functions; the intragranular pH is in the 5-6 range (Johnson and Scarpa, 1976; Russell, 1984). All other previously reported metallocarboxypeptidases have pH optima in the neutral range (Folk et al., 1960; Narahashi and Yoda, 1979; Plummer and Erdos, 1981; Skidgel et al., 1989; Reynolds et al., 1989), also reflecting the environments in which they function (i.e. plasma or the intestine).
Recently, the gene responsible for obesity in the fat/fat mouse has been mapped to the locus on chromosome 8
occupied by the CPE gene, and a point mutation (Ser to
Pro) within the coding region of the CPE gene has been identified
(Naggert et al., 1995). This mutation eliminates the
carboxypeptidase activity and is consistent with our finding that CPE
activity is largely absent from fat/fat mouse pituitary and
pancreatic islets (Naggert et al., 1995). Unexpectedly, some fat/fat mouse tissues (such as brain and adrenal) appeared to
contain moderate amounts of CPE-like enzymatic activity. (
)However, further analysis has found this activity to be
the result of a novel enzyme, named carboxypeptidase D (CPD), which has
many properties in common with CPE. This newly discovered enzyme is the
focus of the studies described in this paper.
Substrates were synthesized by activating a dansylated amino acid (Sigma) with dicyclohexylcarbodiimide and N-hydroxysuccinimide (Aldrich), and then coupling this reaction product to a dipeptide (Bachem) for the synthesis of tripeptides or to Arg (Sigma) for the synthesis of the dansyl-Leu-Arg. The reaction mixture was diluted with 10 mM HCl, washed with chloroform, and then purified on a C18 Sep-Pak Vac cartridge (Waters), using 10-20% acetonitrile to elute the dansylated tripeptide. Purity was determined by thin layer chromatography of the peptides, as described (Fricker and Snyder, 1983).
For kinetic analysis of CPE and -D, 6 ng to 13 µg of purified enzyme was combined with substrate (12.5 µM, 25 µM, 50 µM, 100 µM, 200 µM, 400 µM, and, for some substrates, 1 mM) in a final volume of 250 µl in 100 mM NaAc, pH 5.5. The amount of enzyme used was chosen so that a maximum of 20% of the peptide was hydrolyzed during the incubation. After 1-5 h at 37 °C, the reaction was terminated with 100 µl of 0.5 M HCl and then 2 ml of chloroform were added. The amount (nanomoles) of product was determined from standard curves determined for each peptide. Product for the standard curves was generated from substrate using an excess of CPE. All of the substrates had generally similar conversion factors; approximately 2 fluorescent units corresponded to 1 nmol of product. The kinetic parameters were evaluated by fitting the data using the Cricket Graph programs (Cricket Software, Inc.). Data for CPE are the average of 2 determinations, and data for CPD represent the average of 3 determinations, except for dansyl-Phe-Pro-Arg which was performed once. All determinations were performed in duplicate which typically varied less than 5%.
We have previously developed a single-step purification of CPE to apparent homogeneity using a p-aminobenzoyl-Arg Sepharose affinity resin (Fricker et al., 1990). CPE binds to this resin at acidic pH values (5, 6) and elutes when the pH is shifted to 8 since CPE has low affinity for substrates at neutral pH (Greene et al., 1992); this selective elution of CPE affords a high degree of purification. To test the possibility that another CPE-like enzyme is present in bovine pituitary which binds to the affinity column, but is not eluted by the high pH conditions, we examined whether any enzymatic activity could be eluted from the affinity column by Arg after first eluting the column at pH 8. Although the majority of the CPE-like enzyme activity eluted with the high pH buffer, a substantial amount of activity remained bound to the column at pH 8 and was subsequently eluted when Arg was included in the buffer (Fig. 1). To test whether CPE exhibits this ability to remain bound to the affinity column at high pH, CPE was expressed in the baculovirus system and purified from the insect Sf9 cells using the same affinity column procedure. As expected, virtually all of the baculovirus-produced CPE activity eluted from the affinity column upon high pH treatment (Fig. 1). This result suggests that the activity detected in the Arg elutes is not due to CPE.
Figure 1:
Affinity
column purification of carboxypeptidases from bovine anterior pituitary
and Sf9 cells following infection with CPE-expressing baculovirus.
Tissue was extracted with 1% Triton X-100 and 1 M NaCl at pH
5.5 and purified on 0.7-ml substrate affinity columns as described
under ``Materials and Methods.'' Elute 1 (A-D)
corresponds to the 4 1.8 ml eluates with 50 mM Tris,
pH 8.0, containing 100 mM NaCl and 0.01% Triton X-100. Elute 2 (A and B) corresponds to the subsequent eluates with
25 mM Arg in the same pH 8 elute buffer. For enzyme assays,
samples were diluted 1:25 so that the final concentration of Arg was 1
mM, and activity was determined at pH 5.5 with 200
µM dansyl-Phe-Ala-Arg, as described under ``Materials
and Methods.'' Error bars show the range of duplicate
determinations.
The carboxypeptidase that remains bound to the affinity column at high pH, eluting only when Arg is added, has been designated carboxypeptidase D (CPD). As a first step in the purification, we needed to determine the conditions that extract CPD from the tissue. The majority of CPD activity is membrane-bound and is only extracted in appreciable amounts by the combination of 1% Triton X-100 and 1 M NaCl (Table 1). Only a small fraction of the total CPD activity is solubilized by extraction at pH 9 in contrast to CPE which is efficiently extracted from membranes at this pH (Table 1), as previously reported (Fricker, 1988b; Fricker et al., 1990). Another difference between the enzymes is the relatively low proportion of CPD that is soluble upon extraction at pH 5.5, whereas a large amount of CPE is soluble under this condition (Table 1).
The tissue distribution of CPD and CPE were examined by extracting several bovine tissues directly with 1% Triton X-100 and 1 M NaCl at pH 5.5 in order to compare total amounts (i.e. soluble and membrane-bound forms) of each enzyme. After the two enzymes were physically separated using the affinity column procedure, the amount of enzymatic activity was determined using dansyl-Phe-Ala-Arg. The levels of CPD activity are generally similar in anterior and neurointermediate pituitary and in adrenal medulla and cortex (Table 2). Overall, there is less than a 10-fold difference between highest and lowest levels of CPD. In contrast, CPE shows nearly a 100-fold difference among these tissues (Table 2). To investigate whether CPD is present in secretory vesicles along with CPE, bovine anterior pituitary secretory vesicles were purified on a Percoll gradient (Devi et al., 1991), and extracts were fractionated using the affinity column procedure to separate CPE from CPD. Both CPE and CPD activities are present in the secretory vesicle fraction (Table 2).
Analysis of the proteins present in the various column eluate fractions of bovine pituitary and adrenal shows major bands around 50-56 kDa for the material in the elute 1 fractions (Fig. 2). This size corresponds to that previously reported for CPE (Fricker and Snyder, 1983; Fricker et al., 1990) and the predicted size based on the nucleotide sequence (Fricker et al., 1986). In contrast, there is little 50-56-kDa material which remained bound at pH 8 but subsequently eluted with Arg. Instead, a major band of approximately 180 kDa is detected in these fractions (Fig. 2A). Immunoreactive CPE is detected only in the elute 1 fraction and not in the elute 2 fraction (Fig. 2). Thus, even though there is a significant amount of carboxypeptidase activity in the elute 2 fractions, this activity does not react with antisera directed against either the N- or C-terminal regions of CPE (Fig. 2). Also, the tissue distribution of the 50-56-kDa protein (Fig. 2A) and the immunoreactive CPE (Fig. 2, B and C) generally parallels the distribution of CPE activity, with low levels in the adrenal cortex relative to the other tissues. In contrast, the 180-kDa protein (Fig. 2A) is fairly evenly distributed in the tissues examined, as is CPD activity. (Note: different volumes of the various samples were analyzed on the protein gel and the Western blots, as described in the figure legend.)
Figure 2:
Analysis of affinity column eluates on
denaturing polyacrylamide gels. Bovine tissues were homogenized in 0.1 M NaAc containing 1% Triton X-100 and 1 M NaCl and
centrifuged for 30 min at 50,000 g, and the
supernatant was applied to affinity columns, as described under
``Materials and Methods.'' Elute 1 represents the
material which eluted from the column with the pH 8 buffer; elute 2 represents the material which remained bound at pH 8 and then
eluted with 25 mM Arg. For each sample, the same volume of
elutes 1 and 2 was analyzed on the gels. A, either 40
(pituitary) or 80 (adrenal) µl of elute 1 or 2 were applied to the
polyacrylamide gel. Protein was detected using the silver staining
procedure (Morrissey, 1981). B, either 5 (pituitary) or 40
(adrenal) µl of elute 1 or 2 were fractionated on a denaturing
polyacrylamide gel, transferred to nitrocellulose, and probed with
antisera raised against the N-terminal region of CPE. C,
similar to B, except the Western blot was probed with antisera
raised against the C-terminal region of
CPE.
To confirm that the 180-kDa material is responsible for the observed enzymatic activity, and not some minor 50-60-kDa protein with a very high enzymatic activity, the elute 1 or 2 fractions of bovine adrenal medulla were fractionated on a nondenaturing gel and then assayed for enzymatic activity. The majority of the activity from the elute 2 fraction ran on the native gel with an apparent molecular mass of slightly greater than 200,000 (data not shown). In contrast, the material from the elute 1 fraction ran with an apparent molecular mass of 50,000-60,000 (data not shown). This result strongly suggests that the high molecular weight protein detected in the elute 2 fraction is enzymatically active CPD.
When the arginine eluate from affinity chromatography of bovine pituitary membrane extracts was fractionated on a Mono Q anion exchange column, the majority of enzyme activity was found to elute between 0.2 and 0.35 M NaCl (Fig. 3). This is similar to the elution conditions for CPE, which elutes from this column between 0.25 and 0.35 M NaCl (not shown). Denaturing polyacrylamide gel electrophoresis of the column fractions revealed that the 180-kDa protein co-elutes with the enzyme activity (Fig. 3). A minor band of approximately 130 kDa was also detected in the stock; this protein elutes from the Mono Q column under conditions similar to those that elute the 180-kDa band. It is possible that this 130-kDa protein represents a proteolytic fragment of the 180-kDa band; under conditions where the affinity column elute was concentrated before being applied to the Mono Q column, the amount of the 130-kDa protein was greatly increased, and there was little 180-kDa material (not shown).
Figure 3: Purification of CPD on a Mono Q anion exchange column. Bovine pituitary membranes were extracted with 1 M NaCl and 1% Triton X-100 in 0.1 M NaAc pH 5.5 buffer and purified on a 10-ml p-aminobenzoyl-Arg Sepharose affinity column. CPE activity was eluted with 200 ml of high pH buffer, and then CPD was eluted with 25 mM Arg. This eluate was directly applied to a Mono Q column (Pharmacia). The column buffer was 10 mM bis-Tris chloride, pH 6.0, and the indicated NaCl gradient was used to elute the CPD. The flow rate was 1 ml/min; 1-min fractions were collected. Left, enzyme activity was determined using dansyl-Phe-Ala-Arg at pH 5.5, and the amount of activity is in nanomoles/min/1-ml fraction. Right, denaturing polyacrylamide gel electrophoresis of 40 µl of the indicated fraction or the affinity column eluate (Stock). Protein standards (Bio-Rad) are in kilodaltons.
The optimal pH of CPD, measured with 200 µM dansyl-Phe-Ala-Arg, is around 6 (Fig. 4). Although the optimal pH of CPD is similar to CPE, and different from all other reported metallocarboxypeptidases, the effect of pH values in the 6-7 range differentiate CPE and -D. CPE is virtually inactive at pH 7.3, whereas CPD still has approximately 40% of the maximal activity at this pH. The finding that CPD still has a small amount of activity at pH 8 presumably explains why this enzyme remains bound to the affinity column at pH 8.
Figure 4: Effect of pH on activity of CPE and CPD purified from bovine pituitary membranes. Enzyme was purified by affinity chromatography and FPLC on a Mono Q column, as described under ``Materials and Methods,'' and was assayed with 200 µM dansyl-Phe-Ala-Arg in 0.1 M Tris acetate buffer at the indicated pH (at 37 °C). Error bars show the range of duplicate determinations.
The effects of a variety of protease inhibitors on
CPD activity show that this enzyme is a metallocarboxypeptidase which
is strongly inhibited by the chelating agent 1,10-phenanthroline, but
not by the nonchelating isomer 4,7-phenanthroline (Table 3).
Other chelating agents (EDTA and EGTA) are also effective inhibitors of
CPD, but are less potent than the 1,10-phenanthroline; this is also
found for CPE (Table 3). The serine protease inhibitor
phenylmethylsulfonyl fluoride and the thiol protease inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane
(E-64) do not inhibit either CPE or -D. However, other reagents that
interact with thiol groups are inhibitors of CPE and, to a lesser
extent, CPD. Iodoacetamide and low concentrations (1 µM)
of p-chloromercuriphenylsulfonate partially inhibit CPE, but
not CPD; higher concentrations of p-chloromercuriphenylsulfonate (0.1 mM) strongly
inhibit CPE and show partial inhibition of CPD. Other protease
inhibitors such as aprotonin, leupeptin, and benzamidine are without
substantial effect on either CPE or -D. Also, without substantial
effect on either CPE or -D are benzylsuccinic acid, tosylphenylalanyl
chloromethyl ketone (TPCK), and N-p-tosyl-L-lysine
chloromethyl ketone (TLCK); active site-directed inhibitors of
carboxypeptidase A, chymotrypsin, and trypsin (respectively). Of the
metals tested, the strongest activation of both CPE and -D is observed
with Co
(Table 3). Zn
also
activates both CPE and -D, whereas Ca
,
Mg
, and Mn
are without substantial
effect. Both enzymes are inhibited by Cd
,
Cu
, and Hg
, with the latter two
compounds showing stronger inhibition of CPE than -D.
A variety of
active site-directed carboxypeptidase inhibitors were tested with CPE
and -D. When assayed with 100 µM dansyl-Phe-Ala-Arg at pH
5.5, guanidinoethylmercaptosuccinic acid (GEMSA) is approximately 1
order of magnitude more potent as an inhibitor of CPD than of CPE (Fig. 5). The compound
2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA) is less
potent than GEMSA and shows less of a difference as an inhibitor of CPE
and -D. The lysine-based inhibitor aminopropylmercaptosuccinic acid
(APMSA) is approximately 2 orders of magnitude less potent than the
arginine-based GEMSA, and there is little difference in the IC of APMSA for the two enzymes. Peptides with C-terminal Arg and
Lys residues are less potent than the active site-directed compounds as
inhibitors of CPE and -D. Interestingly, hippuryl-Arg is approximately
2 orders of magnitude and hippuryl-Lys is approximately 1 order of
magnitude more potent as an inhibitor of CPD than of CPE (Fig. 5).
Figure 5: Effect of active site-directed inhibitors on purified CPE and CPD activity. Purified enzyme was added to a mixture of buffer, substrate, and inhibitor to give a final concentration of 50 mM NaAc, pH 5.5, 100 µM dansyl-Phe-Ala-Arg, and the indicated concentration of the inhibitors. The reaction was quenched with 100 µl of 0.5 M HCl, and enzyme activity was determined as described (Fricker, 1995). Open symbols and solid lines, CPE; filled symbols and dashed lines, CPD. Inhibitors (Calbiochem): circles, GEMSA; squares, MGTA; triangles, APMSA. Peptides (Sigma): diamonds, hippuryl-Arg; inverse triangles, hippuryl-Lys.
In addition to comparing the properties of CPE and
CPD, we also compared the properties of CPD and CPM (data not shown).
CPM is a membrane-bound enzyme that is present in many tissues (Skidgel et al., 1989). Human CPM cDNA (obtained from Dr. Randal
Skidgel) was expressed in the baculovirus system and purified by
affinity chromatography on p-aminobenzoyl-Arg Sepharose, as
described for a related resin (Skidgel et al., 1989). The pH
optimum of purified CPM is around 7 when assayed with
dansyl-Phe-Ala-Arg (not shown), which is similar to that previously
reported for CPM and distinct from the pH optimum of CPD. When CPM was
assayed at pH 5.5 (the pH used for the inhibitor studies of CPE and
-D), the IC values were 60 nM for GEMSA, 150
nM for MGTA, 33 µM for APMSA, 800 µM
for hippuryl-Arg, and 22 mM for hippuryl-Lys (not shown).
These properties further distinguish CPD from CPM.
The substrate
specificity of CPD was examined using several substrates. First, to
establish whether CPD was a carboxypeptidase, we tested purified enzyme
with
aminobenzoyl-Arg-Met-Ala-Arg-Ala-Thr-Leu-Gln-ethylenediamine-dinitrophenol.
When cleaved within the peptide chain, the fluorescent intensity of the
substrate increases approximately 20-fold. Incubation of 200 ng of
purified CPD with a 100 µM concentration of this substrate
at pH 6 showed negligible hydrolysis (3%) after 6 h of incubation.
In contrast, this amount of CPD cleaved 50% of a 100 µM solution of dansyl-Phe-Ala-Arg within 1 min when incubated under
identical conditions (data not shown). This result confirms that CPD is
a carboxypeptidase and not an endopeptidase.
Of the synthetic
substrates examined, purified CPD cleaves dansyl-Phe-Ala-Arg with the
highest K and K
/K
(Table 4).
Substitution of the Phe in the P3 position for a Leu or Pro reduces the K
/K
by approximately 50%.
In the P2 position, the order of preference for CPD (K
/K
) is Ala > Leu >
Phe > Gly > Ile > Pro. This order is similar to, but not
identical with, that for CPE, which shows a preference of Ala > Gly
> Leu > Phe > Ile > Pro (Table 4). In addition, the
range of K
/K
for the best
tripeptide substrate to the worst is approximately 45,000-fold for CPD,
but only 10,000-fold for CPE. A dipeptide substrate, dansyl-Leu-Arg, is
poorly cleaved by both CPE and -D, whereas the tripeptide
dansyl-Phe-Leu-Arg is a reasonably good substrate (Table 4).
To test whether CPD can cleave a physiological substrate, the enzyme was incubated with dynorphin B-14 (YGGFLRRQFKVVTR), and the product was analyzed by MALDI-TOF mass spectrometry. The substrate has a mass of 1727.9 on MALDI-TOF mass spectrometry, corresponding to the predicted mass of this peptide. This peptide was converted by CPD into a product with a mass of 1569.7, which corresponds to the mass of dynorphin B-13 (data not shown). Smaller fragments were not detected, indicating that CPD does not remove the nonbasic C-terminal Thr residue from dynorphin B-13, or cleave as an endopeptidase at internal sites.
The partial N-terminal amino acid sequence of CPD purified from bovine pituitary membranes shows 70% identity with an N-terminal region of a protein designated ``gp180'' from duck liver. The N-terminal sequence of CPD also shows approximately 35% identity with a region of bovine and rat CPE near the N terminus (Table 5).
Our previous studies on the fat/fat mouse have
identified a point mutation within a coding region of the CPE gene and
have found that this mutation results in the loss of CPE enzyme
activity produced in the baculovirus system (Naggert et al.,
1995). However, we detected some CPE-like enzymatic activity in fat/fat mouse tissues. Also, the C-terminal processing of
insulin is only partially affected in fat/fat mice (Naggert et al., 1995). One possible explanation is that another
CPE-like enzyme is present within the secretory pathway. A second
enzyme which functions in the same pathway as CPE would also explain
why a null mutation within CPE is not lethal; many physiologically
important neuropeptides and peptide hormones require C-terminal
processing by a carboxypeptidase before the peptide is biologically
active. Thus, there is a strong theoretical basis for a second
carboxypeptidase which is active in the secretory pathway. However,
until recently, there was no evidence for such an enzyme. In the
present study, we have identified a novel carboxypeptidase, designated
CPD, which is present in fat/fat mouse, and in
normal mouse, rat, and bovine tissues.
Many of the properties of
this enzyme are similar to those of CPE, although there are a few
important differences. The finding that CPD is approximately 180 kDa
was unexpected since all known members of the metallocarboxypeptidase
gene family are either 30-36 kDa or 50-60 kDa (Bradshaw et al., 1969; Titani et al., 1975; Tan et
al., 1989; Reynolds et al., 1989; Gebhard et
al., 1989; Eaton et al., 1991). It is unlikely that the
180-kDa band could be an aggregate of smaller subunits that is stable
to the denaturing conditions used in the electrophoresis since
treatment of the purified CPD with millimolar amounts of
dithiothreitol, EDTA, -mercaptoethanol, or SDS did not alter the
molecular weight (not shown). However, treatment of the protein with
peptide N-glycosidase F converted the 180-kDa protein into a
form with an apparent molecular mass of 150 kDa on denaturing gels (not
shown). This result suggests that CPD is N-glycosylated, but
even after removal of these sugars is still much larger than other
known metallocarboxypeptidases.
The size of bovine CPD is similar to
that of gp180, a duck protein identified as a 180-kDa membrane-bound
protein which binds hepatitis B virus particles (Kuroki et
al., 1995). The region of gp180 with homology to bovine CPD
corresponds to the predicted N-terminal sequence of gp180, based on the
nucleotide sequence of a cDNA clone (Kuroki et al., 1995).
Interestingly, gp180 contains three copies of a CPE-like domain, with
the N-terminal copy showing 37% amino acid identity with CPE, the
middle copy showing 47% identity, and the C-terminal copy showing 32%
identity (Kuroki et al., 1995). Other cloned mammalian
carboxypeptidases have less homology to duck gp180; the amino acid
identity is 44% for CPM, 42% for CPN, and 19% for CPB versus the middle domain of gp180. Recently, gp180 has been found to have
carboxypeptidase activity. ()With the exception of skeletal
muscle, gp180 is broadly distributed in duck tissues (Kuroki et
al., 1994); this finding is also consistent with the presence of
CPD in many rat tissues. (
)Based on the N-terminal amino
acid homology, the similar molecular weight, and the broad
distribution, it is possible that gp180 is the duck homologue of bovine
CPD.
CPD is clearly a novel enzyme, based on the enzymatic
properties, physical characteristics, tissue distribution, and partial
amino acid sequence. The similar properties of CPD and CPE help explain
why CPE was discovered 15 years ago, but CPD was discovered only
recently. Also, the levels of CPD are much lower than CPE in the
pituitary; the tissue used as a source for purified CPE for the past
decade. Since CPD did not elute from the affinity column under
conditions which eluted CPE, and accounted for a fraction of the total
carboxypeptidase activity in the pituitary, the existence of CPD was
overlooked for many years. Recently, a membrane-bound CPE-like enzyme
has been identified in a human cell line that does not express CPE
mRNA. ()Although the molecular weight of this enzyme is not
known, the enzyme has an acidic pH optimum, remains membrane-bound upon
extraction with high pH buffers, and does not react with antisera that
recognize CPE; these properties are similar to those of CPD. Further
studies are needed to determine whether this human cell
carboxypeptidase is the same as CPD or if additional CPE-like enzymes
exist.
The finding that a mutation within CPE that inactivates this enzyme is not lethal and does not completely block the formation of insulin raised the possibility that a second carboxypeptidase is present in the secretory pathway. The pH optimum of CPD and its presence in bovine pituitary secretory vesicles are consistent with a role for this enzyme in the processing of secreted peptides and proteins. However, the fact that the fat mutation produces obesity, sterility, and, in male mice of a certain age and genetic background, hyperglycemia, suggests that the second carboxypeptidase cannot completely compensate for CPE in all tissues. Based on the broader tissue distribution of CPD and its greater activity at slightly acidic pH values compared to CPE, it is possible that CPD functions in the trans-Golgi network of neuroendocrine and non-neuroendocrine cells. Proteins such as the insulin receptor are cleaved at multiple basic sites by furin (Bravo et al., 1994), and it is conceivable that CPD would be needed to subsequently remove the C-terminal basic residues remaining after cleavage by furin. Further studies on CPD from both normal animals and from fat/fat mice are needed to understand the relationship of CPD and CPE.