(Received for publication, August 15, 1995)
From the
Protease Ci, a cytoplasmic metalloprotease in Escherichia
coli, has been purified to apparent homogeneity by conventional
chromatographic procedures using I-labeled oxidized
insulin B-chain as a substrate. The purified enzyme behaves as a 54-kDa
protein under both denaturing and nondenaturing conditions, suggesting
that it consists of a single polypeptide chain. It is inhibited by
metal-chelating agents, including o-phenanthroline and NaCN,
but not by inhibitors of serine proteases or thiol-blocking agents.
Furthermore, protease Ci was found to contain 1.1 mol of zinc per mol
of the enzyme upon analysis by HR ICP mass spectroscopy. Thus, protease
Ci must be a zinc metalloprotease. Among the polypeptides tested as
substrates, oxidized insulin B-chain and glucagon are most rapidly
hydrolyzed. Intact insulin is a much poorer substrate than oxidized
insulin B-chain, even though the affinity of the enzyme to intact
insulin is approximately 100-fold greater than that to the B-chain.
Since unlabeled oxidized insulin A-chain is capable of inhibiting the
hydrolysis of
I-labeled insulin B-chain, it also appears
to be a substrate. Protease Ci also degrades lysozyme and lactalbumin,
although to a much lesser extent than oxidized insulin B-chain.
However, it shows little or no activity against proteins larger than 15
kDa (e.g. ovalbumin and denatured bovine serum albumin).
Hydrolysis of oxidized insulin B-chain followed by amino acid
composition analyses of the cleavage products reveals that as many as
10 of its 29 peptide bonds are hydrolyzed by protease Ci. This ability
to hydrolyze relatively small polypeptides suggests that protease Ci
may catalyze the later steps in the pathway for intracellular protein
breakdown.
Soluble extracts of Escherichia coli contain at least
nine distinct endoproteases that appear to be distinct from each other (1, 2) . Proteases Do (DegP), Re (Tsp), Mi, Fa, So, La
(Lon), and Ti (Clp) are serine proteases that hydrolyze relatively
large proteins, such as casein and globin. Of these, proteases La and
Ti require ATP and Mg for activity and appear to
catalyze the rate-limiting steps in the hydrolysis of abnormal proteins
and of certain normal proteins(3, 4) . Two other
enzymes, proteases Ci and Pi, are metalloproteases that degrade smaller
polypeptides, such as insulin and the N-terminal fragment of
-galactosidase with a minimal length of 50 amino acids (called
``auto-
'') that were used for complementation assay of
-galactosidase upon mixing with appropriate acceptor
proteins(2, 5) . Proteases Mi and Pi are periplasmic
enzymes, while all others are localized to the cytoplasm and therefore
may play a role in the degradation of intracellular
proteins(6) .
The periplasmic insulin-degrading protease Pi, also called protease III (5) and pitrilysin(7) , has been extensively characterized, although its physiological function is still unknown. Interestingly, protease Pi shares structural and functional homologies with the cytosolic insulin-degrading enzymes present in most mammalian cells(8) . Overall amino acid sequence similarity between these enzymes is about 50% when conserved amino acid changes are included as matches. Furthermore, both the proteases contain three highly conserved domains, of which the first domain is characterized by the presence of a metal-binding site (9) . Therefore, it has been suggested that the three regions of homology may play an important role in the active site and catalytic mechanism of the proteases(8) .
In addition to sharing sequence similarity, both protease Pi and human insulin-degrading enzyme have a molecular mass of about 110 kDa and require a divalent metal ion for activity(5, 8, 9) . Both proteases have an isoelectric point of 5.3 and are maximally active in the pH range of 6.5-8.5. Both are sensitive to inhibition by bacitracin. However, they differ in sensitivity to sulfhydryl blocking agents, such as N-ethylmaleimide and iodoacetamide. While the human insulin-degrading enzyme is highly sensitive to these reagents, protease Pi is not. An insulin-degrading enzyme similar in its properties to the human enzyme and protease Pi has also been isolated from Drosophila melanogaster(10) .
E. coli in its cytoplasm contains an additional enzyme, protease Ci, that can degrade insulin(1, 2) . Aside from its subcellular location, protease Ci shows remarkable similarities to protease Pi. Like Pi, it appears to be 110-130 kDa, to have a pH optimum at 7.5, and to be sensitive to inhibition by metal-chelating agents and by the antibiotic bacitracin. However, all these properties were determined only on the partially purified protease Ci, whose physiological role is totally unknown. In the present studies, therefore, we purified protease Ci completely to carry out a detailed characterization of its biochemical and physicochemical properties, for determination of its cleavage specificity, and ultimately for elucidation of its physiological function.
Figure 1:
Elution profile of insulin and oxidized
insulin B-chain-degrading activities from a DEAE-Sepharose column. The
crude extract was chromatographed on a DEAE-Sepharose column as
described in the text. Fractions of 20 ml were collected at a flow rate
of 250 ml/h. Proteolysis was assayed at 37 °C using I-labeled insulin and oxidized insulin B-chain as the
substrates as described under ``Experimental Procedures.''
The dotted and slashed lines indicate protein profile
and NaCl gradient, respectively.
The fractions containing high
activity were pooled and applied to a HiLoad Q column (2.6 10
cm) equilibrated with buffer A containing 150 mM NaCl.
Proteins bound to the column were eluted with a linear gradient of
150-300 mM NaCl. The active fractions were pooled,
dialyzed against buffer A, and loaded onto a heparin-Sepharose column
(1.5
8 cm) equilibrated with the same buffer. Proteins that did
not bind to the column were collected, and a saturated
(NH
)
SO
solution (pH 7.8) was added
to a final concentration of 1 M. The sample was then loaded
onto a phenyl-Sepharose column (1
6 cm) equilibrated with
buffer A containing 1 M
(NH
)
SO
. Proteins bound to the
column were eluted by decreasing linearly the salt concentration to
0.01 M. The peak of oxidized insulin B-chain-degrading
activity was eluted at about 0.17 M
(NH
)
SO
(Fig. 2A).
The fractions containing high activity were pooled, adjusted to 1.2 M (NH
)
SO
, and loaded onto
a butyl-Toyopearl column (1
4 cm) equilibrated with buffer A
containing 1.2 M (NH
)
SO
.
Proteins were eluted by decreasing the salt concentration to 0.1 M. Active fractions were pooled, adjusted to 1.5 M
(NH
)
SO
, and again loaded onto the
same butyl-Toyopearl column but equilibrated with 1.5 M
(NH
)
SO
. Proteins were eluted as
above but by decreasing the salt concentration to 0.9 M. The
fractions containing high activity (Fig. 2B) were
pooled, added with glycerol to a final concentration of 20% (v/v), and
kept at -70 °C for further use. Summary of the purification
of protease Ci is shown in Table 1.
Figure 2:
Elution profiles of the protease Ci
activity from phenyl-Sepharose (A) and the second
butyl-Toyopearl columns (B). Proteins obtained from
heparin-Sepharose column (17.2 mg) were chromatographed on a
phenyl-Sepharose column. Fractions of 1 ml were collected at a flow
rate of 10 ml/h. Proteins obtained from the first butyl-Toyopearl
column (0.66 mg) were again chromatographed on the same column after
equilibration with buffer A containing 1.5 M
(NH)
SO
. Fractions were collected as
above, and their ability to hydrolyze
I-labeled insulin
B-chain (
) was then assayed by incubating 5-µl aliquots of
them for 30 min at 37 °C. The dotted and slashed lines indicate protein profile and
(NH
)
SO
gradient,
respectively.
Figure 3: Gel electrophoretic (A) and immunoblot analyses (B) of the cell lysate and the purified protease Ci. E. coli 3302 strain was grown to mid-log phase, harvested, and immediately boiled in 2% SDS. An aliquot of the cell lysate (lane a) and 7 µg of the purified protease Ci (lane b) were electrophoresed in duplicates on 10% polyacrylamide slab gels containing SDS and 2-mercaptoethanol. One of the gels was stained with Coomassie Blue R-250. The other gel was transferred onto a nitrocellulose paper and subjected to immunoblot analysis using the anti-protease Ci antiserum. The arrows indicate the position where protease Ci migrated in the gels.
Figure 4:
Gel
filtration of the protease Ci preparations on a Superose-12 column. The
purified protease Ci () and the enzyme preparation obtained from
the DEAE-Sepharose column of Fig. 1(
) were chromatographed
on a Superose-12 column (1
30 cm) equilibrated with buffer A
containing 100 mM NaCl. Fractions of 0.5 ml were collected at
a flow rate of 20 ml/h, and an aliquot was assayed for hydrolysis of
I-labeled insulin B-chain. Size markers used were as
follows: a,
-amylase (200 kDa); b, alcohol
dehydrogenase (150 kDa); c, BSA (66 kDa); d, carbonic
anhydrase (29 kDa).
However, the native size of protease Ci had been reported to be in the range of 110-130 kDa, although the prior studies used crude preparations of the enzyme for the size estimation(2) . To resolve this discrepancy, the enzyme preparations obtained from the DEAE-Sepharose column (see Fig. 1) were also subjected to gel filtration using the Superose-12 column. As was reported earlier, the activity of protease Ci was eluted in the fractions corresponding to about 110 kDa (Fig. 4, open circles). On the other hand, the enzyme preparation obtained after the HiLoad Q column, which removed more than 98% of total proteins, consistently ran as a 54-kDa protein, just as the purified protease did (data not shown). However, when the gel filtration chromatography was performed in the presence of 8 M urea and the resulting column fractions were subjected to immunoblot analysis using the antiserum raised against the purified protease Ci, the peaks of both the DEAE and purified enzyme preparations were found to elute in the fractions corresponding to about 54 kDa (data not shown). These results suggest that the size change may be due to dissociation of the subunits or removal of certain protein(s) that might have been nonspecifically bound to protease Ci.
We also tested whether the change in the size of protease Ci is due to limited proteolysis of the enzyme during purification. The E. coli cells grown to mid-log phase were harvested, immediately boiled, electrophoresed in the presence of 2-mercaptoethanol, and subjected to immunoblot analysis as above. This antiserum mainly interacted with the 54-kDa protein and not with any protein of 110 kDa (Fig. 3B, lane a). The minor bands seen in the gel could also be detected with the preimmune serum (data not shown). The identical data were obtained when the same experiments were performed under nonreducing conditions. Thus, the change in the size of protease Ci is not due to limited cleavage during purification of the enzyme either by itself or by other contaminating E. coli proteases. These results indicate that the 54-kDa protein responsible for the insulin degradation is protease Ci.
To clarify further the requirement
for metal ions, protease Ci was preincubated with 0.2 mM NaCN
for 20 min, which blocked the initial enzyme activity by about 65%.
Various metal ions were then added to determine whether any of them can
reactivate the enzyme. As shown in Table 3, protease Ci could be
reactivated by Co, Mn
, and
Cu
in the order of their effectiveness but not by
Ca
, Mg
, and Zn
at
the concentrations tested. Without the preincubation, however,
Zn
, Co
, and Cu
strongly inhibited the activity of protease Ci while
Mg
, Ca
, and Mn
were stimulatory (data not shown). Thus, it appeared as if
protease Ci uses Co
as its metal cofactor.
To
determine whether protease Ci indeed contains Co,
HR-ICP mass spectral analyses of two different preparations of the
purified enzyme were performed. The enzyme contained 1.51 and 1.26
µg of Zn
per mg of protein. Assuming a size of 54
kDa, this is equivalent to a mean of 1.1 mol of Zn
per mol of protease Ci. Although the NaCN-treated enzyme could be
reactivated most effectively by Co
and not by
Zn
, it is a well known fact that metalloproteases
that use Zn
as the natural metal cofactor can be
inhibited when incubated with excess Zn
. In addition,
in some cases (e.g. metallocarboxypeptidases), Co
activates the enzyme severalfold over the activity seen with the
native Zn
-containing
enzyme(22, 23, 24) . Thus, protease Ci is
likely to be a Zn
metalloprotease.
Figure 5:
Time-dependent hydrolysis of insulin and
oxidized insulin B-chain by the purified protease Ci. Assays were
performed by incubating 1 µg of I-labeled insulin
(
) and 0.25 µg of the purified protease Ci at 37 °C for
various periods. Incubations were also carried out with 5 µg of
oxidized insulin B-chain (
) and 0.05 µg of the
enzyme.
We then examined whether protease Ci can
also hydrolyze oxidized insulin A-chain. The activity of the purified
enzyme against I-labeled B-chain was measured in the
presence of increasing amounts of the unlabeled A-chain. As shown in Fig. 6, the insulin A-chain (like unlabeled intact insulin and
oxidized B-chain) could competitively reduce breakdown of the
radiolabeled B-chain. These results suggest that protease Ci is also
capable of hydrolyzing oxidized insulin A-chain. In addition, unlabeled
intact insulin molecules were able to inhibit the hydrolysis of
I-labeled B-chain, even more effectively than any of A-
or B-chain. From these data, the K
values of the
competing, unlabeled intact insulin and oxidized insulin B-chain
molecules were estimated to be 0.13 and 12.4 µM,
respectively. These results suggest that the affinity of protease Ci
for intact insulin is about 100-fold higher than that of oxidized
insulin B-chain, despite the fact that the latter polypeptide is much
more rapidly hydrolyzed by the enzyme than intact insulin.
Figure 6:
Effects of unlabeled insulin chains and
BSA on the hydrolysis of I-labeled insulin B-chain by
protease Ci. Hydrolysis of the radiolabeled B-chain (5 µg per
reaction mixture) was assayed by incubating 0.1 µg of the purified
enzyme in the absence and presence of increasing amounts of unlabeled
intact insulin (
), insulin A-chain (
), B-chain (
),
and BSA (
). Incubations were performed for 30 min at 37 °C.
The activity seen without the unlabeled proteins was expressed as 100%
and the others as its relative values.
A variety
of I-labeled proteins of different sizes was also tested
for susceptibility to the purified protease Ci. Glucagon was also
rapidly degraded by the enzyme but approximately 20% as fast as
oxidized insulin B-chain (Table 4). Protease Ci was also capable
of degrading polypeptides with sizes smaller than 15 kDa, such as
lactalbumin and lysozyme, although to a much lesser extent than
oxidized insulin B-chain. However, it showed little or no activity
against any larger proteins tested, such as BSA, denatured BSA,
ovalbumin, carbonic anhydrase, and globin. In addition, protease Ci
cleaved angiotensin I and the synthetic peptide that is used as a
substrate of a tyrosine-protein kinase (RRLIEDAEYAARG) (25) upon analysis of their incubation mixture on HPLC.
However, it failed to cleave Leu-enkephalin and bradykinin. Therefore,
it is tempting to speculate that protease Ci may preferentially
hydrolyze the peptides with amino acids ranging from 10 to 150.
Figure 7:
HPLC separation of the peptide fragments
of oxidized insulin B-chain. The purified protease Ci (1.5 µg) was
incubated with 100 µg of oxidized insulin B-chain at 37 °C for
0, 2, and 15 h. The cleavage products were then separated by
reversed-phase HPLC on a C column as described under
``Experimental Procedures.'' The peaks of peptide fragments
were numbered in the order of their elution. The capital letter B indicates oxidized insulin B-chain.
The cleavage sites on oxidized insulin B-chain were then
determined by analysis of the amino acid composition of each of the
purified peptides. Fig. 8summarizes the amino acid sequences of
the peptides deduced from their amino acid compositions. In accord with
the results from the reversed phase chromatography, a number of
peptides appeared to behave as intermediates in degradation, such as
peptide 8b for peptides 1 and 4a, 12b for 2 and 11, and 14 for 2 and
13. Peptide 5 may also be an intermediate in the generation of peptide
1 and LCGSH. However, the latter pentapeptide was not found among the
14 peaks and may correspond to any of the minor peaks whose amino acid
composition was not determined. These results strongly suggest that
protease Ci preferentially cleaves on the amino side of hydrophobic
amino acids (i.e. the peptide bonds of
His-Leu
, His
-Leu
,
Leu
-Val
, Glu
-Ala
,
Ala
-Leu
, Tyr
-Leu
,
Leu
-Val
). Secondary cleavages occurred on the
amino side of tyrosine (i.e. Leu
-Tyr
,
Phe
-Tyr
). The only exception is peptide 4b
generated by the cleavage of the amino side of glutamic acid (i.e. Val
-Glu
).
Figure 8:
Determination of the cleavage sites on
oxidized insulin B-chain. When the peptide peaks obtained as in Fig. 7were rechromatographed on the same C column
but using a shallow gradient of acetonitrile, each of the peaks 4, 8,
and 12 was further separated into two peaks indicated as a and b. Therefore, total 17-peptide fragments were subjected to
analysis for their amino acid composition as described under
``Experimental Procedures.'' The amino acid sequences of the
peptides (bars) and then the cleavage sites on oxidized
insulin B-chain (arrows) were deduced from the amino acid
composition data.
To examine whether peptide 4b is produced by trace contaminating proteases under the incubation conditions, the same experiment was performed using the purified protease Ci that had been treated with a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mMN-ethylmaleimide, 5 µg/ml leupeptin, and 5 µg/ml E-64), which do not inhibit protease Ci (see Table 2), followed by dialysis against buffer A to remove the inhibitors. However, peptide 4b was consistently generated with the others. On the other hand, when the cleavage of insulin B-chain was carried out with the supernatant fraction obtained after immunoprecipitation of the purified enzyme using anti-protease Ci IgG, no peptide was generated. Therefore, it remains uncertain whether peptide 4b is one of the authentic cleavage products or generated by trace contaminating proteases in the purified protease Ci preparation.
In the present studies, a cytoplasmic metalloprotease in E. coli, protease Ci, was purified to apparent homogeneity
using I-labeled oxidized insulin B-chain as a substrate.
Since E. coli contains an additional insulin B-chain-degrading
enzyme, protease Pi, in the periplasm(2, 6) , we used E. coli 3302 strain, which lacks the periplasmic enzyme. From
140 g of the frozen cells, we obtained 90 µg of purified protease
Ci with a final yield of about 1.5%. Thus, protease Ci accounts for at
least 0.03% of the soluble protein of E. coli.
Protease Ci
was found to have much higher affinity for intact insulin than for
oxidized insulin B-chain, despite the fact that the latter polypeptide
is hydrolyzed approximately 50-fold more rapidly than intact insulin.
Similar results have been reported for protease Pi(7) .
Therefore, it appears possible that dissociation of intact insulin
and/or its cleavage products from the enzyme may be a rate-limiting
step for degradation of additional insulin molecules. In this regard,
the characteristics of low k and low K
are not unlike those of small protein inhibitors
of proteases(26) , as was noted by Barrett and co-workers (7) for protease Pi.
Protease Ci, like protease Pi(5, 9) , is not inhibited by sulfhydryl blocking agents, such as N-ethylmaleimide and iodoacetamide. This insensitivity of the bacterial enzymes distinguishes them from the human and Drosophila insulin-degrading enzymes that are highly susceptible to inactivation by such reagents(9, 10) . The predicted amino acid sequence of protease Pi contains a single Cys residue(27) , whereas the human insulin-degrading enzyme has 12 Cys residues(8) . One of the Cys residues in the human enzyme is present in the metal-binding site (HXCXH) in the first highly conserved domains of insulin-degrading enzymes(28) . Protease Pi also contains the metal-binding motif (HXXXH) but lacks the Cys residue(27) . Protease Ci may contain a similar metal-binding site to that of protease Pi, which might explain its lack of sensitivity to sulfhydryl-modifying agents. Isolation and sequence determination of the DNA clone for protease Ci should reveal whether this is true.
Protease Ci is capable of
hydrolyzing as many as 10 sites among the total 29 peptide bonds in
oxidized insulin B-chain. In contrast, protease Pi cleaves only the
Tyr-Leu
bond(7) , which is also
hydrolyzed by protease Ci. Like protease Ci, the insulin-degrading
enzymes from Drosophila and rat also hydrolyze multiple
peptide bonds in the insulin B-chain(29) , although protease Ci
is able to cleave many more sites. In addition, all the peptide bonds
hydrolyzed by the various eukaryotic insulin-degrading enzymes are also
sensitive to degradation by protease Ci, except for the
Ser
-His
bond and the
Phe
-Phe
bond, which are cleaved only by the
rat enzyme(29) .
Protease Ci appears to preferentially
degrade relatively small-sized polypeptides, in contrast with other
soluble proteases in the cytoplasm of E. coli, proteases Do
(DegP), Re (Tsp), Mi, Fa, So, La (Lon), and Ti
(Clp)(1, 2) . In addition to oxidized insulin B-chain,
protease Ci rapidly hydrolyzed glucagon and degraded lactalbumin and
lysozyme, although at much slower rates. However, little or no
trichloroacetic acid-soluble products are generated by the purified
protease from any protein tested that is larger than 15 kDa.
Interestingly, when proteins larger than 15 kDa, such as casein, were
incubated with the ATP-dependent protease La or Ti in the presence of
protease Ci, the production of acid-soluble products was over 2-fold
faster than in its absence. ()The ATP-dependent proteases
appear to catalyze the initial rate-limiting steps in the degradation
of highly abnormal polypeptides and of certain normal
proteins(3, 4) . Therefore, protease Ci may function
subsequently in the pathway for intracellular protein breakdown and
digest the oligopeptides generated by the ATP-dependent endoproteases.