From the Center for Advanced Biotechnology and
Medicine, the § Graduate Program in Cell and Developmental
Biology, Rutgers University, and the
Department of Pharmacology,
Robert Wood Johnson Medical School, University of Medicine and
Dentistry of New Jersey, Piscataway, New Jersey 08854
Received for publication, September 19, 2000, and in revised form, October 23, 2000
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ABSTRACT |
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The CLN2 gene mutated in the fatal
hereditary neurodegenerative disease late infantile neuronal ceroid
lipofuscinosis encodes a lysosomal protease with tripeptidyl-peptidase
I activity. To understand the enzymological properties of the protein,
we purified and characterized C-terminal hexahistidine-tagged human
CLN2p/tripeptidyl-peptidase I produced from insect cells transfected
with a baculovirus vector. The N terminus of the secreted 66-kDa
protein corresponds to residue 20 of the primary CLN2 gene translation
product, indicating removal of a 19-residue signal peptide. The
purified protein is enzymatically inactive; however, upon
acidification, it is proteolytically processed and concomitantly
acquires enzymatic activity. The N terminus of the final 46-kDa
processed form (Leu196) corresponds to that of mature
CLN2p/tripeptidyl-peptidase I purified from human brain. The activity
of the mature enzyme is irreversibly inhibited by the serine esterase
inhibitor diisopropyl fluorophosphate, which specifically and
stoichiometrically reacts with CLN2p/tripeptidyl-peptidase I at
Ser475, demonstrating that this residue represents the
active site nucleophile. Expression of wild type and mutant proteins in
CHO cells indicate that Ser475, Asp360,
Asp517, but not His236 are essential for
activity. These data indicate that the CLN2 gene product is synthesized
as an inactive proenzyme that is autocatalytically converted to an
active serine protease.
Late infantile neuronal ceroid lipofuscinosis (LINCL, OMIM
204500)1 is a recessive fatal
neurological disease characterized by lysosomal accumulation of
proteinaceous autofluorescent storage material in neurons and other
cell types (reviewed in Ref. 1). The CLN2 gene product was first
identified as a normally abundant 46-kDa mannose 6-phosphorylated
glycoprotein that was absent in brain autopsy specimens from LINCL
patients, leading to the molecular characterization of the disease gene
(2). The encoded protein has significant sequence similarities to two
previously characterized bacterial endoproteases from
Xanthomonas and Pseudomonas (3, 4). These
prokaryotic enzymes have been named bacterial pepstatin-insensitive carboxyl peptidases (BPICPs) based on a series of studies that suggest
that, like classic aspartyl proteases, their catalytic mechanism
involves a pair of amino acids with carboxyl side chains that catalyze
peptide bond hydrolysis at acidic pH but, unlike the classic aspartyl
proteases, they are not inhibited by pepstatin. Based on these sequence
relationships, we demonstrated that the CLN2 protein was a lysosomal
protease that could degrade hemoglobin at acidic pH in the presence of
aspartyl and cysteine protease inhibitors (2, 5). More recently, the
CLN2 protein has been shown to have tripeptidyl exopeptidase activity.
Peptide sequencing of tripeptidyl-peptidase I (TPP-I) purified from rat
spleen demonstrated that it is the rodent ortholog of human CLN2p (6),
and TPPI-I activity is absent in LINCL specimens (7). Recently, Ezaki and colleagues (8) have shown that purified CLN2p has both TPP-I and
endopeptidase activity.
Mechanistic studies on purified CLN2p/TPP-I by a number of groups
indicate that enzymatic activity is not inhibited by standard serine,
cysteine, metallo, or aspartyl protease inhibitors (6, 8, 9). Based on
this, it has been proposed that the CLN2p/TPP-I is a
pepstatin-insensitive carboxyl peptidase. However, Rawlings and Barrett
(10) critically reviewed existing evidence for classification of the
BPICPs and concluded that the catalytic mechanism for these enzymes has
not been rigorously established. These investigators alertly pointed
out that a conserved Gly-Xaa-Ser sequence in CLN2p/TPP-I and the BPICPs
is characteristic of the active site motif of many serine peptidases.
Interestingly, data base searches using the CLN2p/TPP-I sequence with
different BLAST tools (CD-Search and PSI-BLAST) retrieve members of the
S8 subtilisin family of serine peptidases (data not shown).
A thorough understanding of the biochemical properties of the CLN2 gene
product may provide valuable clues toward understanding and developing
therapies for LINCL. In this study, we have characterized the enzymatic
properties of CLN2p/TPP-I. We find that the protein is synthesized as
an inactive zymogen that is autocatalytically converted to an active
serine protease at acidic pH.
Hexahistidine-tagged CLN2p/TPP-I--
Two different human CLN2
cDNAs encoding the His175 and Arg175
variants (GenBankTM accession number AF017456) were
subcloned into pBACgus-1 (Novagen). The constructs encode full-length
CLN2p/TPP-I (residues 1-563) with a C-terminal hexahistidine tag
(residues 564-569). Both constructs were expressed at Kemp
Biotechnologies (Fredrick, MD) using the Sf9 cell baculovirus
system. Although the Arg175 variant has only been observed
in one of several cDNAs sequenced to date, it was used for large
scale production purposes because it gave significantly higher yield.
Aliquots of conditioned medium (1 liter) were stored at Untagged CLN2p/TPP-I--
Mature CLN2p/TPP-I was purified from
frozen human brain autopsy specimens obtained from National Disease
Research Interchange (Philadelphia, PA). Brain mannose 6-phosphorylated
glycoproteins were isolated by chromatography on immobilized
cation-independent mannose 6-phosphate receptor as described previously
(11) except that the column was sequentially eluted with 5 mM mannose 6-phosphate and then with 100 mM
glycine, pH 2.5. The glycine eluate contained the bulk of the mannose
6-phosphorylated CLN2p/TPP-I and was applied to a Mono S HR 5/5 column
(Amersham Pharmacia Biotech). The column was eluted using a 0-1
M sodium chloride gradient in 0.1% Tween 20, 20 mM sodium citrate, pH 5. Fractions containing TPP-I
activity eluted at ~0.5 M sodium chloride and were
further purified by gel filtration on a 1.0 × 30-cm Superose 12 (Amersham Pharmacia Biotech) column eluted using 0.1% Tween 20, 50 mM sodium chloride, 20 mM sodium formate, pH
4.0. The purified 46-kDa protein appeared homogeneous by SDS-PAGE and
Coomassie Blue staining. Total yield of purified protein was typically
20 µg/100 g of human brain. Production of untagged human CLN2p/TPP-I
precursor in CHO cells will be described
elsewhere.2
Enzyme and Inhibitor Assays--
Standard conditions were as
follows unless otherwise noted. All incubation steps contained 150 mM sodium chloride and 0.1% Triton X-100 (saline/Triton)
buffered to the indicated pH as stated in the figure legends. The
proenzyme was activated by dilution into 50 mM formate, pH
3.5, buffered saline/Triton and incubation at 22-37 °C for at least
10 min. For incubations with inhibitors, the concentration of the
enzyme was typically ~50 nM, and the final concentration
of solvent used to prepare the inhibitor stock was <1%. TPP-I
activity was assayed using the kinetic assay described previously (12)
except that the temperature of the fluorescent plate reader was
adjusted so that the input samples were at thermal equilibrium to
eliminate the initial lag period. The final concentration of enzyme was
typically ~5 nM, and the Ala-Ala-Phe-aminomethylcoumarin (AAF-AMC) substrate concentration was 200 µM.
Protein Chemistry--
For peptide mapping, the activation and
DFP reaction conditions were as described above except that volatile
buffers (150 mM ammonium acetate, pH 4.0) were used to
facilitate downstream processing. Samples (typically 100 µg) were
evaporated to dryness in a vacuum centrifuge, resuspended in 100 µl
of 6 M guanidine hydrochloride, 2 mM EDTA, 375 mM Tris, pH 8.1, and incubated for 1 h at 50 °C.
Samples were reduced by adding 4 µl of 1 M dithiothreitol (incubation, 1.5 h at 50 °C) and alkylated by adding 16 µl of 0.5 M iodoacetamide (incubation in the dark for 30 min at
25 °C). The mixtures were desalted on a Superdex Peptide column
(Amersham Pharmacia Biotech) equilibrated with 20% acetonitrile, 0.1%
trifluoroacetic acid. The peak fractions were pooled, evaporated to
dryness in a vacuum centrifuge, resuspended in 100 µl of 150 mM Tris, pH 8.0, and incubated at 37 °C for 4 h
after adding 2.5 µg of trypsin (Worthington, TRSEQZ grade). The
reactions were stopped by addition of trifluoroacetic acid to a final
concentration of ~0.1%. Digests were analyzed on a PE Biosystems
Voyager Pro mass spectrometer using sinapinic acid matrix before and
after fractionation by HPLC on a C18 Vydac protein/peptide column
eluted using an acetonitrile gradient in 0.1% trifluoroacetic acid.
Peptides of interest were further analyzed by Edman degradation using
an Applied Biosystems 477A sequencer.
CLN2p/TPP-I Mutants--
Polymerase chain reaction-based
mutagenesis was used to introduce the S475A (TCG to GCG), D360A (GAC to
GCC), D517A (GAT to GCT), and H236A (CAT to GCT) mutations into the
human CLN2p/TPP-I cDNA sequence. Briefly, for each construct, two
complementary oligonucleotide primers that contained the appropriate
nucleotide changes were used separately with outside primers flanking
either the 5' or 3' coding sequence to generate two polymerase chain reaction products. These were then gel purified, mixed, and used with
the outside primers in a second round of polymerase chain reaction. The
resulting product containing the full-length CLN2 coding sequence was
subcloned into the expression vector pSFFV-neo (13), and the entire
insert was sequenced to confirm the presence of the desired mutations
and absence of unwanted changes. DNA was introduced into CHO cells
using LipofectAMINE (Life Technologies, Inc.), and resistant clones
were selected using G418. For activity assays and Western blotting,
cells were seeded into 6-well plates and cultured in Dulbecco's
modified Eagle's medium/Ham's F-12 medium, 10% fetal bovine serum
until monolayers reached confluence. Cells were washed and cultured in
2 ml of serum-free medium for 24 h, washed with phosphate-buffered
saline, and lysed with a solution (0.5 ml) of 1% Nonidet P-40, 150 mM sodium chloride, 10 mM Tris, pH 7.5. Samples
were clarified by centrifugation at 12,000 × g for 5 min (medium) or 20 min (lysates) and analyzed for TPP-I activity using
the end point assay (12) after preactivation at pH 3.5 for 30 min at
37 °C. Clarified lysates contained between 1.2 and 1.5 mg/ml protein
as determined by the Lowry assay (14). Samples (lysates, 8 µg/lane;
medium, 15.6 µl/lane) were fractionated by SDS-PAGE on a 10% NuPAGE
Bis-Tris gel (Novex). CLN2p/TPP-I was visualized by Western blotting
using a rabbit antiserum prepared against hexahistidine-tagged
CLN2p/TPP-I (antiserum R72-5, contract production by Cocalico
Biologicals) and enhanced chemiluminescence (Renaissance, PerkinElmer
Life Sciences).
Autocatalytic Processing of CLN2p/TPP-I--
We purified a
C-terminal hexahistidine-tagged version of the CLN2p/TPP-I secreted by
an insect cell production system as described under "Experimental
Procedures." When maintained at nearly neutral pH, the protein had an
apparent size of 66 kDa by SDS-PAGE (Fig. 1). Edman degradation revealed that the N
terminus of the 66-kDa species (SYSPE ... ) corresponds to residue
20 of the predicted CLN2p precursor. Differences in the predicted and
actual molecular mass of the His-tagged CLN2p/TPP-I (residue 20-569;
60,118 Da) are probably due to N-linked glycosylation (2,
9). In acidic conditions (pH <4.5), the protein is converted to lower
molecular mass forms (Fig. 1). Note that in addition to the
major species of ~46 kDa, faster migrating bands also appear (~20
kDa), suggesting that the processing occurs through an endoproteolytic
mechanism.
More detailed analysis of the conversion at pH 4.0 indicates that the
66-kDa species is rapidly (t1/2 = 7 min) converted
to a 46-kDa species that is stable upon prolonged (24 h) incubation
(Fig. 2). Note the transient appearance
of lower molecular mass species near the bottom of the gel that appear to decrease in size and eventually disappear at long time points (Fig.
2, lane 14), consistent with endoproteolytic liberation and
eventual degradation of the propeptide. Also, at early time points
there appears to be a ~49-kDa species that transiently appears (Fig.
2, lanes 2 and 3), suggesting that the initial
endoproteolytic cleavage may be upstream of the terminally processed
46-kDa mature form. Edman degradation revealed that the N terminus of
the 46-kDa species (LHLGV) corresponds to residue 196 of the predicted
CLN2p precursor, identical to that of human brain CLN2p (2). Enzyme activity measurements indicate that the 66-kDa form has very low TPP-I
activity and that the time course for acquisition of activity (t1/2 = 6 min) is nearly identical to that of
proteolytic processing (Fig. 2). Similar findings were obtained using
untagged proenzyme purified from CHO cells (data not shown). Taken
together, these data indicate that the CLN2 protein is synthesized as
an inactive proenzyme that upon acidification undergoes autocatalytic
conversion to an enzymatically active species.
Inhibition of CLN2p/TPP-I Activity--
To investigate the
catalytic properties of the CLN2 protein, we incubated the processed
protein at pH 4.25 in the presence of different group-specific protease
inhibitors and then measured TPP-I activity. Essentially identical
results were obtained using purified His-tagged CLN2p/TPP-I produced in
insect cells, untagged CLN2p/TPP-I purified from a CHO cell expression
system, mature CLN2p/TPP-I purified from human brain, and crude human
brain homogenates (Table I). Consistent
with previous studies, there was essentially no effect on activity by
the metalloproteinase inhibitor EDTA, by the cysteine protease
inhibitor E-64, by the aspartyl protease inhibitor pepstatin, or by the
serine protease inhibitor phenylmethylsulfonyl fluoride. A relatively
minor effect was seen with the metalloproteinase inhibitor
1,10-phenanthroline. Also, confirming the results of others (6,
15-17), a tripeptide substrate-based chloromethylketone (AAF-CMK)
inhibited activity (Table I) in a competitive manner (data not shown).
Unexpectedly, TPP-I activity was inhibited by the serine protease
inhibitor DFP (Table I). Similar results were obtained when activity
was assayed using fluorescein isothiocyanate-hemoglobin substrate (data
not shown). In addition, the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) partially inhibited activity (Table I).
Note that at the acidic pH used for these experiments, DCI formed an
insoluble precipitate at millimolar or higher concentrations, thus
complicating analysis of the concentration dependence of this
inhibitor.
We conducted a series of dilution experiments to ascertain the
stability of the enzyme-inhibitor complex. Activated enzyme was
preincubated with inhibitor for 1 h, and then the initial TPP-I
activity was measured immediately after diluting the preparation into a
substrate solution adjusted to contain the same or a 10-fold lower
final concentration of inhibitor (Table
II). This was compared with the activity
of a parallel sample where low concentration of inhibitor was present
in both the preincubation and assay mixture. These experiments revealed
that the inhibition by AAF-CMK was readily reversible, indicating that
the enzyme-inhibitor complex was in rapid equilibrium (Table II,
compare treatments 2 and 4). The inhibition of activity by high
concentrations of DCI was partially reversed following rapid dilution
(Table II, compare treatments 5 and 7), and the reversal was nearly
complete after 3 h (data not shown). In contrast, the inhibition
by DFP was essentially irreversible (Table II, compare treatments 8 and
10), even when measured 24 h after dilution (data not shown). The
DFP inhibition could be prevented by preincubating the enzyme with the
competitive inhibitor AAF-CMK (Table II, compare treatment 9 with
treatments 11 and 4) or by the AAF-AMC substrate (data not shown).
Taken together, these data indicate that a serine in the active site of
CLN2p/TPP-I plays a key role in catalysis.
We investigated different parameters that affected inactivation of
CLN2p/TPP-I by DFP. Inactivation was dependent on time and DFP
concentration (Fig. 3, top
panel). Plots of the log of the residual activity
versus time yielded a series of straight lines for different
DFP concentrations, indicating that the inactivation reaction followed
pseudo-first order kinetics (Fig. 3, middle panel). A double
reciprocal plot of the inverse apparent first order rate constant
versus inverse DFP concentration yielded a straight line
passing near the origin, suggesting that DFP does not form a Michaelis
complex with CLN2p/TPP-I (Fig. 3, bottom panel), and the
second order rate constant (kapp/[DFP]) was
~30 M
The rate of TPP-I/CLN2p inactivation by DFP was most rapid at pH ~4.5
(Fig. 4, top panel). This pH
dependence was similar to that of enzymatic activity assayed using the
AAF-AMC TPP-I substrate (Fig. 4, bottom panel). Experiments
using nonactivated precursor and preactivated enzyme indicated that the
proenzyme reacted very slowly if at all with DFP (data not shown).
Given that the DFP reaction occurs by nucleophilic attack of an
activated serine (18), these data indicate that both proteolytic
processing and acidic pH are required for activation and/or
accessibility of the catalytic serine to external substrates.
Identification of the Active Site Serine--
To determine the
site and stoichiometry of labeling, we compared tryptic digests of
unmodified and DFP-inactivated CLN2p/TPP-I. MALDI-TOF mass
spectrometry revealed that the patterns were essentially identical
except that a 2835.1 m/z fragment disappeared and
a new 3000.6 m/z peak appeared after DFP
treatment (Fig. 5, upper panel). The masses of these peaks indicate that they represent the
same tryptic fragment Val466-Lys492 with or
without the diisopropyl phosphate modification (theoretical m/z, 3000.4 and 2836.2, respectively). Therefore,
modification of CLN2p/TPP-I on a single residue within
Val466-Lys492 is responsible for
inhibition.
HPLC analysis of tryptic digests also demonstrates that one residue
within fragment Val466-Lys492 is
stoichiometrically labeled by DFP. For instance, in Fig. 5 (lower
panel), the two peaks labeled A and A'
present in a tryptic digest of unmodified CLN2p/TPP-I disappear after
DFP inactivation with the concomitant appearance of the two peaks B and
B'. Chemical sequencing and MALDI-TOF analysis reveal that peak A
represents tryptic fragment Val466-Lys492, and
A' represents an incompletely digested tryptic fragment encompassing
Ala448-Lys492, whereas B and B' represent the
corresponding diisopropyl phosphate-labeled fragments. The lack of any
other discernable differences in the modified and unmodified digests
also supports our conclusion that DFP-labeling of a single residue
within Val466-Lys492 is responsible for inhibition.
The Val466-Lys492 CLN2p/TPP-I peptide and the
corresponding regions of the BPICPs align to the active site serine of
subtilisin (Fig. 6). Edman degradation of
the purified DFP-modified tryptic peptide was complicated by poor
retention of the peptide on the polyvinylidene difluoride membrane
during sequencing (data not shown). However, in analysis of three
different preparations, for cycles 1-16 we could clearly assign all
residues to that predicted from the cDNA sequence except for cycle
10, which was predicted to be Ser475. Inspection of the
repetitive yield of serine showed that serine clearly was present at
cycles 7, 12, and 21 but absent at cycle 10. In contrast, serine was
clearly present at cycle 10 in sequencing of the corresponding
unmodified tryptic peptide. Finally, Ser475 represents the
only conserved serine among CLN2p/TPP-I and the BPICPs in the tryptic
peptide. Taken together, these data indicate that Ser475
represents the active site nucleophile of CLN2p/TPP-I and, by extension, the BPICPs.
Expression of Wild Type and Mutant CLN2p/TPP-I--
To directly
investigate the function of Ser475 we expressed wild type
CLN2p/TPP-I and a S475A mutant in CHO cells. The cells expressing wild
type human CLN2p/TPP-I had elevated TPP-I activity and Western blotting
revealed elevated levels of both precursor and processed protein (Fig.
7). The cells expressing the S475A mutant
had activity similar to the neo-transfected control, indicating that
the mutant was inactive compared with the wild type CLN2p/TPP-I protein. Western blotting indicated that the majority of the protein was in the precursor form, suggesting that processing was impaired (Fig. 7). However, the increased amount of ~46-kDa protein compare with the neo-transfected control indicates that other proteases, possibly including endogenous CHO cell CLN2p/TPP-I, are capable of
cleaving the catalytically inactive mutant. We also analyzed two
constructs that had alanine substitutions at Asp360 and
Asp517, which align to residues important for function of
the bacterial pepstatin-insensitive proteases (19, 20). The two
aspartate mutants were similar to the S475A mutation in terms of lack
of enzyme activity and processing (Fig. 7). Finally, in a preliminary attempt to find other catalytically important residues, we also analyzed a H236A mutant and found that it resembled the wild type construct in regard to processing and activity (Fig. 7).
In this study, we find that CLN2p/TPP-I is synthesized as a
catalytically inactive protein that upon acidification is
autocatalytically processed to an active protease. More detailed
kinetic studies indicate that activation can occur both by
intermolecular and intramolecular events, with the latter route
predominating at low proenzyme
concentrations.3 Based on the
sequence of the precursor and mature protein, residues 20-195
(Mr = 19,523) are removed following
acidification. Processing is likely to entail an endoproteolytic
cleavage of the CLN2p/TPP-I as shown by the appearance of small (<20
kDa) fragments that transiently appear after acidification. Consistent
with this, CLN2p/TPP-I has recently been shown to have intrinsic
endoproteolytic as well as tripeptidyl exopeptidase activity (8). The
appearance of a transient 49-kDa species suggests that the initial
endoproteolytic cleavage may occur upstream of the Leu196 N
terminus of the mature form. However, identification of processing intermediates is hindered by the appearance of TPP-I activity that can
further trim various species including the remaining proenzyme,
liberated propeptide, and the processing intermediates themselves.
Although the precise details remain to be elucidated, our in
vitro studies indicate that the CLN2p/TPP-I can undergo autocatalytic processing at conditions that mimic the acidic
environment of the lysosome. This property should be useful when
considering possible enzyme replacement therapy for LINCL patients,
where the recombinant proenzyme could be administered as an inactive prodrug that is converted to active enzyme after proper delivery to its
site of action.
Proteases are typically classified by catalytic type based on their
susceptibility to different types of inhibitors, with DCI and DFP being
diagnostic for serine proteases (21). We find that DCI inhibits
CLN2p/TPP-I in a reversible manner. Although DCI is generally thought
to be an irreversible inhibitor, some bona fide serine
proteases do regain activity following DCI inactivation (22). Our clear
finding that DFP inactivates CLN2p/TPP-I is somewhat unexpected given
the reported insensitivity of TPP-I (6, 8, 17) and a bacterial
pepstatin-insensitive protease (23) to this inhibitor. However, other
studies have reported partial to complete inhibition of TPP-I activity
with high concentrations of DFP (15, 16). These discrepancies may
reflect the kinetics and pH dependence of the DFP reaction. Even at its
optimal pH, DFP inhibition of CLN2p/TPP-I is slow compared with that of
other serine esterases (24), so it is possible that the experimental conditions were not optimized to detect the reaction.
Serine proteases typically contain a catalytic triad of aspartate,
histidine, and serine residues, with the serine hydroxyl group being
activated by the histidine, and the aspartate stabilizing the histidine
imidazole ring as it gains and loses a proton (25). Although most
serine proteases are inactive at acidic pH, this is not true for
carboxypeptidase II and cathepsin A. In both of these enzymes, the
environment of the active site includes a cluster of acid groups that
may lower the histidine pKa (26, 27) and thus allow
it to participate in both general acid and base catalysis at acidic pH
(25). There are also examples of serine proteases that lack the classic
catalytic triad such as members of the SF and SG clan, which have a
catalytic dyad consisting of a lysine and serine (28).
The results presented in this study, as well as our previous detection
of a S475L mutation in a LINCL patient (29), demonstrate that
Ser475 represents the active site nucleophile of
CLN2p/TPP-I. Previous studies reported that diethlypyrocarbonate
inhibited TPP-I activity (6, 16), suggesting that a histidine may be
involved in catalysis. Although His236 is excluded from the
catalytic triad based on our mutagenesis data, it is possible that some
other histidine activates the serine. If so, the alignments among
CLN2p/TPP-I, the BPICPs, and other apparently related proteins (29) do
not reveal an obvious candidate histidine.
The lack of activity of the Asp360 and Asp517
mutants are consistent with a role for these residues in catalysis. One
possibility is that one aspartate participates in a classical catalytic
triad and the other helps decrease the pKa of the
histidine. Alternatively, if a histidine is not important for
catalysis, one or both of the aspartates may assume a novel role in
directly activating the catalytic serine. Protein engineering combined with structural approaches will be required for detailed insights into
the catalytic process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C
until use. All purification steps were conducted at 4 °C, and TPP-I
activity was monitored after preincubating samples at pH 3.5 to
activate the proenzyme (see below). Thawed medium was adjusted to
contain 2 M NaCl/20 mM Tris, pH 8.0, centrifuged at 16,000 × g for 1 h, and the
supernatant was applied to a 53-cm3 butyl-Sepharose 4 Fast
Flow column (Amersham Pharmacia Biotech). The column was eluted with 20 mM Tris, pH 8.0, and the CLN2p/TPP-I precursor peak was
applied to a 10-cm3 TALON immobilized cobalt column
(CLONTECH Laboratories). The column was washed with
50 mM sodium chloride, 20 mM Tris, pH 8.0, and
eluted with the same buffer containing 100 mM imidazole.
The hexahistidine-tagged CLN2p/TPP-I precursor was further purified by
anion exchange chromatography on either a Mono Q HR 5/5 (Amersham Pharmacia Biotech) or an UNO Q-12 (Bio-Rad) column using a 50-500 mM sodium chloride gradient in 20 mM Tris, pH
8.0. The purified protein eluted at ~170 mM sodium
chloride and appeared homogeneous by SDS-PAGE and Coomassie Blue
staining. Protein (1-2 mg/ml) was divided into aliquots and stored at
80 °C until use. The total yield of purified protein was typically
6 mg/liter of culture medium.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The CLN2p/TPP-I precursor is
autocatalytically processed. CLN2p/TPP-I was incubated at the
indicated pH for 10 min at 30 °C. Reactions were initiated by
diluting hexahistidine-tagged precursor protein (1.65 mg/ml protein in
sodium chloride, 20 mM Tris, pH 8, ion exchange buffer)
into 7 volumes of saline/Triton buffer (pH 2.5, 100 mM
sodium citrate; pH 3.0-5.5, 100 mM sodium acetate) and
terminated by addition of reducing SDS-PAGE sample buffer. Samples (1 µg) were fractionated by SDS-PAGE using a 12% Tris-glycine gel
(Novex) and visualized by sequential staining with Coomassie blue and
silver (Novex). The lane labeled pH 8.0 represents starting
material that was diluted directly into SDS-PAGE sample buffer.
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Fig. 2.
Processing of CLN2p/TPP-I precursor is
associated with acquisition of enzyme activity. Purified
hexahistidine-tagged CLN2p/TPP-I precursor was diluted to a
concentration of 0.1 mg/ml in saline/Triton 100 mM sodium
acetate, pH 4.0, buffer and incubated at 23 °C. Aliquots were
removed and either analyzed by SDS-PAGE as described in Fig. 1 or
diluted 20-fold into saline/Triton 100 mM sodium acetate,
pH 4.0, buffer containing 421 µM AAF-AMC and immediately
assayed for TPP-I activity. In the upper panel, lane
1 represents proenzyme diluted directly into SDS-PAGE buffer,
whereas lanes 2-14 represent reactions terminated after
0.5, 5, 10, 20, 30, 40, 50, 60, 90, 120, 15, 25, or 1440 min. The
fraction mature was estimated by scanning the gels and calculating the
background corrected intensities of the broad bands migrating in
regions labeled proform and mature. Data were fit with a one-phase
exponential association equation using Prism 3.0 (GraphPad).
Relative TPP-I activity of different preparations in the presence
of inhibitors
Reversibility of inhibitors
1 min
1 at 25 °C and
pH 4.25 (Fig. 3, bottom panel, inset). Note that this analysis ignores hydrolysis of DFP during the reaction period and
is likely to slightly underestimate the true second order rate constant
(see Fig. 3 legend).
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Fig. 3.
Time and concentration dependence of DFP
inhibition. Top panel, concentration dependence.
Samples were incubated with the indicated concentration of DFP for 10 min (filled squares), 40 min (filled triangles),
2 h (filled diamonds), 6 h (open
squares), or 23 h (open triangles). For clarity of
presentation, the 80- and 250-min time points are not depicted. The
initial rate of TPP-I activity in the presence of inhibitor was
normalized to that of samples that did not contain inhibitor
(v/v0). Curves were fit for the
second order rate constant k2 using the equation
v/v0 = exp( k2t[DFP]). The depicted
theoretical curves were obtained from fits performed independently for
each concentration series at a fixed time point and yielded
k2 values of 81, 54, 39, 39, 31, 27, and 22 M
1 min
1 for the 10-, 20-, 40-, 80-, 120-, 250-, 360-, and 1380-min time points, respectively. The
variation in the calculated rate constants may in part be due to
hydrolysis of DFP during the incubation period. Middle
panel, time dependence. Log transformed relative TPP-I activity
from samples incubated with a fixed concentrations of DFP (1 mM (filled circles), 0.316 mM
(open circles), 0.01 mM (filled
squares), and 0.0316 mM (open squares))
were fit by linear regression with the negative slope of the lines
yielding the apparent first order rate constant. Data values close to 0 have been omitted for clarity. Bottom panel, double
reciprocal plots of the apparent first order rate constant (see above)
versus inhibitor concentration. Inset, plot of
the calculated second order rate constants versus inhibitor
concentration. After preactivation, hexahistidine-tagged CLN2p/TPP-I
was incubated in saline/ Triton 100 mM sodium citrate, pH 4.25, containing the
indicated concentration of DFP. At the indicated times aliquots were
removed and diluted into the same buffer/inhibitor solution containing
AAF-AMC substrate and immediately assayed for TPP-I activity.
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Fig. 4.
pH dependence of DFP inactivation and TPP-I
activity. Top panel, DFP inactivation. Preactivated
CLN2p/TPP-I was incubated at 25 °C in the absence and presence of
0.5 mM DFP in 100 mM formate-buffered
saline/Triton of the indicated pH. At various times (0.17, 1, 2, 3, 4, and 5 h) aliquots were diluted into 250 mM
formate-buffered Triton/saline, pH 4.0, containing AAF-AMC and assayed
for TPP-I activity at 30 °C. Second order rate constants
(kapp/[DFP]) were obtained as described in the
legend to Fig. 3. Bottom panel, TPP-I activity. Preactivated
CLN2p/TPP-I was diluted into 100 mM formate-buffered
saline/Triton of the indicated pH containing AAF-AMC and assayed for
TPP-I activity at 30 °C.
View larger version (27K):
[in a new window]
Fig. 5.
DFP modifies a single site on
CLN2p/TPP-I. Control and DFP-inactivated CLN2p/TPP-I preparations
were digested with trypsin and analyzed by MALDI-TOF mass spectrometry
(top panel) or reverse phase HPLC (bottom panel)
as described under "Experimental Procedures." Reactions were
sampled periodically and assayed to determine residual activity, with
fresh DFP being added and/or the incubation period extended until the
desired degree of inhibition was obtained. Typically, complete
inhibition required 2 mM DFP for 4 h. This lower
reactivity than predicted in the kinetic experiments described in Figs.
3 and 4 may reflect differences in experimental conditions
(e.g. presence of ammonium versus sodium) that
may result in increased DFP hydrolysis.
View larger version (18K):
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Fig. 6.
Primary sequence of
Val466-Lys492 CLN2p/TPP-I tryptic peptide
aligned with the corresponding regions of
Xanthomonas BPICP (accession number Q60106),
Pseudomonas BPICP (accession number P42790), and
subtilisin (accession number P00780). Residues identical in three
or more of the four sequences have a black background,
whereas those that are conserved have a gray
background.
View larger version (70K):
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Fig. 7.
Analysis of CLN2p/TPP-I mutants. Cells
and media from clones transfected with pSV2neo control (lanes
1 and 7), wild type human CLN2p/TPP-I (lanes
2 and 8), S475A mutant (lanes 3 and
9), D360A mutant (lanes 4 and 10),
D517A mutant (lanes 5 and 11), and H236A mutant
(lanes 6 and 12) were analyzed directly for
CLN2p/TPP-I expression by Western blotting. TPP-I activity was measured
after incubating the samples at pH 3.5 to convert proenzyme to the
active form as described under "Experimental Procedures." Data are
representative of at least two independent clones for each
construct.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Joanne Widom and Jon Clardy (Cornell University, Ithaca, NY) and Chris Kemp (Kemp Biotechnologies, Fredrick, MD) for help in producing recombinant histidine-tagged CLN2p/TPP-I. We also thank Steven Anderson for helpful discussions and David Sleat for valuable advice and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants NS37918, DK45992, and RR13824.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.
¶ These authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 732-235-5032; Fax: 732-235-5289; E-mail: lobel@cabm.rutgers.edu.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M008562200
2 L. Lin and P. Lobel, manuscript in preparation.
3 I. Sohar and P. Lobel, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: LINCL, late infantile neuronal ceroid lipofuscinosis; BPICP, bacterial pepstatin-insensitive carboxyl peptidase; TPP-I, tripeptidyl-peptidase I; AAF-AMC, Ala-Ala-Phe-aminomethylcoumarin; AAF-CMK, Ala-Ala-Phe-chloromethylketone; DFP, diisopropyl fluorophosphate; DCI, 3,4-dichloroisocoumarin; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Goebel, H. H., Mole, S. E., and Lake, B. D. (eds) (1999) The Neuronal Ceroid Lipofuscinoses (Batten Disease): Biomedical and Health Research , Vol. 33 , IOS Press, Amsterdam |
2. |
Sleat, D. E.,
Donnelly, R. J.,
Lackland, H.,
Liu, C. G.,
Sohar, I.,
Pullarkat, R. K.,
and Lobel, P.
(1997)
Science
277,
1802-1805 |
3. |
Oda, K.,
Takahashi, T.,
Tokuda, Y.,
Shibano, Y.,
and Takahashi, S.
(1994)
J. Biol. Chem.
269,
26518-26524 |
4. | Oda, K., Ito, M., Uchida, K., Shibano, Y., Fukuhara, K., and Takahashi, S. (1996) J. Biochem. (Tokyo) 120, 564-572[Abstract] |
5. | Sohar, I., Sleat, D. E., Jadot, M., and Lobel, P. (1999) J. Neurochem. 73, 700-711[CrossRef][Medline] [Order article via Infotrieve] |
6. | Vines, D., and Warburton, M. J. (1998) Biochim. Biophys. Acta 1384, 233-242[Medline] [Order article via Infotrieve] |
7. | Vines, D. J., and Warburton, M. J. (1999) FEBS Lett. 443, 131-135[CrossRef][Medline] [Order article via Infotrieve] |
8. | Ezaki, J., Takeda-Ezaki, M., Oda, K., and Kominami, E. (2000) Biochem. Biophys. Res. Commun. 268, 904-908[CrossRef][Medline] [Order article via Infotrieve] |
9. | Junaid, M. A., Wu, G., and Pullarkat, R. K. (2000) J. Neurochem. 74, 287-294[CrossRef][Medline] [Order article via Infotrieve] |
10. | Rawlings, N. D., and Barrett, A. J. (1999) Biochim. Biophys. Acta 1429, 496-500[Medline] [Order article via Infotrieve] |
11. |
Sleat, D. E.,
Sohar, I.,
Lackland, H.,
Majercak, J.,
and Lobel, P.
(1996)
J. Biol. Chem.
271,
19191-19198 |
12. |
Sohar, I.,
Lin, L.,
and Lobel, P.
(2000)
Clin. Chem.
46,
1005-1008 |
13. |
Fuhlbrigge, R. C.,
Sheehan, K. C.,
Schreiber, R. D.,
Chaplin, D. D.,
and Unanue, E. R.
(1988)
J. Immunol.
141,
2643-2650 |
14. | Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 |
15. | McDonald, J. K., Hoisington, A. R., and Eisenhauer, D. A. (1985) Biochem. Biophys. Res. Commun. 126, 63-71[Medline] [Order article via Infotrieve] |
16. | Page, A. E., Fuller, K., Chambers, T. J., and Warburton, M. J. (1993) Arch. Biochem. Biophys. 306, 354-359[CrossRef][Medline] [Order article via Infotrieve] |
17. | Watanabe, Y., Kumagai, Y., and Fujimoto, Y. (1992) Biochem. Int. 27, 869-877[Medline] [Order article via Infotrieve] |
18. | Powers, J. C., and Harper, J. W. (1986) in Proteinase Inhibitors (Barrett, A. J. , and Salvesen, G., eds), Vol. 12 , pp. 55-152, Amsterdam, Elsevier Science Publishing Co., Inc., New York |
19. | Ito, M., Narutaki, S., Uchida, K., and Oda, K. (1999) J. Biochem. (Tokyo) 125, 210-216[Abstract] |
20. |
Oyama, H.,
Abe, S.,
Ushiyama, S.,
Takahashi, S.,
and Oda, K.
(1999)
J. Biol. Chem.
274,
27815-27822 |
21. | Barrett, A. J. (1994) Methods Enzymol. 244, 1-15[CrossRef][Medline] [Order article via Infotrieve] |
22. | Harper, J. W., Hemmi, K., and Powers, J. C. (1985) Biochemistry 24, 1831-1841[Medline] [Order article via Infotrieve] |
23. | Oda, K., Sugitani, M., Fukuhara, K., and Murao, S. (1987) Biochim. Biophys. Acta 923, 463-469[Medline] [Order article via Infotrieve] |
24. | Cohen, J. A., Oosterbaan, R. A., and Berends, F. (1967) Methods Enzymol. 11, 686-702 |
25. | Fersht, A. (1999) Structure and Mechanism in Protein Science , W. H. Freeman, New York |
26. | Liao, D. I., Breddam, K., Sweet, R. M., Bullock, T., and Remington, S. J. (1992) Biochemistry 31, 9796-9812[Medline] [Order article via Infotrieve] |
27. | Rudenko, G., Bonten, E., d'Azzo, A., and Hol, W. G. (1995) Structure 3, 1249-1259[Abstract] |
28. | Rawlings, N. D., and Barrett, A. J. (1994) Methods Enzymol. 244, 19-61[Medline] [Order article via Infotrieve] |
29. | Sleat, D. E., Gin, R. M., Sohar, I., Wisniewski, K., Sklower-Brooks, S., Pullarkat, R. K., Palmer, D. N., Lerner, T. J., Boustany, R. M., Uldall, P., Siakotos, A. N., Donnelly, R. J., and Lobel, P. (1999) Am. J. Hum. Genet. 64, 1511-1523[CrossRef][Medline] [Order article via Infotrieve] |