From the New York State Institute for Basic Research in Developmental Disabilities, Department of Developmental Neurobiology, Staten Island, New York 10314
Received for publication, November 21, 2002
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ABSTRACT |
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Human tripeptidyl-peptidase I (TPP I, CLN2
protein) is a lysosomal serine protease that removes tripeptides from
the free N termini of small polypeptides and also shows a minor
endoprotease activity. Due to various naturally occurring mutations, an
inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2).
In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the
endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells.
Human TPP I was initially identified as a single precursor
polypeptide of ~68 kDa, which, within a few hours, was converted to
the mature enzyme of ~48 kDa. Compounds affecting the pH of
intracellular acidic compartments, those interfering with the
intracellular vesicular transport as well as inhibition of the fusion
between late endosomes and lysosomes by temperature block or
3-methyladenine, hampered the conversion of TPP I proenzyme into the
mature form, suggesting that this process takes place in lysosomal
compartments. Digestion of immunoprecipitated TPP I proenzyme with both
N-glycosidase F and endoglycosidase H as well as treatment
of the cells with tunicamycin reduced the molecular mass of TPP
I proenzyme by ~10 kDa, which indicates that all five potential
N-glycosylation sites in TPP I are utilized. Mature TPP I
was found to be partially resistant to endo H treatment; thus, some of
its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser475 on human TPP I
maturation in cultured cells demonstrated that although TPP I zymogen
is capable of autoactivation in vitro, a serine protease
that is sensitive to AEBSF participates in processing of the proenzyme
to the mature, active form in vivo.
Degradation of polypeptides requires the collective action of
various endo- and exopeptidases, finally releasing free amino acids and
dipeptides reused in the cell cytoplasm according to the metabolic
needs of the cell. Two tripeptidyl peptidases identified to date in
mammalian cells sequentially cleave tripeptides from the N termini of
oligopeptides: tripeptidyl peptidase I (TPP
I,1 CLN2 protein) and
tripeptidyl peptidase II (TPP II) (for a recent review, see Ref. 1).
TPP II is a cytosolic enzyme that belongs to the subtilisin subclass of
serine peptidases (2). TPP I (EC 3.4.14.9) is a lysosomal exopeptidase
with an acidic pH optimum (3, 4) and a minor endoprotease activity (5). Naturally occurring mutations in TPP I are associated with a fatal lysosomal storage disorder, the classical late infantile form of
neuronal ceroid lipofuscinosis (CLN2, Jansky-Bielschowsky disease) (6,
7). This autosomal recessive disorder starts at the age of 2-4 years
with poorly controllable seizures and dementia, followed by visual loss
and cerebellar and pyramidal and extrapyramidal signs, leading to death
in the second decade of life. Rare, atypical cases with later onset of
the disease and more protracted course also have been documented (for a
recent review, see Ref. 8). Curvilinear profiles, lysosomal inclusions
typical for CLN2, have been demonstrated ultrastructurally in amniotic
fluid cells from around 16 weeks' gestation and in fetal skin and
lymphoblasts from around 20 weeks' gestation (9), which correlates
well with the early expression and developmental regulation of TPP I
(10-12).
TPP I in humans is encoded by a gene mapped to chromosome 11p15 (13).
The deduced amino acid sequence of TPP I consists of 563 amino acid
residues and includes a 19-amino acid signal sequence and a 176-amino
acid propeptide removed during the maturation process to yield a mature
enzyme of 368 amino acid residues (6, 14, 15). By SDS-PAGE, the mature
enzyme, which was purified from human osteoclastomas (3), rat spleen
(4, 5) and kidney (16), and bovine and human brain (15, 17), has an apparent molecular mass of 46-48 kDa, whereas the proenzyme has a mass
of 66 kDa (15, 18). However, by nondenaturing PAGE and gel filtration,
the molecular mass of the rat TPP I was calculated to be 280 kDa and
290 kDa in the absence and presence of Natural substrates of TPP I are unknown; however, it appears that this
peptidase is involved in degradation of small unstructured polypeptides
with unsubstituted N terminus and uncharged amino acid in the P1
position (4, 19). In vitro, TPP I cleaved peptide hormones
such as angiotensin II, glucagon (4), substance P (17), angiotensin
III, and neuromedin B (16) as well as synthetic amyloid- The deduced amino acid sequence of TPP I zymogen has five potential
N-glycosylation sites at amino acid positions 210, 222, 286, 313, and 443. Like many other lysosomal hydrolases, TPP I proenzyme is
able to autoactivate in the acidic pH in vitro (15, 22).
However, the role of glycosylation for the biology of TPP I and the
process of maturation of TPP I zymogen in vivo have not yet
been examined. Here we addressed these issues by analyzing the
biosynthesis, glycosylation, and processing of hTPP I overexpressed in
Chinese hamster ovary (CHO) cells. Our data suggest that maturation of
TPP I takes place in lysosomal compartments and that glycosylation enables intracellular transport and maturation of TPP I. Furthermore, we show that a serine protease that is sensitive to
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF)
participates in processing of the proenzyme to the mature, active form
in vivo.
Materials--
Cell culture medium components were from
Invitrogen. [35S]methionine/cysteine (>1000
Ci/mmol, Tran35S-label) was purchased from ICN. Protease
inhibitor mixture (Complete), AEBSF (Pefabloc SC Plus), E64,
endoglycosidase H (endo H), and N-glycosidase F (PNGase F)
and FuGENE 6 transfection reagent were from Roche Molecular
Biochemicals. Protein A-Sepharose and the BCA kit were from Pierce.
Peroxidase-conjugated secondary antibodies and ECL reagents were from
Amersham Biosciences. Monoclonal antibodies to lysosome-associated
membrane protein (LAMP I) (hamster-specific) were from the
Developmental Studies Hybridoma Bank (University of Iowa). Polyclonal
antibodies against calreticulin were from Affinity Bioreagents. Human
brain cDNA library was from Clontech. pcDNA3.1Hygro vector was from Invitrogen, and pET22b vector was from Novagen. Secondary antibodies conjugated to Alexa Fluor 488 were
from Molecular Probes, Inc. (Eugene, OR), and secondary antibodies conjugated to Cy3 were from Jackson ImmunoResearch. Vectashield mounting medium was from Vector. All other chemicals were from Sigma.
TPP I Cloning and Cell Transfection--
Open reading frame
encoding full-length TPP I was amplified by polymerase chain reaction
from human brain cDNA library (Marathon Ready) by using the primers
forward (ccggtaccagaatgggactccaagcctgc) (F) and reverse
(ccgcggccgctcaggggttgagtagag) (R) and subcloned into the
KpnI/NotI site of pcDNA3.1Hygro. CHO cells
were grown in F-12 medium supplemented with 10% fetal calf serum (FCS)
at 37 °C in a humidified atmosphere with 5% CO2. One
day before transfection, the cells were seeded on 35-mm culture dishes.
Cells were transfected by using the FuGENE 6 transfection reagent,
according to the manufacturer's recommendations. Stably transfected
cells were selected by using Hygromycin B (200 µg/ml).
Hygromycin-resistant individual colonies were picked up, expanded into
cell lines, and maintained in selective medium.
To obtain a high level of secretion of TPP I proenzyme for in
vitro studies, we used CHO cells deficient in dihydrofolate reductase (CHO-DHFRneg) (ATCC CRL-9096) transfected with
plasmid encoding the dihydrofolate reductase (DHFR) and TPP I. The
EcoRI/SphI fragment encompassing mouse
dhfr cDNA cassette was isolated from plasmid
pSV2-dhfr (ATCC), blunt-ended, and ligated in the same
direction as the cytomegalovirus promoter into
SspI-digested plasmid pcDNA3.1Hygro to make plasmid pcDNA-DHFR. cDNA encoding full-length hTPP I was PCR-amplified from the cDNA library, as above, by using primers F and R,
restricted with KpnI and NotI, and introduced
into corresponding restriction sites of pcDNA-DHFR expression
vector to make pcDNA-DHFR-TPP I. The structural integrity of the
insert was verified by dideoxy-mediated sequencing of the entire
insert. Purified and linearized plasmid was transfected into
CHO-DHFRneg cells grown in Iscove's modified Dulbecco's
minimal essential medium supplemented with 0.1 mM
hypoxanthine and 0.016 mM thymidine and 10% FCS by using
FuGENE 6 transfection reagent, as above. Two days after transfection,
cells were subcultured and incubated in selection medium
(ribonucleotide-free), supplemented with 10% FCS and hygromycin B (200 µg/ml). Single colonies were picked up by use of cloning rings,
expanded, and tested for TPP I by using Western blotting and enzymatic
assay. The highest expressors were then used for selection and
amplification with methotrexate, as described (23). That optimization
of cell culture conditions allowed us to obtain around 59 µg of
recombinant TPP I per 1 ml of serum-free medium (OPTI-MEM I)
conditioned for 4 days.
Antibodies--
The cDNA sequence encoding the mature TPP I
enzyme (amino acids 196-563) was expressed in E. coli in
pET22b vector, where it was found mostly in inclusion bodies (not
shown). Recombinant TPP I was purified from inclusion bodies by means
of gel filtration and ion exchange chromatography in the presence of 8 M urea. Purified protein was devoid of any enzymatic
activity toward TPP I substrate Ala-Ala-Phe-aminomethylcoumarin
(AAF-AMC), unstable in aqueous solutions without detergents, and easily
precipitated. Purified TPP I was used to raise both monoclonal and
polyclonal antibodies in mice and rabbits, respectively. Monoclonal
antibodies 8C4 and 2E12 were described by us previously (11). Antiserum
RAS307 raised in rabbits against TPP I was affinity-purified on
recombinant TPP I immobilized on CNBr-activated Sepharose (Amersham
Biosciences). RAS307 does not recognize hamster TPP I (results not shown).
Cell Cultures--
Primary skin fibroblasts from CLN2 subjects
and controls as well as Epstein-Barr-transformed CLN2 lymphoblasts were
from the Cell and Tissue Culture Repository at the Institute for Basic Research. Mouse neuroblastoma cells (N2a) (ATCC CCL-131) were obtained
from the American Type Culture Collection. Cells were maintained at
37 °C in a humidified atmosphere with 5% CO2 either in
Dulbecco's modified Eagle's medium (fibroblasts, N2a) or RPMI 1640 medium (lymphoblasts), supplemented with 10% FCS, 2 mM
glutamine, and antibiotics. For uptake experiments, cells were
transferred to serum-free medium (OPTI-MEM I).
SDS-PAGE and Western Blotting--
Cells were lysed in a buffer
containing 50 mM Tris, pH 7.4, 1% Triton X-100, and
protease inhibitor mixture (lysis buffer). The protein content was
measured by using a BCA method and bovine serum albumin as a standard.
Cell lysates were solubilized in sample buffer, and 2-40 µg of
protein per lane was loaded onto 10% Tris/Tricine SDS-PAGE.
Electrophoretically separated proteins were electrotransferred onto
nitrocellulose membranes. Membranes were subsequently blocked with 5%
nonfat dry milk in phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBST), incubated overnight with primary antibodies, washed extensively
in PBST buffer, incubated with peroxidase-conjugated secondary
antibodies diluted 1:5,000, and developed using the ECL kit.
In Vivo Labeling and Immunoprecipitation--
Subconfluent
(70-90% confluence) cell cultures (35- or 60-mm dishes) were starved
in methionine- and cysteine-free medium for 1 h and then labeled
with 100-250 µCi/ml of Tran35S-label. After a pulse, the
cells and media were either harvested or subjected to chase in full
medium, at the periods indicated. Afterward, the cells were lysed in
lysis buffer, frozen, thawed, and centrifuged to remove nuclei. Lysates
were adjusted to 0.5% Triton X-100, 500 mM NaCl, 0.5×
RIPA buffer (1× RIPA buffer: 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS). Immune complexes were collected by using affinity-purified RAS307
IgG bound to Protein A-Sepharose, washed four times with RIPA buffer
and once with 50 mM Tris, pH 7.4, boiled for 5 min in a
sample buffer, and separated on 10% Tris-Tricine gels. Gels were
electrotransferred, and proteins were visualized by autoradiography.
Deglycosylation Experiments--
hTPP I was in vivo
labeled and immunoprecipitated, as described above. For PNGase F
digestion, after four washes with RIPA buffer, beads were suspended in
0.5% SDS, 1% TPP I Activity Measurement--
Cultured cells were lysed in
0.1% Triton X-100, 20 mM ammonium formate, pH 3.5, and
protease inhibitor mixture. Protein concentration was determined by
using BCA assay and bovine serum albumin as a standard. The reaction
mixture contained 10 µg of cell lysate protein or 10 µl of
clarified cell culture medium and 0.25 mM substrate
(AAF-AMC), 0.1% Triton X-100 in a total volume of 100 µl of 0.1 M sodium acetate, pH 3.5. The reaction was carried at 37 °C for 30 min and was terminated by the addition of 50 µl of 10% SDS. These reaction conditions allowed for efficient activation of
the immature TPP I present in the sample (if any) as well as reliable
measurement of specific activity toward the reporter substrate.
Liberated 7-amino-4-methylcoumarin was measured fluorometrically on
CytoFluor (Applied Biosystems) (excitation 360 nm, emission 460 nm)
after alkalizing the solution by adding 50 µl of 1 M
Tris-HCl, pH 9.0.
Immunofluorescence Microscopy--
For double immunostaining,
CHO cells grown in LabTek chamber slides were fixed with methanol for
20 min at 4 °C. Nonspecific binding sites were blocked with 10% FCS
in PBS for 1 h. After incubation with primary antibodies, either
mAb 8C4 against TPP I and pAb to calreticulin or pAb RAS307 and mAb to
LAMP I in 10% FCS in PBS overnight at 4 °C, cells were washed in
PBS and incubated for 1 h at room temperature with
species-specific secondary antibodies conjugated with fluorescent dyes:
Alexa Fluor 488 (for TPP I and RAS307; green fluorescence) and Cy3 (for
LAMP I and calreticulin; red fluorescence). The cover slips were
mounted with Vectashield and viewed with a Nikon Eclipse E600
laser-scanning confocal microscope. Omission of the primary antibodies
was used as a control of the method.
Biosynthesis of hTPP I--
hTPP I expressed in CHO cells under
the control of the cytomegalovirus promoter was present in the
lysosomes, as revealed by double immunostaining and laser-scanning
confocal microscopy (Fig. 1A),
thus similar to endogenous enzyme in human cells (5, 11). On
immunoblots of cell lysates, the majority of hTPP I appeared as a
48-kDa species representing a mature form of the enzyme and as a minor
species with a mass of ~68 kDa, corresponding to the proenzyme (Fig.
1B, lane 1). hTPP I proenzyme also was found on immunoblots of conditioned media (Fig. 1B,
lane 2), but its mass was about 2 kDa higher than
that of its cellular counterpart. However, after PNGase F treatment
(endoglycosidase cleaving off all asparagine-linked oligosaccharides),
the SDS-PAGE mobility of intracellular and secreted proenzyme was
identical (Fig. 1C). Thus, the observed difference in mass
was caused by dissimilar oligosaccharide structures on secreted and
intracellular proenzyme of hTPP I, a finding that was reported for
other overexpressed mature acid hydrolases (24, 25) or their proforms
(26). CHO cells also secreted very small amounts of mature enzyme,
which could be visualized only after prolonged exposure of
autoradiograms and immunoblots following immunoprecipitation (see
below).
To analyze the biogenesis of hTPP I, CHO cells were pulse-labeled with
[35S]methionine/cysteine and harvested after various
periods. hTTP I was immunoprecipitated using RAS307 (affinity-purified
rabbit anti-hTPP I antibody), separated by SDS-PAGE,
electrotransferred, and autoradiographed. Autoradiographic images of
the respective blots showed that hTPP I is synthesized as a precursor
protein with an apparent molecular mass of ~68 kDa (Fig.
2A). Time resolution of this
process by pulse/chase experiments revealed that the precursor disappeared slowly, whereas a species of ~50 kDa corresponding to the
newly synthesized mature enzyme increased in intensity. At 8 h of
chase, a band with mass of ~50 kDa started to thicken and form a
distinct doublet, which at 24 h of chase appeared as two well
separated bands with masses of 50 and 48 kDa. The estimated half-life
(t1/2) of the proenzyme is ~2.3 h, and the
estimated t1/2 of the mature form is ~20 h.
Given that under steady-state conditions, mature hTPP I appears as a
single band with a mass of ~48 kDa (see Fig. 1B),
posttranslational modification giving rise to two species of the
processed hTPP I observed on autoradiograms could result either from
oligosaccharide trimming or additional proteolytic cleavage of a small
fragment of maturing enzyme. Because conversion of the 50-kDa to the
48-kDa species started late during the biosynthesis of hTPP I (after 8 h of chase), we reasoned that most likely it takes place in the
lysosome. hTPP I proenzyme, like many other acid hydrolases labeled
with mannose 6-phosphate (Man-6-P) recognition marker, is internalized
by cells from extracellular milieu mostly through mannose 6-phosphate
receptor (MPR)-mediated endocytosis (22). Thus, to more closely
characterize the nature and subcellular localization of this late event
of hTPP I maturation, we analyzed intracellular processing of the
proenzyme endocytosed by CLN2 lymphoblasts. These cells are devoid of
endogenous hTPP I as a result of disease-associated mutation (27). As
shown in Fig. 2B, hTPP I proenzyme added to the culture
medium was taken up by CLN2 lymphoblasts and converted first to
a 50-kDa species and then, between 8 and 24 h after
administration, to a 48-kDa species. We obtained similar results for
mouse neuroblastoma cells (N2a) (not shown). Because under normal
conditions endocytosed ligands need ~30 min to reach lysosomes (28),
these data provide further evidence that conversion of the 50-kDa to
the 48-kDa species does in fact occur in lysosomes. This experiment
also revealed that the final trimming of maturing enzyme is not
restricted to overexpressing cells but represents part of the normal
processing of the enzyme.
To examine whether the 2-kDa trimming of maturing hTPP I results from
the action of lysosomal glycosidases, CLN2 lymphoblasts were maintained
for 8 and 24 h in media supplemented with hTPP I proenzyme and
then lysed and subjected to PNGase F treatment. Longer running of the
gel allowed better separation of the proteins in the range of 50 kDa
and visualization of two species of maturing hTPP I with masses of
~50 and ~48 kDa (Fig. 2C). The higher band was
distinctly stronger after 8 h of cell exposure to the proenzyme, whereas the lower band was much more prominent 24 h after
proenzyme administration. Upon PNGase F treatment, the apparent
molecular mass of deglycosylated mature hTPP I analyzed at both time
points was the same (Fig. 2C). Thus, we conclude that the
late step of maturation of hTPP I includes carbohydrate trimming in the
lysosome. Trimming of oligosaccharide residues on acid hydrolases in
the lysosomes was also documented for glucocerebrosidase (29) and Intracellular Transport and Maturation of hTPP I--
Most acid
hydrolases are synthesized as preproenzymes in the rough endoplasmic
reticulum (ER), where the signal peptide is cleaved co-translationally
and the precursors undergo asparagine-linked glycosylation and
carbohydrate processing, which continues in the Golgi apparatus (for a
review, see Ref. 30). In the ER-Golgi intermediate and in the
cis-Golgi compartments, lysosomal hydrolases acquire a
Man-6-P marker. In the trans-Golgi network, the
"uncovering" enzyme removes the covering GlcNAc residues (31),
which allows for the specific, high affinity binding of the Man-6-P
label of acid hydrolases to one of the two MPRs and their further
vesicular transport to the endolysosomal system (for a review, see Ref. 32). In the trans-Golgi network, some acid hydrolases enter the constitutive secretory pathway and are secreted. Following dissociation from MPRs in late endosomes, which is
pH-dependent and proceeds at pH below 5.5, acid hydrolases
are converted to their mature forms either in prelysosomes or
lysosomes. Studies in cells devoid of MPRs or deficient in the enzyme
involved in the first step of generation of the Man-6-P marker
(UDP-N-acetylglucosamine:lysosomal enzyme
phosphotransferase) evidenced the existence of other, MPR-independent and cell type-specific pathway(s) for delivery of acid hydrolases to
lysosomes (33).
To investigate intracellular transport and the site of maturation of
hTPP I, first we examined the effect of brefeldin A (BFA), monensin,
and bafilomycin A1 (bafA) on the processing and secretion of the enzyme. All of these compounds alter intracellular vesicular transport of lysosomal enzymes; however, their major site(s) action and
molecular mechanisms differ. BFA produces disassembly and redistribution of the Golgi complex into the ER, most probably due to
the inhibition of some of the proteins that activate ADP-ribosylation factors (for a recent review, see Ref. 34). Monensin is a carboxylic proton ionophore that neutralizes the intracellular acidic organelles. However, the major effect of monensin appears to be associated with the
inhibition of the function of the trans cisternae of the
Golgi apparatus, often near the point of exit of secretory vesicles or
at low monensin concentration or short exposure time in the midregion
of the stacked cisternae (for a review, see Ref. 35). bafA is a
specific inhibitor of vacuolar ATPase, a major proton pump that
generates H+ gradients across the membranes of
intracellular acidic compartments (36). Thus, like weak bases, monensin
and bafA disturb normal trafficking of lysosomal enzymes via a
pH-dependent MPR-mediated pathway by alkalizing
intracellular acidic compartments. As a consequence, normal sorting,
secretion, proteolytic processing, and endocytosis of lysosomal enzymes
are altered, and protein degradation within lysosomes is inhibited
(37-40). At least some of the effects exerted by bafA can be
attributed to inhibition of fusion between late endosomes and lysosomes
(41). It has not yet been documented whether monensin produces a
similar effect. However, monensin prevents the formation of complex
oligosaccharides on lysosomal enzymes, which can result either from
bypassing of the trans-Golgi compartment by lysosomal
enzymes or from inhibition of the oligosaccharide-processing enzymes in
this compartment (42). Thus, the biological effects of bafA and
monensin partially overlap, despite their molecular mechanisms being different.
CHO cells were pulsed for 1 h and chased for 3 h in the
presence of BFA, bafA, and monensin, and hTPP I from both cell lysates and media was immunoprecipitated and analyzed by autoradiography and
Western blotting. Treatment by all compounds tested caused a
significant inhibition of hTPP I maturation, with increased cellular
levels of hTPP I proenzyme (Fig.
3A, upper
panel). When the maturation rate was expressed as a ratio of
the precursor to the mature form by scanning the pixel density of the
autoradiograms, the following values were obtained: for control cells,
2; for BFA-treated cells, 8; for bafA-treated cells, 6, and for
monensin-treated cells, 5. These findings indicate that BFA was the
most potent inhibitor of hTPP I maturation. However, neither of the
compounds tested completely inhibited maturation of hTPP I. We believe
that this was due to incomplete block of the MPR-mediated lysosomal pathway by tested compounds under the experimental conditions used.
Consistent with our data, monensin incompletely blocked the
segregation, transport to lysosomes, and maturation of another lysosomal proteinase, cathepsin D, in human skin fibroblasts, hepatoma
cells, and monocyte cell line (38). However, we cannot exclude the
possibility that CHO cells are able to utilize an alternative pathway
of acid hydrolase delivery to lysosomes that is less sensitive to the
drugs applied. Higher doses of monensin and bafA and longer incubation
time led to significant degradation of hTPP I (not shown), which
indicates that like TPP I in vitro (17), hTPP I is unstable
under alkalizing conditions also in vivo.
All tested compounds also affected secretion of hTPP I proenzyme into
the medium (Fig. 3A, lower
panel). BFA completely inhibited secretion of hTPP I
proenzyme. In contrast, monensin and bafA increased secretion of hTPP I
proenzyme. Increased secretion or even induction of secretion of the
proenzymes of acid hydrolases is a well documented effect of alkalizing
compounds including monensin (37, 38) and bafA (39), which divert
enzymes normally destined to lysosomes into the secretory pathway. The
level of hTPP I proenzyme in cell secretions was slightly increased by the presence of Man-6-P in the culture media. Thus, inhibition of
MPR-mediated endocytosis after bafA and monensin treatment could not
significantly contribute to increased levels of hTPP I proenzyme in the
media after treatment with both these compounds. Of note, the apparent
molecular mass of hTPP I proenzyme secreted to the culture media by
monensin-treated cells was slightly lower than in controls. This most
likely reflects altered terminal glycosylation of hTPP I in
monensin-treated cells, an effect elicited by monensin on numerous
other glycoproteins (for a review, see Ref. 35).
Analysis of autoradiograms also showed very small amounts of mature
enzyme with a mass of ~50 kDa in cell secretions (Fig. 3A,
lower panel), which were slightly increased after
bafA treatment. The presence of trace amounts of partially processed
species of hTPP I with a mass of ~50 kDa in conditioned media most
likely results from limited proteolysis of the proenzyme in the
extracellular milieu.
To see whether the compounds studied also affected the steady-state
pool of hTPP I, the enzyme was immunoprecipitated from CHO cells and
culture media and visualized on immunoblots by using mAbs to hTPP I. All compounds tested increased the cellular level of hTPP I proenzyme,
most prominently BFA (Fig. 3B). In conditioned media, hTPP I
proenzyme was absent after BFA treatment, whereas its level was
increased after monensin administration. Of interest, in four
independent experiments, both bafA and monensin also increased the
secretion of fully processed mature enzyme with a mass of ~48 kDa;
however, the effect of bafA was much more prominent. This finding,
together with our data indicating that processing of hTPP I leading to
the formation of a ~48-kDa species takes place in lysosomes, implies
that bafA and, to a lesser extent, also monensin induce secretion of
mature hTPP I from lysosomes to the culture media. The similar effect
of monensin and bafA was previously reported also for other acid
hydrolases (37, 43).
Our data showing that maturation of hTPP I was hampered by inhibition
of the intracellular transport system or alkalization of the acidic
compartments suggested that in vivo, this enzyme is
processed to the mature form in the post-Golgi compartments, most
likely endosomes or lysosomes. In an attempt to more closely characterize the intracellular site of hTPP I maturation, we analyzed processing of endocytosed proenzyme after temperature block and 3-methyladenine (3MA) treatment. It is well documented that at temperatures around 20 °C, the receptor-ligand complexes are
endocytosed, but they accumulate in the prelysosomal compartments (28).
3MA, like bafA, blocks the transport from late endosomes to lysosomes but does not affect the pH of these compartments (44). In the presence
of 3MA, the normal fusion between late endosomes and lysosomes was
inhibited in vitro by 50-80% (45).
To analyze the effect of temperature block and 3MA on maturation of
hTPP I, we used N2a cells. Under standard conditions, these cells
internalize hTPP I by using MPR-mediated endocytosis more efficiently
than CLN2 fibroblasts and lymphoblasts (not shown). Because endocytosis
proceeds more slowly at 20 °C than at 37 °C (28), we expected
that any differences in processing of hTPP I at 20 °C could be
visualized more easily in N2a cells than in diseased cells. Cells were
incubated for 4 h at 20 °C and 37 °C and then lysed and
analyzed by Western blotting. Immunoblots of lysates of cells
maintained at 37 °C showed that the majority of internalized
proenzyme underwent maturation, whereas the maturation of internalized
proenzyme was completely inhibited in cells maintained at 20 °C
(Fig. 4A). To confirm that the
proenzyme was indeed internalized and not associated with the cell
membrane after temperature block, cells maintained for 4 h at
20 °C were extensively washed and incubated for an additional 3 h either at 20 or 37 °C in fresh medium without hTPP I. As shown in
Fig. 4B, maturation of the proenzyme was almost completely
inhibited in cells maintained even for 7 h at 20 °C, whereas
the mature form predominated in cells incubated for 4 h at
20 °C followed by 3 h at 37 °C. Strong inhibition of
maturation of endocytosed hTPP I proenzyme was also observed in N2a
cells treated with 3MA (Fig. 4C). Thus, impairment of fusion
between late endosomes and lysosomes by both temperature block and 3MA
inhibited processing of hTPP I proenzyme into the mature form, which
strongly suggests that maturation of hTPP I in vivo takes
place in lysosomal compartments.
Glycosylation of hTPP I--
To characterize glycosylation of hTPP
I, we performed in vivo labeling and in vitro
deglycosylation experiments. Cells were labeled in vivo with
[35S]methionine/cysteine, and hTPP I was
immunoprecipitated and in vitro digested with endo H, which
removes only the high mannose type of asparagine-linked
oligosaccharides, and with PNGase F, cleaving off all
asparagine-linked oligosaccharides.
Immediately after a pulse of [35S]methionine/cysteine,
hTPP I proenzyme was prone to digestion by both endo H and PNGase
F, which reduced the mass of the precursor to ~58 kDa (Fig.
5A). This indicates that the
proenzyme contains only high mannose type, unmodified oligosaccharides
and that this modification accounts for ~10 kDa of the proenzyme
total mass. After a chase of 3 h, mature enzyme was found to be
partially resistant to endo H digestion, demonstrating that some of its
oligosaccharides had been converted to the complex or hybrid type.
However, the proenzyme visualized on autoradiograms was still sensitive
to endo H, which suggests that it represents a portion of the immature
enzyme not yet delivered to the trans-Golgi compartments,
where glycoconjugates of the complex type are formed. Consistent with
this, hTPP I proenzyme secreted to the culture medium was
partially resistant to endo H digestion (not shown).
When the cells were pulse-chased in the presence of tunicamycin, an
inhibitor of N-type protein glycosylation, the cellular hTPP I appeared
as a ~58-kDa species (Fig. 5B), further confirming that
N-type glycoconjugates add 10 kDa to the TPP I mass. Moreover, upon
tunicamycin treatment, maturation of the proenzyme was completely inhibited, whereas its secretion was only slightly reduced, which suggests that hTPP I is targeted to lysosomes but not to the
extracellular milieu via the glycoconjugate-dependent (MPR)
pathway. Of interest, upon tunicamycin treatment, the level of
unglycosylated hTPP I precursor migrating at ~58 kDa was distinctly
higher than the level of hTPP I proenzyme in untreated cells. By
analyzing subcellular distribution of hTPP I after 24 h of
tunicamycin treatment by using confocal microscopy, we observed that a
portion of hTPP I is present in large vesicular structures associated
with the ER compartments, colocalizing with calreticulin, an
ER-resident protein involved in protein folding (Fig. 5C).
These findings suggest that tunicamycin produces retention of a
substantial portion of hTPP I in the ER compartments.
Proteolytic Processing of hTPP I--
It was demonstrated that
autoactivation of TPP I in vitro is a
pH-dependent process, being the most efficient in a narrow pH range of 2.5-4 (15). However, most of the studies indicate that the
lysosomal pH value is around 4.3-5 (46-48). Thus, we hypothesized that because the lysosomal milieu does not provide a favorable environment for spontaneous processing of TPP I zymogen in
vivo, another protease could be involved in this process.
To investigate the enzymatic activity capable of in vivo
processing of hTPP I, CHO cells overexpressing hTPP I were in
vivo labeled and chased in the presence of inhibitors of all major classes of proteases, and TPP I was immunoprecipitated and analyzed by
autoradiography. Neither E64 (an inhibitor of cysteine proteases), pepstatin A (an inhibitor of aspartic proteases), leupeptin (an inhibitor of serine and cysteine proteases), nor phosphoramidon (an
inhibitor of metalloproteases) significantly affected the intracellular
processing of hTPP I (Fig. 6,
A and B). However, AEBSF, a specific, potent, and
irreversible inhibitor of serine proteases, applied at 0.4 mM, almost totally inhibited the proteolytic maturation of
hTPP I (Fig. 6A, lane 3). In some
experiments, the amount of immature enzyme also was moderately reduced
(up to 30%) upon AEBSF treatment in comparison with untreated cells,
whereas the mature hTPP I was invariably absent. Based on morphological criteria, under the experimental conditions used, AEBSF did not produce
cell toxicity.
Because AEBSF may cause nonspecific covalent modification of proteins,
in a separate experimental approach, cells were labeled and chased for
3.5 h in a cold medium to allow the proenzyme to mature, and then
the inhibitors were applied for an additional 20 h of cold chase.
Neither AEBSF nor the other inhibitors studied affected the level of
mature enzyme (Fig. 6, A, lanes 5-8,
and B, lanes 4-6). Thus, it appears
that AEBSF inhibits the process of maturation of hTPP I proenzyme but
does not induce any detectable deleterious effect on enzyme already processed.
Nonlinear regression fit of the inhibition data of hTPP I maturation
in vivo versus AEBSF concentration produced a
hyperbolic curve with an estimated IC50 of ~144
µM (Fig. 6C).
We next compared the effect of AEBSF on proteolytic processing of hTPP
I with that exerted by another serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), as well as AAF-CMK, a specific inhibitor of tripeptidyl peptidases. In contrast to AEBSF (at 0.4 mM), neither PMSF (at 0.6 mM) nor AAF-CMK (at 1 µM) significantly affected the intracellular processing
of hTPP I (Fig. 7A). The level
of secretion of hTPP I was not changed by AEBSF treatment, which
indicates that the absence of the mature form of the enzyme in cells
upon AEBSF administration was not caused by its increased secretion.
AEBSF shows higher stability at physiological pH value and superior
effectiveness in inhibiting a broad range of serine proteases in
comparison with PMSF. Furthermore, specific inhibitory activity of
AEBSF only partially overlaps with leupeptin (e.g. leupeptin
does not inactivate chymotrypsin and related proteases). These factors
can explain the lack of inhibitory effect of PMSF and leupeptin on the
proteolytic processing of hTPP I we observed. The lack of AAF-CMK
effect on this process suggests that the mature hTPP I already present
in lysosomes is not involved in transactivation of hTPP I proenzyme
in vivo.
As an inhibitor of serine proteases, AEBSF could potentially also
interact directly with TPP I and affect its proteolytic processing
in vivo by inhibiting its autoactivation. To investigate this possibility, we examined the effect of AEBSF on hTPP I
autoactivation under in vitro conditions and compared it
with that exerted by PMSF and AAF-CMK. Inhibitors were first
preincubated with the immature hTPP I at pH 7.0 for 30 min and then
incubated at pH 3.5 for 30 min at 37 °C, and the samples were
analyzed by SDS-PAGE and Western blotting by using mAb against hTPP I. Neither PMSF (at 0.6 mM) nor AEBSF (at 0.4 mM)
inhibited self-activation of the hTPP I zymogen in vitro
(Fig. 7B). AAF-CMK (at 1 µM) inhibited autoactivation of hTPP I by ~5%. We also analyzed the ability of
AEBSF to inhibit the activity of hTPP I in vitro toward a
reporter substrate. hTPP I proenzyme was preincubated at pH 3.5 for 20 min to allow for self-activation and then incubated with various concentrations of AEBSF for an additional 30 min. Afterward, TPP I
activity was measured, as described under "Experimental
Procedures." As illustrated in Fig. 7C, AEBSF did not
inhibit activity of hTPP I in vitro toward the reporter
substrate at the concentrations used for in vivo studies
(0.4 mM). Slight inhibition (below 20%) of hTPP I activity
was found only at higher AEBSF concentrations. Thus, the inhibition of
hTPP I processing by AEBSF we observed in vivo could not be
attributed either to its effect on autoactivation or activity of hTPP I.
As an additional control, we examined the effect of
4-(2-aminoethyl)-benzene-sulfonamide (AEBSNH2) on
maturation of endocytosed hTPP I proenzyme in N2a cells.
AEBSNH2 is a structural analogue of AEBSF, which is
inactive in proteolysis, in which the fluoride group is substituted
with -NH2 (49). AEBSF significantly inhibited the
proteolytic processing of endocytosed proenzyme in N2a cells, similar
to its effect on biosynthetically labeled hTPP I in CHO cells. In
contrast, AEBSNH2 did not affect the maturation of
internalized proenzyme (Fig. 7D). We obtained similar
results for CLN2 lymphoblasts and fibroblasts (not shown). This
observation further substantiates our thesis that the effect of AEBSF
on hTPP I processing in cultured cells is associated with its
antiproteolytic activity.
Of interest, AEBSF treatment was not followed by cellular accumulation
of the unprocessed proenzyme, which we observed after inhibition of
hTPP I maturation by tunicamycin, BFA, monensin, or 3MA. In some
experiments, we even observed decreased amounts of the proenzyme in
cells treated with AEBSF. This finding suggested that if not processed
in lysosomes, hTPP I zymogen was rapidly degraded by lysosomal
proteases. To examine this possibility, N2a cells were incubated with
hTPP I proenzyme in the presence of AEBSF and inhibitors of all major
classes of proteases. As illustrated in Fig. 7E, both E64
and pepstatin A co-incubated with AEBSF significantly increased the
amount of hTPP I proenzyme in N2a cells in comparison with that
observed in cells incubated with AEBSF alone. This experiment confirms
that unprocessed hTPP I proenzyme is rapidly degraded in lysosomes and
that cysteine and aspartic proteases participate in this process.
It was recently reported that Ser475 represents the active
site nucleophile of hTPP I (15). Thus, we reasoned that this mutation should disturb the normal processing of TPP I, assuming that it is an
autocatalytic process. Hence, we analyzed the processing and specific
activity of TPP I in primary fibroblasts from a CLN2 subject with a
missense mutation in the active site serine (S475L) in one allele and a
splice site junction mutation (3556G
In summary, on the basis of the experiments presented above, we
conclude that a proteolytic enzyme of the serine type that is sensitive
to AEBSF is involved in processing of hTPP I proenzyme in
vivo.
Biosynthesis and Intracellular Transport of hTPP I--
The
present study demonstrates that hTPP I is synthesized as a zymogen with
an apparent mass of 68 kDa, which is converted within a few hours to a
~50-kDa species and then to a fully processed mature enzyme with a
mass of 48 kDa. The mature hTPP I expressed in CHO cells is a stable
protein with a half-life of ~20 h. In an attempt to identify the
subcellular compartment in which the proenzyme of hTPP I matures, we
used compounds that interfere with ER to Golgi transport (BFA), Golgi
structure and function (monensin), and pH of intracellular acidic
organelles (bafA, monensin) as well as inhibitors of fusion between
late endosomes and lysosomes (bafA, temperature block, 3MA). All of
these treatments inhibited the conversion of hTPP I zymogen to the
mature form. Thus, although some acid hydrolases acquire enzymatic
activity and can function already in the prelysosomal compartments,
such as late endosomes (28, 50), early endosomes (51), or even ER (52),
our data suggest that maturation of hTPP I takes place in the lysosomes.
We also observed trimming of oligosaccharides on the maturing enzyme in
the lysosomes, producing a ~2-kDa reduction in mass, as a late event
of hTPP I maturation in biosynthetically labeled CHO cells and after
endocytosis of the proenzyme by CLN2 fibroblasts, lymphoblasts, and N2a
cells. The role of this late posttranslational modification of hTPP I
function is unclear at present. A detailed analysis of the potential
functional significance of oligosaccharide removal by lysosomal
exoglycosidase in another lysosomal enzyme, glucocerebrosidase, led to
the conclusion that oligosaccharide removal simply reflects further
maturation of the enzyme and is of no importance to its function
(29).
It is well documented that overexpressed acid hydrolases are
enzymatically active; thus, they are correctly targeted and processed into mature forms. However, production of large amounts of
overexpressed enzyme in cells may affect some steps of its normal
biosynthetic pathway. One of the biological effects associated with
overexpression of acid hydrolases is their increased or induced
secretion. Secretion of hTPP I expressed in the CHO cells we observed
was also reported by others (22), although under standard conditions,
endogenous hTPP I was not released by cultured primary cells
(18).2 Thus, selective
secretion of hTPP I appears to result from its overexpression. The
cellular mechanisms responsible for secretion of overexpressed
hydrolases are still not understood. An aggregation-secretion model
proposed in the past (53) suggested that the majority of overexpressed
lysosomal enzymes aggregates in the trans-Golgi network,
becomes inaccessible for MPRs, and is released from cells by default
via the constitutive secretory pathway. However, both our data and the
results of others (22) indicate that even if overexpressed hTPP I
aggregates in the trans-Golgi network, this is a temporary
and reversible process. Another example of posttranslational modifications related to the overexpression of lysosomal hydrolase that
we also observed is the different structure of oligosaccharides on the
secreted and intracellular glycoforms of lysosomal enzymes (24-26). It
is most likely caused by the relative inefficiency of the glycosylation
machinery in the trans-Golgi network to complete the
oligosaccharide chains on enzymes synthesized at a high rate (25).
Because overexpressed hydrolases retain the Man-6-P label and are
active after endocytosis, they represent a good source of enzyme for
the development of enzyme replacement therapy (22, 23, 53).
Of interest, bafA and, to a lesser extent, also monensin not only
increased secretion of hTPP I proenzyme, which is a well documented
effect of these compounds on acid hydrolases (37-39) but also led to
the appearance of the mature, fully processed 48-kDa species in the
culture media. Two possible mechanisms could be responsible for this
phenomenon. First, bafA and monensin could induce secretion of mature
hTPP I from lysosomes by initiating regulated exocytosis of lysosomal
content. In support of this hypothesis, release of mature cathepsin D
and
Until recently, the release of acid hydrolases into the extracellular
environment was believed to be confined to specialized secretory cells
such as mast cells, neutrophils, or cytotoxic T lymphocytes. A
compelling line of evidence indicates that numerous other types of
cells such as fibroblasts, myoblasts, and epithelial cells including
CHO cells release lysosomal acid hydrolases after the fusion of
conventional lysosomes with plasma membrane in a Ca2+-dependent manner (54). The molecular
mechanisms of this process, termed regulated exocytosis, are still not
entirely understood (for a review, see Ref. 55). Recent data suggest
that the lysosomal synaptotagmin isoform Syt VII, a member of the
synaptotagmin family of Ca2+-binding proteins, is involved
in regulated exocytosis of lysosomal enzymes mediating plasma membrane
repair (56). Monensin facilitates the entry of Ca2+ to the
cell by a Na+-out/Ca2+-in exchange (35). Thus,
by increasing the influx of Ca2+ into the cell, monensin
could potentially induce Ca2+-regulated exocytosis of
lysosomal hydrolases. The results of the studies presented below
suggest that bafA also could alter calcium homeostasis in cells. First,
at least in certain types of cells, the vacuolar H+-ATPase
is expressed not only on intracellular vesicular structures but also on
the cell membrane (for a review, see Ref. 57). Second, experiments in
yeast (58) and mammalian cells (59) suggest that vacuolar
H+-ATPase activity may be essential for proper
Ca2+ homeostasis. Third, it appears that at least some
subunits of vacuolar H+-ATPase interact with calcium
channels (60). However, more experiments are needed to determine
whether, indeed, inhibition of activity of vacuolar
H+-ATPase by bafA could affect intracellular ionic
gradients and facilitate Ca2+ entry to the cell, thus
allowing initiation of a series of events leading to exocytosis of
lysosomal enzymes.
Glycosylation of hTPP I--
hTPP I has five potential
N-glycosylation sites (14). Treatment of cell
immunoprecipitates with both endo H, which removes only the high
mannose type of asparagine-linked oligosaccharides, and PNGase F, which
cleaves off all asparagine-linked oligosaccharides, immediately after
pulse with [35S]methionine/cysteine reduced the mass of
TPP I proenzyme by ~10 kDa. This finding indicates that newly
synthesized hTPP I is modified only by high mannose-type glycans. The
same reduction in mass of hTPP I was observed after tunicamycin
treatment, which, by inhibiting UDP-GlcNAc:dolichyl-phosphate
GlcNAc-1-phosphate transferase, precludes N-glycosylation
in vivo. The apparent molecular mass of the completely
deglycosylated hTPP I we observed is in agreement with the estimated
molecular mass of hTPP I deduced from the cDNA sequence. Thus,
taking into account that the molecular mass of a high mannose
N-glycosylation residue is 1,800-2,000, all potential N-glycosylation sites are utilized in hTPP I. Mature hTPP I,
after 3 h of chase, was found to be partially resistant to endo H
treatment, which indicates that some N-linked
glycoconjugates are of the complex/hybrid type.
Glycosylation is a common feature of lysosomal proteins. Carbohydrates
contribute to the proper folding and assembly of newly synthesized
nascent proteins in the lumen of the ER and promote interaction(s) with
the components of the quality control machinery (for a recent review,
see Ref. 61). Oligosaccharides also ensure stability and resistance to
protease digestion of some lysosomal hydrolases and lysosomal membrane
proteins (62-64) as well as intrinsic enzyme activity (65) and
solubility (66), although the removal of the carbohydrate moiety from a
variety of glycoproteins had no apparent effect on their biological
activity or chemical properties (67).
The formation of Man-6-P residues on N-linked carbohydrate
moieties on lysosomal hydrolases is also essential for their binding to
the MPRs in the trans-Golgi and further transport to the
lysosome via the MPR-dependent pathway. According to our
data, the lack of N-linked carbohydrates on hTPP I after
tunicamycin treatment inhibited intracellular trafficking and the
maturation process of hTPP I. This observation demonstrates the
importance of oligosaccharide modification for proper lysosomal
transport and subsequent maturation of hTPP I. Of interest, upon
tunicamycin treatment, the level of unglycosylated hTPP I precursor
migrating at ~58 kDa was distinctly higher than the level of hTPP I
proenzyme in untreated cells. By using confocal microscopy, we observed
that in cells exposed to tunicamycin, a portion of hTPP I is present in
large vesicular structures associated with the ER compartments,
colocalizing with calreticulin, an ER-resident protein involved in
protein folding. It is well documented that inhibition of glycosylation
may alter the proper folding of proteins in the ER, leading to their
aggregation (for a recent review, see Ref. 68). Tunicamycin is able to
trigger the unfolded protein response associated with induction of
numerous ER chaperone proteins such as calreticulin, BiP, or GRP94
(69), leading to stabilization of ER-retained proteins (70). Thus, both
aggregation in the ER and association with ER chaperone proteins could
contribute to the increased stability of the ~58-kDa unglycosylated hTPP I species we observed in cells exposed to tunicamycin. It should
be noted that a portion of unglycosylated immature hTPP I was still
secreted into the media after tunicamycin treatment. This observation
indicates that unglycosylated immature hTPP I was released from the
cells by using the MPR-independent, constitutive secretory pathway.
Proteolytic Processing of hTPP I--
Proteolytic processing
leading to the removal of prodomains of zymogens is a part of the
maturation pathway of most, if not all, lysosomal proteases. Prodomains
of proteases assist in protein folding and inhibition of enzymatic
activity; thus, they prevent nonspecific protein degradation and enable
spatial and temporal regulation of proteolytic activity (71, 72).
Previous studies showed that in vitro, hTPP I zymogen is
capable of self-activating at acidic pH (15, 22). A detailed analysis
of this process at pH 4.0 showed that the zymogen was rapidly
(t1/2 = 7 min) converted to a mature form with a
transient appearance of lower molecular mass species, which disappeared
at longer time points (15). However, because autoactivation of TPP I
in vitro is most efficient in a narrow pH range of 2.5-4
(15), thus at lower pH values than those reported for lysosomes
(46-48), we reasoned that in vivo, another protease could
be involved in this process.
The results of our studies strongly suggest that in vivo,
hTPP I is indeed proteolytically processed by a serine-type protease that is sensitive to AEBSF. According to our data, AEBSF, a potent serine protease inhibitor, did not inhibit hTPP I activity toward a
reporter substrate or its autoactivation in vitro. However, it was capable of inhibiting cleavage of hTPP I proenzyme into the
mature form in cultured cells with IC50 of ~144
µM. Inhibition of maturation of both newly synthesized
hTPP I and of endocytosed hTPP I by AEBSF in lysosomes caused
degradation of unprocessed proenzyme by aspartic and cysteine proteases.
AEBSF is an irreversible and cell-permeable inhibitor of a broad range
of serine proteases. Its charged aminoethyl moiety acts as a substrate
analogue, forming ionic complexes with proteases, whereas the reactive
sulfonyl fluoride group enables formation of a stable covalent bond
with the enzyme (73). Experiments with AEBSNH2, a
structural analogue of AEBSF, provided further support to our thesis
that inhibition of hTPP I maturation in cells treated with AEBSF
resulted specifically from its antiproteolytic action. As we have
shown, AEBSNH2, which is inactive in inhibiting serine
proteases, was unable to prevent proteolytic cleavage of hTPP I
proenzyme in cultured cells.
It was shown previously that a S475A mutant expressed in CHO cells was
catalytically inactive, despite its being proteolytically processed,
although less efficiently than wild-type protein (15). This suggested
that either other protease(s) or CHO cell TPP I could be involved in
the cleavage of the catalytically inactive mutant. We had the
opportunity to analyze proteolytic processing and activity of hTPP I in
fibroblasts from a CLN2 subject heterozygous toward a missense point
mutation in the active site nucleophile Ser475. A splice
site junction mutation present in the other allele of the
cln2 in this individual produces a frameshift after
Phe169, which precludes production of the active enzyme
(27). As we demonstrated, mutant TPP I was processed normally in
cultured fibroblasts, despite the fact that mutation of the
Ser475 produced inactive enzyme. This finding clearly
illustrates that catalytically inactive TPP I could not be responsible
for its self-activation, emphasizing that the cleavage must have been accomplished by an enzyme other than TPP I.
All of these data strongly suggest that although TPP I is capable of
self-activating in vitro, a serine-type protease that is
sensitive to AEBSF is involved in proteolytic processing of TPP I
proenzyme into the mature form in vivo. It should be noted, however, that even if our study implicates a serine protease in TPP I
maturation in vivo, it does not preclude the possibility that under certain conditions, such as sufficiently low pH, maturation of TPP I could also proceed via autoactivation in vivo.
TPP I is up-regulated under various pathological conditions such as
malignancy, neurodegeneration, ischemia, or inflammation (11, 74); its
deficiency causes a fatal lysosomal storage disorder, and to date, it
is the only identified tripeptidyl peptidase acting in lysosomes, which
emphasizes the important role of TPP I for the biology of cells. It is
tempting to postulate that participation of another serine protease in
TPP I maturation could additionally control the functional availability
of TPP I for the cell, apart from its potential regulation at the
transcription level (14), and ensure a cell type-specific response
adequate to the metabolic requirements of the cell under normal and
pathological conditions.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, respectively, which suggests that the enzyme is composed of six identical subunits (16).
peptide
1-42 and 1-28 (17) and most probably collagen (4) and subunit c of
mitochondrial ATP synthase (17, 18), a proteolipid that accumulates in
all types of neuronal ceroid lipofuscinoses except for the infantile
form (20). The activity of TPP I can be inhibited efficiently by the
tripeptide analogue of the substrate Ala-Ala-Phe-chloromethylketone
(AAF-CMK) (3, 4, 17, 21). Recent data have demonstrated that TPP I is a
serine protease inhibitable by 3,4-dichloroisocoumarin and diisopropyl
fluorophosphate, with Ser475 representing the active site
nucleophile and Asp360 and Asp517 being
involved in catalytic activity (15).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol and boiled for 5 min, and the
solution was adjusted to 1% Nonidet P-40, 50 mM sodium
phosphate, pH 7.5, and 0.6 units of PNGase F and incubated overnight at
37 °C. For endo H digestion, immunoprecipitated TPP I was denatured
as above and digested in 50 mM sodium acetate, pH
6.5, overnight with 15 milliunits of endo H, followed by SDS-PAGE and
autoradiography, as described above. Cold hTPP I in cell lysates and
cell secretions was denatured and digested with PNGase F, as above.
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Fig. 1.
hTPP I in CHO cells. A,
subcellular vesicular structures visualized in CHO cells by pAb
RAS307 anti-hTPP I (a, green), colocalize with
structures labeled by mAb to LAMP I (b, red) on a
composite image (c, yellow) generated by a
laser-scanning confocal microscope. Original magnification is ×1000.
B, immunoblot analysis of hTPP I in CHO cell lysates
(lane 1) and culture medium (lane
2). Lane 1, 2 µg of protein per
lane; lane 2, 10 µl of serum-free medium
conditioned for 48 h. Blot developed with mAb 8C4. The
arrow indicates the proenzyme. C, lysates of CHO
cells and cell secretions were either mock-digested or treated with
PNGase F, as indicated, and immunoblotted with mAb 8C4. Ten µg of
protein of cell lysates are shown per lane; 10 µl of serum-free
medium conditioned for 48 h were loaded per lane. The
arrow indicates deglycosylated proenzyme.
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Fig. 2.
Biosynthesis and maturation of hTPP I. A, CHO cells overexpressing hTPP I were pulse-labeled with
[35S]methionine/cysteine for 1 h, chased for the
indicated periods, immunoprecipitated with pAb RAS307 against hTPP I,
electrophoresed, electrotransferred, and analyzed by autoradiography.
The arrow indicates the hTPP I proenzyme; an
arrowhead indicates the mature form. B, CLN2
lymphoblasts with a splice site junction (SSJ) mutation (3556G > A; 3556G > C) in hTPP I were maintained for the indicated periods
in serum-free medium supplemented with 5 µg/ml of hTPP I proenzyme,
lysed, and analyzed by immunoblotting with mAb 8C4. Thirty µg of
protein were loaded per lane. C, CLN2 lymphoblasts with a
SSJ mutation (3556G > A; 3556G > C) were maintained for the
indicated periods in serum-free medium supplemented with 5 µg/ml hTPP
I proenzyme, lysed, and either mock-digested or treated with PNGase, as
indicated, and analyzed by immunoblotting with mAb 8C4. The
arrowhead indicates deglycosylated mature enzyme.
Thirty µg of protein were loaded per lane.
-galactosidase A (25).
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Fig. 3.
Intracellular trafficking of hTPP I. A, cells were pulse-labeled for 1 h with
[35S]methionine/cysteine and chased for 3 h in a
medium containing 10 µg/ml BFA, 0.1 µM bafA, 10 µM monensin, and 5 mM Man-6-P. hTPP I was
immunoprecipitated from cell lysates and conditioned media with pAb
RAS307 and analyzed by autoradiography. To determine whether the
secreted proenzyme was not reuptaken by the cells, pulse-chase
experiments were also done in the presence of Man-6-P. B,
hTPP I was immunoprecipitated with RAS307 from lysates and media of CHO
cells treated for 4 h with 10 µg/ml BFA, 0.1 µM
bafA, 10 µM monensin, and untreated controls, as
indicated, and analyzed by Western blotting with mAb 8C4.
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Fig. 4.
The effect of temperature block and 3MA on
maturation of hTPP I endocytosed by N2a cells. A, cells
were maintained in serum-free medium supplemented with 5 µg/ml of
hTPP I proenzyme for 4 h at 20 °C (lane
1) or 37 °C (lane 2), lysed, and
analyzed by Western blotting with mAb 8C4. B, cells were
maintained in serum-free medium supplemented with 5 µg/ml of hTPP I
proenzyme for 4 h at 4 °C, extensively washed, and incubated
for an additional 3 h either at 4 °C (lane
1) or 37 °C (lane 2). Lysates of
cells were analyzed on immunoblots. C, cells were maintained
in serum-free medium supplemented with 5 µg/ml of hTPP I proenzyme
for indicated periods in the presence of either vehicle only
(Me2SO) or 5 mM 3MA, as indicated, and then
lysed and analyzed by Western blotting with mAb 8C4. Forty µg of
protein are shown per lane.
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Fig. 5.
Glycosylation of hTPP I. A,
cells were pulse-labeled for 1 h with
[35S]methionine/cysteine and chased for 0 and 3 h,
as indicated. hTPP I was immunoprecipitated, denatured, and digested
with endo H or PNGase F or mock-digested, electrophoresed, and analyzed
by autoradiography. The asterisks indicate the mature form.
B, hTPP I was immunoprecipitated from cell lysates and media
of untreated cells and cells treated with tunicamycin (5 µg/ml) after
1 h of pulse with [35S]methionine/cysteine and
3 h of chase, as indicated, and analyzed by autoradiography.
C, untreated CHO cells (a-c) and cells exposed
for 24 h to tunicamycin (2 µg/ml) (d-f) were
double-labeled with pAb RAS307 to hTPP I (a and
d) followed by secondary antibodies conjugated to Alexa
Fluor 488 (green fluorescence) and calreticulin (b and
e) visualized with secondary antibodies conjugated to Cy3
(red fluorescence). Merged images (c
and f) were generated by laser-scanning confocal microscopy.
Original magnification is ×1000.
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Fig. 6.
The effect of protease inhibitors on hTPP I
maturation in vivo. Cells were pulse-labeled with
[35S]methionine/cysteine for 1 h and chased in the
presence of protease inhibitors for 3 h (lanes
1-4 in A and lanes 1-3 in
B), or the inhibitors were excluded from pulse and 3.5 h of cold chase and added for 20 h afterward (lanes
5-8 in A and lanes 4-6 in
B). A, lanes 1 and
5, pepstatin A (1 µM); lanes
2 and 6, E64 (20 µM);
lanes 3 and 7, AEBSF (0.4 mM); lanes 4 and 8,
control untreated cells. B, lanes 1 and 4, phosphoramidon (10 µM);
lanes 2 and 5, leupeptin (10 µM); lanes 3 and 6,
control untreated cells. hTPP I was immunoprecipitated with pAb RAS307,
electrotransferred, and autoradiographed. C,
dose-dependent inhibition of hTPP I maturation in
vivo by AEBSF. Cells were exposed to various concentrations of
AEBSF for 3 h, and then hTPP I was immunoprecipitated and analyzed
by autoradiography and densitometry. Curve fitting was performed with
the help of GraphPad Prism 3.0 (GraphPad Software).
View larger version (36K):
[in a new window]
Fig. 7.
The effect of serine protease inhibitors on
hTPP I maturation in vivo and in
vitro. A, CHO cells were pulse-labeled with
[35S]methionine/cysteine for 1 h and chased for
3 h. hTPP I was immunoprecipitated from lysates and media of cells
treated with 0.4 mM AEBSF, 1 µM AAF-CMK, 0.6 mM PMSF and untreated controls and analyzed by
autoradiography. B, 100 ng of hTPP I zymogen was mixed, on
ice, with AAF-CMK, AEBSF, and PMSF or incubated alone (control) in 10 µl of 5 mM Tris, pH 7.0, preincubated at room temperature
for 30 min, and then diluted 2-fold with 0.1% Triton X-100, 100 mM sodium acetate, pH 3.5 (final concentrations) and
incubated at 37 °C for 30 min. The reaction was terminated by the
addition of sample buffer. Afterward, the samples and nonactivated
control (input) were boiled and loaded onto SDS-PAGE,
electrophoresed, electrotransferred, and analyzed by immunoblotting.
The following final concentrations of inhibitors (in activation buffer,
pH 3.5) were used: 1 µM AAF-CMK, 0.4 mM
AEBSF, 0.6 mM PMSF. C, 5 nM
activated hTPP I was mixed on ice with serially diluted AEBSF in 50 µl of 5 mM Tris, pH 7.0, and incubated for 30 min at room
temperature. Afterward, 50 µl of 200 mM sodium acetate,
pH 3.5, 0.2% Triton X-100, and 250 µM AAF-AMC was added
to each microplate well. The activity of hTPP I was measured as
described under "Experimental Procedures." D, N2a cells
were maintained for 4 h in serum-free medium supplemented with 5 µg/ml of hTPP I proenzyme alone (control) or in medium supplemented
with 0.4 mM AEBSF or 0.4 mM
AEBSNH2, lysed, and analyzed by immunoblotting with mAb
8C4. Forty µg of protein are shown per lane. E, N2a cells
were maintained for 2 h in serum-free medium supplemented with 5 µg/ml hTPP I proenzyme either alone (control) or supplemented with
0.4 mM AEBSF in the absence or presence of 20 µM E64, 1 µM pepstatin A, and 10 µM phosphoramidon and then lysed and immunoblotted with
mAb 8C4. Forty µg of protein are shown per lane.
C) on the other allele
(7). A splice site junction mutation at this position is one of the
most common mutations in CLN2 subjects, and according to our data,
homozygotes for this mutation display no TPP I activity and no
detectable protein on Western blot (27). By enzymatic assay, specific
TPP I activity in control fibroblasts was 1.51 nmol·min
1·mg
1. In fibroblasts from the
compound heterozygote toward S475L mutation, TPP I activity was
undetectable, as in another CLN2 cell line studied (homozygote toward
R208STOP) (Fig. 8A). However,
when we analyzed fibroblast lysates from the subject with S475L
mutation on immunoblots, we observed normally processed TPP I (Fig.
8B). The level of mature enzyme was lower than in control
cells, as anticipated, given that all detected protein derived from
only one allele. Thus, a disease-associated mutation in the active site
of hTPP I has not prevented proteolytic processing of hTPP I proenzyme,
which renders questionable the role of autoactivation for maturation of
hTPP I proenzyme in vivo.
View larger version (12K):
[in a new window]
Fig. 8.
hTPP I in fibroblasts from a CLN2 subject
with a missense mutation in the active site Ser475.
A, specific TPP I activity in fibroblasts from the subject
with the active site Ser475 mutation (S475L), CLN2 subject
homozygous toward R208STOP mutation, and control fibroblasts.
ND, not detected. B, immunoblot of fibroblasts
from the subject with the active site Ser475 mutation
(S475L) (lane 1), fibroblasts from the CLN2
subject homozygous toward the R208STOP mutation (lane
2), and control fibroblasts (lane 3).
Thirty µg of protein are shown per lane (mAb 8C4).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hexosaminidase from lysosomes of human fibroblasts after
monensin treatment (37) as well as induction of
N-acetyl-
-glucosaminidase secretion by mouse macrophages
after bafA treatment (43) has already been reported. Second, hTPP I
proenzyme secreted after bafA and monensin treatment could be processed
into the mature form in the conditioned media, either autocatalytically
or enzymatically (see below). At present, neither of these mechanisms
can be definitively confirmed or excluded on the basis of data we have
collected. However, because hTPP I precursor is synthesized at a
relatively slow rate, the amount secreted during the 3-h chase period
is small, and its steady-state level in cells is distinctly lower than
that of the mature enzyme, it appears that the first scenario is more probable.
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Acknowlegment |
---|
We thank Maureen Stoddard-Marlow for copy editing the manuscript.
![]() |
FOOTNOTES |
---|
* This research was supported in part by the Batten Disease Support and Research Association and by the New York State Office for Mental Retardation and Developmental Disabilities.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: New York State
Institute for Basic Research in Developmental Disabilities, 1050 Forest
Hill Rd., Staten Island, NY 10314. Tel.: 718-494-5208; Fax:
718-982-6346; E-mail: a.golabek@att.net.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M211872200
2 A. A. Golabek, E. Kida, M. Walus, P. Wujek, P. Mehta, and K. E. Wisniewski, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: TPP I, tripeptidyl peptidase I; TPP II, tripeptidyl peptidase II; AAF-CMK, Ala-Ala-Phe-chloromethylketone; CHO, Chinese hamster ovary; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; endo H, endoglycosidase H; PNGase F, N-glycosidase F; DHFR, dihydrofolate reductase; FCS, fetal calf serum; AAF-AMC, Ala-Ala-Phe-aminomethylcoumarin; PBS, phosphate-buffered saline; BFA, brefeldin A; ER, endoplasmic reticulum; bafA, bafilomycin A1; MPR, mannose 6-phosphate receptor; Man-6-P, mannose 6-phosphate; 3MA, 3-methyladenine; PMSF, phenylmethylsulfonyl fluoride; LAMP, lysosome-associated membrane protein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; RIPA, radioimmune precipitation assay; mAb, monoclonal antibody; pAb, polyclonal antibody; hTPP I, human TPP I; AEBSNH2, 4-(2-aminoethyl)-benzene-sulfonamide.
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