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INTRODUCTION |
The
-thymosins are a group of acidic peptides (pI 3.5-4.5)
present in a calf thymus extract denominated thymosin fraction 5 (TF5)1 (1), which shows
immunoregulatory properties in several in vitro and in
vivo assay systems (2). Thymosin
1
(T
1; 28 amino acids) is the most abundant
-thymosin
in TF5 and was the first to be isolated and sequenced (3). A less
abundant TF5 component named thymosin
11
(T
11) has subsequently been characterized (4); this
peptide comprises the T
1 sequence plus an additional 7 residues at its C terminus (i.e. 35 residues in total). Both T
1 and T
11 show immunoregulatory
properties similar to those of TF5 (5).
A polypeptide including T
1 in its structure has been
detected among the translation products of calf thymus mRNAs (6, 7). This apparent precursor of the
-thymosins was later isolated from thymus (8) and other mammalian tissues (9, 10). Sequencing indicated that it comprised 109-111 amino acids, including the sequence of T
11 (and thus T
1) at its N
terminus (11, 12). This protein was denominated prothymosin
(ProT
) (8). ProT
is a highly acidic protein (pI 3.55) with a
highly conserved sequence (13). Its wide distribution in mammalian
tissues suggested that its function was probably not immunological,
despite the apparent immunoregulatory properties of T
1
and T
11. Subsequent studies indicated that ProT
has a
generalized role in the proliferation of mammalian cells, by mechanisms
involving migration to the nucleus (14-16) and cytosolic
phosphorylation of ProT
(17, 18). Reports from our group and others
over recent years have provided increasing evidence that the nuclear
function of ProT
involves interactions with histones (19-21) and
with other proteins related to DNA metabolism, including proliferating
cell nuclear antigen, Cdk2, and cyclin A (22), arguing strongly
for a role in chromatin remodeling. In further support of this view,
ProT
enables nucleosome assembly (20) and modulates the activity of
histone acetyltransferase p300 (23).
Independently of ProT
and its function, uncertainty remains about
the status of T
1 and T
11. When ProT
was isolated in 1984 from thymus extracts prepared under conditions in
which proteolytic activity was prevented, T
1 and
T
11 were not detected in reverse-phase HPLC separation
of the extracts (8). This led to the suggestion that the presence of
ProT
-derived
-thymosins in TF5 was an artifact resulting from
uncontrolled proteolysis during preparation of TF5. In contrast,
experiments performed in our laboratory, using isoelectric focusing
rather than HPLC for
-thymosin detection, have indicated that
T
1 is indeed present in extracts of this type in diverse
mammalian tissues (10).
To resolve this controversy, we have been performing experiments
designed to detect a protease in mammalian cells that is capable of
processing ProT
to generate the
-thymosins. In this paper, we
report the characterization of a lysosomal asparaginyl endopeptidase
with the required specificity. The characteristics of this enzyme match
those of legumain, a cysteine endopeptidase initially known only from
plants (24) and the trematode Schistosoma (25) but later
found in mammals (26). Experiments designed to assess the possible
biological significance of the processing of ProT
by this enzyme are
also presented.
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EXPERIMENTAL PROCEDURES |
Cells and Subcellular Fractionation--
Cells used were
transformed human B lymphocytes (NC-37). For subcellular fractionation,
cells were washed and resuspended (about 2 × 108
cells/ml) in 0.25 M sucrose, 10 mM acetic acid,
10 mM triethanolamine, and 1 mM EDTA, pH 7.4, and then homogenated (15 strokes) in a Potter Teflon glass blender and
then centrifuged (2000 × g for 10 min) to yield the
nuclear pellet. The supernatant was centrifuged at 20,000 × g to obtain the cytoplasmic organelle fraction (mitochondria and lysosomes) as pellet, and the supernatant was centrifuged again at
100,000 × g to obtain the microsome fraction as pellet and cytosol fraction as supernatant.
The nuclear pellet was resuspended (2 × 108
nuclei/ml) in 10 mM Tris-HCl buffer, pH 8.0, containing 10 mM KCl, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 1 mM dithiothreitol, and 0.5 mM PMSF, and then
homogenated (10 strokes) in a Potter Teflon glass blender. After
centrifugation at 20,000 × g for 15 min at 4 °C,
the supernatant was dialyzed against buffer A (50 mM
Tris-HCl buffer, pH 7.5, 5% (v/v) glycerol, 0.5 mM PMSF, 1 mM DTT) to yield the nucleoplasm fraction. The pellet was
resuspended in 10 mM Tris-HCl buffer, pH 7.4, with 0.1 mM MgCl2, 420 mM NaCl, 1 mM DTT, and 0.5 mM PMSF containing 0.5 M NaCl and then incubated for 5 min at 0 °C and
centrifuged at 20,000 × g for 15 min; the supernatant
was then dialyzed against buffer A to yield the nuclear membrane fraction.
The lysosome and mitochondria fractions were purified from the
cytoplasmic organelle fraction by density gradient centrifugation in
two sequential Percoll/metrizamide density gradients (27). Lysosomes,
mitochondria, microsomes, and nuclear membranes were extracted in
citrate/phosphate buffer pH 4.5 or phosphate buffer pH 7.5, both
containing 1% Triton X-114, for 30 min at 4 °C and then centrifuged
at 13,000 × g for 5 min.
Digitonin permeabilization was carried out as described (28). Cells
were resuspended in ice-cold PBS (30 volumes), and the suspension was
brought to 0.02% (w/v) digitonin (diluted from a 14 mg/ml stock
solution). Cells were allowed to permeabilize for 2 min on ice, and the
released cytosolic components were recovered by centrifugation at
800 × g.
Obtention of
-Thymosins--
A heat-stable acidic polypeptide
fraction (including the
-thymosins) was obtained from whole cell
extracts or from subcellular fractions by a modification of the
procedure of Goldstein et al. (1), as previously
described (10). Briefly, frozen cells or subcellular fractions were
pulverized under liquid nitrogen with a chilled pestle and mortar,
dispersed in 10 volumes of boiling 0.15 M NaCl, and
homogenized in a Waring blender. The homogenates were centrifuged, the
supernatants were brought to pH 2.5, and insoluble material was removed
by centrifugation. The resulting supernatants were brought up to 70 mM NaCl, 50 mM Tris-HCl, pH 8, and then loaded
into a DEAE-cellulose chromatography column, which was eluted with 0.4 M NaCl to give the fraction of acidic polypeptides.
ProT
was purified from the acidic polypeptides fraction by
reverse-phase HPLC (RP-HPLC) on a Nucleosyl 300-C18 column (Sugelabor) (5 µm; 4.6 × 250 mm) in a Beckman HPLC System. Elution was done with a programmed gradient of n-propyl alcohol
(0-50% v/v) in a 0.1% aqueous dilution of trifluoroacetic acid. The
resulting purified ProT
was dephosphorylated by incubation for 30 min at room temperature with 1 unit of calf intestine alkaline
phosphatase per µg of ProT
, in 20 mM Tris-HCl, pH 7.8, containing MgCl2 (1 mM) and ZnCl2
(0.1 mM). Dephosphorylated ProT
was obtained
from this reaction mixture by RP-HPLC as indicated above.
Phosphorylation of ProT
with radioactive orthophosphate was done
using ProT
protein kinase, purified as described (18). Briefly, a
phosphorylation reaction mixture containing 50 µg of dephosphorylated
ProT
, 50 mM Tris-HCl, pH 7.4, 150 mM KCl, 26 mM MgCl2, 1.6 mM EGTA, 3.3 mM DTT, 80 ng/ml protamine, 83 mM
-glycerol
phosphate, and 100 mM [32P]ATP (3,000 Ci/mmol) in a total volume of 250 µl was incubated at 37 °C for 45 min. [32P]ProT
was then obtained by RP-HPLC as
described above. T
1 and T
11 were a gift
from Dr. Heimer (Hoffman-Roche).
Isoelectrofocusing and SDS-PAGE--
Isoelectrofocusing was
carried out as described previously (10). Briefly, samples were applied
to PAG plates with a pH range of 4.0-6.5 (Amersham Biosciences)
and electrofocused for 2.5 h (2000 V, 25 mA, 25 watts) in a LKB
Multiphor isoelectric focusing cell thermostatted at 10 °C. The
isoelectrofocused gel was fixed with trichloroacetic/sulfosalycilic
acid and stained with Coomassie Brilliant Blue G. Slab gel
electrophoresis was carried out on 18% polyacrylamide gels by the
method of Laemmli (29).
ProT
-processing Assays--
The reaction mixture contained
different concentrations of ProT
(15-30 µg) plus crude or
purified cell extract (1-15 µg) in 45 µl of 0.1 M
ammonium acetate buffer, pH 4.5, plus 0.5 mM DTT or 0.1 M phosphate buffer, pH 7.5, plus 0.5 mM DTT. In
some experiments, protease inhibitors were added. The mixture was
incubated at 37 °C for 8 h. The reaction products were then
analyzed by SDS-PAGE, RP-HPLC, or isoelectrofocusing, as indicated
under "Results." The structures of the processing products,
obtained by RP-HPLC or from extracts of bands in SDS-PAGE, were
established by analysis of the amino acid composition of their tryptic
peptides, as previously described (10).
Purification of the Lysosomal Asparaginyl
Endopeptidase--
Lysosomes of NC-37 cells were extracted with 20 mM sodium acetate, pH 5.0, 1 mM EDTA containing
1% Triton X-114; the mixture was centrifuged at 13,000 × g for 5 min, and the supernatant was passed through
0.22-µm nitrocellulose filters. The filtrate was then applied to an
Amersham Biosciences Mono-S FPLC column (type HR 5/5) equilibrated with
20 mM sodium acetate, pH 5.2, 10 mM NaCl.
Elution was with a NaCl gradient (10-1000 mM) in the same buffer. 1-ml fractions were collected and tested for asparaginyl endopeptidase activity by fluorimetric assays with the synthetic peptide benzyloxycarbonyl-Val-Ala-Asn-7-amido-4-methylcoumarin (Bachem) as substrate, as described (30). ProT
processing activity was also assayed in the different fractions, as detailed above.
Deglycosylation of the purified protease was carried out by incubation
of aliquots of purified protease in 250 µl of buffer containing 0.1 M citric acid, 0.2 M NaHPO4, 1 mM EDTA, 0.025% CHAPS, pH 7.2, at 100 °C for 5 min.
After cooling, 0.53 milliunits of N-glucidase-F (Roche
Molecular Biochemicals) was added, and the solution was incubated at
37 °C for 24 h. The protease was then precipitated with 10%
trichloroacetic acid and analyzed by immuno-Western blotting as
indicated below.
Immunoassays--
Western blotting assays were performed by
transferring the proteins separated by SDS-PAGE to polyvinylidene
difluoride membranes. A polyclonal legumain antiserum was
custom-produced by Neosystem Laboratories (Strasbourg, France) against
the human legumain sequence fragment KGIGSGKVLKKSPQ, as previously used
for antibody production (30). Neosystem Laboratories also supplied
preimmune serum from the same rabbit.
The effect of the legumain antiserum antibodies on the
ProT
-processing activity of the different cell fractions was
investigated by previous incubation of the fraction of interest with
different concentrations of immune or nonimmune serum, in the absence
or presence of the peptide KGIGSGKVLKKSPQ in PBS, for 1 h at room temperature. ProT
processing was then assayed as described above.
 |
RESULTS |
ProT
-processing Activity in Subcellular Fractions--
To
investigate the intracellular processing of ProT
, we performed a
systematic study of ProT
-proteolytic activities of various subcellular fractions of human lymphoma cells (NC-37), including nucleoplasm, cytosol, nuclear membranes, cytosolic organelles, and
microsome fractions. The effect of pH on proteolytic activity was also
investigated, on the basis of assays at physiological and acid pH.
Analysis by SDS-PAGE of the various reaction mixtures (Fig.
1A) indicated that proteolysis
of ProT
occurred only in reaction mixtures containing the cytosolic
organelle fraction. This proteolytic activity was dependent on pH,
being detected only at pH 4.5. To further localize the proteolytic
activity in the cytosolic organelle fraction, we separated
mitochondrial and lysosomal subfractions from this fraction by density
gradient centrifugation and assayed ProT
-proteolytic activity in
extracts of these subfractions at different pH values. As indicated in Fig. 1B, proteolytic activity was localized only in the
lysosomal subfraction and again required acid pH. Since the lysosomal
extracts were originally prepared at pH 7.5, we investigated the
possible influence of pH during extract preparation. To this end,
lysosomes were extracted at pH 4.5, and proteolytic activity was then
assayed at pH 4.5 or at higher pH. As shown in Fig. 1C,
ProT
-proteolytic activity assayed at pH 4.5 was markedly higher in
extracts prepared at pH 4.5 than in extracts prepared at pH 7.5 (see
Fig. 1B) and markedly higher when assayed at pH 4.5 than
when assayed at pH 6.0 or 8.0. Note that nucleoplasm, microsome,
nuclear membranes, mitochondria, and cytosolic fractions prepared at pH
4.5 still did not show ProT
-proteolytic activity (data not shown).
Moreover, ProT
-proteolytic activity was detected only when disrupted
lysosomes were assayed, not in assays without previous lysosome
disruption (data not shown). An important characteristic of the pattern
of proteolysis of ProT
by the acidic lysosomal lysates is that two of the fragments of ProT
had the same electrophoretic mobility as
synthetic T
1 and T
11 (Fig.
1C).

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Fig. 1.
SDS-PAGE analysis of ProT
proteolysis by different subcellular fractions of human
lymphocytes. Aliquots of ProT (about 20 µg) were incubated
with different NC-37 cell fractions, and each reaction mixture was then
analyzed by SDS-PAGE under the conditions indicated under
"Experimental Procedures." A, analysis of ProT
proteolysis by the nucleoplasm, cytosol, nuclear membrane, cytosolic
organelle, and microsome fractions. Aliquots of about 10 µg of the
nucleoplasmic and cytosolic fractions and that of nuclear membranes,
microsomes, and cytosolic organelles extracted at pH 7.5 were used to
assay the proteolytic activity at pH 7.5 or 4.5. The relative mobility
of ProT is indicated. B, analysis of ProT proteolysis
by the mitochondria and lysosome fractions. Extracts of these
organelles were prepared in phosphate buffer, pH 7.5, and assayed
(aliquots of about 5 µg of extract) at pH 7.5 or 4.5. The
Mr of ProT is indicated. C, effect
of pH on the proteolysis of ProT by the lysosome fraction. The
lysosomes were extracted in citrate buffer, pH 4.5, and assayed
(aliquots of about 5 µg of extract) at pH 4.5, 6.0, or 8.0. The
relative mobilities of ProT and synthetic T 1 and
T 11 are shown in the right lane.
D, effect of different protease inhibitors on ProT
proteolysis by the lysosome fraction. Lysosomal extracts were prepared
in citrate buffer, pH 4.5, and assayed (aliquots of about 5 µg of
extract) at pH 4.5 in reaction mixtures containing (a)
pepstatin (10 µg/ml) plus E-64 (0.1 mg/ml), (b) PMSF (1 mM) plus leupeptin (10 µg/ml), (c) pepstatin
plus E-64 plus PMSF plus leupeptin at these concentrations, or
(d) iodoacetamide (1 mM).
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To further investigate processing of ProT
, we investigated the
influence of various protease inhibitors on the pattern of fragments
obtained with the lysosomal lysate. Analysis of the different reaction
mixtures showed rather surprising results (Fig. 1D), since
the ProT
fragmentation pattern was not modified by any of the broad
spectrum protease inhibitors used (including serine, cysteine, and
aspartic protease inhibitors) but was markedly modified by protease
inhibitor iodoacetamide, which efficiently prevented the processing.
This indicated that a particular cysteine protease activity is involved
in the processing of ProT
.
Since ProT
is phosphorylated by a cytosolic threonine protein kinase
with unknown structure (18), we next investigated the influence of
ProT
phosphorylation on the proteolysis pattern. Specifically, we
compared proteolysis of dephosphorylated ProT
and ProT
phosphorylated in vitro by the purified ProT
protein kinase with [32P]ATP as cosubstrate. Results of these
experiments (data not shown) indicate that phosphorylation of ProT
did not affect its proteolysis by the lysosomal lysates.
The above results suggest the existence of a specific lysosomal
protease activity that is highly dependent on pH and that is able to
cleave ProT
, even in the presence of broad spectrum protease
inhibitors, yielding four fragments, two of which have the same
electrophoretic mobility as T
1 and
T
11.
Characterization of the Products of Processing of ProT
--
To
characterize the different products derived from the processing of
ProT
by the lysosomal protease activity, we used in the first
instance RP-HPLC. The elution pattern for ProT
-processing products
obtained with the lysosomal extract at pH 4.5 showed four peaks (Fig.
2A). Analysis by SDS-PAGE of
these four peaks (inset in Fig. 2A) showed that a
polypeptide with the same electrophoretic mobility as T
1
is the main component in peak 1; peaks 2 and 3 are mixtures of this
component and the other ProT
fragments, and peak 4 is whole ProT
.
Similar elution patterns were obtained when chromatographic conditions
were varied. Characterization of the component in peak 1 was
accomplished by determining the amino acid composition of its tryptic
peptides, as separated by RP-HPLC (Fig. 2B). The tryptic map
and amino acid composition of the peak-1 polypeptide proved identical
to that of T
1 (Fig. 2B). Structural analysis
of the other ProT
-processing products (those eluting in peaks 2 and
3) was carried out by tryptic digestion of the respective bands excised
from the SDS-PAGE gels in which the components of the processing assay
reaction mixtures had been separated. The amino acid compositions of
the tryptic peptides derived from these products and separated by
RP-HPLC (data not shown) indicate that the component with the same
electrophoretic mobility as synthetic T
11 is indeed
T
11, whereas the two fragments with higher
electrophoretic mobility correspond to residues 29-109 and 36-109 of
the ProT
sequence. In view of these structural analyses, summarized
in Fig. 2C, we conclude that the lysosomal protease cleaves
ProT
at Asn-Gly residues located at positions 28-29 to yield
T
1 and at positions 35-36 to yield T
11,
whereas the resulting C-terminal portions of ProT
, positions 29-109
and 36-109, remain undigested. To judge by the concentrations of the respective bands in the SDS-PAGE gel (Fig. 2C), both
T
1 and T
11 are produced in similar
proportions by the lysosomal protease, whereas the concentrations of
fragments 29-109 and 36-109 appear to be lower. However, this
discrepancy should probably be attributed to low efficiency in the
Coomassie staining of the larger (highly acidic) fragments of ProT
,
rather than to a lower concentration in the processing products, since
extracts of the respective bands (from SDS-PAGE) separated by RP-HPLC
prior to structural analysis showed similar spectrophotometrically
determined concentrations to T
1 and T
11
(data not shown). In view of its specificity, then, this protease can
be considered as an asparaginyl-glycyl endopeptidase, and this
specificity is consistent with its ability to generate
-thymosins
in vivo.

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Fig. 2.
A, separation of the ProT -processing
products by reverse-phase HPLC. The products of processing of ProT
by the lysosome fraction were analyzed by RP-HPLC on a Vydac column (5 µm, 300 Å, 4.6 × 150 mm) with a programmed acetonitrile
gradient (0-30%) in 0.1% trifluoroacetic acid in water (flow 0.7 ml/min). Peaks 1-4 were concentrated, and aliquots were analyzed by
SDS-PAGE (inset). B, tryptic mapping of peak 1. 5 µg of the peak 1 concentrate was digested with trypsin, and the
resulting peptides were separated by RP-HPLC. The structures of the
tryptic peptides deduced from their amino acid composition are shown,
together with the T 1 sequence. C, summary of
the structural analysis of the ProT processing products.
Bottom, arrows indicate sites in the ProT
sequence that are cleaved in vitro by the lysosomal
asparaginyl endopeptidase. Top, concentrations of the
different fragments observed in assays in vitro, as
determined by densitometric analysis of SDS-PAGE bands (means of three
assays).
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Identification of the Lysosomal Protease That Processes
ProT
--
The characteristics of the lysosomal protease processing
ProT
in vitro to yield T
1 and
T
11 (including its specificity, its response to pH
variations, and its resistance to protease inhibitors) strikingly
resemble those of the mammalian form of legumain, a protease originally
described in plants and recently reported to be involved in major
histocompatibility complex-restricted antigen presentation in mammalian
cells (30). We therefore performed experiments to investigate whether
the lysosomal asparaginyl endopeptidase that processes ProT
might be
legumain. These experiments included legumain activity assays,
immunodetection assays, and structural characterization. Fractionation
of the lysosomal extracts by ion exchange FPLC demonstrated that
the fractions showing ProT
-processing activity likewise showed
legumain activity, as determined by fluorimetric assay using the
synthetic substrate
benzyloxycarbonyl-Val-Ala-Asn-7-amido-4-methylcoumarin (Fig.
3A). For immunodetection
assays, we used antibodies raised against the peptide
KGIGSKVCCKSCPQ (a fragment of the human legumain sequence that is
coincident with that in other mammals) (30). Western blotting analysis
of the FPLC-purified lysosomal lysates, shown in Fig. 3B,
detected a protein with the same size (46 kDa) as human legumain, at a
concentration similar to that in crude extract. Since active human
legumain is glycosylated (31), we investigated the effect of previous
deglycosylation. In the FPLC-purified lysosomal lysate treated with
N-glycosidase F, the legumain antiserum detected a protein
with lower apparent molecular mass (41 kDa; Fig. 3B); this
decrease is consistent with that reported after deglycosylation of
other mammalian legumains (26).

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Fig. 3.
Characterization of the lysosomal asparaginyl
endopeptidase. A, fractionation of the asparaginyl
endopeptidase activity by FPLC. The lysosomal extract at pH 4.5 (5 mg)
was chromatographed on a Mono-S FPLC column, and proteolytic activity
with ProT (red) or
benzyloxycarbonyl-Val-Ala-Asn-7-amido-4-methylcoumarin
(Z-Val-Ala-Asn-AMC) (blue) as substrate was assayed in
20-µl aliquots of the different fractions collected, as detailed
under "Experimental Procedures." Fractions with the higher
proteolytic activity were combined (purified protease activity).
B, immunoblotting analysis of the lysosomal asparaginyl
endopeptidase. A legumain antiserum (1:400) and a nonimmune serum at
the same dilution were used to probe the lysosomal extracts (15 µg)
(Crude), the purified protease activity (25 µl)
(FPLC-Purified), or the same purified protease activity
following treatment with N-glucidase-F (0.2 milliunits), as described
under "Experimental Procedures." C, effect of the
legumain antiserum on the ProT -processing activity. ProT
processing was assayed with aliquots of the purified protease activity
(25 µl) previously incubated with legumain antiserum (1:2), alone or
in the presence of 20 µg of the peptide KGIGSGKVLKSGPQ used to
raise the serum (see "Experimental Procedures"), with nonimmune
serum (1:2) or with an equivalent volume of reaction mixture buffer
(control). The different reaction mixtures were separated by SDS-PAGE,
and the relative concentrations of ProT and its derived peptides
T 1 and T 11 were determined by
densitometric analysis (means of three experiments).
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To complete the characterization of the enzyme processing ProT
, we
investigated whether the legumain antiserum blocks the proteolysis of
ProT
by the lysosomal protease. As indicated in Fig. 3C,
the antibodies efficiently blocked processing of ProT
, whereas the
corresponding nonimmune serum had no effect. However, no blockade
occurred when the synthetic peptide used to obtain the immune serum was
included in the processing assay reaction mixture (Fig. 3C).
Similar results were obtained using crude lysosomal extracts (data not
shown). These results provide further support for the view that the
asparaginyl endopeptidase responsible for the processing of ProT
in vitro to generate T
1 and
T
11 is legumain.
Processing of ProT
in Vivo--
To further study the processing
of ProT
, we decided to investigate relationships between the
observed proteolysis of ProT
by the legumain in vitro
with the processing seen in vivo. To this end, we compared
the polypeptide pattern observed after ProT
processing in
vitro with the
-thymosins pattern observed in lymphocytes, in
which both ProT
and legumain are known to be present. Whole cell
extracts and subcellular fractions were obtained from lymphocytes (NC-37 cells), in all cases under conditions in which proteolytic activity was strictly prevented.
-Thymosins in the acidic
polypeptide fraction obtained from the diverse extracts were detected
by isoelectrofocusing (IEF), a technique that is highly effective for
the separation and identification of these peptides in different cell
extracts (10). IEF analysis of the
-thymosin components from
whole-cell extracts of NC-37 cells is shown in Fig.
4A. This analysis indicates that NC-37 cells contain components with the same pI as ProT
and
T
1 but not components with the same pI as
T
11 or the acidic fragments 29-109 and 36-109 of
ProT
detected after in vitro processing. Structural
analysis confirmed that the more acidic components were ProT
and
T
1.

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Fig. 4.
-Thymosin presence and
subcellular distribution in vivo. The heat-stable
acidic polypeptide fraction containing the -thymosins was obtained
and separated by IEF as detailed under "Experimental Procedures."
A, -thymosins in whole-cell NC-37 extracts. An aliquot of
the acidic polypeptide fraction (150 µg) from whole-cell extract (2 g
of cells) was separated by IEF, and the Coomassie-stained bands of the
more acidic components were quantified by densitometry, extracted, and
structurally analyzed as detailed under "Experimental Procedures."
Histograms indicate the concentration of ProT and T 1
identified by the structural analysis. Mobilities of synthetic
T 11 and the ProT fragments 29-109 and 36-109
(previously determined by IEF analysis of the respective bands excised
from SDS-PAGE gels in which the components of the processing assay were
separated) are also indicated in the gel. Similar results were obtained
in IEF analyses of heat-stable acidic polypeptide fractions obtained
from five different whole cell extracts. B, subcellular
distribution of -thymosins (fractions obtained by differential
centrifugation). NC-37 cell homogenates (2 g of cells) were
fractionated by differential centrifugation; the heat-stable acidic
polypeptide fraction was obtained in each case, and aliquots of 150 µg were separated by IEF. The more acidic components were analyzed,
as for A. Histograms indicate the concentration of products
identified as ProT and T 1 (means of three
experiments). C, subcellular distribution of -thymosins
(fractions obtained by digitonin permeabilization). NC-37 cell
homogenates (2 g of cells) were fractionated by digitonin
permeabilization as detailed under "Experimental Procedures,"
giving the cytosolic (digitonin-released) and the noncytosolic
(digitonin-retained) fractions; the heat-stable acidic polypeptide
fraction was obtained in each case, and aliquots of 150 µg were
separated by IEF. The more acidic components were analyzed, as for
A. Histograms indicate the concentration of products
identified as ProT and T 1 (means of three
experiments).
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|
This IEF pattern and the similar concentrations of ProT
and
T
1 determined by densitometry (Fig. 4A) are
in agreement with our previous findings indicating that
T
1 is naturally present in various mammalian tissues
(10) and in line with the view that ProT
is processed in
vivo by legumain or another protease with similar specificity to
yield T
1.
We next investigated the intracellular location of T
1 in
NC-37 cells. IEF analysis of
-thymosins in the different subcellular fractions separated by differential centrifugation (Fig. 4B)
and by digitonin permeabilization (Fig. 4C) indicated that
T
1 is located in the cytosolic fraction and is not
associated with any cell organelle. The observed cytosolic location of
ProT
(Fig. 4, B and C) is in agreement with
the tendency of ProT
to leak out of isolated nuclei (15).
It is worth pointing out that the
-thymosins obtained from both
whole-cell extracts and from organelle fractions showed rather "messy" chromatographic behavior; thus, in the RP-HPLC analysis ProT
emerged as a clear peak, but T
1 was retained for
longer by the column and eluted as mixtures with other components in the
-thymosinic fraction (data not shown). Similar behavior was observed under diverse HPLC conditions, both reverse-phase and ion exchange.
 |
DISCUSSION |
Data reported here strongly suggest that the presence of
T
1 in lymphocytes and other mammalian cells is due to
the processing of ProT
by a lysosomal asparaginyl endopeptidase
identified as legumain. This enzyme was recently discovered in mammals
(26), having been known previously only from plants (24) and
invertebrates (25). Mammalian legumain shows high specificity for a
small subset of asparaginyl bonds; it appears to have no specific
requirement for amino acids other than the asparagine, although it
shows a preference for Ala or Pro at position P3 (i.e. the
third residue in the N-terminal direction) (32). The specificity of
legumain with ProT
as substrate is in line with these
characteristics, but it also shows a special requirement for a Gly at
P1'. Specifically, in vitro the enzyme only cleaves Asn-Gly
bonds at positions 28-29 (with an Ala residue at P3) and 35-36 (with
a Pro residue at P3) and appears not to cleave the Asn-Gly bond at
positions 42-43 (which has a Glu at P3) or Asn bonds with other amino
acids (Ala at position 38, Glu at positions 40 and 50).
To judge from the analysis of the
-thymosins in cell extracts
prepared under conditions in which proteolysis was absolutely prevented
(Fig. 4), this protease shows even higher specificity in
vivo, since only T
1 (i.e. cleavage at
positions 28-29) was detected at a concentration similar to ProT
,
whereas T
11 (i.e. cleavage at positions
35-36) was not detected, in line with previous studies of other
mammalian tissues (10). However, we cannot rule out the possibility
that the apparent absence of T
11 is due to rapid
degradation of this peptide in vivo. The nondetection of the
larger ProT
fragment 29-109 (or 36-109) in cell extracts is
likewise presumably indicative of rapid degradation of this fragment
in vivo.
The presence of T
11 at low concentrations in the calf
thymus fraction TF5 (4) might be due to the characteristics of the procedure used for its preparation. In fact, we did not detect this
polypeptide in the
-thymosin fractions obtained (from calf thymus
and other mammalian tissues) under the conditions used here (10,
33).
As noted, the characteristics of the in vivo processing of
ProT
suggest that it is accomplished by legumain. It is worth noting
that additional confirmation of this conclusion by in vivo enzyme blockade is difficult, since specific inhibitors of this enzyme
are not currently available and since the use of Asn-containing peptides as competitive inhibitors is inefficient when incubations of
over 1 h are necessary (30).
In light of the present results, previous failures to detect
T
1 in extracts prepared under drastic denaturing
conditions (8) may perhaps be attributable to the "messy"
chromatographic behavior of
-thymosins prepared under these
conditions, due to their tendency to aggregate, as evidenced in the
present and previous reports (10, 33). In fact, original isolation of
T
1 included gel filtration in the presence of 6 M guanidium chloride (3). In the present and previous
studies (10, 33), we have used isoelectrofocusing, which, unlike the
HPLC procedures used by other authors (8), is effective for separating
and identifying the
-thymosins. In this connection, it is also worth
noting that antibodies raised against fragments of the N-terminal
region of ProT
(the most immunogenic region; such antibodies have
been widely used for immunodetection of ProT
) do not differentiate between ProT
and its N-terminal derivatives T
1 and
T
11.
To judge by the wide distribution of both T
1 (10) and
legumain (26) in different tissues, we would suggest that ProT
processing to yield T
1 is a generalized process in
mammalian tissues. Interestingly, tissues showing high legumain
activity such as lymphoid tissues (26, 30) also show high levels of T
1 (10). The highly conserved primary structure of
ProT
, especially around the asparagine/glycine bonds cleaved by
legumain (13), provides further support for the view that ProT
processing to yield T
1 is generalized. Moreover, the
present results indicate that processing of ProT
in vitro
is independent of its phosphorylation state (ProT
is phosphorylated
at specific residues when cells are activated to proliferate) (17, 18).
The presence of phosphorylated T
1 in proliferating cells
and the inability of the ProT
protein kinase to phosphorylate
T
1 (34) thus suggest that phospho-ProT
may be
processed in vivo to yield phospho-T
1.
Proteolysis of ProT
at the Asp99 residue by caspase 3 has recently been reported to occur in HeLa cells in which apoptosis has been induced (35, 36). This proteolysis has so far not been
demonstrated to be a generalized process occurring in other cell types
undergoing apoptosis and, in any case, occurs in an entirely different
biological context (i.e. apoptosis) from that of the
generation of T
1 in proliferating or nonproliferating cells.
Independently of this, the question remaining is why ProT
is processed by legumain to yield T
1: simply as a
step in the catabolism of ProT
, or in view of some biological
function of T
1? Our current knowledge about mammalian
legumain may perhaps shed some light on this question. Legumain shows
strict specificity for a restricted subset of asparaginyl bonds, which
certainly suggests that it is unlikely to contribute to the gross
catabolism of proteins but rather that it will tend to play more
specific roles in protein processing (32). Of particular interest is the identification of legumain as a key protease in class-II MHC antigen processing (30). In view of these considerations, the proteolysis of ProT
in lymphocytes and other mammalian cells is in
our view likely to be selective processing rather than nonspecific degradation. The fact that T
1 is present at high levels,
similar to those of ProT
, suggests that T
1 may have
some biological function. The cytosolic location of T
1,
its demonstrated incapacity to migrate to the nucleus (15), and its
lack of any known secretion signal (note that ProT
is synthesized in
free polysomes) (37) argue for a nonnuclear intracellular function.
This putative function may or may not be related to that of
ProT
.