From the Department of Biochemistry, Indian Institute
of Science, Bangalore 560012, India
Received for publication, August 4, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin
(Suc-LLVY-AMC), a fluorogenic endopeptidase substrate, is used to
detect 20 S proteasomal activity from Archaea to mammals. An
o-phenanthroline-sensitive Suc-LLVY-AMC hydrolyzing
activity was detected in Escherichia coli although it lacks
20 S proteasomes. We identified PepN, previously characterized as the
sole alanine aminopeptidase in E. coli, to be responsible
for the hydrolysis of Suc-LLVY-AMC. PepN is an aminoendopeptidase.
First, extracts from an ethyl methanesulfonate-derived PepN mutant,
9218, did not cleave Suc-LLVY-AMC and
L-Ala-para-nitroanilide (pNA).
Second, biochemically purified PepN cleaves a wide variety of both
aminopeptidase and endopeptidase substrates, and
L-Ala-pNA is cleaved more efficiently than
other substrates. Studies with bestatin, an aminopeptidase-specific
inhibitor, suggest differences in the mechanisms of cleavage of
aminopeptidase and endopeptidase substrates. Third, PepN hydrolyzes
whole proteins, casein and albumin. Finally, an E. coli
strain with a targeted deletion in PepN also lacks the ability to
cleave Suc-LLVY-AMC and L-Ala-pNA, and
expression of wild type PepN in this mutant rescues both activities. In
addition, we identified a low molecular weight Suc-LLVY-AMC-cleaving peptidase in Mycobacterium smegmatis, a eubacteria
harboring 20 S proteasomes, to be an aminopeptidase homologous to
E. coli PepN, by mass spectrometry analysis.
"Sequence-based homologues" of PepN include well characterized
aminopeptidases, e.g. Tricorn interacting factors F2 and F3
in Archaea and puromycin-sensitive aminopeptidase in mammals. However,
our results suggest that eubacterial PepN and its homologues displaying
aminoendopeptidase activities may be "functionally similar" to
enzymes important in downstream processing of proteins in the cytosol:
Tricorn-F1-F2-F3 complex in Archaea and TPPII/Multicorn in eukaryotes.
Dynamic changes in the proteome of a cell depend on rates
of protein synthesis and their degradation. The past few decades have
witnessed enormous strides in identifying the molecules and understanding the mechanisms involved in intracellular protein degradation. There is increasing evidence of the involvement of protein
degradation in diverse biological activities, e.g. cell cycle progression, transcriptional activation, antigen processing, disease progression, etc. (1-5).
Broadly, cytosolic protein degradation is categorized into four steps.
(i) Proteins targeted for degradation are initially unfolded into
polypeptides by ATP-dependent proteases belonging to the
Lon/Clp family in bacteria or 26 S proteasomes in higher organisms
(1-5). (ii) These enzymes also make the initial endoproteolytic "cuts" in the polypeptide. Interestingly, in both Escherichia coli (6) and higher organisms (7) the average length of peptides
released by these enzymes range from 3 to 25 amino acids. (iii) These
longer peptides are trimmed into smaller peptides (less than 10 amino
acids) by the action of endopeptidases (8-11), tripeptidyl- and
dipeptidylpeptidases (12-15). (iv) Finally, aminopeptidases and
carboxypeptidases digest these peptides into amino acids (2, 9, 16). In
general, aminopeptidases involved in protein degradation act on short
but not long peptides (9, 16).
In eukaryotes, functional 26 S and 20 S proteasomes are responsible
for the majority of non-lysosomal protein degradation and are essential
for cell survival (1, 3). Prokaryotes, on the other hand, possess
redundant proteolytic systems. E. coli strains lacking
lon and clp (2) and Mycobacterium
smegmatis lacking 20 S proteasomes (17) are viable, whereas
Thermoplasma 20 S proteasomes are important only under
conditions of heat shock (18). Both prokaryotes and eukaryotes possess
oligopeptidases and exopeptidases that act during the later (steps iii
and iv) stages of protein degradation. In Thermoplasma, the
combination of 20 S proteasomes together with the
Tricorn-F1-F2-F3 complex is important in recycling amino acids
(9). Similarly, mouse cells possess
TPPII,1 which integrates with
the ubiquitin-proteasome pathway for efficient breakdown of proteins
(15). Interestingly, MHC class I-binding antigenic peptides are
generated primarily by proteasomes (1). In some cases, these peptides
are trimmed at the N terminus by peptidases, e.g. leucine
aminopeptidase (19), PSA, bleomycin hydrolase (20, 21), and endoplasmic
reticulum aminopeptidase associated with antigen processing (22).
Recently, characterization of enzymes involved in downstream processing
of peptides during degradation (9-11, 14-16) and trimming of MHC
class I-binding peptides (19-22) and their destruction (23) has gained importance.
Suc-LLVY-AMC, a chymotryptic peptidase substrate, is cleaved by
eubacterial (24, 25), archaeal (26), and mammalian (7, 27) 20 S
proteasomes. Although the E. coli genome does not encode "true" 20 S proteasome subunits (28), an LMW Suc-LLVY-AMC
hydrolyzing activity was detected. There are reports in two bacteria,
Rhodococcus (24) and Frankia (25), and mouse
liver (27) where an LMW Suc-LLVY-AMC-cleaving peptidase was also
detected. However, the enzyme(s) responsible for this activity have not
been identified. In this report, we directly identified the LMW
Suc-LLVY-AMC peptidase as PepN or a PepN homologue from two different
bacteria, E. coli and M. smegmatis. Recent
studies have demonstrated the role of enzymes possessing aminopeptidase
and endopeptidase activities in Thermoplasma (8, 9), yeast
(11), and mouse (14, 15) to act in downstream processing of peptides
during intracellular protein degradation. Our results suggest that PepN
and functionally similar enzymes may play a similar role in eubacterial
protein degradation.
Strains, PCR, Plasmids, and Overexpression of PepN--
Wild
type E. coli K12RV strain (29), E. coli strain
9218, an EMS-derived pepN mutant (30), and 9218 harboring
plasmid pBM15 encoding an E. coli genomic fragment
containing pepN with its endogenous promoter (31) were grown
in LB media in the absence or presence of 30 µg/ml tetracycline
(Himedia, India). 9218 transformed with pBR322 or pBM15 is referred in
the text as 9218/pBR322 or 9218/pBM15. E. coli K12RV genomic
DNA was used as template to amplify pepN using high fidelity
DyNAzyme (Finnzymes, Finland) and gene-specific primers: forward
5'-AAAACTGCAGGGATCCCATATGACTCAACAGCCACAAGCC-3' and reverse
5'-AAAACTGCAGCTCGAGTCAATGGTGATGGTGATGGTGAGCCAGTGCTTTAGTTATCTT-3'. The amplified PCR product (~2.6 kbp) was gel-eluted and cloned into
pGEM®-T Easy vector (Promega). Full-length pepN was
excised using NcoI and XhoI from this plasmid and
subcloned into pBAD24 (32), referred as pBAD/EcPepN, for expression analysis.
To generate an E. coli DH5 Fractionation of Cellular Extracts--
E. colicells were grown in LB for 10 h with 0.5% inoculum, washed, and
sonicated, and cytosolic extracts were prepared in 10 mM
Tris-HCl, pH 8, by collecting the supernatant after centrifuging at
100,000 × g for 1 h. Extracts were loaded on
10-40% SDG in 10 mM Tris-HCl, pH 8.0, and centrifuged at
151,000 × g at 4 °C in an ultracentrifuge (Beckman
Instruments). After 20 h, 1-ml fractions were collected and
assayed for Suc-LLVY-AMC hydrolysis. Bovine serum albumin (69 kDa;
4.5 S) and thyroglobulin (669 kDa; 20 S) were used as molecular
weight markers. Protein amounts were estimated by Bradford's method
with bovine serum albumin as the standard.
Purification of the E. coli LMW Suc-LLVY-AMC-cleaving
Peptidase--
E. coli DH5 Purification and Identification of the LMW Suc-LLVY-AMC-cleaving
Peptidase from M. smegmatis--
M. smegmatis SN2 strain
was grown in Youmans Karlson's medium with 0.5% inoculum at 37 °C
for 50 h. Cells were harvested by centrifugation at 10,000 × g at 4 °C, and extracts were prepared by resuspending the
cell pellet in 10 mM Tris, pH 8, followed by sonication.
The sonicate was centrifuged at 100,000 × g for 1 h and used for SDG or further purification. The extract was concentrated using sucrose and PEG sequentially, dialyzed, and fractionated using Sepharose CL6B gel filtration. The LMW active fractions were pooled, bound to DEAE-cellulose, and eluted with 150-350 mM NaCl gradient. Active fractions were dialyzed
and bound to a Q-Sepharose column followed by elution with 200-450
mM NaCl. Active fractions were pooled, equilibrated with 1 M ammonium sulfate, loaded on a butyl-Toyopearl column, and
eluted using 1 to 0.01 M ammonium sulfate gradient. Active
fractions were subjected to SDS-PAGE, and the highly enriched protein
as observed by Coomassie Blue staining was cut and sent for
identification. In-gel trypsin digestion, analysis of the released
peptides by capillary LC-MS and MS/MS, and identification was performed
at The W. M. Keck Biomedical Mass Spectrometry Laboratory,
University of Virginia.
Enzyme Assays, Inhibitors, and Kinetic Characterization--
All
fluorogenic (0.5 mM) and chromogenic (1 mM)
peptide substrates were obtained from Sigma. For SDG and PepN
purification steps, peptidase assays were performed by incubating
peptide substrates and enzyme at 37 °C for appropriate periods in
assay buffer (50 mM Tris-HCl, pH 8, 0.2 M
MgCl2, and 1 mM In Vitro Protein Degradation Assays--
Protein degradation
assay (39) was performed by incubating purified 1 µg of PepN with 100 µg of FTC-albumin, FTC-casein, or FTC-insulin (Sigma) for different
times in 40 mM phosphate buffer, pH 8. The assay was
terminated by precipitating the reaction mixture with 5%
trichloroacetic acid overnight at 4 °C. Trichloroacetic acid-precipitable proteins were pelleted by centrifugation, and the
supernatant was diluted with phosphate buffer. Fluorescence was
measured with excitation wavelength of 490 nm and emission wavelength
of 525 nm. Net fluorescence due to PepN activity was calculated after
subtraction with appropriate controls. One fluorescence unit is the
amount of fluorescence reading obtained with a 48 µM
solution of quinine sulfate (39).
A Metalloprotease Is Probably Responsible for the Hydrolysis of
Suc-LLVY-AMC in E. coli--
E. coli cytosolic extracts
were fractionated using SDG ultracentrifugation, and the Suc-LLVY-AMC
hydrolyzing activity was studied in individual fractions. A single peak
of activity was observed at an LMW range in fraction 4 (Fig.
1A). In the same experiment,
the major high molecular weight peak of Suc-LLVY-AMC peptidase activity
by 20 S proteasomes in mouse liver was in fraction 8 (data not shown).
Next, the effect of a panel of inhibitors on the activity of SDG
fraction 4 was studied. Incubation with o-phenanthroline
displayed significant inhibition of Suc-LLVY-AMC hydrolysis (Fig.
1B). Several zinc-dependent metallopeptidases are sensitive to o-phenanthroline (12, 16, 40, 41), and these results suggested that the Suc-LLVY-AMC-hydrolyzing enzyme in
E. coli could be a metallopeptidase.
PepN Is Responsible for the Hydrolysis of Suc-LLVY-AMC--
As the
E. coli proteolytic system is well characterized (2) and the
genome sequence is available (28), we resorted to a functional genomics
approach to identify the probable gene responsible for Suc-LLVY-AMC
hydrolysis. A list of annotated metalloproteases was made using the
E. coli genome data base, and a comparative analysis was
performed (data not shown). Two metallopeptidases, PepN and PqqL, had
similar features as the Suc-LLVY-AMC-hydrolyzing peptidase:
cytosolic localization and a molecular mass of ~100 kDa. PepN was previously identified as the sole alanine aminopeptidase in E. coli (42-44) belonging to the M1 family, whereas PqqL
was annotated as a putative insulinase belonging to M16 family of metallopeptidases (35). pqqL was amplified from E. coli genomic DNA by PCR, cloned, and overexpressed in E. coli BL-21 after
isopropyl-1-thio- Purified E. coli PepN Is an Aminoendopeptidase--
The LMW
Suc-LLVY-AMC-cleaving peptidase was purified from a DH5
The cleavage specificity of the purified enzyme was studied using
different chromogenic and fluorogenic peptide substrates. As shown in
Table II, it cleaved a wide variety of
aminopeptidase and endopeptidase substrates. However,
L-Ala-pNA was cleaved very efficiently,
~100-fold more efficiently than other aminopeptidase substrates
tested, consistent with the role of PepN as the sole alanine
aminopeptidase in E. coli (42-44). We also tested AAF-AMC, a fluorogenic aminoendopeptidase substrate (11, 14), and we observed
that PepN hydrolyzed this substrate. This suggests that PepN is very
efficient as an aminopeptidase in cleaving after alanine, leucine,
tyrosine, and phenylalanine. The cleavage of endopeptidase substrates
by PepN was much slower than aminopeptidase substrates. This was most
evident while comparing cleavage of AAF-AMC and Suc-AAF-AMC as blocking
the N-terminal amino acid resulted in reduced cleavage of the
endopeptidase substrate by ~65-fold. PepN hydrolyzed several
endopeptidase substrates, revealing a preference for hydrophobic and
basic residues. Further detailed kinetic characterization of purified
E. coli PepN was performed with an endopeptidase
(Suc-LLVY-AMC) and an aminopeptidase (L-Ala-pNA) substrate. Values for kinetic parameters (Table
III) revealed that Suc-LLVY-AMC bound
PepN with lower Km and had a slower rate of turnover
as evident by the low kcat value. The
kcat/Km value was 230-fold
more for L-Ala-pNA hydrolysis than Suc-LLVY-AMC hydrolysis (Table III), implying that PepN is a more efficient aminopeptidase than an endopeptidase.
Differential Effects of Bestatin on the Aminopeptidase and
Endopeptidase Activities of PepN--
The effect of different
inhibitors on PepN activity was studied. As shown in Fig.
4A, both endopeptidase and
aminopeptidase activities were inhibited by
o-phenanthroline, 4-(2-aminoethyl)benzenesulfonyl fluoride,
and N-ethylmaleimide. The lack of inhibition by EDTA and
EGTA was surprising; however, there is a report (41) of a metalloenzyme
that is sensitive to o-phenanthroline but not to EDTA or
EGTA. A previous study had also observed 20 and 80% inhibition of
aminopeptidase activity of PepN with phenylmethylsulfonyl fluoride and
a sulfhydryl group modifier, respectively (44). Interestingly, bestatin
(45), an aminopeptidase-specific competitive inhibitor, showed a
differential inhibition profile and inhibited L-Ala-pNA activity completely, whereas
Suc-LLVY-AMC hydrolysis was reduced by 60%. As this result allowed us
to differentiate these two activities of PepN, we titrated PepN with
bestatin and studied hydrolysis of Suc-LLVY-AMC and
L-Ala-pNA (Fig. 4C). Inhibition of
L-Ala-pNA hydrolysis by 50% required 0.02 mM bestatin which is 7-fold lower than that required to
attain 50% inhibition of Suc-LLVY-AMC hydrolysis. Even at the highest
concentration of bestatin used, complete inhibition of Suc-LLVY-AMC
hydrolysis was not observed (Fig. 4B). However, comparable
amounts of o-phenanthroline were required at 0.8 and 0.3 mM for 50% inhibition of L-Ala-pNA and Suc-LLVY-AMC hydrolysis (Fig. 4B). The above results
suggested that there are intrinsic differences in the mechanisms by
which aminopeptidase and endopeptidase substrates are hydrolyzed by E. coli PepN.
Hydrolysis of Whole Proteins by PepN Is
Bestatin-independent--
As most aminopeptidases cannot cleave long
peptides or native proteins (9, 16), we tested whether PepN, being an
aminoendopeptidase, could hydrolyze oxidized insulin B chain or native
proteins. Interestingly, PepN hydrolyzed oxidized insulin B chain in a
time-dependent manner, and the mass spectrometric analysis
of peptides released by insulin B chain hydrolysis suggested
endopeptidase cuts (data not shown). We further tested the ability of
PepN to hydrolyze native proteins. PepN was incubated with model
protein substrates for different times, and the hydrolysis was
monitored. Purified PepN hydrolyzed all three protein substrates tested
although FTC-casein and FTC-albumin were hydrolyzed better than
FTC-insulin (Fig. 5A). These
results demonstrate that PepN can cleave intact proteins, although the kinetics of cleavage of casein was slower than that observed for 20 S
proteasomes (9). We studied the sensitivity of FTC-casein hydrolysis by
PepN to o-phenanthroline and bestatin. The hydrolysis of
FTC-casein was inhibited significantly (49%) with the highest concentration of o-phenanthroline. On the other hand,
bestatin did not display any significant effect on the ability of PepN to hydrolyze FTC-casein (Fig. 5, B and C),
suggesting that PepN was degrading casein using its endopeptidase
activity but not its aminopeptidase activity.
Rescue of Both Activities by Expression of Cloned PepN in a Strain
with a Targeted Deletion in PepN--
As 9218 is an EMS-derived
mutant, we could not rule out the possibility of a mutation in another
gene in addition to pepN. To confirm that PepN is
responsible for Suc-LLVY-AMC hydrolysis in E. coli, we
generated a DH5 An LMW Suc-LLVY-AMC-cleaving Enzyme Is a PepN Homologue in M. smegmatis--
During the characterization of 20 S proteasomes from
two actinomycetes, an LMW Suc-LLVY-AMC-cleaving peptidase was observed (24, 25). Therefore, we wished to identify an LMW Suc-LLVY-AMC-cleaving peptidase from another actinomycetale, M. smegmatis, which
also encodes 20 S proteasomes (17). Interestingly, a Suc-LLVY-AMC hydrolyzing activity was observed in M. smegmatis lacking
20 S proteasomes (17), although the identity of this enzyme(s) was not
established. Cellular extracts from M. smegmatis after SDG fractionation displayed low and high molecular weight peaks of activity
(Fig. 7A). The LMW major
activity was sensitive to o-phenanthroline, EDTA, and EGTA
(Fig. 7B), suggesting the involvement of a metallopeptidase. Next, we purified this low molecular weight Suc-LLVY-AMC-hydrolyzing peptidase activity, and the highly enriched protein (Fig.
7C) was identified by homology from the M. smegmatis genome data base as a possible aminopeptidase (~94.4
kDa), homologous to E. coli PepN. The putative translated
sequence and identified peptides (underlined) are shown in Fig.
7D. Thus, we have demonstrated that PepN and a PepN
homologue were responsible for the LMW Suc-LLVY-AMC hydrolyzing
activity in two bacteria, E. coli and M. smegmatis.
In this study, we demonstrate that E. coli PepN is the
major enzyme responsible for cleaving Suc-LLVY-AMC, a substrate widely used to characterize 20 S proteasomes from all organisms. The first
report on PepN characterized it as an aminoendoprotease (43) using
125I-casein; however, a later study (44) did not detect any
endopeptidase activity. Based on genetic and biochemical studies, we
demonstrate that E. coli PepN, a single polypeptide, is a
soluble metalloaminoendoprotease. First, an EMS-derived PepN mutant,
9218, lacked the ability to cleave both Suc-LLVY-AMC and
L-Ala-pNA; however, expression from a genomic
fragment encoding E. coli PepN rescued both these activities (Fig. 2). Second, we purified the LMW Suc-LLVY-AMC-cleaving enzyme from
E. coli, and purity was demonstrated by a single band after silver staining and overlapping enzymatic peaks of both activities after fast protein liquid gel filtration chromatography (Fig. 3). Also,
the thermal denaturation curves for both activities were identical
(Fig. 3). This purified enzyme cleaved L-Ala-pNA more efficiently than Suc-LLVY-AMC. In fact the
kcat/Km value was ~230-fold
more for L-Ala-pNA hydrolysis than Suc-LLVY-AMC and varied from 196- to 260-fold between different purified enzyme preparations. Although the ability of PepN to cleave Suc-LLVY-AMC was
slow (Vmax 3.6 µmol/h/mg; Table III), this
rate compared well with reports of other Suc-LLVY-AMC-cleaving
peptidases, including 20 S proteasomes. For example, the
Vmax value of Thermoplasma 20 S
proteasome is 0.054 µmol/h/mg (46), 8.8 µmol/h/mg by
Frankia 20 S proteasomes (25), and 0.6 µmol/h/mg by an
Entamoeba 11 S protease (47). It does not appear that PepN
directly cleaves after tyrosine in Suc-LLVY-AMC as Suc-LY-AMC was not
hydrolyzed (Table II). Therefore, it is most likely that PepN cleaves
Suc-LLVY-AMC (somewhere before Tyr) by acting as an endopeptidase
followed by release of AMC due to its aminopeptidase activity. PepN
cleaved L-Ala-pNA very efficiently, ~100-fold
better than other aminopeptidase substrates (Table II), consistent with
its role as the sole alanine aminopeptidase in E. coli
(42-44). Third, the kinetics of hydrolysis of casein by PepN was
slower than that observed with Thermoplasma 20 S
proteasomes (9). In fact, PepN is probably a better endopeptidase than
a protease, as suggested by the kinetics of cleavage of oxidized insulin B chain (data not shown) and casein (Fig. 5). Fourth, we
generated an E. coli DH5 PepN belongs to the M1 family of metallopeptidases, whose members are
well characterized aminopeptidases that contain a gluzincin motif (34, 35). A motif search revealed that PepN does not have any
known ATPase motifs suggesting that the enzyme is probably ATP-independent, which is consistent with its lack of ATP requirement in enzyme assays. PepN homologues are ubiquitous and present in all
kingdoms (data not shown). The wide distribution of PepN homologues suggests that it is important in normal cellular functions. In fact, a
PepN homologue is present in the pathogen Mycobacterium leprae which has minimal number of genes (48). We compared the active site sequences of PepN and its homologues that are well characterized. PepN homologues in Thermoplasma acidophilum,
F2 and F3, are well characterized aminopeptidases important in protein degradation (9). PepN homologues from Lactococcus lactis
(49), Streptococcus thermophilus (40), and Aspergillus
niger (41) are, primarily, lysine aminopeptidases. The PepN
homologue in yeast, AAP1, is not essential for viability but affects
glycogen accumulation (50). Also, a mouse PepN homologue, PSA, is
important in reproduction (51-53) and trimming of MHC class I
antigenic peptides (20, 21). As observed in Fig.
8A, PepN and its homologues possess the conserved exopeptidase, GXMEN motif
(36). The known three Zn2+-binding ligands present within
the
HEXXHX18E motif (34, 35, 37) are also conserved as are the two identified catalytically important glutamic acid residues: one in the
GXMEN motif (36) and another which is next to the
first Zn2+-binding histidine in the
HEXXH motif (34, 35, 37). Although PepN and its homologues contain key amino acids
important for Zn2+ binding and aminopeptidase activity,
there are differences with respect to endopeptidase activity. PepN
homologues from S. thermophilus (40) and F2 and F3 from
T. acidophilum (9) do not display endopeptidase activity,
whereas E. coli PepN and its homologue from M. smegmatis display endopeptidase activity (Table II and Fig. 7). To
our knowledge, there are only seven aminoendopeptidases reported:
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain with a targeted
deletion in pepN, we used the following strategy (33).
pGEM®-T Easy vector containing pepN was digested with
NruI to remove 312 bp (620-932 bp) encompassing
catalytically important residues in the M1 family (34-37). This DNA
was eluted and ligated to a gene encoding kanamycin resistance,
previously eluted after PvuII digestion from pUC4K vector
(Promega). From a positive clone, pepN disrupted with the gene encoding kanamycin resistance was released using NdeI
and NcoI (~3.85 kbp) and transformed into DH5
containing pKD46 encoding
Red (33) by electroporation. Positive
clones were selected on LB plates containing 100 µg/ml ampicillin and
30 µg/ml kanamycin at 30 °C and later cured of pKD46 by overnight
growth at 42 °C (33). The E. coli DH5
strain
containing a targeted deletion in pepN, referred to as
DH5
PepN, was confirmed by PCR and enzyme assays. To study the
effect of expression of wild type PepN, overnight cultures of
DH5
PepN transformed with pBAD24 or pBAD/EcPepN were diluted
100-fold in LB medium containing 100 µg/ml ampicillin and different
concentrations of L-arabinose (Himedia). Cells were harvested after 4 h followed by preparation of extracts and
determination of enzymatic activities.
cells transformed with pBM15
were grown, and cytosolic extracts were prepared and loaded on a
DEAE-cellulose column. Bound proteins were eluted with 100-250
mM NaCl gradient in 10 mM Tris-HCl, pH 8. Peak
fractions of Suc-LLVY-AMC hydrolysis were pooled, loaded on a
Q-Sepharose column, and eluted with a linear gradient of 150-300
mM NaCl. Active fractions were pooled, equilibrated with 1 M ammonium sulfate, and loaded on a Butyl-Toyopearl column
followed by elution with a gradient of 1 to 0.01 M ammonium sulfate. Active fractions were pooled, stored at 4 °C, and used in
experiments as purified PepN. Proteins were separated by SDS-PAGE and
visualized by staining gels with Coomassie Brilliant Blue G-250 or
silver nitrate staining. The apparent molecular weight of PepN was
determined using a precalibrated Superdex 200 FPLC gel filtration
column (Amersham Biosciences).
-mercaptoethanol), whereas assays were performed in 40 mM phosphate buffer, pH 8, for
experiments with the purified enzyme. Assays were terminated by adding
100% ethanol, and fluorescence was measured with excitation
wavelengths of 370 and 335 nm and emission wavelengths of 430 and 410 nm for AMC-based and
NA-based substrates, respectively, using a
spectrofluorimeter (Shimadzu, Japan). Similarly, assays for
chromogenic substrates were performed, and the product formed was
measured by taking absorbance at 410 nm in a spectrophotometer
(Shimadzu). Net increase in AMC or pNA released due to PepN
activity was calculated after subtraction with appropriate controls.
Standard curves were plotted using known amounts of AMC or
pNA to calculate the amounts of AMC or pNA
released. Specific activity was calculated as nanomoles of AMC or
pNA released for 1 µg of protein per h at 37 °C, unless otherwise mentioned in the figure legends. Inhibition experiments were
performed by incubating the enzyme with different inhibitors as
follows: 5 µM antipain, 5 µM leupeptin, 1 mg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride (serine protease); 5 µM pepstatin (aspartate protease); 5 mM EDTA,
5 mM EGTA, 4 mM o-phenanthroline
(metalloprotease); 10 µM lactacystin, 20 µM
Cbz-LLL-H, 50 µM Cbz-LLL-vinylsulfone (20 S proteasome);
1 µM Cbz-LHVS (cathepsin S); 5 µg/ml E-64 (cathepsins B
and L, papain, and cysteine protease); 1 mM
N-ethylmaleimide (sulfhydryl protease); 280 µM
bestatin (aminopeptidase) or as mentioned in the respective legends.
The specificities of the respective inhibitors were given above in
parentheses. The sucrose gradient fraction containing maximal activity
or purified PepN was incubated with different inhibitors for 15 min at
room temperature followed by enzyme assay using appropriate substrates.
Appropriate solvent controls were used to calculate the percent
activity. Based on standardization experiments, endopeptidase assays
were performed with 1 µg of enzyme for 2 h, and aminopeptidase
assays were performed with 7.5 ng of enzyme for 1 h for kinetic
studies. PepN was incubated with different concentrations of
substrates, and the rate of hydrolysis was measured. Kinetic parameters
were determined graphically by the direct linear plot (38).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A metallopeptidase is probably responsible
for the Suc-LLVY-AMC hydrolyzing activity in E. coli. A, cytosolic extracts were
fractionated by SDG and assayed for hydrolysis of Suc-LLVY-AMC.
B, fraction number 4 was incubated with different protease
inhibitors, and their effect on Suc-LLVY-AMC hydrolysis was studied.
Mean values with standard deviations of six independent experiments are
shown.
-D-galactopyranoside induction.
However, no significant difference in Suc-LLVY-AMC hydrolysis was found
after overexpression of PqqL (data not shown). E. coli K12
strain 9218, a PepN mutant (30), was transformed with pBR322 or pBM15,
which harbors pepN (31). As shown in Fig. 2A, extracts from 9218/pBM15
overexpressed a protein at ~90 kDa, which was absent in the
9218/pBR322 extract. Next, cytosolic extracts from these strains were
fractionated by SDG ultracentrifugation. As observed in Fig.
2B, Suc-LLVY-AMC hydrolyzing activity was detected in the
E. coli K12RV extract but not in 9218/pBR322. However,
cytosolic extracts from 9218/pBM15 displayed greater Suc-LLVY-AMC
hydrolyzing activity compared with the E. coli K12RV extract. This pattern was identical for
L-Ala-pNA, as reported earlier (30). However,
hydrolysis of Cbz-LLE-
NA, a substrate used to detect
post-glutamylpeptidyl activity, was unaltered in the mutant or after
PepN overexpression (Fig. 2). Together, these results suggested that
PepN was primarily responsible for Suc-LLVY-AMC and
L-Ala-pNA hydrolysis in E. coli.
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Fig. 2.
A PepN mutant strain of E. coli is not able to hydrolyze Suc-LLVY-AMC.
A, cytosolic extracts of E. coli strains K12RV,
9218/pBR322, and 9218/pBM15 were separated on 10% SDS-PAGE and stained
with Coomassie Blue. An asterisk shows the overexpressed
PepN protein band. B, cytosolic extracts were fractionated
by SDG, and individual fractions were assayed for their ability to
hydrolyze Suc-LLVY-AMC (top),
L-Ala-pNA (middle), and Cbz-LLE- NA
(bottom).
strain
transformed with pBM15 using several chromatographic steps (Table
I), and a single band corresponding to
~85 kDa was visualized after the final step of purification (Fig.
3A). The purified protein was
subjected to gel filtration chromatography, and fractions were assayed
(Fig. 3B) for Suc-LLVY-AMC and
L-Ala-pNA hydrolysis. The peak of
L-Ala-pNA hydrolysis was found to overlap with
the peak of Suc-LLVY-AMC hydrolysis, corresponding to ~85 kDa. The
ratios of these two activities in different fractions were in the range
of 2,500 ± 380, suggesting that the enzyme was responsible for
both activities. Also, this enzyme migrated as a monomer with a
molecular mass of ~85 kDa under both SDS-PAGE and gel filtration
chromatography. The thermal denaturation profile of the purified enzyme
was studied by incubating it at different temperatures for 30 min and
testing its ability to hydrolyze Suc-LLVY-AMC and
L-Ala-pNA (Fig. 3C). Both the
endopeptidase and aminopeptidase activities displayed overlapping
curves suggesting a similar thermal denaturation pattern.
Purification of overexpressed PepN
/pBM15 was monitored by
following hydrolysis of Suc-LLVY-AMC in different fractions. The
details of purification are described under "Experimental
Procedures."
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Fig. 3.
Overlapping profiles of aminopeptidase and
endopeptidase activities by purified E. coli
PepN. A, overexpressed PepN was purified (Table
I), and protein profiles of the crude extract and purified PepN
(asterisk) were visualized after silver staining.
B, purified PepN was subjected to gel filtration
chromatography, and individual fractions were assayed for hydrolysis of
Suc-LLVY-AMC (filled squares) and
L-Ala-pNA (open squares).
C, purified PepN was incubated for 30 min at various
temperatures and assayed for the hydrolysis of Suc-LLVY-AMC
(filled squares) and L-Ala-pNA
(open squares) at 37 °C.
Hydrolysis of chromogenic and fluorogenic peptide substrates by
purified E. coli PepN
Kinetic characteristics of hydrolysis of Suc-LLVY-AMC and
L-Ala-pNA by purified E. coli PepN
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Fig. 4.
Effect of inhibitors on activities of
PepN. A, purified PepN was preincubated with various
inhibitors, and the ability to hydrolyze Suc-LLVY-AMC (filled
bars) and L-Ala-pNA (open bars)
was studied. Specific activity was calculated as nanomoles of AMC or
pNA released for 1 µg of protein per h at 37 °C. The
asterisk represents bestatin, which inhibited the
aminopeptidase activity completely but not the endopeptidase activity.
This result is representative of two independent enzyme preparations.
The ability of PepN to hydrolyze Suc-LLVY-AMC (filled
squares) and L-Ala-pNA (filled
diamonds) was studied after incubating with increasing
concentrations of o-phenanthroline (B) or
bestatin (C).
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Fig. 5.
Degradation of whole proteins by purified
PepN is bestatin-insensitive. A, FTC-casein
(squares), FTC-albumin (triangles), and
FTC-insulin (diamonds) were incubated with purified 1 µg
of PepN for different time points at 37 °C. Purified PepN was
incubated with varying concentrations of o-phenanthroline
(B) or bestatin (C) followed by addition of
FTC-casein. After 12 h at 37 °C, the reactions were terminated,
and the effect of different amounts of inhibitors on casein degradation
was studied.
PepN strain by replacing the endogenous pepN gene with a homologous disrupted pepN
harboring the kanamycin resistance cassette in place of catalytically
important M1 family residues. As shown in Fig.
6A, genomic DNA from DH5
amplified a band of ~2.6 kbp, whereas the identical primers amplified
a band of ~3.8 kbp from DH5
PepN, consistent with the expected results. Next, we performed peptidase assays from extracts with these
two strains. Extracts from DH5
PepN were unable to cleave both
Suc-LLVY-AMC and L-Ala-pNA, although hydrolysis
of Cbz-LLE-
NA was not affected (Fig. 6B). These results
agree with that obtained with 9218 (Fig. 2). Finally, induced
expression of only PepN in DH5
PepN rescued both Suc-LLVY-AMC and
L-Ala-pNA cleaving activities in a
dose-dependent manner (Fig. 6C). Together, these
experiments directly demonstrate that PepN is the major enzyme involved
in cleaving Suc-LLVY-AMC in E. coli.
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Fig. 6.
The loss of Suc-LLVY-AMC and
L-Ala-pNA hydrolysis in the E. coli DH5 PepN
strain is rescued by expression of PepN. A,
genomic DNA from DH5
(lane 2) or DH5
PepN
(lane 3) was used to amplify pepN by PCR.
Lanes 1 and M represent no template control and
1-kb ladder (Fermentas), respectively. B, cellular extracts
were prepared from DH5
or DH5
PepN, and enzyme assays were
performed. C, DH5
PepN transformed with either pBAD24
(diamonds) or pBAD24/EcPepN (squares) was induced
with different concentrations of L-arabinose. Cellular
extracts were tested for their ability to hydrolyze Suc-LLVY-AMC
(top), L-Ala-pNA (middle),
or Cbz-LLE-
NA (bottom).
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Fig. 7.
The LMW Suc-LLVY-AMC-hydrolyzing peptidase in
M. smegmatis is a PepN
homologue. A, cytosolic extracts of M. smegmatis were fractionated by SDG and assayed for hydrolysis of
Suc-LLVY-AMC. B, fraction number 4 was incubated with
different protease inhibitors, and their effect on Suc-LLVY-AMC
hydrolysis was studied. Mean values with standard deviations of eight
independent experiments are shown. C, the LMW
Suc-LLVY-AMC-hydrolyzing peptidase from M. smegmatis
extracts was purified, separated by SDS-PAGE, and stained by Coomassie
Brilliant Blue. D, the protein band after purification was
cut and in-gel-treated with trypsin, and the peptides generated
(underlined) were identified by mass spectrometry to belong
to a PepN homologue.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain with a targeted deletion
in PepN. This strain also lacked the ability to cleave
L-Ala-pNA and Suc-LLVY-AMC (Fig. 6),
corroborating our findings with 9218. Furthermore, expression of a
PCR-amplified wild type E. coli PepN in this strain (Fig. 6)
harboring the targeted deletion in pepN rescued both these
activities (i.e. cleaving L-Ala-pNA
and Suc-LLVY-AMC). This experiment, using a defined genetic mutant in
pepN and rescue of both activities by inducing expression of
a single cloned gene, PepN, unequivocally proves that PepN, a single
polypeptide, encodes both aminopeptidase and endopeptidase activities.
Finally, to demonstrate the presence of low molecular weight
Suc-LLVY-AMC-cleaving peptidases in another eubacterial organism, we
purified an enzyme displaying this activity from M. smegmatis, a Gram-positive actinomycetale harboring 20 S
proteasomes. We identified the LMW Suc-LLVY-AMC-cleaving enzyme to be
an aminopeptidase homologous to E. coli PepN, by mass
spectrometry analysis. Also, this enzyme cleaved
L-Ala-pNA and other aminopeptidase substrates
(data not shown), suggesting that the LMW Suc-LLVY-AMC-cleaving enzyme
is an aminoendopeptidase in M. smegmatis. Thus, E. coli PepN and its homologue in M. smegmatis are
aminoendopeptidases, capable of cleaving Suc-LLVY-AMC. However, two
differences were observed. The SDG peak fraction of the LMW Suc-LLVY-AMC hydrolysis in M. smegmatis displayed greater
specific activity and was inhibited by EDTA and EGTA compared with
E. coli (Figs. 1 and 7), suggesting biochemical differences
between these two enzymes.
-N-benzoylarginine-
-naphthylamide hydrolase (54), hydrolase H (55, 56), PepN (43), bleomycin hydrolase (57), cathepsin H
(58), Multicorn (11), and TPPII (14). Interestingly, the latter five
are involved in protein degradation, suggesting an important role of
aminoendopeptidases in this process. However, the critical residues
involved in aminopeptidase versus endopeptidase activities
in these enzymes have not been identified. Bestatin, a transition state
analogue and aminopeptidase inhibitor, inhibited primarily the
aminopeptidase activity of PepN (Fig. 4). This differential inhibition
profile suggests the possibility of subdomains/motifs within a single
active site or the involvement of two distinct active sites for the
aminopeptidase and endopeptidase activities. In addition, it is also
possible that the high affinity for endopeptidase substrates (Table
III) results in absence of complete inhibition of hydrolysis of these
substrates by the competitive aminopeptidase inhibitor, bestatin.
Further studies are required to identify the critical amino acids
involved in aminopeptidase and endopeptidase activities of PepN and its
homologues.
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Fig. 8.
Homologues of PepN are widely conserved and
play a role in intracellular protein degradation. A,
multiple sequence alignment of the region harboring the catalytically
important residues from well characterized PepN homologues was
performed using ClustalW. Asterisks represent identical
residues, whereas colons and single dots
represent conserved and semi-conserved substitutions. Boxes
highlight the exopeptidase motif (broken line) and the M1
family metallopeptidase motif (solid line). The conserved
Zn2+-binding residues are boldface
italic, and the glutamate residues important in catalysis
are shown in boldface. Each organism with its PepN homologue
is indicated in front of the sequence, and the
GenBankTM accession numbers of sequences are as
follows: EcPepN, E. coli PepN (M15273); MsPepN, M. smegmatis PepN (not annotated); StPepN, S. thermophilus
PepN (AJ007700); LlPepN, L. lactis PepN (X61230); AnApsA,
A. niger ApsA (AJ292570); TaF2, T. acidophilum
Tricorn interacting factor F2 (NP_393781.1); TaF3, T. acidophilum Tricorn interacting factor F3 (NP_394276.1); ScAap1,
Saccharomyces cerevisiae Aap1 (L12542); MmPSA, Mus
musculus puromycin-sensitive aminopeptidase (BC009653).
B, table of enzymes involved in various steps in
intracellular protein degradation in eubacteria, Archaea, and
Eukarya.
The key proteases and peptidases important in cytosolic protein degradation from eubacteria, Archaea, and eukaryotes are shown in Fig. 8B. In eukaryotes, 26 S proteasomes degrade proteins via the ubiquitin- and ATP-dependent pathway (1, 3). Unfolded proteins and large peptides are further processed by 20 S proteasomes followed by TPPII/Multicorn (11, 13-15) and prolyl oligopeptidase (59). Finally, amino acids are recycled back by the exopeptidase actions of DPPIII (12), PSA (20, 21), leucine aminopeptidase (19), degradation by Thimet oligopeptidase (23), etc. In Archaea, the role of ATP-dependent proteases is unclear although Lon and Clp homologues are present (9). The role of processing of unfolded proteins and peptides by the combined action of 20 S proteasomes and the Tricorn-F1-F2-F3 complex is well demonstrated (9). Peptides are further processed by the F1/F2/F3 aminopeptidases themselves (9) or other exopeptidases, e.g. TET (16). The role of ATP-dependent proteases, e.g. Lon and Clp family proteases in eubacterial cytosolic protein degradation is well established (2). Ci and Fa (2) are well known ATP-independent endoproteases, and further processing probably requires PepN and other endopeptidases. Finally, small peptides may be degraded to amino acids by exopeptidases, Dcp, PepA, PepB, PepD, PepE, PepM, PepN, PepQ, etc. (2). Previously, PepN was thought to play a role as an aminopeptidase (2, 42, 43) or a dipeptidase (60) in protein degradation. Our data suggest that PepN plays a role in the latter three steps of protein degradation (Fig. 8B) by acting as an aminoendopeptidase. However, PepN is not essential in E. coli (61), probably due to the presence of redundant peptidases (62). In fact, mutations in all four peptidases (PepN, PepA, PepB, and PepD), but not single peptidase genes, in Salmonella typhimurium result in a significant decrease in cytosolic protein degradation (63). Thus, PepN and functionally similar enzymes play important roles in different steps of protein degradation in eubacteria.
PepN has two homologues, Tricorn interacting factors F2 and F3,
in T. acidophilum based on "sequence homology." These
two aminopeptidases, along with a proline iminopeptidase F1, interact with tricorn, an endopeptidase, and degrade peptides released from 20 S proteasome (9). Together the Tricorn-F1-F2-F3 complex, but
not individually, hydrolyzes Suc-LLVY-AMC or insulin B chain efficiently (9). On the other hand, the eukaryotic functional homologues of Tricorn, i.e. Multicorn (11) and TPPII
(13-15), display both endopeptidase and aminopeptidase activities.
Thus, there are differences in the downstream steps of protein
degradation in archaebacteria and eukaryotes. E. coli PepN
utilizing its endopeptidase and exopeptidase abilities hydrolyzes both
Suc-LLVY-AMC and insulin B chain, thus acting as a "functional
homologue" of the Tricorn-F1-F2-F3 complex. Moreover, PepN can also
hydrolyze intact proteins like casein and albumin. These observations
suggest that PepN may act as a functional eubacterial counterpart of
enzymes involved in the three downstream processing steps of cytosolic
protein degradation (Fig. 8B). Also, the downstream
processing steps during eubacterial protein degradation are probably
similar to eukaryotic protein degradation. Similar to multicorn
(11) and TPPII (14), the endopeptidase activity of PepN is much lower
than its aminopeptidase activity. However, unlike these two enzymes,
PepN is not a multimeric enzyme. In summary, we have demonstrated that
the major LMW Suc-LLVY-AMC-cleaving peptidase in E. coli and
M. smegmatis to be PepN or a PepN homologue, which displays
aminoendopeptidase activities similar to the Tricorn-F1-F2-F3 complex
in Archaea and TPPII/Multicorn in eukaryotes.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. S. Mahadevan and members of his
laboratory for E. coli K12RV and help with generating the
E. coli DH5PepN strain; Dr. M. Foglino for 9218 and
pBM15; Dr. J. Gowrishankar and Dr. P. Sadhale for pBAD24; Dr. Corey
for lactacystin; and Dr. Ploegh for Cbz-LLL-H, Cbz-LLL-vinylsulfone,
and Cbz-LHVS. We appreciate the assistance of the DBT-IISc
instrumentation facilities and encouragement for this project by Drs.
D. N. Rao, U. Varshney, J. Monaco, and P. Ajit Kumar.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the Department of Science and Technology, Government of India.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.
§ Supported by a fellowship from the Council of Scientific and Industrial Research.
¶ To whom correspondence should be addressed. Tel.: 91-80-3943051; Fax: 91-80-3600814; E-mail: nandi@biochem.iisc.ernet.in.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M207926200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TPPII, tripeptidylpeptidase II;
AMC, 7-amino-4-methylcoumarin;
NA,
-naphthylamide;
EMS, ethyl methanesulfonate;
FTC, fluorescein
thiocarbamoyl;
LMW, low molecular weight;
pNA, para-nitroanilide;
PSA, puromycin-sensitive aminopeptidase;
SDG, Sucrose density gradient;
Suc-LLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin;
Cbz, benzyloxycarbonyl;
MHC, major histocompatibility complex.
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